Alfonso N. Maeda-Martínez

Phenoloxidase activity in larval and juvenile homogenates and adult plasma and haemocytes of bivalve molluscs.

Phenoloxidase (PO) activity was studied in larval and juvenile homogenates and in the plasma and haemocytes of adult Crassostrea gigas, Argopecten ventricosus, Nodipecten subnodosus, and Atrina maura. Samples were tested for the presence of PO activity by incubation with the substrate L-3, 4-dihydroxyphenylalanine using trypsin, alpha-chymotrypsin, laminarin, lipopolysaccharides (LPS), and sodium dodecyl sulphate (SDS) to elicit activation of prophenoloxidase (proPO) system. PO activity was not detected in larval homogenate. In juvenile homogenate, PO activity was found only in C. gigas and N. subnodosus. PO activity was present in adult samples and was enhanced by elicitors in the plasma of all species tested, but in haemocyte lysate supernatant (HLS) of only N. subnodosus. Activation of proPO by laminarin was suppressed by a protease inhibitor cocktail (P-2714) in plasma and HLS of all species tested.

Ontogenetic variations of hydrolytic enzymes in the Pacific oyster Crassostrea gigas.

Occurrence and level of hydrolytic enzymatic activity (proteases, glycosidases, phosphatases, lipases, and esterases) were studied in oocytes, larvae, juveniles, and adult haemolymph of the Pacific oyster Crassostrea gigas. Samples were obtained as oocyte lysate supernatant, larval homogenate supernatant, juvenile homogenate supernatant, haemocyte lysate supernatant, and plasma. The presence of enzymes was demonstrated by colorimetric and lysoplate assay techniques. Between stages, significant differences in enzymatic activity determined by the colorimetric technique were found. Higher levels of enzymatic activity were found in the adult stage. Lysozyme-like activity was not found in oocytes, but was present in larvae, juveniles, and adults. In larvae, the highest lysozyme-like activity was in 3-d larvae. Juveniles had a 48-fold higher level of lysozyme-like activity, compared with 20-h larvae and was six-fold higher compared with 3-d larvae. In adults, lysozyme-like activity had a five-fold higher level in haemocyte lysate supernatant compared with plasma and was 98-fold higher compared with 20-h larvae. As determined with the API ZYM kit, 19 hydrolytic enzymatic activities were present, in oocytes, larvae, juveniles, and adult haemolymph of C. gigas. The presence of important lysozyme-like activity was confirmed from trochophora larvae (20 h) to adult stages.

Suspension culture of catarina scallop Argopecten ventricosus (=circularis) (Sowerby II, 1842), in Bahia Magdalena, Mexico, at different densities

Abstract Hatchery-reared spat (5 mm) of catarina scallop ( Argopecten ventricosus (=circularis )) were cultured in plastic trays suspended from a long line at Rancho Bueno, Baja California Sur, Mexico. The mean surface temperature range was 20 to 29 °C and salinity remained steady at 37 ppt during the 320-day experiment. Every 1 or 2 months, the trays and the scallops were cleaned, the scallops measured, and survival recorded. Three densities were tested on each of the culture stages; called nursery (from day 1 to day 80), intermediate (80 to 136) and growout (136 to 320). Average densities were 1500, 2500, and 4000 scallops/m 2 for the nursery stage; 400, 700, and 1000 for the intermediate; and 150, 250, and 400 for the growout stages. At the end of nursery and intermediate stages, scallops of the modal size class were selected to begin the following stage using scallops of homogeneous size. In the nursery stage, growth rate was higher at low density. At the intermediate stage, growth rates were highest in low and medium densities ( P P 91% at the end of the experiment and did not correlate with density. Mortality began to occur in animals older than 8 months, during the spawning season at the onset of summer. Two hypothetical growth curves were constructed from the data taking for each stage those densities producing the fastest and slowest growth. Results indicate maximum shell length could be attained in 273 (fastest) and 398 (slowest) days. A cost analysis indicates that, regardless of the longer time, culturing scallops at high density would be less expensive than at low density, taking into account the muscle-weight loss caused by crowding.

Scallop growout using a new bottom-culture system

Abstract A new bottom-culture system was tested at a commercial level to grow catarina scallops (Argopecten ventricosus) in the Rancho Bueno tidal channel in Bahia Magdalena, Mexico. The system consisted of a 50×1-m sleeve of 19-mm mesh polyethylene netting placed on the sea floor of selected growout areas. A total of 448 sleeves were deployed at various times from October 1994 to April 1995 in four zones from the mouth to the head of the tidal channel. Each sleeve contained 10,000 scallops (initially 32 mm shell height) at a density of 200 scallops/m2. Fifty-four percent of the spat were produced in a hatchery and the rest were collected using onion-bag collectors. Hatchery and wild spat were deployed separately. Water parameters were measured monthly in each zone: temperature, salinity, dissolved oxygen and total suspended solids. There were better water conditions towards the mouth of the channel. The scallops were harvested from 27 July to 18 August 1995: a total of 2.87 million scallops from the original 4.48 million. Their mean shell height was 56.2 mm and the mean weight of their adductor muscles was 6.8 g. The production was 19.3 t of adductor muscles. Statistically significant differences in mean shell height and mean adductor weight were found between scallops grown in different zones, but no statistically significant differences were found comparing yields from hatchery vs. wild scallops. To find the best culture conditions, a scallop relative value (SRV) was calculated by multiplying survival by adductor muscle weight and relative market price, and dividing by the growout duration. There were higher SRVs for scallops cultured in zones closer to the channel mouth. The highest SRV was found in a group from this zone, with 86% survival, 6.25 g mean adductor weight, and a growout duration of only 3.6 months. A new successful method for growing scallops in shallow areas is, thus presented here. It gave better results than suspension methods tested in the same area.

