Growth and pigmentation of marron (Cherax tenuimanus) fed a reference ration supplemented with the microalga Dunaliella salina

Aquaculture, 99 ( 199 1) 285-295 Elsevier Science Publishers B.V., Amsterdam

285

Growth and pigmentation of marron ( Cherax tenukmanus) fed a reference ration supplemented with the microalga Dunalielia salina T.R. Sommef”, N.M. Morrissybq2 and W.T. Potts” aWesternBiotechnologyLimited, 2-6 Railway Parade, Bayswater, W.A. 6053, Australia bDepartmento$ Fisheries, WesternAustralian Marine ResearchLaboratories, P.O. Box 20, North Beach, WA. 6020, Australia

(Accepted 28 March 1991)

ABSTRACT Sommer, T.R., Morrissy, N.M. and Potts, W.T., 1991. Gruwth and pigmentation of marron (Cherax tenuimanrrs) feC a reference diet supplemented with the microalga Dunaliella salina. AquacultureS 95: 285-295. The Western Austri;rlianfreshwater crayfish Cherax tenuimanus, called marron, loses dark exoskeletal colouration when fed artificial diets in indoor tanks, suggesting a carotenoid deficiency in these foods. In the present study the international Halifax crustaceau lifcrence diet (HEX CRD) was used as a control against tee ltments consisting of HFX CRD to which was added either a powder or fresh slurry of the carotemoid-rich microalga, Dunaliella salina. Juvenile marron housed in an indoor battery culture system were fed to the diets for 100 days. Thin layer chromatography of pigment extracts revealed that all three experimental groups contained astaxanthin, mono-esterified astaxanthin, diesteritied astaxanthilt and beta-carotene. However, canthaxanthin, adonirubin and esterified adonirubin were observed additionally in the algal treatment groups only. Pigment analyses of marron at 2% day inter& showca elevated levels of total carotenoid arid beta-carotene in thi algal treatment groups as compared to the control group. Marron cn the algal slurry treatment also showed a significantly higher growth rate.

INTRODUCTION

For aggressive species of freshwater crayfish and homarid lobsters, indoor battery systems (intensive individual housing in modular units) provide for controlled experimentation and have possible commercial application. Among the experimental parameters, supply of a formulated pelleted diet can be ensured as the sole source of nutrition by maintaining a “clean” battery (Mcr‘Present address: California Department of Water Resources, 325 1 S Street, Sacramento, CA 95816, USA. ‘To whom correspondence should be addressed.

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rissy, 1984). I-Iowever,exclusive use of pelleted feeds has led to progressive loss of exoskeletal pigmentation through successive moult cycles of the marran Chevax tenuimanus (Morrissy, 1984) and other species (Conklin et al., 1975; Huner and Meyers, 1979). Loss of shell colour detracts from acceptance of crayfish by commercial markets. This change suggests a dietary carotenoid deficiency. Crustaceans are incapable of de novo synthesis of carotenoids and astaxanthin is the primary carotenoid in freshwater crayfish (e.g. Wolfe and Cornwell, 19’65;Czeczuga, 1971) . Supply of plant material maintains crustacean shell pigmentation and may correct other nutritional deficiencies due to use of artificial diets, thereby improving growth rate (Huner, 1984; Narpaz and Schmalbach, 1986). D’Abramo et al. ( 1983) found that juvenile lobsters, Humarus americanus, were able to utilize a wide range of synthetic and natural carotenoids for shell pigmentatisn. Use of spec%c refined sources of carotenoids is desirable for incorporation into pelleted diets for nutritional research. IIowever, recent progress in improving growth performance on artificial diets has ireen made by including shrimp or crayfish extracts which contain carotenoids and essential growth factors, such as polyunsaturated fatty acids, and reduce loss of watersoluble vitamins and amino acids (Omara-Alwala et al., 1985; Bordner et al,, 1986). irnfortunately these crustacean products are unavailable, commercially, in Australia. However, Tanaka ( i9i8 ) found that inclusion of pigment extracts from the blue-green alga, Spirulina, in feed produced astaxanthin deposition in Penaeusjaponicus.For the present study, concentrates of Dunaliellasalincl (Theodoreso), a halotolerardtgreen alga, were available in Western Australia where the alga is cultured commercially in open ponds at salt lakes or works in Australia (Borowitzka and Borowitzka, 1988). A major characteristic of this spr;;ciesof alga in hypersaline ponds is mass:ve accumulation of beta-carotene. Beta-carotene is a biosynthetic precursor of astaxanthin in crustaceans (D’Abramo et al., 1983). METHQDSANDMATERIALS

