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Copepods as live feed

- optimisation and use in aquaculture

PhD Thesis by Per Meyer Jepsen

Department of Environmental, Social and Spatial Changes, Roskilde University, Denmark

Januar 2014

© 2014 Per Meyer JepsenLayout: Per Meyer Jepsen and Ritta Juel BitschFront page: Per Meyer Jepsen and Ritta Juel BitschPrint: Prinfo Paritas Digital Service

ISBN 978-87-7349-858-3

About the author:

Per Meyer Jepsen was born in South Jutland in Denmark, and raised in Haderslev. In 1999 he graduated from high school and moved to Copenhagen working as a construction worker at the Copenhagen Metro. In 2001 he entered Roskilde University with the faith to become a game warden in an African national park, preferable TSavo East National Park in Kenya. But in 2003 he wrote a bachelor project about the calanoids copepod Acartia tonsa, and faith took a new direction revealing a fascinating world of copepods and aquaculture under gentle advisory by Professor Benni Winding Hansen. 10 years later the fascinations is still bur-ning strong.

5TABLE OF CONTENTS

Table of contents

About the author: ......................................................................................3

Acknowledgement ......................................................................................7

Funding .....................................................................................................9

Abstract - English .....................................................................................11

Abstrakt – Dansk .....................................................................................13

1.0 Introduction ......................................................................................15 1.1 Prey size ............................................................................................ 17 1.2 Prey behaviour .................................................................................. 19 1.3 Biochemical profiles .......................................................................... 20 1.4 Copepod order and specie relevant for aquaculture? .......................... 21 1.5 The calanoid copepod Acartia tonsa................................................... 24 1.6 Eggs storage of Acartia tonsa ............................................................ 25

2.0 Presentation of the PhD project .........................................................29 2.1 Semi-intensive ................................................................................... 29 2.2 Intensive ............................................................................................ 31

3.0 Systems to produce copepods as live feed ..........................................33 3.1 Semi-intensive cultures ..................................................................... 33 3.2 Intensive cultures ............................................................................... 35

4.0 Feeding and light regimes for Acartia tonsa .......................................45

5.0 Conclusion and future persepctives ...................................................53

References ................................................................................................59

6 TABLE OF CONTENTS

Manuscripts overview ..............................................................................67

Manuscript 1. ..........................................................................................69A seasonal study on turbot larvae Scophthalmus maximus (Linnaeus, 1758) reared in a semi-intensive outdoor system I: Plankton composition and biomass development .................................................................................. 71

Manuscript 2. ........................................................................................101A seasonal study on turbot larvae Scophthalmus maximus (Linnaeus, 1758) reared in a semi-intensive outdoor system II: Larval growth, prey selection and survival until fry. .................................................................................. 103

Manuscript 3. ........................................................................................133Total egg harvest by the calanoid copepod Acartia tonsa (Dana) in intensive culture – effects of high stocking densities on daily egg harvest and egg quality. Aquaculture Research. ..................................... 135 Manuscript 4. ........................................................................................169Tolerance of un-ionized ammonia in live feed cultures of the calanoid copepod Acartia tonsa Dana .................................................... 171

Manuscript 5. ........................................................................................183Expression of hsp70 and ferritin in embryos of the copepod Acartia tonsa (Dana) during transition between subitaneous and quiescent state ............. 185

ACKNOWLEDGEMENT

Acknowledgement

I am indebted to many people who have been an integral part of my research and private sector carriers. I have had the honour of being exposed to three mentors in my life. My life mentor and daddy, Poul Jepsen, my scientific mentor, Benni W. Hansen, and my private sector mentor, Bent Højgaard. To all of you overcoming my bad jokes still managing an everlasting support and enthusiasm for my thesis or work is an accomplishment in itself, well done!

I am sincerely grateful for professor Benni Winding Hansen for constructive guidance as my Ph.D. supervisor. You have managed the delicate balance of guidance while letting me grow independently with my research.

Bent Højgaard you gave me a chance of showing my worth, before I even finished my master studies. A job with you as my boss, started as an adventure with several travels to Borneo and later around the whole wide-world. Dear Bent, thanks for showing me the world and taught me all the dirty tricks at the rough export market. Thanks to the copepod PhD-student group Eleonora Bruno, Elisa Blanda, Jakob K. Højgaard, Mark Holm, Minh Vu and Thomas A. Rayner. We have laughed and cried together at several occasions, being at months of field work, conferences or just another ordinary day in the lab. PhD-student at ENSPAC in general, thanks for talks, parties and different fun activities, it is always a pleasure. Co-worker at environmental biology thanks for creating a super positive and open work environment.

Dr Drillet, thanks for you never enthusiasm about copepods and aquaculture, I hope you will find peace in your work life, rather sooner than later.

Hans H. Jakobensen, for all you support in the field and unofficially being my second supervisor, if you keep up the work, you just might get a big hug one of these days. Furthermore, a small gratitude towards co-workers and friends in the private Jonas K. Højgaard, Morten B. Mortensen, Christian Selv-Dolph, Esben Holm and other consulters from AKVAgroup.

ACKNOWLEDGEMENT

From Roskilde University specially Anne Faaborg and Rikke Guttesen has been and still are an enormous help. Without your attitude and help, practical env-ironmental biology would be non-existing at the institute.

And finally, I could not have done this without the love and support of my family. Thank to my mom for making me realise that vacuum cleaning can be fun. My two lovely little sisters Puk and Andrea. Puk and Hans-Oluf, thanks for always opening your home for me when travelling back “home” on holidays, birthdays or several other occasions. It is highly appreciated, and thanks for including me in your life with your two lovely kids, Thomas and Theis, it is always fun to be Uncle Per. Andrea, starting in same footsteps as me, but finding your passion within the health sector becoming a nurse. I hope your mission will succeed and that you will heal the world. Furthermore, it is always lovely to drink good red wine together and discuss higher cuisine, with you and Morten. My farther and mentor, Polle, has been my biggest supporter through thick and thin, on his own way. In early life you showed me what a life would be without education, which left a big impression on me, and gave me strength and a good work ethic throughout my studies. Furthermore, you are always so proud of me. Thank you for that, Dad!

9FUNDING

Funding

This thesis was possible with funding from IMProvement of AQuaculture high quality fish fry production (IMPAQ). Further I have received an EliteForsk-rejsestipendium.

Thanks are also due to Roskilde University – Department of Environmental, Social and Spatial Change (ENSPAC) and the Graduate School of Environmental Stress Studies (GESS) for hosting me.

11ABSTRACT ENGLISH

Abstract - English

This thesis addresses the topic of copepods as live feed and how to optimise and use copepods in semi intensive and intensive systems. In order to analyse the use of copepods as live feed a two string approach where used; investigating a semi intensive and an intensive copepod system.

The semi intensive system utilized copepods as live feed for turbot larvae and was evaluated during three outdoor production cycles in 2011 (from May to September). The semi intensive experiment was divided into two studies one investigating the lower trophic levels (phytoplankton and copepods) and their interactions and the other part investigating the higher trophic levels (Copepod and fish larvae). In the semi intensive system a phytoplankton – copepod – turbot larvae food web were established. Decreasing peak values of chlorophyll a were observed from spring to fall. The main governor of the observed phytoplankton was limitations of inorganic nutrients. The lowered phytoplankton peaks trans-lated into a decreasing amount of nauplii, copepodites and adults, over the pro-ductive season. Also successions of copepod species were observed from Acartia spp. in spring to Centropages hamatus during the summer and fall production cycles. When temperature decreased a decrease in egg hatching success were ob-served and it is speculated that the copepods started production of resting eggs, but a later study will investigate these effects. Turbot larvae survival decreased during the productive season and exhibited a higher rate of abnormalities in fall compared to the spring production. Due to the decreased copepod abundances the nutritional demands of the turbot larvae were not meet. These lacks of prey items were more profound during summer and fall. And active selections of nau-plii were observed during the first 10 days of the turbot larvae ontogeny, where after they switched to prey on copepodites and later adult copepods. High daily demand from turbot larvae of copepods as prey were observed, which could not be meet in the current semi intensive system. We therefore recommended a lower stocking of turbot larvae than current practices. The observed differences during the productive season were probably a combined effect of lower inorganic nutrients, phytoplankton and prey field abundance. The main conclusion is that turbot larvae that received a good start, with abundant prey during their early life, will benefit in their later post metamorphic life. This was exhibited, clearly,

12 ABSTRACT ENGLISH

by vital traits, such as increased survival, increased growth, and less deformities in market sized juveniles.

For intensive copepod cultures the focus was on the calanoid copepod Acartia tonsa. The main focus was on understanding the limiting factor to be able to obtain high productivity dense A. tonsa cultures. One of the weaknesses for intensive copepod cultures is that they have not yet been raised in very dense cultures. A literature review showed that densities in cultures are not consistently studied. A study with densities up to 5000 adult A. tonsa L-1 was setup, to investigate effects on mortality and egg quality and production. It showed no density depend mortality or egg quality, although an egg production with ~12,000 egg L-1 d-1 at 2,500 adult A. tonsa L-1 was observed. Therefore 2,500 adult A. tonsa L-1 is suggested to be the optimal density for intensive A. tonsa cultures. Furthermore a lack of studies on water quality in cultures was identified. Therefore a study investigated NH3 and how it restricts A. tonsa nauplii and adults. The focus on NH3 is due to that it is the most toxic compound that occurs in cultures due to excretion from the co-pepods. Nauplii were found to be more sensitive than adults and responses were observed both in mortality and behaviour. Another study was setup to investigate the molecular mechanisms behind the transition of A. tonsa eggs from subitaneous to quiescent eggs. The physiological changes are well studied and most stressors and how the egg physiological responds are known. But the molecular protection mechanism behind is not understood or shown in any calanoid copepods. We showed that the egg especially up regulates ferritin in the transition to quiescence followed by a decline to same levels as before entering into quiescence.

13ABSTRAKT DANSK

Abstrakt – Dansk

PhD afhandlingen handler om optimering af produktionen og brugen af vandlop-per som levende foder. I analysen er todelt og omhandler tildes vandlopper i et semi intensive produktions system og i et intensivt produktions system.

I det semi intensive system blev 3 produktions cyklusser fulgt i 2011 hvor vand-lopper og deres udnyttelse som foder for pighvar larver blev moniteret fra maj til medio september. Studiet blev opdelt i 2 del studier, et der følger de lavere trofiske niveauer (planteplankton og vandlopper), det andet der følger de højere trofiske niveauer (vandlopper og pighvar larver). De forskellige fødekæder, plan-teplankton – vandlopper – pighvar larver blev etablerede og det overordnede fødenet blev moniteret og interaktionerne imellem de forskellige fødekæder blev fulgt. Vi observerede en faldende mængde klorofyl a over produktionssæsonen, hvilket var resultat af at de naturligt forekommende uorganiske næringsstoffer var opbrugt. Grundet den manglende planteplankton faldt vandloppe populationer også, både nauplii, copepoditter og voksne vandlopper blev færre jo længere hen på sæsonen vi kom. Der blev også observeret en succession i vandloppe population hvor Acartia spp. blev udskiftet med Centropages hamatus, der især var domi-nerede i de sidste to produktions cyklusser. Vandloppernes æg klækningssucces faldt især i den sidste produktions cyklus, hvilket sandsynligvis er et resultat af at temperaturen faldt og derfor begyndte vandlopperne at producere hvile æg. På grund af den tidligere konstaterer faldende vandloppe population over sæsonen faldt pighvar larvernes overlevelse også. Endvidere havde de pighvar larver der overlevede flere deformiteter i den sidste produktions cyklus, i forholdet til de to foregående. Dette skyldes at det ernæringsmæssige behov for pighvar larverne især ikke blev opfyldt i produktions cyklus 2 og 3. Vi konstaterede også at pighvar larverne aktivt selekterede for naupliier i de første 10 dage af deres liv. Derefter skifter de over til at spise copepoditter og voksne vandlopper. Generelt havde pighvar larverene en dagligt større behov for føde i form af vandlopper som det semi intensive produktionssystem ikke kunne imødekomme. Vi anbefaler derfor at man starter ud med færre pighvar larver så det semi intensive system kan følge med og opfylde pighvar larvernes ernæringsmæssige behov. Hovedkonklusionen er at pighvar larver der får en god start på livet med rigelige mængder af vandlopper i de korrekte størrelser som fødeemner vil have gavn af det i deres senere liv efter

14 ABSTRAKT DANSK

metamorfosen. Dette var især tydeligt at fiskelarverne der havde haft den bedste start i produktions cyklus 1 havde en øget overlevelse, bedre vækst samt mindre deformiteter da de nåede til salgsstørrelsen. Fokus for den intensive vandloppe kultur var på den calanoide vandloppe Acartia tonsa. Det primære fokus var at forstå den begrænsende faktor for at kunne opnå en høj produktivitets tæthed af A.tonsa kulturer. En af svaghederne for intensiv vandloppe kulturer er at de endnu ikke er blevet dyrket i meget tætte kulturer. En litteratur gennemgang viste at der ikke er nogen konsekvent undersøgelse af vandloppe tætheder i kulturerne. En undersøgelse med tætheder på op til 5000 voksne A.tonsa L-1 blev benyttet for at undersøge effekter på dødelighed, æg kva-litet og æg produktion. Det viste at der ikke er en effekt af densitet på vandloppe dødelighed eller vandloppernes æg kvalitet. Der blev fundet en optimal densitet ved 2.500 voksen A.tonsa L-1 hvor de producerer ~ 12.000 æg L-1 d-1. Endvidere afslørede litteratur studiet at der også er en mangel på undersøgelser af vandkva-liteten i vandloppe kulturer. Derfor blev NH3 og hvordan det begrænser A.tonsa naupliier og voksne undersøgt. Grunden til at fokusere på NH3 er at det er det mest giftige stof der naturligt forekommer i vandloppe kulturer på grund af at vandlopper udskiller det. Undersøgelsen afslørende at naupliier mere sensitive end voksne vandlopper hvilket gav sig til udtryk både i dødelighed og adfærd. En anden undersøgelse undersøgte de molekylære mekanismer A.tonsa æg benytter i transitionsperioden ved overgang fra subitaneous til hvile æg (quiescent). I tran-sitions perioden er de fleste stressfaktorer velundersøget og vandloppe æggenes fysiologiske reaktion er kendt. Imens den molekylære beskyttelse mekanismen bag endnu ikke hverken er forstået eller vist i nogen calanoide vandlopper. Vi viste at ægget især opregulerer ferritin i transitions perioden hvorefter niveauet igen falder tilbage til samme niveau som før.

15CHAPTER 1. INTRODUCTION

Chapter 1

1.0 Introduction

The terminology “Aquaculture” is covering culture of aquatic organisms, including fish, crustaceans, molluscs, aquatic plants etc. Aquaculture is one of the world’s fastest growing food production sectors, creating great potential for food supply and economic benefits. But aquaculture is closely dependent on aquatic ecosystems, inputs of natural resources, and requires continuing support from this natural re-source base (Tacon and Forster, 2003). When aquaculture is compared to capture production, the aquaculture share is about 1/3 of the total world production of fish (Figure 1). In 2030, aquaculture products are predicted to supply of 50% of the world demand (Sofia, 2010).

The world aquaculture production is mainly driven by Asia with China (~36.7x106 tonnes) and India (4.7x106 tonnes) as the biggest producers in the world. Outside

Figure 1. The world fish production divided into the share from fisheries and from aquaculture (Sofia, 2010).

16 CHAPTER 1. INTRODUCTION

Asia the largest producer in the world is Norway (1.0x106 tonnes). The Aquacul-ture production in Asia mainly consists of freshwater species and crustaceans, whereas in Norway the majority is Atlantic salmon (Salmo salar). The majority of aquaculture production is in freshwater (60%), some in marine (30%) and last 10% in brackish waters (Sofia, 2010). For the world capture fisheries production a reverse pattern is revealed, with 14% caught in freshwater and 86% is captured in marine waters (Sofia, 2010). This reveal that there is a high demand for ma-rine fishes and an increasing percentage of this demand has to be supplied from aquaculture, since natural resources are depleted (fig. 1). Nevertheless there is an overall growth in marine aquaculture, the relative share has decreased during the last 20 years from 40% to present 30% (Figure 2).

The major bottleneck for marine aquaculture is supply of fish larvae for the pro-ductions. At current aquaculture hatchery practices, the marine fish larvae have high mortalities and some species cannot be breed. Cultivation of many marine fish species for commercial purposes is limited by the lack of suitable food for the first feeding fish larvae. The majority of live feed for marine fish larvae in aqu-aculture are rotifers (Brachionus spp) and Artemia. In nature, Artemia is never a part of fish larvae prey items, since they are brine shrimps, that live in an extreme

Figure 2. The world aquaculture production (stacked area graph) and relative share (line charts) from the different culture environments.


environment were fish larvae cannot survive. Rotifers are found in gut contents of fish larvae; nevertheless copepods are more prevalent (Munk & Nielsen, 1994). Naturally, this has encouraged an intense research in copepods as live feed in aquaculture, because copepods are nature’s choice. Recently different conference sessions, reviews etc. have highlighted the importance of copepods in aquaculture (e.g. Conceicão, 2010; Drillet et al. 2011; Lee et al. 2005; Schipp, 2006; Støttrup, 2006). In the literature, there is a general agreement that the major benefits using copepods compared to Artemia and rotifers are related to:

• Prey size• Prey behaviour• Biochemical profiles

1.1 Prey size

One of the limitations for the first feeding of many marine fish larvae in aquacul-ture is that the larvae are unable to feed on formulated diets (Cahu and Infante, 2001). This is mainly true for altricial marine fish larvae, which have an undevelo-ped digestive system when the yolk sac is exhausted. For an example diadromous species, such as salmon and trout, where larvae life start in freshwater has large eggs, large larval size and a fully developed digestive system when first feeding is initiated (table 1). Due to the large larval size they are capable of start feeding on formulated feed pellets, and do not need live feed, which has been a contributing factor to their success as an aquaculture specie.

Most marine larvae are small when hatched, altricial and dependent on live feed. When fish larvae have depleted their yolk sack, it is important that relevant prey sizes are available to feed on to avoid starvation and death. In terms of prey size, the relationship between fish larval mouth size and prey size is a decisive factor in the capacity of fish larvae to deal with prey (Cunha and Planas, 1999). In general, prey size is a matter of two factors either prey is considered too small to be energetic profitable to feed or prey size are too large for first feeding fish larvae to be able to feed on. Prey sizes are often measured as both a length and a width, but since live prey are oblongate they are swallowed head first (Cunha and Planas, 1999). Thereby, the prey width is the determined prey measurement.


Table 1 shows different sizes of marine and freshwater fish larvae and eggs.

Size of eggs and larval lenght at hatching in different fish species (modified from Jones and Houde, 1981)

Species (Common & latin name) Egg diameter (mm)

Lenght of lar-vae (mm)

Larval envi-ronment

Atlantic salmon (Salmo salar) 5.0 - 6.0 15.0 - 25.0 FreshwaterRainbow trout (Oncorhynchus mykiss) 4.0 12.0 - 20.0 FreshwaterCommon carp (Cyprinus carpio) 0.9 - 1.6 4.8 - 6.2 FreshwaterSea bass (Dicentrarchus labrax) 1.2 - 1.4 7.0 - 8.0 SaltwaterSeabream (Sparus aurata) 0.9 - 1.1 3.5 - 4.0 SaltwaterTurbot (Scophthalmus maximus) 0.9 - 1.2 2.7 - 3.0 SaltwaterSole (Solea solea) 1.0 - 1.4 3.2 -3.7 SaltwaterGrey mullet (Mugil cephalus) 0.9 - 1.0 1.4 - 2.4 SaltwaterGrouper (Epinephelus tauvia) 0.8 - 0.9 1.4 - 2.4 SaltwaterBream (Acanthopagrus cuvieri) 0.8 - 0.9 1.8 - 2.0 Saltwater

Figure 3 (left): Relationship between the gape width of Sciaenops ocellatus and the consumed prey width of different prey types, zooplankton, rotifers and Artemia (Turingan et al. 2005). (right): Different copepod species, Artemia and rotifer and their corresponding width (Chesney, 2005).


For S. ocellatus larvae, there is a linear increased prey width as a relationship of gape width (Figure 3 (left)). Secondly, the consumed prey has to be smaller than the total gape width (left at Figure 3). However, the gape width increased faster than the prey width, highlighting the importance of prey with small width for the first feeding larvae stage. Chesney (2005), showed a relationship between dif-ferent copepod prey and the initial width of Artemia nauplii and rotifers (Figure 3 (right)). In comparison copepod nauplii (N1) has a smaller width than Artemia and rotifers. Additionally, different copepod species has both different width and length relationships, so ideal species can be selected for different fish larvae. So in terms of size copepods are superior compared to the rotifers and Artemia and utilizing nauplii sizes would allow breeding of small marine larvae that currently are impossible with rotifers and Artemia.

1.2 Prey behaviour

Evolutionary, marine fish larvae feed on zooplankton, and feeding responses are triggered by two primary components for predator-prey interactions (encounter and attack) (Buskey, 2005; Hoilling, 1959).

Encounter between predator and prey is either by direct contact or by remote detection. Different factors affects the encounter for fish larvae but the most important are speed of predator and prey speed, and the detection range of the predator, also known as the encounter radius. In relation to speed, mean swimming speed of copepods increases as they develop and a faster prey and more difficult to catch for fish larvae (Buskey, 1994). Although if an equal stage distribution of copepods are ensured when they are used as live feed in aquaculture, swimming speed should have a relatively small effect on encounter probability (Buskey, 2005). The same logic applies for Artemia and the free swimming rotifers, which both are slower swimmers than copepods. Artemia and some copepods are known to per-form swarming behaviour (Ambler, et al. 1991; Guldbrandsen, 2001). For visual predators, swarming behaviour of the prey can reduce the encounter rate due to a confusion effect. Therefore, it is important to ensure a heterogeneous distribution of prey in the feeding strategy in rearing tanks. A way to ensure a heterogeneous distribution of prey items is by applying moderate turbulence which has been shown to enhance fish larvae feeding rates (Buskey, 2005). But encounter are not always enough to trigger an attack from a fish larvae.


Attacks occur due to different circumstances. For example many fish larvae are visual predators and need to be able to detect and recognise a prey to exhibit an attack (Buskey, 2005). Many copepod nauplii are not feeding in their first nauplii stage, and this gives them a much lower colour contrast compared to older cope-pod stages (Buskey, 1994). When copepods initiate feeding, they often become more visible because the algae will colour their guts. But in general both Artemia and rotifers are a more visible prey compared to copepods. Another important attack response is prey motion patterns. For example, both copepod and Artemia nauplii exhibit a “jerky zig-zag” behaviour, which has been contributed to be an important triggering factor in marine fish larvae (Buskey 2005; Marcus, 2005; Schipp, 2006). Rotifers mainly exhibit epi-benthic, but also free swimming beha-viour. When live feed are detected and attacked by the fish larva, the attack success depends by the potential escape behaviour exhibited by the prey item. Compared to both Artemia and rotifers, copepods exhibit vigorous escape responses in order to avoid predators. This is either because they sense a hydrodynamic response or due to a photic stimulus (Buskey, 2005; Buskey and Hartline, 2003). Nevertheless copepods have a vigorous escape responds, increased prey consumption is often triggered for marine fish larvae by introducing copepod nauplii, as a complement to either rotifers or Artemia. This is mainly benefitted to that copepods, by their presence either triggered hunting behaviour as a visual response or maybe as che-mical stimuli (Støttrup and Norsker, 1997).

Further the fish larvae have to be able to capture and ingest the prey. But since copepods are marine fish larvae natural prey I consider they are able to do that, and are not discussing this further.

1.3 Biochemical profiles

Nutritional quality can be divided into what is important for copepods themselves and what is the nutritional quality of copepods as prey item.

In terms of Amino acids, Arginine, Histidine, Isoleucine, Leucine, Lysine, Methio-nine, Phenylalanine, Threonine, Tryptophan and Valine have been regarded as essential (Cowie, and Corner, 1963). For Fatty acids in general all the 18-, 20 and 22-carbon chain acids are important, and Linolenic acids (18:3) and Eicosapen-taenoic acids (20:5) are thought to be essential acids for copepods (Kleppel et al.


2005 and references within). An important feature of microalgae used as food is that a high nutritional quality food not always is preferable if the algal cell size is too small, since copepods feed more efficiently on certain particle sizes (Berggreen et al. 1988). There is a cost benefit relationship between copepods and their food items: that the nutritional deliveries per unit of energy spend on capture has to be as high as possible. Therefore, a less high quality food item can be preferable if the cell size for example is 2-3 folds larger than a smaller but more nutritional food item. This is inherent in their stage specific retention spectra (Berggreen et al. 1998). Then again, if copepods are feed a nutritional sufficient and within the correct cell size range, they do not need to be enriched as compared to both Artemia and rotifers. They will keep a high relevant biochemical profile that is relevant for most marine fish larvae. Especially rotifers have a problem with su-staining a high nutritional quality and will lose their nutritional value ~6 hours after enrichment (Hoff, 1996).

In terms of fatty acids copepods are rich in essential fatty acids that are extremely important for larval fish survival and growth (Olivotto et al. 2008; Støttrup and Norsker, 1997; Sun and Fleeger, 1995). For example highly unsaturated fatty acids (HUFA) are essential in marine fish larvae diet (Sargent et al. 1999). Deficient amounts of HUFA results in decreased fish larvae health, anomalities, stress to-lerance, development, pigmentation and growth (Bell et al. 2003; Coperman et al., 2002; Furuita et al., 1999; Olivotto et al., 2006; Vagelli, 2004).

1.4 Copepod order and specie relevant for aquaculture?

Even though copepods are numerous, the number of species cultivated for aqu-aculture is limited to three orders, Cyclopoida, Harpacticoida and Calanoida (Støttrup, 2006). For Cyclopoida around half are marine and the other half inhabits freshwater environments (Mauchline, 1998). Cyclopoida are both com-mersal, parasitic and pelagic associated (Mauchline, 1998). A common trait for cyclopoida is that they are egg carriers (Støttrup, 2006). Cultures of Cyclopoida are the least studied of the three orders and the genera Oithona spp., Paracyclopina spp. and Apocyclops spp. seems to be the best candidates for live feed for marine fish larvae (Lee et al, 2013: Støttrup, 2006). Harpacticoida are primarily marine species (~90%) (Mauchline, 1998). They are mainly epibenthic associated, egg carriers, and the best candidates for aquaculture appear to be the genera Euterpina


spp., Tigriopus spp., and Tisbe spp. (Støttrup 2006). Around 75% of the Cala-noida are marine, and they are mainly pelagic (Mauchline, 1998). Most calanoids are broadcasters, shedding their eggs individually directly in the water column (Støttrup, 2006) (Figure 4). Calanoid copepods are the most studied copepod order, containing the most studied genera in the world, Acartia spp. Acartia spp. is closely followed by Calanus spp., together with Temora spp., Centropages spp., Psedocalanus spp. and Paracalanus spp. (Støttrup, 2006). There is advantages and disadvantage of all three orders, I will not elaborate on this within the frame of this PhD thesis, but for interested the reviews by Støttrup (2006), Schipp (2006) and Drillet et al. (2011) can be recommended. I have choosed to focus upon Ca-lanoid copepods in the present PhD thesis. The reason is that calanoid copepods have the interesting trait that the eggs can be provoked into a controllable resting stage, where the egg can be stored, similar to brine shrimp (Artemia) cysts. This method is successfully applied for Artemia and is one of the major features that ease the distribution of Artemia as live feed for marine aquaculture. The question is then what specie to cultivate?

Figure 4: Phylogenetic relationship between the 10 orders of Copepoda (Mauchline, 1998 and referances within).


The choice of specie is difficult and argues for or against specie is many. For me, an important feature is that the specie is fairly easy to cultivate and that its biology and ecological and use as live feed is well studied. An example of a species that has been cultivated intensively is Gladioferens imparipes (Payne and Rippingale, 2001). Gladioferens imparipes were cultivated for 420 days with the production goal to achived as many nauplii as possible, since they wanted to feed nauplii directly to fish larvae. Other species cultivated in a setup modified from Payne and Rippingales (2001) system is Temora stylifera, Centropages typicus and Calanus helgolandicus (Buttino et al. 2012; Carotenuto et al. 2012). All of the studies are focusing on nauplii production, but have all achieved lower productions than showed by Payne and Rippingale (2001). In general best productions were ob-tained with the neritic species whereas the open water C. helgolandicus obvious performed worst in a confined recirculated production tank. The reason that G. imparipes seems to thrive better that the other species could be that it in its older later stages attaches to surfaces, which is a similar behaviour to most harpactoid copepods. Thereby reserving energy for reproduction instead of swimming activity (Payne and Rippingale, 2001).

In this PhD study, I have approached the topic; copepods as live feed for marine fish larval with 2 angles. First by studying the success of marine turbot larval rea-ring, reared in a semi-intensive outdoor rearing system in Denmark and secondly by focussing on Acartia tonsa intensive culturing.

In the semi-intensive system the species present were the calanoid copepods Acartia spp. and Centropages hamatus. The choices of these species were out of my reach since they had been selective breed by the fish farm Maximus A/S more than a decade ago, and used since. Although the calanoid copepod Temora longicornis has also been reported from the system non where present during our studies in 2011 and 2012 (Engell-Sørensen, 2004). In the intensive system the specie choice was the calanoid copepod Acartia tonsa. I will only describe A. tonsa since it is an active selection for my intensive studies whereas C. hamatus was what was present in the semi-intensive and could change season to season.


1.5 The calanoid copepod Acartia tonsa

Acartia tonsa is a cosmopolitan distributed species found in the Atlantic, Indian and Pacific Oceans, the Baltic-, Black-, Caspian-, Mediterranean-, and North Seas, and the Gulf of Mexico (Mauchline, 1998) (Figure 5).

The planktonic A. tonsa can tolerate temperatures from 1 to 32°C, and salinities from 5 to 72, and can survive sudden change is these conditions (Cervetto, et al. 1999; Chinnery and Williams, 2004; Højgaard, et al. 2008; Ohs, et al. 2009; Paf-fenhöfer and Stearns, 1988). The normal depth distribution of A. tonsa is from 0 to 50m and they exihibit a daily vertical migration from bottom (at day) to surface (at night), as a behavoural repsonse to avoid predators (Cervetto, et al. 1995). Although in the current laboratury culture used in the intensive experiments this trait is lost (Tiselius, et al. 1995).

Acartia tonsa development time is from egg to adult at 17 °C around 14 days, or ~1 stage day-1. Although, the delveopment time is temperature dependent, with faster development time as a function of increased temperature (Hansen, et al. 2010). Acartia tonsa goes trough 1 egg stage, 6 nauplii stages and 5 copepodite stages before reaching adult. Eggs normal development time (duration of embryo-

Figure 5: The distribution of Acartia tonsa, green dots represent one or more sampling stations (OBIS (2014). Data from the Ocean Biogeographic Information System. Intergovernmental Oceanographic Commission of UNESCO. Web. http://www.iobis.org (consulted on 05-01-2014), courtesy to Mark Holm for extraction of data (n=2323)).


genesis) is 48 hours at 17 °C but this is also temperature dependent (Hansen et al. 2010). It is worth noticing that in nauplius stage I A. tonsa do not feed. Acartia tonsa reproduce by sexual reproduction, where the male sense the females hydromechanical signals and start chacing her (Bagøien and Kiørboe, 2005). The male produce 1 spermatopore day-1, and there seems to be evendence that Acartia tonsa performes sexual selection, with larger males and females prefering to mate with each other (Ceballos and Kiørboe, 2010). The life time of A. tonsa is from 14 days to 2-3 months, with a longer female survival (Mauchline, 1998). The average egg production is ~28 eggs female-1 day-1 (Table 2).

1.6 Eggs storage of Acartia tonsa

Acartia tonsa eggs can be categorised into 4 different classes: 1) Subitaneous- 2) Delayed hatching- 4) Quiescent- and 4) Diapause eggs. Egg viability is affected

Egg female-1 day-1 (mean ± S.D.) Reference50.5 ± 1.9 Drillet et al. 2008b37.2 ± 2.6 Drillet et al. 2008b41.6 ± 1.7 Drillet et al. 2008b44.6 ± 2.3 Drillet et al. 2008b10.0 ± 7.1 Drillet et al. 2008b7.4 ± 4.6 Drillet et al. 2006

11.6 ± 7.4 Drillet et al. 200628.5 ± 9.2 Drillet et al. 200632.2 ± 17.8 Drillet et al. 200622.5 ± 8.8 Jepsen et al. 200725.0 ± N/A Støttrup et al. 1986*21.9 ± 5.9 Zhang et al. 201321.9 ± 0.1 Peck & Holste, 2006**25.6 ± 7.5 Peck & Holste, 2006**41.8 ± 4.9 Peck & Holste, 2006**26.9 ± 0.6 Peck & Holste, 2006**17.4 ± 1.2 Peck & Holste, 2006**

*Fed with a mix of R. baltica and Isochrysis spp.**Different salinities, fed with Rhodomonas spp.

Table 2: Litterature reports of laboratory cultures egg production for Acartia tonsa, fed with Rhodomonas baltica.


by different parameters such as temperature, salinity and oxygen. Therefore, the following are assumed to be under standard conditions 17ºC, salinity, 30 and 80-100% O2 saturation.

1. Subitaneous eggs; eggs that hatch within 72 hours at standard conditions.2. Delayed hatching eggs; eggs that hatch after 72 hours and gradually for

weeks at standard conditions.3. Quiescent eggs; subitaineous eggs that are manipulated into an arrested

development by changing different environmental conditions. Quies-cent eggs will resume embryogenesis when they are re-provided with standard conditions. Known manipulators: temperature (Drillet, et al. 2006), salinity (Højgaard, et al. 2008), oxygen (Holmstrup, et al. 2006) and sulphide (Invidia, et al. 2004; Nielsen et al 2006).

4. Diapause eggs; is eggs that are cued into diapause by changes in large scale slowly developing environmental factors, but they will not hatch again even if environmental factors are back to standard conditions. They require a refractory phase of months to years before their internal clock allows them to hatch. Diapause eggs only start to redevelop when the diapause is broken (Marcus, 1996).

Quiescence and Diapause were also defined by Danks (1987) as following:

• Quiescence: “An immediate direct response to a limiting factor”.• Diapause: “A more profound interruption that routes the metabolic

programme of the organism away from direct developmental pathways and into much more clearly organised break in development”.

For Aquaculture purposes only subitaneous and quiescent eggs are interesting, since these two stages are “controllable” whereas delayed hatching and diapause eggs are considered uncontrollable at least with current knowledge. Focus on producing subitaneous eggs that are induced into quiescent ads flexibility into the production since days, weeks or even months of egg production can be poo-led together, stored, and sent out to end users, marine hatcheries. Furthermore, Drillet et al. (2006) has shown that A. tonsa eggs can be “quiescently” cold stored for up to one year, still being viable, and without a significant loss of unsaturated fatty acids. The eggs can also be quiescently stored at room temperature when manipulated into quiescence by transferring the eggs to freshwater (Højgaard et


al. 2008). Last a novel study has showed some of the regulative processes when A. tonsa eggs enters into quiescence (Nilsson et al. 2013) (Figure 6).

The well documented quiescent technique is considered the most promising storage technique for distribution of copepod eggs to aquaculture facilitates world-wide. The eggs can be hatched and the nauplii larvae can be fed to marine fish larvae while still obtaining a high nutritional quality (Drillet et al. 2006; Højgaard et al. 2008). For marine fish hatcheries this gives them the ability to purchase enough copepod eggs to cover their production window were they use live feed for their fish larvae. Therefore, focus on intensive copepod egg producing facilities will create an analogue product to Artemia cyst, which is already proven as being an easy applicable product for marine fish larvae hatcheries. Although it is important to document that delayed or diapause eggs is not unintentionally produced in copepod cultures, since e.g. high densities and bad water quality besides low food availability can induce delayed hatching copepod eggs (Camus and Zeng, 2009; Drillet et al.2011).

Figure 6: Acartia tonsa eggs regulation of Hsp70 and Ferritin during the transition period from subitaneous eggs to quiescent eggs. Grey bars are subitaneous eggs (control) and white bars are eggs in transition (Nilsson et al. 2013).

29CHAPTER 2. PRESENTATION OF THE PHD PROJECT

Chapter 2

2.0 Presentation of the PhD project

I used a two string structure for my PhD thesis, one monitoring a semi-intensive production system during the productive season, another working with some of the challenges for intensive Acartia tonsa cultures.

