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Enrichment of Chironomid Larvae withAscorbic AcidAli Hamidoghlia, Bahram Falahatkara, Majidreza Khoshkholgha &Ahad Sahragardb

a Fisheries Department, Faculty of Natural Resources, University ofGuilan, Sowmeh Sara, Guilan, Iranb Plant Protections Department, Faculty of Agriculture, University ofGuilan, Rasht, Guilan, IranPublished online: 21 Aug 2014.

To cite this article: Ali Hamidoghli, Bahram Falahatkar, Majidreza Khoshkholgh & Ahad Sahragard(2014) Enrichment of Chironomid Larvae with Ascorbic Acid, Journal of Applied Aquaculture, 26:3,216-224, DOI: 10.1080/10454438.2014.934165

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Journal of Applied Aquaculture, 26:216–224, 2014Copyright © Taylor & Francis Group, LLCISSN: 1045-4438 print/1545-0805 onlineDOI: 10.1080/10454438.2014.934165

Enrichment of Chironomid Larvae withAscorbic Acid

ALI HAMIDOGHLI1, BAHRAM FALAHATKAR1,MAJIDREZA KHOSHKHOLGH1, and AHAD SAHRAGARD2

1Fisheries Department, Faculty of Natural Resources, University of Guilan, Sowmeh Sara,Guilan, Iran

2Plant Protections Department, Faculty of Agriculture, University of Guilan, Rasht,Guilan, Iran

Due to the importance of ascorbic acid (AA) in fish larval develop-ment and performance, chironomid (midge) larva were enhancedwith AA using chicken manure mixed thoroughly with a sta-ble form of ascorbic acid (L-ascorbyl-2-polyphosphate). AA wasused in three doses (0, 100, and 1,000 mg/kg) each with threereplicates. The chironomid culture was maintained for 18 days.There were no significant differences (P > 0.05) in averagebiomass and growth of chironomid larva among the treatments.Spectrophotometric analysis of AA content of chironomids demon-strated that 1,000 mg/kg produced larvae with significantly (P <

0.05) higher AA content (779.86 ± 31.81 µg/g) than 0 (74.2 ±15.45 µg/g) or 100 mg/kg (325.03 ± 116.61 µg/g). This resultindicated that using chicken manure as substrate mixed by highdoses of stable form of AA is an efficient method of enriching andenhancing the nutritional value of this kind of live feed.

KEYWORDS Bloodworm, mass production, ascorbic acid,enrichment

Address correspondence to Bahram Falahatkar, Fisheries Department, Faculty of NaturalResources, University of Guilan, P. O. Box 1144, Sowmeh Sara, Guilan 1144, Iran. E-mail:falahatkar@guilan.ac.ir

Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/wjaa.

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Vitamin C Enrichment in Bloodworm 217

INTRODUCTION

Unsuccessful attempts at fish hatcheries have been the result of poor larvaland juvenile survival because of their inability or failure to accept artificialdiets. Therefore, a successful hatchery production is largely dependent onthe availability of suitable live feed (Lim et al. 2003). Chironomids areone of the most distributed organisms in freshwater, with representativesin both terrestrial and marine environments. They have the ability to tol-erate a wide range of physico-chemical conditions. These insects have ahigh reproductive capacity, each female lays one gelatinous tear like matrixthat contains approximately 2000 eggs (Armitage et al. 1995). Chironomidae(midge) larvae are rich in protein (approximately 55.7% in dry weight) andmost essential amino acids (Bogut et al. 2007) and are an important feed formany fishes and cultured invertebrates (Tidwell et al. 1997; Volkman et al.2004). Mass culture of chironomids for commercial rearing and/or fish stock-ing programs has developed in several countries, including the United States(McLarney et al. 1974), China (Shaw & Mark 1980), Thailand (Armitage et al.1995), and Russia (Vedrasco et al. 2002).