Experimenral diets The Malifax Cmstacean Reference Diet (HFX CRD) (Table 1) was used

as a control diet and as a base for two treatment diets supplemented with either algal powder or algal slurry ingredients. RFX CRD was obtained as i /S” pellets from Marine Lobster Farms Ltd, P.O. Box 1028 Charlottetown, P&.1., Canada ClA 7M4 (CRD 86, Lot 86 0110 MLF). Initial development of this diet, based on crab protein, is described by Boghen et al. ( 1982). A recznt summary of the development of the diet appeared in Caste11et al 1989)) and preliminary results of use for Cherax tenuimanus have been described (Morrissy, 1989).

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TABLE 1 Composition of Halifax crustacean reference diet (HFX CRD) Ingredient’ Percent composition Crab protein concentrate Wheat gluten Corn starch Dextrin Celufil (non-nutritive bulk) Cod liver oil Corn oil Cholesterol Mineral mix (modified Bernhart-Tomarelli) Vitamin premix CRD Vitamin E (DL alpha tocopherol) Choline chloride (70%) IU or mg/kg of diet Thiamine- HCl

RiboflavZn Yicotinamide d-Biotin DLCa-pantothenate Pyridoxine*HCI Folic acid Menadtone sodturn btsulEiz Cyanocobalamin ( B Iz) Inositol Cholecalciferol (D,) {SSC000 NJ/g) Vitamin A acetate (500 000 NJ/g) L-ascorbic acid Butylated hydroxyanisole Butylated hydroxytoluene Para-amino benzoic acid Cehdiil

HFX CRD

40.0 5.0 15.0 5.0 17.8 6.0 3.0 1.0 4.0 2.0 0.2 1.0 64 144 520 1.6 286 48 19.4 16 s4 2540 340 51 000 1220 15.2 15.2 40.4

14 554

‘After Caste11et al. ( 1989).

The algal supplements were obtained from a commercial culture facility in Western Australia (Western Biotechnology Limited, 2-6 Railway Parade, Bayswater, W.A. 6053). Dunalielfa salina, cultivated In open ponds, was harvested as cell concentrates (Moulton et al., 1983a ). Part of the harvested slurry was spray &-ied in a Niro dryer to produce the algal powder and both forms were stored at - 20 “C for 2 months until pelletizatiun, described below. The control and treatment diets were prepared bv grinding HFX CRT9pellets to a powder. The algal ingredient for each treatment was added as ag_ proximately 10% by weight. The diets were rebound with 23% Kelgin algin ate (Kelco ) , gelled in 0.1% CaCl, after a cold exrlusion process using a 2 mm die, air dried, and stored at - 20°C until daily use. The original unbound

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HFX CRD was 93% DW. The control diet was 68% DW and the treatment diets containing the slurry and powder ingredients were 55% and 68% DW, respectively. Diets were fed at a daily rate of 5% DW per mean individual live body weight of marron in each experimental group. The total amount fed to each group was recalculated as required to account for changes due to growth, mortality, and removal of samples of marron for pigment tii&ysis.

