Aquaculture Nutrition 1997 3; 55–63
Influence of squid extracts on the triggering of secondaryvitellogenesis in Penaeus vannamei R. MENDOZA & A. REVOL Universidad Autonama de Nuevo Léon, Facultad de Ciencias Biológicas, San Nicolás de los Garza, Nuevo Léon, Mexico C. FAUVEL IFREMER DEVA-SUD, Palavas – les Flots, France J. PATROIS IFREMER – BREST, Plouzané, France J.-C. GUILLAUME IFREMER TAHITI, Taravao-Tahiti, French Polynesia are a precocious maturation and a reduced period between spawnings, but it also leads to other physiological anomalies, due to the removal of an organ producing and stocking a number of A series of five experiments were carried out to find an alterna- hormones controlling moulting, carbohydrate metabolism, tive to eyestalk ablation for inducing and controlling vitellogen- cardiac frequency, pigmentation, etc. (Browdy 1992). Among the esis in penaeid shrimps. Several extracts from squid were tested main drawbacks attributed to this operation, provoking a sudden as supplements to a basal diet. Polar components of hydro- mobilization of energy resources directed towards reproduction, alcohol (ethanol/dicloromethane/water, 2:2:1:8) soluble and lipid are: the scarce mating rate of eyestalk-ablated females (Browdy squid fractions (Bligh & Dyer), when incorporated in formulated 1992), the deterioration with time of spawning quality feed at low doses, trigger secondary vitellogenesis in 15–35 g (Emmerson 1983) and irregularity of yolk deposition in oocytes female Penaeus vannamei, showing maturations of the same (Anilkumar & Adiyodi 1980).
order of magnitude as the eyestalk-ablated controls.
On the other hand, some authors (Brown et al. 1979; Achievement of vitellogenesis was estimated by a homologous Chamberlain & Lawrence 1981a; Tan-Fermin 1991) have ELISA-vitellogenin test. Even though the nature of the active reported that eyestalk-ablated females show a regular ovarian molecules was not completely elucidated, the results obtained development and that they produce good-quality larvae in indicate that they may probably be steroid-like molecules of reasonable periods of time. Certainly there is a great demand for cephalopods, acting in a heterologous way.
energy associated with multiple spawning during a short period of time: this implies new and high nutritional requirements that KEY WORDS: feed, induction, shrimp, squid, vitellogenesis must be covered by feeding and/or by body reserves of the animal. The commercial operations rely on fresh food in a Received 21 February 1996, accepted 25 June 1996 proportion varying from 20% to 50% of the maturation diets in Correspondence: Dr Roberto Mendoza, Universidad Autonoma de Nuevo order to obtain successful maturations (Bray & Lawrence 1992).
León, Facultad de Ciencias Biológicas. Apartado postal F-56, CiudadUniversitaria, San Nicolás de los Garza, Nuevo León, CP 66450, Mexico Among the fresh feed most commonly fed to the adults, molluscs and particularly squid have been used to promote maturation and enhance egg quality, as complement in semipurified diets or incorporated in these (AQUACOP 1977; Lumare 1986). Some of these studies have concluded the superiority of squid compared Eyestalk ablation is the only endocrinological control practised with other animals (Chamberlain & Lawrence 1981b; Rodriguez- on penaeid shrimp to stimulate the onset of vitellogenesis. This Marín et al. 1986). In addition, the positive effect of squid on method allows the triggering and development of secondary development of vitellogenesis seems to have a wide range of vitellogenesis in species in which maturation cannot be obtained zoological specific action, as has been demonstrated in Pagrus in captivity. At present most of the commercial production of major (Luquet & Watanabe 1986).
postlarvae relies on this technique, which enables producers to From these different points emerges the need for an alternative coordinate maturation, artificial insemination and spawning, to to the practice of eyestalk ablation, to stimulate and control vitel- adapt the production to a regular schedule.
logenesis in order to allow a better management of the brood- The immediate consequences of unilateral eyestalk ablation stock. For this purpose, a series of experiments was carried out Effects on Atlantic Salmon fed what starch 1997 Blackwell Science Ltd R. Mendoza et al. directed at elucidating the effect and nature of different squid provided to assure oxygen saturation. Salinity remained around fractions on triggering secondary vitellogenesis in Penaeus 35 g L , and the pH was near to 8.2. A 14:10 h light/dark artificial photoperiod was maintained.
