Celia Vázquez-Boucard

Feasible predictive criteria for reproductive performance of white shrimp Litopenaeus vannamei: egg quality and female physiological condition

The present study analyzed the effects of consecutive spawnings on egg quality in a homogeneous domesticated population of Litopenaeus vannamei under optimal maturation conditions. A multidisciplinary approach was used to evaluate egg quality and female condition, using production, morphometric, biochemical, and histological criteria. For this purpose, 106 individually tagged females were stocked with males in maturation tanks, and productivity variables were recorded over 36 days. Biochemical composition of eggs, hemolymph, hepatopancreas, and ovaries was also analyzed. Results indicate that about half (48%) of females did not spawn, 18% spawned once, 15% spawned twice, 11% spawned three times, and 8% spawned four times or more. Variables associated with reproductive quality, such as number of eggs per spawn, fertilization rate, and egg diameter, were not affected by consecutive spawnings. Fourth-spawn eggs had significantly higher levels of total lipids, triacylglycerides, and total proteins than first-spawn eggs. First-spawn eggs from females that by the end of the production period had spawned four or more times had higher triacylglycerides and vitellin levels than first-spawn eggs from females that had three or less spawns. This indicates that triacylglycerides and vitellin could be used as predictors of female reproductive performance. Significantly higher condition index (weight to length ratio) was observed for females that spawned four or more times. No significant difference was observed in biochemical composition of hepatopancreas and ovaries as a function of the effect of number of spawns. This study indicates that egg quality and physiological condition of females were not affected by consecutive spawnings, and that females with multiple spawn capability are desirable for greater reproductive performance. Production variables and biochemical composition of first-spawn eggs and some morphometric characteristics of females may be used as indicators of multiple spawn capability.

Yolk proteins during ovary and egg development of mature female freshwater crayfish (Cherax quadricarinatus).

Vitellins from ovaries and eggs at different stages of development in freshwater crayfish (Cherax quadricarinatus) were examined by chromatography, PAGE and SDS-PAGE. With these methods, two forms of vitellin (Vt1 and Vt2) were observed in ovaries and eggs (stages I and V). In ovaries in secondary vitellogenesis, native molecular mass was 470 (Vt1) and 440 (Vt2) kDa. The electrophoretic pattern of the eggs proved to be more complex. The protein molecular mass depend on the development stage of the egg: stage I, 650 kDa (Vt1) and 440 kDa (Vt2); stage V, 390 kDa (Vt1) and 340 kDa (Vt2). The identified vitellins appear to be lipo-glycocarotenoprotein. A similar vitellin polypeptide composition was observed in the two forms of vitellin from ovaries and eggs in stage V. In ovaries the SDS-PAGE analysis showed four subunits with molecular weights of approximately 180, 120, 95 and 80 kDa (Vt1 and Vt2). The polypeptide composition in the two forms of vitellins in stage I and stage III eggs were different at 195, 190, 130 and 110 kDa (Vt1) and 116 and 107 kDa (Vt2). On the other hand, in stage V eggs, 110, 95, 87 and 75 kDa (Vt1 and Vt2) were identified. Two antibodies (Ab1 and Ab2) were prepared against the purified proteins of stage V eggs and their specificity was demonstrated by radial immunoprecipitation, and Western blotting analysis. Two forms of vitellins were also found in stage V eggs after chromatography on Sepharose CL-2B column and hydroxylapatite and polyacrylamide gel electrophoresis.

Detection of white spot syndrome virus (WSSV) in the Pacific oyster Crassostrea gigas.

Oysters Crassostrea gigas were placed at water supply canals of three shrimp farms in Guasave, Mexico where WSSV outbreaks occur. Animals were sampled through April-August and September-December to detect WSSV DNA. By using three different PCR protocols, only oysters from a farm undergoing a WSSV outbreak were found WSSV-positive in gills and digestive gland. Two WSSV amplicons were sequenced and they corresponded over 99% to WSSV genome segments. Results showed that oysters can capture WSSV particles suspended in water. Susceptibility of oysters to WSSV infection and their role as a carrier remain to be determined.

Subchronic organismal toxicity, cytotoxicity, genotoxicity, and feeding response of pacific oyster (Crassostrea gigas) to lindane (γ‐HCH) exposure under experimental conditions

This study evaluated organismal toxicity, cytotoxicity, and genotoxicity and the filtration rate in response to different concentrations of subchronic lindane (gamma-hexachlorocyclohexane [gamma-HCH]), exposure (12 d) in adult Pacific oysters Crassostrea gigas. Oysters were exposed in vivo in laboratory aquaria to 10 different concentrations (0.0-10.0 mg/L) of gamma-HCH. The median lethal concentration (LC50) after 12 d was calculated as 2.22 mg/L. Cytotoxic effects were observed in hemocytes, where the mean cell viability was significantly decreased at 1.0 mg/L of gamma-HCH after 12 d. Genotoxicity of gamma-HCH measured by single cell gel electrophoresis assay, in hemocytes was evident at 0.7 mg/L of gamma-HCH after 12 d. After 4 h of exposure to gamma-HCH, filtration rates were reduced compared with controls to 65.8 and 38.2% at concentrations of 0.3 and 0.7 mg/L, respectively, and after 11 d of exposure, filtration rates were reduced to 60.4 and 30.9% at concentrations of 0.1 mg/L and higher. These results show the subchronic effects of gamma-HCH at different concentrations and effect sensitivities are categorized as filtration rate < genotoxicity < cytotoxicity < mortality. The relevance of integral toxicity evaluation, considering different endpoints from molecular, cellular, and individual levels is discussed.

