BIOCHEMICAL COMPOSITION AND TISSUE WEIGHT OF Chorus giganteus (GASTROPODA: MURICIDAE) EXPOSED TO DIFFERENT DIETS AND TEMPERATURES DURING REPRODUCTIVE CONDITIONING

Claudio A. Carrasco, Jorge M. Navarro and Germán E. Leiva

Claudio A. Carrasco. Marine Biologist, Universidad Austral de Chile (UACH), Chile.

Jorge M. Navarro. Ph.D. Memorial University of Newfoundland, Canada. Professor, (UACH), Chile. Address: Instituto de Biología Marina "Dr. Jürgen Winter", Universidad Austral de Chile, Valdivia, Chile. e-mail: jnavarro@uach.cl

Germán E. Leiva. Marine Biologist (UACH), Chile. Biologist, Austral, Servicios & Tecnología Marina, Chile.

Summary

A study was made of the effects of temperature and diet on the dry soft tissue weight and biochemical composition of the gastropod Chorus giganteus (trumulco) over a 133 day period of reproductive conditioning. Adult specimens measuring 9-13cm in total length obtained from the subtidal habitat at Chaihuin (Valdivia, Chile) were maintained in seawater tanks at 13, 15 and 18ºC, and fed ad libitum in parallel systems with either mussels, Mytilus chilensis, or razor clams, Tagelus dombeii. Two groups of snails were simultaneously installed in suspended culture at Metri Bay on the eastern shore of Reloncavi Sound using the same diets, in order to provide a comparative group of specimens exposed to environmental temperatures (11.0-15.4ºC). The results show that all tissues of the snails fed with T. dombeii increased in dry weight and in biochemical components, while the second group of snails showed low preference for M. chilensis and no tissue gains. The most favourable temperatures for the reproductive conditioning were 13 and 15ºC, as well as the ambient temperature, with no significant differences among these treatments.

COMPOSICIÓN BIOQUÍMICA Y PESO DE LOS TEJIDOS DE Chorus giganteus (GASTROPODA: MURICIDAE) EXPUESTO A DIFERENTES DIETAS Y TEMPERATURAS DURANTE EL ACONDICIONAMIENTO REPRODUCTIVO

Resumen

Se realizó un estudio para determinar los efectos de la temperatura y dieta sobre el peso y composición bioquímica de los tejidos blandos del gastrópodo Chorus giganteus durante un periodo de acondicionamiento reproductivo de 133 días. Especímenes adultos obtenidos desde la zona submareal de Chaihuin (Valdivia, Chile), con tamaños entre 9 y 13cm de longitud total, fueron mantenidos en estanques de agua de mar a 13, 15 y 18ºC y alimentados ad libitum en sistemas separados con el bivalvo Mytilus chilensis o con la navajuela Tagelus dombeii. Otros dos grupos de caracoles fueron instalados en cultivos suspendidos en la bahía de Metri, al Este del seno de Reloncaví, utilizando las mismas dietas, con el fin de tener un grupo comparativo de especímenes expuestos a la temperatura ambiente (11,0-15,4ºC). Los resultados demuestran que todos los tejidos de los caracoles alimentados con T. dombeii aumentaron su peso y componentes bioquímicos, mientras que el grupo de caracoles alimentados con M. chilensis mostró una baja preferencia por esta dieta, sin presentar ganancia de tejidos. Las temperaturas más favorables para el proceso de acondicionamiento reproductivo fueron 13 y 15ºC, al igual que la temperatura ambiental, sin diferencias significativas entre estos diferentes grupos.

COMPOSIÇÃO BIOQUÍMICA E PESO DOS TECIDOS DE Chorus giganteus (GASTROPODA: MURICIDAE) EXPOSTO A DIFERENTES DIETAS E TEMPERATURAS DURANTE O ACONDICIONAMENT

