Slide 1

Comparative Study of the ReproductivePerformance of Four Strains of Artemia

Robert J. Mayer, Eythan Moren, Alexandra Mndez andLiza V. Jimnez

University of Puerto Rico at AguadillaOctober 5, 2006

1This presentation evaluates important aspects of the ecological physiology of the genus Artemia inPuerto Rico

Discussion of research data and conclusions

Background on the genus Artemia in Puerto RicoInternational study on Artemia

Artemia are Anostracan Branchiopods that are well adapted to the extreme conditions of hypersaline environments Synthesis ofhemoglobinsHeat toleranceFlexibility in dietProduction of resistant stagesEfficient osmoregulation

3Artemia habitats are:

1. Coastal thalassic or inland athalassic (natural or man made)2. Usually fishless3. Salinity ranging from 15 to 3004. Variable anionic composition, climatic condition and altitude5. Found in every continent except Antarctica

Branchiopods are thought to have been widely distributed globally, in many different aquatic environments (marine, freshwater and ephemeral), until the occurrence of the Mid-Mesozoic Marine Revolution (150 mya). This era brought about an evolutionary increase in the intensity of predation of zooplankters by bony fishes, probably due to the development of suction feeding, causing the extinction of many groups of Branchiopods. This resulted in the gradual restriction of extant large bodied forms (e.g. Notostracans, Anostracans and Conchostracans) to fish-free environments (Kerfoot and Lynch, 1987). A similar event probably took place in freshwater habitats but there is much less evidence for this.

oviparousovoviviparousLife cycle of Artemia is tightly coupled tothe environment Continuous reproductionAlternation of reproductive mode

There are sexual and parthenogeneticspecies

4Read the text and discuss the life cycle stressing the fact that these organisms have two reproductive modes (oviparous and ovoviviparous). Talk very briefly (2 minute max. for this slide)Artemia is a very important organism from an ecological and economic perspective:

Artemia biomass is used inaquaculture (fish food)

Artemia are important for the production of salt and for thesurvival of many species of birds in salt flat systems

What is the importance of locating and characterizing populations of Artemia?

5Basically read the headline (yellow) and the rest of the textAn eco-physiological characterization of local Artemia populations is very important Development of management strategies for the Cabo Rojo salt flat system

Development of culturemethods for the commercialproduction of Artemia biomass

6Read the headline and then read text in white. These are the two main reasons for characterizing these populations of brine shrimp

Artemia is widely distributed Generated by Gilbert Van Stappen, Artemia Reference Center - Belgium

The identification and characterization of Artemia species is part of an international initiative

A collaborative international effort is essential for the study of Artemia urgent need for new sources !Interdisciplinary collaboration is very importantUniformity of the methodsDissemination of results

8Read the headline and then read text in white. These are the two main reasons for characterizing these populations of brine shrimp

How do we characterize thedifferent strains of Artemia?

Cross breeding testsBiometrics (cysts and nauplii)Reproduction and life-spanAllozyme studiesCytogeneticsMolecular markers

Molecular methods

Molecular markersDNA polymorphismNuclear and mitochondrial DNARFLPRAPDAFLPDNA sequencing

What do we know about the biology of thepopulations of Artemia from Puerto Rico ?

There are currently three populations of Artemia in Puerto Rico (Cabo Rojo Salterns, La Parda Saltern in Gunica and PapayoSalterns in La Parguera)There is probably one populationin ViequesInhabit the waters of man made solarsalterns

11Say this Artemia Research at UPR Aguadilla has three main components (read each component in white) and say this: today I will be talking about the third research topic What do we know about the biology of thepopulations of Artemia from Puerto Rico ?Mophometrically different from type population and amongthemselves

No indication of an intrinsic barrier to gene exchangebetween local and the type population

Differences in cyst eclosion profiles between thepopulations from PR and the type population

All the populations from Puerto Rico are sexual and diploid

Main questionWhat are the effects of temperature, salinity and predator-derived chemical compounds on the reproductive performance of different strains of Artemia?

Ventilationchamber Nylon mesh (100 m) Water levelIsolation chamberPlankton isolation chambers were used toisolate reproductive couplesAnimals grown in mass culture until sexual maturity

Introduced in isolation chamberupon reaching sexual maturity

14In order to do this experiment we designed isolation chambers using pvc with a diameter of 5 cm. Ivan go ahead and explain the parts of the chamber

Read the two captions in blue saying animals were grown in mass cultures in the laboratory until sexually mature and were introduced into the isolation chambers upon developing secondary sexual characteristics say this also this took place approximately 49 days after eclosion of the larvae from cystsA plankton recirculation apparatus was used in the life history experiments

Control chamberExperimental chamberPumpPumpWater intake(100 m mesh)Water intake(100 m mesh)

Isolation chambersIsolation chambersWater distribution pipeWater distribution pipeRigid plastic tubingRigid plastic tubingClear plastic partitionsSpigotSpigotHeaterHeater

TOP VIEW23 C

Salinity of 4515Explain this apparatus (we will talk about it tomorrow) and mention the experimental conditions in blue. Stress the fact that the apparatus was designed and built at UPR-AGUADILLA by us!!!

