Evidence of the Existence of Site-Specific Female Contact Pheromones Involved in the Sexual Interaction Behavior of the Pacific Whiteleg Shrimp Penaeus vannamei.

Although the presence of female contact sex pheromones in P. vannamei has been hypothesized, to date its existence has not been proven. To gather more evidence of their existence, cuticular liposoluble extracts were obtained from the following samples of adult females to be used as the experimental treatments: (1) ventral exoskeleton of immature female (VI), (2) dorsolateral exoskeleton of immature female (DI), (3) ventral exoskeleton of mature female (VM), and (4) dorsolateral exoskeleton of mature female (DM). Polyvinyl chloride tubes (artificial females; AF) were coated with each extract and the behavior displayed by sexually mature males in contact with the AF was recorded and classified as follows: 0 = no response; 1 = contact; 2 = pushing; and 3 = prolonged contact (≥10 s). To test the hypothesis that the extracts collected from the ventral portion of the abdomen exoskeleton have a higher effect on the behavior of males than the extracts collected from the dorsolateral portion of the abdomen exoskeleton, the experiment was divided into two bioassays: Bioassay I (VI vs. DI) and Bioassay II (VM vs. DM). In each bioassay, all experimental treatments were significantly different (p > 0.05) from the CTL group (AF coated with hexane). Notably, the pushing behavior was significantly higher (p < 0.05) in the VI treatment compared to the CTL and DI treatment. These results provide evidence of the existence of contact female sex pheromones with sexual recognition function located primarily in the ventral portion of the abdomen exoskeleton of P. vannamei.

Water quality and the CO2-carbonate system during the preconditioning of Pacific oyster (Crassostrea gigas) in a recirculating aquaculture system.

The continued increase of the demand for seed of the Pacific oyster (Crassostrea gigas) has driven the aquaculture industry to produce land-based hatcheries using broodstock conditioning. This has led to the need to create closed systems to control the main factors involved in reproduction (temperature and food). Additionally, reproductive synchronization of broodstocks may be considered to ensure homogeneous maturation and spawning among the organisms. In this work, we synchronized the broodstock reproductive stage of Pacific oysters in a recirculating aquaculture system (RAS) using a "preconditioning" process and evaluated the effect of the water quality and the CO2-carbonate system on preconditioned broodstock. The oysters were kept at 12 °C for 45 days in a RAS containing a calcium reactor (C2) or without a calcium reactor (C1, control). Water quality parameters were measured daily, and the oyster's condition and reproductive development were monitored using condition index, biometrics, and histology, on Days 0, 20, and 45. C1 and C2 systems kept the water quality within the ranges reported as favorable for bivalves. The calcium reactor kept the pH (8.03-8.10), alkalinity (200 mg/L as CaCO3), CO32- (≤ 80 µmol/kg), and Ω aragonite (≤ 1) closer to the ranges reported as optimal for bivalves. However, no significant differences were detected in the total weight and the condition index in C1 and C2. The preconditioning allowed to maintain the organisms in early reproductive development, allowing gametogenesis synchronization to start maturation.

Characterization and localization of primordial germ cells in Totoaba macdonaldi.

The totoaba, Totoaba macdonaldi, is an endangered fish of the Gulf of California with high economic and ecological potential. Therefore, our purpose was to characterize the Primordial Germ Cells (PGCs) of this Sciaenid with two objectives: (1) to provide the basis for PGCs cryopreservation to preserve the genetic resources and (2) to take the first step to know the gonadal genesis and sex differentiation of totoaba. Immunofluorescence analysis performed from 2-cell stage to 8-day after hatch (DAH) shows that Vasa protein is specific for PGCs. These cells were first observed in the peripheral and dorsal regions of the blastodisc at approximately the 50%-epiboly stage and migrated to both sides of embryo body during the development. Finally, at 7 DAH the PGCs of the hatching embryo reached the place where the gonad will be developed. Histology analysis of larvae showed a genital ridge with enclosed PGCs on the dorsal side of the peritoneum at 9 DAH, gonadal primordium growth was observed at 11 DAH as a result of the interaction between PGCs and somatic cells derived from the peritoneum. Results of qPCR showed that vasa expression was restricted to the embryonic and early larval development, highest values were observed in 2-cell and mid-blastula stage suggesting the maternal inheritance of vasa mRNA. These findings support the hypothesis of preformation in T. macdonaldi PGCs. The migration pattern of PGCs allow us to recommend the isolation and subsequent cryopreservation of these cells before 7 DAH when the embryonic and larval development is given at 21 °C.

Assessment of gamete quality for the eastern oyster (Crassostrea virginica) by use of fluorescent dyes.

Evaluation of sperm motility is the single most widely used parameter to determine semen quality in mammals and aquatic species. While a good indicator for fresh sperm viability, post-thaw motility is not always effective at predicting fertilizing ability. Techniques using fluorescent dyes can assess functionality of mammalian sperm, but have not been widely applied in aquatic organisms. The eastern oyster Crassostrea virginica is an important mollusk in the United States, and cryopreservation protocols have been developed to preserve sperm and larvae to assist research and hatchery production. In this study, protocols were developed to assess sperm cell membrane integrity and mitochondrial function by flow cytometry and to assess viability of eggs by fluorescence microscopy. The fluorescent dyes SYBR 14 and propidium iodide (PI) (to assess membrane integrity) and rhodamine 123 (R123) (to assess mitochondrial membrane potential) were used to evaluate the quality of thawed oyster sperm previously cryopreserved with different cryoprotectant and thawing treatments. Membrane integrity results were correlated with motility of thawed sperm and mitochondrial membrane potential with fertilizing ability. Fluorescein diacetate (FDA) was used to assess cytotoxicity of cryoprotectant solutions and post-thaw damage to oyster eggs. The results indicated that membrane integrity (P=0.004) and thawing treatments (P=0.04), and mitochondrial membrane potential (P=0.0015) were correlated with motility. Fertilizing ability was correlated with cryoprotectant treatments (P=0.0258) and with mitochondrial membrane potential (P=0.001). The dye FDA was useful in indicating structural integrity of fresh and thawed eggs. Exposure of eggs, without freezing, to dimethyl sulfoxide yielded higher percentages of stained eggs and fertilization rate than did exposure to propylene glycol (P=0.002). Thawed eggs were not stained with FDA (<1%) and larvae were not produced.