O REPRODUTIVO

Resumo

Realizou-se um estudo para determinar os efeitos da temperatura e dieta sobre o peso e composição bioquímica dos tecidos brandos do gastrópode Chorus giganteus durante um período de acondicionamento reprodutivo de 133 dias. Especímenes adultos obtidos desde a zona submareal de Chaihuin (Valdivia, Chile), com tamanhos entre 9 e 13cm de longitude total, foram mantidos em estanques de água de mar a 13, 15 e 18ºC e alimentados ad libitum em sistemas separados com o bivalve Mytilus chilensis ou com a almeja Tagelus dombeii. Outros dois grupos de caracóis foram instalados em cultivos suspendidos na bahía de Metri, ao Leste do seno de Reloncaví, utilizando as mesmas dietas, com o fim de ter um grupo comparativo de especímenes expostos à temperatura ambiente (11,0-15,4ºC). Os resultados demonstram que todos os tecidos dos caracóis alimentados com T. dombeii aumentaram seu peso e componentes bioquímicos, enquanto que o grupo de caracóis alimentados com M. chilensis mostrou uma baixa preferência por esta dieta, sem apresentar ganho de tecidos. As temperaturas mais favoráveis para o processo de acondicionamento reprodutivo foram 13 e 15ºC, ao igual que a temperatura ambiental, sem diferenças significativas entre estes diferentes grupos.

Keywords / Biochemical Composition / Chorus giganteus / Muricidae / Reproductive Conditioning /

Received: 06/28/2005. Modified: 03/16/2006. Accepted: 03/23/2006.

Introduction

Optimal conditioning of molluscan broodstock is required for the production of high quality gametes for intensive culture. Adequate storage of energetic reserves and gonad development has been obtained by optimization of holding temperature (Uki and Kikuchi, 1984; Chaparro, 1990; Navarro et al., 2000) and diets (Uki and Kikuchi, 1984; Chaparro, 1990). Responses of broodstock to experimental attempts at conditioning may be measured by the increase in body weight and tissue biochemical composition, as these parameters respond to treatments for short periods, of weeks or months (Barber and Blake, 1981).

The subject of the present study, Chorus giganteus, is a commercially valuable snail which is undergoing overexploitation in its natural habitat. Despite increasing efforts, the landing of C. giganteus decreased from 2800 metric tons in 1980 to less than 100 tons in 1997 (Ram et al., 2000), and due to favourable characteristics of its biology has shown promise as a potentially cultivable marine resource (Gutiérrez and Gallardo, 1999; Navarro et al., 2002). This species is endemic to the Chilean coast, inhabiting waters between 8 and 30m in depth (Amín et al., 1984) on sandy bottoms (Gallardo, 1981). It occurs (Osorio et al., 1979) from Antofagasta (23º48'S, 70º32'W) to Valdivia (39º25'S, 73º10'W). The reproductive characteristics of this species include a continuous annual reproductive cycle, with asynchronic maturation (Jaramillo and Garrido, 1990). Eggs are laid in capsules and provided with nutritive eggs as an extra-embryonic source of nutrition (González and Gallardo, 1999). The eggs hatch after about 80-87 days in the capsule and the larvae have a 2-5 day lecithotrophic period in the plankton (González and Gallardo, 1999). Competent larvae are sensitive to metamorphic induction with 20mM KCl, showing a greater percentage of postlarval survival (González, 1997) and greater rates of growth as juveniles (Muñoz and Leiva, 1994).

Some research has been done on broodstock conditioning under different regimes of diet and temperature (Navarro et al., 2002); however, no studies have evaluated the effects of different methods of conditioning on dry soft tissue weight and biochemical components of the adult tissues. The present study evaluates the effects on these parameters of four experimental temperatures (three fixed, plus ambiental), and two different diets, a knowledge required for optimal reproductive conditioning of this species prior to mass culture.

Material and Methods

Experimental specimens

300 adult specimens of C. giganteus measuring 9-13cm in total length were obtained by diving in the subtidal zone at Chaihuin (39º52'S, 73º25'W; Figure 1) and transported to the Aquaculture and Marine Sciences Center of the Universidad de Los Lagos on Metri Bay (41º36'S, 72º42'W), near Puerto Montt, Chile, in April 1998.

Prey species for use as experimental diets for C. giganteus included razor clams (Tagelus dombeii) obtained from intertidal sand/mud flats at Coihuin (41º29'S, 72º54'W) and mussels (Mytilus chilensis) from suspended cultures in Metri Bay. These species were selected because the razor clams were common natural prey in the habitat of C. giganteus (Osorio et al., 1979) and the mussels appeared to be a potentially useful prey species for mass snail culture, due to their ready availability in large quantities and low cost.