Changes in oxidative stress parameters in relation to age, growth and reproduction in the short-lived catarina scallop Argopecten ventricosus reared in its natural environment.

Increase in oxidative damage and decrease in cellular maintenance is often associated with aging, but, in marine ectotherms, both processes are also strongly influenced by somatic growth, maturation and reproduction. In this study, we used a single cohort of the short-lived catarina scallop Argopecten ventricosus, to investigate the effects of somatic growth, reproduction and aging on oxidative damage parameters (protein carbonyls, TBARS and lipofuscin) and cellular maintenance mechanisms (antioxidant activity and apoptosis) in scallops, caged in their natural environment. The concentrations of protein carbonyls and TBARS increased steeply during the early period of fast growth and during reproduction in one-year-old scallops. However, oxidative damage was transient, and apoptotic cell death played a pivotal role in eliminating damage in gill, mantle and muscle tissues of young scallops. Animals were able to reproduce again in the second year, but the reduced intensity of apoptosis impaired subsequent removal of damaged cells. In late survivors low antioxidant capacity and apoptotic activity together with a fast accumulation of the age pigment lipofuscin was observed. Rates of oxygen consumption and oxidative stress markers were strongly dependent on somatic growth and reproductive state but not on temperature. Compared to longer-lived bivalves, A. ventricosus seems more susceptible to oxidative stress with higher tissue-specific protein carbonyl levels and fast accumulation of lipofuscin in animals surviving the second spawning. Superoxide dismutase activity and apoptotic cell death intensity were however higher in this short-lived scallop than in longer-lived bivalves. The life strategy of this short-lived and intensely predated scallop supports rapid somatic growth and fitness as well as early maturation at young age at the cost of fast cellular degradation in second year scallops.

Influence of environmental variables and reproduction on the gonadal fatty acid profile of tropical scallop Nodipecten nodosus.

We examined the influence of the reproductive cycle and environmental variables on the gonadal fatty acid profile in the tropical scallop Nodipecten nodosus. Individuals from a natural population in north-eastern Venezuela (Caribbean Sea) were sampled monthly during one year. The gonadosomatic index (GSI) and a visual assessment of gonadic status are also reported. The environmental conditions characterized by chlorophyll a, temperature and seston concentration were measured in parallel. The highest reproductive activity, with a GSI of 13-18%, was observed from November 2003 to April 2004, and a decrease in GSI, taken as an indication of three spawning periods, was recorded in October, January and May-June. The GSI was 5-8% outside these intervals. A similar trend was observed in maturity stage frequency. Statistical analysis (MDS-ANOSIM) established two different periods; one dominated by upwelling (January to July) and the other characteristic of non-upwelling conditions (August to December). The fatty acids 16:0, 22:6n-3, 20:5n-3 and 16:1n-7 were responsible for the differences between two periods and explained 50% of the observed variance. Accordingly, during the upwelling period, fatty acids such as 22:6n-3 were positively correlated with temperature, organic seston and bivalves in the spent sexual stage and inversely related to the GSI. Furthermore, individuals in the ripe sexual stage were positively related to 20:5n-3 and 18:3n-3, whereas chlorophyll a was directly related to 18:1n-7 and 16:1n-7. In the non-upwelling period, a significant and positive correlation was obtained between fatty acids such as 20:5n-3 and 22:6n-3 and the GSI and the bivalves in the ripe sexual stage, whereas these acids were inversely correlated with the spent sexual stage. In addition, chlorophyll a was positively related to 18:1n-9 and 18:2n-6. Changes in the gonadal fatty acid profile of N. nodosus were influenced by the environmental variables (exogenous factors) during the upwelling and non-upwelling periods and by reproductions (endogenous factors).

The effect of predator exposure and reproduction on oxidative stress parameters in the Catarina scallop Argopecten ventricosus.