Material and methods
Vitellogenin determination: Haemolymphatic vitellogenin concentration was determined by a homologous vitellogenin All the experiments were performed following the same protocol.
immunoassay (ELISA), according to the method developed by Different fractions extracted from squid were incorporated into a Mendoza et al. (1993), the principle of which is the following.
basal diet (Table 1) in amounts corresponding to 100 g kg Purified vitellogenin (VTG) is first immobilized in a solid phase, freeze-dried squid. Care was taken to maintain constant the lipid then free VTG contained in samples or standards is added and protein components as well as the energy value. Animals together with a polyclonal antibody (anti-VTG). The free VTG were fed each diet, twice a day at 3% of the biomass during a 15- will then compete with coated VTG for the binding sites of the day experimental period.
antibody, thus preventing a certain fraction of this antibody from Animals: Adult Penaeus vannamei Boone originally from Tahiti being immobilized. The amount of antibody is measured in a were maintained in captivity at the facilities of the IFREMER subsequent step by an enzyme-labelled second antibody. The Center at Brest, France and Tahiti, French Polynesia, where the enzyme activity detected is inversely related to the VTG concen- experiments took place. Only those females not yet in vitellogen- tration in the sample. The assay is calibrated using dilutions of esis were selected. The animals were tagged individually with a standard VTG as a competitor.
coloured silicon ring around the eyestalk, then they were randomly Quantification of vitellogenin was performed on individuals in allotted to the experimental tanks at a density of 10 females per intermolt (stage ‘C') in order to avoid haemolymphatic variations tank. The assignment of the different treatments (diets) and the which might influence the VTG concentration. VTG was tanks was done at random. Unilaterally eyestalk-ablated females measured before and after each trial and the percentage of (positive controls) were maintained in separate tanks.
females entering secondary vitellogenesis was determined.
Experimental conditions: The tanks (3 m ) were supplied with Experimental design: To study the effect of squid fractions, sea water pumped through a sand filter and thermoregulated to randomized screening experiments (experiments 1 and 2) were reach a constant temperature of 28°C. A water-renewal rate of performed. Then for subsequent experiments (experiments 3, 4 was maintained and a supplementary aeration was and 5) a randomized-block design with two replicates was estab- lished. As replicates were carried out at different times they were Table 1 Maturation basal diet
considered as blocks. Comparisons of the mean concentrations of different treatments were accomplished by ANOVA and Duncan's new multiple range test (Steel & Torrie 1980), additionally a χ2test was applied to assess the difference between proportions of Fish protein hydrolysate individuals that responded to the fractions.
Experiment 1 Gelatinized potato starch Three main biochemical groups obtained from squid which might potentially be implicated in the promotion of secondary vitello- genesis were tested. The three different classes were separated in a first approach by Blight and Dyer's lipid extraction technique from freeze-dried whole squid, using a mixture of ethanol/dichloromethane/water (2:2:1.8) (modified by Beninger aVitamin mix, per kg: vitamin A palmitate, 200 000 IU; vitamin D , 200 000 IU; α-tocopherol acetate, 3.75 g; menadione bisulphite, *Only those females with a vitellogenin concentration above a threshold 200 mg; thiamine, 0.45 g; riboflavin, 2.5 g; Ca pantothenate, 5 g; pyridoxine, 0.4 g; niacin, 10 g; folic acid, 0.43 g; biotin, 0.5 g; were considered. This level was defined taking into vitamin B , 1 mg; meso-inositol, 20 g; choline chloride, 37.5 g.
account the results reported by Quackenbush (1989) with P. vannamei bCod liver oil, 38 mL kg–1; soybean lecithin, 19 g kg–1.
and those that have found with P. indicus (Mendoza & Fauvel 1989).