Buttery witwe system The table-top battery system with 100 compartments ( 100~ 75 mm) at the Western Australian Marine Research Laboratories, Perth, has been previously described by Morrissy ( 1984) j Groundwater for the well-aerated, single pass ( 100 changes per day) water supply was micron-filtered and UV treated, to eliminate possible extraneous food supplements. The supply was heated to

22.5OC which is clot to the temperature of 24°C for maximum growth of 0 f year old marron (Morrissy, 1990). The battery compartments were rigourously cleaned every 2 days and then the marron were refed, as above. Exuviae were ieft to be-consumed;ecdyses and mortality were recorded daily. Fluorescent lights gave a photoperiod of 12L: 12D (L, 06.00-l 8.00 h) with the intensity of overhead lighting reduced by one layer of shadecloth to 0, l0.2 microeinsteins m-” s-l (LI- 185 meter, Lambda Instr. Corp., Nebraska). Marron Two hundred 0+ year old ( 1986-87 year-class) pondobred marron (released in mid-February at 0.06 g) of mean size 0.32 g (three random samples

of ten; mean size, 0.31,0.30 and 0.35 g, respectively) were transported from the Pemberton Hatchery (Department of Fisheries, Western Australia) to the Western Australian Marine Research Laboratories on 6 March 1987 and 99 individuals were placed into the battery. Experimental design

The experiment was conducted for 100 days commencing on 16 March 1987. Marron within each of the three experimental groups (each comprising 33 marron initially ) were weighed individually to 0.0 1 g, on days 50 and 100. Marron were sacrificed for pigment analysis on day 0 (n = 15 from stock), day 25 (n=8 from each group), day 50 (n=5), day 75 (11=3) and day 100 (y1=8 ) . At day 100 a sample ( n = 8 ) also was collected from the Pemberton stock pond. Samples ( 10 g ) of the stock algal supplements and the three diets were taken a? intervals for pigment analyses. Pigment values for marron are derived from 2-3 individuals, pooled for extraction, in the first half of the experiment and later, at a larger crayfish size, from l-2 individuals. Repli cates were derived from different pooled marron samples. Moult stage was

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289

not identified and this is iikely to have contributed to variability in pigment levels ( D’Abramo et al., 1983 ) :

Pigment extraction A/gal extracts. Carotenoids were extracted from the algal powder and slurry by suspending replicate samples in 90% acetone, homogenizing for 1 min using an Ika-Werk homogenizer, and then centrifuging for 10 min at 2 100 g leaving a coloumess precipitate (Moulton et al., 1987b).

Diets. Beta-carotene concentrations were estimated from approximately 5 g samples, ground into a homogenous powder. Replicate 0.5 g subsamples were suspended in 20 ml methanol and 10 ml acetone, then homogenized for 1 min. Concentrations of minor carotenoids, lutein and zeaxanthin, were determined on replicate 5.0 g samples rather than 0.5 g. Carotenoids were then extracted into toluene by adding 10 ml of the solvent, mixing by inversion for 3-5 min, followed by centrifugation for 10 min at 2 100 g. Traces of acetone in the supematant were removed using three successive washes with deionised water.

Marron. All marron were stored at - 20’ @ until pigment analysis. stomach and gut were dissL?ctedfrom all marron to avoid contamination with pigments remaining in the digestive tract. Samples were then weighed and homogenized fqr f min in 10 ml acetone. The pigments were extracted as described for the diet samples except that 5 ml of toluene was used.

Pigmeirt analysis Thin-layer chromatography.Major carotenoids in extracts from day 100 marron were identified chromatographically by comparison with authentic standards (Troche, Basle, Switzerland) on TLC (Whatman Silica Gel 6OA, 250 pm) in hexanelethyl acetate/ethanol (N/49/ 1).

Spectrophotometric analysis. Total carotenoid concentration of the toluene extracts of the animals was estimated by scanning the visible spectrum using a Varian DMS 100 spectrophotometer and determining the pe& abtiorbaudc, around 474 nm (Sommer et al., 1991). An extinction coefficient (E:Tm ) 01 1900 was used for calculation of carotenoid concentration. This coefficienl was determined using a synthetic astaxanthin standard (Roche Carophyll Pink) in toluene; the presence of other minor carotenoids may have caused some error. In acetone extracts of algal powder and slurry, the total levels oi carotenoid were calculated f ,:om the peak absorbance (452 nm) using an ex. tinction coefficient ( EjT,n ) of 2590.