Predation is known to impact growth and reproduction, and the physiological state of the prey, including its susceptibility to oxidative stress. In this study, we investigated how prolonged exposure to predators modulates tissue specific antioxidant defense and oxidative damage in the short-lived epibenthic scallop Argopecten ventricosus (2years maximum lifespan). Scallops that were experimentally exposed to predators had not only lower antioxidant capacities (superoxide dismutase and catalase), but also lower oxidative damage (protein carbonyls and TBARS=thiobarbituric acid reactive substances including lipid peroxides) in gills and mantle compared to individuals not exposed to predators. In contrast, oxidative damage in the swimming muscle was higher in predator-exposed scallops. When predator-exposed scallops were on the verge of spawning, levels of oxidative damage increased in gills and mantle in spite of a parallel increase in antioxidant defense in both tissues. Levels of oxidative damage increased also in the swimming muscle whereas muscle antioxidant capacities decreased. Interestingly, post-spawned scallops restored antioxidant capacities and oxidative damage to immature levels, suggesting they can recover from spawning-related oxidative stress. Our results show that predator exposure and gametogenesis modulate oxidative damage in a tissue specific manner and that high antioxidant capacities do not necessarily coincide with low oxidative damage.

Monoamine fluctuations during the reproductive cycle of the Pacific lion's paw scallop Nodipecten subnodosus

The Pacific lions paw scallop Nodipecten subnodosus has been one of the most important commercial species of mollusc in the Baja California peninsula in Mexico since 1990. This species is a functional hermaphrodite with tropical and sub-tropical distributions and experiences wide annual temperature oscillations influencing its physiological functions. We determined norepinephrine (NE), dopamine (DA), and serotonin (5-HT) concentrations in different organs (female and male gonads, digestive gland, adductor muscle, gill, mantle, and foot) of N. subnodosus, at 6 reproductive stages (resting, initial, maturing, mature, partially spent and fully spent). Monoamine concentrations were determined by HPLC fitted with an electrochemical detector. Results indicated that monoamine concentrations increased during maturing stage, peaked at mature stage and declined after spawning. NE concentrations were higher than the rest of monoamines followed by DA, and 5-HT. NE was present in all organs at all reproductive stages. DA concentrations were higher in the gill and digestive gland during all stages. 5-HT was only detectable in the male gonadic portion at all stages except at spent stage. NE was the most abundant neurotransmitter found in the female gonad of N. subnodosus, while 5-HT was the most abundant neurotransmitter found in the male gonad. Furthermore, these two neurotransmitters accumulated in the respective gonad tissues during the initial reproductive stages I to IV and then declined after spawning (stages V and VI). This suggests that this species utilized different neurotransmitters specific for each gender and that this utilization was related to the reproductive cycle.

Effect of different light spectra on the growth and biochemical composition of Tisochrysis lutea

We determined the effects of various light spectra (white, green, blue, and red) on the growth rate, biochemical composition, and fatty acid content of Tisochrysis lutea (Haptophyta, Isochrysidales) maintained in batch cultures. The growth rate peaked with white and blue light, and the lowest rate was observed with green and red light. The chlorophyll a content differed significantly between light spectra and growth phases—higher values were recorded with blue and red light in both growth phases. The proximal composition varied significantly with growth phases and light spectrum. In the exponential growth phase, protein content was significantly greater with blue light and in the stationary phase with green light. The level of carbohydrates in the exponential growth phase was significantly higher for white light, but unchanged in the stationary growth phase between light spectra. The lipid percentages were similar in the exponential phase but differed significantly in the stationary growth phase. The lipid percentages peaked in the stationary growth phase with red and green light. The highest eicosapentaenoic acid (EPA) levels were seen in white light in the exponential growth phase and under green light in the stationary growth phase. Docosahexaenoic acid (DHA) levels were greatest in the exponential growth phase with red light and in the stationary growth phase with green light. Blue light increased the DHA content in both growth phases. We conclude that T. lutea alters its metabolic pathways and experience shifts in growth rate, proximate composition, and fatty acid content, depending on the type of light used.

Histological characterization of the spawning process in the catarina scallop, Argopecten ventricosus (Sowerby II, 1842) induced by thermal shock and serotonin injection

Summary The histology of the spawning catarina scallop Argopecten ventricosus was studied to characterize cellular and anatomical changes occurring along the gonad-kidney pathway. Broodstock were conditioned for 25–30 days and induced to spawn with two methods: thermal shock and serotonin by intracardiac injection. Samples of the kidney-gonad-digestive gland complex were fixed at the sperm and oocyte stages of gamete emission and at the end of spawning. Both induction methods elicited gametes, and no particular difference in the microscopic anatomy of the acini-kidney pathway was detected. However, serotonin injection, apart from producing a rough valve opening, sporadic movement (opening and closure of valve), and pronounced foot extension, induced gamete emission in less time than thermal shock. Primary gonad ducts opened to acini to permit sperm emission; at the same time, germinal vesicles broke down in female acini and meiosis restarted. Shortly afterward, oocytes gradually changed form from typically poly-hedral to a more malleable form. This probably contributed to oocyte transport through ciliary activity along the gonad and kidney ducts. Nucleoli disintegration, chromosome condensation, and appearance of meiotic figures were observed along the acini-kidney pathway. Although sperm and oocytes were sometimes observed in gonad or kidney ducts, self-fertilization was rarely observed.