Life history experiments showed that the totalnumber of offspring produced was higherfor APAR

The production of naupliiwas lower than that of cysts

Production of mixed broodsRandom alternationbetween reproductivemodes

The lifespan of APAR individuals was higherthan that of the other populations

Higher reproductivepotential for APAR

There was a significant time - dependent reduction in reproductive potential for all populations

Senescence?

Bet hedging strategyEffects of predator derived chemicals on the reproduction of ArtemiaUsually live in predator-free environments

Many of these habitats experience frequent predator invasions Few studies in the literature on predator prey interactions involving Artemia Mid-Mesozoic Marine Revolution (150 mya) evolutionary increase - intensity of predation ofzooplankters by bony fishes (development of suction feeding)

19Causing the extinction of many groups of Branchiopods. This resulted in the gradual restriction of extant large bodied forms (e.g. Notostracans, Anostracans and Conchostracans) to fish-free environments (Kerfoot and Lynch, 1987).

Most of the work involving inducible defenses involving zooplankton has been done on freshwater cladocerans and has received a lot of attention in zooplankton ecology in the past two decades

Previous work involving inducible defenses Predation can have effects on : functional morphology, behavior, life history, and distribution of zooplankton

Stanley I. Dodson

20Inducible defenses Common in unpredictable environments (intermittent predator attacks) prey genotypes that produce a defense only in the presence of a predator should be fovored.

Dodson, Kerfoot, O Brien, Weider and Pijanowska: have shown that predation can have effects on : the functional morphology, behavior, life history, and distribution of zooplankton Studies on Artemia photoresponses Forward and Rittschof, 1999 Enzymatic degradation products of sulfated and acetylated external mucus of fish can increase the photosensitivity of first instar Artemia nauplii Lower photoresponse thresholds causing larvae to remain deeper in the water column where low light levels reduce the effectiveness of visual predators

Defenseless??

Kairomones - interspecific signal chemicals which only benefit the receivingorganism in the context of the signal transmission21The components of fish mucus that activate photoresponses involved in DVM of larval brine shrimp were characterizedby testing 1) fish mucus 2) products of digestion of fish mucus with heparinase and chondroitinase 3) heparin, chondroitin sulfate, and hyaluronic acid dissacharides (4) subunits of these dissacharides

These studies suggest that Artemia has receptors for the amino sugar breakdown products of mucopolysacharides found in external mucus of fish.

This study challenges the idea that Branchiopods are defenseless slow moving organisms that inhabit simplified ecosystems where predators and competitors develop weakly or are completely absent

No other studies that consider the influence of chemical signals on other aspects of the behavior, life history characteristics and morphology of Artemia.

From Borror, Triplehorn, Johnsons INSECTS (sixth edition)

Aquatic insects found in the saltern pondsTrichorixa reticulata

22Order HenipteraCorixidae (waterboatmen)Notonectidae (backswimmers)Gerridae (water striders)

Order Diptera

Ephydridae (Brine flies)

Ephydra (brine flies)

Experimental conditionThe presence of T. reticulata increases total number of nauplii produced by cohort

CorixidsOffshore waterNumber of nauplii produced by cohort

23

Experimental conditionThe presence of T. reticulata increases the number of nauplii produced per BOTPR female Number of nauplii produced per femaleCorixidsOffshore water

Stunning mechanism ?Increase the probability that some animals survive to a reproductive age ?24Experimental conditionThe presence of T. reticulata has no effect on number of cysts produced per BOTPR female

Number of cysts produced per femaleCorixidsOffshore water

25ConclusionsThe reproductive performance of Artemia varies between strains and is affected byenvironmental conditions

A. parthenogenetica had the highest reproductiveperformance of all the population tested

Artemia can modify its reproductiveperformance and behavior in response towaterborne chemical compounds (practical applications?)

26Future direction of the research

Determination of the degree of genetic variation among the PR populations and GSL using the Restriction Fragment Length Polymorphism (RFLP) fingerprinting technique Completion of the reproductive characterization of PR populations assesment of potential use in aquaculture

Effect of other chemical compounds on the reproduction and genetics of local strainsAnalysis SDS-PAGE analysis of heat shock protein patternsin the cysts of Artemia from local strains

This interdisciplinary project involves undergraduate students from UPR-Aguadilla

Helping revitalize our nations talent pool in STEM Acknowledgements

R. B. Forward and D. RittschofDuke University Marine LaboratoryUPR Sea Grant College Program Award Number NA16RG2278

University of Puerto Rico at AguadillaUS Fish and Wildlife Service Caribbean Islands NWR

Artemia Reference Center Gent, Belgium

Development of a pilot plant for the small scale production of Artemia biomass in Cabo Rojo, Puerto RicoGreat outreach and educational opportunityPilot project to study the possibility of the development of a production operation of a larger scaleGood way to raise funds for conservation projects and maintenance of the Centro Interpretativo de las Salinas de Cabo Rojo

Great training for undergraduate and graduate students30El primer cuadro y el segundo son iguales y miden 15 pies por 39.5 pies. El tercer cuadro mide 88 pies por 39.5 pies. Al verse unidas mide 118 pies de largo, 39.5 pies de ancho y 3 pies de profundo