Vitamin C or ascorbic acid (AA) insufficiency impairs several biologicalprocesses. AA-deficient fish are more susceptible to stress (Ren et al. 2010)and suffer from impaired immune systems (Dabrowski 2001; Xie et al. 2006;Azad et al. 2007). The dietary requirements of AA for normal physiologi-cal functions in animals, including fish, has been widely studied (Mæland& Waagbø 1998; Wang et al. 2006; Darias et al. 2011). Most fish do nothave the L-gulono-1, 4-lactone oxidize enzyme that converts glucose toAA (Dabrowski 1990), and thus must obtain AA from exogenous sources(Dabrowski 2001).

Generally, midge larvae contain insufficient amounts of vitamin C tosupport good larval fish growth (Banjjo et al. 2006). Enrichment of live preysuch as Artemia with AA has been reported to improve fish larval perfor-mance (Merchie et al. 1995; Monroig et al. 2007), but no data are availablefor AA enrichment of chironomids. The aim of the present study was to eval-uate the indoor production of chironomid larva fed chicken manure withrespect to their enrichment with different levels of AA to meet the needs offish hatcheries.

MATERIALS AND METHODS

Wild midge eggs, larvae, and pupae were collected in 1 kg samples of mudfrom a fish pond located near the Faculty of Natural Resources, Universityof Guilan (Sowmeh Sara, Guilan, Iran). The insects and the sediment weretransferred to an indoor concrete rectangular basin (120 × 60 × 40 cm)filled with 140 L of fresh water. To this mess was added 2 kg of chicken

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218 A. Hamidoghli et al.

manure, and the basin filled with dechlorinated tap water (approximately upto 20 cm depth). Aeration was carried out with an air compressor attachedto an air-stone, and the water temperature was adjusted to 25 ± 1.2◦C witha thermostatically controlled heater. The basin was located in a room witha window and enough space (10 m3) so that adult midges hatching fromthe basin could readily perform their mating flights under a natural daylightcycle of 12L:12D (Armitage et al. 1995). The mating flight was performeddirectly on top of the basin, and after impregnation female midges laid theiregg masses on the surface of the water, where they attached by a gelatinousanchor to the edge and corners of the basin.

These egg masses (10 individuals) were detached from the basin sidesby forceps and transferred to circular white plastic rearing tanks (41 cmdiameter and 15 cm height) filled with 10 cm of dechlorinated tap water(13.2 L). Sifted chicken manure (90 g) for larval feeding was added toeach tank (687 g m−2). Water temperature, oxygen, and light were regu-lated as for the basin. Culture conditions during the experiment are given inTable 1.

A stable form of ascorbic acid (L-ascorbyl-2-polyphosphate; Stay-C,Roche, Basel, Switzerland) was used to enrich the substrate. Concentrationsof 0 (C0), 100 (C100), and 1,000 mg (C1000) of AA were mixed thoroughly with1 kg of sifted chicken manure and then were added to the plastic rearingtanks, three tanks per treatment. On day 2–3 after hatching, the larvae leftthe egg mass, burrowed into the organic matter (Koehler 2003), and fed onthe enriched substrate.

Chironomids were harvested when most larvae reached 1–1.5 cm inlength, indicated by the first observation of pupa on water surface. Full wormsize was achieved in 18 days after the introduction of egg masses at 25◦C. Ascoop net with 1 mm mesh size was used to harvest the chironomid larva.Larvae buried in the manure were collected in the net and washed into freshwater for cleaning. Total harvested larval biomass was transferred to a pieceof filter paper with forceps and weighed to the nearest 0.01 mg. Averagegrowth rate (AGR) and biomass (B) per m2 of substrate were calculated foreach group according to these formulae:

TABLE 1 Culture conditions of chironomid larva during theexperimental period (18 days for each phase of production).