1997 Blackwell Science Ltd Aquaculture Nutrition 3; 55–63
Induction of shrimp vitellogenesis by squid extracts 1982); the fractions obtained were: a lipid fraction (LIP), a Experiment 3 hydro-alcohol soluble fraction (HAS) and a protein fraction For this experiment, both the HAS and the lipid fractions were (Proteins-B&D). Due to the denaturation of the protein fraction refractionated according to their polarity. This was accomplished we chose to include also squid proteins obtained by precipitation by partition chromatography with butanol/water (1:2) according with ammonium sulphate (AS-Proteins). Soluble proteins from to Mangold (1984). The butanol extracts were combined, washed freeze-dried squid were first extracted in a phosphate buffer twice with Na CO aqueous solution 20 g L before being evapo- M, pH 7.4) and then were precip- rated. The residues were partitioned in methanol/hexane (1:1).
itated with a saturated solution of ammonium sulphate (80% final The methanol extracts (polar fraction) and the residuals (aqueous concentration). Precipitated proteins were dialysed against the phase and hexane phase) were incorporated in the basal feed.
same buffer and were continuously monitored with BaC1 to indicate any residues of ammonium sulphate.
Experiment 4 Each fraction was included in a previously tested basal diet (Table 1) that had not shown any positive effect on the onset of In this case, the polar fractions of HAS and lipids were obtained P. vannamei vitellogenesis. In this experiment, six treatments based on the eleutropic series for the extraction of steroids were compared in a completely randomized experimental design (Makin 1984). The HAS and lipid fractions were saponified and with no replicates, just to identify the most interesting fractions.
the non-saponifiable phases were extracted first with chloroform The treatments are those specified in Table 2.
and then with ethylacetate. The fractions obtained from the last solvent were pooled and dried. The rest of the fractions (acetone Experiment 2 non-soluble fraction, non-saponifiable fraction in chloroform) were pooled to constitute the ‘residual fraction'. Treatments were Due to difficulties in performing the ammonium sulphate precipi- constituted as in the former experiment (Table 2).
tation on a large scale, proteins and peptides were obtained by Once identified, fractions showing positive effects were used precipitation with water and ethanol following the technique in an additional experiment aimed to further elucidate their described by Roberts et al. (1980), which allowed the separation of insoluble or precipitated proteins (PP) from soluble peptides and proteins (SP). In this experiment the fractions were extracted Experiment 5 from freeze-dried squid, Loligo sp., whereas the fresh squid used was Nototodarus sloani Gray. A totally random design was The HAS fraction was partitioned into subfractions by established with two replicates per treatment.
chromatography on Sephadex G-25 (Pharmacia). The sequential Table 2 Description of the experimental series and treatments
1Treatments are coded as follows:BD: BD + lipids.
BD + hydro-alcohol soluble fraction after Bligh & Dyer's extraction.
BD + protein fraction after Bligh & Dyer's extraction.
BD + protein fraction after ammonium sulphate precipitation.
BD + precipitated peptides after precipitation by the technique of Roberts et al. (1980).
BD + soluble peptides after precipitation by the technique of Roberts et al. (1980).
freeze-dried squid (Loligo sp.).
fresh squid (Nototodarus sloani).
BD + polar fraction from the HAS.
BD + residual fraction from the HAS.
BD + polar fraction from the LIP.
BD + residual fraction from the LIP.
BD + non-polar fraction from the HAS.
BD + reconstituted HAS fraction (HAS-polar fraction & HAS-non-polar fraction).
1997 Blackwell Science Ltd Aquaculture Nutrition 3; 55–63
R. Mendoza et al. fractionating was carried out in an 80 3 2.5 cm column.
The following elution sequence was used: chloroform/ methanol (19:1), chloroform/methanol/acetic acid (19:1:4), Experiment 1: In the first screening experiment no significant chloroform/ methanol/acetic acid (9:1:3), methanol/water (1:1), differences regarding the VTG concentration of individuals were water. The lipid components were obtained in the first two noted (Table 3, Fig. 1) but a large variation of the percentage of fractions and the hydrosoluble ones in the last three. The subfrac- individuals stimulated was recorded (P < 0.05). These percent- tions obtained were pooled to reconstitute the HAS fraction. This ages were larger for the treatments containing proteins obtained was done to know if a complementary factor of the non-polar by ammonium sulphate precipitation (AS-proteins), squid (SQ), fraction (NPF) was involved in the activity of the polar fraction.
and the hydro-alcohol soluble fraction after the Bligh & Dyer The experimental design to test these treatments was based on extraction (HAS). These fractions were also those which had induced the highest VTG levels. There was a clear difference in response between the protein fractions obtained after the Bligh & Table 3 Results of the six experiments. Within experiments, means with the same superscript letters belong to homogeneous groups (separated by
Duncan's new multiple range test) 1see Table 2.
