The multi-omics analysis in the hepatopancreas of Eriocheir sinensis provides novel insights into the response mechanism of heat stress.

Under global warming, heat stress can induce the excessive production of reactive oxygen species, causing irreversible damage to aquatic animals. It is essential to predict potentially harmful impacts on aquatic organisms under heat stress. Eriocheir sinensis, a typical crustacean crab, is widely distributed in China, American and Europe. Parent E. sinensis need migrate to the estuaries to reproduce in winter, and temperature is a key environmental factor. Herein, we performed a comprehensive transcriptomic and proteomic analysis in the hepatopancreas of E. sinensis under heat stress (20 °C and 30 °C), focusing on heat shock protein family, antioxidant system, energy metabolism and immune defense. The results revealed that parent E. sinensis generated adaptative responses to maintain physiological function under 20 °C stress via the transcriptional up-regulation of energy metabolism enzymes, mRNA synthesis and heat shock proteins. The transcriptional inhibition of key enzymes related to energy metabolism implied that 30 °C stress may lead to the dysfunction of energy metabolism in parent E. sinensis. Meanwhile, parent E. sinensis also enhanced the expression of ferritin and phospholipase D at translational level, and the glutathione s-transferase and heat shock protein 70 at both transcriptional and translational levels, speculating that parent E. sinensis can strengthen antioxidant and immune capacity to resist oxidative stress under 30 °C stress. This study elucidated the potential molecular mechanism in response to heat stress of parent E. sinensis hepatopancreas. The preliminary selection of heat tolerance genes or proteins in E. sinensis can provide a reference for the population prediction and the study of evolutionary mechanism under heat stress in crabs.

High-throughput sequencing and fatty acid profile analyses of the Black Amur bream (Megalobrama terminalis) reveal variation in dietary niche associated with geographic segregation.

Fish dietary niche is a core focus, and it reflects the diversity of resources, habitats, or environments occupied by a species. However, whether geographic segregation among different populations triggers dietary diversification and concomitant fish niche shift remains unknown. In the present study, we selected the Black Amur bream (Megalobrama terminalis) is a migratory fish species that plays an important role in the material transfer and energy cycling of river ecosystems, inhabiting southern China drainage with multiple geographic populations. Here, we utilized the combined analyses of 18S rDNA high-throughput sequencing in fish gut contents and fatty acid (FA) in muscle tissues to evaluate potential spatial patterns of habitat and resource use for M. terminalis in three rivers of southern China. Our results showed that prey items of the Xijiang (XR) population (Pearl River) exhibited the highest species diversity and richness among the three geographic populations. Moreover, diet composition of M. terminalis was affected by spatial differences associated with geographic segregation. Analyses of FA biomarkers indicated that the highest levels of C16:0, C18:3n-3, and C18:2n-6c were found in Wanquan (WS) population (Wanquan River). The XR population exhibited a distinct FA profile characterized by higher amounts of arachidonic acid (ARA) and docosahexaenoic acid (DHA). The Moyang (MY) population (Moyang River) acted as the linkage between WS and XR populations and consisted of middle levels of saturated FAs (SFAs) and polyunsaturated FAs (PUFAs). The XR population displayed a greater FA niche width compared with WS population. Furthermore, we observed a close positive relationship between the niche width and α-diversity indices of dietary resources for FA proflies. Our study provides valued information to develop different conservation strategies among different populations and improve fisheries management for M. terminalis and other endemic species in local rivers.

The Influence of Shelter Type and Coverage on Crayfish (Procambarus clarkii) Predation by Catfish (Silurus asotus): A Controlled Environment Study.

Procambarus clarkii is adept at using natural shelters and caves to evade attacks from predators. However, the concealment abilities and mechanisms of P. clarkii for different types of shelters under predation pressure have not yet been reported. In this study, laboratory experiments were carried out to determine the effects of different coverages (25%, 50%, and 75%) and different combinations (I-VII) of three types of shelters (PVC pipes, water grass, and stone) on the predation rhythm, behavior, and abilities of Silurus asotus on P. clarkii. The results indicated that the predation of S. asotus on P. clarkii exhibited significant rhythmicity under shelter conditions, excluding PVC pipes, 75% stone, and combination VI. Among the three types of shelters, PVC pipes provided the strongest concealment, followed by stone and water grass. With the increase in shelter coverage, the anti-predation ability of P. clarkii continued to increase, and the optimal shade rate for water grass was 50%. In the different shelter combinations, the environmental complexity had little effect on the predation activity of S. asotus on P. clarkii. These findings demonstrated that the type and abundance of shelters in the wild environment can affect the predation rhythm and activities of S. asotus on P. clarkii.