Parameter Mean ± SE

pH 6.8 ± 0.5Water temperature (◦C) 25 ± 1.2Air temperature (◦C) 24 ± 1.0Lighting regime 12L:12DLight intensity (lux) 700 ± 46.0

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Vitamin C Enrichment in Bloodworm 219

AGR(mg day−1

) = Total wet weight gain/days

B(g m−2

) = Total biomass/tank surface area

From each replicate 25 specimens were removed randomly for lengthand weight measurements to the nearest 0.1 mm and 0.0001 g, respectively.A 2 g sample of larvae was taken from each tank and stored at –80◦C for totalascorbic acid analysis, by the DNPH (2,4-dinitrophenylhydrazine) methodaccording to Dabrowski and Hinterleitner (1989). Briefly, 0.2 g of samplewas weighed, 2 ml precipitation solution (17.9 ml HClO4, 25 g TCA and0.4 g EDTA) was added and homogenized on ice (about 20–30 s) using ahigh speed homogenizer (IKA, Staufen, Germany). Then samples were cen-trifuged for 30 min in 12,600 g at 4◦C. Stock solution and standards (10,25, 50, 100, 250 and 500 ppm) were made for determination of standardcurve (Figure 1). Blank solution (only TCA), standards and sample super-natants were added to test tubes. Tubes were duplicated for repetition ofthe experiment. Then 25 µL of 0.2% dichlorophenolindophenol (DCIP) wasadded to test tubes, vortexed, incubated at room temperature for 20 minand, 25 µL distilled water was added. After that, 250 µL of 2% Thioureain 5% meta-phosphoric acid and 250 µL of 2% DNPH in 12M H2SO4 wereadded to the tubes, vortexed and incubated at 60◦C for three hours in waterbath. Incubated tubes were placed on ice water to cool down, caps wereremoved and 500 µL ice-cold 18M H2SO4 was added, recapped and then vor-texed. Standard absorbance was read at 524 nm and samples were scannedfrom 400–600 nm using a compact double-beam UV-1800 spectrophotometer(Shimadzu, Kyoto, Japan).

All data were examined for normality and homogeneity of variancesusing the Kolmogorov-Smirnov and Levene’s tests, respectively. To detect

FIGURE 1 Calibration curve for standards of AA at 524 nm. Equation (y) and R-square (R2)values are displayed on the chart.

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220 A. Hamidoghli et al.

differences between groups in growth, biomass, and the amount of AA inlarvae, data were analyzed by one-way ANOVA using SPSS 17.0 software(SPSS, Chicago, IL, USA). The null hypothesis was that there would be nosignificant differences (P > 0.05) in production biomass and total ascorbicacid of chironomids enriched with different levels of AA. When significantdifferences were detected, a post hoc Tukey’s test was used for multiplecomparisons. Data are presented in mean ± standard error (SE).

RESULTS

After 18 days of culture, chironomids larvae reached a suitable size for har-vest. The average total biomass of chironomid larvae was 10.06 ± 0.04,9.72 ± 0.39, and 10.22 ± 0.31 g for the C0, C100, and C1000 groups respec-tively, with no significant differences among groups (Table 2; F = 0.102,df = 2, P = 0.904). Average weights and lengths of individual larvae fromeach tank were 2.64 ± 0.07 mg and 10.22 ± 0.45 mm in C0, 2.61 ± 0.01 mgand 9.56 ± 0.24 mm in C100, and 2.72 ± 0.03 mg and 10.54 ± 0.14 mm inC1000 respectively. AGR was higher in C1000 (0.153 ± 0.002 mg d−1) amongthe treatments, but with no significant differences.

Total AA concentration of chironomid larvae in the different groups isillustrated in Figure 2. The average total AA concentration of C1000 group(779.86 ± 0.3 µg/g) was significantly higher than C0 and C100 groups (P =0.001). But the average total AA concentrations of C100 (325.03 ± 0.4 µg/g)and C0 (74.2 ± 0.0 µg/g) were not significantly different.

DISCUSSION

Chironomid larvae can be successfully cultured in circular plastic tanks. Suchculture for fish feed has been reported previously, and many authors haveindicated that chicken manure is the best sediment for chironomid culture.Alston and Dendy (1974) cultured chironomidae larvae in circular plasticpools, harvesting a maximum dry weight of 17 g lavae/m2 of surface with224 g of chicken manure per m2. Also, Shaw and Mark (1980) reportedan outdoor culture of chironomids in Hong Kong using 2,130 g of chickenmanure per m2, yielding about 20.7 g of larvae per m2 per cycle of 50 days.Our method required 687 g of chicken manure per m2 of tank area for max-imum production, and the biomass of larvae produced per m2 was 76.79 ±1.08 g (C0), 74.19 ± 1.7 g (C100), and 77.98 ± 2.74 g (C1000).