2Percentage of individuals stimulated.
1997 Blackwell Science Ltd Aquaculture Nutrition 3; 55–63
Induction of shrimp vitellogenesis by squid extracts Vitellogenesis induction by this particular fraction was equiva- lent to that observed in eyestalk-ablated animals (Table 3). The lipid fraction was the next in order of importance, but the propor- tion of shrimps attaining maturation was lower (about one-third of the HAS fraction). Paradoxically the treatments involving fresh squid (SQ and basal diet + SQ) generated only very weak responses. The protein fractions evoked a response comparable only to the basal diet.
Vitellogenin (mg mL
Response (%)
Experiment 3: The overall effect of the different fractions was superior in terms of stimulation over the negative control (Fig. 3). The best result were obtained with the HAS BD - H A S BD-P. B & D BD-P. AS SQUID
fraction, hydro-alcohol soluble/polar fraction (HAS/PF) and Figure 1 Effect of the main biochemical groups issued from squid on
lipid fraction (LIP): these provoked almost a double con- Penaeus vannamei vitellogenesis. Each mark represents an individual centration as that produced by other fractions. It is to be responding to the treatments, marks C are individuals not responding or noted that the HAS/PF generated a response equivalent to that of showing only basal level and were not considered in the statistical the eyestalk-ablated organisms. In this experiment, the analysis. (BD, basal diet; BD–LIP, BD + lipids; BD–HAS, BD + hydro- alcohol soluble fraction; BD–P.B&D, BD + proteins after Bligh & Dyer's percentage of individuals responding positively was homo- extraction; BD–P.AS, BD + proteins after ammonium sulphate precipita- Experiment 4: The effects of fractions obtained by the protocol of Dyer extraction and those obtained by precipitation with steroids extraction were in general similar to those of the former ammonium sulphate.
experiment (Fig. 4). The fractions HAS and HAS/PF again showed a marked effect. However in this case the lipid fraction Experiment 2: Figure 2 shows the difference in magnitude (VTG and its polar component induced VTG levels lower than 50% of concentration) and in percentage of individuals stimulated in the those obtained by the fractions of experiment 3.
second screening experiment. Even though no significant differ- ences were observed, the impact caused by the HAS fraction when compared with the rest of the treatments in noteworthy.
Vitellogenin (mg mL
Vitellogenin (mg mL
Response (%)
Response (%)
Figure 3 Effect of fractions of different polarity from the HAS and
lipidic extracts, partitioned according to the protocal of Kaplanis for the Figure 2 Effect of an insoluble and soluble peptides and proteins.
extraction of ecdysteroids. (BD, basal diet; BD–HAS, BD + hydro- Conventions as in Fig. 1. (BD, basal diet; BD–LIP, BD + lipids; alcohol soluble fraction; HAS/PF, HAS polar fraction; HAS/RF, HAS BD–HAS, BD + hydro-alcohol soluble fraction; BD–PP, BD + precipi- residual fraction; BD–LIP, BD + lipids; LIP/PF, lipids polar fraction; tated peptides; BD–SP, BD + soluble peptides; BD–SQD, BD + squid; LIP/RF, lipids residual fraction; EABL, unilaterally eyestalk-ablated EABL, unilaterally eyestalk-ablated females.) 1997 Blackwell Science Ltd Aquaculture Nutrition 3; 55–63
R. Mendoza et al. The results of the different bioassays indicate the possibility of stimulating the vitellogenic process merely by incorporating squid extracts at low doses in the feed, thus achieving maturation levels of the same order of magnitude as the positive controls (unilaterally eyestalk-ablated females) without performing the eyestalk ablation. Another important finding was the induction of vitellogenesis in females (15–25 g) below the size currently Vitellogenin (mg mL
considered as minimal for maturation (30–45 g) (Bray & Lawrence 1992). The shrimps used were from intensive cultures, Response (%)
while normally those animals destined to constitute a broodstock are raised under special conditions.