Effects of Antarctic Krill Meal in Diet on Reproductive Performance and Embryo Quality of Eriocheir sinensis.

A 60-day feeding trial was conducted to evaluate the impact of dietary Antarctic krill meal on the reproductive performance and embryo quality of the Chinese mitten crab, Eriocheir sinensis. Three diets were formulated, incorporating varying levels of Antarctic krill meal at 0% (Diet K0), 10% (Diet K10), and 20% (Diet K20), with a control group fed razor clam Sinonovacula constricta. Each diet was randomly assigned to three replicate tanks, each stocked with 5 males and 10 females. Male and female weights were 145.38 ± 8.01 and 102.57 ± 9.73 g, respectively. The results revealed no significant differences in weight gain rate, specific growth rate, and survival rate. However, the hepatopancreatic weight and hepatopancreas index of female crabs in each group decreased, while gonadal weight and gonadosomatic index increased significantly after 60 days, with Diet K20 showing the highest values. Egg production and fecundity of female crabs reached their peak in Diet K20, with no significant differences in reproductive indices among all groups. The phospholipid content in Diet K20 was significantly higher than in the other groups (P < 0.05). Cholesterol contents in Diet K0 and the control group were significantly higher than in Diet K10 and K20 (P < 0.05). No significant differences were observed in egg diameter, egg weight, moisture, crude protein, and crude fat between the groups. The content of C20 : 2 and C20 : 4n6 was highest in Diet K0, with a significant difference compared to Diet K10 (P < 0.05). However, no significant differences were found in the total content of saturated fatty acids, monounsaturated fatty acids, and polyunsaturated fatty acids among all groups. Based on the research findings, it is recommended that the optimal level of Antarctic krill meal in diets is 20%.

Effects of Hypoxia Stress on Survival, Antioxidant and Anaerobic Metabolic Enzymes, and Related Gene Expression of Red Swamp Crayfish Procambarus clarkii.

The red swamp crayfish Procambarus clarkii is the most reared shrimp in China, but it is often affected by hypoxia stress in the process of seedling culture and adult crayfish culture. The oxygen consumption rate and asphyxiation point of juvenile crayfish (1.17 ± 0.03 g) and subadult crayfish (11.68 ± 0.11 g) at different temperatures (20, 22, 24, 26, and 28 °C) were studied. The survival, glycolysis, and expression of antioxidant genes were compared under 24 h acute hypoxia stress (1, 2, and 3 mg/L) and normal dissolved oxygen (7.5 mg/L). The results showed that the oxygen consumption rate and asphyxiation point of juvenile and subadult crayfish increased with increasing temperatures (20-28 °C). At the same temperature, the oxygen consumption rate and asphyxiation point of juvenile crayfish were significantly higher than those of subadult crayfish (p < 0.05). Within 24 h, the three hypoxia stress environments did not lead to the death of crayfish, indicating that P. clarkii has a strong ability to adapt to hypoxia. Hypoxia stress significantly affected the activities of antioxidant and anaerobic metabolic enzymes and gene expression in juvenile and subadult crayfish. The activities of the superoxide dismutase (SOD), catalase (CAT), and lactate dehydrogenase (LDH) and the content of lactic acid (LD) in the hepatopancreas of juvenile and subadult crayfish in the hypoxia stress groups increased significantly. The expression levels of SOD mRNA, CAT mRNA, Hsp70 mRNA, and crustin 4 mRNA in the hepatopancreas of juvenile and subadult crayfish in the hypoxia stress groups were significantly higher than those in the control group (p < 0.05), and the higher the degree of hypoxia stress, the higher the expression of each gene. The results showed that the antioxidant system of juvenile crayfish was more sensitive to hypoxia environments, and hypoxia stress resulted in increased stress levels in juvenile crayfish and subadult crayfish.

Co-Self-Assembled Monolayers Modified NiOx for Stable Inverted Perovskite Solar Cells.