2.1 Semi-intensive

In 2004 a study were published about rearing of flounder (Platichthys flesus) from yolk-sac larvae and until metamorphose in an outdoor semi-intensive rearing system (Engell-Sørensen et al. 2004) (Figure 7). During my master study at Ros-kilde University in 2006/7 I heard about the outdoor part of this system, since some of my supervisors, PhD-student, were conducting a study their (Sørensen et al. 2007). Later when I worked in a private sector aquaculture company I heard

Figure 7 (left): Maximus A/S fish farm. a) Zooplankton settling pond, b) Outdoor production ponds, c) Indoor RAS, d) Water intake from the nearby estuary. (right): Turbot fry at selling size. (Source left figure: “Maximus A/S”, Limfjorden, Dragstrup Vig, in Denmark (N 56.8; E 8.5). Goggle Earth, June 2011. Accessed 04-01-2014).

30 CHAPTER 2. PRESENTATION OF THE PHD PROJECT

about the indoor part of the system. The Recirculated Aquaculture System (RAS) installed in 1990, at the fish farm where one of the first pioneer RAS in Denmark. In 2011 I got the chance to follow three production cycles with turbot larvae in the system, from arriving at the outdoor system, until leaving the RAS system at fry selling size. This was an unique chance, to get access to an operational turbot farm, using copepods as live feed for fish larvae.

Unique in the term, that traditionally marine fish larvae producers are very secret about their production techniques. Furthermore, in Denmark only two fish farms has more or less continuously produced marine fish larvae at a commercial scale since the early 1990ties. One is Venø fish farm and the other is Maximus A/S, mainly focusing on P. flesus and turbot (Scophthalmus maximus). Their production strategy is divided into fish fry for further aquaculture production in mainly Spain and France, the other part is for restocking of natural populations in Denmark. Restocking with flounder and later turbot has occurred since 1993 (Figure 8).

The challenge for Maximus A/S is that during the outdoor productive season, Marts to September, they traditional experience lesser survival of the fish larvae as an effect of season. The production technique in semi-intensive systems relies

Figure 8: Numbers of P. flesus and S. maximus since 1993 in Danish waters (Source: www.fiskepleje.dk/Kyst/udsaetning. Accessed 04-01-2014).

0

20000

40000

60000

80000

100000

120000

1993

1995

1996

1997

1998

1999

2000

2001

2002

2003

2004

2005

2006

2007

2008

2009

2010

Fish

rest

ocke

d [in

divi

dual

s]

Year

Turbot

Flounder

31CHAPTER 2. PRESENTATION OF THE PHD PROJECT

on natural phytoplankton, copepods and other bioic and abiotic factors changing during the productive season. This production method is a way to mimic the natural occurring marine pelagic food web. The question is what is the main factor that affects the decreasing survival of the fish larvae during the productive seasons? To investigate this three production cycles were monitored, where the lower trophic levels (nutrients, phytoplankton, copepods) is described in Jakobsen et al. (submitted), and the higher trophic levels (copepods, fish larvae) in Jepsen et al. (submitted). The two papers to be submitted as back to back campaign papers to the international journal aquaculture.

2.2 Intensive

My interest for intensive copepod farming started in 2003/04 during my bachelor project. We conducted some initial investigations on copepod stocking density and egg production (Jepsen et al. 2007). From here on I ventured into investigation on a novel way to provoke copepods eggs into quiescent stage (Højgaard et al. 2008). Furthermore, four different strains of the same copepod were compared in a common garden experiment, to investigate if some strains had more optimal traits for aquaculture intensification than other (Drillet et al. 2008a; Drillet et al. 2008b). Then in 2011 I was co-author on a review paper named: “Status and recommendations on marine copepod cultivation for use as live feed” published in the international journal Aquaculture by Drillet et al. (2011). Some of the reviews recommendations were (modified from Drillet et al. 2011):

• We suggest that water recirculation in copepod cultures is essential for further development because water quality is essential in cultures.

• We recommend feeding dense copepod cultures continuously using automatic procedures, rather than feeding once or twice daily to ensure constant optimal food concentrations in the water.

• We recommend using light regimes that are not known to increase the production of resting eggs (e.g. 12D/12L and UV free) for producing copepods, although this should be adapted for individual species.

Same year (2011) the paper was published I initiated my PhD study. The study was started with investigation on semi-extensive copepods systems turbot larvae farm (Maximus A/S) in Northern Jutland, Denmark. During one of the 6 hours long

32 CHAPTER 2. PRESENTATION OF THE PHD PROJECT

trips to the fish farm Maximus A/S I had an interesting talk with senior scientist Josianne Støttrup from DTU-aqua, Denmark, who criticised the recommendation paper. Her main criticism were that the above statements were undocumented and the danger was that they would become “common knowledge” for a science and industry standard without really being proved. She had a valid point; therefore I initiated a literature review on culture parameters for A. tonsa, since this was the chosen species for the present thesis. Result from this can be seen in table 5.

It was soon clear that some of the water quality parameters were well established for cultures like; oxygen, temperature and salinity. Whereas inorganic nutrients and their toxicity for copepod cultures were understudied. The most toxic natural occurring compound in marine copepod cultures are probably NH3. Therefore a study was setup during my PhD to investigate the limitations for intensive cope-pod cultures, with focus on both nauplii and adults (Jepsen et al. 2013). Another limitation is stocking densities of copepods. In Jepsen et al. (2007) we found that 600 adult copepods L-1 posed no limits for cultures and speculated that the limitations could be thousand or more pr. Liter. Another study was setup as part of my PhD to investigate limitations for copepods cultures with denser cultures ranging from 10 to 5000 copepods L-1 (Drillet et al. (in revision). Investigating the effects of feeding and light, these factors were combined in another study (Jepsen et al. (unpublished data). Preliminary result will be presented later in this PhD thesis. Cold-storage of copepod eggs are a well-established technique where storage capacity are known (Drillet et al. 2006). But the mechanism behind how the eggs are able to enter into storage is not understood yet. In Nilsson et al. (2013) we investigated some of the protection mechanism that copepod eggs uses when they enter into storage and further when the re-enter into normal development.

33CHAPTER 3. SYSTEMS TO PRODUCE COPEPODS AS LIVE FEED

Chapter 3

3.0 Systems to produce copepods as live feed

Copepod cultures in this thesis are divided into semi-intensive and intensive systems. The difference between the two production methods are that when the farming system intensify the use of resources increases (Tacon and Forster, 2003). In this thesis semi-intensive systems are defined as systems with usage of land, water, aeration, exogenous food input and simple monitoring, but with simple or no water treatment. Intensive systems are defined with same parameters as for semi-intensive but also as closed indoor systems with complete control of input and output.

3.1 Semi-intensive cultures

Almost all laboratory copepod cultures are considered as semi-intensive. Together with a few more industrialised big scale outdoor copepods systems, either used only for copepod production or in combination with fish larvae production. For laboratory cultures many variations are used but most of them consist of a rearing tank, addition of water, aeration and a separate system for rearing of algae used as food for the copepods. For outdoor semi-intensive systems an example is shown from Taiwan were earth ponds are excavated and used for rearing copepods. Water is added to the ponds together with feed/nutrients for either algae or the copepods. Paddle wheels are used for aeration in the ponds, but also to harvest the copepods by applying a fine meshed net downstream to the paddle wheel (Jacob K. Højgaard, personal comments) (Figure 9 (left)).

34 CHAPTER 3. SYSTEMS TO PRODUCE COPEPODS AS LIVE FEED

The system in Taiwan poses different environmental risks, for example they use different compounds of unknown origin both to feed the ponds put also as pro-biotics, disease treatment etc. (Figure 9 (right)).

Other more advanced semi-intensive inland tank systems has been reported (Engell-Sørensen et al. 2004; Evjemo, et al. 2003; Ludwig, (2003); Sørensen et al. 2007; Toledo et al. 1999; van der Meeren et al. 2008 ). In these systems either natural copepod populations are reared or populations from laboratory cultures are added, to large outdoor tanks (Figure 10). Often nutrients are used to stimulate a phytoplankton bloom, either natural occurring in the intake water or added in form of fertiliser, to stimulate copepod growth. In some of the systems fish larvae are added to prey upon the copepods, whereas in other systems the copepods are harvested by different filtrations systems and used as live feed for fish larvae in separate systems. Semi-intensive copepod systems have been reported to reach high production of copepods and nauplii, which again can support rearing of a high amount of marine fish larvae. A problem with these production systems is that they are outdoor systems and has the risk of being contaminated from a variety of sources e.g. toxic algae, parasites, bacteria etc. Similar to the problems experienced for many extensive system.

Figure 9 (left): Picture from an earth pond semi-intensive system in Taiwan, Tunggang. The pond is mainly dominated by the tropical copepod Pseudodiaptomus annandalei. Ponds are either used solitarily to rear copepods that are harvested and uses as live feed at other farms, or in combination with mainly grouper fish larvae. (right): Picture of some of the environmental issues from the semi-intensive system (Courtesy to Jacob K. Højgaard).


The investigation at Maximus A/S revealed profound differences in the seasonal dynamics, and their effects on the rearing success of turbot. A general decrease in phytoplankton over the season was observed that resulted in lower copepod abundance and finally a lower survival of the turbot larvae (Figure 11). Although, a lower survival was observed over the season, the fish larvae that did survive all had a similar specific growth rate and low rates of abnormalities. Although the fish larvae nutritional demand were not fulfilled (Figure 12).

In conclusion semi-intensive systems are valid for production of economical high valued fish larvae. Since an adequate food in early life is essential and the juveniles fish will benefit past later in life. Furthermore semi-extensive systems could be backed up by harvesting the copepods eggs from the bottom of the production tanks, after ending a production cycle. Keeping them cold stored, and later hatch nauplii when in shortage, late in the productive season. This would add flexibility to the semi-intensive production system.

3.2 Intensive cultures

Intensive systems have been reported for more than 30 years, although without yet having provided a production and cost efficient alternative to rotifer and Artemia cultures. Within the three orders relevant for aquaculture following large intensive cultures was reviewed in table 3.

Figure 10 (left): Picture of a production tank at Maximus A/S with the two inlet filtration system in the left of the pictures. (right): Zooplankton sampling from a production tank at Maximus A/S during one of the production cycles.


Figure 11: Development in zooplankton abundance (ind. L-1 of calanoid eggs, nauplii, copepodites and adults, during three production cycles. Bars are means of Acartia spp. and C. hamatus polled together ± S.D.

production cycle 3

Fish larval age (day)

0 5 10 15 20 250

20

40

60

80

100

production cycle 2

0 5 10 15 20 25

Abu

ndan

ce (i

nd. L

-1)

0

20

40

60

80

100

production cycle 1

0 5 10 15 20 250

20

40

60

80

100

Acartia spp. Centropages hamatusCalanoid nauplii


Figure 12: The total turbot larvae’s populations calculated daily carbon demand (solid line) and the total daily available standing stock of prey items (punctuated line). In data before day 10, only the nauplii fraction of the available prey is included, since the turbot larvae can only predate on nauplii. (Δ) Represent the turbot larvae`s average specific growth rate ± standard deviation (n=3). At first sampling point in production cycle 1 data only represented one tank, since no turbot were caught in the other 2 tanks.


Table 3 Large scale intensive mass cultures of copepods.Culture specie Order Culture size Densities Productivity Reference

Acartia tonsa Calanoida 200-450L 50-100 L-1200-220 eggs L-1

Støttrup et al. 1986

Acartia tonsa Calanoida 1500L

679 adult L-1, 1225 nauplii L-1 N/A

Turk et al. 1982

Acartia tonsa Calanoida 1900L

232 adults L-1, 820 nauplii L-1 N/A Ogle, 1979

Acartia tonsa Calanoida 100L 425 L-140 eggs fem-1 day-1

Peck and Hol-ste, 2006

Acartia

tranteri Calanoida 1000L 1 ml-1

25 eggs fem-1 day-1,

Nauplii 20-80 L-1,

Copepodites and

adult 10-60 L-1

Morehead et al. 2005

Acartia spp. Calanoida 1000L 2000 L-11000 ind. day-1

Schipp et al., 1999

Acartia sp. Calanoida 400L N/A

500,000 eggs or nauplii day-1

Knuckey et al. 2005

Centropages

helgolandicus Calanoida 1000L 15 ind. L-110-20 eggs fem-1 day-1

Carotenuto et al. 2012

Centropages

typicus Calanoida 1000L 100 ind. L-1100 nauplii L-1

Buttino et al. 2012

Gladioferens

imparipes Calanoida 500-1000L 1 adult 1ml-1900 nauplii day-1

Payne and

Rippingale, 2001

Parvocalanus sp. Calanoida 400L2.5 adult ml-1, 8 nauplii ml-1

3750 nauplii L-1 day-1

Shields et al. 2005

Temora

styfifera Calanoida 1000L 200 ind. L-1370 nauplii L-1

Buttino et al. 2012

Table 3: list the existing intensive systems reported in the literature. Only cultures above 100L volume has been chosen and with some degree of water treatment or other water purifying control connected to the cultures.


A recent tendency within culturing techniques of calanoid copepods is to apply recirculation technologies (Buttino et al. 2012; Carotenuto et al. 2012; Payne and Rippingale, 2001). Another example is the prototype recirculated aquaculture system installed at Roskilde University (Figure 13).

Apocyclops

panamensisCyclopoi-da 100L

6500 adults L-1

18,000 nau-plii L-1

Phelps et al. 2005

Amphiascoides

atopusHarpacti-coida 4m3 N/A

2 to 4 million ind. day-1

Sun and Fleeger, 1995

Tigriopus

japonicasHarpacti-coida 210m3 10-22ml-1 4-5kg Fukusho, 1980

Tisbe

holothuriaeHarpacti-coida 100L N/A

500,000 ind. day-1

Støttrup and

Norsker, 1997

Tisbe sp.Harpacti-coida 250L N/A N/A

Morehead et al. 2005

Figure 13: A principal diagram of the recirculated aquaculture system installed at Roskilde University. The water treatment consists of a protein skimmer, a biofilter, a pump sump, an ultraviolet filter (UV) and 3 cartridge filters. Four 300L production tanks are used for copepod cultivations. Arrows indicates water flow in the system.


The reason for applying RAS as the above described is that they have advantages compared to extensive and semi-intensive copepod production systems. A RAS ensures stability in production parameters and they are closed systems where env-ironmental input and output are controlled. A problem with the studies published is that the reported daily production capacities of nauplii are highly variable, and are not stable enough for industrialised marine hatcheries. In modern aquaculture stability in the production is essential and if one chain in the value chain “breaks” the rest of the production is wasted. However, different issues with intensive copepod farming have to be solved. These challenges are in particular: Copepod stocking densities (Drillet et al. (in revision)), water quality (Jepsen et al. 2013). Feeding regimes for cultures (Chapter 4) and farming protocols that will reduce labour cost by automatic control of different processes (Drillet et al. 2011).

In terms of stocking densities a reason study showed a correlation between ma-ximum stocking density and egg production (Drillet et al. (in revision)) (Figure 14). Egg production is a measure of the reproductive capacity in the population, but also an indirect measure of the growth rate in the population (Berggreen et al. 1988). The conclusion form Drillet et al (in revision) is that at stocking densities

Figure 14: Acartia tonsa total egg harvested L-1 d-1 (± SD) as an average of 5 days of production for different initial stocking densities. Additional data reported by Jepsen et al (2007) and Peck and Holste (2006) are also presented, for comparison.


above 2,500 ind. L-1, the population recruitment and growth is ceased, and with increasing stocking densities no more population productivity will be achieved.

This initial investigation confirmed density stockings found in other studies with A. tonsa (Jepsen et al. 2007; Medina and Barata, 2004; Peck and Holste, 2006). Another study with Acartia sinjiensis also confirmed that egg production were not affected at stocking densities up to 2000 ind. L-1, although they observed an inducement of delayed hatching in the eggs (Camus and Zeng, 2009). This delay in hatching success do not seem to be the case for A. tonsa, were an inherit trait of ~10% of the eggs appears to be coded for delayed hatching, independent from the stocking density (Figure 15).

Conclusive for copepod stocking densities it is recommended to keep copepods at a maximum stocking density of 2500 ind. L-1. Furthermore, studies should focus on potential effects of cannibalism and nauplii densities. Nauplii densities are expected to be much higher due to their smaller size and personal I have observed very dense nauplii cultures.

Water quality has often been recognised as a restriction for copepods cultures (Drillet et al. 2011). Unionized ammonium is the most toxic and therefore the limiting factor for cultures of species in recirculated aquaculture. Jepsen et al. (2013) investigated the toxic effect of unionized ammonia on A. tonsa and what the limitations for cultures were.

Figure 15 (left): Proportion of delayed hatching eggs of Acartia tonsa as a function of the initial copepod stocking density in the incubation chambers (mean ± S.D.) (Drillet et al. (in revision)). (right): Hatching success of Acartia sinjiensis after 48h (dark bars) and after 96h (white bars) (Camus & Zeng, 2009).


It is clear that unionized ammonia not is the limiting factor for A. tonsa cultures. Ammonia is not a practical problem for cultures, and if it is Jepsen et al. (2013) has provided the limitations that can be applied to design the dimensions of biofilters in recirculated aquaculture systems (Table 4).

Culture parameters for Acartia tonsaWithin recent year’s studies of intensive calanoid cultures have been published (Buttino, et al. 2012; Payne and Rippingale, 2001; Carotenuto, et al. 2012) and a few on Acartia spp. (Støttrup, et al. 1986; Schipp, et al. 1999). From different studies protocols on how to rear copepods can be derived. Concerning Acartia tonsa cultures many parameters are well established e.g. temperature (Hansen, et al. 2010), salinity range (Calliari, et al. 2006; Hansen, et al. 2012; Peck and Holste 2006), water quality (Jepsen, et al. 2013), photo period (Peck and Holste 2006), densities (Jepsen, et al. 2007; Drillet, et al.(in revision)) and different strains has been compared in common garden experiments (Drillet, et al. 2008a; Drillet, et al. 2008b). Although A. tonsa has been cultivated since 1986, a summary of culti-vation parameters and their application to aquaculture is still missing (Støttrup et al. 1986). Rippingale and Payne (2005) summarised the biological features of G. impairs in the book “Copepods in Aquaculture” and what implications these has on aquaculture. A similar table is presented to highlight Acartia tonsa biological features and their implications for aquaculture (Table 5).

Table 4: Shows No Observed Effect Concentration (NOEC) and Lowest Observed Effect Concentration (LOEC) of adult and nauplii Acartia tonsa. The adult and nauplii densities are calculated from the equation and excretion data in Jepsen et al. (2013).

pHNOEC adult

[µgNH3 L-1]

LOEC adult

[µgNH3 L-1]

Adult den-sity [Ind.

L-1]

NOEC nau-plii [µgNH3

L-1]

LOEC nauplii [µgNH3

L-1]

Nauplii den-sity [Ind. L-1]

7.5 477 1,789 170* 106 30 81 10.8*106

8.0 477 1,789 55*106 30 81 3.5*106

8.5 477 1,789 18*106 30 81 1.2*106

9.0 477 1,789 7*106 30 81 444,223

9.5 477 1,789 3.4*106 30 81 213,262


Biological fea-tures of Acartia tonsa

Implications for aqua-culture

Recommendations for in-tensive cultures of Acartia tonsa

References

Wide tempera-ture range from 4 to 32°C

• Cultures can be adapted to local conditions

• Eggs can be cold stored

17 to 25°C, for optimal egg production and devel-opment time

(Chinnery & Wil-liams, 2004; Drillet et al. 2006; Hansen et al. 2010; Paffenho-fer & Stearns 1988)

Wide salinity range 5 to 72, tolerate rapid salinity change

• Cultures can be adapted to local conditions

• Salinity change can be used to suppress invasive pathogenic and other nuisance organisms

• Abrupt salinity change can be used to store eggs

From 15 to 36 for cul-tures, depending on strain

For egg storage transfer from ambient culture salinity to milliQ water

(Cervetto, et al. 1999; Chinnery & Williams, 2004; Højgaard, et al. 2008; Ohs, et al. 2009)

Light regime for cold stored eggs, nauplli to adult 0L:24D. For egg hatch-ing 24L

• Cost for artificial light above cultures can be saved

• Darkness can sup-press invasive organ-isms

• Some reports about light influence on egg hatching success

No light for cultures and cold stored eggs.

Hatch eggs at dim light

(Andreas Hagemann (personal comment); Jepsen et al. (Unpub-lished data); Peck & Holste 2006)

Stronger pigmentation if exposed to ultra violet light (UV)

• UV-radiation can enhance copepod pigmentation, and thereby visibility for predator (fish lar-vae).

For cultures keep UV low to avoid radiation damage

For use as live feed UV can be used in moderate doses to enhance pig-mentation and thereby visibility as prey

(Hansson, 2000)

Table 5: Recommendations on how to utilize A. tonsa biological features in intensive cultures.


Body size and somatic growth can be regulated by temperature

• Suitable size ranges can be “constructed” for different types of marine fish larvae

• Nauplii, copepo-dites and adults can be stored for later use for fish larvae

For cultures keep tem-perature as recommended above

For use as live feed smaller cephalothorax size with higher temperature

Cold storage of nauplii can be done with 60% survival to day 9.

(Ambler, 1985; Chinnery & Wil-liams 2004; Hansen, et al. 2010; Støttrup, 2006)

NH4/NH3 levels from 0.1 to 0.4 mg L-1, with no observed effect on cultures

• Maximum stocking density for batch cultures can be calculated

• Cultures can be maintained with biofilter technology

For nauplii keep below 30µg NH3 L

-1

For adult keep below 477µg NH3 L

-1

(Jepsen et al. 2013; Sullivan & Ritacco, 1985)

Tolerant to low oxygen 1.5ml L-1

• Cultures can be maintained by aera-tion, no need for ap-plying liquid oxygen

• Cold storage of eggs can be improved with anoxic condi-tions

• Keep cultures around 80 to 100% satura-tion

• For cold storage of eggs strip oxygen by bubbling with nitro-gen

(Marcus et al. 2004; Holmstrup et al. 2006)

Tolerant to low change in pH

• Cultures can be maintained without pH control

• Eggs are not effected by pH

• Keep cultures within 7.5 to 9.0

• For cold storage of eggs strip oxygen by bubbling eggs with nitrogen

(Hansen et al. In prep.; Sullivan & Ritacco, 1985)

Fast generation time from 14 to 19 days

• Selection

• Restocking of cul-tures not critical

• Fast adaption to en-vironmental factors

• Physiologic plastic-ity

• For batch cultures for egg production keep old cultures since females outlives males 2-12 weeks

• Selection should be toward large males and females to in-crease egg production

(Ceballos & Kiørboe, 2010)

45CHAPTER 4. FEEDING AND LIGHT REGIMES FOR ACARTIA TONSA

Chapter 4

4.0 Feeding and light regimes for Acartia tonsa

From Zhang et al. (2013) Rhodomonas baltica is found as an ideal algal diet, and Peck and Holste (2006) investigated the effect of photoperiod and egg produc-tions, and showed no effects, from 8L:16D to 20L:4D for A. tonsa, respectively. Although, Peck and Holste´s (2006) study missed the extremes, complete darkness and total light. Another study revealed a nocturnal feeding pattern in A. tonsa which maybe result in better feed uptake in A. tonsa and thereby egg production in dark cultures (Stearns 1986).

Furthermore, feeding regimes are rarely reported and if, the copepods are feed by one spiked concentration of algae day-1 (Buttino, et al. 2012; Støttrup et al. 1986). Therefore, a 20 days study was setup to investigate the effect of the two extremes total darkness and total light, fed the same species, bio volume of algae but with three different feeding regimes; spiked, pulsed and continues algal addition.

From figure 16 it is observed that the daily feeding regimes were different. For the two spiked concentrations, a daily spike elevated algae concentrations which again decreased due to grazing and sedimentation, before the feeding regime was repeated with a new spike, 24 hours later. This feeding pattern is the “normal” feeding regime for most laboratory batch cultures of copepods. For pulsed treat-ments, four daily pulses were added every 6 hour, giving four immediate responses in algae concentrations. The continuous treatments are almost constant adding of algae during the feeding cycle. Although, the daily algae concentrations were different over time, when testing every 3 hour, the average mean concentration during the entire experiment were not statistically different, when calculated ac-cording to Frost (1972). If food availability is similar for all developmental stages of A. tonsa, the development time should follow the rates reported in Berggreen et al. (1988), with a maximum carbon conversion rate into body carbon of 0.45.

46 CHAPTER 4. FEEDING AND LIGHT REGIMES FOR ACARTIA TONSA

Figure 16: Daily addition of 1L Rhodomonas baltica with same concentration, and its fate in experimental beakers. Light background represent treatments in light, dark backgrounds represent treatments in dark. Each symbol represents average of 4 replicates, and each error bar is the standard error between the 4 replicates (Jepsen et al. unpublished data).

Therefore, A. tonsa development rate were investigated with the above culture parameters. The different development stages were analysed by ZooImage, a fre-eware software program, according to the method described in Vu et al. (2014).


The development stages of Acartia tonsa were investigated in relationship to feeding regime and light/dark treatment (Figure 17). The development stage, eggs, were removed after day 5. To investigate the development time before a switch to next stage, each intersection between two stages were located. The data points between the intersections were individually plotted as two linear graphs and math were used to solve the intersection point between the two lines.

Figure 17: Temporal development of the different development stages of A. tonsa from egg to adult. Data points represent the means of four replicates. Light background represent treatments in light, dark backgrounds represent treatments in dark (Jepsen et al. unpublished data).


Table 6: Development time of Acartia tonsa as an effect of different feeding regimes and light/dark treatment. Light background represent treatments in light, dark backgrounds represent treatments in dark (Jepsen et al. unpublished data).

As seen in table 6 no clear patterns on development time as a function of either feeding regime or light/dark treatments, were found. Although, there was a ten-dency to that treatments in light developed differently than treatments in dark; however, this was not a significant pattern. All the dark treatments developed with the same pattern, independent from which food treatment they were offered (Figure 17 and table 6).

To investigate the development in specific terms, the individual specific growth rate and the specific egg production were calculated. This was possible since the instantaneous rates of mortality (Z d−1) could be calculated from the analyses from the ZooImage data, and then corrected for the sampling mortality by ap-plying equations from Klein Breteler, et al. (2004). The specific egg production were calculated by using, female carbon content from ZooImage, together with measurement of the egg diameter from ZooImage and manual egg counting (Vu et al., (2014)). From the egg diameter the eggs carbon weight can be derived using the formula for a volume of a sphere multiplied with 0.14*10-6 µg C µm-3 (Kiørboe, et al. 1985) (Table 7). Table 7: Individual specific growth rate (S.G.R.) are average growth during the 20 days experiment (n=4). Specific egg production (S.E.P.) were obtained during the last three days of the experiment, since all were adult at this time point, data are average (n=4). Light background represent treatments in light, dark backgrounds represent treatments in dark (Jepsen et al. unpublished data).

Mean duration (days ± S.E.) before switch to next development stage (n = 4).Treatment Spike Light Pulse Light Continues Light Spike Dark Pulse Dark Continues Dark

Nauplii IV-VI 5,26 ± 0,01b 4,72 ± 0,06a 4,76 ± 0,07a 4,71 ± 0,10a 4,98 ± 0,07ab 4,95 ± 0,10abCopepodite I-III 8,28 ± 0,03a 8,51 ± 0,07a 10,27 ± 0,16b 8,27 ± 0,04a 8,62 ± 0,09a 8,44 ± 0,09aCopepodite IV-VI & Adult* 10,82 ± 0,06a 9,75 ± 0,14b 14,28 ± 0,11c 10,34 ± 0,05ab 10,57 ± 0,15ab 10,83 ± 0,05aWithin rows, there are no statistically significant differences between means marked with the same letter (One-Way ANOVA & Holm-Sidak, p<0.05) & (One-Way ANOVA on Ranks & Tukey, p<0.05*)

Treatment Spike Light Pulse Light Continues Light Spike Dark Pulse Dark Continues Dark

S.G.R. ± S.E S.G.R. ± S.E S.G.R. ± S.E S.G.R. ± S.E S.G.R. ± S.E S.G.R. ± S.E

Individual 0.19 ± 0.19 0.17 ± 0.13 0.19 ± 0.16 0.18 ± 0.15 0.19 ± 0.07 0.19 ± 0.15 Population 0.08 ± 0.14 0.07 ± 0.09 0.05 ± 0.08 0.03 ± 0.10 0.09 ± 0.07 0.05 ± 0.09 Time[Days] S.E.P. ± stdev S.E.P. ± stdev S.E.P. ± stdev S.E.P. ± stdev S.E.P. ± stdev S.E.P. ± stdev

18 0.18 ± 0.13 0.21 ± 0.17 0.17 ± 0.09 0.05 ± 0.04 0.26 ± 0.07 0.10 ± 0.12 19 0.21 ± 0.12 0.16 ± 0.12 0.19 ± 0.14 0.07 ± 0.05 0.16 ± 0.09 0.08 ± 0.07 20 0.18 ± 0.12 0.09 ± 0.02 0.15 ± 0.10 0.11 ± 0.06 0.23 ± 0.05 0.10 ± 0.08


Figure 18: Bars represents the average total egg production observed in the six different treatments, error bars are S.E. (n=4). White bar colour represent treatments that has been exposed to light, dark grey bar colour represents treatments that has been kept in darkness. No bar fill patterns are the Spike treatments, fill pattern of coarse diagonal stripes are the Pulse treatments and bars with dotted fill patterns are the Continues treatments (Jepsen et al. unpublished data).

Investigating both the specific growth rate and the specific egg production shows similar pattern independent of both light and feeding regime treatments. Furthermore, the total egg production and the subsequent hatching success were investigated (figure 18).


High variability was observed in the total egg production. On day 18 statistically differences, in the total egg production, were observed between pulse dark versus spike light and dark (One-Way ANOVA: F5;22=3.533, p=0.023). No other signi-ficant differences were observed, on any of the other days. The hatching success were also not statistically different form each other.

With the observed data there is no differences either on light or darkness or if you apply spike, pulse or continuous feeding regimes. The copepods were fed to half saturation concentrations, and they respond as expected to this availability in food concentrations. This was true both for specific growth rates (~0.20 d-1) and specific egg production (~0.20 d-1) (table 6). The specific growth rates are similar to the specific growth rates at feeding level 123 ± 18 µg C L-1 and 222 ± 19 µg C L-1 in Berggreen et al. (1988). Furthermore, the specific egg production is also as expected since female egg productions and specific growth rates have been shown to be similar (Berggreen et al. 1988). The reason why no differences are observed between light and darkness is probably that the diel feeding rhythm is lost in this specific A. tonsa strain (Tiselius et al. 1995). So it seems that when the diel rhythm is lost then the feeding is only affected by the maximum ingestion rate and food availability. In regards of food availability one should expect that continues or pulse feeding are preferable since food in principle should be more available in these treatments (figure 16). But no profound development (figure 17 and table 6), growth (table 7) or egg production/hatching effects (figure 18) were observed as an effect of the three different feeding regimes. Acartia tonsa has been shown to be able to adapt their feeding behaviour to patchy food environments (Tiselius, 1992). This is confirmed in the obtained data, that even when the copepods are fed in the spike treatments they feed in patchiness when food is available, resulting in similar production rates as in the pulse and continues feeding regimes. Feeding in patchiness is an ecological adaption since utilizing a patchy food environment is fundamental to the survival of planktonic organisms (Tiselius, 1992).

In conclusion, feeding regimes do not affect the productivity of A. tonsa when fed at their half saturation concentrations. Furthermore, it is expected that the same effects applies for concentrations above half saturation levels. No effects of light or darkness are observed, therefore one could recommend to turn the light off and save money on electricity in continuous A. tonsa cultures. Further studies will show if A. tonsa are affected at feeding concentrations lower than half saturation levels, which are relevant in an ecological perspective but not in


aquaculture. In aquaculture you would always feed a saturations levels to obtain maximal productivity.

53CHAPTER 5. CONCLUSION AND FUTURE PERSEPCTIVES

Chapter 5

5.0 Conclusion and future persepctives

In the past 10 years I have focused on copepods as live feed for aquaculture. In this PhD thesis I followed two different production methods, semi-intensive and intensive copepod farming.

Semi Intensive copepod culturesThe semi-intensive system offered the opportunity to investigate a commercial operational turbot farm, which utilized copepods as live feed for turbot larvae. The pelagic marine food web were investigated and due to its complexity it was divided into two papers, one with the lower trophic levels (Jakobsen et al. (sub-mitted)) and another with the higher trophic levels (Jepsen et al. (submitted)). The semi-intensive Maximus A/S system do have success with rearing of turbot larvae in especially in spring, but later in the productive season the fish larvae survival is decreased. The study revealed that to be able control the outdoor ponds you need

ANALYSES (Laboratory)NutrientsChlorophyllCopepodsNaupliiPhytoplanktonTemperaturePrimary productionpHOxygenCO2

SYSTEM MANIPULATIONBioticCopepods (in/out)Algae ((in/out)AbioticNutrients (in)OxygenCO2Water (in/out)

EXTRA ANALYSES

MODELS(Concerning)Algae BacteriaCopepodsDay-lengthFish larvae

COMPUTERConsequence analyses

DECISIONSFish farm manager

Figure 19: Schematic drawing of a proposed management and control program for controlling Maximus A/S (Urup, 1994).

54 CHAPTER 5. CONCLUSION AND FUTURE PERSEPCTIVES

a quite intensive monitoring program, and even then it is difficult to predict and control the system. Urup, (1994) proposed a monitoring program with many of the same parameters that we measured in 2011 (Figure 19).

If you apply the suggested monitoring program, I agree that you can predict the development of the system, but even though there will be several parameters that you cannot control (Urup, 1994). For example, in the current system you cannot prevent intake of toxic algae, parasites below 50µm and bacteria (intake filtration is below). This could potentially be solved by implementing water treatment on intake water (Ultra Violet / Ozone filtration). But then you kill all intake algae and would have to seed the tanks from arsenic algae stocks, adding on comple-xity. Further if the current system were reliable why is there a high variance in fish larvae produced from season to season as reported in Engell-Sørensen (2004) (from 13,000 to 153,000 larvae year-1)? The production record for Maximus A/S is a production of 750,000 turbot larvae achieved in 2008 (Anders T. Pedersen, personal comment). During the monitoring program at Maximus A/S 2011 we worked 3 people full time, taking care of the monitoring program alone, whereas at the fish farm 2½ people worked with their daily procedures. To return an eco-nomic surplus you need to produce at the maximum every year. Since Maximus A/S has not been able to do this, the consequence is that daily management is 2½ people and the monitoring schedule is limited to daily oxygen, temperature and secchi dish measurements, together with weekly copepod counting. Therefore, for an economical sustainable future for the fish farm the monitoring program has to be easy and automatic. A suggestion could be to implement a fluroprobe at the farm, giving you a daily proxy for chla a levels together with an estimate of which algae groups that are present. Implementing this procedure would remove secchi measurements. Furthermore probes measuring in situ light, oxygen, tem-perature, CO2 and pH levels could be installed in each tank and connected to a centralised computer, which again could decide to manipulate O2 and CO2 as suggested by Urup (1994). Copepod biomass could be automatic analyses using an office scanner and the freeware software ZooImage (Vu et al. 2014). A predictive tool could help the farm manager to take decisions, as the “computer consequence analyse” according to figure 19. (Urup, 1994). This could be done by developing a model with input data from Engell-Sørensen, (2004), Jakobsen et al. (submitted), Jepsen et al. (submitted), Sørensen et al. (2007) and Jakobsen et al. (in prep.) and Blanda et al. (in prep), last two from a manipulative study in same system conducted in 2012.


I believe that semi intensive system future in Denmark is in automatic procedures and farming of high value species, with a well developed market.

Intensive copepod cultureI have studied limitations for intensive cultures of Acartia tonsa in two different studies (Drillet et al. (in revision); Jepsen et al. 2013). I have also reveal some of the protective mechanism that the egg uses, when exposed to cold storage (Nilsson et al. 2013). At industrial scale SINTEF in Norway and Stiching Zeeschelp in Holland have intensive aquaculture facilities with A. tonsa as the targeted culture species. Furthermore, Roskilde University has a new RAS with the purpose to rear A. tonsa. The intensive rearing systems design, densities and other physiological parameters are still not documented in the scientific literature. So for now, the link between laboratory scale experiments and industrial scale are missing. It could be interesting to see if the found densities in Drillet et al. (in revision) are applicable for industrial scale experiments, or if unforeseen problems occur at up-scaling? Furthermore, it could confirm the model predictions from Drillet and Lombard (2013), about improving copepod cultivation. In terms of water quality it was natural to focus on NH3 since it is the most toxic “natural occurring” compound in copepod cultures and thresholds for NH3 is essential to know before up-scaling of intensive cultures. Furthermore, culture limitations of NH3 are the most essen-tial when designing and dimensioning biofilters for RAS. Although, other culture parameters such as, pH, nitrite, nitrate, CO2, Suspended Solids (SS), Dissolved Oxygen (DO) and alkalinity are also relevant. One has to remember that for RAS, each individual parameter is important, but it is the additive and combined inter-relationship, and the understanding of all the parameters that is really important for copepod cultures health and growth (Timmons and Ebeling, 2007). From table 5 it can be derived that NH3, DO and pH are relatively known culture parameters for A. tonsa cultures. Whereas, at least to the authors’ knowledge no studies are investigating effects of nitrite, nitrate, SS and CO2, which should be a future study area. When limits of the single parameters are established, studying the combined or cocktail effects of the parameters would be a natural focus area.