The heterogeneity of responses in the case of protein fractions prepared by different methods points to the hypothesis that the Figure 4 Effect of fractions of different polarity from the HAS and
lipidic extracts, partitioned according to the eleutropic series of Makin for beneficial effect observed with the precipitated proteins might be the extraction of esteroids. (BD, basal diet; BD–HAS, BD + hydro- due to molecules coprecipitated with or bound to proteins.
alcohol soluble fraction; HAS/PF, HAS polar fraction; HAS/RF, HAS The results of the experiments in which different fractionation residual fraction; BD–LIP, BD + lipids; LIP/PF, lipids polar fraction; protocols were applied showed the same tendencies, the greatest LIP/RF, lipids residual fraction; EABL, unilaterally eyestalk-ablated stimulation being observed with the HAS and lipid extracts.
Nevertheless, the intratreatment and intertrial variability observed was high and complicated the statistical analysis.
Experiment 5: No significant differences were found regarding The variation observed may be attributed to the squid species the proportion of individuals showing signs of maturation.
and/or origin. This could be the reason for the absence of stimula- Nevertheless differences were obtained when comparing the tion observed when fresh squid (Nototodarus sloani, from the VTG concentration of shrimps (Fig. 5). There was an especially South Pacific region) was used, and when the basal diet was good performance in the treatment in which the HAS polar complemented with it (experiment 2), while the extracts obtained fraction was assayed (HAS/PF), in contrast to the non-polar one from the freeze-dried squid (Loligo sp. from the English Channel (HAS/NPF). The original HAS fraction and that reconstituted region) were shown to be active. The composition of squid may (HAS/RE) showed equivalent results. Only the positive control change during the season and as a consequence of the different (EABL) outperformed the HAS/PF.
stages of their reproductive cycle. Moreover, Middleditch et al. (1980) showed that many lipid components pass unaltered through the trophic chain, thus the ability of squid to trigger maturation may be based equally on its own feed, and this may vary over the seasons and according to its geographical location.
The intratreatment variations noticed in the course of the experiments led us to consider some factors that might explain the differences. One of these factors is dominance. Indeed there is a certain hierarchy among the individuals sharing the same tank, Vitellogenin (mg mL
as has also previously been pointed out by researchers of the Oceanic Institute (Newsline 1988). Dominance could imply a Response (%
difference in consumption and thus a concentration of the stimu- lating effect. This factor would be accentuated by the asynchrony of the moulting cycle among the individuals during the experi- mental period. Finally, the low proportion of the extracts incor- Figure 5 Effect of the polar and apolar fractions of the HAS extract and
porated in the basal diet could have caused an uneven distribution the joint effect of both fractions. (BD, basal diet; BD–HAS, BD + hydro- in the feed.
alcohol soluble fraction; HAS/PF, HAS polar fraction; HAS/NPF, HAS Considering the stimulation observed with the HAS and the non-polar fraction; HAS–RE, reconstituted HAS fraction (BD-HAS/NPF; EABL, umilaterally eyestalk-ablated females.) lipid fractions, we aimed our further experiments at elucidating 1997 Blackwell Science Ltd Aquaculture Nutrition 3; 55–63
Induction of shrimp vitellogenesis by squid extracts the nature of the active molecule(s). In the case of the HAS Kanazawa 1971). Since then, an increasing number of enzymes fraction, the component responsible for triggering the vitellogen- has been found (review: Swevers et al. 1991), revealing the esis appears to be of polar nature. In a similar way the results importance of this specific enzymatic equipment.
obtained after refractioning the lipid extract indicate as well that The third point supporting the hypothesis of a steroid-like the active component would be rather polar in nature. However molecule is the possibility of induction. There are many demon- the second extraction has shown that it is not a phospholipid strations of sexual steroids showing positive stimulating results (because it is acetone-soluble).
with different kinds of crustaceans (Caillouet 1972; Kulkarni The fact that the activity was found in different fractions and et al. 1979; Nagabhushanam et al. 1980; Yano 1987). However, that this was not evenly conserved may be due to the following: their physiological functions have not yet been elucidated. The when separating the fractions by chromatography the active part triggering of vitellogenesis by gonadotrophic hormones allows may have been shared between the HAS and lipid extracts envisaging the existence of a steroid relay as in vertebrates according to its solubility in each one of them, or there might (Bomirski & Klek-Kawinska 1976; Zukowska-Arendaczyck have been a partial destruction of the active factor. The possible 1981; Souty & Picaud 1984).