Experimental design

Prior to reproductive conditioning tests, the snails were measured, separated by sex, and randomly allocated in eight 75L plastic aquaria for acclimation to test temperatures for a one-week period. Snails in one set of three tanks, with water at 13, 15 and 18ºC, were fed ad libitum with T. dombeii (>3cm length). Snails in a second set of three tanks, also kept at 13, 15 and 18ºC, were fed with M. chilensis (>3cm length). Water in all tanks was constantly replaced with 50µm filtered seawater at a rate of 1.0L·min-1. To control for the effects of temperature, snails in two other tanks were each fed with the corresponding bivalve diet but maintained in open tray cultures, in Metri Bay, where ambiental temperatures during the test period, between June and November of 1998, fluctuated between 11.0 and 15.4ºC (Table I).

Over the experimental period of 133 days, sampling for analyses included 6 snails from the natural population prior to conditioning, 6 snails at the time conditioning was begun (1 week of post-acclimation) and 6 snails per each temperature/diet combination in each of 4 samplings made over the time period of conditioning (Table II). At each sampling, male and female groups of C. giganteus were randomly selected from each experimental temperature and diet treatment, as well as from the field tests. Because of logistic reasons (i.e. remote locations of this research and limited laboratory space) little control was exerted over the experimental design and no true replications were included. This did not allow to test the tank effects, reducing the accuracy of the measurements.

Dry soft tissue weight and biochemical composition

For determination of the dry soft tissue weight and subsequent biochemical analysis, individual snails of each sex were dissected from the shell and the soft tissues separated into foot, gonad and rest of the tissue. Separate tissues were dried at 60ºC for 48h, weighed and grounded in a ball mill for biochemical analyses. The gonadosomatic index was calculated as the percentage of the gonad dry weight over the total dry soft tissue weight.

Protein, carbohydrates and lipids were determined on subsamples of dried, homogenized tissue. Protein was determined in 3-5mg of tissue using the Pierce Laboratories BCA method, which uses a bicinchoninic acid solution for colorimetric determination of total protein. Carbohydrates were extracted by boiling 8-10mg of homogenized tissue in trichloroacetic acid with silver sulphate (Barnes & Heath, 1966) and quantified with glucose as the standard using the phenol-sulphuric acid method (Dubois et al., 1956). The gravimetric method of Bligh and Dyer (1959) was used for determination of lipids in 40-50mg of foot tissue, while gonad tissue was assayed using the method described by Marsh and Weinstein (1966) on 1-3mg samples using tripalmitin as a standard. The latter method was employed due to the very small amounts of gonad tissue present in the samples during the test period.

Statistical analysis

All data were standardized to a specimen of 11.5cm in total length, which represented the mean size of individuals used in the experiment. This standardization was done, according to Bayne et al. (1987), as

WS = (LS / LE)b × WE

where WS: standardized dry weight of the specimen, LS: total standard length (11.5cm), LE and WE: actual total length and dry tissue weight, respectively, of the snail, and b: weight exponent of the relation between the length and weight.

Analysis of variance (ANOVA) and an a posteriori Scheffe test were used to detect significant differences among treatments defined by diets, temperatures, sexes, and conditioning times. Kruskal-Wallis tests were used in cases where the distribution of variables was significantly different from normal. All statistical tests were carried out at the 5% level (p £0.05), and the data was processed using the "S-plus 4.0" package for Windows.

Results

Dry soft tissue weight

The dry weight of the foot in C. giganteus showed a slight tendency to increase over the experimental period when fed with T. dombeii, compared with specimens fed with M. chilensis which demonstrated a slight decrease (Figure 2). This observation was more pronounced during the latter months of conditioning. Over the entire study, the dry weight of the foot was significantly greater in snails fed with the razor clams than with mussels. Significant differences were also observed in dry weight of the foot with the time-diet interaction. This showed that the conditioning time used accentuated the difference in weight of the foot between the snails on the two diets.

The dry weight of the foot showed no significant differences with regard to experimental temperature used in the laboratory and ambient temperature in the field (Figure 2). A significant effect was observed of diet on the weight of the gonad, where greater gonad weight was achieved on a diet of T. dombeii (Figure 3).