One of the most demanding tasks for copepod cultures are estimation of copepod abundance and biomass. The present practices for copepod abundance and biomass estimation is manual microscopy, measuring cephalothorax length and applying a length vs. weight regressions, e.g. Berggreen et al. (1988). By applying a computer and an office scanner, automatic scanning and biomass estimation for A. tonsa is


possible (Vu et al. (2014). Combining Vu et al. (2014) automatic scanning with an automatic sampling method e.g. Alver et al. (2007) would provide essential culture parameters for intensification of cultures (Figure 20).

When the culture biomass is known you need to be able to automatic measure the carbon value and amount of feed in algae to cultures. Several probes or instruments can give a direct or proxy of algae carbon content e.g. coulter counter, fluoroprobes, optical density etc. Although, this should be integrated into an automatic process and multiplied with the known carbon content for the selected algae, e.g. 47.4 pg C cell-1 for R. baltica (Berggreen et al. 1988). When algae and copepod biomass are known a Feed Conversion Ratio (FCR) can be applied for your culture. For optimal A. tonsa growth rate (0.45 d-1) this ratio would be ~2.3, so every time 2.3g C of alga is fed to the culture you will get a conversion into copepod biomass of 1g C (Berggreen et al. 1988). These steps toward automatic culture control provide the basic for automatic feeding of intensive copepod cultures, which can be adapted into a Programmable Logic Control (PLC) systems (Figure 21).

Future effort for intensive copepod production should be on automatic quantitative sampling of copepods in cultures that could automatic be analysed by ZooImage.

Figure 20: Pictures modified from Vu et al. (2014). G: copepodite IV-V-male A. tonsa. H: Adult female A. tonsa scanned on an Epson Perfection V500 Photo colour scanner (6400 dpi, 16 bit gray scale in the positive film mode).


Same should be done for algal culture measurements both in stock algae cultures and in copepod culture tanks. When this is established the different parame-ters/variables should be integrated into a PLC system and applied to cultures. Furthermore culture parameters for A. tonsa cultures now are established in the scientific literature, so the focus should be on up scaling and testing parameter in industrialised systems. An advantage with intensive A. tonsa cultures is that you have a two string solution for your intensive production. You can either choose to have decentralized, local productions at the marine fish farm, or you can have a centralised production focussing on A. tonsa egg production that can be shipped world-wide, being hatched, and the nauplii used as fish larvae live feed (Drillet et al. 2011). I personally believe most in the latter centralised production, since decentralised production requires extra skilled staff at all the marine fish hatcheries. Hopefully at least one of the two strings will succeed with an economical viable intensive copepod production within the next 5 to 10 years.

Closing remarksMy research managed to establish some of the critical parameters for intensive A. tonsa cultivation, in terms of copepod density (Drillet et al. In revison), water quality (Jepsen et al. 2013), feeding and light regimes (Jepsen et al. (Unpublished)). Last, we established a relationship between maximum stocking densities versus egg production and survival. We found a plateau at 2,500 individuals L-1. First steps are initiated towards automatic procedures of A. tonsa cultures by implementing

Figure 21: Left part of the figure is an overview with input and output (arrows) to the culture tank, together with constant parameters (volume, temperature and threshold (Th.). Right part is a suggestion of a feedback protocol used to control copepod feeding. The previous and current algae concentration is X0 and X1, respectively. The predefined required level of algae or threshold is Th. For A. tonsa the Th. is 950µg C L-1, respectively (Berggreen et al. 1988).

Tank volumeThreshold valueCopepod biomass

Sample result (cell ml-1)

Maintain feed

In situ feed measurement

Start feeding

X1 > Th.

X1- X0 < Th.

Residual feed

Feeding rate Yes

Yes

No

No


ZooImage scanning of all the copepod stages (Vu et al. (2014)). Procedures for cold storage of A. tonsa eggs are well known, but the physiological mechanisms behind are still a black box. Nilsson, et al. (2013) showed some of the protective physiological mechanism that A. tonsa eggs/embryons use to protect itself against environmental stressors. Understanding the mechanism behind cold storage can potentially improve the storage capacity both by a prolongation of the storage period and by removing current temperature restrictions.

I sincerely hope that my small puzzle pieces will help solve the overall puzzle, and the goal to establish intensive A. tonsa culture will be achieved in a near future.

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Manuscripts overview

SEMI INTENSIVE SYSTEMS

Manuscript 1: Jakobsen, H.H., Jepsen, P.M., Blanda, E., Jørgensen, N.O.G., Novac, A., Engel-Sørensen, K. Hansen, B.W. (in preparation). A seasonal study of turbot larvae Scophthalmus maximus (Linnaeus, 1758) reared in a semi-intensive outdoor sy-stem I: Plankton composition and biomass development - the role of inorganic nutrients limitation.

Manuscript 2:Jepsen, P. M., Jakobsen, H. H., Blanda, E., Novac, A., Engell-Sørensen, K., and Hansen, B. W. (in preparation). A seasonal study on turbot larvae Scophthalmus maximus (Linnaeus, 1758) reared on copepods in a Danish semi-intensive outdoor system. II: Larval growth, prey selection and survival until fry.

INTENSIVE SYSTEMS

Manuscript 3:Drillet, G., Rais, M., Novac, A., Jepsen, P.M., Mahjoub, M-H., Hansen, B.W. (in revision). Total egg harvest by the calanoid copepod Acartia tonsa (Dana) in intensive culture – effects of high stocking densities on daily egg harvest and egg quality. Aquaculture Research.

Manuscript 4:Jepsen, P.M., Andersen, C.V.B., Schjelde, J., Hansen, B.W. (2013). Tolerance of un-ionized ammonia in live feed cultures of the calanoid copepod Acartia tonsa Dana. Aquaculture research, Article first published online: 9 APR 2013, DOI: 10.1111/are.12190.

Manuscript 5:Nilsson, B., Jepsen, P.M., Rewitz, K., Hansen, B.W. (2013). Expression of hsp70 and ferritin in embryos of the copepod Acartia tonsa (Dana) during transition between subitaneous and quiescent state. Journal of Plankton Research. Article first published online: 1–10. doi:10.1093/plankt/fbt099

Manuscript 1.

Jakobsen, H.H., Jepsen, P.M., Blanda, E., Jørgensen, N.O.G., Novac, A., Engel-Sørensen, K. Hansen, B.W. (in preparation). A seasonal stu-dy of turbot larvae Scophthalmus maximus (Linnaeus, 1758) reared

in a semi-intensive outdoor system I: Plankton composition and bio-mass development - the role of inorganic nutrients limitation.

MANUSCRIPT 1 71

A seasonal study on turbot larvae Scophthalmus maximus (Linnaeus, 1758) reared in a semi-intensive outdoor system I:

Plankton composition and biomass development

- the role of inorganic nutrients limitation

Jakobsen, Hans H.1*; Jepsen, Per M.2; Blanda, Elisa2; Jørgensen Niels O. G.3; Novac, Aliona4; Engel-Sørensen Kirsten5; Hansen, Benni W.2

• Department of Bioscience. Aarhus University. DK-4000 Roskilde. Den-mark

• Department of Environmental, Social and Spatial Change. Roskilde Uni-versity. DK-4000. Roskilde.

• Denmark. • Department of Plant and Environmental Sciences, Science Faculty, DK-

1871 Frederiksberg, Denmark• “Alexandru Ioan Cuza” University of Iasi, Faculty of Biology, 20A, Carol

I Blvd., RO-700505, Iasi, Romania.• Fishlab, Terp Skovvej 107B, DK-8270 Højbjerg, Denmark

*Corresponding author

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Abstract

The plankton food web dynamics were followed during a full production season in a semi intensive land based rearing facility for rearing of turbot (Scophthalmus maximus) larvae. The production season was divided into three 3-5 week pro-duction cycles. Each production cycle was conducted in three replicate 280 m3 tanks. A production cycle was initiated by using screened water from a nearby estuary added to the tanks. The estuary water used, experienced deceasing nitro-gen concentrations from spring to fall. A maximal chlorophyll a peak developed upon initiating of a production cycle, but displayed decreasing peak values from ca. 18 µg Chl a L-1 in spring to ca. 7 µg Chl a L-1 in the fall. This decrease was concurring with the nutrient concentration in the estuary water, indicating the available nutrient concentration was the main governor of the phytoplankton dynamics. The tanks were seeded with an increasing number of copepods, from a storage tank during the season, to establish a phytoplankton – copepod food web. However, we observed an opposite and decreasing nauplii abundance from spring to fall in the rearing tanks. Parallel laboratory incubations of individual copepod females, Acartia spp. and Centropages hamatus, conducted during the production season indicated a low but similar daily specific egg production rate (mean of both Acartia spp. and Centropages hamatus summarised: 12.2 ± 10 d-1. Hatching remained constant but displayed large standard variation during the production season (mean of both Acartia spp. and Centropages hamatus summa-rised 58.4% ± 45%). The decreasing nutrient load yielded increasing carbon to chlorophyll a ratios (weigth:weight) in the seston, pointing towards increasing nitrogen deficiency during the production seasons. Although, nitrogen loading was decreasing, the pool of amino acids was constant. The amino acids maintained homeostasis among the essential amino acids in the particulate material during the season, and only lowered concentration in total essential amino acids was observed in the 3rd production cycle. We suggest that the decreasing nitrogen input lower the chlorophyll a concentrations and changes in the underlying quality of their phytoplankton food. In addition, the copepod species composition was changing due to the cascading effect governed by the reduced nitrogen loading.

Keywords: aquaculture, turbot larvae, live feed, nitrogen deficiency, phytoplankton

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1.0 INTRODUCTION

Over the past 2 – 3 decades, commercial aquaculture rearing of turbot (Scopht-halmus maximus) for human consumption has been increasing, and there is an unutilised potential for further production increase. However, one of the bottle-necks that challenge the industry is maintaining high and stable survival of newly hatched and first feeding larvae. Unlike in most other high value fish productions, the newly hatched turbot larvae are typically reared in semi intensive land based systems (Engell-Sørensen et al., 2004). In these systems, a classical phytoplankton – zooplankton –food web is established, nourishing the turbot larvae until they are ready for metamorphosis. Prior to metamorphosis, while the turbot larvae are pelagic, the turbot larvae are transferred to closed or semi closed, often indoor systems where they are reared until further processed (Paulsen et al., 1985).

A Danish semi intensive system situated at 56°N, rearing turbot larvae, has an outdoor productive season from May until September (late spring to early fall). The commercial rearing system consists of a number of 280m3 open circular outdoor concrete tanks. At this latitude, the rearing systems typically undergo 2 to 4 outdoor production cycles, each of 3-5 weeks during the productive season (Engel-Sørensen et al., 2004). As the productive season progress, the survival of turbot larvae tend to decrease, being lowest in the last production cycle (Engell-Sørensen et al., 2004; Jepsen et al., in prep.).

A production cycle is initiated by filling rearing tanks with 50 µm screened estua-rine water, eventually enriched by inorganic nutrients (Engell-Sørensen et al., 2004). After a few days, growth of naturally occurring phytoplankton is achieved and zooplankton is added from a ‘storage’ tank of naturally occurring species of especially calanoid copepods. The copepod assemblage has been selected over the last 15-20 years and exhibits a similar repeated species succession pattern throug-hout the annual production period (Sørensen et al., 2007).

When the copepods begin reproducing, after a few days, turbot yolk sack larvae are acclimatised and applied to the tanks, establishing a full pelagic food web. After growing for 3-5 weeks, the larvae are ready for metamorphosis into benthic associated juvenile turbot. At this point, the turbot larvae are collected from the water column and transferred to indoor tanks for veining to pelleted food.The estuarine water used to fill the tanks undergoes a natural seasonal dynamics

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typical for coastal productive systems, where nutrient are high in winters, de-creasing during spring and low through the reaming part of the productive cycle. Therefore, the growth potential for phytoplankton in rearing tanks may be con-strained during summers due to depletion of inorganic nutrients such as nitrogen.There is a well-recognised link between the concentration of dissolved inorganic nitrogen and amino acids in phytoplankton cells e.g. Eppley (1971). Moreover, when the amount of amino acids in marine microalgae decreases due to nitrogen limitation, the fatty acid composition changes rapidly, suggesting that an increasing part of the photosynthetic acquired carbon was allocated from growth to storage compounds such as fatty acids and aldehydes (Flynn et al., 1992; Ribalet et al., 2007). Hence, nitrogen dynamics and availability, changes the overall nutritional value of phytoplankton, and will ultimately challenge the biochemical composition of the copepod diet.

A nitrogen containing diet that meets the requirements for somatic growth in nauplii and copepodites is important in copepod nourishment (Ambler, 1986; Checkley, 1980). At the same time essential amino acids are displaying fixed ratios (homeostasis) in copepods (Guisande et al., 1999). Thereby, not only does the total pool of amino acids govern growth in copepods, but also the absence of single essential amino acids may lead to an additive negative effect on copepods in nitrogen limited food webs. Thereby the decreasing nitrogen loads is most likely cascaded further to limited prey available for feeding turbot larvae, yet this remains to be addressed in semi intensive fish rearing tanks.

The aim of this work is to describe the development of prey phytoplankton and calanoid copepods in a land based aquaculture turbot larval rearing system during a production season, covering 3 production cycles. We hypothesise that the decrease in turbot larval survival reported in a companying paper by Jepsen et al. (in prep.) is linked to inorganic nutrients and in particular to the nitrogen concentration in the initial tank water, used to initiate each of the production cycles. That is, the seasonally decreasing in-put of nitrogen to the system upon initiating a production cycle affects phytoplankton biomass, growth and feed quality, leading to decreased copepod recruitment and abundances.

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2.0 METHODS

2.1 Stocking

2.1.1 Phytoplankton The phytoplankton development was followed during 3 production cycles of Scophthalmus maximus larvae in the periods from late May to medio September 2011. The three production cycles are here from named PC1 for production cycle 1, PC2 for production cycle 2 and PC3 for production cycle 3.The experiments were conducted in triplicate land based outdoor open top semi-intensive circular concrete tanks. Each tank holds 280 m3 and has a diameter of 12 m with a water depth of approximately 2 m. The tanks were filled with estuarine water pumped from the adjacent Dragstrup Vig (Limfjorden, Dk, N 56.8; E 8.5). All intake water was 50 µm screened by a drum filter (Hydrotech HDF 2007-2H http://www.hydrotech.se/solutions/drumfilters/) to remove unwanted organisms such a barnacle larvae, ascidians etc. No additional inorganic nutrients were amended initially to the tanks.

2.1.2 CopepodsCopepods and nauplii were collected by a dish filter (UNIK Rotating Wheel Filter Type 1200 http://www.unikwater.com/hjulfiltre/) from a storage tank where a seeding culture of copepods are maintained free of parasites. The copepods and nauplii were added to each of the tanks in concentrations according to table 1. The tanks were left to acclimatise for 2 to 5 days before yolk sack turbot larvae were added. However, this study only focuses on primary producers and copepods and more details on the linkage between copepods and fish larvae is presented in a companion paper by Jepsen et al. (in prep.).

Table 1. Initial concentrations of the copepods L-1

added to the tanks. Values are means of tanks ± S.D.

Production cycleAdult and copepodites Nauplii

1 1.4 ±0.5 1.2±1.02 5.2±2.4 0.1±0.23 8.6±6.2 4.8±2.5

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2.2 Sampling

The various process variables relevant for this study were sampled with different intervals accordingly to the schedule in table 2. The experimental water for analysis was collected by a 3L heart-valve sampler in half-meter water depth in each of the three production tanks. Samplings were conducted at four different points to collect a representative sample of the tank. The sampled water was gently pooled into a 30L dark bucket. A sub-sample representing the “total” was collected for microscopically estimates of phytoplankton species compositions and biomas-ses. The remaining part was inversely filtered by slowly submerging an open-end bucket fitted with 50 µm NITEX mesh size into the 30L bucket to collect water for analysis of free amino acids and combustible particulate carbon, hydrogen and nitrogen (CHN). We used the 50 µm because the size fraction 0-50 µm is most relevant as feed for all development stages of copepods (Berggreen et al., 1988).

2.2.1 Light and temperature Light and temperature were logged in 1 meter depth using HOBO Pendant ® dataloggers http://www.onsetcomp.com. Data was logged with 5 min. intervals and collected at the termination of each campaign.

2.2.2 Nutrient analysis Samples for nutrient was collected form the 50µm screened fraction and stored in 20 mL polyvinyl bottles frozen at -20°C. Dissolved NO2

-, NO3-, NH4

+ and orthophosphate were determined from the total sample by colorimetric continuous

Table 2. Sampling frequency of the different parameters measured during the production cycles.Parameter frequencyIrradiance 5 min interval.Temperature 5 min interval.In vivo fluorescence DailyNutrients 5-7 d.Phytoplankton biomass 5-7 d.Seston amino acids 5-7 d.Seston C:N < 50µm 5-7 d.Zooplankton biomass and copepod egg production 5-7 d.

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flow analysis by a Skalar SanPlus auto analyser. Analysis procedures were done on routine basis following a standard ISO17025 accredited procedure (http://webtool.danak.dk/Plone/) according to the methods described by Grasshoff (Grasshoff, 1976). The nitrogen species analysed was summarised into the dissolved inorganic nitrogen (DIN) pool whereas orthophosphate is found on the dissolved inorganic form PO4

-2 (DIP).

2.2.3 In vivo fluorescenceA fluoroprobe from bbe Moldaenke (http://www.bbe-moldaenke.de/chlorop-hyll/fluoroprobe/) was used to determine in vivo fluorescence during the three PC’ s. The fluoroprobe is a multi-channel fluorometer that uses group specific fluorescence and quantifies phytoplankton into 4 main groups namely crypt-ophytes, green algae, cyanobacteria and brown algae (Beutler et al., 2002). The brown algae group is a composite of diatoms, dinoflagellates, heterokontophyta and haptophyta. Data were collected from vertical casts sampled between 10 and 12 noon.

2.2.4 CHN-Analysis In each of the triplicate tanks, five hundred mL of the 50 µm Nitex filtered wa-ter were collected on a 25 mm diameter 0.2 µm GF/F Whatmantm filters (www.sigmaaldrich.com). Individual filters were placed in pre-burned tin capsules (muffle furnace 550°C) and analysis by a CE Instruments EA 1110 elemental analyser (http://www.ceinstruments.co.uk), and C and N were determined against methionine standards.

2.2.5 Phytoplankton biomass Water samples were collected from the unfiltered “total” tank water. Samples were fixed in acid Lugols (1% final concentration), measured and counted in an inver-ted microscope (Utermöhl, 1958). The analysis followed the general guidelines given in the Danish environmental monitoring program (Henriksen and Kaas, 2004; Jakobsen, 2012). Briefly, all cells > 2-3µm were identified to at least genera and lowest possible taxa. At least 50 cells and preferentially more than 100 cells were counted of the dominant species. A total of at least 500 cells were counted. Bio-volumes of 10 cells or more of each phytoplankton species were estimated by applying cell dimensions to appropriate simple geometric shapes (Edler, 1979). The estimated bio-volumes were converted into carbon biomasses using the con-version factors of 0.13 and 0.11 pg C µm-3 for thecate dinoflagellates and other

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phytoplankton, respectively (Edler, 1979). Diatom cell carbon was estimated accordingly to Strathmann (1967).

2.2.6 Free extractable amino acids in seston < 50 µm. In each of the triplicate tanks, five hundred mL water was filtered onto a 25 mm diameter 0.2 µm GF/F Whatmantm filters (http://www.sigmaaldrich.com). The filters were immediately stored frozen in liquid nitrogen, and stored at -80°C until further analysis. The seston amino acids collected on the filters were extracted in 5 mL 99.8% HPLC grade methanol and analysed by high performance liquid chro-matography (HPLC). Specific amino acids were identified from retention times in the chromatograms (Jørgensen et al., 2013). Published extraction procedures for free amino acids in marine particulate material have involved organic solvents, such as toluene and methanol mixtures, and hot water (Bölter and Dawson, 1982). To test for extraction efficiency by the present methanol addition, concentrations of extractable free amino acids in cells of three different algal cultures (filtered onto 0.2 µm cellulose nitrate filters) were compared after analysis by high performance liquid chromatography (HPLC) according to Jørgensen et al. (1993). No stati-stical difference in composition or concentration of amino acids extracted by the two treatments was found (Jørgensen, unpublished observation). Hence, it was assumed that the extraction of amino acids in the algal cells were representative to amino acids in the present seston material.

2.2.7 Copepod, abundance, biomass and productivity measurements Copepods were collected from four vertical hauls with a 0.25 m diameter 45µm mesh size plankton net with closed cod-end. From the depth, diameter of the net and the number of hauls the sampled water volume was calculated assuming 100% retention efficiency by the net. The samples were fixed in acid Lugols´ (final conc. 1%) and stored until later analysis. Diversity and biomass of 300-500 copepods were determined using dissection microscopy (Nielsen and Møhlenberg, 2004). Biomass determinations were based on individual length: weight constants for particular stages and species. In absence of length: weight constants, biomasses were based on volume calculations of 10 individuals using a factor of 0,12 pg C µm-3.

2.2.8 Secondary production experiments Live copepods from each tank were collected by hauling a 200 µm mesh size plankton net (diameter: 0.25 m) with a closed cod-end through the tanks. The

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cod end was emptied into a 10 L cooler carefully maintaining similar temperature conditions as in the tanks. When only one copepod specie were dominant 15 females were individually transferred into each 600 mL acid rinsed polycarbonate Nalgene® bottles filled with 50 µm screened tank water (Acartia spp. in PC1 and Centrophages hamatus in PC2 and PC3). When both species were present 15 females of each species were individually incubated. The bottles were incubated at temperatures of ca. 18 °C thus providing comparable rates independent of ambient tank temperatures. The bottles ware incubated for 24 h. at dim light fol-lowing the solar photoperiod. The bottles were regularly rotated to maintain food particles in suspension. After 24 h the bottle content were filtered through a 50 µm and a 200 µm filter and the eggs where collected and counted. Females where collected from the 200 µm filter fraction, and prosome lengths were measured. The prosome length was used in calculating carbon specific biomass. The eggs were transferred into plastic Petri dishes with filtered tank water and incubated under similar conditions to the egg production. After 48 h the content of the Petri dishes were fixed in Lugol´s and the number of hatched nauplii and eggs were counted to estimate hatching success. The specific egg production for Acartia spp. were calculated from Berggreen et al (1988) and carbon of C. hamatus were calculated accordingly length: weight scaling from Blanda et al (in prep.). The egg carbon content for both copepod species were obtained from bio volume of eggs measured on 10 eggs of each species, from each PC, and the carbon density given in Kiørboe et al (1985).

3.0 RESULTS

3.1 Study site

The nearby monitoring station at Løgstør Bredning Denmark (N 56.6; E 9.0) display (fig 1) 20 years of monthly means of dissolved N, P and Si (http://www.dmu.dk/vand/havmiljoe/mads/plankton/fytoplanktondata/) and is a proxy of the intake water available for the fish farm. The concentration of nitrogen is initially non-limited in May but decrease throughout the production period to a low and constant level, close to the half saturation concentration of 1.6 µmol L-1 for nitrogen uptake in diatoms (Sarthou et al., 2005). Likewise nitrogen, evidence of phosphorous limitation appears during spring as values below threshold levels of 0.24 µmol L-1 are found (Sarthou et al., 2005). Phosphate concentrations in

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the estuarine remain above limitation while nitrogen remains low during summer and towards the early fall in September.

3.2 Statistics

Because of the different numbers of sampling in the different PCs an unbalanced test design is used and we used a mixed model test. That is; PC is the fixed ef-fect that is compared while data and tank are the random effects. We addressed this by using a mixed general linearized model (PROC MIXED) by the software package SAS 9.2™. Data was log transformed prior to testing to make variances comparable between sampling days.

3.3 Abiotic and biotic factors

3.3.1 Abiotic factorsDaily mean temperature was 16.5 ± 2°C for PC1, 19.9 ± 1.5 °C for PC2 and 11.5 ± 0.8 °C for PC3 in 1 meters depth.

3.3.2 Inorganic nutrientsThe initial nutrient concentrations in the PCs reflected the monthly means for May, August and September and hence followed the general observation of the estuarine water (compare fig.1 and table 3). NO3

- was initially the most dominant nitrogen species in PC1 whereas NO3

- was absent in the 2nd PC and very low (0.7 µmol L-1) in the 3rd PC (table 3). DIP and silicate (Si) were above limitation initially in all PCs. During the first week of PC1, DIN decreased to ca. 2 µmol

Fig 1. Monitoring data showing relevant environmental parameters from the nearby monitoring station in Løgstør Bredning, covering same time period as the monitored production season. Salinity (•, PSU), temperature (---, ºC) , dissolved inorganic phosphate (DIP,,µmo L-1) and dissolved inorganic nitrogen (DIN, --, µmo L-1). Data are means from 20 years measurement data ± S.D.

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L-1 and remained low during the rest of the PC1 (fig 2a). However, Si increased slightly towards the end of the PC1 to a value of 4.5 µmol L-1, most likely due to re-mineralisation of the initial phytoplankton bloom. The fish farm manager at-tempted to correct for the nutrient deficiency by topping the tanks with screened bay water in two occasions in PC2 (marked with arrows on fig 2b). Although fresh bay water was added, the DIN level stayed below 1 µmol L-1 during most of the PC. In the 3rd PC most of the DIN was in the form of ammonium initially (table 3). DIP and Si was likewise higher than the previous PCs. However, the DIN fell quickly to less than 0.5 µmol L-1 within the first 5 days. In contrast to DIN, DIP and Si remained higher during the 3rd entire PC. An attempt to raise DIN was likewise in the 2nd PC, and inorganic nutrients were added (marked by the arrow on fig 2c). The added DIN was composed of 5 and 6 µmol L-1 NO3

- and NH4+

(data not shown) respectively and DIN thus reached a concentration of 11 µmol L-1 (Fig 2c). The following day NH4

+ fell down to 2.5 µmol L-1 and NO3- fell to

4.5 µmol L-1. The change in relative ratios between the two main nitrogen species suggests a higher affinity of phytoplankton to NH4

+.

3.3.3 Primary producersThe total chl a identified by the fluoroprobe increased initially in all three PCs from about 2 µg chl a L-1 towards a maximal value that was reached approximately 1 week after filling of the tanks. The strongest bloom development was found in the 1st PC, where a maximal value of 18 µg chl a L-1 was reached (fig 3a). A slightly lower bloom peak developed in the 2nd PC (10 µg chl a L-1, fig 3b) and almost no initial response in Chl a in the 3rd PC was observed (7 µg chl a L-1, fig 3C).

Table 3. Initial nutrient concentrations of production cycle 1, 2 and 3. DIN is the sum of all inorganic nitrogen species. Values are µmol L-1 ± S.D. of the 3 tanksProduction cycle Date (2011) NH4

+ NO3+ NO2

+ DIP DIN Si1 20/5 7/6 5.5±1.5 10.5±0. 6 0.8±0.0 0.5±0.1 16.8 18.7±0.62 27/76/8 1.5±0. 6 0.0±0.0 0.00±0.0 0.8±0.0 1.5 4.0±0.93 19/83/9 3.5±0.4 0.7±0.1 0.2±0.0 2.4±0.03 4.3 17.0±2.7

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Fig 2. Nutrient dynamics during the 3 production cycles. Values are given in µmol L-1 as means of dissolved inorganic nitrogen (DIN; --), dissolved inorganic phosphate (DIP;) and silicate (). Error bars are S.D. of the 3 tanks used in production cycle 1, 2 and 3. Arrows indicated where the fish farm manager added either water from the bay (production cycle 2) or fertiliser (inorganic nutrients) as in production cycle 3.

This also yielded differences between PCs (Mixed model: F2;6=14.76, P=0.0048). Comparing PCs pairwise gave significant similar means in PC2 and PC3 (t-test; t=-1.23, df=6, P=0.26) whereas PC1 was higher than PC2 (t-test; t=516, P <0.002) and PC3 (t-test; t=3.93, P <0.008). The phytoplankton composition determined by the fluoroprobe was dominated by diatoms and algae of the brown group whereas cryptophytes and cyanobacteria were present in very low concentrations (data not shown). A common trait for all PCs was a levelling chlorophyll a at around 6 chl a L-1 after the initial phytoplankton bloom increase (fig 3).

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Fig 3. Flouroprobe data showing total chlorophyll a ( ), the contribution from green algae () and brown algae () to the total chlorophyll a. All units are µg chlorophyll a L-1 and are calculated as a means of all 3 tanks ± S.D. The term green and brown algae include several phytoplankton genera; please see text for further explanation of the definitions. Note that diatoms are included in the brown group. The stipulated line is the mean temperature of all 3 tanks ± S.D. (vertical lines) collected with HOBO loggers. Arrows indicate similar as in figure 2.

The mean phytoplankton biomass of the 3 replicate rearing tanks obtained by inverted microscopy analysis displayed an initial bloom response in biomass in all 3 PCs with the lowest response in the 2nd PC (fig 4). In the 1st PC, the first biomass value recorded was May the 24th i.e. 4 days after the tanks were started suggesting that a lower initial value may have been missed by the sampling (see initial low Chl a at fig 3). The initial total biomass recorded was 436 µgC L-1which decreased during the PC to a final value of ca. 180 µgC L-1.

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Diatoms, mainly Skeletonema costatum dominated the initial diatom biomass and represented approximately 50% of the total initial phytoplankton biomass. Un-identified flagellates (<14 µm) made up approximately 80 – 100 µgC L-1 of the

Fig 4. Left panels a,b,c, are the phytoplankton community divided into algal classes. Color codes for the left panel are : Dinophyceae, : Cryptophyceae, : Chlorophyceae, : unknown flagellates, : Bacillariophyceae, : Prymnesiophyceae,: Prasinophyceae. Right panels d,e,f are the diatom species composition. Color codes for the right panel are : Chaetoceros sp., : Dactyiosolen sp., : Nitzchia longissima, : Skeletonema costatum, : Leptocylindricus danicus, : Thalassiosira nordenskioldi.

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available phytoplankton biomass throughout the 1st PC. However, the dominance of S. costatum decreased during the 1st PC and had almost disappeared at the end, whereas Cheatoceros spp. maintained a more stable biomass (fig 4d). In the end of the PC1, un-identified flagellates was dominating (fig 4a). In the 2nd PC phytoplankton biomass was initially ca. 250 µgC L-1 and constituted as in PC1 mostly of S. costatum (fig 4e). The other phytoplankton were either prasinophytes or un-identified > 14µm flagellates (fig 4b). When the fish farm manager added extra estuarine water a small increase in the chl a concentrations was observed and a more pronounced peak to the 2nd addition (arrows on fig 3b). At the end of the 2nd PC the initial population of S. costatum had almost vanished and was replaced by a dominant population of Dactyliosolen sp. (fig 4e). In the 3rd PC bio-mass was initially ca. 360 µgC L-1. Likewise, during the previous PCs S. costatum (fig 4f ) displayed the highest biomass initially, which decreased during the PC3. In contrast, Dactyliosolen sp. remained in the water column; thus becoming the relatively most important diatom in the end of the 3rd PC.

We estimated the ratio between phytoplankton carbon biomass and the concentra-tion of Chl a determined by the flouroprobe (µgC L-1 (µgChl a L-1)-1). Significant differences between PCs were found (Mixed model: F2;6=19.1, P=0.025) see table 4. Pairwise comparison revealed that the PC1 was lower than both PC2 (t-test, t=-6.05 P =0.009) and PC3 means (t-test, t=-3.2 P =0.019) whereas no differences could be identified between the 2nd PC and the 3rd PC (t-test, t=0.99 P =0.26).

3.3.4 Seston carbonSeston carbon (<50µm) increased initially in parallel to the increase in chl a, to a maximal value of 1500 µgC L-1 the 27th of May, where after it decreased to ap-proximately 700 µgC L-1 in the end of the PC1. In the 2nd PC seston (<50µm) ranged between 600 and 1000 µgC L-1. Data for the 3rd PC was accidently lost and data for PC3 is therefore unavailable. The C:N ratio maintained almost constant elemental proportions close to 7 in all measurements (fig 5).

Table 4 Carbon to Chl a ratio (µgC L-1 /µgChl a L-1) of PC1, PC2 and PC3.Production cycle C: Chl a1 23.8±14.82 84.3±45.23 73.9±66.1

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3.3.5 Amino acidsSeston free amino acids (AA) were divided into essential amino acids (EAA) and non-essential amino acids (non-EAA) (table 5). EAA were identified as being es-sential for Acartia sp. according to Kleppel (1993) and Van der Meeren (2008), and included lysine, threonine, methionine, tryptophan , valine, isoleucine, leucine, argenine and histidine. Methionine and tryptophan are shown together due to co-elution. Phenylalanine is recognised as an impotent and essential amino acid our method was not adapted towards this particular AA. In the first production cycle, the concentration of AA increased from 344 nmol L-1 on Day 1 to a maximum of 771 nmol L-1 on Day 4 (mean values of three tanks; Table 5). After 10 days, a minimum concentration of 124 nmol L-1 occurred. In the subsequent 2 week period, the concentrations varied from 201 to 314 nmol L-1. In the 2nd production cycle, the AA pool increased from initially 436 to 612 nmol L-1 five days later (mean concentrations). In the 3rd production cycle, the AA pools varied from 95 to 184 nmol L-1 (mean concentrations) during the 12 day period. A statistical test including abundance of all AA and EAA, and normalised essential amino acids in

Fig 5. Particulate carbon () and the ratio of C to N (). Error bars are S.D. of the three tanks used in production cycle 1 and 2. Data for C:N determination in production cycle 3 was unfortunately lost.

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Tabl

e 5.

Am

ino

acid

s by

date

of p

rodu

ctio

n cy

cle

1, 2

and

3 fr

om th

e 50

µm fr

actio

n. E

AA

, non

-AA

and

AA

are

abs

olut

e va

lues

(nm

ol L

-1±

stan

dard

dev

iatio

n). T

he

spec

ified

am

ino

acid

s are

giv

en in

per

cent

ages

of A

A±

stan

dard

dev

iatio

n. W

here

stan

dard

dev

iatio

n is

mis

sing

onl

y da

ta fr

om o

ne ta

nk w

as a

vaila

ble.

Dat

e(2

011)

PCEA

A(n

mol

L-1)

non-

AA

( nm

olL-1

)A

A( n

mol

L-1)

Thio

nine

His

tidin

eA

rgen

ine

Val

ine

*Try

ptop

han/

Met

hion

ine

Leuc

ine

Iso-

leuc

ine

Lysi

ne

20-0

51

50.0

±4.7

294±

9534

4±97

3.2±

3.0

1.0±

3.4

0.5±

0.5

0.2±

0.2

0.3±

0.4

1.0±

0.7

5.3±

2.7

3.1±

1.9

24-0

51

127±

4164

4±21

177

1±24

64.

8±9.

64.

0±3.

11.

0±0.

70.

2±0.

10.

8±1.

21.

8±2.

12.

3±0.

61.

7±0.

427

-05

182

.4±8

114

3±86

226±

168

8.5±

12.9

10.7

±15.

73.

7±4.

40.

5±0.

53.

0±3.

98.

4±11

.60.

9±0.

20.

8±0.

330

-05

123

.4±1

110

0±64

124±

763.

3±1.

32.

9±2.

52.

0±1.

80.

4±0.

12.

0±2.

44.

9±6.

21.

7±0.

21.

7±0.

302

-06

163

.3±5

425

1±22

731

4±28

12.

1±1.

92.

0±1.

82.

4±2.

60.

2±0.

12.

4±2.

45.

7±5.

72.

7±2.

72.

6±2.

207

-06

145

.915

520

11.

81.

31.

91.

01.

23.

17.

74.

914

-06

158

.4±7

.317

5±39

233±

461.

9±5.

40.

6±0.

82.

0±4.