existence of two independent factors acting at different levels These arguments point to the existence of a component of should also be considered.
steroid nature in the squid which very probably is located in the Even if the nature of the molecules was not completely eluci- gonads. If this is true, this finding would explain the results dated, according to the kind of extraction achieved, it is probable reported by AQUACOP (1977) regarding the stimulation of that the active fractions contain, among other components, sexual penaeid shrimps by the ingestion of sexually mature molluscs, steroids that may have had a direct or indirect influence on shrimp vitellogenesis. The work of Boticelli (cited by De Another possibility would be that the gonadotrophic hormone Longcamp et al. 1974) supports this hypothesis. This author controlling vitellogenesis in cephalopods and emitted by the showed that alcoholic extracts of the gonads of Pecten have optical glands (Wells & Wells 1959; Richard & Lemaire 1975) oestrogenic and prostagenic properties. On the other hand, even could have stimulated the vitellogenesis in female shrimps in a if the lipids from squid represent a good source of cholesterol heterologous way. This hormone has been postulated to be a (93% of sterols) (Blanchier et al. 1986), which shrimps are not steroid (Froesh 1979; Mangold 1987).
able to synthesize and is needed as a substrate of the synthesis of Equivalent results concerning the stimulation of shrimps, steroids, it should be noted that the basal diet already contained Penaeus japonicus Bate, with the incorporation of mollusc extracts ample amounts of cholesterol. Nonetheless, it is worth consid- in the feed were reported by Kanazawa (1989) employing a ering that cephalopods contain a broad spectrum of steroids in similar protocol of extraction to the one that we have used, the their gonads and an important amount of these is also found in main difference being that Kanazawa used the extracts in combi- their haemolymph (Carreau & Drowsdowsky 1977; Nikitina nation with eyestalk ablation in order to achieve vitellogenesis.
et al. 1977). Recent research results provide us with three These results open new perspectives from the standpoint of arguments supporting the functional role of steroids in the control applied aquaculture, because they could eventually lead to the of vitellogenesis. First is the presence of these molecules in the development of a ‘maturation feed' that would allow aquacultur- various tissues involved in the vitellogenesis of crustacea (ovary, ists a better control of reproduction.
haemolymph, mandibular organ, hepatopancreas, etc.) Different steroids (testosterone, 11-ketotestosterone, 17-β-oestradiol, 17- The authors wish to express their gratitude to the National en3-one, etc.) have been detected by sensitive methods like RIA, Council of Science and Technology of Mexico (CONACyT) and GC-MS and SIM (Ollevier et al. 1986; Couch et al. 1987; Fairs to the Institut Français de Recherche pour 1'Exploitation de la et al. 1989; Novak et al. 1990).
Mer (IFREMER) for their financial assistance to carry out this Secondly there is strong evidence relative to the correlation research. Armelle Sevère, Patrick Quazuguel and Peva Levy are between the concentration of these molecules and the develop- gratefully acknowledged for skilfully performed analysis and ment of the ovary (Van Beek & De Loof 1988; Fairs et al. 1989; immense help in the practical work.
Novak et al. 1990). Additionally there is a great body of evidence of many enzymatic systems that participate in the metabolism of steroids. Indeed, the capacity of bioconversion of cholesterol into Anilkumar, G. & Adiyodi, K. (1980) Ovarian growth, induced by progesterone and other steroids has been demonstrated in the eyestalk ablation during the pre-breeding season, is not normal in the ovaries of crustaceans (Kanazawa & Teshima 1971; Teshima & crab, Paratelphusa hydrodromus. Int. J. Invertebr. Reprod., 2, 95–105.
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Free Radical Biology & Medicine, Vol. 22, No. 5, pp. 885 – 888, 1997 Copyright q 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/97 $17.00 / .00 THE ORIGIN OF THE HYDROXYL RADICAL OXYGEN IN THE Roger V. Lloyd,* Phillip M. Hanna and Ronald P. Mason National Institute of Environmental Health Sciences, National Institutes of Health, P.O. Box 12233,

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