Vitellogenin in black turtle (Chelonia mydas agassizii): Purification, partial characterization, and validation of an enzyme‐linked immunosorbent assay for its detection

Black turtle plasmatic vitellogenin (VTG) was purified from 17beta-estradiol-induced males using ion-exchange chromatography. The isolated protein was identified as VTG by its glycolipoprotein nature and amino acid sequence homology with other vertebrate VTG. It was characterized as a 500-kDa dimer composed of two identical, 200- to 240-kDa monomers. Polyclonal antibodies raised against black turtle VTG showed high titer and specificity, as demonstrated by enzyme-linked immunosorbent assay and Western blot analysis, respectively. The range of the assay was estimated to be between 15 ng/ml and 2 microg/ml, and the inter- and intra-assay coefficients of variation were 9.4 and 7.3%, respectively. Black turtle antibody cross-reacted with VTG of two other sea turtle species, Caretta caretta (loggerhead) and Eretmochelys imbricata (hawksbill), extending the applicability of the assay as part of a sea turtle health assessment program.

Effects of exposure to oxamyl, carbofuran, dichlorvos, and lindane on acetylcholinesterase activity in the gills of the Pacific oyster Crassostrea gigas.

Acetylcholinesterase (AChE) activity has been used to test the exposure of mollusk bivalves to pesticides and other pollutants. The Pacific oyster Crassostrea gigas is a species with a worldwide distribution, and it has a high commercial value. The use of this species as a bioindicator in the marine environment, and the use of measurements of AChE activity in tissues of C. gigas require prior evaluation of organisms exposed to several toxic compounds in the laboratory. In our study, the effects of pesticides on AChE activity in the gills and mantle tissues of C. gigas were analyzed by exposing animals to organophosphate (dichlorvos), carbamate (carbofuran and oxamyl), and organochlorine (lindane) pesticides. Adult Pacific oysters were exposed to several concentrations (0.1–200 μM) of dichlorvos, carbofuran, and oxamyl for 96 h, and lindane (1.0 and 2.5 μM) was applied for 12 days. In gill tissues, all pesticides analyzed caused a decrease in AChE activity when compared to the control unexposed group. The mean inhibition concentration (IC50) values were determined for dichlorvos, carbofuran, and oxamyl pesticides. Dichlorvos had the highest toxic effect, with an IC50 of 1.08 μM; lesser effects were caused by oxamyl and carbofuran, with IC50s of 1.67 and 3.03 μM, respectively. This study reports the effects of pesticides with several chemical structures and validates measurement of AChE activity in the gill tissues of C. gigas for use in environmental evaluations or food quality tests.

Pesticide Residues, Heavy Metals, and DNA Damage in Sentinel Oysters Crassostrea gigas From Sinaloa and Sonora, Mexico

Pesticides and heavy metals were analyzed in sentinel Crassostrea gigas oysters placed in six aquaculture sites close to a contaminated agricultural region. Each site was sampled twice. Tests revealed the presence of organochlorine (OC) pesticides in the oysters at concentrations varying from 31.8 to 72.5 μg/kg for gamma-hexachlorocyclohexane (γ-HCH); from 1.2 to 3.1 μg/kg for dichlorodiphenyldichloroethylene (4,4-DDE); from 1.6 to 2.3 μg/kg for endosulfan I; and from 1.4 to 41.2 μg/kg for endosulfan II, as well as heavy metals in concentrations that exceeded Mexican tolerance levels (405.5 to 987.8 μg/g for zinc; 4.2 to 7.3 μg/g for cadmium; and 7.2 to 9.9 μg/g for lead). Significant levels of DNA damage in oyster hemocytes were also detected. There was a significant, positive correlation between genotoxic damage and concentration of nickel or the presence of endosulfan II. Cellular viability evaluated by cytotoxic analyses was found to be high at 80%. Marked inhibition in activity of acetylcholinesterase (AChE ) and induction of glutathione S-transferase (GST) activity was noted. Data demonstrated a significant relation between AChE activity inhibition and presence of endosulfan II, γ-HCH, copper, lead, and 4,4-DDE, as well as between AChE and GST activity at different sites.

Crassostrea gigas oysters as a shrimp farm bioindicator of white spot syndrome virus

This study explored whether Crassostrea gigas oysters can be used as a bioindicator of white spot syndrome virus (WSSV) in shrimp farm water canals. Bioassays showed that C. gigas can accumulate WSSV in their gills and digestive glands but do not become infected, either by exposure to seawater containing WSSV or by cohabitation with infected shrimp. The use of a WSSV nested PCR to screen oysters placed in water canals at the entry of a shrimp farm allowed WSSV to be detected 16 d prior to the disease occurring. The finding that C. gigas can concentrate small amounts of WSSV present in seawater without being harmed makes it an ideal sentinel species at shrimp farms.

Validation of an enzyme‐linked immunosorbent assay for measuring vitellogenin in california halibut (Paralichthys californicus)

Vitellogenin (VTG) is the major protein present in the plasma of females undergoing oogenesis. In males, the VTG gene normally is suppressed; however, synthesis of VTG can be induced by exposure to xenoestrogenic compounds. In the present study, an enzyme-linked immunosorbent assay was developed and validated to evaluate VTG levels in the California halibut (Paralichthys californicus). Vitellogenin and lipovitellin (LV) were identified in the plasma of 17 beta-estradiol-induced females and in the ovaries of wild females, to our knowledge for the first time. Purified VTG from the plasma of induced females was obtained, and polyclonal antibodies against the LV of mature female ovaries was prepared and their specificity assessed by Western blot analysis. At Bahia Magdalena, Baja California Sur, Mexico, quantitative measurements of VTG in the plasma of female specimens were made during one reproductive cycle.