40.

7±1.

81.

1±3.

22.

5±7.

87.

5±3.

48.

6±2.

005

-08

297

.4±3

933

9±94

436±

133

3.9±

4.9

1.7±

3.9

1.5±

4.0

0.5±

1.1

1.2±

3.7

3.6±

11.5

5.1±

1.6

4.9±

1.5

10-0

82

186±

146

426±

329

612±

475

6.4±

6.5

7.6±

9.7

2.2±

2.7

0.4±

0.6

2.3±

3.5

5.4±

7.6

3.3±

0.3

2.8±

0.5

19-0

83

44.2

140

184

5.5

2.8

2.9

0.9

1.7

5.2

2.8

2.1

25-0

83

53.9

224

277

5.9

3.1

2.4

0.3

1.3

2.9

1.9

1.6

31-0

83

27.8

±7.9

66.9

±5.4

94.8

±13

6.7±

12.5

4.8±

37.5

3.0±

9.1

0.4±

1.5

1.6±

4.2

4.0±

15.2

5.0±

6.3

3.9±

1.8

*Try

ptop

han

and

Met

hion

ine

show

n co

mbi

ned

due

to c

o-el

utio

n

MANUSCRIPT 188

the three production cycles showed that all AA and EAA in absolute terms were similar between the three production cycles (Mixed model: F2;6=2.1, P=0.2). The normalised EAA tabulated in table 5 displayed similar proportions “homeostasis” across the all three production cycles (Mixed model: F2;6=2.59, P=0.15).

3.3.6 ZooplanktonThe metazoan plankton biomasses were mostly made up by calanoid copepods of the genera Acartia spp. and Centropages hamatus that both occurred in variable concentration with high standard deviations of the mean between tanks and days (fig 6 and table 6). Acartia spp. biomass did not show significant differences bet-ween PCs (Mixed model: F2;6=2.53, P=0.16). C. hamatus on the other hand was abundant in increasing biomasses between the three PCs (Mixed model: F2;6=12.38, P=0.007) (table 6). A further test comparing PCs pairwise, showed that PC1 was significant lower than PC2 (t-test, t= -3.27 P=0.017) and PC3 (t-test, t= -4.60 P=0.0037) whereas PC2 and PC3 were similar (t-test, t= -1.39 P=0.21).

The average nauplii biomass (Mixed model: F2;6=0.88, P=0.46) and the number of eggs found in the water column (Mixed model: F2;6=0.14, P=0.88) was constant by PC during the production season (table 6). Comparing non log transformed ratios of eggs female-1 (Mixed model: F2;6=0.78, P=0.49), eggs nauplii-1 (Mixed model: F2;6=1.13, P=0.38) and nauplii female-1 (Mixed model: F2;6=0.46, P=0.65) showed no significant difference between PC means.

Table 6. Production cycle means of zooplankton biomasses.

Values except eggs are means in µgC L-1 ± S.D. Eggs are given

in numbers L-1.

Production

cycle Acartia spp. C hamatus Nauplii eggs

1 8.32±8.0 1.29±1.7 3.5±3.0 1.44±1.5

2 2.67±3.8 9.19±5.1 1.9±1.9 1.86±2.8

3 0.86±0.2 13.6±8.7 1.4±1.6 0.58±0.3

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No statistical difference in the specific egg production experiments conducted in the laboratory was found for Acartia spp (Mixed model: F2;6=0.86, P=0.47) or Centrophages hamatus (Mixed model: F2;6=0.61, P=0.57). Hatching success in Acartia spp. (Mixed model: F2;6=0.29, P=0.76) or in Centrophages hamatus (Mixed model: F2;6=1.16, P=0.38) was even and not different among PCs (fig 7).

Fig 6. Copepods life stage biomass development over time for the 3 PC’s. Values are tank means (µgCL-1± S.D). Color codes are : Acartia spp., : Centrophages hamatus, : calanoide nauplii : eggs.

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4.0 DISCUSSION

Our goal in this study is to identify the most likely parameters in the lower trophic levels responsible for the lowered survival of turbot larvae recorded by Jepsen et al. (in prep.).There are several governing parameters such as irradiance, temperature and nutrients that could have created the different phytoplankton food web pat-

Fig 7. Panel a, c and e is specific egg production (egg female-1 day-1) normalised to carbon and copepod egg hatching success (panel b, d and f ) for the 3 production cycles. Acartia spp. and Centrophages hamatus . Values are tank means ± S.D.

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terns observed, that cascaded in the production and population development in copepods and ultimately cascaded into the reduced turbot larval survival observed in Jepsen et al (in prep.) in PC1, PC2 and PC3.

Phytoplankton is the basis for copepod growth and reproduction. Different phy-toplankton communities where developing, with an overall decreasing peak chl a value as the production season progressed. That is; we measured a maximal chl a concentration around 19 µg chl a L-1 in the 1st PC, a maximal peak values of ca. 7 µg chl a L-1 in PC2 and hardly any chlorophyll a peak beyond the initial chl a was observed in PC3 (fig 3).

Specific egg productions were measured under constant temperature conditions and provide a measure of the potential secondary production in the system. However, the observed secondary production conducted in the laboratory was similar over the entire PCs and that egg specific egg production in Acartia spp. and Centrophages hamatus was less 25% of the maximal estimated in the field, thus indicating food limitation (Kiørboe and Nielsen, 1994).

The temperatures in the rearing tanks were averages of 17 °C and 21 °C in the 1st and 2nd PCs respectively, but decreased to 12 °C in the 3rd PC. There is no doubt that decreasing temperatures affects the metabolic processes in the food web. Assuming a Q10 of 2.8 (Hansen et al., 1997), a decrease in temperature from 20 °C to 12 °C may reduce secondary production and growth of nauplii and copepodites by a factor of 0.55.

A lowered copepod production was expected during PC3 by the fish farm manager. To circumvent this, 6 fold more copepods were initially added to PC3 compared to PC1. This increased addition of copepods had no effect on the final copepod net biomass. The final biomass measured in PC3 did not scale proportionally in comparison to the final copepod biomass in PC1 and PC2 (fig 6). In contrast was the copepod biomass increasing 10 fold (fig 6a) during PC1. Although, the temperature reduces the production in PC3, the potential for a strong copepod population was present during the first part of PC3. For example was a strong top down control by turbot larvae predation reported in this set up (for details Jepsen et al. (in prep.)). However, top down control on copepods by turbot larvae is not the only source of arrested development in the copepods population. The decreased abundance in phytoplankton chl a also bottom up limit copepod recru-

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itment and secondary production. DIP increased during the production season eliminating phosphate as the limiting parameter and DIN is most likely is the limiting nutrient. Not only do the concentrations of DIN govern phytoplankton growth, but phytoplankton display different affinity for NH4

+, and NO3, and the shift in the ratio of NH4

+ and NO3- could potentially be a structuring force (Som-

mer, 1983). For an example responded diatoms to additions of nitrogen in an enclosure experiment (Hauss et al., 2012). Similar to these finding did we observe that the diatom Dactyliosolen sp. grew well in PC 2 and PC 3 where N was low, in a similar fashion as observed by Hauss (2012) in the treatments without N. This observation is pointing at nutrient stoichiometry and species depended nitrogen affinity among diatoms as a governor in shaping species composition. Such nu-trient stoichiometry driven species evolution may also explain the shift from the NO3

- using Skeletonema costatum that is reported as high quality food for copepod production (Jónasdottir et al., 2011; Koski et al., 2012) towards e.g. Dactyliosolen sp. of less known food quality. Moreover, the shift in diatom species composition concurred with increasing dominance of unknown small flagellates that made up about 50% of the biomass at the end of all PCs. Flagellates are by their size less suitable in the diets of copepods than e.g. larger diatoms (Berggreen et al., 1988). Another component that potentially could play a role in shaping phytoplankton food quality is the duration of nutrient starvation i.e. the level of senescence of the used algae inoculum (Ribalet et al., 2007; Vidoudez et al., 2011). The phyto-plankton stock that was used in initiating the three PCs had experienced different periods of nutrients depletion. The 1st PC was inoculated with phytoplankton growing in DIN, DIP and Si repleted water; the three main macro nutrients ne-eded for diatom growth. The elevated NH4

+ and DIP recorded at the beginning of PC3 (table 3) most likely originates from oxygen depleted remineralisation in the estuarine sediment and seems to reoccur in the estuarine annually (fig 1). The seeding inoculum was collected from the nutrient deplete estuary was aging in a similar fashion as reported in the literature (Ribalet et al., 2007; Vidoudez et al., 2011) and did not seem to respond well to the added nutrient pulses. Inability to respond and take immediate advantage of a nitrogen pulse by the phytoplankton community is indeed indicated in PC3 where a strong pulse of DIN and DIP of 11 µmol L-1 and 2.2 µmol L-1 respectively was injected to the rearing tanks. This injection do not result in responses in chl a development and only a quick short pulse in phytoplankton biomass (see arrows in fig 3c and compare with 4c,f ). We speculate that the shift in nitrogen removal in PC3 is mostly due to opportunistic bacterial uptake rather than to uptake by phytoplankton.

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The available C:N ratios for PC1 and PC2 was close to the Redfield ratio required for algal production. This was unexpected because no external N source was added in PC1 and only limited nitrogen was added in PC2. That is, C:N rations was expected to increase after the initial phytoplankton peak had developed (Geider and La Roche, 2002; Van Nieuwerburgh et al., 2004). Nitrogen limitations slow down the development time from nauplii to the adult. Arrested development has been observed for up to 4 fold in the calanoid copepods Temora longicornic and Pseudocalanus elongatus when fed nitrogen depleted phytoplankton (klein Breteler et al., 2005). On the other hand, laboratory reared A. tonsa maintained egg production at C:N rations similar to the C:N ratios recorded in the present study (Kiørboe, 1989) suggesting that the degree of nitrogen depletion observed in terms of C:N ratios apparently had less impact on egg production.

Exposure to nitrogen limitation affects phytoplankton and arrest photosynthetic growth (Flynn et al., 1992). That is; reduced nitrogen supply forces the algae syn-thesis mechanisms to change from cell division towards carbon storage material such as fatty acids (Flynn et al., 1992; klein Breteler et al., 2005).

Amino acids such as the listed in table 5 were all present among the free amino acids measured in our study. Most or all of these essential amino acids have previously been detected in the pool of free amino acids in algal cultures, e.g. in dinoflagel-lates in a coastal region (Flynn et al., 1994) and in laboratory cultures (Carmento et al., 2013; Zygmuntowa, 1972) underpinning the role of phytoplankton as the main amino acid source for copepods. A comparison of the observed free amino acid concentrations in particulate organic matter <50 µm, relative to expected content of free amino acids in the phytoplankton, shows that the extracted amino acids made up from about 40 to 140% of the expected free amino acid content in the phytoplankton, if applying free amino acid content in various algae and cyanobacteria by Zygmuntowa (1972) and C content of 24% of dry weight of cells (Platt and Irwin, 1973). Based on data in another study (free amino acids in four different algal species; Carmento et al.(2013)), content of free amino acids in our study corresponds to 117 to 613% of the estimated amino acid content in the algal cells. Thus, our results appear to support that phytoplankton were major contributors of free amino acids, in comparison to alternative sources.

Copepod nauplii have indeed high demands of amino acids for somatic growth, and need a nitrogen rich diet to increase their body biomass. For an example,

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growing nauplii were shown to have a weight-specific incorporation rate of 18% of their body protein (Roman, 1991). Free amino acids in seston <50 µm con-stitute therefor a potential and valuable nutrient source to the copepods. Impor-tance of free amino acids in the phytoplankton cells, relative to the total nutrient content of the phytoplankton, can be estimated from C content of the amino acids and the phytoplankton biomass. Assuming an average of 3.6 C atoms per molecule amino acid in the free, extractable pool (average C content of the 12 most abundant AA; total AA concentrations given in Table 6) and applying C content of the phytoplankton (Fig. 4), free amino acids were determined to make up 2-5% of the phytoplankton C in PC1 and PC2, but only about 1% in PC3. In laboratory cultures of selected species of diatoms, green algae and cyanobacteria, Zygmuntowa (1972) observed that amino C constituted 1-2% amino acid C if assuming a C content of the algae of 24% (mean C content of various algal cells; Platt and Irwin (1973)). The higher contributions of amino acid C in PC1 and PC2 indicates that the phytoplankton had a higher content of amino acids, or that the amino acids originated from other organic matter in the seston. In a study of the German Elbe estuary, Kerner and Yasseri (1997) observed that particle-bound amino acids made up 12% of the C content of POC in seston. Although content of free amino acids was not determined in seston in our study, organisms such as bacteria, protists and vertebrate larvae may have contributed intracellular free amino acids and hereby sustained feeding of the copepods.

We found that the pools of AA in seston remained fairly constant, except for the peaks in May and August to about 700 nmol L-1. Moreover, the proportions bet-ween EAAs in the <50 µm plankton seston fraction was stable, indicating home-ostasis among phytoplankton concurring with observations made on copepods (Guisande et al., 1999; Jeffries, 1969).

Shortage in nitrogen arrests cell growth in phytoplankton and the cell meta-bolism is directed toward manufacturing of storage compounds (Flynn et al., 1992; Harrison et al., 1990). Among these are polyunsaturated aldehydes (PUA) commonly found in diatoms cultures under nutrient stress (Ribalet et al., 2009; Ribalet et al., 2007) along with other age dependent metabolites (Vidoudez et al., 2011), the potential negative functions of these compounds are still cryptic and debated (Casotti et al., 2005; Lauritano et al., 2012; Miralto et al., 1999; Miralto et al., 1996). The phytoplankton used in the 3rd PC had been utilising remineralised nitrogen for 2 -3 months and we cannot exclude that the seeding

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diatoms already at the time of PC3 initiation already had built up storages that promote stress responses. However, this hypothesis needs to be verified in future effort. Moreover, adverse food conditions has been proposed as one of the cues that promotes female formation of resting egg in calanoid copepods (Mauchline, 1998) and the low hatching success of the eggs could be an indicator of a shift in reproduction strategy toward resting eggs.

In a Norwegian study, where copepods were sampled from pond managed with frequent fertilisation over a 2 yr. period, very little variation in the copepods bio-chemical constituents was found. However, the control samples from the nearby fjord that underwent seasonal variation in nutrients and hence phytoplankton dynamics, was the variation in the biochemical constituents of the copepods much more dynamic (van der Meeren et al., 2008). That is, nutrient levels in the ponds had a significant and positive impact on the nutritional quality. The system studied in the present work is by en large unfertilised and the nutritional stress that we observe in terms of lowered chl a and the change in species composition towards the end of the production season is apparently cascaded into the nutritional quality in a similar fashion as fjord controls in Van der Meeren (2008).

Closing remarks Some conspicuous differences from PC1 to PC3 are: 1. The increasing nitrogen depletion. 2. The decrease in the maximal chl a peak and 3. The appearance of Centrophages hamatus in PC2 and PC3. 4. Fixed proportion in C:N and in the essential amino acids in the seston in contrast to the high variability in the phy-toplankton species composition. 5. The specific secondary copepod production measured by egg production in the lab show similar production levels in the 3 PCs. To maintain higher and predictable copepods biomasses maintaining higher Chl a levels > 15 µg Chl a L-1 prior to adding copepods to the rearing tanks is important. This can be achieved by administering inorganic nitrogen. In the system that was used by Van der Meeren (2008), a Secci depth of 1.5 m was the proxy for when to add nutrients. A careful selection towards an Acartia spp. dominated community in the storage tank which seemed to be linked with the highest turbot survival Jepsen et al (in prep.). Moreover, if the added phytoplankton and copepods are allowed a longer acclimation period than the present practice before turbot larvae are added, then a stronger and more viable pelagic food web will develop, in a fashion that mimic the spring bloom conditions natural turbot larvae are experi-encing (Kiørboe and Nielsen, 1994) .

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ACKNOWLEDGEMENTS

This work was supported by the Danish Strategical Research council grant #10-093522 (IMPAQ). We are indebted to the fish farm manager Anders Thinggaard Pedersen and fish manager assistants Allan Andersen and Gunnar Thinggard Pedersen at MAXIMUS for hospitality during the 3 productions cycles; without their positive and enthusiastic support, this work could never have been executed. HHJ received support from the VELUX foundation to procure optical equipment that benefited this project in the post analysis (VKR022608). AN would like to give thanks to “Transnational Network for Integrated Management of Postdoctoral Research in Communicating Sciences. Institutional building (postdoctoral school) and fellowships program (CommScie)” – POSDRU/89/1.5/S/63663, financed under the Sectoral Operational Programme Human Resources Development 2007–2013. PMJ were supported by EliteForsk travel grant (J. no. 11-116388). Jacob Carstensen at Bioscience provided suggestions and insightful ideas on sta-tistical analyses. We also thank Louise H. Nørremark from FishLab who assisted in laboratory plankton analysis. Kristian Filrup, Katrine Lørup, Pernille Due Poulsen, Anne Busk Faarborg and Rikke Guttesen provided valuable assistance in the field and in the lab during the field campaigns.

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Manuscript 2.

Jepsen, P. M., Jakobsen, H. H., Blanda, E., Novac, A., Engell-Sørensen, K., and Hansen, B. W. (in preparation). A seasonal study on turbot larvae Scophthal-

mus maximus (Linnaeus, 1758) reared on copepods in a Danish semi-intensive outdoor system. II: Larval growth, prey selection and survival until fry.

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A seasonal study on turbot larvae Scophthalmus maximus (Linnaeus, 1758) reared on copepods in a Danish semi-intensive outdoor system. II:

Larval growth, prey selection and survival until fry.

Jepsen, Per M.1*; Jakobsen, Hans H.2; Rayner, Thomas A.1; Blanda, Eli-sa1; Novac, Aliona3; Engel-Sørensen, Kirsten4; Hansen, Benni W.1

1. Department of Environmental, Social and Spatial Change. Universitetsvej 1, POBox, 260. Roskilde University. DK-4000 Roskilde. Denmark.

2. Department of Bioscience. Aarhus University. Frederiksborgvej 399, DK-4000 Roskilde. Denmark.

3. Faculty of Biology,”Alexandru Ioan Cuza” University of Iasi, Carol I Bou-levard 22, RO-700505 Iasi. Romania.

4. Fishlab, Terp Skovvej 107B, DK-8270 Højbjerg, Denmark

*Corresponding author

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ABSTRACT

Turbot were reared from yolk sack larvae to juvenile in outdoor semi intensive, circular open top, 280 m3 concrete tanks. Historically, the fish farmer experien-ced a decreased turbot larval survival, during the productive season (Early May to September). To investigate the survival and growth pattern, three (3-5 weeks) outdoor production cycles were followed from early May to September, in 2011. In the tanks, a pelagic food chain consisting of phytoplankton, copepods, and turbot larvae was established. Abiotic and biotic parameters and quantification of lower trophic levels were monitored together with turbot larval survival, development, prey electivity and growth. The effort revealed a decrease in larval survival from 18.4% in May to 12.1% in July and 5.0% in September. Phytoplankton and the prey field (copepods) abundance also decreased during the season. However, despite of the differences in survival the larvae obtained non statistically different specific growth rates from 0.22 d-1 in May to 0.21 d-1 in July and 0.12 d-1 in September. Furthermore, the observed stomach content was similar over the three production cycles. From present data, the turbot larvae’s population carbon demands were modelled. In all three production cycles, the turbot experienced food shortage, especially in production cycle 2 and 3. This was probably a combined effect of lower phytoplankton and prey field abundance, although the fish farm manager stocked with less turbot larvae and more copepods in production cycle 2 and 3. Therefore an even lower stocking of turbot larvae is recommended. The main conclusion is that turbot larvae that received a good start, with abundant prey during their early life, will benefit in their later post metamorphic life. This was exhibited, clearly, by vital traits, such as increased survival, increased growth, and less deformities in market sized juveniles.

KeywordsScophthalmus maximus, Acartia spp., Centropages hamatus, Aquaculture, Marine turbot larvae, Live feed.

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1.0 INTRODUCTION

Aquaculture is an expanding industry (FAO, 2010). In Europe, marine aquacul-ture science began as a production of fry to restock declining species in the sea and has therefrom evolved into a food industry (Purdom, et al. 1972). Plaice (Pleuronectec platessa), sole (Solea solea) and turbot were among the pioneer marine fish species produced during the 1960s and 1970s. Jones, et al. (1974) suggested that turbot would be a suitable candidate with attractive market prices. Hence, turbot has been reared in captivity for decades and has, since the early start of the 1980s, increased its yearly production to 9,500 tons in 2008 (FAO, 2010). Historically, the production was initiated in France and Spain, with Spain as the main producing country until the early 2000s wherefrom China has dominated the world turbot market (FAO, 2010).

Paulsen (1985) considered turbot as one of the most promising species, but empha-sised that the methods for rearing turbot larvae at that time were a limiting factor (Jones et al. 1974; Urup, 1994). Today almost three decades after the pioneering work, still many potential bottlenecks in the large-scale industrial production of marine fish species exists (Planas and Cunha, 1999). The Danish production of turbot located at 56ºN, is marginal in comparison to the world production. One substantial difference worth noting regarding Danish turbot production strategies is that the production is mainly focused upon larval rearing from yolk sack larvae to metamorphosis into juvenile fry turbot (5g turbot) and thereby weaning the first feeding larvae from live feed to pelleted feed (FAO, 2010; Urup, 1994). Hence, the Danish turbot production has a small tonnage but actually covers a relatively large numerical production of turbot fry. In the past 20 years turbot larvae in Den-mark are mainly reared in semi-intensive outdoor enclosures and concrete tanks (Engel-Sørensen, et al. 2004; Urup, 1994). This production method is based on mimicking the pelagic food chain dynamics allowing the turbot larval functioning as top predators. It differs from the traditional marine hatchery methods, where live feed items are rotifers (Brachionus sp.) and brine shrimps (Artemia spp.), since the turbot larvae here are feed with copepods as prey (Van der Meeren, 1991). Using copepods as live feed is challenging, but is advantageous in terms of nutritional superiority and enhanced turbot larval survival and growth (Urup, 1994; Shields, et al. 1999; Van der Meeren, 1991). Turbot has been intensively studied, but only a few papers report the dynamics of the copepod prey field and how this governs fish larvae survival, ontogeny and growth in semi-intensive production system

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(Engel-Sørensen, et al. 2004; Paulsen, 1985; Urup, 1994). To our knowledge, no well accepted production standard aiming at optimised larval ontogeny and survival exists yet. From experiences collected over the past two decades by fish farmers, lowered rearing success in the late summer productions compared to spring productions is reported (Engel-Sørensen, 2004; Anders Thingaard Pedersen pers. comm.). This study presents data from three production cycles of turbot larvae and juvenile, from spring to late summer in 2011.

This work is the second part of two companion contributions. The first part by Jakobsen et al. (in prep.) reports the phytoplankton and zooplankton dynamics. The second part reports the turbot larval survival, development, growth and food consumption. The focus of the second study is therefore to understand the governing parameters for the ontogenetic development of turbot larvae and their carbon demand in semi-intensive outdoors systems. It is the foundation for recommendations to improved management procedures for future turbot larvae and juvenile rearing.

2.0 MATERIAL AND METHOD

The investigation took place on a land based fish farm located at the estuary Limfjorden, Dragstrup Vig, in Denmark (N 56.8; E 8.5). Three successive pro-duction cycles were monitored in the periods from late May to medio September 2011. The three production cycles were named production cycle 1, production cycle 2 and production cycle 3, and henceforward referred to as PC1, PC2 and PC3, in the text respectively. The triplicate outdoor open top concrete tanks were initiated and stocked with phytoplankton, copepods and yolk sac turbot larvae as described in Jakobsen et al. (in prep.). Before turbot larvae reached metamorphosis, the tank water level was reduced to less than 1m depth and the larvae were caught using a seine net and gently transferred to an indoor Recirculated Aquaculture System (RAS) for further development. Turbot larvae from each outdoor tank were transferred to separate indoor 2m3 RAS tanks where they were reared until juvenile (approx. 5g).

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2.1 Environmental conditions in the turbot rearing tanks

The planktonic components leading to turbot larval feed are only described in brief; more details are reported in the companion paper by Jakobsen et al. (in prep.). Phytoplankton biomass was monitored daily using an in situ multichannel flourometer, quantifying plant pigment concentration as a proxy for phytoplank-ton biomass. Phytoplankton biomass and species composition was sampled at frequencies and method as described in Jakobsen et al. (in prep.).

Copepod abundance, species composition and egg production (secondary pro-duction) were collected at the frequencies and with the methods as described in Jakobsen et al. (in prep.) and copepod biomass were analysed according to the NOVANA standard (Andersen et al., 2011-2015).

2.2 Turbot larvae

Turbot larvae used in all three PCs were obtained from Stolt Sea Farm Turbot Norway A/S. The yolk sack larvae arrived less than 48 hours after hatching. Upon arrival, yolk sack larvae were immediately gently oxygenated to keep the turbot larvae in suspension. The following day, the turbot larvae were acclimatised to production tank conditions by slowly adding tank water to their transport boxes. This process took half a day and the larvae were thereafter gently released into the production tanks.

The turbot larvae were enumerated prior to release by subsampling and manual counting. In PC1, the three tanks were stocked with 30,000 ± 2,300 turbot larvae tank-1 on May 27, 2011. In PC2, the same triplicate tanks were stocked with 27,000 ± 8,600 turbot larvae tank-1 on July 27, 2011. In PC 3, the triplicate tanks were stocked on August 22, 2011 with 17,000 ± 2,400 turbot larvae tank-1.

2.2.1 Dry weight, carbon and nitrogen content and growth of turbot larvaeTurbot larvae were anaesthetised with Metomidate (5 mg L-1 seawater) and indi-vidually imaged on a microscope slide. Images were taken of the turbot’s lateral or ventral side depending on the development stage. For each sampling time, at least images of 10 turbot larvae were acquired from each tank. A NIKON Coolpix digital camera, mounted on a calibrated Olympus SZ40 dissection microscope

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(Olympus Optical (Europe) GmBH, Hamburg, Germany) at a magnification of 40X, was used. Image were analysed with ImageJ software. Mouth size was measured according to the method by Shirota (1970). Standard lengths (SL) were measured instead of total length to ensure that turbot larvae could be measured even if the caudal fin was damaged (Cunha and Plannas, 1999). Development was analysed using the following life-history stages: 1. Yolk sac larvae, 2. Preflexion, 3. Flexion, 4. Postflexion and 6. Juvenile (Miller and Kendall, 2009). After pictures were acquired, the turbot larvae were processed further to either Dry Weight (DW) analysis, elemental composition analysis of Carbon and Nitrogen content (CN), Fatty Acid analysis (FA), or stomach content analysis. Carbon Weight (CW) was derived from the CN analyses.

2.3.2 Dry weight Ten turbot larvae from each tank and at each sampling time were collected for DW determinations. Turbot larvae were individually placed in pre-weighed and pre-burned tin capsules (muffle furnace at 550°C). Thereafter the tin capsules with larvae were placed in an oven at 105°C for 24h to obtain DW.

2.3.3 Carbon and nitrogen contentCN determinations were following the same protocol as for DW. Briefly, 10 tur-bot larvae from each tank and each sampling time were collected in tin capsules and processed in a CE Instruments EA 1110 CHNS elemental. A methionine standard curve was used to obtain concentrations of C and N.

2.3.4 Allometry of turbot larvaeStandard length versus DW or CW were analysed to identify the allometric development of the fish larvae. A power function were used to describe the rela-tionship between turbot larvae and DW or CW (eq 1). eq. (1)

Where i is the intercept with the ordinate and the slope constant k is the slope that describes its biomass either in CW or DW. The data of the three replicate tanks, in all three PCs were fitted to eq. 1 giving 9 k slope constants. The 9 k constants were grouped by PCs and statistically tested against each other using a One-Way ANOVA test. If no statistical significance were found between the PCs, then the data for all PCs were pooled together and eq.1 was again fitted to the pooled data.

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2.3.5 Specific growth rate of turbot larvaeTurbot larval Specific Growth Rates (SGR) was calculated from the increase in DW and CW versus time from eq 3. eq.(3)

Where SGR W is DW or CW initially and Wi is the weight at the following sample time. td is the duration (days) between two sampling points.

2.3.6 Fatty acid content of turbot larvae The FA extraction procedure in turbot larvae is based on the method used in Folch et al. (1957) and the trans-esterification process as described in Lund et al. (2007). For each sampling time FA were determined on 10 turbot larvae from each tank stored individually in cryo-vials at -80ºC until further process. A volume of 3ml of 2:1 (v:v) chloroform: methanol (CHCl3:MeOH) mixture was added to each cryo-vial. A known amount of internal standard (20µl of 1000µg/ml tricosanoic FA methyl ester [C23:0 FAME]) was added for FA quantification. The samples were homogenized by a ULTRA-TURRAX® (IKA® T10 Basic) mounted with a dispersing element S10N-8G and subsequently left for 24h at -20ºC. Thereafter, around 90% of the extraction solvent was gently removed while avoiding bottom debris, transferred to GC vials and evaporated by a gentle stream of nitrogen at 65ºC. For acid trans esterification of the lipids, the GC vials were added 1 ml of MeOH: Toluene: Acetylchloride (22:28:5, v:v:v) and placed into a heating block at 95ºC for 2h. Thereafter, the vials were added 0.5ml of 5% NaHCO3 in MilliQ water and the upper phase was transferred to new GC vials. The remaining bot-tom phase in each vial was washed with 0.5 ml heptane twice and transferred to the new GC vial, which was then evaporated under a stream of nitrogen at 65ºC. Finally, 0.5ml of CHCl3 was added to the vials and sealed thereafter. The GS-MS instrumental setup was the same as described in Lund et al. (2007).

2.3.7 Stomach content of turbot larvaeTurbot larval stomach content were analysed by gently catching individual larvae from each tank with a 100µm mesh sized, hand held, zooplankton net and were anaesthetised immediately with Metomidate (5mg L-1 seawater). The gut content was determined by dissecting the stomach and examining the feed items under an inverted microscope (NIKON DIAPHOT 300) at x200. Total numbers of

SGR =

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nauplii were derived from the posterior part of the exoskeletons, and copepodites and adult copepods were derived from whole exoskeletons. From these data, total number of consumed copepods per turbot larvae was determined.

2.3.8 Electivity index To calculate the ontogenetic selectivity in the turbot larvae among the different life stages of copepods, Ivlevs index of electivity was used (Strauss, 1979). eq. (4)

Where E is the electivity, ri is the proportion of the abundance of prey item I in the gut in relation to the total gut content, pi is the proportion of the abundance of the same prey item in the prey field in relation to the total prey field. The index ranges from -1 to 1 with negative values indicating avoidance or inaccessibility to a given prey item. Zero value indicates random selection, and positive values indicate active selection of a prey.

2.3.9 Carbon required to sustain growth of turbot larvae The daily requirement of carbon for one turbot larvae is calculated by multiplying the carbon biomass of turbot larvae with the obtained average SGR of the turbot larvae for the three PCs, while taking into account that 29% of the biomass con-sumed is converted into new fish biomass (Houde 1989).

eq. (5)

This value is calculated by multiplying turbot specific growth rates with the cor-responding turbot carbon weight. By knowing the initial and terminal number of turbot larvae and fry in the tanks for each PC, the instantaneous rates of mortality (Z d−1) were calculated (Breteler, et al. 2004). eq. (6)

N0 is the number of individuals at time 0 and Nt is the number of individuals at time t.

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By multiplying the individual turbot larval carbon demand (Ci) with the estimated current standing stock of turbot larvae, calculated from the mortality estimate (Nt), the instantaneous total carbon demand for the turbot larval populations at different times was derived.

The total standing stocks (summarised from all three tanks) of all copepods including all life stages were used. Subtracting the total carbon demand for the turbot larvae from the total carbon biomass of copepods indicates whether prey was sufficient. That is, positive values indicate excess of copepods whereas negative values indicate limitations of copepods as food. In data before day 10, only the nauplii fraction is included, since the turbot larvae can only predate on nauplii.

2.4 Quality of turbot juveniles

Turbot juvenile weight and abnormalities were rated when the juvenile turbot were ready for sale (~ 5g wet weight fish). All juvenile turbot from each of the indoor tanks were inspected visually, sorted and counted by a 4 channel Apollo fish sorter (Apollo A/S, Denmark, http://www.apollo.dk/index.php?id=5) and a TPC fish counter, FLAC 1000 (IMPEX Agency, Hørning, Denmark, http://www.impexagency.dk). Number of sorted turbots can be derived from table 1. During the visual inspection, turbot juveniles with abnormalities were discarded and their percentages of the total number of turbot calculated.

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3.0 RESULTS

The tanks were stocked with a decreasing amount of turbot larvae during the three PCs, due to a lack of availability in supply. In PC1, PC2 and PC3 the tanks were stocked with 30,000, 27,000 and 17,000 turbot larvae tank-1, respectively. The survival of turbot at juvenile size decreased over the three PCs, with highest survival and juvenile size in PC1 (table 1). Overall, the juvenile sizes were largest in PC1 & PC2, whereas in PC3 all juveniles were only between 3-4g. The turbot from one of the triplicate tanks of PC3 exhibited a gill infection in the indoor RAS, therefore juveniles from this tank were discarded and not included in the result in table 1. The juvenile qualities, judged by the fraction of abnormalities, were similar in PC1 and PC2 whereas the abnormality fraction increased 3 fold in PC3.

3.1 Abiotic conditions and phytoplankton (Chl a)

The temperature mean in PC1 was 16.7 ± 2.0°C. PC2 was warmest 20.5 ± 1.8°C. PC3 was the coldest with a mean temperature of 11.7 ± 1.0°C.

In brief a peak phytoplankton of 18µg chl a L-1 was observed in PC1. In contrast, the peak development in PC2 and PC3 were 10 µg chl a L-1 and 7 µg chl a L-1, respectively. The phytoplankton amount in PC1 was statistically significantly larger than in PC2 and PC3. No statistical significant difference were observed between PC2 and PC3 (Jakobsen et al. in prep.).

Table 1

Production cycle

Total start number of fish larvae

Total end number of fish larvae

Fish larvae survival

Fish larvae abnormalities

1 90,000 16,514 18.4 ± 3.8% 3.5 ± 2.3%2 81,000 8,892 12.1 ± 5.0% 2.4 ± 0.8%3 51,000 1,876* 5.0 ± 5.8% 9.6 ± 5.1%

Total numbers are sums of the three replicate tanks.Fish larvae survival and abnormalities are average of the three replicate tanks ± S.D.*Total of 2 tanks. One tank were lost due to decease when the fish larvaewere in the indoor system

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Figure 1: () Bar plots are total chl a ± S.D. (n=3). () Temperature is daily average °C ± S.D. (n=3). (-) Line plots are average Dissolved Inorganic Nitrogen (DIN) in µmol L-1 (n=3).

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The highest concentrations of inorganic nutrients were observed in PC1, whereas a inorganic nutrient depletion were observed in PC 2 and PC3, for further details refer to Jakobsen et al. (in prep.).

3.2 Copepods

Figure 2: Abundance (individuals L-1) of calanoid nauplii, Acartia spp. and Centropages hamatus, during the 3 production cycles. Bars are average ± S.D.

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The copepods community were generally dominated by calanoid copepods, with Acartia spp. and Centropages hamatus as the predominant species. No statistically significant differences were observed in nauplii abundance (both species pooled) between any of the PCs (One-Way ANOVA: F2;6=0.543, p=0.607). The copepods in PC1 were dominated by Acartia spp. However, a statistically significant suc-cession took place where Acartia spp. was replaced by Centropages hamatus as the dominant species in PC2 and PC3 (One-Way ANOVA: F2;6=38.640, p=<0.001). The total abundance of copepods (Nauplii + copepodites + adults) were not sta-tistically significantly differences between any of the three PCs (Kruskal-Wallis One-Way ANOVA: H2=5.422, p=0.071).

3.2.1 Egg production and hatching success of copepods

Figure 3: () Egg production of Acartia spp. and () Centropages hamatus during the 3 production cycles, bars are average ± S.D. (n=10). () Average hatching success of Acartia spp. eggs, and () average hatching success of Centropages hamatus eggs. Error bars are S.D. (n=10).