Gonadosomatic index (GI)

The GI evaluated the relation between weight of the gonad and total soft tissue weight of the individual. At the beginning of the conditioning experiment the values for this index were 3.33 +2.1% in males and 2.10 +0.6% in females. By the end of the conditioning period these values had increased to 9.70 +9.5% in males fed with T. dombeii at ambient temperature, and 6.30 +1.2% in females at 13ºC and fed on the same diet (Figure 4). There were no significant differences observed either between sexes nor among the different treatments of diet and temperature.

Biochemical composition

Proteins. Protein content of the foot (Figure 5) was significantly greater in snails fed with T. dombeii than those fed with M. chilensis. The tendency of the protein curves (eight combinations of diet and temperature) were similar, repeating the same pattern of higher values in August, decrease in September, rise in October and stabilization nearing the end of the experimental period. Foot protein varied over time with significant differences between the samples of August-September and September-October (Figure 5). The protein content of the gonad also showed significant differences between diets, with an increase in this component in specimens fed with T. dombeii (Figure 6).

 

Carbohydrates. A high degree of variation was observed in carbohydrate content in the foot tissue of C. giganteus. The T. dombeii diet produced a larger increment in this component, with significant differences found in comparison with the M. chilensis diet. Significant differences were found in the diet-time interaction in the last two months of the experiment (Figure 7). The gonad tissue also showed significant differences in carbohydrates related to diet. Again, the T. dombeii diet produced greater levels of carbohydrates. In contrast, the M. chilensis diet showed highly reduced carbohydrate values in the gonad at the end of the experiment (Figure 8).

Lipids. The lipid content of the foot showed significant differences related to diet (Figure 9), with greater values produced when feeding T. dombeii. Significant differences were also found between temperatures, with greater lipid accumulations recorded for the snails maintained at 13ºC. In gonad tissue (Figure 10) snails fed T. dombeii showed lipid content values significantly greater than those fed with M. chilensis. Lipids in gonad tissue demonstrated some significant differences with regard to temperature; when maintained at ambient temperatures snails developed significantly greater amounts of gonad lipids than when maintained at 18ºC. No significant differences in lipid content were observed between snails of different sex in any of the tissues examined.

Discussion

When C. giganteus was fed with M. chilensis, the dry weight of all its tissues decreased gradually during the 8 months of the conditioning period, which reflects the low preference of the snail for this species. The study of Navarro et al. (2002), under similar experimental conditions, yielded similar results: each individual of C. giganteus fed on average upon 0.77 to 1.32, 3 to 5cm long mussels per month. These results may be due to the fact that in its natural habitat, C. giganteus does not prey on M. chilensis, which occupies a different habitat. Cristian Manque (personal communication) found a contrasting behaviour in C. giganteus enclosed in the laboratory and fed on prey species that did not occur in its typical habitat; the snails were first fed with Semimytilus algosus and then with M. chilensis, and after four months of feeding with the latter, each snail consumed about 3.8 individuals of M. chilensis per month.

Tagelus dombeii was well accepted as diet of C. giganteus, leading to increment in the weight of the snail tissues. Navarro et al. (2002) found that one individual of C. giganteus reached monthly consumptions between 5.24 and 7.26 T. dombeii of 4-6cm in length. Conversely to M. chilensis, T. dombeii is a common bivalve species in the habitat of C. giganteus (Osorio et al., 1979), representing a common prey species in the diet of this snail. The higher rate of food consumption with T. dombeii than with M. chilensis may be related to the fact that the soft tissues of T. dombeii are not totally enclosed by the shell, allowing the snail to consume this bivalve without spending energy in penetrating the shell. Thus, feeding on this prey would imply a low energy cost for the snail by the optimal foraging theory (Hughes, 1986), as compared to M. chilensis, a prey that closes its valves tightly.

When T. dombeii was used, C. giganteus mainly consumed the gonad and digestive gland of the clams, discarding foot and siphons. Hughes and Dunkin (1984) describe a similar behavior in the gastropod Nucella lapillus, which consumes the digestive gland and discards the mantle and foot when preying on mytilids. Selective consumption was also observed in Thais haemastoma, which discarded non-muscular tissues of oysters (Gunter, 1979). According to Hughes (1986), when high quality preys are abundant, selective ingestion would maximize the net energy gain acquired by the predators, which could ingest the most favourable organs (those least difficult to ingest, with the highest energetic and/or nutritional value).