The grow-out period for midge larvae is related to temperature andculture conditions. Koehler (2003) reported that the larval stage can last fromless than 2 to 7 weeks, depending on water temperature. In our experiment,18 days at 25◦C was sufficient for larval growth. Credland (1973) stated that at24◦C Chironomus riparius eggs hatched in about 36 h, and the adults began

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TAB

LE2

Ave

rage

bio

mas

s(g

),in

div

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g),in

div

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lengt

h(m

m),

aver

age

grow

thra

te(A

GR),

bio

mas

s(B

)per

m2

ofsu

bst

rate

and

num

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ofpro

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dch

ironom

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thro

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18day

sofpro

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with

diffe

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ofas

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icac

id.Res

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thre

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.

Asc

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idle

vel(m

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−1)

Bio

mas

s(g

)In

div

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wei

ght(m

g)In

div

idual

lengt

h(m

m)

AG

R(m

gday

−1)

Num

ber

of

larv

ae/

tank

B(g

m−2

)

010

.06

±0.

042.

64±

0.07

10.2

2±

0.45

0.14

6±

0.00

438

12±

95.7

980

.09

±1.

1210

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72±

0.39

2.61

±0.

019.

56±

0.24

0.14

5±

0.00

037

11±

143.

5577

.38

±1.

7710

0010

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±1.

302.

72±

0.03

10.5

4±

0.14

0.15

3±

0.00

237

44±

471.

181

.34

±2.

86

AG

R(m

gday

−1)

=to

talw

etw

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tga

in/to

talex

per

imen

talday

s.B

(gm

−2)

=to

talbio

mas

s/ta

nk

surf

ace

area

.

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222 A. Hamidoghli et al.

b

b

a

0

100

200

300

400

500

600

700

800

900

C0 C100 C1000

Asc

orb

ic a

cid

co

nte

nt

(ug

/g)

Ascorbic acid levels (mg/kg)

FIGURE 2 Total ascorbic acid (mean ± S.D.) concentration (µg/g) in chironomid larvaenriched with 0, 100, and 1000 mg (C0, C100, and C1000) of AA per kg of manure. Columns withdifferent letters are significantly different (P < 0.05).

to emerge after another 28–30 days. Development time of C. riparius larvareared in aquaria at 26◦C was 22 days, and developmental rate increasedwith increasing temperature (Sahragard & Rafatifard 2010).

Our results showed that AA content in chironomids can be increasedusing this enrichment method. Though higher amounts of L-ascorbyl-2-phosphate to the sediment resulted in higher concentrations of AA inchironomidae larvae, these differences did not significantly affect the growthperformance of chironomids during the 18 days period of the experiment.No published literature was found on the effects of vitamin C on growthperformance of midge larvae. Generally, insects are incapable of L-ascorbicacid synthesis (Dutta Gupta et al. 1972), but perhaps their AA require-ments are so low that even severe shortages of this vitamin in their mediawould not influence growth performance. Further studies in this subject areneeded.

Spectrophotometric results indicated that unenriched midges had lowerquantities of AA. Chironomidae larvae are bottom feeders, feeding on sedi-ment that has no or insufficient vitamin C. The 74.2 µg/g AA in the controlgroup (C0) probably would not satisfy the requirements of fish for this nutri-ent: 50–500 mg AA/kg diet (NRC 2011). The average AA value of the C1000

group (779.86 µg/g) could possibly satisfy larval nutritional requirements ofthis vitamin.

ACKNOWLEDGMENTS

The authors are indebted to Dr. J. Fattahi Moghadam, M. Kia Eshkevariyan,and M. Alipour at the Iran Citrus Research Institute for their assistance invitamin C analysis. Special thanks to Dr. Y. Hamidoghli and S. R. Akhavanfor their helpful advice during the project.

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Vitamin C Enrichment in Bloodworm 223

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