[4-(3,6-dimethyl-9H-carbazol-9yl)butyl]phosphonic acid (Me-4PACz) self-assembled molecules (SAM) are an effective method to solve the problem of the buried interface of NiOx in inverted perovskite solar cells (PSCs). However, the Me-4PACz end group (carbazole core) cannot forcefully passivate defects at the bottom of the perovskite film. Here, a Co-SAM strategy is employed to modify the buried interface of PSCs. Me-4PACz is doped with phosphorylcholine chloride (PC) to form a Co-SAM to improve the monolayer coverage and reduce leakage current. The phosphate group and chloride ions (Cl-) in PC can inhibit NiOx surface defects. Meantime, the quaternary ammonium ions and Cl- in PC can fill organic cations and halogen vacancies in the perovskite film to enable defects passivation. Moreover, Co-SAM can promote the growth of perovskite crystals, collaboratively solve the problem of buried defects, suppress nonradiative recombination, accelerate carrier transmission, and relieve the residual stress of the perovskite film. Consequently, the Co-SAM modified devices show power conversion efficiencies as high as 25.09% as well as excellent device stability with 93% initial efficiency after 1000 h of operation under one-sun illumination. This work demonstrates the novel approach for enhancing the performance and stability of PSCs by modifying Co-SAM on NiOx.

Integration of Transcriptomics and Proteomics Analysis Reveals the Molecular Mechanism of Eriocheir sinensis Gills Exposed to Heat Stress.

Heat stress is an increasingly concerning topic under global warming. Heat stress can induce organisms to produce excess reactive oxygen species, which will lead to cell damage and destroy the antioxidant defense of aquatic animals. Chinese mitten crab, Eriocheir sinensis, is sensitive to the change in water temperature, and parent crabs are more vulnerable during the breeding stage. In the present study, the multi-omics responses of parent E. sinensis gills to heat stress (24 h) were determined via transcriptome and proteome. The integrative analysis revealed that heat shock protein 70 (HSP70) and glutathione s-transferase (GST) were significantly up-regulated at gene and protein levels after heat stress, indicating that HSP70 and the antioxidant system participated in the regulatory mechanism of heat stress to resist oxidative damage. Moreover, the "Relaxin signaling pathway" was also activated at gene and protein levels under 30 °C stress, which implied that relaxin may be essential and responsible for reducing the oxidative damage of gills caused by extreme heat stress. These findings provided an understanding of the regulation mechanism in E. sinensis under heat stress at gene and protein levels. The mining of key functional genes, proteins, and pathways can also provide a basis for the cultivation of new varieties resistant to oxidative stress.

Triazophos exposure on maternal Daphnia magna at environmental-related concentrations revealed toxic effects to its offspring.

Due to chemical and photochemical stability, triazophos has been frequently detected in rivers and oceans over the years with extensive use for pest control in agriculture, and it has become a worldwide ecological concern to the aquatic environment. Until now, fewer data are available regarding the potential long-term adverse effects of triazophos on aquatic invertebrates, which plays an essential role in aquatic food webs, as a key group for water ecosystems. In this experiment, the F1- and F2 progenies of Daphnia magna were recovered when daphnias (F0) exposure to triazophos at environmental-related concentrations (0.1 and 1.0 µg/L) for 21 d; and the indexes related to phenotypic traits, reproduction and gene expression were measured in tested animals. The results showed that heart rate and total number of neonates in exposed F0-daphnias were significantly lower than those of control group, and the detoxification genes (HR96 and P-gp) were up-regulated while genes related reproduction (Vtg) and molting (Nvd and Shd) were significantly down-regulated. The heart rate and individual size of F1-daphnias (<24 h) were significantly reduced in the treatment group. After 21-d recovery, the heart rate and expression of HR96, P-gp, Vtg, Nvd and Shd were declined in F1-daphnias. There was no obvious difference of morphological traits and heart rate between treatment and control in F2-daphnias (<24 h). In summary, daphnias (F0) exposure to triazophos with environmental dose could raise toxic effects on its offspring (F1), which is mainly manifested by reduced heart rate, the accumulated number and individual size of offspring and decreased expression of genes related to molting and reproduction.

A novel C-type lectin (SpccCTL) suppresses MCRV replication by binding viral protein and regulating antiviral peptides in Scylla paramamosain.