The temperature of 18ºC was deleterious to the snails as, in addition to failure to gain weight, a massive mortality of the experimental specimens was observed in the last month of the experiment. In a concurrent study carried out by Merino (2000) with this species, only 15% of the organisms survived at 18°C. Thus, it seems that adults of C. giganteus are unable to survive for a long time in water at >17°C, as they would lose their capacity for thermal compensation outside their zone of tolerance (Newell and Branch, 1980). In a great variety of marine organisms the maximum temperature limit may be related to natural conditions in their habitat (Henderson, 1929 in Newell et al., 1971). Based on this, and with the knowledge that C. giganteus inhabits the subtidal zone below 8m depth (Lépez, 1981 in Amín et al., 1984), it is assumed that C. giganteus never experiences temperatures as high as 18°C during long periods of time, and thus its upper compensation limit must be below this value. However, it seems also reasonable to assume that if this species is able to survive near 8 months at 18ºC, it can easily survive shorter periods of high temperatures that can occur at their natural environment. Also, the high mortality of the test specimens may be attributed to the short period of acclimation afforded to the snails before experimentation. Newell et al. (1971) found in the prosobranchs Littorina littorea and Monodonta lineata that the upper lethal temperature was significantly affected by thermal acclimation.

Similarly to results on the dry soft tissue weight in C. giganteus, the biochemical composition of the different tissues was affected by variations in the conditioning process. The protein content, as well as the content of the other biochemical components, showed that the diet had a significant effect on the foot and gonad: high rates of consumption of T. dombeii resulted in weight increment of these structures. The relatively constant content of protein in the muscular tissue (foot) of specimens fed with mussels indicated that structural integrity of this body tissue was maintained to the end of the experiment. This situation was observed by Holland et al. (1975) in Littorina littorea after 14 weeks without feeding. These authors also observed that during the time the snails were maintained without food, the lipid and carbohydrate contents of the tissues decreased in greater proportion than the protein. According to these results, L. littorea as well as C. giganteus prioritize the catabolism of carbohydrates and lipids rather than proteins when deprived of food.

The gonad of C. giganteus contained greater quantities of lipids than carbohydrates, in contrast to the foot tissues. This distribution of lipids and carbohydrates in gonad and muscular tissues holds as a characteristic pattern in prosobranchs (Webber, 1977). The results of gonad histology by Merino (2000) in snails from the present study showed no significant differences associated with the type of diet. Merino (2000) reported that even though fed with the diet of lesser consumption, mature snails deposited egg capsules when held in the natural environment. It thus appeared that the group of snails fed with M. chilensis carried out the formation of gametes primarily at the expense of reserved energy available from body tissues, which was subsequently measured as a decrease in biochemical components. In contrast, snails fed with Tagelus dombeii could obtain sufficient energy for the process of gametogenesis, considering that they were able to divert energy reserves as carbohydrates in the foot tissues, in coincidence with observations made for L. littorea (Holland et al., 1975).

This study indicates that the best reproductive conditioning for the snail C. giganteus will be strongly determined by suitable temperatures, no higher than those experienced in the natural habitat, and by appropriate diets represented by those bivalves which can be easily preyed upon. Thus, when C. giganteus was fed with the bivalve T. dombeii, it increased its weight and the concentration of the biochemical components in its tissues. Furthermore, greater amounts of carbohydrates than lipids were found in the foot tissue, whereas the contrary was observed in the gonads, suggesting that the foot represents an important energy reservoir in C. giganteus. The results obtained are relevant to the development of more efficient culture methods of this commercially valuable snail, especially in relation with the selection of the more suitable places and diets to be supplied during the reproductive conditioning process.

ACKNOWLEDGEMENTS

The authors thank Cristian Manque, Rodrigo Merino, Maritza Araneda, and personnel of CEACIMA-Metri for field and laboratory help. This study was financed by the Aquaculture and Marine Biology Program 1 (97), Invertebrate Subprogram, FONDAP, Chile and for the DID-UACH.

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