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A two-way ANOVA tested for the effect of egg productions of Acartia spp. and Centropages hamatus between the three PCs, and no statistically significant dif-ferences were observed between PCs (F2;12=2.961, p=0.09). Within production cycle 3 Centropages hamatus had a statistically significantly higher egg production than Acartia spp. (F1;12=15.049, p=0.002). No statistical effects were found when testing PCs vs. species (F2;12=0.329, p=0.726). Data for egg hatching success were Arcsin transformed. Hatching success was tested with a two-way ANOVA. Within PC1 a statistically significant difference in hatching success was found between Acartia spp. and C. hamatus (F1;16=15.265, p=0.002). The overall hatching success in PC2 was higher and statistically significant difference from PC3 (F2;16=6.227, p=0.016). Whereas when testing within species the hatching success for Acartia spp. were higher in PC1 and significant different from PC3 (F2;16=8.826, p=0.002). For C. hamatus, same pattern was observed but with an even more profound lower hatching success in PC3 that was significantly different from both PC1 and PC2 ((F2;16=8.826, p=0.003 (1vs.3), p=0.022 (2vs.3)).

3.3 Dry weight, carbon weight and growth of turbot larvaeThe DW and CW were modelled against SL by an exponential growth model.

Figure 4: A) Turbot larvae dry weight versus standard length during the 3 production cycles. B) Turbot larvae carbon weight versus standard length during the 3 production cycles. Functions are results from fitted two parameter exponential models.

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No statistical differences were observed between the PCs for dry weight vs. SL (One-Way ANOVA: F2;6=0.0425, p=0.959). DW vs. SL was remodelled by pooling data from all the 3 PCs (fig. 4A). When testing carbon weight vs. SL, no statistically significant differences were observed between the tanks in any of the three PCs ((Kruskal-Wallis One-Way ANOVA: H2=0.800, p=0.721). Therefore, all data for CW vs. SL from each PC were pooled and a new model was fitted to the data (Fig. 4B).

3.3.1 Specific growth rates of turbot larvae The average SGR ± S.E. for the turbot during the three PCs were 0.22 ± 0.13d-1, 0.21 ± 0.12d-1 and 0.12 ± 0.10d-1, respectively.

The observed SGR during the three PCs were not statistically different (One-Way ANOVA: F2;6=0.516, p=0.621).

3.3.2 Fatty acid content of turbot larvae

Figure 5: Specific growth rates (d-1) from the 3 production cycles. Bars represent average of three replicate tanks ± S.E. (n=3).

Table 2 PC1 turbot larvea Fatty Acid Profile (%) PC2 Turbot larvea Fatty Acid Profile (%) PC3 Turbot larvea Fatty Acid Profile (%)

Fatty Acid 06-06-2011 10-06-2011 13-06-2011 15-06-2011 06-08-2011 11-08-2011 16-08-2011 06-09-2011 12-09-2011C20:4 n-6(ARA) 0.01 ± 0.00 0.01 ± 0.00 0.01 ± 0.00 0.01 ± 0.00 0.03 ± 0.01 0.03 ± 0.00 0.02 ± 0.00 0.01 ± 0.00 0.01 ± 0.00C20:5 n-3(EPA) 0.07 ± 0.01 0.08 ± 0.03 0.08 ± 0.03 0.09 ± 0.02 0.11 ± 0.02 0.08 ± 0.01 0.10 ± 0.00 0.08 ± 0.00 0.07 ± 0.01C22:6 n-3(DHA) 0.49 ± 0.04 0.61 ± 0.11 0.67 ± 0.06 0.64 ± 0.05 0.48 ± 0.09 0.53 ± 0.07 0.56 ± 0.01 0.63 ± 0.00 0.56 ± 0.06Total µg Fatty Acid pr. mg DW

212.01 ± 80.43 257.56 ± 79.98 246.16 ± 197.10 129.04 ± 9.92 230.60 ± 26.32 154.86 ± 20.51 259.13 ± 153.60 423.02 ± 190.25 161.66 ± 43.67

DHA/EPA 7.00 ± 0.60 7.63 ± 2.82 8.38 ± 2.22 7.11 ± 1.57 4.36 ± 0.69 6.63 ± 0.33 5.61 ± 0.39 7.88 ± 0.35 8.00 ± 0.18PUFA 0.64 ± 0.02 0.72 ± 0.04 0.77 ± 0.02 0.76 ± 0.03 0.66 ± 0.04 0.70 ± 0.04 0.74 ± 0.04 0.77 ± 0.01 0.72 ± 0.03

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The lowest levels of arachidonic acid (ARA) were observed in PC1 and they were statistically different from both PC2 and PC3 (Kruskal-Wallis One-Way ANOVA: H2=67.895, p<0.05). Eicosapentaenoic acid (EPA) fractions were highest in PC2 and ranging from 0.08 to 0.11. The observed high EPA fractions in PC2 was statistically significant different from PC3, no other differences were observed (One-Way ANOVA on Ranks: H2=7.998, p<0.05). Fractions of docosahexaenoic acid (DHA) increased over time in PC1 and PC2, whereas a decrease was observed in PC3. The lowest levels of DHA were observed in PC2 (table 2) and they were statistically significance differences from PC3 (Kruskal-Wallis One-Way ANOVA: H2=15.836, p<0.05). When DHA/EPA ratios were compared no differences were found between any of the three PCs (Kruskal-Wallis One-Way ANOVA: H2=5.800, p<0.05). The fractions of polyunsaturated fatty acid (PUFA) increased with fish larval age, except in PC3. 3.3.3 Stomach content

Figure 6: Bars represent the stomach content observed in the turbot larvae during the 3 production cycles. Bars are average prey ind.-1 ± S.D. (n=10).

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No statistical differences was detected in the turbot larval prey content in their stomach between the three PCs (One-Way ANOVA: F2;8=0.355, p=0.711) (fig. 6).

The turbot larvae mouth size increased as a function of turbot larval age. This was also reflected in the prey sizes consumed by the turbot larvae (fig. 6). The stomach content reveals that young turbot larvae mainly prey upon nauplii and later switches to copepodites followed by a later switch to adult copepods. That is, the turbot larvae cannot catch either copepodites or adult because of mouth size restrictions (fig. 6 and fig. 7). To further investigate this, Ivlevs index of electivity was used to confirm ontogenetic observations in stomach content and relate it to the observed prey field (fig. 8).

Figure 7: The increasing mouth-size during development of the turbot larvae (n=485) versus prey size development. Acartia tonsa prosome length versus development time is modified from Berggreen et al. (1988), and Centropages hamatus length is from Blanda et al. (unpublished data) versus development time modified from Fryd et al. (1991).

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3.3.4 Electivity index

Generally, it was observed that young turbot larvae aged 7-9 days exhibited an active selection for nauplii, whereas older larvae, from day 10 and forward selected actively for copepodites and adult copepods (fig. 8).

Figure 8: Prey electivity for the turbot larvae during the 3 production cycles. The index range from -1 to 1, where negative values indicate avoidance or inaccessibility of a prey, zero indicate random selection or no preference of a prey above another, and positive values indicate an active selection of a given prey (n=10).

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3.3.5 Carbon required to sustain growth of turbot larvae

Figure 9: The total turbot larval populations calculated daily carbon demand (solid line) and the total daily available standing stock of copepod prey items (punctuated line). In data before day 10, only the nauplii fraction of the available prey is included, since the turbot larvae can only predate on nauplii. (Δ) Represent the turbot larval average specific growth rate ± S.D. (n=3). At first sampling point in production cycle 1 data only represented one tank, since no turbot larvae were caught in the other 2 tanks.

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The individual turbot larvae’s carbon demand per unit time increases with larval age. The largest carbon demand was observed in the end of PC3. The turbot larvae are only able to feed on copepodites and adults from day 10 and onward. Therefore, only nauplii carbon biomass data’s are included in the available zooplankton prey biomass in data before day 10 in any of the three PCs. In PC1, the turbot larvae carbon demands are satisfied until day 19. The turbot larvae are potentially starving during most of PC2. In PC3, a starvation period is observed in the preflexion stage, since a shortage of nauplii is observed. Furthermore a potential starvation period is observed in the end of PC3. The specific growth rate in PC1 increased from -0.02 to 0.47 d-1. These two very different data for specific growth rates in fact succeeded each other in time, reflecting a weight increase after a period with negative growth. During the rest of PC1, the SGR were in average 0.2 d-1. The same average SGR of 0.2 d-1 were also observed in both PC2 and PC3. Figure 9 is a proxy to estimate the needed copepod biomass required for growth of a pelagic turbot larvae from hatching to just before me-tamorphosis over the season.

DISCUSSION

Our observation on decreased turbot larval mortality during the season concur with earlier observations and probably present a systematic problem associated with the particular production form (Engel-Sørensen et al. 2004). Hence, our goal in this study is to identify the parameters responsible for the observed lowered survival of the turbot larvae, in PC1 (18.4%) and PC2 (12.1%), compared to the survival in PC3 (5.0%) (table 1), besides SGR of in average 0.2 d-1. Furthermore, the proportion of turbot juvenile abnormalities observed in PC3 was higher than in PC1 and PC2 (table 1). There are several governing parameters influencing turbot larvae and juvenile survival and development, which will be analysed and discussed in the following chapter. The abundance of copepods decreased from PC1 to PC2 and PC3. Both adults and nauplii are more abundant in PC1 as compared to PC3 in particular, but also in PC2. A decrease in copepod abundance was expected during PC3 by the fish farm manager. The fish farmer therefore added 6 times the number of copepods initially in PC3 compared to what was added in the other two PCs (Jakobsen et al. in prep.). This increased addition of copepods had, however, no effect on the final

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copepod abundance in contrast to in PC1, where copepod abundance increased 10 fold (Jakobsen et al. in prep.) (fig. 2). The decrease in copepod abundance can partly be explained by the lack of phytoplankton and can as we propose in Jakobsen et al. (in prep.) potentially be solved by addition of sufficient inorganic nutrients. The copepod egg productions were not different between the PCs, although a decreased hatching success was observed in PC3 when compared to PC1 and PC2. It is speculated that the lower hatching reflects that Acartia spp. and C. hamatus initiated resting egg production due to the observed temperature decrease (Lindley, 1990; Støttrup et al. 1999). The observed decreased hatching will prevent copepod population recruitment and consequently the latest obser-ved data (September, temperature 11°C) is at the limit of the outdoor productive season. Potentially resting eggs could be harvested and utilized when low cope-pod abundance are observed (Marcus and Murray, 2001); this perspective will be pursued in a future study (Benni W. Hansen personal comment).

Other important sources for the decrease in copepod abundances are the added turbot larvae, which exhibit a significant predation on nauplii, copepodites and later on adult copepods, thereby limiting copepod community development. For a turbot larva, the absolute abundance of prey is important. If the availability of prey is too low, the larva will starve and potentially die. The required carbon needed to support the estimated carbon demand for the turbot larvae was not met completely in any of the three PCs. In PC1, the turbot larvae experienced a surplus in food from the time of stocking and until post-flexion at age 19 days. From day 19 and until the larvae were transferred to the indoor RAS (day 23) the turbot larvae population as a hole most likely experienced starvation in PC1. Turbot larvae carbon limitations were more profound during PC2 and PC3. In the end we suggest it resulted in initially high turbot larvae mortality. This finding is supported by another study where 90% of larval mortality occurs before day 10, due to turbot larval starvation (Christensen and Hansen, 1980). The larvae that survived the first critical phase seem to maintain similar SGR across all three PCs (fig. 9). This is probably a result of that the slow growers are dying, increasing the overall population growth rate (Rosenberg and Haugen, 1982). The obtained SGR in all PCs are within the ranges reported in literature references (Christensen and Hansen, 1980); van der Meeren, 1991). The first observed SGR in PC1 (day 6) displays a negative value. This can either be a result of insufficient zooplankton supply (van der Meeren, 1991) or due to that the turbot larvae had just absorbed their yolk-sac. At this point in the turbots life, it is about to initiate first feeding,

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resulting in a weight loss compared to the earlier data point where they were weighed with their yolk-sacs.

The newly hatched turbot larvae are restricted by mouth size and body immaturity to feed solely on the smallest and less motile members of the copepod community (fig. 7) (Cuhna and Planas, 1999). The relationship between fish larval mouth size and prey size is one of the most governing factors in fish larvae to catch prey (Cunha and Planas, 1999). Cuhna and Planas (1999) found that the optimal prey width for first feeding turbot larvae was 144µm (2 days post hatching). This prey width relation is in correspondence with the present findings in turbot larvae’s stomach content (fig. 6), and their prey selection (fig. 8). Van der Meeren (1991) assumed that turbot larvae were able to consume copepodites at day 17. We found that the turbot larvae were able to prey on copepodites from day 10 (fig. 6) (Eleonora Bruno, personal comment). This earlier selection for copepodites seems more realistic than day 17, since consuming largest possible prey, is a predator way to optimise its energy input. Hence, an earlier selection for copepodites is therefore more energy efficient (Van der Meeren, 1991). This suggests that before addition of yolk sac larvae to the tanks, it is important to ensure an excess of nauplii and a later succession of older copepod development stages. It is therefore recommended to add adult copepods into the tanks at least one week before addition of yolk sac larvae, instead of the common practice of 2-5 days before. This enables the adult copepods to reproduce, securing plenty of nauplii stage I-III for the benefit of the first feeding turbot larvae.

Other factors such as biochemical profile, size and behaviour of the prey, would also influence turbot larval capture success. Since copepods are superior in bioche-mical profiles and have ideal size for first feeding turbot larvae, these factors are neglected here (Chesney, 2005; Cuhna and Planas, 1999; Shields et al. 1999). In terms of prey behaviour, e.g. Acartia tonsa mean swimming speed increases with age, which could explain that preflexion turbot larvae only catch early stage nauplii and are not able to catch older copepod stages (Buskey, 1994). Furthermore, it has been argued that C. hamatus is a better prey than Acartia spp., since Acartia spp. is faster than C. hamatus and more transparent (Van der Meeren, 1991). This is contradicted by the present study, where the highest survival of the turbot larvae is observed in PC1, which mainly was dominated by Acartia spp. There are dif-ferent pros and cons for the two copepod prey species, but we do not believe that the succession from Acartia spp. to C. hamatus is the factor that determines the

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survival of the turbot larvae. Nevertheless, turbot larvae have an active selection for C. hamatus compared to Acartia spp. after day 23 according to Van der Me-eren (1991). However, we do not consider selection essential in our experiment, since the turbot larvae were transferred to indoor system at the development stage where the selection was reported in Van der Meeren, (1991). Moreover, the prey abundance was low as compared to the study by van der Meeren (1991). Hence, we believe the decrease in prey-size selectivity observed and lack of nauplii at the first feeding turbot larvae stage (Munk, 1995) as the most likely drivers that governs survival. Furthermore, no differences in stomach content were observed between the productions cycles, (fig. 6), even though we had a succession in the copepod community between PC1 (Acartia spp.) and PC2 and 3 (C. hamatus). Thereby, suggesting that the intraspecific competition on the food source (copepods) bet-ween the turbot larvae leave some turbot larvae to starvation and consequentially death (fig.6). Additionally, no statistical differences were found in growth rates between the three productions (fig. 5). The turbot larvae exhibited high initial mortality in one of the tanks in PC3. Subsequently, a high abundance of copepods was observed, suggesting a strong top down control by the fish larvae in the other experimental tanks. The fish farmer initially added 6 fold more adult copepods than in the other PCs. Because the phytoplankton biomass in terms of Chl a already was low, limited effect was observed of the enhanced copepod stocking. That is; the timing between additions of algae → copepods → turbot larvae, is a delicate balance that has to be well monitored and managed eventually with a feed-back monitoring program.

The rearing tanks are outdoor tanks, reflecting both the natural phytoplankton assemblages and the inorganic nutrients levels. This “green water” technique has demonstrated an increased fish larval survival and vitality in other green water systems with DHA rich algae (for review see Van der Meeren and Naas, 1997; Engel-Sørenesen et al. 2004; Reitan, et al. 1993; Shields, 2001; Støttrup et al., 1998). DHA is an important essential FA, especially during the first larval stages (Watanabe, 1993). High levels of the DHA in live feed diets have been shown to reduce stress and enhance pigmentation of S. maximus before metamorpho-sis. Dhert et al. (1994) observed best pigmentation, with less than ~30% non-pigmented turbot larvae, at DHA levels of 55% and EPA levels of 15%. In our study the highest level of abnormalities (mainly from pigmentation errors) was 9.6% (table 1), with DHA levels of 57.4% ± 6.7 S.D. and EPA levels of 8.4% ± 1.3 S.D., respectively. This suggests that the observed DHA and EPA fractions

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in the present study are more ideal, especially in PC1 where lowest levels of ab-normalities are observed (table 1). In PC2 the highest levels of EPA are observed together with the lowest levels of DHA, since DHA is an elongate of EPA this is expected (table 2). The DHA/EPA ratios in the present study are similar between the PCs. Since the three PCs have the same assemblage of copepods the DHA/EPA ratios is expected to be quite similar between PCs. The dietary ratio of DHA/EPA has been considered as a determinant of pigmentation, especially during pre-metamorphic stages (Rainuzzo, et al. 1994). Since DHA/EPA is fairly high in all three PCs in this study (>4) this is not considered a problem.

Scrutinising the different abiotic and biotic data, there is no clear picture of which factor that might results in the decrease in turbot larvae survival. Most of the observed data are not significantly different between PCs, which is typical for outdoor mesocosm system, which often exhibits a high variability in the collected data (Pilson et al. 1979). Nevertheless, the terminal numbers of turbot larvae (table 1) were higher in the 1st PC, and the final turbot juveniles were larger. This could be due to that in PC1 the largest initial algal bloom was observed (fig. 1). The initial phytoplankton bloom effectuated the largest observed initial nauplii production and the highest observed abundance of copepods throughout PC1. This resulted in high survival rates, high specific growth rates, lowered abnorma-lities on the turbot larvae and juveniles. Similar observations have shown that flounder larval survival depends on timing between prey needs of the fish larvae and the onset of the microalga bloom and the subsequent copepod productivity (Engel-Sørensen et al, 2004).

Recommendations We recommend that the production in the semi-intensive rearing system is initi-ated with a substantial algae bloom (> 15 µg C L-1). Such algal bloom potentially requires an addition of inorganic nutrients to support growth of phytoplankton. Instead of the current practice of adding copepods 2-5 days before addition of the turbot larvae, we recommend initiating the algal bloom before stocking copepods. Furthermore, we suggest leaving the copepods un-grazed by turbot larvae for at least a week, prior to adding the yolk sac larvae. Following this management proce-dure, all life stages of the copepods will be present with nauplii in excess providing a fully developed prey field for first feeding fish larvae. Another practice could be to stock with less turbot larvae securing survival of the turbot larvae by decreasing the total predation pressure on the copepod population. Although survival in the

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semi-intensive production system seems low, it is within other findings when com-pared to other systems (Van der Meeren 1991). Higher survival as high as 36% is reported in systems supported by enriched rotifers and Artemia nauplii. However the trade-off of the enhanced survival was a high degree of mal-pigmentation (1-85%) and lowered SGR 0.12-0.13 d-1 (Estéves et al. 1999). In the present study the turbot larvae did not exhibit high rates of abnormalities (table 1), and showed a higher SGR (~0.2 d-1). Copepods have advantages compared to other live feeds (Shields et al 1999). But the value of semi-intensive production method depends on the production of high value fish larvae that cannot be reared on traditional live feeds. Moreover the advantage in term of reduced abnormalities and better growth compensate for the complexity of the rearing technique. Furthermore, marine larvae exposed to inadequate food (formulated diets) in early larval stage have been shown to get high size variability, later life skeletal abnormalities and growth retardation (Ruyet et al. 1993). Therefore, an adequate food source such as copepod food in early life is essential and will benefit turbot past larval stages. AcknowledgementsThe help from Anders Tinggaard Pedersen, Allan Jørgensen and Gunnar Thing-gaard at the fish farm Maximus A/S is greatly appreciated. Also thanks are due to the staff at Venø fish farm A/S and Louise Nørremark from FishLab. From Roskilde University thanks are due to the student workers Katrine Lørup, Pernille Due, Tore S. Reyhe and Kristian S. Kryhlmand. We are also in debt to Ph.D. student Thomas Rayner and research assistant Kristian Filrup for assisting at Maximus A/S. Last but not least thanks are due to the laboratory technicians Anne Busk Faarborg and Rikke Guttesen for practical help in the laboratory at Roskilde University. This work was funded by IMPAQ grant (J. no. 10-093522) to Benni W. Hansen and grant (J. no. 11-116388) to Per M. Jepsen.

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marin overvågning. Chapter 2.7 - Mesozooplankton. By Nielsen, T.G., Møhlenberg, F. Pp.12 [in Danish].

Berggreen, U., Hansen, B., Kiørboe, T., (1988). Food size spectra, ingestion and growth of the copepod Acartia tonsa during development: implications for determining copepod production. Marine Biology. 99, 341–352.

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Blanda E., Bruno E., Højgaard J., Jakobsen H.H., Jepsen P.M., Mahjoub M.S., Pedersen A.T., Pedersen M.F., Rayner T.A., Hansen B.W. (in preparation). Nitrogen Addition in Semi-Extensive Aquaculture Enclosures: Effects on Phytoplankton and Zooplankton Composition.

Breteler, W., Koski, M., Rampen, S., (2004). Role of essential lipids in copepod nutrition: No evidence for trophic upgrading of food quality by a marine ciliate. Marine Ecology Progress Series. 274, 199–208.

Buskey, E.J. (1994). Factors affecting feeding selectivity of visual predators on the copepod Acartia tonsa: locomotion, visibility and escape responses. Hydrobiologia 292/293, 447-453.

Chesney, E.J. (2005). Copepods as Live Prey: A Review of Factors That Influence the Feeding Success of Marine Fish Larvae. In: Lee, C-S., O´Bryen, P.J., Marcus, N.H. (2005). Copepods in Aquaculture. Blackwell Publishing Ltd. Pp. 133-150. ISBN 13:978-0-8138-0066-0.

Christensen, P.M., Hansen, H.H. (1980). Vækst og overlevelse for larver of pighvar (Scophthalmus maximus). Danmarks Fiskeri og Havundersøgelser, pp. 0-83 Charlottelund, Denmark.

Cuhna, I., Planas, M. (1999). Optimal prey size for early turbot larvae (Scophthalmus maximus L.) based on mouth and ingested prey size. Aquaculture 175, 103–110.

Dhert, P., Lavens, P., Dehasque, M., Sorgeloos, P. (1994). Improvement in the larviculture of turbot Scophthalmus maximus: Zootechnical and nutritional aspects, possibility for disease control. Reprint from: European Aquaculture Society, Special publication No. 22, Gent, Belgium. Pp. 73-87.

Engell-Sørensen, K., Støttrup, J.G., Holmstrup, M., (2004). Rearing of flounder (Platichtys flesus) juveniles in semi-extensive systems. Aquaculture 230, 475–491.

Estevez, A., McEvoy, L.A., Bell, J.G., Sargent, J.R., (1999). Growth, survival, lipid composition and pigmentation of turbot (Scophthalmus maximus) larvae fed live-prey enriched in Arachidonic and Eicosapentaenoic acids. Aquaculture 180(3-4), 321-343.

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Ianora, A., Miralto, A., Poulet, S.A., Carotenuto, Y., Buttino, I., Romano, G., Casotti, R., Poh-nert, G., Wichard, T., Colucci-D’Amato, L., Terrazzano, G., Smetacek, V. (2004). Aldehyde suppression of copepod recruitment in blooms of a ubiquitous planktonic diatom. Nature 429, 403-407.

Ianora, A., Romano, G., Carotenuto, Y., Esposito, F., Roncalli, V., Buttino, I., Miralto, A. (2011). Impact of the diatom oxylipin 15S-HEPE on the reproductive success of the copepod Temora stylifera. Hydrobiologia 666(1), 265-275.

Jakobsen, H.H., Jepsen, P.M., Blanda, E., Jørgensen, N.O.G., Novac, A., Engel-Sørensen, K. Hansen, B.W. (in prep.). A seasonal study of turbot larvae Scophthalmus maximus (Linnaeus, 1758) reared in a semi-intensive outdoor system I: Plankton composition and biomass devel-opment - the role of inorganic nutrients limitation.

Jeannine, P.-L.R. (2002). Turbot (Scophthalmus maximus) Grow-out in Europe: Practices, Results, and Prospects. Turkish Journal of Fisheries and Aquatic Sciences. 2, 29-39.

Jones, A., Alderson, R., Howell, B. R. (1974). Progress towards the Development of a Successful Rearing Technique for Larvae of the Turbot, Scophthalmus maximus L. p. 731-737. In The Early Life History of Fish, Springer-Verlag Berlin Heidelberg. ISBN 978-3-642-65854-9.

Knuckey, R.M., Semmens, G.L., Mayer, R.J., Rimmer, M.A. (2005). Development of an optimal microalgal diet for the culture of the calanoid copepod Acartia sinjiensis: Effect of algal species and feed concentration on copepod development. Aquaculture 249(1-4), 339-351.

Lindley, J.A. (1990). Distribution of overwintering calanoid copepod eggs in sea-bed sediments around southern Britain. Marine Biology 104, 209-217.

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Miller, B.S., Kendall A.W.Jr. (2009). Early Life History of Marine Fishes. University of California Press Ltd. London, England. pp. 0-364. ISBN 978-0-520-24972.

Munk, P. (1995). Foraging behavior of larval cod (Gadus morhua) influenced by prey density and hunger. Marine Biology 122, 205– 212.

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Pilson, M.E. Q., Oviatt, C.A., Vargo, G.A., Vargo, S.L. (1979). Replicabillity of MERL micro-cosms: initial observations. In: Advances in marine environmental research, Narragansett, RI. pp. 0-409.

Planas, M., Cunha, I. (1999). Larviculture of marine fish: problems and perspectives. Aquaculture 177, 171-190.

Purdom, C.E., Jones, A., Lincoln, R.F. (1972). Cultivation trials with turbot (Scophthalmus maximus). Aquaculture, 1(2), 213–230.

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Rainuzzo, J.R., Reitan, K.I., Olsen, Y. (1994). Lipid-Composition in Turbot Larvae Fed Live Feed Cultured by Emulsions of Different Lipid Classes. Comparative Biochemistry and Physiology a-Physiology 107(4), 699-710.

Reitan, K.I., Rainuzzo, J.R., Øie, G., Olsen, Y. (1993). Nutritional Effects of Algal Addition in 1st Feeding of Turbot (Scophthalmus maximus L) Larvae. Aquaculture 118(3-4), 257-275.

Rosenberg, A.A., Haugen, S.A. (1982). Individual Growth and Size-Selective Mortality of Larval Turbot (Scophthalmus maximus) Reared in Enclosures. Marine Biology 72, 73-77.

Ruyet, J.P.L., Alexnadre, J.C., Thébaud, L., Mugnier, C. (1993). Marine Fish Larvae Feeding: Formulated Diets or Live Prey? Journal of the World Aquaculture Society 24(2), 211-224.

Shelbourne, J.E., (1964). The artificial propagation of marine fish. Advances in Marine Biology 2, 1-83.

Shields, R.J., Bell, J.G., Luizi, F.S., Gara, B., Bromage, N.R., Sargent, J.R. (1999). Natural copepods are superior to enriched Artemia nauplii as feed for halibut larvae (Hippoglossus hippoglossus) in terms of survival, pigmentation and retinal morphology: relation to dietary essential fatty acids. Journal of Nutrition 129, 1186–1194.

Shields, R.J. (2001). Larviculture of marine finfish in Europe. Aquaculture 200, 55– 88.

Strauss, R.E. (1979). Reliability Estimates for Ivlev´s Electivity Index, the Forage Ratio, and a Proposed Linear Index of Food Selection. Transactions of the American Fisheries Society 108, 344-352.

Sørensen, T.F., Drillet, G., Engel-Sørensen, K., Hansen, B.W., Ramløv, H. (2007). Production and biochemical composition of eggs from neritic calanoid copepods reared in large outdoor tanks (Limfjord, Denmark). Aquaculture 263(1-4), 84-96.

Støttrup, J.G., Bell, J.G., Sargent, J.R. (1999). The fate of lipids during development and cold-storage of eggs in the laboratory-reared calanoid copepod, Acartia tonsa Dana, and in response to different algal diets. Aquaculture 176(3-4), 257-269.

Støttrup, J.G., Shields, R., Gillispie, M., Gara, M.B., Sargent, J.R., Bell, J.G., Henderson, R.J., Tocher, D.R., Sutherland, R., Næss, T., Mangor Jensen, A., Naas, K., Van der Meeren, T., Harboe, T., Sanchez, F.J., Sorgeloos, P., Dhert, P., Fitzgerald, R., (1998). The production and use of copepods in larval rearing of halibut, turbot and cod. Bulletin Aquatic Association of Canada 4, 41–46.

Urup, B. (1994). Methods for the production of turbot fry using copepods as food. In Lavens, P., Remmerswaal, R.A.M (Eds.), Turbot culture: Problems and Prospects. Special Publication. – European Aquaculture Society, vol. 22. European Aquac. Soc., Gent, Belgium, pp. 47-55.

Van der Meeren, T. (1991). Selective feeding and predicyion of food consumption in turbot larvae (Scophthalmus maximus L.) reared on the rotifer Brachionus plicatilis and natural zooplankton. Aquaculture 93, 35-55.

Van der Meeren, T., Naas, K.E. (1997). Development of rearing techniques using large enclosed ecosystems in the mass production of marine fish fry. Reviews in Fisheries Science. 5 (4), 367– 390.

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Vigilant, V.L., Silver, M.W. (2007). Domoic acid in benthic flatfish on the continental shelf of Monterey Bay, California, USA. Marine Biology. 151, 2053-2062.

Watanabe, Y. (1993). Importance of docosahexaenoic acid in marine larval fish. Journal of the world aquaculture society. 24(2), 152-161.

Wilcox, J. A., Tracy, P. L., Marcus, N. H. (2006). Improving Live Feeds: Effect of a Mixed Diet of Copepod Nauplii (Acartia tonsa) and Rotifers on the Survival and Growth of First-Feeding Larvae of the Southern Flounder, Paralichthys lethostigma. Journal of the world aquaculture society 37(1), 113-120.

Zhang, J., Wu, C., Pellegrini, D., Romano, G., Esposito, F., Ianora, A., Buttino, I. (2013). Effects of different monoalgal diets on egg production, hatching success and apoptosis induction in a Mediterranean population of the calanoid copepod Acartia tonsa (Dana). Aquaculture 400, 65-72.

Manuscript 3.

Drillet, G., Rais, M., Novac, A., Jepsen, P.M., Mahjoub, M-H., Han-sen, B.W. (In revision). Total egg harvest by the calanoid copepod Acar-

tia tonsa (Dana) in intensive culture – effects of high stocking densi-ties on daily egg harvest and egg quality. Aquaculture Research.

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Aquaculture Research


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1

2

3

Guillaume Drillet1*, Mouloud Rais1, Aliona Novac2, Per M. Jepsen3, MohamedSofiane 4

Mahjoub1, Benni W. Hansen3 5

6

1DHI Water and Environment, 1 Cleantech Loop, #0305 CleanTech One, 637141 Singapore, 7

Singapore 8

2Faculty of Biology, ”Alexandru Ioan Cuza” University of Iasi, Romania 9

3Department of Environmental, Social and Spatial Changes, Roskilde University, Denmark 10

11

12

corresponding author: [email protected]

14

15

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16

Understanding the factors limiting copepod productivity in dense cultures is a prerequisite for 17

the partial or entire replacement of and rotifers as live feed for finfish larvae. In 18

dense cultures high encounter rates between individuals may increase stress, cannibalism 19

incidents and potentially trigger resting egg production. 20

We conducted an experiment to evaluate the potential egg production and egg quality of 21

stocked at densities ranging from 10 to >5,000 ind.L1. Egg Production (EP), 22

Delayed Hatching Eggs production (DHE), hatching success (HS), egg mortality and water 23

quality were used as end points. 24

In the present system, was raised at >5,000 ind.L1 without affecting the mortality, 25

confirming that attaining this high density in culture is possible. However, egg harvest 26

reached an optimum of 12,000 eggL1d1 at ~2,500 ind.L1 indicating that increasing stocking 27

density above this level is not of practical interest. Calculations showed that the loss in egg 28

harvest at stocking densities <2,500 ind.L1 is of 1.3% for every additional 100 adult 29

copepods L1. The increasing adult density did not affected the proportion of DHE produced 30

(~10% of harvest) but decreased significantly the HS, though not to a point that would be 31

problematic in a commercial production. 32

Understanding the biology of copepods when stocked at high density is important to improve 33

copepod culture systems and increase egg harvest yields. Technical solutions such as the 34

continuous separation of eggs from adults in the water column, recirculation, and the 35

continuous provision of food are seen as potential solutions. 36

37

: Fecundity, live feed, aquaculture, crowding, cannibalism, resting stage, water 38

quality 39

40

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41

Copepods are wellknown for being a high quality live feed for marine fish larvae in 42

aquaculture and in the ornamental trade industry where it is increasingly used to replace 43

partially or entirely more commonly used live feeds such as and rotifers ( 44

spp) Wilcox (2006). However, one of the weaknesses in the development of sustainable 45

copepod production systems is that, unlike rotifers and , copepods have not been 46

raised successfully in very dense cultures (see reviews by Støttrup 2003; Drillet . 2011a, 47

Mahjoub 2013). 48

High copepod densities in cultures may affect water quality negatively to levels where 49

toxicity can occur, as well as it may quickly deplete food resources (Støttrup & Norsker 50

1997, Jepsen . 2013; this paper). High stocking density also causes increased interactions 51

among individuals and with their eggs, potentially affecting the overall egg production, the 52

total harvest as well as the quality of the eggs. These effects are hitherto not well described 53

and understood, but they potentially hold the keys for the improvement of production yields 54

in intensive cultures. By understanding the biological and physiological limitations of 55

copepods stocked at high densities and counteracting them with technical solutions, as well as 56

by improving vital rates of cultured species, the development of the ideal cultivation systems 57

is feasible. 58

Under stress due to high stocking density, copepods may produce resting eggs that do not 59

hatch immediately after being laid. This maternallycontrolled reaction to density stress has 60

been reported for the harpacticoid copepod where a delay in the egg 61

hatching was observed at densities ranging between 1,000 and 40,000 ind.L1 (Kahan . 62

1988). In addition, similar effects were reported for the calanoid copepods 63

andat densities as low as 500800 ind.L1 (Ban & Minoda 1994; Camus & 64

Zeng 2009). 65

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In the case of the cosmopolitan calanoid copepod , inconsistent results on the effects 66

of stocking density on egg production and hatching success have been published (Medina & 67

Barata 2004; Jepsen . 2007; Peck & Holste 2006). The dissimilarities in the conclusions 68

of these studies can be attributed to inherent differences in the populations used for the 69

experiments (see Drillet . 2008), but may also be due to the experimental setup used in 70

each of the studies. Peck & Holste (2006), as well as Medina & Barata (2004) used 71

tanks/beakers for their experiments where the volume in which the copepods were stocked 72

was equal to the entire volume of the system, similar to a batch system. Jepsen . (2007) 73

on the other hand, used eight 250mL incubation chambers held submerged in 60L large tanks 74

and therefore the eventual effects of physical and chemical interactions among the copepods 75

where practically decoupled (because the chemical cues are diluted up to 30 times). 76

Because of these differences in the experimental setups, it is not possible to argue whether 77

the isolated effects of physical interactions due to crowding (increased encounter rates, 78

cannibalism etc.) are a main factor affecting copepod egg production and their eggs hatching 79

success or whether chemical cues perceived by the copepods represent the main drivers. We 80

reviewed the previous studies that deal with this subject and present these results in table 1. 81

In this compilation, it is evident that reports on the effect of stocking density of calanoid 82

copepods above 2,000 ind.L1 are scarce. In a review of cultures held around the world, the 83

maximum stocking density reported for calanoid cultures were reported to be 3,000 ind.L1 84

(; Støttrup 2003 and references therein) supporting the need for additional 85

studies on the subject. 86

In the present study, the production capabilities of when stocked at densities ranging 87

from 10 to >5,000 ind.L1 were tested and the quality of the produced eggs was monitored. 88

89

90

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91

92

The copepod (strain code DFHATI, Støttrup . 1986) was cultured continuously 93

in 70 L dark tanks filled with 0.2m filtered sea water (FSW) and feed daily with 94

the chrytophyte that were raised on B1 Media (Hansen 1989) and 95

harvested on their exponential growth phase. Other algal or nonalgal feed could have been 96

used to grow and result in different Specific Growth Rates (see Drillet 2011a) 97

but to enable comparison with other studies; only was used in this experiment. The 98

salinity of the water was kept at 34 and the temperature was 17±1ºC. Dim light was provided 99

continuously as described in previous studies (Drillet . 2006ab; Jepsen . 2007). 100

Adults and late copepodites (C4C6) used for the experiment were collected from the cultures 101

using a 400m mesh. These animals were subsequently transferred to a 70L tank with FSW. 102