Pattern recognition receptors (PRRs) play a crucial role in the recognition and activation of innate immune responses against invading microorganisms. This study characterizes a novel C-type lectin (CTL), SpccCTL. The cDNA sequence of SpccCTL has a full length of 1744 bp encoding a 338-amino acid protein. The predicted protein contains a signal peptide, a coiled-coil (CC) domain, and a CLECT domain. It shares more than 50 % similarity with a few CTLs with a CC domain in crustaceans. SpccCTL is highly expressed in gills and hemocytes and upregulated after MCRV challenge, suggesting that it may be involved in antiviral immunity. Recombinant SpccCTL (rSpccCTL) as well as two capsid proteins of MCRV (VP11 and VP12) were prepared. Pre-incubating MCRV virions with rSpccCTL significantly suppresses the proliferation of MCRV in mud crabs, compared with the control (treatment with GST protein), and the survival rate of mud crabs is also significantly decreased. Knockdown of SpccCTL significantly facilitates the proliferation of MCRV in mud crabs. These results reveal that SpccCTL plays an important role in antiviral immune response. GST pull-down assay result shows that rSpccCTL interacts specifically with VP11, but not to VP12. This result is further confirmed by a Co-IP assay. In addition, we found that silencing SpccCTL significantly inhibits the expression of four antimicrobial peptides (AMPs). Considering that these AMPs are members of anti-lipopolysaccharide factor family with potential antiviral activity, they are likely involved in immune defense against MCRV. Taken together, these findings clearly demonstrate that SpccCTL can recognize MCRV by binding viral capsid protein VP11 and regulate the expression of certain AMPs, suggesting that SpccCTL may function as a potential PRR playing an essential role in anti-MCRV immunity of mud crab. This study provides new insights into the antiviral immunity of crustaceans and the multifunctional characteristics of CTLs.

Correlation analysis of migration in Eriocheir sinensis of the Yangtze River estuary based on salinity preference.

Reproduction and development are the most important stages in the life of aquatic animals, which are also the most sensitive stages to environmental stress. As a migratory crab, parent Eriocheir sinensis need to migrate to estuary (brackish water) for reproduction, and megalopas need to migrate to freshwater for development. Yangtze River estuary is located at the junction of the Yangtze River and the East China Sea, and the salinity of different regions varies greatly (salinity 0-30‰). Therefore, salinity is one of the key environmental factors affecting E. sinensis. In this study, the salinity preference of parent and megalopas E. sinensis was observed and analyzed by the six-chambered and seven-chambered devices of laboratory experiments. Two groups of alternative salinity were set for parent crabs (7, 14, 21, 28, and 35 ‰) and megalopas (0, 5, 10, 15, 20, 25, and 30‰). The results indicated that males showed no obvious preference for salinity, while females had obvious preference for different salinity and stronger dependence and tolerance to saltwater. Megalopas showed a significant preference for low salinity which was beneficial to migrating to freshwater quickly. It was speculated that the behavior of megalopas tending to freshwater was an inherited behavior in the evolutionary process rather than influenced by environment. The salinity preference of E. sinensis might change the position of spawning grounds and migratory routes, and then the annual fluctuation of salinity in Yangtze River estuary will affect the change of E. sinensis resources.

Correlation between the Density of Acipenser sinensis and Its Environmental DNA.

Since the construction of the Gezhouba Dam, Chinese sturgeon (Acipenser sinensis) numbers have gradually declined, rendering this species critically endangered according to the International Union for the Conservation of Nature. Environmental DNA (eDNA) technology plays an important role in monitoring the abundance of aquatic organisms. Species density and biomass have been proven to be estimable by researchers, but the level of accuracy depends on the specific species and ecosystem. In this study, juvenile A. sinensis, an endangered fish, were selected as the research target. Under controlled laboratory conditions in an aquarium, one, two, four, six, and eight juvenile A. sinensis were cultured in five fish tanks, respectively. Water samples were filtered at eight different time points for eDNA content analysis. Additionally, eDNA yield was tested at six different time points after a 0.114 ind./L density of A. sinensis was removed, and the employed degradation model was screened using the Akaike information criterion (AIC) and the Bayesian information criterion (BIC). The results showed that eDNA content remained stable after 3 days and exhibited a significant positive linear correlation with the density of A. sinensis (R2 = 0.768~0.986). Furthermore, eDNA content was negatively correlated with the 3-day period after the removal of A. sinensis. The power function had the smallest AIC and BIC values, indicating better fitting performance. This study lays a momentous foundation for the application of eDNA for monitoring juvenile A. sinensis in the Yangtze Estuary and reveals the applicability of eDNA as a useful tool for assessing fish density/biomass in natural environments.