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HPLC. The major pigments present in marron were esters of astaxanthin,

which were not quantified because of the difficulties in separating esterified forms of carotenoids. However, the concentrations of the non-esterified carotenoids beta-carotene, lutein and zeaxanthin in marron were determined on the toluene extracts using a Waters HPLC system described in Sommer et al. ( 199 1) . Toluene extracts of feed and acetone extracts of algae were analysed similarly. Separations on marron extracts were performed using a Zorbax ODS column and acetonitrile/dichloromethane/methanol (79/20/0.2) for the mobile phase at 1.5 ml min- *. In feed and algal samples, lutein and zeaxanthin were run using a mobile phase of acetonitrile/dichloromethane/methano1 (U/15/2). Pigment 1evelsTMnarron over the course of the experiment were tested for significant trends by standard regression analysis. Sizes of marron were examined for differences in growth rate by analysis of variance. RESULTS

Pigment concentration of diets No carotenoids were detected in the HFX CRD control diet, while betacarotene was the major carotenoid component in the treatment diets along with less than 5% lutein and zeaxanthin (Table 2). The levels of beta-carotene in the algal powder and slurry supplemented HFX CRD remained constant over the experiment. Alarron pigment composition TLC analyses on the marron at day 100 revealed that all study groups contained astaxanthin, astaxanthin monoester, astaxanthin diester and beta-carTABLE 2 Carotenoid composition of algal pawder, algal slurry and experimental diets ..-* Algal supplements ( mg!g) Average concentrations

Powder Slurry Diets’ @g/g) Control Algal meal Algal s:urq ‘Pooled sample of days 0.5 1 and 100. N.D., not detectable.

-

Lutein

Zeaxanthin

Beta-carotene

0.18 0.17

0.22 0.18

15.0 4.0

N.D. 12.75 14.46

N.D. 16.0 16.8

N.D. 1280 646

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291

90 80

70 i 80

60 1 4o I 30 t 20 I-

10 0 t 0

q ALGAL

CONTROL

60 DAY

26 MEW

76

100

q ALGALSLURRY t%dPOND GROWN

Fig. 1. Mean total carotenoid levels in battery cultured marron fed control, algal powder (meal) or algal slurry diets and pond-grown marron. Standard errors were 3.0- 10.9.

6

0 Cl

CONTROL

26 lIza ALGAL!EAL

60 DAY

76

IBI ALGALSLURRY

100 POND GROFN

Fig. 2. Mean beta-carotene levels in battery cultured marron fed control, algal powder (meal) or algal slurry diets and pond-grown marron. Standard errors were O-3-2.4.

T.R. SOMMER ET AL.

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Yiean weights and survival of marron over 100 days HFX CRD control diet

Mean marron weight (g) + s.d. At day 50 (n) At day 100 (n) Survival (%) 0 -25 days 26-50 51-75 76-100 Total deaths

0.46f0.14 (24) 0.64 f 0.2 1 (16)

91 96 100 100 4

HFX CRD + 10% algal

HFX CRD f 10% algal

pi?WCkt

Sh.llQ

0.44kO.14 (23) oxi3ao.17 (15)

0.67 + 0.28 (25) 0.87 +,0.32 (16)

100

100

92 100 100 2

96 100 94 2

otene. Lutein and zeaxanthin were undetected. Canthaxanthin, adonirubin and esterified adonirubin were found additionally in the algal slurry and algal powder groups, but not in control and pond-grown groups. Results of pigments analyses for the different groups of marron are summarized in Figs. 1 and 2. There was a significant decrease (PC 0.05 ) in total carotenoid levels in the animals fed the pigment-deficient control diet (Fig. 1). Moreover, the pigment levels at day 100 in the control group were very similar to pond-grown animals at the Pemberton Hatchery. The results for the algal powder and algal slurry were highly variable over the course of the study, although it is clear that these animals maintained high levels of total carotenoid (Fig. 1) . Beta-carotene showed a similar trend, with markedly higher levels in the groups fed microalgae (Fig. 2). Marrorngrowthand survival Survival was high in all groups (Table 3 ) , as is usual in battery experiments (Morrissy, 1984). Mortality during the first 25 days in the control group was probably due to stress or injury during transport from the Pemberton hatchery. At days 50 and 100 there was no significant difference in mean crayfish weights between the control and the algal powder treatment groups. However, the algal slurry treatment group was sign.ificantlylarger (PcO.05) at both sampling dates (Table 3 ) . DISCUSSION

