Host Species and Environment Shape the Gut Microbiota of Cohabiting Marine Bivalves.

Pacific oysters (Crassostrea gigas) and Mediterranean mussels (Mytilus galloprovincialis) are commercially important marine bivalves that frequently coexist and have overlapping feeding ecologies. Like other invertebrates, their gut microbiota is thought to play an important role in supporting their health and nutrition. Yet, little is known regarding the role of the host and environment in driving these communities. Here, bacterial assemblages were surveyed from seawater and gut aspirates of farmed C. gigas and co-occurring wild M. galloprovincialis in summer and winter using Illumina 16S rRNA gene sequencing. Unlike seawater, which was dominated by Pseudomonadata, bivalve samples largely consisted of Mycoplasmatota (Mollicutes) and accounted for >50% of the total OTU abundance. Despite large numbers of common (core) bacterial taxa, bivalve-specific species (OTUs) were also evident and predominantly associated with Mycoplasmataceae (notably Mycoplasma). An increase in diversity (though with varied taxonomic evenness) was observed in winter for both bivalves and was associated with changes in the abundance of core and bivalve-specific taxa, including several representing host-associated and environmental (free-living or particle-diet associated) organisms. Our findings highlight the contribution of the environment and the host in defining the composition of the gut microbiota in cohabiting, intergeneric bivalve populations.

Methylmercury bioaccumulation in water flea Daphnia carinata by AIEgen.

Mercury ion (Hg2+) is a toxic heavy metal ion and Hg2+ is convertible to methylmercury (MeHg) by many aquatic microorganisms, leading to bioaccumulation and biomagnification in aquatic organisms, which can interfere with brain development and function in humans. This study employs a newly developed AIEgen (Aggregation-induced emission fluorogen) to quantify and visualise the process of MeHg bioaccumulation in vivo on the species of water flea Daphnia carinata. Two approaches to MeHg absorption were taken, either by direct incubation in a MeHg solution or by indirect consumption of algae contaminated with MeHg. We analysed the relationship between the ratio of photoluminescence (PL) intensities (I585/I480) and MeHg concentration (CMeHg) and generated a master curve for determining MeHg concentration based on the measurement of PL intensities. Fluorescent image analysis showed the occurrence of MeHg in D. carinata to be mainly in the compound eyes, optic nerve and carapace. This study indicates that MeHg absorption can be quantified and visualised in the body of zooplankton, and the MeHg transfer to zooplankton is more likely through direct exposure than via indirect food intake. The accumulation of MeHg in the eye and the nervous system could be the cause of the high mortality of D. carinata exposed to MeHg in water.

Comparison of partial replacement of fishmeal with soybean meal and EnzoMeal on growth performance of Asian seabass Lates calcarifer.

This study explored the impact of fishmeal replacement by commercial soybean meal (SM) and EnzoMeal (EZM) on Asian seabass Lates calcarifer growth performance using six diets. The six diets comprised two sources of plant proteins with three levels each, including 300 g kg-1 soybean meal (SM30), 300 g kg-1 EnzoMeal (EZM30), 400 g kg-1 soybean meal (SM40), 400 g kg-1 EnzoMeal (EZM40), 500 g kg-1 soybean meal (SM50), and 500 g kg-1 EnzoMeal (EZM50). The soybean level was shown to significantly affect the final fish weight, weight gain, specific growth rate (SGR), survival, feed intake, feed conversion ratio (FCR), and apparent digestibility coefficient (ADC). Further, the plant meal type significantly affected the final weight, weight gain, feed intake, ADC, and body lipid content. The highest final weight was observed in the SM30 group, and the lowest final weight was in the EZM50 group. Fish fed EZM had lower body weight than those fed soybean meal at the same inclusion level. However, once the fish had adapted to the EZM diet the fish weight variation was low. At the 300 g kg-1 and 400 g kg-1 inclusion levels the fish fed EZM showed significantly higher ADC than those fed soybean. The pepsin activity of fish fed EZM at 300 g kg-1 and 400 g kg-1 was higher than those fed soybean meal at the same levels. The enterocyte height in the hindgut of fish fed SM40 and SM50 was significantly higher than those fed EZM40 and EZM50, respectively. This study indicates that EZM could be a potential source of plant protein to replace fishmeal in fish feed as it contains high protein and low anti-nutritional factors. However, the major endpoint measurements on fish performance suggest that low feed intake constrains further EZM inclusion beyond 300 g kg-1 in the diet.

Improvement of post-thaw sperm survivals using liquid nitrogen vapor in a spermcasting oyster Ostrea angasi.