Salinity-Driven Changes in Behavioral Responses of Catadromous Eriocher sinensis.

The effects of salinity on behavior are far-reaching, and Eriocheir sinensis showed disparate behaviors under different salinity conditions. Female crabs were more active in saline water, especially low salinity stress, which is beneficial for female crabs to escape from the low-salinity environment quickly. Then, antennal movement indicated that antennae might be the main osmoreceptors in E. sinensis, and 65 min might be a good starting time for salinity stress to analyze osmoregulation in crabs. Interestingly, E. sinensis had obvious behavioral differences in the high and low salinity stress, and behaviors were more intense in a salinity dip from salinity 18 to salinity 0. This study analyzed the osmoregulatory process of catadromous E. sinensis in different salinity from the point of osmoregulatory organ and behavioral response. These results will provide a scientific basis for the osmoregulatory mechanism of E. sinensis, which are conducive to evaluating and analyzing the impact of saltwater intrusion in the Yangtze River estuary on resource fluctuation.

Preference for Shelters at Different Developmental Stages of Chinese Mitten Crab (Eriocheir sinensis).

A shelter is a good habitat for aquatic organisms, which could aid in avoiding cannibalism and facilitate predation. Chinese Mitten Crab (Eriocheir sinensis) is an important aquaculture species with troglodytism and nocturnal habit. To clarify the preference for shelters at different developmental stages of E. sinensis, different shelters (mud, sand, grass and rocks) were selected for comparison. These results indicated that juvenile crabs had a significant preference for grass; button-sized crabs preferred to hide in mud; and the favorite shelters for parent crabs were rocks, followed by mud. E. sinensis in three stages all showed concealing behavior. The concealing behavior of juvenile crabs was the most significant, followed by button-sized and parent crabs. Additionally, E. sinensis held a low hiding rate at night but a high hiding rate during the daytime due to nocturnal habits. These findings will help to better understand the habits of E. sinensis and provide a reference for resource restoration, habitat construction and the restoration of E. sinensis.

PvML1 suppresses bacterial infection by recognizing LPS and regulating AMP expression in shrimp.

Toll and Toll-like receptors (TLRs) play essential roles in the innate immunity of Drosophila and mammals. Recent studies have revealed the presence of Toll-mediated immune signaling pathways in shrimp. However, the recognition and activation mechanism of Toll signaling pathways in crustaceans remain poorly understood due to the absence of key recognition molecules, such as peptidoglycan recognition proteins. Here, a novel MD2-related lipid-recognition (ML) member named PvML1 was characterized in Penaeus vannamei. We found that PvML1 shared a similar 3D structure with human MD2 that could specifically recognize lipopolysaccharides (LPS) participating in LPS-mediated TLR4 signaling. PvML1 was highly expressed in hemocytes and remarkably upregulated after Vibrio parahemolyticus challenge. Furthermore, the binding and agglutinating assays showed that PvML1 possessed strong binding activities to LPS and its key portion lipid A as well as Vibrio cells, and the binding of PvML1 with bacterial cells led to the agglutination of bacteria, suggesting PvML1 may act as a potential pathogen recognition protein upon interaction with LPS. Besides, coating V. parahemolyticus with recombinant PvML1 promoted bacterial clearance in vivo and increased the survival rate of bacterium-challenged shrimp. This result was further confirmed by RNAi experiments. The knockdown of PvML1 remarkably suppressed the clearance of bacteria in hemolymph and decreased the survival rate of infected shrimp. Meanwhile, the silencing of PvML1 severely impaired the expression of a few antimicrobial peptides (AMPs). These results demonstrated the significant correlation of bacterial clearance mediated by PvML1 with the AMP expression. Interestingly, we found that PvML1 interacted with the extracellular region of PvToll2, which had been previously shown to participate in bacterial clearance by regulating AMP expression. Taken together, the proposed antibacterial model mediated by PvML1 might be described as follows. PvML1 acted as a potential recognition receptor for Gram-negative bacteria by binding to LPS, and then it activated PvToll2-mediated signaling pathway by interacting with PvToll2 to eliminate invading bacteria through producing specific AMPs. This study provided new insights into the recognition and activation mechanism of Toll signaling pathways of invertebrates and the defense functions of ML members.