Density and sex ratio (Sex = female / (total adults)) were estimated from six (6) subsamples of 103

40mL taken after a gentle homogenisation of the copepod distribution in the 70L tank using a 104

paddle. 105

106

107

Incubation chambers 108

The chambers consisted of a set of two meshes placed on the top of each other. The upper 109

mesh (150m) retained the copepods in the upper chamber (volume (Vc) of 1.11±0.003L), 110

while the eggs were allowed to settle down on the lower mesh (50 m) for the subsequent 111

daily harvest. The set of meshes was inserted into a 2L beaker (Figure 1). The entire volume 112

of the beaker (Vb) was 1.95±0.06 L and peristaltic pumps (Ole Dich, Instrument makers APS. 113

Denmark) were used to circulate the water within the beakers at a rate of 48ml.min1. The 114

entire water volume including algal feed was therefore recirculated every 40 min minimizing 115

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sedimentation (Figure 1). Twelve different incubation chambers were inoculated with initial 116

copepod densities (CD) of 10, 238, 713, 1188, 1664, 2139, 2971, 3565, 4159, 4753, 5062, and 117

5347 ind.L1. The experimental duration was 10 days. 118

119

Water quality 120

Oxygen and temperature were measured daily in each beaker using a handheld Oxyguard 121

Polaris 2 (Oxyguard International A/S, Birkerød, Denmark), and pH with a pH meter (PHM 122

210 Standard pH Meter, Meter Lab., Radiometer Copenhagen). Samples for the 123

determination of inorganic nutrients concentration were taken from each treatment. Two 124

samples of 5 mL were taken daily, to determine Total Ammonia Nitrogen (TAN) and nitrite 125

(NO2) levels with MERCK Spectroquant® tests 1.14752.0001 and 1.14776.0001, 126

respectively. On day 1, 2, 3, 6 and 10, 2 ml from each beaker were analysed for nitrate (NO3) 127

(MERCK Spectroquant® test 1.14773.0001). All analyses were carried out using a MERCK 128

Spectroquant NOVA 60. 129

130

Food availability 131

cell density (cell.ml1) in the beakers was estimated daily using a particle 132

counter (Beckman MultisizerTM 3 Coulter Counter ®, Miami, FL, USA). The density was 133

adjusted to 80,000 cell.mL1 when it was higher than 10,000 cell.mL1 after one day of 134

incubation or to 100,000 cell.mL1 when lower than 10,000 cell.mL1 after one day of 135

incubation. To calculate the average food availability in the beakers, we considered an 136

exponential decrease of the algae over the 24 hours of incubation and calculated the average 137

food availability after 12 hours. 138

The specific ingestion rate (SIR in ngC of food grazed per ngC of copepod per day) was 139

calculated for each treatment and for each day based on the amount of algae grazed over 24 140

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hours of incubation and the size of the adults. To perform this, 114 females and 54 males’ 141

cephalothorax lengths were measured and the adult’ carbon content was estimated using 142

Berggreen . (1988) relationship between cephalothorax length (L m) and carbon content 143

(Ccf in ngC.female1; CCm ngC.male1). The carbon content of (Ac ; 47.4 pgC.cell1) 144

was also estimated from Berggreen . (1988). Because the incubations were carried out 145

under dim light and with a continuous recirculation of the water we ignored algal growth. 146

1.11 10 . Berggreen . (1988) (1) 147

(2) 148

149

Mortality and sex ratio 150

The mortality rates (Z d1) for each treatment were assumed to follow an exponential decay 151

between the initial and the final number of copepods (Drillet . 2006a) and computed as 152

follow: 153

ln ⁄ ) (3) 154

Where N10 is the number of copepods at day10 and N0 is the number of copepods at day 0. In 155

subsequent calculations the mortality is taken into account and therefore the densities 156

appearing in the graphs are changing over time for each treatment and as follow: 157

(4) 158

Where t is the time in days and Nt is the number of copepods in the chamber at day t. 159

When mortality is not taken into account (to make the data more readable), graphs refer to the 160

initial stocking density (e.g. Figure 4). 161

The sex ratio was measured prior to the start of the experiment in the 70L tank as well as at 162

the end of the experiment for each treatment (as previously described). 163

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Egg production / Egg harvest 164

Every day, all the produced eggs from the incubation chambers were harvested and counted 165

(Eh). For samples with <10,000 eggs all eggs were counted; for samples with >10,000 eggs 166

subsamples representing 1/6th to 1/8th of the harvest were counted. For day 1, the incubation 167

lasted only 12h so these data were either ignored (Fig 3) or multiplied by two in subsequent 168

calculations (other figures) because the stress level endured by the organisms while being 169

sorted is considered pretty high over the first few hours of incubation. 170

Every day the set of egg harvested vs. actual copepod densities, were fitted using a 171

MichaelisMenten model with SigmaPlotTM and the theoretical maximum total egg 172

production (Vmax) was derived in order to evaluate the changes in total egg production 173

during the 10 days experiment. 174

The observed egg production (EPobs) per female was calculated as follow: 175

(5) 176

To estimate the theoretical egg production per female (EPth), Berggreen . (1988) 177

relationship between SGR and SIR was used. SGR for female copepods is not exhibited as 178

somatic growth but is transferred solely into egg production. Egg carbon content (Ec) of 179

45.7ngC per egg was used (Kiørboe . 1985). 180

0.44 0.081 Berggreen . (1988) (6) 181

Based on (6) (7) 182

However, equation (6) is linear and the potential loss of algae cells in the beaker due to 183

sedimentation, and lysis would lead to unrealistic ingestions and thus EPth1 (equation 7). 184

Therefore, the Maximum SGR possible (MAXSGR) as proposed by Berggreen . (1988, 185

Table 4) (MAXSGR =0.45) was used to calculate the theoretical maximum egg production per 186

individual and per day. This value was used as a cap that physiologically cannot 187

exceed: 188

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(8) 189

When EPth1> EPth2, the copepods are considered to be fed (food saturated) while 190

when EPth1< EPth2 , the copepods are considered to face suboptimal food condition (food 191

limited). 192

193

Hatching success 194

Every second day, 307 ± 130 (Mean ± SD) of the harvested eggs were used to prepare a 195

hatching experiment for each copepod density in 20mL Petri dishes, when possible in 196

triplicate. Eggs were counted at the beginning of the experiment and nauplii and eggs 197

remaining were counted at the end of the experiment. The hatching was carried out for 72 198

hours, at 17ºC, salinity 34, constant dimlight, and remaining eggs were considered as resting 199

eggs (delayed hatching eggs Drillet . 2011b) though one cannot exclude that a few 200

may have been nonviable (or unfertilized); eggs that disappeared were considered dead. 201

202

203

204

205

During the experiment, the pH varied from 7.31 to 8.37 and the oxygen levels were always 206

>7mg.L1. The quantity of inorganic nutrients tended to increase over time and this tendency 207

was more obvious for NO2 than for other nutrients (data not presented). The TAN, NO2

and 208

NO3 values ranged from 0 to 3.8 mg.L1, 0 to 0.3 mg.L1, and 0 to 3.5 mg.L1, respectively 209

(Table 2). 210

211

212

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The copepod mortality (Z) during the incubations was low and ranging from 1 to 5.5%.d1, 213

and was not correlated to the adult stocking density (Pearson Product Moment Correlation, 214

P>0.05). The sex ratio at the beginning of the experiment and at the end was 0.45±0.10 and 215

0.45±0.11 (Mean ± SD), respectively, and therefore we used an average sex ratio of 0.45 for 216

all subsequent calculations. The female and male cephalothorax lengths were 789±30 m and 217

703±21m (mean ± SD) respectively, equivalent to carbon weights of 3193±12 and 218

2282±68ng individual1 (mean ± SD) (equation 1). 219

220

221

The food availability changed over the 24h of incubation for every stocking density of 222

copepods ranging from 80,000/100,000 Cell.ml1 at the beginning of an incubation to as low 223

as 1,000 Cell.ml1 after 24 hours. This change was stronger in the treatments where the 224

copepod stocking densities were higher but the average food concentration was kept most of 225

the time above 20,000 Cell.ml1 (Figure 2). The lowest average food concentration over 10 226

days of incubation was 19,633±9,684 (mean ± SD).227

Eh in all copepod density chambers changed slightly over time due to acclimation time and 228

formed two groups as observed when fitting the egg harvest (Vmax) from MichaelisMenten 229

equations against the days of incubation (figure 3). Day 1 data were ignored because the 230

incubation period lasted 12h only. Days 25 had a lower Eh than days 610 and therefore the 231

results from these two production periods were subsequently separated (Figure 4 and 5). Eh 232

reached a plateau at adult stocking densities >2,500 ind.L1 (Figure 4). Data from day 2 to 5 233

and 6 to 10 were fitted with separated MichaelisMenten equations and Vmax (maximum egg 234

production; eggs L1d1) values were extracted. The Vmax values was 6,828 for days 2 to 5 235

data (R2=0.43) and 11,807 for day 6 to 10 data (R2=0.80). 236

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EPobs decreased with the increasing stocking density starting with a production of 20 to 25 237

egg female1d1 at relatively low copepod densities and decreasing to values around 5 egg 238

female1d1 at copepod stocking densities >3,500 ind.L1 (Figure 5). Based on the adult sizes 239

and their carbon content (Equation 1), the maximum possible egg production EPth2 (equation 240

8) was calculated to be 31.4 egg female1d1 and EPth1 was calculated according to the SIR of 241

the copepods in the experiment. Out of 120 theoretical egg production data based on 242

estimated food intake, 51 egg production data were capped at 31.4 egg.female1d1 showing 243

that food limitation occurred at densities >~2,800 ind.L1. By dividing EPobs with EPth2 in sets 244

of data where copepods were stocked at densities < 2,500 ind.L1, it is possible to present the 245

effects of density on the proportion of the theoretical egg harvest (Figure 7). The increasing 246

copepod stocking density affected negatively the EPobs and therefore the EPobs/EPth2 ratio 247

(Pearson Product Moment Correlation, P<0.05; Figure 7). In this range of stocking density 248

(02,500 ind.L1) the harvest loss per female due to the increased copepod density is of 1.3% 249

every time the density is increased by 100 ind.L1 (Equation, Figure 7). 250

251

success252

The average egg hatching success was high, ranging from 82±3 to 94±1% (mean ± SD). 253

Inversely, the proportion of DHE was low ranging from 5±1 to 9±1% (mean ± SD) of the 254

total eggs produced. The proportion of dead eggs was low, always ranging from 1±1 to 9±3% 255

(mean ± SD) of the total eggs produced. All days considered, there was a negative correlation 256

between the HS and the adult stocking density as well as a positive correlation between the 257

proportion of dead eggs and the stocking density (Pearson correlation, n=60, P<0.05) (Figure 258

8). The decline of hatching due to density was low (1.728% for every increase of 1,000ind.L259

1 in the beakers; Figure 8). However, there were no correlations between stocking density and 260

the proportion of DHE (Pearson correlation, n=60, P>0.05). 261

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12

262

263

The present experiment was carried out in order to understand the effects of increasing 264

stocking density of adult on the total daily egg harvest and egg quality of a 265

culturing system. 266

267

268

The increased density of copepods is increasing the quantity of faeces in the beakers. This in 269

turn generates an increased microbial mineralization of organic nitrogen resulting in the 270

accumulation of inorganic nitrogen in aquaculture systems (Avnimelech & Ritvo 2003). The 271

TAN contents represent the sum of NH3 and NH4+ in the system; these two forms being in pH 272

dependant equilibrium. Ammonia (NH3) has been reported to be toxic to aquatic species 273

including the copepod (Sullivan & Ritacco, 1985; Buttino 1994, Jepsen . 274

2013) and therefore, it is important to monitor. Under the most extreme conditions in the 275

present experiment (pH 8.37, TAN = 3.8mg.L1) only 6.1% of the TAN is in the NH3 form 276

(0.23mg.L1) which is lower that the no observed effect concentration (NOEC) reported by 277

Jepsen . (2013) for and Buttino (1994) for and support that there was 278

no toxicity from NH3 no matter the copepod density during our experiments. 279

Levels of nitrates (NO3) and nitrites (NO2) tended to increase during our experiments 280

because there was no water renewal. Following a thorough literature review, no studies 281

conclusively reported toxicity levels of NO3 and NO2 for copepods, but a recent review on 282

the toxicity effects of ammonia, nitrite and nitrate on several species and developmental 283

stages of decapods showed that lethal toxicity occurs at levels much higher than those 284

observed in the present study (Romano & Zeng 2013). However, although no drastic water 285

quality conditions were reached during our experiments, it might occur when feeding the high 286

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density cultures more food in order to optimize food uptake by copepods because algal 287

cultures contain a high amount of unused nutrients as well as exudates that will add to the 288

rearing systems, under these conditions, the water quality will probably become a more 289

significant limiting factor and recirculation or water exchange may then be necessary. 290

291

292

The recorded mortalities are comparable to mortalities observed in previous experiments with 293

the same strain incubated at lower densities (Berggreen . 1988; Drillet . 294

2006a). This confirms that it is in fact possible to stock cultures of at densities up to 295

>5,000 ind.L1 while keeping a low mortality and supports that the copepods had access to 296

sufficient food to support their basic metabolism. Working on the same strain at 297

densities ranging from 100 to 600 ind.L1, Jepsen . (2007) observed daily mortalities of 298

17.0±1.7% and a tendency to an increased mortality with increasing copepod densities. 299

However, this may have been due to stress provoked by handling of the copepods every 12 300

hours in their experiment. 301

302

303

The obtained maximum individual egg production (EPobs) is comparable to values reported in 304

the literature where copepods were incubated individually or in small batches in bottle 305

experiments (e.g. Kiørboe . 1985; Berggreen . 1988; Drillet . 2006a, 2008). 306

The total egg harvest observed in the present study is influenced by copepod density and 307

reaches a maximum of 11,00012,000 eggs.L1d1 in our system. The acclimation period 308

which lasted over the five first day of our incubations correspond to the period necessary for 309

in the experimental setup to reach it maximum egg harvest ( Kiørboe et al. 1985). This 310

is partly due to the acclimation of the individuals to their new environment and food intake; 311

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14

but should also be due to that the experiments were started with adults that may not have 312

been fertilized as well as late copepodites (see ‘Plankton culture’) which have eventually 313

moulted over the course of the experiment and were subsequently fertilized prior to the 314

initiation of their egg production. In the present experiment adults had sufficient access to 315

food to support their basic metabolism but females stocked at high densities (>2,500 ind.L1) 316

were not fed (Figure 6) and would have produced more eggs if fed higher amount 317

of algae. In a commercial production, this food limitation can easily be avoided by providing 318

a continuous supply of algae. In the range of density where adults were not food limited there 319

is a negative correlation between the egg harvest and the number of adults confirming that 320

increasing the copepod stocking density does affects negatively the egg harvest. This can be 321

explained partly by egg cannibalism which is known to occur in copepods (Daan ., 1988; 322

Uye & Liang, 1998; Camus & Zeng 2009) including in (Lonsdale . 1979, Drillet 323

. In Press). The latter study (Drillet In Press) showed that under certain conditions 324

optimal for cannibalism (high starved adult and egg density, 3h incubations) a single adult 325

could ingest up to 20 eggs hour1. Considering that eggs sediment in the chambers to 326

reach the harvesting mesh (fig 1), there must be a tradeoff between the depth of the chamber, 327

the flow through the chamber and the density of adults in the culture (Drillet & Lombard 328

2013) and therefore it has been argued that continuous egg harvest (or separation from adults) 329

is necessary to improve egg harvest yield per adult (Drillet . 2011a and ref therein). 330

Nevertheless, the copepods stocked at high densities may also cause physical disturbance 331

between adults and this may limit their feeding time, change their swimming behaviour 332

(Hansen . 1991; Dur . 2011) as well as their metabolism (Razouls 1972) but may not 333

affect sexual pickiness between males and females at densities up to 640 ind.L1 (Mahjoub 334

MS , unpublished data). However, these two hypotheses which may explain the present 335

observations have not been investigated in the present experiment. Increasing the 336

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understanding of the biology and physiology of copepods under high density conditions is 337

therefore an area where research should be focussed as it will guide the development of 338

technical solutions to improve mass copepod culturing capacities. 339

340

341

The egg hatching success decreased significantly with copepod stocking density in the 342

present experiment however this decrease was low (1.7% for every additional 1,000 ind.L1) 343

and would not be an issue in a large scale copepod producing firm. The increasing copepod 344

density did however not affect the proportion of DHE produced as it was shown for other 345

species (Ban & Minoda 1994; Camus & Zeng 2009; Kahan . 1988). At >5,000 ind.L1 in 346

the incubation chamber (Vc) the chemical cues generated by the adults in the water are 347

diluted in the entire volume of the beaker. However, this is still equivalent to the 348

accumulation of cues from an equivalent copepod density of 2,800 ind.L1 ( 5000 ). 349

This is much higher than the density at which chemical cues/encountering disturbances 350

induced diapause or delayed hatching in and (500 to 351

800 ind.L1; Ban & Minoda 1994; Camus & Zeng 2009). The present population (strain) of 352

has been cultured in the laboratory for more than 30 years and was shown to produce 353

significantly less resting eggs in culture than another laboratory strain of the similar 354

mitochondrial clade isolated in the Baltic Sea in the early 2000’s (Drillet . 2011b; Drillet 355

. 2008). Despite that the copepods in the high density treatments where not fed 356

(Figure 6), there were no observation of resting egg production as previously described 357

(Drillet . 2011b) confirming that the copepods were not stressed by food limitations. 358

Potentially, time of cultivation selectively removed some of the copepod life history traits 359

(Tiselius . 1995) as in our case, the capacity to produce resting eggs. 360

361

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362

total egg harvest in the present test culture system was limited by adult 363

stocking density most likely due to physical contact between conspecifics and their eggs. Egg 364

productions between 11,000 to 12,000 egg.L1.day1 in such a system, when the copepods are 365

saturated with food can be expected. This could most likely be further increased depending 366

on the system design with respect to e.g. depth of tanks, recirculation of water and other 367

technical improvements. The hatching success of eggs produced at high copepod densities 368

was statistically lower than those produced at lower densities but not to a level that can be 369

considered dramatic. Moreover, there were no effects of the adult stocking density on the 370

production of resting eggs in this copepod strain. Hence, the eggs produced exhibited a 371

promising hatching pattern. The assessment of physiological and biological changes that 372

occur when copepods are stocked in high density is lacking to explain all the present 373

observations and should be; together with zoo technical solutions (e.g. tank design, 374

automatic process control, and egg storing techniques), the focus of future research in this 375

field. 376

377

378

Thanks are due to Anne Busk Faarborg and Rikke Guttesen for assisting with laboratory 379

work. We also thank two anonymous reviewers for their constructive comments. 380

The present work was supported by the Danish ministry for independent research, Ung Elite 381

forsk grants 10093759 and 10094773 to GD and IMPAQ grant (10093522) to BWH and 382

Elite forsk travel Grant (J. no. 11116388) to PMJ. 383

384

385

386

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387

Avnimelech, Y. & Ritvo, G. (2003) Shrimp and fishpond soils: processes and management. 388

, 549–567. 389

Ban, S. & Minoda, T. (1994) Induction of diapause egg production in by 390

their own metabolites. (293), 185–189. 391

Berggreen, U., Hansen, B. & Kiørboe, T. (1988) Food size spectra, ingestion and growth of 392

the copepod during development, implications for determination of 393

copepod production. , 341–352. 394

Buttino, I. (1994) The effect of low concentrations of phenol and ammonia on eggproduction 395

rates, fecal pellet production and egg viability of the Calanoid copepod . 396

, 629634. 397

Camus, T.& Zeng, C. (2009) The effects of stocking density on egg production and hatching 398

success, cannibalism rate, sex ratio and population growth of the tropical calanoid 399

copepod . , 145–151. 400

Chuntapa, B., Powtongsook, S. & Menasveta, P. (2003) Water quality control using 401

in shrimp tanks. , 355366. 402

Daan, R., Gonzalez, S.R. & Breteler, W. (1988) Cannibalism in omnivorous calanoid 403

copepods. , 45–54. 404

Drillet, G., Iversen, M.H., Sørensen, T.F., Ramløv, H., Lund, T. & Hansen, B.W. (2006a) 405

Effect of cold storage upon eggs of a calanoid copepod, (Dana) and 406

their offspring. , 714–729. 407

Drillet, G., Goetze, E., Jepsen, P.M., Højgaard, J.K. & Hansen, B.W. (2008a) Strainspecific 408

vital rates in four cultures, I: strain origin, genetic differentiation and 409

egg survivorship. , 109–116. 410

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Drillet, G., Jepsen, P.M., Højgaard, J.K., Jørgensen, N.O.G. & Hansen, B.W. (2008b) Strain411

specific vital rates in four cultures II: life history traits and biochemical 412

contents of eggs and adults. , 47–54. 413

Drillet, G., Frouël, S., Sichlau, M.H., Jepsen, P.M., Højgaard, J.K., Joarder, A.K. & Hansen, 414

B.W. (2011a) Status and recommendations on marine copepod cultivation for use as 415

live feed. , 155166 416

Drillet, G., Hansen, B.W. & Kiørboe, T. (2011b) Resting egg production induced by food 417

limitation in the calanoid copepod . , 2064418

2070. 419

Drillet, G. & Lombard, F. (2013) A first step towards improving copepod cultivation using 420

modelling: the effects of density, crowding, cannibalism, tank design and strain 421

selection on copepod egg production yields. dOI: 422

10.1111/are.12317 423

Drillet, G., Maguet, R., Mahjoub, MS., Roullier, F. & Fielding, M.J. (In Press). Egg 424

cannibalism in : effects of stocking density, algal concentration, and egg 425

availability. . DOI: 10.1007/s1049901397471 426

Dur, G., Souissi, S., Schmitt, F.G., Michalec, F.G, Mahjoub, M.S. & Hwang, J.S. (2011) 427

Effects of animal density, volume, and the use of 2D/3D recording on behavioral 428

studies of copepods. ,197214. 429

Hansen, P.J. (1989) The red tide Dinofagellate Effects on behavior 430

and growth of aTintinnid ciliate. ,105116. 431

Hansen, B. W., Hansen, P. J. & Nielsen, T. G. (1991) Effects of large nongrazable particles 432

on clearance and swimming behaviour of zooplankton. 433

, 257269. 434

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Jepsen, P.M., Andersen, N., Holm, T., Jørgensen, A.T., Højgaard, J.K. & Hansen, B.W. 435

(2007) Effects of adult stocking density on egg production and viability in cultures of 436

the calanoid copepod (Dana). , 764–772. 437

Jepsen, P.M., Andersen, C.V.B., Schjelde, J. & Hansen, B.W. (2013) Tolerance of unionized 438

ammonia in live feed cultures of the calanoid copepod Dana. 439

doi:10.1111/are.12190. 440

Kahan, D., Berman, Y. & Barel, T. (1988) Maternal inhibition of hatching at high population 441

densities in (Copepoda, Crustacea). ,139442

144. 443

Kiørboe, T., Møhlenberg, F. & Hamburger, K. (1985) Bioenergetic of the copepod 444

: relation between feeding, egg production and respiration and composition of 445

specific dynamic action. , 8597. 446

Lonsdale, D., Heinle, D. & Siegfried, C. (1979) Carnivorous feeding behaviour of the adult 447

calanoid copepod , Dana. 448

, 235248 449

Mahjoub, MS., Schmoker, C. & Drillet G. (2013) Live feeds in Larval fish rearing: 450

Production, use, and the future. In Larval Fish Aquaculture. Quin JG Eds. Nova 451

Science Publishers. New York. 230p. 452

Medina, M. & Barata, C. (2004) Staticrenewal culture of (Copepoda: 453

Calanoida) for ecotoxicological testing. , 203–213. 454

Miralto, A., Ianora, A., Poulet, S.A., Romano, G. & Laabir, M. (1996) Is fecundity modified 455

by crowding in the copepod ? , 456

10331040. 457

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Peck, M.A. & Holste, L. (2006) Effects of salinity, photoperiod and adult stocking density on 458

egg production and egg hatching success in (Calanoida: Copepoda): 459

Optimizing intensive. , 341–350. 460

Razouls, S. (1972) Influences des conditions expérimentales sur le taux respiratoire des 461

copépodes planctonique. , , 462

145153. 463

Romano, N. & Zeng, C. (2013) Toxic Effects of Ammonia, Nitrite, and Nitrate to Decapod 464

Crustaceans: A Review on Factors Influencing their Toxicity, Physiological 465

Consequences, and Coping Mechanisms. ,121 466

Støttrup, J.G. (2003) Production and nutritional value of copepods. In: Støttrup, J.G., 467

McEvoy, L.A. (Eds.), Live Feeds in Marine Aquaculture. Blackwell Science, Oxford, 468

UK, p. 318. 469

Støttrup, J.G., Richardson, K., Kirkegaard, E. & Pihl, N.J. (1986) The cultivation of 470

Dana for use as a live food source for marine fish larvae. , 8796 471

Støttrup, J. G. & Norsker, N. H. (1997) Production and use of copepods in marine fish 472

larviculture. , 231247. 473

Sullivan, B.K. & Ritacco, P.J. (1985) Ammonia toxicity to larval copepods in eutrophic 474

marine ecosystems – a comparison of results from bioassays and enclosed 475

experimental ecosystems. , 205–217. 476

Tiselius, P., Hansen, B., Jonsson, P., Kiørboe, T., Nielsen, TG., Piontkovski, S. & Saiz, E. 477

(1995) Can we use laboratory reared copepods for experiments?: A comparison of 478

feeding behaviour and reproduction between a field and a laboratory population of 479

. , 369–376. 480

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Uye, S. & Liang, D. (1998) Copepods attain high abundance, biomass and production in the 481

absence of large predators but suffer cannibalistic loss. , 482

495501. 483

Wilcox, J.A., Tracy, P.L. & Marcus, N.H. (2006) Improving live feeds: effect of a mixed diet 484

of copepod nauplii () and rotifers on the survival and growth of first485

feeding larvae of the Southern Flounder, . 486

37:113– 120 487

488

489

490

: Summary of studies investigating the effect of copepod stocking densities on 491 reproductive parameters (√: studied; : not reported; na: not applicable). 492

493

494

: Mean values (±SE), Maximum and minimum values of TAN, NO2 and NO3 over 495 the 10 days of incubation for the different copepod stocking densities. 496

497

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: Setup of each 2L beakers. A represents the upper chamber (Vc) with a mesh 498

bottom of 150m. B represents the lower mesh (50m) where eggs are harvested. The water 499

is recirculated from the bottom of the beakers (particles <50m) to the upper chamber using a 500

peristaltic pump. 501

502

503

: Average food availability in the beakers depending on the copepod stocking 504

density. In dotted line, the saturation and half saturation levels as described by Berggreen 505

. (1988) (1500g C.L1 and 750 g C.L1, equivalent to 31,645 Cell.ml1 and 15,822 506

Cell.ml1, respectively) 507

508

509

: Total egg harvest as a function of days of incubation (Values of Vmax ± SE). Data 510

are extracted from fitting of MichaelisMenten equations to every day egg harvest. The 511

ellipsoids help to visualise the acclimation period (day 25) and the optimal production period 512

(day 610). 513

514

515

: total eggs harvested per litre and per day (± SD) as an average of 5 516

days of production (for days 1 to 5 and 6 to 10) for different initial copepod stocking 517

densities. Additional data reported by Jepsen . (2007) and Peck & Holste (2006) are 518

included. MichaelisMenten fits are also presented. 519

520

521

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egg production per female and per day (± SD) as an average of 5 522

days (day 1 to 5 and 6 to 10) at different initial copepod stocking densities. 523

524

525

Theoretical egg production in our experiment based on EPth1 and EPth2 data. The 526

dashed line indicates the limit between food saturated conditions and limited food availability 527

conditions. 528

529

530

: Proportion of theoretical egg harvest under nonlimiting food conditions. Fitting 531

for the linear regression line is 1.264 10 0.712 (r2=0.3821). 532

533

534

: Proportion of eggs hatching, egg dying and delayed hatching eggs as a function of 535

the initial copepod stocking density in the incubation chambers (±SD). Fitting for the linear 536

regression lines for hatching success, delayed hatching eggs and dead eggs are: Hatching 537

success: 1.728 10 92.66 (r² = 0.60); Delayed hatching eggs: 1.856 538

10 6.591(r² = 0.08) and Dead eggs: 1.135 10 2.242(r² = 0.50), 539

respectively. 540

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Authors Species Density: tested effect

Cop. Densities

(L-1) Food

Survival or

mortality

Sex ratio

Egg production

Hatching success

Resting eggs or delayed hatching

Present experiment

A. tonsa (DIFMR, Denmark)

Mixed Physical & diluted chemical

10 - 5300 Rhodomonas salina √√√√ √√√√ √√√√ √√√√ √√√√

Walton, 1985 Onychodiaptomus birgei

Mixed: Physical & chemical

133, 400, 1333. Cryptomonas ozolini - - √√√√ - √√√√

Kahan et al 1988

Trigriopus japonicus

Mixed Physical & chemical 1 - 40,000 Wheat germ - - - - √√√√

Ban and Minoda, 1994

Eurytemora affinis (Lake Ohnuma, Japan)

Chemical 500

Chlamydomonas reinhardtii : Cryptomonas tetrapyrenoidosa (1:1)

√√√√ √√√√ √√√√ - √√√√

Miralto et al 1996

Centropages typicus

Mixed Physical & chemical / Chemical

40, 200, 1000

Prorocentrum minutum - na √√√√ - -

Medina and Barata, 2004



500, 1000, 2000.

Marinure 25 + Isochrysis galbana : Rhinomonas reticulata (1:1)

√√√√ √√√√ √√√√ √√√√ -

Peck and Holste 2006

A. tonsa (Southern Baltic, Gemany)


50, 200, 400. Rhodomonas sp. - - √√√√ √√√√ -

Jepsen et al., 2007


Mixed: Physical & diluted chemical

100, 200, 300, 400, 600.

Rhodomonas salina √√√√ √√√√ √√√√ √√√√ -

Camus and Zeng, 2009

A. sinjiensis (Queensland, Australia)


125, 250, 500, 1000, 2000.

Isochrysis sp. : Tetraselmis chuii (13:1)

√√√√ √√√√ √√√√ √√√√ -

Page 32 of 33



MANUSCRIPT 3168

For Review Only

Table 2

Treatment Mean TAN Max. & Min. TAN Mean NO2 Max. & Min. NO2 Mean NO3 Max. & Min. NO3

[Ind. L-1] [TAN ± SD] d1-10 [TAN] d1-10 [NO2 ± SD] d1-10 [NO2] d1-10 [NO3 ± SD] d1-10 [NO3] d1-10 10 0.10 ± 0.26 0.79 - 0.00 0.006 ± 0.007 0.020 - 0.000 0.42 ± 0.52 1.30 - 0.00

238 0.04 ± 0.12 0.36 - 0.00 0.014 ± 0.012 0.040 - 0.002 0.42 ± 0.56 1.21 - 0.00 713 0.18 ± 0.22 0.76 - 0.04 0.026 ± 0.015 0.057 - 0.011 1.16 ± 0.86 2.29 - 0.21

1188 0.60 ± 0.88 2.86 - 0.03 0.029 ± 0.020 0.067 - 0.009 1.01 ± 1.42 3.50 - 0.00 1664 0.78 ± 0.77 2.55 - 0.14 0.048 ± 0.044 0.138 - 0.004 1.06 ± 1.27 3.24 - 0.26 2139 0.80 ± 0.71 1.86 - 0.09 0.090 ± 0.082 0.254 - 0.014 1.12 ± 1.42 3.50 - 0.00 2971 1.12 ± 0.91 3.24 - 0.3 0.083 ± 0.076 0.246 - 0.016 1.37 ± 1.29 3.50 - 0.37 3565 0.89 ± 0.80 2.65 - 0.16 0.082 ± 0.063 0.183 - 0.018 0.88 ± 0.94 2.29 - 0.00 4159 1.26 ± 1.06 3.80 - 0.31 0.104 ± 0.103 0.310 - 0.007 1.19 ± 1.44 3.50 - 0.00 4753 1.37 ± 1.05 3.67 - 0.22 0.102 ± 0.092 0.301 - 0.022 1.20 ± 1.46 3.50 - 0.00 5347 1.28 ± 1.11 3.80 - 0.18 0.089 ± 0.075 0.239 - 0.018 1.18 ± 1.43 3.50 - 0.00 5062 1.47 ± 1.11 3.80 - 0.35 0.072 ± 0.048 0.166 - 0.029 1.19 ± 1.38 3.50 - 0.24

Page 33 of 33



Manuscript 4.

Jepsen, P.M., Andersen, C.V.B., Schjelde, J., Hansen, B.W. (2013). Tolerance of un-ionized ammonia in live feed cultures of the ca-lanoid copepod Acartia tonsa Dana. Aquaculture research, Article first published online: 9 APR 2013, DOI: 10.1111/are.12190.

MANUSCRIPT 4 171

Tolerance of un-ionized ammonia in live feed cultures

of the calanoid copepod Acartia tonsa Dana

Per M Jepsen, Claus V B Andersen, Johannes Schjelde & Benni W Hansen

Department of Environmental, Social and Spatial Change, Roskilde University, Roskilde, Denmark

Correspondence: Per M Jepsen, Department of Environmental, Social and Spatial Change, Roskilde University, Universitetsvej 1,

Building 12.1, Postbox 260, Roskilde DK-4000, Denmark. E-mail: [email protected]

Abstract

Optimal water quality is considered as being a

restriction for marine copepod cultures for live

feed. There is a lack of knowledge on the water-

quality conditions in copepod cultures and the

effect on copepods. Few studies have investigated

the effect of ammonia on copepods, and fewer

reports No Observed Effect Concentrations (NOEC)

and Lowest Observed Effect Concentrations (LOEC),

which provides safety levels before cultures are

affected. This study investigates the tolerance of

Acartia tonsa nauplii and adults to ammonia, using

mortality as the endpoint after 24, 48 and 72 h of

exposure. Nauplii were exposed to levels from 0 to

5127 lg NH3 L�1 and adults to levels from 0 to

8481 lg NH3 L�1. Nauplii NOEC was 30 lgNH3 L�1 and LOEC was 81 lg NH3 L�1. Adult

NOEC was 477 lg NH3 L�1 and LOEC was

1789 lg NH3 L�1. 50% Lethal Concentrations

(LC50) for nauplii of 48 and 72 h was 1257 and

220 lg NH3 L�1. LC50 for adults was 2370

(24 h), 972 (48 h) and 770 (72 h). Combining

NOEC with excretion rates of NH4/NH3 a model

was developed to calculate densities in batch cul-

tures. We recommend that for batch cultures of

A. tonsa, NH3 is kept below NOEC for nauplii and

that levels of NH3 together with pH are monitored

weekly.

Keywords: ammonium, Acartia tonsa, copepod

culture, NOEC, LOEC, copepod density

Introduction

A threshold for succeeding with farming of marine

finfish larvae is the availability of suitable feed

items. The feeding strategies for fish larvae are

either dry feed/extruded pellets or live feed. Dry

feed is used widely within freshwater hatcheries,

but to date there is no dry feed/formulated pellets

alternative in first feeding of most marine fish spe-

cies. Therefore, within marine hatcheries live feed

are the only option for the first life stages, and

thus of paramount importance for the fish farming

in general (Shields, Bell, Luizi, Gara, Bromage &

Sargent 1999; Cahu & Infante 2001).

Today, the main live feeds for marine fish larvae

are enriched rotifers (Brachionus plicatilis) and

brine shrimps (Artemia sp.). In many cases,

enriched Artemia and rotifers have proven to be

inadequate prey compared with copepods when

trying to breed marine species of fish (Shields et al.

1999; Kortner, Overrein, Øie, Kjørsvik & Arukwe

2011). Copepods, and in particular their larval

stages – nauplii, are well known to be a more

appropriate diet for many marine fish larvae. In

the past 30 years calanoid species as those belong-

ing to the Acartia genus have been studied inten-

sively. An interesting trait of the species Acartia

tonsa is that the eggs can be collected and stored

for a prolonged time before hatched. This feature

makes A. tonsa eggs an analogue product to the

Artemia cysts (Støttrup, Bell & Sargent 1999; Mar-

cus & Murray 2001; Drillet, Jørgensen, Sørensen,

Ramløv & Hansen 2006). Although cold-stored

eggs of A. tonsa hold one of the keys to the future

for supplying marine hatcheries with live feed,

problems derives with difficulties in rearing suffi-

cient quantities (Payne & Rippingale 2001).