Low survival of cryopreserved sperm impedes the application of cryopreservation technique in spermcasting oyster species. This study developed a simple method of liquid nitrogen vapor freezing to improve post-thaw sperm survival in the spermcasting oyster Ostrea angasi. The results indicate that the permeable cryoprotectants, dimethyl sulfoxide (DMSO), ethylene glycol (EG) and propylene glycol (PG) were non-toxic to sperm up to 20% concentration and 90 min exposure whereas methanol at 10% or higher was toxic to sperm for any exposure over 30 min. Among the treatments with permeable cryoprotectants, 15% EG produced the highest post-thaw sperm motility. Sperm motility was further improved by the addition of non-permeable cryoprotectants (trehalose and glucose), with 15% EG + 0.2 M trehalose resulting in the highest post-thaw sperm motility among all the combinations evaluated. The durations of 20, 30 and 60 min equilibrations produced a higher post-thaw sperm motility and plasma membrane integrity (PMI) than 10 min. Higher post-thaw motility and PMI were achieved by freezing sperm at the 8 cm height from the liquid nitrogen surface than at the 2, 4, 6, 10 or 12 cm height. Holding sperm for 10 min in liquid nitrogen vapor produced higher post-thaw motility and PMI than for 2, 5 or 20 min. The cryopreservation protocol developed in this study improved both post-thaw motility and PMI of O. angasi sperm at least 15% higher than those cryopreserved using programmable freezing method. Liquid nitrogen vapor freezing might have greater applicability in improving post-thaw sperm quality of spermcasting oyster species.

Sperm cryopreservation in the spermcasting Australian flat oyster Ostrea angasi by a programmable freezing method.

Cryopreservation offers long-term storage of gametes without constraint from seasonal gamete maturation, provides opportunities to improve the efficiency of breeding and genetic programs, and protects endangered species from extinction due to epidemic diseases and natural disasters. In this study, a protocol for cryopreserving sperm of the spermcasting Australian flat oyster Ostrea angasi was developed by optimizing key factors influencing the quality of cryopreserved sperm. Dimethyl sulfoxide (DMSO) was non-toxic to sperm within the concentration and duration assessed in the toxicity experiment whereas 10% methanol or a higher concentration was toxic to sperm from the exposure duration of 30 min onwards. DMSO produced higher post-thaw sperm motility among the treatments with a single cryoprotectant. The inclusion of trehalose or glucose with DMSO further increased the post-thaw sperm motility (%) and plasma membrane integrity (PMI). Sperm equilibrated for 30 min showed higher post-thaw motility and PMI than those for 10 or 50 min. Higher post-thaw sperm motility and PMI were achieved at the freezing rate of -3 °C/min than at -7 °C/min. Sperm packaged in 0.5 ml straws had a higher post-thaw motility and PMI than those packaged in 0.25 ml straws. In this study, 44.4% post-thaw sperm motility and 49.2% PMI were achieved when sperm were equilibrated in 10% DMSO +0.45 M trehalose for 30 min, packaged in 0.5 ml straws, frozen at -3 °C/min from 4 °C to -80 °C, and thawed at 40 °C for 8 s. The availability of viable cryopreserved sperm would open an option for future breeding and genetic improvement programs for the spermcasting Australian flat oyster.

Spermatozeugmata structure and dissociation of the Australian flat oyster Ostera angasi: Implications for reproductive strategy.

Variation in reproductive strategy is one of the key factors contributing to recruitment success of molluscs in different habitats. Spermcasting is a unique mode in mollusc reproduction where males produce spermatozeugmata, a radially arrayed sperm cluster wrapped by gelatinous membrane. In this study, spermatozeugmata structure and their dissociation in the Australian flat oyster Ostrea angasi were investigated to elucidate the reproductive strategy in spermcasting molluscs. The histological observation indicated that spermatogonia gradually aggregated in the gonad follicle at the early gonad development stages and developed into spermatozeugmata and became tightly packed at the advanced stages. Even though mature male and female gametes could be found in a hermaphroditic individual, the animal may prevent self-fertilization by shedding different sex gametes at different time. The O. angasi sperm are similar in size and shape to broadcasting oysters, but have one additional mitochondrion. Variations in maintaining spermatozeugmata integrity and sperm motility between individuals depended on the level of masculinity or femineity. The durations of spermatozeugmata dissociation and sperm viability were longer in males than in hermaphrodites. The unique structure and capability for spermatozeugmata to maintain the functional integrity after spawning have adaptive significance for fertilization and gamete dispersal in this species.

Sperm cryopreservation of the Indian major carp, Labeo calbasu: effects of cryoprotectants, cooling rates and thawing rates on egg fertilization.

A sperm cryopreservation protocol for the Indian major carp, Labeo calbasu, was developed for long-term preservation and artificial fertilization. Milt collected from mature male fish were placed in Alsever's solution (296mOsmolkg(-1)) to immobilize the sperm. Cryoprotectant toxicity was evaluated by motility assessment with dimethyl sulfoxide (DMSO) and methanol at 5, 10 and 15% concentrations. DMSO was more toxic at higher concentrations than methanol, and consequently 15% DMSO was excluded from further study. A one-step cooling protocol (from 5 to 80°C) with two cooling rates (5 and 10°C/min) was carried out in a computer-controlled freezer (FREEZE CONTROL(®) CL-3300; Australia). Based on post-thaw motility, the 10°C/min cooling rate with either 10% DMSO or 10% methanol yielded significantly higher (P=0.011) post-thaw motility than the other rate and cryoprotectant concentrations. Sperm thawed at 40°C for 15s and fresh sperm were used to fertilize freshly collected L. calbasu eggs and significant differences were observed (P=0.001) in percent fertilization between cryopreserved and fresh sperm as well as among different sperm-to-egg ratios (P=0.001). The highest fertilization and hatching rates were observed for thawed sperm at a sperm-to-egg ratio of 4.1×10(5):1. The cryopreservation protocol developed can facilitate hatchery operations and long-term conservation of genetic resources of L. calbasu.