SpSR-B2 functions as a potential pattern recognition receptor involved in antiviral and antibacterial immune responses of mud crab Scylla paramamosain.

Although class B scavenger receptors (SR-Bs) in mammals are multifunctional molecules, the functions of SR-Bs in invertebrates remain largely unknown. In this study, we characterized an SR-B homolog, namely SpSR-B2, from Scylla paramamosain. SpSR-B2 shared high similarity with mammalian SR-Bs, and exhibited specific binding activity to ac-LDL, indicating that it may be a new member of SR-B class in invertebrates. SpSR-B2 was upregulated after challenge with white spot syndrome virus (WSSV) or bacteria. Binding assays showed that SpSR-B2 specifically interacted with WSSV envelope protein VP24. Besides, SpSR-B2 could bind to all tested bacterial cells and agglutinate these bacteria. SpSR-B2 also exhibited a strong binding activity to LPS but weak binding activities to other tested polysaccharides. These findings indicated that SpSR-B2 was a potential recognition molecule for viral protein VP24 and bacterial LPS. Knockdown of SpSR-B2 resulted in dramatically decreased expressions of certain antimicrobial peptides (AMPs), and overexpression of SpSR-B2 led to the increased expression of the AMP of SpALF2, suggesting that SpSR-B2 could regulate the expression of AMPs. Taken together, this study revealed that SpSR-B2 functioned as a potential pattern recognition receptor participating in antiviral and antibacterial immunity, and provided new insights into the immune functions of invertebrate SR-Bs.

Transcriptome analysis of pacific white shrimp (Penaeus vannamei) intestines and hepatopancreas in response to Enterocytozoon hepatopenaei (EHP) infection.

Penaeus vannamei is the most economically important species of shrimp cultured worldwide. Enterocytozoon hepatopenaei (EHP) is an emerging pathogen that severely affects the growth and development of shrimps. In this study, the transcriptome differences between EHP-infected and uninfected shrimp were investigated through next-generation sequencing. The unigenes were assembled with the reads from all the four libraries. The differentially expressed genes (DEGs) of intestines and hepatopancreas were analyzed. There were 2,884 DEGs in the intestines and 2,096 DEGs in the hepatopancreas. The GO and KEGG enrichment analysis indicated that DEGs were significantly enriched in signaling pathways associated with nutritional energy metabolism and mobilizing autoimmunity. Moreover, the results suggested the downregulation of key genes in energy synthesis pathways contributed greatly to shrimp growth retardation; the upregulation of immune-related genes enhanced the resistance of shrimp against EHP infection. This study provided identified genes and pathways associated with EHP infection revealing the molecular mechanisms of growth retardation.

Effects of vitrification protocol on the lactate dehydrogenase and total atpase activities of chinese mitten crab Eriocheir sinensis embryos.

BACKGROUND: Vitrification is the most promising option for the cryopreservation of fish embryos but requires high concentrations of potentially toxic cryoprotectants that can also cause cell injury, and affect cell division, enzymatic activities and cell metabolism. The effect of cryopreservation on the enzyme activity in crustacean embryos has not been reported. OBJECTIVES: The aim of this study was to investigate the effects of a vitrification protocol on the enzyme activity of different stages of Eriocheir sinensis embryos. The goal was to select an appropriate stage and vitrifying solution for the cryopreservation of embryos from this crustacean. MATERIALS AND METHODS: Embryos from E.sinensis at five development stages (cleavage, blastula, gastrula, eyed stage and heart beating stage) were submitted to six kinds of cryoprotectant incorporation protocol with a five stepwise method. Six vitrifying solutions were prepared by combining cryoprotectants PG, MeOH, Me(2)SO and DMF in different proportions (A: 20 % PG + 20 % DMF; B: 20 % MeOH+20 % DMF; C: 20 % PG+20 % MeOH; D:20 % PG+10 % MeOH+10 % DMF; E:20 % Me2SO+20 % PG;F:20 % Me(2)SO+20 % MeOH). After incubation in the six kinds of vitrification solutions, embryos were loaded into cryo-tubes and plunged into liquid nitrogen. The activities of two cytoplasmic enzymes, LDH and total ATPase were analyzed in control embryos, those subjected to the cryoprotectant solutions and in frozen/thawed embryos. RESULTS: The cryoprotectant incorporation protocol had important effects on the enzymatic activities, and different vitrifying solutions had distinct effects on the enzymatic activities. The early stage embryo was sensitive to the toxic effect of the cryoprotectants, with a significant drop in total ATPase in comparison with fresh, control embryos. Enzymatic activities dropped significantly after vitrification, indicating cell damage and loss of cytoplasmic enzymes. CONCLUSION: The composition of vitrifying solutions had different effects on the loss of the enzyme activity, and the later stage embryo was more resistant to the effect of vitrification.