The original aim of this study was to test the use of algal products in feed as a means of correcting the abnormal pale pigmentation which is observed

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to develop in marron fed artificial diets. Addition of carotenoid-rich alga] ingredients to such a diet did indeed result in superior levels oftotal carotenoid and beta-carotene; However, the carotenoid levels did not necessarily reflect the perceived shell colouration of the marron groups studied. Control animals were a translucent blue colour, as compared to a dark brown colour in algal slurry and powder groups, and black in pond-grown individttals.. Yet the total carotenoid level at day 100 in pond-grown individuals was fairly similar to that of the control animals and much lower than those of groups fed algae (Fig. 1). The visual differences may reflect the way in which carotenoids are incorporated in?o complexes or the presence of another pigmenting group. Carotene proteins may alter to provide appropriate shell colouration in response to substrate colour (Chessman et al., 1967). Those carotenoid pigments present in the Lhmakda supplements and responsible for carotenoid accumulation in marron remain unidentified. The biosynthetic pathway desrGbed by Goodwin ( 1984) indicates that oxygenated carotenoids such as zeaxanthin would probably be a more effective pigment source than beta-carotene. Based on the very high levels of carotenoids supplied in the present diets, zeaxanthin could have contributed most or all of the carotenoid observed in the marron at, the end of the study. Similar dietary levels of oxygenated carotenoids are e tive for lobsters (D’Abramo et al., 1983 ). However, the high levels of beta-carotene observed in the experimental groups, by contrast to no detectable zeaxanthin or lutein, indicate that beta-carotene is probably absorbed by marron and may be converted to astaxanthin at low efficacy. The low efficacy of beta-carotene conversion to astaxanthin with crustaceans relative to other carotenoids such as zeaxanthin and canthaxanthin has been previously noted by D’Abramo et al. ( 1983 ) . The presence of a growth stimulating factor in the alga was indicated by the higher growth of marron fed HFX CRD supplemented with algal slurry. The factors responsible did not appear to be the carotenoids, which were supplied at a higher level in the algal powder supplemented diet. This factor may be heat-labile since the algal powder was heat-dried. Other studies comparing growth on fresh, shock frozen, dried or conventionally frozen food have suggested an unstable, fat-soluble, growth stimulating factor (Fluchter, 1982 1. The factor more likely serves as a micronutrient rather than a (water-soluble) feed attractant. Significant levels of 18C : 3 : ai3 and 20C : 5 : 0~3fatty acids have been measured in Dunalielfaby Tornabene et al. ( 1980) and Ben-Amotz et al. ( 1985 ) , but others have not detected 20C: 5 : ~3 (Fried et al., 1982 ). Trace amounts of these polyunsaturated fatty acids have been increasingly recogsised as essential in crustacean diets, particularly those of the 2OC: 5 : ~3 and 22C: 6: ~3 forms (D’Abramo et al., 1980; Castell, 1982; Kanazawa et al., 1985). Polyunsaturated fatty acids are susceptible to oxidation during feed ma.nufacture and storage. In addition, Tornabene et al. ( 1980) and Evans

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and Kates ( 1984) reported a complex array of other compounds in the lipid fraction from Dunaliella.Similar compounds such as sterols and phytosterols have been shown to be required by freshwater crayfish (D’Abramo et al., 1985). The commercial value of Dunaliellaas a pigment source and growth stimulant remains unproven since the current study tested relatively high levels of algal supplements ( 10%). Other commercially available pigment sources such as astaxanthin are effective for crustaceans at Mach lnwerQiettaqlevels. Astaxanthin-rich crayfish extracts have also been shown to stimulate both growth and pigmentation (Bordner et al., 1986). Further research is needed to examine the relative value of reduced algal supplementation rates. ACKNOWLEDGEMENTS

Chris Bird provided technical support in operating the experimental marron system. The assistance of Frances B’Souza, Riekie Fraietta and Michele Burford of Western Biotechnology Limited with the carotenoid analyses is gratefully acknowledged. Dr. Nick Caputi of the Western Australian Marine Research Laboratories provided the statistical analyses.

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