Although culturing of calanoid copepods has

become increasingly more reliable and approxi-

mately 60 copepod species have been raised in the

laboratory, they are still reared in small-scale cul-

ture systems (Reviewed by Drillet, Frouel, Sichlau,

Jepsen, Højgaard, Joarder & Hansen 2011). How-

ever, the so far reported systems are almost all

based upon batch cultures of copepods. Only a few

© 2013 Blackwell Publishing Ltd 1

Aquaculture Research, 2013, 1–12 doi:10.1111/are.12190

MANUSCRIPT 4172

continuous cultures have been reported (Støttrup

& Norsker 1997; Buttino, Ianora, Buono, Vitiello,

Malzone, Rico, Langellotti, Sansone, Gennari &

Miralto 2012). Regarding water quality in copepod

cultures, to our knowledge, only Payne and Ripp-

ingale (2001) and Støttrup and Norsker (1997)

touch the subject. Payne and Rippingale (2001)

conclude that in particular ammonia seems to

adversely influence the cultures fitness, especially

with regard to survival from nauplii to adulthood,

and the copepods fecundity. The few available

studies of ammonia toxicity for copepod cultures

mainly focus on the lethal toxicity effects of NH3′s

in terms of 50% Lethal Concentration (LC50) with

emphasis on copepod survival in natural environ-

ments. However, accepting 50% mortality of a

farmed species is not interesting within aquacul-

ture intensive mass production. Instead, knowing

the lowest concentration before mortality is an

issue is indeed valuable information, because keep-

ing the culture parameters below this limit

will ensure the farmed species better survival and

fitness.

Several author’s report excretion rates of ammo-

nia by calanoid copepods and also by Acartia spp.

(Nival, Malara, Charra, Palazzoli & Nival 1974;

Miller & Landry 1984; Kiørboe, Møhlenberg &

Hamburger 1985; Checkley, Dagg & Uye 1992;

Miller & Roman 2008). By combining the knowl-

edge of the threshold for when NH3 causes adverse

conditions and the nitrogen budgets for A. tonsa,

one has a direct method to determine the accept-

able culture density limit for A. tonsa before experi-

encing growth limitations induced by the water

quality in the culture itself.

Materials and methods

Cultures

The experimental cultures of A. tonsa were

established from Danish Technical University

DTU-AQUA. The strain originated from individuals

isolated in 1981 from the Øresund (N 56°/E 12°;Denmark) and were assigned the identification

code DFH-ATI (Støttrup, Richardson, Kirkegaard &

Pihl 1986). The strain has been maintained under

constant salinity, temperature and light conditions

for 30 years (salinity 32 psu., 17°C, no light), and

has been fed a monoalgal diet of Rodomonas salina.

Eggs were routinely harvested and cold stored for

later use (see Drillet et al. 2006).

Cultures were maintained under constant labora-

tory conditions for more than 2 months for accli-

matization. Temperature and salinity were kept at

17.0 � 1.0°C and salinity 32 psu. Copepod cul-

tures were reared in black 70 L tanks with 0.2 lmfiltrated seawater. Copepods were fed in excess

(>950 lg C L�1) (Kiørboe et al. 1985; Berggreen,

Hansen & Kiørboe 1988) with the cryptophyte

R. salina (8.0 lm equivalent spherical diameter).

R. salina was cultivated at 17.0°C on B1 media

(Hansen 1989) in logarithmic growth phase under

constant light (66 lmol m�2 s�1).

Copepod egg and nauplii experiment

This experiment was based on two egg batches,

successively harvested from the same maternal

copepod culture. For biotic and abiotic data for the

experiment refer to Table 1.

Eggs were harvested from the spawning tank by

syphon and cleaned using the method described in

Hansen, Drillet, Kozm�er, Madsen, Pedersen and

Sørensen (2010). The eggs were transferred into a

600 mL acid-washed polycarbonate bottle. The

egg batch was subsampled into 10 9 20 mL Petri

dishes using a 10 mL kip-automate (NS 29.2/32;

Buch & Holm, Witeg, Germany) and counted

using a dissection microscope [Olympus SZ 40;

Olympus Optical (Europe) GmBH, Hamburg, Ger-

many]. The average number of eggs in the 10

subsamples was used to estimate the total number

of eggs in the 600 mL polycarbonate bottle. Fur-

thermore, the 10 9 10 mL subsample for each

egg batch was left for hatching in 17°C. After

72 h, the nauplii and the remaining eggs were

fixed with 1% final concentration of acid Lugol

solution and counted. The initial population size

for the experiment was obtained by multiplying

the number of counted eggs with the egg hatching

success.

Before the experimental setup, the crystalline glass

(100 mL, Ø = 8.0 cm 9 H = 4 cm) were acid-

washed. After acid-wash the crystalline glass and

polycarbonate bottle were flushed with filtrated sea-

water.

Subsequently the eggs were subsampled (10 mL)

into the crystalline glass in five replicates for each

concentration. All treatment concentrations were

prepared from a stock concentration of NH4Cl

(10 000 lg NH3 L�1 at 17°C, pH 8.0 and 32 psu,

29.69 mg NH4Cl dissolved in 1 L of 0.2 lm filtered

seawater), and prior to the experiment adjusted and

© 2013 Blackwell Publishing Ltd, Aquaculture Research, 1–122

Ammonia and Acartia tonsa P M Jepsen et al. Aquaculture Research, 2013, 1–12

MANUSCRIPT 4 173

stabilized to pH 8.0 using NaOH (Buttino 1994). As

pH was not stable during the 72 h experiment, and

even small changes in pH have impact on the frac-

tion of NH3 the average concentrations are reported

and were 0 (control), 30, 81, 131, 183, 212,

452 lg NH3 L�1 for egg batch 1 and 0 (control),

468, 616, 944, 1193 and 5127 lg NH3 L�1 for

egg batch 2 respectively. Two egg batches were pre-

pared since mortality effects were at higher ammo-

nium concentration than expected when designing

experiment 1. Therefore, a subsequent experiment

with eggs from same adult cohort was prepared

with individual controls and overlapping concentra-

tions, 452 lg NH3 L�1 for batch 1 and 468 lgNH3 L�1 for batch 2 respectively. Before the

experiment, every replicate was sampled for max.

1 mL volume of seawater. The samples were spec-

trophotometrically analysed for ammonia with the

Salicylate-Hypochlorite method described by Bower

and Holm-Hansen (1980), using a Shimadzu (UV-

1601), UV-visible spectrophotometer. The crystal-

line glasses were covered with a Petri dish lid

(Ø = 8.5 cm) to avoid ammonia evaporation and

exchange with the surroundings and the eggs were

left at 17.0°C for 72 h for hatching. At 24, 48 and

72 h dead nauplii were counted in each of the crys-

talline glasses and mortalities were derived from

these data. At 72 h the total content in the crystal-

line glasses was fixed with acid Lugol and all dead

nauplii and eggs were counted. After 0, 24, 48 and

72 h oxygen and temperature were measured using

a HACH Portable LDOTM HQ20 Dissolved Oxygen

metre and pH using PHM 210 Standard pH Meter

(Meter Lab.; Radiometer Copenhagen, Copenhagen,

Denmark).

The pH and temperature-dependent relative con-

centration of NH4+ and NH3 can be described by

the equilibrium equations in Erickson (1985) and

Emerson, Russo, Lund and Thurston (1975). By

knowing pH and temperature of each replicate the

fraction can be calculated of un-ionized ammonia

using the equation derived from Clement and Mer-

lin (1995). The calculation method is summarized

in Korner, Das, Veenstra and Vermaat (2001).

Adult copepod experiment

The experiment was carried out using adults from

the same batch culture as used for the copepod

egg and nauplii experiment.

Adult A. tonsa were harvested from the culture

tanks using a 400 lm Nitex screen filter. Gender of

each individual adult was determined under a dis-

section microscope (Olympus SZ 40). A stock con-

centrations were prepared from NH4Cl using the

method described by Buttino (1994) (100 000 lgNH3 L�1 at 17°C, pH 8.0 and 32 psu, 296.90 mg

NH4Cl dissolved in 1 L of 0.2 lm filtered seawater)

and prior to the experiment adjusted and stabilized

to pH 8.0 using NaOH. Since pH was not stable

Table 1 Biotic and abiotic data from the Acartia tonsa copepod egg and nauplii experiment. Values for oxygen and pH

are shown as minimum and maximum � SD values for the entire 3-day experiment (0–72 h). Values for ammonia and

ammonium concentrations are shown as mean � SD for all data during the 3-day experiment (0–72 h). Hatching suc-

cess is shown as total hatching after 72 h experiment. Anodes in the first column show the batch number. ○ belongs to

batch 1 and ● belongs to batch 2

Mean NH3

[lg L�1 � SD] t0–72

Mean NH4+

[lg L�1 � SD] t0–72

Min. & Max.

Oxygen [mg O2 L�1 � SD] t0–72

Min. & Max.

pH � SD t0–72

Mean hatching

[% � SD] t0–72

○ 0 � 0 0 � 0 7.20–7.62 � 0.19 8.07–8.17 � 0.04 24.7 � 2.8

● 0 � 0 0 � 0 7.16–7.76 � 0.09 7.98–8.19 � 0.01 52.8 � 9.5

○ 30 � 4 669 � 121 7.32–7.74 � 0.13 8.09–8.27 � 0.02 24.7 � 5.4

○ 81 � 9 1825 � 193 7.50–8.00 � 0.16 8.09–8.26 � 0.01 18.0 � 3.8

○ 131 � 20 2963 � 322 7.46–7.92 � 0.22 8.10–8.27 � 0.01 18.9 � 2.0

○ 183 � 37 4055 � 390 7.38–7.86 � 0.38 8.10–8.30 � 0.01 15.5 � 2.7

○ 212 � 40 4871 � 540 7.40–7.60 � 0.15 8.08–8.30 � 0.05 19.9 � 9.2

○ 452 � 99 10 174 � 955 7.44–7.70 � 0.09 8.07–8.30 � 0.01 28.9 � 7.0

● 468 � 134 12 472 � 1447 7.42–7.60 � 0.12 7.95–8.18 � 0.04 65.1 � 9.7

● 616 � 143 15 839 � 1069 7.32–7.66 � 0.11 7.98–8.19 � 0.02 61.9 � 5.5

● 944 � 222 25 528 � 2401 7.30–7.60 � 0.14 7.97–8.19 � 0.04 78.4 � 8.8

● 1193 � 211 34 276 � 7463 7.24–7.68 � 0.22 7.96–8.17 � 0.02 83.4 � 12.0

● 5127 � 751 154 469 � 8334 7.00–7.58 � 0.19 7.90–8.11 � 0.04 80.7 � 5.8

© 2013 Blackwell Publishing Ltd, Aquaculture Research, 1–12 3

Aquaculture Research, 2013, 1–12 Ammonia and Acartia tonsa P M Jepsen et al.

MANUSCRIPT 4174

during the 72 h experiment, and even small

changes in pH has impact on the fraction of NH3

the average concentration is reported. Concentra-

tions were prepared by dilution from the stock solu-

tion and were 0 (control), 318, 477, 1789, 3035,

4349 and 8481 lg NH3 L�1. Four 600 ml

acid-washed polycarbonate Nalgene® (Sybron Co.,

Rochester, NY, USA) bottles were each randomly

inoculated with 9–11 adults, with a sex ratio

between 1:4 and 1:1 male: female. The dilemma that

adult copepods need algal feed during several days of

incubation, and at the same time that live algae

takes up ammonia, and thereby influence the expo-

sure concentrations of ammonia, lead us to use

Shellfish diet 1800® (Reed Mariculture Inc., Camp-

bell, CA, USA) as feed for the copepods. Immediately

after Shellfish diet 1800® and adult copepods were

added to the incubation bottles, ensuring fed in

excess, the bottles were completely filled with the dif-

ferent concentrations of ammonium and closed

using household film and a lid to avoid air bubbles

potentially disturbing the copepods. Bottles were

then randomly fixed at a plankton wheel (0.5

round min�1), and placed in a temperature con-

trolled room at 17°C with constant dim light. At 24,

48 and 72 h the entire bottle volume was gently

emptied through a 180 lm Nitex screen filter, and

into a 1 L acid-washed glass beaker. The adults were

immediately after gently flushed into a 20 mL Petri

dish for visual inspection. Adults in the 20 mL Petri

dishes were categorized into three categories

0 = dead, 1 = slow and 2 = normal behaviour.

Dead individuals were counted and removed, cepha-

lothoraxes length and sex was determined when pos-

sible. Slow individuals were categorized as slow

when only swimming legs and antennae were mov-

ing or when individuals exhibited an elusive jump

only after being gently poked with a glass Pasteur

pipette. Other individuals were categorized as nor-

mal behaviour. The bottles were refilled with the

standard solution from the 1 L beaker glasses, Shell-

fish diet 1800® were added to ensure food in excess

and the adults categorized as slow and normal were

retransferred. At 72 h the adults that exhibited slow

or normal behaviour were fixed with acid Lugol in

the Petri dish and the cephalothorax lengths were

measured. Each bottle was sampled for un-ionized

ammonia. A 1 mL sample was sampled by syphon-

ing through a submerged 45 lm Nitex filter to

avoid sampling eggs or nauplii. Samples were taken

initially and after 48 and 72 h. To determine

ammonia the Salicylate-Hypochlorite method as

described earlier were used. At each sampling time

pH and temperature were determined in all repli-

cates, using PHM 210 Standard pH Meter (Meter

Lab.; Radiometer Copenhagen). At 24 h oxygen

was determined in the bottles using a HACH Porta-

ble LDOTM HQ20 Dissolved Oxygen/pH metre

(6.9 � 0.1 mg O2 L�1). Earlier experiments in our

laboratory with the same setup have shown similar

oxygen concentration range and that oxygen con-

centration is not the limiting factor for copepod fit-

ness in these bottle experiments (Drillet, Jepsen,

Højgaard, Jørgensen & Hansen 2008). Therefore,

oxygen was not further monitored in the present

experiment.

Rhodomonas salina′s effect upon ammonia

concentration

To investigate the effect of R. salina upon ammo-

nium concentration a 24 h experiment was con-

ducted. Four 133 mL Blue Cap bottles for each

treatment were inoculated with 0.2 lm filtered

seawater and a concentration of 100, 250, or

2000 lg NH3 L�1 and with or without 4700

cell mL�1 of R. salina. Bottles were then randomly

fixed at a plankton wheel (0.5 round min�1), and

placed in a temperature-controlled room at 17.0°Cwith constant dim light. The algae concentrations

were determined using a Coulter Counter� Multi-

cizerTM Z3 from Beckman Coulter (Miami, FL,

USA). At 0, 2, 4, 6 and 24 h a 1 mL sample was

taken in each bottle and used to determine NH3

using the Salicylate-Hypochlorite method described

earlier. Initially all concentrations were buffered to

pH 8.0 with NaOH. At each sampling time, pH

and oxygen were determined, using a HACH Por-

table LDOTM HQ20 Dissolved Oxygen/pH metre and

a PHM 210 Standard pH Meter (Meter Lab.; Radi-

ometer Copenhagen). Algal concentrations were

determined initially and after 24 h at the termina-

tion of the experiment.

Statistical and model analysis

The model for determining ammonia effect was

fitted with Log (agonist) vs. response – Find EC

anything according to GraphPad Prism version

5.04. The Top and Bottom plateau′s have been

constrained to 0 and 100, since the mortality is

ranging from 0 to 100%. (GraphPad Prism version

5.04 for model details and Figs 1 and 2, for model

application).



MANUSCRIPT 4 175

Statistical comparison of the effect of ammonia

was examined using one-way ANOVA employing the

P < 0.001 level of significance. If a one-way ANOVA

showed statistical significance, the Holm–Sidak vs.

control method was used as a post hoc test

employing P < 0.05 to test the differences. All sta-

tistical tests were performed using SigmaStat v.

3.5. LOEC is defined as the first result that is statis-

tically different from the control, whereas NOEC is

the last concentration that is not statistically sig-

nificant from the control.

Results

Copepod egg and nauplii experiment

The highest nauplii mortality after 24 h NH3 expo-

sure was at concentration 131 lg L�1 with

23.5 � 4.1%. The nauplii mortality was generally

Figure 1 Acartia tonsa nauplii cumulated mortality

during 3-day exposure to 11 different concentrations of

un-ionized ammonia and two controls. ○ = Egg batch

1 and ● = Egg batch 2, error bars are 95% confidence

levels and punctuated lines are 95% confidence band.

Model values for 24 h: (Hill Slope = 0.072 � 0.043

SE, d.f. = 61, r2 = 6% and SS = 3427); 48 h (Hill

Slope = 0.459 � 0.046 SE, d.f. = 61, r2 = 76% and

SS = 5492) and 72 h (Hill Slope = 0.539 � 0.065 SE,

d.f. = 61, r2 = 74% and SS = 11 372).

Figure 2 Acartia tonsa adult cumulated mortality dur-

ing 3-day exposure to seven different concentrations of

un-ionized ammonia and a control. Error bars are 95%

confidence levels and punctuated lines are 95% confi-

dence bands. Model values for 24 h (Hill

Slope = 1.359 � 0.289 SE, d.f. = 25, r2 = 74% and

SS = 8522); 48 h (Hill Slope = 0.833 � 0.226 SE,

d.f. = 25, r2 = 59% and SS = 13 596) and 72 h (Hill

Slope = 0.839 � 0.266 SE, d.f. = 25, r2 = 61% and

SS = 11 704).



MANUSCRIPT 4176

low in the first 24 h where there was no statistical

significant difference between the control and the

concentrations of 30, 468, 616, 944 and 1193 lgNH3 L�1, the first statistical difference is between

control and 81, 131, 183, 212, 452 and 5127 lgNH3 L�1 (P < 0.05; Holm–Sidak) (Table 1 for

detailed abiotic and biotic factors). Effect Concentra-

tion was not used after 24 h since the fitted model

had an r2 of only 6% giving EC estimates a high risk

of false values (Fig. 1). After 48 h exposure the

highest nauplii mortality was in nominal concen-

tration 5127 being 68.8 � 1.9%. The lowest nau-

plii mortality was in the control with 9.3 � 5.7%.

Both the control and the 30 lg NH3 L�1 differed

statistically significant from all the other concentra-

tions within 48 h (P < 0.05, Holm–Sidak). Since

the first observed effect on mortality after 48 h,

occurs between concentration 30 and 81 lgNH3 L�1, the NOEC after 48 h is 30 lg NH3 L�1

and the LOEC after 48 h is 81 lg NH3 L�1. The

highest mortality after 72 h was in concentration

5127, and was 98.7 � 1.6%. The mortality in the

control was 14.8 � 6.2%. Hence, the NOEC after

72 h is 30 lg NH3 L�1 and the LOEC after 72 h is

81 lg NH3 L�1, as both the control and the 30 lgNH3 L�1 differed statistically significantly from all

other concentrations within 72 h (P < 0.05, Holm–

Sidak) (Table 3 for summary of NOEC and LOEC

result).

Adult copepod experiment

After 24 h of NH3 exposure all adults were

observed dead in concentration 8481 NH3 L�1

(Table 2 and Fig. 3). In the first 24 h, there was

no statistical significant difference between the

control and the nominal concentrations 318 and

477 lg NH3 L�1, but the control and concentra-

tions 318 lg NH3 L�1 and 477 lg NH3 L�1 all

were significantly different from the five other con-

centrations (P < 0.05; Holm–Sidak). The first sig-

nificance was observed between 477 lg NH3 L�1

and 1789 lg NH3 L�1, thereby the NOEC after

24 h is 477 lg NH3 L�1 and the LOEC after 24 h

is 1789 lg NH3 L�1(P < 0.05; Holm–Sidak).

Interestingly, the same patterns were observed in

a behaviour experiment, where more adult

A. tonsa exhibited slow behaviour or were dead

after 24 h exposure, between the 477 and

1789 lg NH3 L�1. After 48 h exposure an ele-

vated mortality was observed in all ammonia con-

centrations and the control compared to 24 h.

The lowest mortality was observed in the control

with 14.4 � 10.9%. Testing statistically after 48 h

exposure time the same pattern as after 24 h

exposure was revealed, with a NOEC after 48 h of

477 lg NH3 L�1 and a LOEC after 48 h of

1789 lg NH3 L�1 (P < 0.05; Holm–Sidak). An

even higher elevated mortality was observed after

72 h when compared to 24 and 48 h exposure

time (Figs 2 and 3). Testing statistically after 72 h

exposure time a difference between the control

and concentration 318 and 477 lg NH3 L�1 was

observed. There was not observed any statistical

significant difference between the control and the

other five concentrations (P < 0.05; Holm–Sidak).

Therefore, the NOEC after 72 h is 477 lg NH3 L�1

and the LOEC after 72 h is 1789 lg NH3 L�1

(P < 0.05; Holm–Sidak) (Table 3 for summary of

NOEC and LOEC result). The behaviour study

underlined the same tendencies that were observed

in the chronic exposure experiment, with adults

exhibiting slow behaviour as an effect of both

increased ammonia and exposure time (Fig. 3).

Table 2 Biotic and abiotic data from the Acartia tonsa adult experiment. Data for ammonia and ammonium concentra-

tions are shown as mean � SD for all data during the 3-day experiment (0–72 h). Oxygen is shown as mean � SD for

the first 24 h. Values for pH are shown as minimum and maximum � SD values for the 3-day experiment (0–72 h).

Algae data are mean cells mL�1 � SD in the bottle during the 72 h experiment

Mean NH3

[lg L�1 ± SD] t0–72 h

Mean NH4+

[lg L�1 ± SD] t0–72 h

Mean Oxygen

[Mg O2 L�1] t0–24 h

Mean algae

[Cell mL�1 ± SD] t0–72 h

Min. & Max. pH

[pH ± SD] t0–72 h

0 � 0 0 � 0 6.70 � 0.00 7290 � 2383 7.88–8.12 � 0.08

318 � 84 8.357 � 767 6.80 � 0.00 7950 � 3411 7.89–8.07 � 0.07

477 � 174 15 475 � 1138 6.90 � 0.00 7677 � 3030 7.90–8.09 � 0.08

1789 � 892 58 078 � 8833 6.80 � 0.00 5785 � 3105 7.90–8.07 � 0.07

3035 � 756 98 545 � 8512 7.03 � 0.21 7659 � 3517 7.88–8.04 � 0.06

4349 � 1362 111 500 � 7621 6.96 � 0.05 7041 � 3793 7.90–8.08 � 0.08

8481 � 1392 282 710 � 10008 6.90 � 0.00 6025 � 3444 7.89–7.92 � 0.01



MANUSCRIPT 4 177

The prosome length of dead adult A. tonsa was

667 � 63 lm and of survived adult A. tonsa was

668 � 58 lm, there was observed no statistical

difference between the sizes of dead and alive adult

A. tonsa. (Student’s t-test P > 0.05). Moreover, no

gender-specific mortality was observed, 92 dead

females and 90 dead males were sex determined in

total (Student’s t-test P > 0.05) (data not shown).

Production of TAN and density calculation

The maximum stocking density in a small-scale

A. tonsa batch culture can be predicted when

taken the following assumptions into consider-

ation: batch culture, no seawater shift, no algal

feed, by using calculated TAN (Total Ammonia

Nitrogen) production and using copepod ammo-

nium excretion rates from the literature, using the

following equation:

Acartiatonsa density L�1 ¼ NOECX=PTAN � f

ð1Þ

where NOECx is NH3 No Observed Effect Concen-

tration obtained from the literature, or if from the

present study, 30 lg NH3 L�1 is used for nauplii

and 477 lg NH3 L�1 for adults. PTAN is levels of

TAN production often reported as excretion rates

reported in the literature. ƒ is fraction of TAN that

is un-ionized ammonia, which depends on the

actual culture levels of salinity, temperature and

pH [Table 3 for result from Eqn (1)].

Figure 3 Acartia tonsa adult

behavioural response during 3-day

exposure to seven different concen-

trations of un-ionized ammonia

and a control. Response was cate-

gorized into normal, slow and dead

behaviours. Values are cumulated

percentage of survivors.



MANUSCRIPT 4178

Rhodomonas salina′s effect upon ammonia

concentration

Algal concentration decreased in all treatments

from 4600 cell mL�1 to a range between 3300

and 4000 cells mL�1. The pH fluctuated from

7.99 to 8.10 between the bottles, therefore the pH

was standardized to pH 8.00 by multiplying with

a factor given from the measured pH. The effects

of R. salina upon the ammonium concentration

were a decrease in ammonium with addition of

algae. In 100 lg NH3 L�1 an 11.3% decrease, in

250 lg NH3 L�1 a 53.3% decrease and in

2000 lg NH3 L�1 a 23.7% decrease in ammo-

nium concentration were observed.

Discussion

Chronic exposure to elevated levels of NH3 has

shown Acartia clausi to increase the egg production

rate and the total reproductive capacity (Verriopo-

ulos & Mora€ıtou-Apostolopoulou 1981; Buttino

1994). It is, however, unknown if the increased

egg production response is solitarily an effect of

NH3 exposure, or is a general reproduction strat-

egy for A. tonsa exposed to stressors in the envi-

ronment, or a combination of effects. Relating the

egg hatching success vs. ammonia exposure, Sulli-

van and Ritacco (1985) discussed that the per-

centage that hatched A. tonsa eggs had a tendency

to be reduced at increasing treatment levels. This

finding is supported by Buttino (1994), where

A. clausi eggs showed a reduced hatching suc-

cess as a function of female exposure time. In the

present study, an increasing hatching success with

increasing ammonium concentrations is observed

as presented in Table 1. Buttino’s (1994) findings

show a decrease in hatching success after

6–7 days of exposure, which we never reach since

in the present experiment we only exposed for

3 days. In line with Sullivan and Ritacco’s (1985)

findings we conclude that the hatching was not

clearly related to the treatment levels, since we at

present do not have an explanation for the poten-

tial physiological response pattern behind.

Nauplii mortality

The mortality of nauplii for the first 24 h was not

significantly different between the exposure concen-

trations and the control. We have no solid explana-

tion for this phenomenon, but have observed it for

both egg batches in the present study. One could

hypothesize that the reason for a non elevated mor-

tality as an effect of ammonium in the first 24 h is

that the nauplii digestive system is not fully devel-

oped (Mauchline, Blaxter, Southward & Tyler

1998). Thereby the non-feeding nauplii stage I is

most likely only susceptible to the ammonia that

penetrates over their body surface. However, if this

was the case we would expect an even higher mor-

tality in the higher concentration. Other experi-

ments could clarify the mechanism behind mortality

at nauplii stage I and effect upon hatching rates.

Sullivan and Ritacco (1985) reported 48 h LC50values ranging from 180 to 260 lg NH3 L�1 for

Acartia hudsonica nauplii and from 180 to 224 lgNH3 L�1 for A. tonsa nauplii. In the present study

Table 3 Shows no observed effect concentrations (NOEC) and lowest observed effect concentrations (LOEC) for Acartia

tonsa adults and nauplii. Adult and nauplii densities are model results using Eqn (1). In the calculations NOEC for 24 h

exposure of NH3 for adults and nauplii are used and at five different pH scenarios. The salinity is 32 psu, the tempera-

ture is 17.0°C, and therefore in pH 7.5, 0.996% of total ammonia nitrogen (TAN) is at the NH3 fraction. At pH 8.0,

3.08% is NH3 and at pH 8.5, 9.14% is NH3 and at pH 9.0, 24.1% is NH3 and at pH 9.5, 50.2% is at the NH3 fraction.

Adult copepod ammonia excretion level used is 13.5 ng PTAN ind�1 h�1 which is an average value of excretion rates

reported in the following studies (Miller & Glibert 1998; Ikeda, Kannoand & Shinada 2001; Miller & Roman 2008;

Saba, Steinberg & Bronk 2009, 2011). The adult and the nauplii densities are the number of adults excreting NH3 into

a batch culture before they reach NOEC for adults or nauplii, under the above specified variables

pH

NOEC adult

[lg NH3 L�1]

LOEC adult

[lg NH3 L�1]

Adult density

[Ind. L�1]

NOEC nauplii

[lg NH3 L�1]

LOEC nauplii

[lg NH3 L�1]

Nauplii density

[Ind. L�1]

7.5 477 1789 170 9 106 30 81 10.8 9 106

8.0 477 1789 55 9 106 30 81 3.5 9 106

8.5 477 1789 18 9 106 30 81 1.2 9 106

9.0 477 1789 7 9 106 30 81 444 9 223

9.5 477 1789 3.4 9 106 30 81 213 9 262



MANUSCRIPT 4 179

with A. tonsa nauplii, the 48 h LC50 is 1257 [95%

confidence level (CL95) 915–1725] and 72 h LC50is 220 (CL95 160–303). The 72-h LC50 in the

present study is similar to the findings of Sullivan

and Ritacco (1985) after 48 h. From the present

study it is clear that A. tonsa nauplii are more sus-

ceptible to ammonia than adult copepods, hence

lower NOEC and LOEC for nauplii than for adults

(Table 3). This is supported by Sullivan and Ritacco

(1985) that states that: ‘Larval Acartia is more sen-

sitive to NH3 than other marine species and life

stages that have been tested’. In terms of NOEC and

LOEC as presented in Table 3 elevated levels are not

observed over time. Therefore, it is recommended

that NH3 is kept below 30 lg NH3 L�1 and moni-

tored weekly in nauplii cultures.

Adult copepod mortality

Data for A. tonsa mortality for acute and chronic

effects of ammonia are limited. Considering 1%

lethal concentration (LC1) of NH3 for A. tonsa, to

our knowledge only Buttino (1994) reports a

model value of 24 h LC1 = 400 lg NH3 L�1. In

the present study the NOEC for adult A. tonsa is

477 lg NH3 L�1, which are similar to Buttino′s

findings. Behaviour of exposed adults showed the

same pattern supporting that exposure at 477 lgNH3 L�1, for up to 72 h is not considered harmful

for the adults. Our data suggest that exposure time

for ammonia has a significant effect upon the sur-

vival of adult of A. tonsa. This is clear from Fig. 2

that cumulated mortality rapidly increases as a

function of ammonia levels and exposure time.

Further this is supported by Fig. 3 where adult

over time first exhibits normal behaviour, and

then slow behaviour and in the end they die, as

function of the same parameters as above. High

pH and exposure time will limit batch cultures. As

seen in the rapid decrease in LC50 values for

adults, where 24 h LC50 is 2370 (CL95 1692–

3319), 48 h LC50 is 972 (CL95 494–1509) and

for 72 h LC50 is 770 (CL95 368–1284). High den-

sities are rarely reported for A. tonsa in batch

cultures (Medina & Barata 2004; Peck & Holste

2006; Jepsen, Andersen, Holm, Jørgensen,

Højgaard & Hansen 2007). This could potentially

be an effect of that the adults at high densities

limit their own offspring′s environment and

thereby decrease the recruitment success of the

population since A. tonsa nauplii are more suscep-

tible to ammonia than adult copepods. Støttrup

and McEvoy (2003) suggest maximum densities

for cultivated calanoids within the range of 100–

1000 adults L�1. However, our data suggest that

densities could be higher without limiting the abi-

otic environment for A. tonsa. For laboratory cul-

tures, NH3 will rarely create a problem, although

if cultures are unstable it is recommended to inves-

tigate inorganic nutrients and pH. For small-scale

high density batch cultures we recommend that

pH is as a minimum measured weekly together

with NH3. In large-scale intensive aquaculture

facilities, daily pH and inorganic nutrient measure-

ment are recommended, analogue to normal farm

management practices in fish facilities. Especially

care should be taken if pH starts increasing above

8.0 as the NH3 fraction then rapidly will increase

to toxic levels (at pH 8.0 = 3.08% at NH3 at pH

9 = 24.1% at NH3 fraction) (Emerson et al. 1975).

Also during experiments it is recommended to doc-

ument the inorganic nutrient levels and if below

NOEC adverse effects of NH3 can be neglected to

affect the results. Monitoring ammonia becomes

more important the longer the experiment is due

to its accumulation. The observed NOEC and LOEC

limits for both nauplii and adults allow that direct-

reading colorimetric tests can be used for analy-

sing ammonium and ammonia. This is possible as

ammonia levels are above detecting limits of

these readily available, relatively cheap and easy

to operate instruments, thereby easing the daily

culture management. In fish aquaculture water

exchange is often the solution when elevated lev-

els of NH3 are observed. Other preventing actions

than water exchange are by decreasing the pH

level and thereby decreasing the fraction of

nitrogenous waste products at the NH3 form by

addition of HCl. However, for copepod cultures

decreasing the pH by adding acids is not the opti-

mal good solution. A more ‘copepod friendly’ sug-

gestion could be to increase the addition of

phytoplankton feed (in the log growth phase) to

the copepod culture since pH is mainly controlled

by the phytoplankton production (Sullivan & Rit-

acco 1985). Thereby, ammonia will decrease both

as a result of the reduction in pH and because

ammonia is an easily acceptable nutrient for phy-

toplankton. It was demonstrated that the addition

of algae could reduce the amount of ammonium

with >50% within 24 h, proving the method via-

ble for daily culture management, although this

did not take copepod grazing and excretion into

account.



MANUSCRIPT 4180

Ammonia in copepod cultures

Thuy (2011) showed a maximum concentration of

NH3 in both an A. tonsa flow-through and a recir-

culated culture systems of 10 lg NH3 L�1. The

systems reached densities of 20 000 nauplii L�1

and 5000 adults L�1. These ammonia levels will,

according to findings in Buttino (1994) and in the

present study, not be a practical culture problem

for nauplii or adult A. tonsa. Ammonia levels in

Thuy (2011) study are therefore not considered a

practical culture problem as the effect on nauplii or

adult mortality is expected to be negligible. Recent

experiments in a small batch culture showed that

concentrations of ammonium increases with adult

A. tonsa densities and time. The densities were

approximately 5000 adult L�1 and NH4+-N

increased from an initial concentration of 100 to a

final concentration of 500 lg NH4+ -N L�1 at day

5 (G. Drillet, M. Rais, N. Novac, P. M. Jepsen,

S. Mahjoub & B. W. Hansen, in preparation). The

study showed a steady increase in ammonium

over time; therefore levels in high densities could

potentially over time be toxic for the nauplii cul-

ture. To calculate the potential limitation Eqn (1)

was developed.

Equation (1) is a tool to predict small-scale

batch copepod density limits before the nitrogen

waste excretion from adult A. tonsa limits the

copepod culture environment. The limitation of

the equation is, however, that it does not include

excretion from the nauplii themselves. This is at

present not possible as to our knowledge no such

data have been reported. Also it does not take into

account the amount of NH3 removed by

phytoplankton feed, regular seawater exchange

and/or eventual biofilter installations. This needs

to be further investigated, especially when consid-

ering commercial recirculated aquaculture sys-

tems. Ammonia is the most toxic decomposition of

the nitrogen waste products in aquaculture biofil-

ters. But when converted in nitrification and deni-

trification processes in biofilters, nitrate and nitrite

will potentially reach toxic levels. Further studies

could focus on developing A. tonsa’s tolerance lev-

els towards nitrate and nitrite.

Feeding of copepods to hatchery water in fish

larval tanks

Timmons and Ebeling (2007) suggest that NH3

limits for commercial fish farms should be below

50 lg NH3 L�1 for chronic exposure to avoid

adverse effects. Other authors suggest levels from

1 to 147 lg NH3 L�1 for marine fish to be consid-

ered a safe rearing practice (Meade 1985; Russo &

Thurston 1991; Tucker 1998). Brownell (1980)

found 24 h first-feeding 10% effective concentra-

tion (EC10) ranges from 10 to 220 lg NH3 L�1

and 24 h LC10 ranged from 290 to 430 lgNH3 L�1 for different species of marine fish larvae.

In the first-feeding stages for most marine fish lar-

vae they prey mainly upon feed items like rotifers

(Small) and/or copepod nauplii (Kiørboe, Munk &

Støttrup 1985; Wilcox, Tracy & Marcus 2006). As

shown in the present study nauplii are after 48 h

more susceptible to ammonium stress than adult

A. tonsa. Feeding nauplii into hatchery water char-

acterized by the above mentioned ammonia levels

will potentially affect them after 48 h of exposure;

hence the 30 lg NH3 L�1 NOEC. Therefore, we

suggest a feeding strategy where eggs from either

the centralized (cold stored eggs) or decentralized

(local produced eggs) strategy as suggested in Dril-

let et al. (2011) are used. Eggs are left for hatching

in sufficient numbers for 24 h in clean saltwater,

and nauplii stage I thereafter fed into fish larval

tanks in bulk or intervals depending on fish species.

Thereby ammonia toxicity effects on live feed cope-

pod nauplii are ensured to be avoided.