Effects of extracellular environment factors on the motility in Japanese eel spermatozoa.

We have examined the effects in vitro of the ionic composition, pH and temperature on the motility by the spermatozoa of the Japanese eel (Anguilla japonica). Milt was obtained from 10 males that had been artificially matured by repeated injections of hCG. Sperm motility was monitored with a VHS video recorder and a video camera connected to a microscope. The results showed that most of the sperm were highly motile in 250-700 mM NaCl, 250-650 mM KCl and 350-550 mM CaCl2 solution. The longest duration of sperm motility recorded in 500 mM NaCl, 250 mM KCl and 350 mM CaCl2 solution. Sperm was not motile when suspended at pH 2, sperm motility was observed at pH 3, there was a relatively higher percentage of motile sperm in solutions at pH 4-12 (above 80%). The motility and duration increased within 18-24°C and decreased at the range of 24-30°C. Appropriate K+ ion concentration in the active medium could enhance the percent motility and duration of eel spermatozoa.

[Lead accumulation and elimination in juvenile Acipenser sinensis tissues].

A semi-static test of Pb exposure (0, 0.2, 0.8, and 1.6 mg L(-1)) was carried out to examine the Pb accumulation and elimination in different tissues of juvenile Chinese sturgeon (Acipenser sinensis). With increasing Pb concentration in water, the Pb concentration in juvenile A. sinensis tissues increased, showing a clear dose-response relationship. The Pb accumulation had a trend of being higher in bone (dorsalbone plate and cartilaginous bone) and muscle, followed by in stomach, intestine, and skin, and relatively low in liver, gill, and notochord. A 6-week duration test after Pb exposure showed that in low dose group (0.2 mg x L(-1)), the Pb concentration in all tissues had no significant differences to the control group, but in medium dose group (0.8 mg x L(-1)), the Pb concentration in most tissues except gill stomach, cartilaginous bone, and muscle was significantly higher than the control. In high dose group (1.6 mg x L(-1)), the Pb concentration in the tissues other than liver, intestine and skin was significantly higher than the control. Through the comparison of accumulation and elimination, it was found that after low- and medium dose exposure, the Pb concentration in skin, gill, and liver was higher than its accumulation, presuming that Pb was absorbed from gill, skin, and alimentary canal, and eliminated from gill and skin.

The bioaccumulation of fluoride ion (F(-)) in Siberian sturgeon (Acipenser baerii) under laboratory conditions.

A 90-d growth trial was conducted to determine the accumulation of fluoride ions (F(-)) in juvenile Siberian sturgeon (Acipenser baerii). Fish were exposed to nominal F(-) concentrations of 4, 10, 25, 62.5 mg L(-1) (added as NaF), along with a control group. Fish growth was determined and F(-) concentrations in bone, cartilage, skin, gill, muscle, liver, gut and pylorus were analyzed by fluoride specific electrode method. Fish growth was inhibited for groups exposed to 10, 25, and 62.5 mg F(-) L(-1) compared to the control group. Significant increases in F(-) concentrations were observed in bone, cartilage, skin and gill of Siberian sturgeon exposed to 4 mg F(-) L(-1). The highest F(-) concentration was in bone (3204.4 mg kg(-1), dry weight), followed by cartilage (1401.2 mg kg(-1)), gill (389.4 mg kg(-1)), skin (100.1 mg kg(-1)). Muscle, liver, gut and pylorus did not show increasing accumulation with increasing F(-) levels in the water, but liver, gut, pylorus accumulated higher F(-) concentration compared to muscle. It is summarized that F(-) accumulates mainly in bone and cartilage in Siberian sturgeon, with 10 mg F(-) L(-1) in water harmful to growth. Even 4 mg F(-) L(-1) may be harmful to Siberian sturgeon growth during a long-term waterborne exposure.