Acknowledgments

This work was funded by IMPAQ grant (J. no.

10-093522) to Professor Benni W. Hansen and

grant (J. no. 11-116388) to Ph.D. student Per M.

Jepsen. Thanks are due to Thomas A. Rayner,

Kristian S. Kryhlmand and Anna Johansson for

enthusiastic participation in collecting experimen-

tal data. Laboratory technician Anne B. Faarborg

is indebted for guidance and practical laboratory

help, Dr Troels M. Pedersen and Dr Guillaume

Drillet for comments on earlier drafts and advice

on model simulation, and Dr Morten F. Pedersen

for statistical advice and help. Special gratitude’s

are sent to two unknown reviewers for good and

constructive feedback.

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Manuscript 5.

Nilsson, B., Jepsen, P.M., Rewitz, K., Hansen, B.W. (2013). Expression of hsp70 and ferritin in embryos of the copepod Acartia tonsa (Dana) during

transition between subitaneous and quiescent state. Journal of Plankton Re-search. Article first published online: 1–10. doi:10.1093/plankt/fbt099

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Expression of hsp70 and ferritin in embryosof the copepod Acartia tonsa (Dana) duringtransition between subitaneous andquiescent state

BIRGITTE NILSSON1,2, PERMEYER JEPSEN1, KIM REWITZ2,3 AND BENNIWINDING HANSEN1*1DEPARTMENT OF ENVIRONMENTAL, SOCIAL AND SPATIAL CHANGE, ROSKILDE UNIVERSITY, UNIVERSITETSVEJ 1, ROSKILDE DK-4000, DENMARK, 2DEPARTMENT OF

NATURE, SYSTEMS AND MODELS, ROSKILDE UNIVERSITY, UNIVERSITETSVEJ 1, ROSKILDE DK-4000, DENMARK AND3DEPARTMENT OF BIOLOGY, CELL AND

NEUROBIOLOGY, UNIVERSITY OF COPENHAGEN, COPENHAGEN, DENMARK

*CORRESPONDING AUTHOR: [email protected]

Received February 8, 2013; accepted September 13, 2013

Corresponding editor: Marja Koski

Subitaneous eggs of the neritic calanoid copepod Acartia tonsa (Dana) are capable ofentering a resting state called quiescence to overcome adverse environmental condi-tions. Although physiological changes associated with this transition have beendescribed, the molecular mechanisms are thus far not studied in any calanoidcopepod. Two stress-related proteins, hsp70 and ferritin, were cloned from A. tonsaand their expression determined in embryos of A. tonsa during transition betweensubitaneous and quiescence eggs. Expression of hsp70 remained low during quies-cence despite environmental stress. However, ferritin expression exhibited a strong in-crease from days 3 to 5 of quiescence followed by a declined. After 2 weeks ofquiescence, development of the embryos recovered by the addition of oxygen andincreased temperature. The expression of both genes increased 222-fold for hsp70and 52-fold for ferritin during the recovery phase toward hatching. This suggests thatferritin is a protein needed when embryos of A. tonsa enter quiescence. Both hsp70and ferritin are required during recovery from quiescent to subitaneous state whenembryogenesis continues toward egg hatching.

KEYWORDS: Acartia tonsa; quiescence; hsp70; ferritin; gene expression; copepodeggs

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J. Plankton Res. (2013) 0(0): 1–10. doi:10.1093/plankt/fbt099

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INTRODUCTION

The ubiquitous marine pelagic copepods from higherlatitudes experience seasonal environmental changes thatare not favorable for their survival (Dahms, 1995). Eggsof the calanoid copepod Acartia tonsa are able to enter aresting state, called quiescence, to overcome these un-favorable environmental conditions (Sørensen et al.,2007). Under favorable conditions, subitaneous eggs areproduced that are characterized by hatching within afew days after spawning (Marcus, 1996). In response toadverse environmental conditions, subitaneous eggsenter a quiescent state, where embryonic development isdelayed until exposure to more favorable environmentalconditions. Quiescence can be defined as a direct inhib-ition of development due to adverse conditions (Danks,1987). Quiescence is a disruption of the embryogenesisthat is hypothesized to be associated with transcriptionalquiescence and reduced protein synthesis (Hofmann andHand, 1994; Stuart and Brown, 2006). Thus, the meta-bolic rate is suppressed during quiescence (Pedler et al.,1996; Clegg, 1997; Nielsen et al., 2006). Quiescence canbe induced in copepod eggs by several adverse condi-tions. Abrupt changes in salinity (Holmstrup et al., 2006;Højgaard et al., 2008), low temperatures (Drillet et al.,2006) and anoxia (Holmstrup et al., 2006; Nielsen et al.,2006) have been shown to induce quiescence in order tostore A. tonsa eggs over time.When a copepod embryo undergoes quiescence, it

requires a number of stabilizing factors. Heat shockprotein 70 is a family of proteins where some of whichare constitutively expressed, while others are induced byseveral types of stress conditions, such as high and lowtemperatures (Burton et al., 1988; Voznesensky et al.,2004; Rhee et al., 2009), anoxia (Stensløkken et al., 2010),reactive oxygen species (ROS) (Bellmann et al., 1996),high culture density (Lee et al., 2012) and osmotic stress(Spees et al., 2002). Hsp70 functions as a chaperone mol-ecule that prevents stress-induced misfolding of proteinsby facilitating correct folding pathways (Feder andHofmann, 1999). Under adverse environmental condi-tions, increased synthesis of hsp70 is important for thesurvival of organisms, such as A. tonsa, as it stabilizes pro-teins and prevents them from misfolding.Little is known about the different heat shock proteins

in copepods. Aruda et al. (Aruda et al., 2011) identifiedseveral, including four forms of hsp70 in the calanoidcopepod Calanus finmarchicus. Hsp70 have shown to beinduced in active C. finmarchicus in surface waters, but notin the diapausing animals in deeper waters (Aruda et al.,2011). Voznesensky et al. (Voznesensky et al., 2004) andRhee et al. (Rhee et al., 2009) found that hsp70 gene ex-pression is elevated when copepods are exposed to

elevated temperatures. The heat shock response of C. fin-marchicus in shallow waters protects proteins against thehigher temperatures experienced under these environ-mental conditions (Aruda et al., 2011).

Changes in available cellular oxygen can result inhigher levels of ROS, which in turn causes oxidativestress (Harrison and Arosio, 1996). ROS includes superoxide (O2

2), hydrogen peroxide (H2O2) and the highlyreactive hydroxyl radical (†OH) that is responsible formost damage to cellular macromolecules (Harrison andArosio, 1996; Mates, 2000). Because iron is a catalyst inthe production of potentially damaging ROS, theamount of free iron in the cell must be minimized toreduce the amount of cellular damage. To achieve this,cells store excess iron in iron-storage proteins (Harrisonand Arosio, 1996; Hintze and Theil, 2006). One suchprotein is ferritin, a globular protein capable of mineral-izing and storing iron in its cavity (Theil, 2003; Liu andTheil, 2005). Hsp70 is induced due to a broad range ofstressors; ferritin is only induced at higher levels either ofiron or increased levels of ROS. High levels of ferritinexpression have been reported in the snail, Littorina littorea,during anoxia, when oxidative stress damages macromo-lecules in cells (Larade and Storey, 2004).

Hsp70 and ferritin are indicators of cellular stresswhich respond dynamically to different types of stress. Sofar, no studies have investigated the molecular mechan-ism and changes that underlie this transition phase forA. tonsa.

Physiological changes associated with transitionbetween subitaneous and quiescent eggs have beendescribed, but the molecular mechanisms underlyingthese changes have not been identified in any calanoidcopepod. The aim of this study was to measure geneexpression of hsp70 and ferritin during transition betweensubitaneous and quiescent egg stages for A. tonsa.Furthermore, we measured subitaneous gene expressionin A. tonsa in order to compare it with the gene expressionduring transition between subitaneous and quiescentstage.

METHOD

Cultures

The population of A. tonsa used in this study was suppliedby DTU-AQUA (Institute of Aquatic Resources,Denmark; identity code: DFH.AT1) (Støttrup et al.,1986). Cultivation of the copepods took place understable conditions in a walk-in climate room in cylindricalflat-bottomed polyethylene tanks (60 L) containing 25 Lof saltwater (salinity 30–35) with gentle oxygen supply.

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The copepod cultures were kept dark due to the assump-tion that adult A. tonsa exhibit optimal food uptake atnight and thereby have an optimal egg production(Stearns et al., 1989). The diet for A. tonsa consisted of the�8 mm diameter microalgae Rhodomonas salina which wasfed to the copepods ad libitum. Algae were cultivated in2 L round-bottom glass flasks with B1 medium.Cultivation took place under constant temperature(178C), CO2 supply and light (PAR �80 mE m22 s21).Eggs were harvested and cleaned by the method

described by Hansen et al. (Hansen et al., 2010). The ageof eggs was max. 2 h, and after harvesting and cleaningthey were distributed to the different treatments by pipet-ting. Each sample contained a known number of eggsbetween 20 and 214. Four replicate samples of eggs wereleft in 20 mL Petri dishes for hatching at 178C for 24 h,thereby obtaining hatching of subitaneous eggs. Threereplicate samples were, after 24 h of anoxic cold storage,transferred into 3 � 20 mL petri dishes and left forhatching for 48 h. The same procedure was used after72 h of cold storage and after 336 h of cold storage. Fivereplicate egg batches were used to check the recoveryphase and, after 336 h of cold storage, they were trans-ferred to aerobic conditions at 178C and left to hatchfor 24 h.Hatching success was estimated by fixing the nauplii

and the remaining eggs with 1% final concentration ofacid Lugol solution and counting them using a dissectionmicroscope [Olympus SZ 40; Olympus Optical (Europe)GmBH, Hamburg, Germany].

RNA purification and cDNA synthesis

Bulk samples (.1000) of A. tonsa eggs of different ageswere obtained from the bottom of copepod tanks, where-after the eggs were separated through a 100 and 70 mmmesh to separate eggs from other copepod stages. Eggswere thereafter thoroughly rinsed with salinity 30 salt-water. This procedure took place on the surface of a52 mm mesh filter. Eggs were scraped from the 52 mmmesh filter and put directly into 350 mL RLT buffer sup-plied with the RNeasy Mini Kit (Qiagen). The eggs werehomogenized using a disposable pestle. Total embryoRNA was isolated using the RNeasy Mini Kit (Qiagen)on column DNAse treated to remove genomic DNA andstored at 2208C until further use.First-strand cDNA was reverse transcribed from 20 ng

of total RNA using the SuperScript III First-StrandSynthesis System for RT-PCR kit with the oligo(dT)20primer (Invitrogen). First-strand cDNA for hsp70, ferritinand b-actin was amplified by the polymerase chain reac-tion (PCR) using the HotStarTaq Master Mix Kit(Qiagen) or the Pfu polymerase kit (Fermentas) using

degenerated primers against conserved regions of thegenes. Degenerated primers for hsp70 were as describedpreviously for the copepod C. finmarchicus (Voznesenskyet al., 2004). Primers for ferritin and b-actin (Table I)were generated from conserved regions identified bysequence alignment of ferritin and b-actin orthologsfrom different species (GenBank accessions numbers:EU371046; AJ245734; JN194149). Putative hsp70 cDNAwas amplified by the PCR using the degeneratedprimers. The amplification conditions for the PCR reac-tion were 958C15min (948C30s þ 528C30s þ 728C1min) �40 cycles þ 728C10min for hsp70 and b-actin, and958C15 min (948C30 s þ 458C30 s þ 728C1 min) � 40cycles þ 728C10 min for ferritin. PCR products were testedfor molecular size and purity by agarose gel electrophor-esis. PCR products were excised, purified and extractedusing the QIAquick Gel Extraction Kit Protocol(Qiagen). Purified PCR products were then ligated intopCRw2.1 TOPOw vector and transformed into OneShotw Mach1-T1R competent cells (InvitrogenTM). Sixhsp70, five ferritin and eight b-actin positive clones wereselected and sequenced. The sequencing resulted inthree identical hsp70, two identical ferritin and four identi-cal b-actin partial cDNA sequences. The amplicons ofhsp70, ferritin and b-actin encoded ORF of 200 aminoacids, 94 amino acids and 54 amino acids, respectively.Alignments with orthologs from other species confirmedthat these sequences represent hsp70, ferritin and b-actinfrom A. tonsa. The top nine BLAST results, together withe-values, are presented in the Results section. The align-ment graphics were made using Jalview 2 (Waterhouseet al., 2009).

Gene expression analysis

Adult females were transferred to a clean 70 L polyethyl-ene tank with saltwater (salinity 30–35, 178C, lightoxygen supply, darkness). After 2 h, eggs produced wereharvested and isolated into respective samples by mouthpipetting. Time 0 samples consisted of eggs laid between0–2 h, time 2 h samples consisted of 2–4 h old eggs andso forth. The experiment was separated into threephases; a subitaneous, a quiescent and a recovery phase.

Table I: Degenerated primers for hsp70 andferritin, and primers for b-actin

hsp70 Forward: 50-GCNAARAAYCARGTNGCNATGAA-30

Reverse: 50-YTTYTCNGCRTCRTTNACCAT-30

ferritin Forward: 50-CATYAACAAGCARATCAA-30

Reverse: 50-AGYACAAYYCTTCCWCCWCG-30

b-actin Forward: 50-TCCATCATGAAGTGCGATGT-30

Reverse: 50-TTGATCTTGATGGTGGATGG-30

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The subitaneous phase consisted of eggs treated withsaturated oxygen at 178C that were sampled at time 0 h(five replicates); 2 h (three replicates); 4 h (three repli-cates); 6 h (three replicates) and 24 h (three replicates). Inthe quiescent phase, eggs were kept at 38C and N2 wasadded to the water to remove all O2, in order to inducequiescence (sensu Holmstrup et al., 2006). After 336 h ofquiescence, eggs were supplied with O2 saturated salt-water with a temperature of 178C to initiate the recoveryphase. Sampling frequencies are given in Table II. Ateach frequency, three biological replicates were made.Each sample contained �100 eggs, which were flashfrozen in liquid N2 and then stored at 2808C until totalRNA isolation. Primers for quantitative real-time PCR(Table III) were constructed from the sequenced PCRproducts by the usage of Universal Probe Library AssayDesign Center software (Roche Applied Science).Quantitative real-time PCR was performed as

described (Rewitz et al., 2009, 2010) by using theMx3005P QPCR System (Stratagene) and theQuantiTectw SYBRw Green PCR kit (Qiagen). Samples(three replicates of each sample) were prepared accordingto the manufacturer’s protocol with the following modifi-cation: a total volume of 15 mL per sample (1 �QuantiTect SYBR Green PCR Master mix; 0.3 mM of

each primer; 2 mL cDNA and 3.5 mL RNase-free water).The quantitative real-time PCR amplification conditionswere: 958C10min (958C15s þ 608C15s þ 728C15s.) � 45.All samples were subjected to melting curve analysis toensure homogeneity of the amplified products.Expression of hsp70 and ferritin was normalized to thelevel of the housekeeping gene b-actin as described inRhee et al. (Rhee et al., 2009). The expression of b-actinshowed minimal variability under our different experi-mental conditions (data not shown) making it a suitablereference gene. Gene expression was calculated accord-ing to 2DDCt method and mRNA levels were given asrelative units normalized to the mRNA levels of b-actin.

Statistical analysis

Data were ln transformed and tested for normality(Kolmogorov–Smirnov) and for equal variance (Levenemedian test) (all data). For both genes, a one-way analysisof variance (ANOVA) was used on the ln transformedsubitaneous eggs and quiescent eggs data to comparebetween samples. Since data were significantly different,a post hoc test was applied to test for the significant differ-ences (all pairwise multiple comparison procedure by theHolm–Sidak method P, 0.05) (Figs 4 and 5). TheHolm–Sidak post hoc test was also used to test if therewere significant differences in gene expression betweensubitaneous and quiescent eggs up till 24 h (0 h subita-neous versus 1

2 h quiescence; 2 h subitaneous versus 1 hquiescence; 4 h subitaneous versus 4 h quiescence; 24 hsubitaneous versus 24 h quiescence) of embryogenesis(Figs 4 and 5). The P-level of 5% CL was used. For recov-ery data of both genes, a Pearson correlation analysis(P, 0.001) was used to see if there were any correlationsover development time for both genes (Fig. 6). Theprogram used to prepare Figures and for statistical ana-lysis was SigmaPlot version 12.0, Systat Software# 2006.

RESULTS

Hatching success

Hatching success of subitaneous eggs in the controlbatches that were used in the experiment was between24% (time 0 batch) and 29% (recovery batch) after 24 h;and 65% (24 h and 72–288 h batches) after 48 h (Fig. 1).Eggs treated with N2 had a hatching success of 28% after48 h. According to Hansen et al. (Hansen et al., 2010)acclimatized eggs stored at 178C would have an esti-mated hatching success at around 55% after 24 h andaround 90% after 48 h. The A. tonsa culture used in thepresent experiment was acclimatized to 178C and the

Table II: Experimental design

Treatment Sampling

Subitaneous eggs 178C;saturatedO2

First sampling at time 0 h. Took outsamples at 2, 4, 6 and 24 h

Inducingquiescence

38C; 0% O2 Sampling at 0.5, 1, 4 and 24 h afterremoving the O2 from theseawater

Quiescence 38C; 0% O2 Quiescence induced eggs weresampled at 48, 72, 96, 120, 144,192, 312 and 336 h

Recovery fromquiescence

178C;saturatedO2

After 336 h of quiescence, the eggswere added seawater withsaturated O2 and a temperature of178C. Sampling occurred at 1, 8,24, 48, 72 and 96 h after additionof O2

Every sample contained �100 Acartia tonsa eggs.

Table III: Quantitative real-time PCR primersfor hsp70, ferritin and b-actin

Quantitative real-time PCR primers

hsp70 Forward: 50-TTCAATGATTCACAGAGACAAGC-30

Reverse: 50-TCCTTGTGATGTTAAGACCAGCTAT-30

ferritin Forward: 50-ACGCTTGCACTGATAATCCA-30

Reverse: 50-AGTTCTACCGTGACGCATCC-30

b-actin Forward: 50-CTTCTGCATACGGTCAGCAA-30

Reverse: 50-ACCCGTACGCCAACACTG-30


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hatching success obtained was 26–31% lower after 24 hand 25% lower at 48 h compared with Hansen et al.(Hansen et al., 2010). Less than 2% nauplii were observedduring the quiescent phase. This indicated that the eggssuccessfully entered quiescence by removal of O2 and in-cubation in darkness at a low temperature (38C). Nofurther nauplii were observed until collection of eggsfrom the recovery samples. After 24, 48, 72 and 96 h ofrecovery, the hatching success was 57, 74, 78 and 84%,respectively (Fig. 1). Only eggs were used for qPCR ana-lysis. By the time 96 h of the recovery period had past,only 14% of the embryos were still in the egg stage andthe remaining hatched. Here, the maximal hatching after96 h was close to the maximal hatching around 90%reported by Hansen et al. (Hansen et al., 2010). The in-creasing number of hatched eggs during the recoveryphase indicated the eggs were perfectly viable after quies-cence and recovery as well.There can be some concerns about non-viable eggs

adding RNA to the samples. Eggs are presumably hatch-ing continuously during the experimental time period forthe subitaneous and recovery experiments, meaning thatthe proportion of non-viable eggs changes with time. Atthe start of the experiments, there will be a lot of viableeggs, but as the experimental time extends, the proportionof non-viable eggs will become larger. RNA, however,degrades quickly in non-viable eggs, and will thereforenot be considered as a problem because these eggs onlywould add little to the RNA in the samples; the expressionlevels would therefore be representative of the viable eggs.The expression comparison is furthermore valid, giventhat we are using ratios in the gene expression analysis.

Identification of hsp70, ferritin and bb-actin

Hsp70, ferritin and b-actin were cloned from A. tonsa toobtain sequences for gene expression analysis. Acartiatonsa hsp70 had the highest sequence similarity (88%identical amino acids) to hsp70 from the mite Tetranychuscinnabarinus (accession no. ACG60424.1), followed by85% identity with different crustacean species includingArtemia franciscana (AF427596.1). The ferritin sequencefrom A. tonsa had the highest sequence identity of 61%with the lancelet Branchiostoma belcheri tsingtauense(AAO18672.1) followed by sequence identity of 59–55%with different crustacean species, including Artemia francis-cana. b-actin sequence from A. tonsa was the most con-served sequence exhibited identity of 94% with thebutterfly Papilio canadensis (AAF81599.1). This shows thatthe sequences for A. tonsa had high sequence identity tohsp70, ferritin and b-actin orthologs from other species, ofwhich many are arthropods including crustaceans.Sequence alignments with orthologs from other species(Fig. 2) clearly demonstrate that the obtained sequencesencode A. tonsa orthologs of hsp70, ferritin and b-actin.Phylogenetic analyses of hsp70, ferritin and b-actin fromA. tonsa clearly demonstrate that these protein sequencescluster together with orthologs from other species (Fig. 3).

Gene expression analysis

Expression over time of hsp70 and ferritin in subitaneouseggs was generally low compared with expression in qui-escent eggs and during the recovery phase. A generaldecline in the expression level of both genes was observedduring 24 h after spawning of the subitaneous eggs(Figs 4 and 5). Statistical significance was found for subi-taneous eggs of hsp70 between 0 h to 6 h and 24 h,respectively (P, 0.05, marked with ‘a’ on Fig. 4). For fer-ritin, differences in gene expression for subitaneous eggswere between 0 h and 24 h (P, 0.05, marked with thesymbol a in Fig. 5). The relative mRNA levels of hsp70over time for eggs entering and staying in quiescenceexhibited some fluctuations with a minor increase at72 h. However, there were no significant differencesduring the quiescent period (Fig. 4). Ferritin expressionexhibited a major increase (P, 0.001) from 72 to 120 hof quiescence compared with earlier time points (Fig. 5,marked with the symbol b). The relative mRNA level offerritin increased between 16-fold and 32-fold at h 72–120 compared with subitaneous 0 h samples. Thus, therewas a significant peak in ferritin gene expression from72 h till 120 h. Between 120 and 336 h, expression of fer-ritin declined dramatically. Hsp70 and ferritin expressionduring quiescence appeared to be higher than for subita-neous eggs (Figs 4 and 5). Especially ferritin seemed to be

Fig. 1. The bars show percentage hatching success+SEM of eggsfrom the different treatments. The subitaneous eggs (n ¼ 4) andrecovery eggs (n ¼ 5) were incubated for 24 h. The three batches usedfor quiescent eggs (n ¼ 3) were incubated for 48 h before inspected forhatching, respectively.


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induced at higher levels during the first 24 h of quies-cence, compared with the subitaneous stage. Hsp70 alsoexhibited slightly higher expression levels after 0.5 h ofquiescence.When comparing subitaneous and quiescent hsp70

gene expression, there was a significant differencebetween subitaneous (grey bars) data after 24 h com-pared with quiescent (white bars) data after 1

2 h, 1 h, 4 hand 24 h (P, 0.002, marked with the symbol b onFig. 4). Hsp70 expression obtained after 24 h of

quiescence was significantly different from 4 and 6 h insubitaneous eggs (P, 0.002, marked with the symbol con Fig. 4). Ferritin gene expression in quiescent (whitebars, Fig. 5) eggs was consequently significantly differentfrom subitaneous (grey bars, Fig. 5) eggs after 24 h(P, 0.002, marked with the symbol c on Fig. 5).

When analyzing the gene expression during the recov-ery phase, a large increase in expression was found forboth hsp70 and ferritin (Fig. 6). The observed positive cor-relation was confirmed for both hsp70 (r2 ¼ 0.93, P: 0.05)

Fig. 2. (A) Alignment of putative hsp70 amino acid sequences encoded by the partial cDNA sequence from Acartia tonsa compared with sequencesfrom nine different species. Tetranychus cinnabarinus hsp70-3 (ACG60424.1, E-value: 1e2125); Philodina roseola hsp70 (ACI90341.1, E-value: 5e2124);Trichinella spiralis hsp70 (CBX25718.1, E-value: 8e2124); Hypena tristalis hsp70 (AFO70209.1, E-value: 6e2121); Moina macrocopa hsp70 (ACB11341.1,E-value: 3e2121);Marsupenaeus japonicus hsp70 (ABF83607.1, E-value: 1e2123); Solenopsis invicta hsp70 (EFZ09406.1, E-value: 2e2122); Harmonia axyridishsp70 (ABR92405.1, E-value: 2e2122); Homarus americanus hsp70 (ABA02165.1, E-value: 2e2121). (B) Alignment of putative ferritin amino acidsequences encoded by the partial cDNA sequence from Acartia tonsa compared with sequences from nine different species Branchiostoma belcheritsingtauense ferritin (AAO18672.1, E-value: 1e219); Eisenia andrei ferritin (ACL14179.2, E-value: 3e217); Fenneropenaeus indicus ferritin (AEQ53930.1,E-value: 4e219); Litopenaeus vannamei ferritin (AAX55641.1, E-value:6e219); Ostrea edulis ferritin (AFK73708.1, E-value: 4e220); Epinephelus coioides ferritinheavy subunit (ACH73080.1, E-value: 5e219); Procambarus clarkii ferritin (AEB54659.1, E-value:1e218); Artemia franciscana ferritin (ABK91815.1,E-value: 3e217); Pagrus major ferritin heavy chain (AAP20171.1, E-value:2e218). (C) Alignment of putative b-actin amino acid sequences encoded bythe partial cDNA sequence from Acartia tonsa compared with sequences from nine different species. Papilio canadensis actin A1 (AAF81599.1, E-value:7e226), Lepeophtheirus salmonis actin(ABU41027.1; E-value: 1e-25); Calanus finmarchicus actin (Q92192.1, E-value: 1e224); Lonomia obliqua actin 1(AAV91408.1, E-value: 1e224); Rhodnius prolixus actin (ABY47893.1, E-value: 6e226); Lissorhoptrus oryzophilus b-actin (ADH95740.1, E-value: 8e226);Apolygus lucorum b-actin (AEP31949.1, E-value: 8e226); Harpegnathos saltator actin (EFN78404.1, E-value: 1e225); Maconellicoccus hirsutus actin(ABN12061.1, E-value: 1e225). Sequences were obtained from GenBank and BLAST E-values are given. Dark background indicates conservedareas amino acids, whereas lighter background indicates less conserved residues.


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and ferritin (r2 ¼ 89, P: 0.05) by a Pearson correlationanalysis. Maximum relative mRNA levels were observedafter 96 h of recovery with a 222-fold increase for hsp70and 52-fold increase for ferritin compared with subita-neous 0 h samples. Expression levels of both genes weremuch higher during the recovery phase than observedduring the subitaneous and quiescence stages.

DISCUSSION

In the present study, gene expression levels of hsp70 andferritin were measured during the transition between subi-taneous and quiescent stage of A. tonsa eggs. The hsp70response towards stress has been shown to vary amongspecies during dormancy. Hsp70 expression can beinduced in some species (Yocum et al., 2005), while itdecreases in others (Tungjitwitayakul et al., 2008;Gkouvitsas et al., 2009). In some cases, the expression ofhsp70 does not show any differences between dormantand active animals (Goto et al., 1998; Tachibana et al.,2005; Zhang and Denlinger, 2010), which is also the casefor embryos of A. tonsa that did not show any significantdifferences in hsp70 gene expression between subitaneousand quiescent eggs.

However, 72–120 h after initiation of quiescence, apeak in gene expression was observed for ferritin. Thiscould be due to the fact that the eggs normally wouldhatch .24–72 h after spawning (Hansen et al., 2010) andthe results indicate that the embryogenesis of A. tonsa con-tinues for some time after exposure to adverse environ-mental conditions. It can be proposed that the embryosfirst sense the full consequences of the environment at thetime of hatching, which induces quiescence. However, thisremains to be verified. High ferritin expression has beenshown to indicate initiation of a dormant state in embryosof Artemia franciscana and copepodites of the calanoidcopepod Calanus finmarchicus (Chen et al., 2003; Tarrant

Fig. 3. Three phylogenetic trees showing the relationship of hsp70,ferritin and b-actin sequences from the calanoid copepod Acartia tonsawith orthologs from species with high sequence identity compared withA. tonsa. The species is as follows for hsp70 GenBank (accession no.):Tetranychus cinnabarinus hsp70-3 (ACG60424.1), Marsupenaeus japonicushsp70 (DQ663761.1), Moina macrocopa hsp70 (EU514495.1), Homarusamericanus hsp70 (DQ173923.1), Artemia franciscana hsp70 (AF427596.1),Litopenaeus vannamei hsp70 (AY645906.1), Macrobrachium rosenbergii hsp70(AY466445.1), Tigriopus japonicas hsp70 (EU162749.1), Paracyclopina nanahsp70 (HQ115581.1). The hsp70 out-group is a heat shock protein cognate 5(Hsc-70-5) from Metanalges sp. (AFJ22482.1). For ferritin: Branchiostoma

belcheri tsingtauense ferritin (AY175376.1), Fenneropenaeus indicus ferritin(JN651914.1), Mercenaria mercenaria ferritin (JQ691632.1), Epinepheluscoioides ferritin heavy-chain subunit (EU714166.1), Litopenaeus vannameiferritin (AY955373.1), Artemia franciscana ferritin (AY062897.1), Eiseniaandrei ferritin (FJ516398.3), Procambarus clarkii ferritin (HQ414585.1),Osmerus mordax ferritin heavy-chain subunit (BT075303.1) andDicentrarchus labrax heavy-chain subunit (FJ197145.1). The ferritinout-group is artemin (Artn) from Artemia franciscana (AY062896.1). Forb-actin: Sitobion avenae actin (AY581122.1), Phaedon cochleariae actin(EF134404.1), Papilio canadensis actin (AF277457.1), Lepeophtheirus salmonisactin (EF490842.1), Lonomia obliqua actin 1 (AY829794.1), Grapholita molestaactin (JN857938.1), Lissorhoptrus oryzophilus b-actin (HM060307.1),Gastrophysa atrocyanea actin (AB306273.1) and Artemia franciscana actin(DQ641940.1). The b-actin out-group is an actin-related protein 66B(ARP66B) from Drosophila melanogaster (AAF50488.1).


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et al., 2008). Ferritins in embryos of Artemia sp. have beenhypothesized to inhibit development during the restingstate due to chelation of iron stores (Chen et al., 2003).During dormancy, ferritin functions as a chaperone mol-ecule that protects against oxidative stress and helps main-tain cellular hydration (Chen et al., 2003). When ferritinlevel decreases, iron becomes available for processes thatrequire iron, allowing embryogenesis to continue (Tarrantet al., 2008). Thus, the elevated expression levels of ferritinin A. tonsa must likely indicate the initiation of the quies-cent state, consistent with the possibility that embryos arefirst sensing and reacting to the environment when theyapproach hatching. Oxidative stress could also be a factorthat requires a higher level of ferritin, when eggs are sub-jected to anoxia.Expression of hsp70 and ferritin during the recovery

from quiescent to subitaneous state in embryos of A. tonsashowed a profound increase as the development of theeggs progressed. The observed positive correlation ofhsp70 and ferritin expression over time toward hatching ofthe eggs exceeds what would be expected for an increasein general transcription. This is also supported by the factthat expression of both genes is normalized to b-actinmRNA levels. The increase during recovery most likelyreflects a specific requirement of these proteins during

the developmental transition from quiescence to subita-neous. During the recovery, the eggs were transferredfrom 3 to 178C which could affect the level of gene

Fig. 5. Quantitative real-time PCR analysis of ferritin in subitaneousand quiescent embryos of the calanoid copepod Acartia tonsa at followingtime points: 0, 0.5, 1, 2, 4, 6, 24, 48, 72, 96, 120 and 336 h. First, aone-way ANOVA was used to check significance between thesubitaneous eggs. A significant difference was observed between 0 and24 h and all are significantly different from each other (P, 0.05) andare market with the letter a. Secondly, a one-way ANOVA was used toinvestigate the quiescent eggs. The test showed that quiescent eggs of72, 96 and 120 h were significantly different (P, 0.001) from eachother and from all other quiescent eggs. The differences are markedwith the letter b. The same test revealed that the quiescent eggs from 0to 24 h were not significantly different from each other, but they weresignificantly different (P, 0.002) from all other quiescent eggs, markedwith the letter c. When comparing subitaneous eggs with quiescenteggs, a one-way ANOVA observed that subitaneous eggs (24 h) weresignificantly different (P, 0.002) from quiescent eggs (24 h), markedwith letter d. Values are means+SEM (n ¼ 3–5). Grey bars:subitaneous data. White bars: quiescent data.

Fig. 6. Quantitative real-time PCR analysis of hsp70 and ferritin duringrecovery from quiescence in embryos of the calanoid copepod Acartiatonsa. Eggs were kept in water with no O2 at 38C in a quiescent period of336 h before transferring the eggs to oxygenated water at 178C. Sampleswere taken 1, 6, 24 and 96 h after addition of O2 and a temperature of178C. A Pearson correlation analysis showed (R ¼ 0.93, P-value: 0.007)for hsp70 and (R2 ¼ 89, P-value: 0.019) for ferritin, respectively.

Fig. 4. Quantitative real-time PCR analysis of hsp70 in subitaneousand quiescent embryos of the calanoid copepod Acartia tonsa at followingtime points: 0, 0.5, 1, 2, 4, 6, 24, 48, 72, 96, 120 and 336 h. First, aone-way ANOVA was used to check significance between thesubitaneous eggs. Significant differences were observed between 0, 6and 24 h, and all are significantly different from each other (P, 0.05)and are market with the letter a. No significant differences wereobserved among the quiescent eggs. When comparing subitaneous eggswith quiescent eggs, a one-way ANOVA observed that subitaneous eggs(24 h) were significantly different (P, 0.002) from quiescent eggs at 0.5,1, 4 and 24 h. These significant differences are marked with the letterb. The same one-way ANOVA revealed that quiescent eggs (24 h) weresignificantly different (P, 0.002) from subitaneous eggs of 4 and 6 h,and is marked with the letter c, respectively. Values are means+SEM(n ¼ 3–5). Grey bars: subitaneous data. White bars: quiescent data.


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expression due to a general increase in metabolism.Nielsen et al. (Nielsen et al., 2007), however, calculated therespiratory Q10-value of A. tonsa eggs to be 2.51 (+0.15SD). Thus, the observed increase in gene expression isnot only due to the raised temperature during recovery,but indicates that hsp70 and ferritin are needed. Otherarthropods have shown similar up-regulation of hsp70(Rinehart et al., 2000; Hayward et al., 2004; Zhang andDenlinger, 2010; Altiero et al., 2012). There is a clear dif-ference among species regarding expression patterns ofhsp70 due to different stressors, even among copepodspecies (Lauritano et al., 2012).High ferritin expression in Artemia sp. should delay de-

velopment during dormancy and protect macromole-cules from oxidative stress (Chen et al., 2003). In thepresent study, during recovery, expression exhibited veryhigh levels as embryogenesis of A. tonsa continues afterquiescence. Therefore, the high expression of ferritinduring recovery of A. tonsa embryos cannot be associatedwith delayed development as described in Chen et al.(Chen et al., 2003) and Tarrant et al. (Tarrant et al., 2008).Instead, the levels of ferritin required during recoverymust have a role in protecting cells from oxidativedamage of macromolecules. Since hsp70 is also known toprotect against ROS (Bellmann et al., 1996), it is plausiblethat both proteins are required at very high levels as theembryo needs to stabilize and continue developmentafter quiescence, leading to nauplii hatching. The low butstill detectable expression of both hsp70 and ferritin in sub-itaneous eggs is expected to be due to their natural role innon-stressed cells (Theil, 2003).In conclusion, this study represents the first identifica-

tion of hsp70, ferritin and b-actin as well as the character-ization of hsp70 and ferritin expression associated withtransition between the subitaneous and quiescent state inembryos of the calanoid copepod A. tonsa. Using gene ex-pression analysis of stress-related proteins on embryos,we have contributed important new knowledge on thecomplex life strategy, including transition and recoverybetween subitaneous and quiescence eggs, of a neriticcalanoid copepod.

FUNDING

The work is part of IMProvement of AQuaculturehigh quality fish fry production (IMPAQ) funded bythe Danish Strategic Research Council project no.10-093522 to B.W.H. and was supported by the DanishCouncil for Independent Research, Natural Sciences toK.F.R. We are indebted to the laboratory technicians,Anne Busk Faaborg and Mette Juel Riisager, at thetwo research groups at ENSPAC and NSM, Roskilde

University, to Thomas Allen Rayner for linguistic correc-tion, and to two anonymous reviewers that contributedwith very constructive input to an earlier version of themanuscript.

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