Abstract:Scallops belong to bivalves of the mollusc phylum and are important shellfish culture species in China. Important varieties of scallops and their complex reproductive regulation mechanisms have been the focus of research in shellfish biology. Many genes have been reported to be involved in the sex differentiation and gonadal development of scallops, whereas the regulation of these genes has rarely been reported. MicroRNA is a class of important endogenous regulatory factors that can participate in the regulation of gene expression. To explore the role of microRNAs in the gonadal development of shellfish, this study took the Zhikong scallop (Chlamys farreri), a hermaphroditic and sex-stable bivalve species and thus good material for such a mechanism exploration, as the research material. In a previous study, we found that miR-124-3p_4 was highly expressed in the testes of juveniles during the early stage of sexual differentiation, and thus took this microRNA as the research object. The sequence of miR-124-3p_4 was compared with other species and the core sequence was consistent. The genes targeted by miR-124-3p_4 were predicted through RNAhybrid and miRanda, from which 729 and 436 target genes were obtained, respectively, and the intersection was 260. Combining with the transcriptome data of C. farreri, eight ovary bias genes and twelve testis genes were obtained. Based on the binding site and binding-free energy analysis, all eight ovary bias genes matched perfectly with miR-124-3p_4 and their free energy was all below –20 kcal/mol, indicating the interaction activities between them. Among these genes, dpgn-like was found to have the strongest interaction with miR-124-3p_4. Sequence and structure analyses of the dpgn-like gene showed that its cDNA full length was 4526 bp, coding 559 amino acids. Four MFS, two KAZAL_FS, and one OATP conserved domain were identified in the DPGNL protein. Phylogenetic results clarified its identity and evolutionary status. The expression of the dpgn-like gene was mainly in the mantle, foot, gill and gonads, of which the expression in the ovaries was significantly higher than that in the testis with a fold change of 4.4. In situ hybridization was performed in mature ovaries and results showed that the dpgn-like gene expression was localized in oogonia and oocyte cytoplasm, but not in follicle cells, indicating its potential involvement in ovary development. To test whether miR-124-3p_4 and the dpgn-like gene were directly interacted, a dual luciferase reporter gene analysis was conducted. Through co-transfer of miR-124-3p_4 and the dpgn-like gene into HEK293T cells, the dpgn-like gene expression was significantly reduced by 64%. Furthermore, in vivo overexpression of miR-124-3p_4 in the ovary was explored by the microinjection of miR-124-3p_4 agomir. After 3 d of microRNA overexpression, a 16.28-fold increase of miR-124-3p_4 to the control group was confirmed by RT-qPCR. Meanwhile, the dpgn-like gene expression was reduced to 0.74, again indicating that miR-124-3p_4 could combine and down-regulate dpgn-like gene expression in ovaries. In summary, this study screened for the potential target genes of miR-124-3p_4, identified the dpgn-like gene which had the strongest interaction with it, revealed the sequence and structure characteristic of the dpgn-like gene, and uncovered its expressional patterns. These results suggest that the dpgn-like gene may be involved in gonadal development. In addition, both in vitro and in vivo analyses indicated that miR-124-3p_4 could directly target the dpgn-like gene. Collectively, these outcomes reveal that male-biased miR-124-3p_4 negatively regulated ovary-biased dpgn-like gene expression in the testis, implying its potential role during the development of gonads.

Abstract:To explore whether strontium otolith-marking technology can be applied to evaluate the effect of grass carp stocking in aquaculture, 9-d-old larval grass carp (Ctenopharyngodon idellus) were used. They were immersed for 2 d in 80 mg/L strontium chloride hexahydrate (SrCl₂•6H₂O). After the immersion, the fish were sampled continuously during the rearing process, and the pre-treated otoliths examined by electron probe micro-analyzer. We used mapping and line analysis to investigate the elemental fingerprinting effect of strontium on the otoliths of grass carp larvae and captured the exact time of formation on the first day and the time lag of strontium marking based on counting of the daily rings of the three otolith types in grass carp. The results of the quantitative line analysis showed that immersion with 80 mg/L SrCl₂•6H₂O solution for 2 d caused strontium-marking peaks to form on the grass carp otoliths. The strontium-marking peaks of lapillus and sagitta were higher than those of asteriscus and gradually decreased to normal levels with an increase in recovery feeding time. Both lapillus and sagitta also had greater Sr and Sr/Ca ratios than asteriscus during the phase when the three otolith types did not show an increase in Sr values or Sr/Ca ratios. The mapping analysis results were consistent with the line analysis, and the marked areas in the three otolith types showed obvious red patterns, while the unlabeled low-strontium otolith areas showed blue patterns. In addition, through the comparative analysis of strontium-marking characteristics and the number of otolith growth days, it was found that among the three otolith types, the formation time of asteriscus was later than that of lapillus and sagitta, with the first daily increment formed at the 12th day of hatching, while lapillus and sagitta formed the first daily increment at the 2nd day of hatching. There was a 1-d time lag between the beginning of strontium-marker formation and the start of immersion and the disappearance of strontium markers and the end of immersion for lapillus and sagitta, respectively. Strontium markers were observed to appear in the core of the asteriscus, and the disappearance of the strontium-marker signals was observed with a time lag of 1 d from the end of immersion. Here, in terms of fish mortality and behavior, the Sr dose had no significant difference (P＞0.05) between the control and experimental groups. Considering the effects of marking due to the absorption of strontium by different otoliths and the differences in the morphology and structure of otoliths, as well as the difficulty of sampling, it is recommended that lapillus be used as the most suitable otolith for the observation of Sr markings of grass carp larvae. This can provide a theoretical basis and technological support for marking and detection of the stocking of grass carp and other cyprinids.

Abstract:To explore the nutrient enrichment effect of Antarctic krill meal to Eriocheir sinensis brood stocks, a 60-d feeding trial was conducted 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%, 10% and 20%, with a control group fed razor clam Sinonovacula constricta. The hepatopancreatic weight (HW), hepatopancreas index (HSI), gonadal weight (GW), and gonadosomatic index (GSI) were detected after 60 d of nutritional enrichment. Each treatment had three replicate tanks stocked with five males and 10 females. The reproductive performance of broodstock and embryo quality—such as egg production, fecundity, reproductive index, egg diameter, weight, and biochemical and fatty acid compositions—were analyzed. The results showed: (1) The HW and HSI of female crabs in each group significantly decreased (P＜0.05), while GW and GSI increased significantly (P＜0.05) after 60 d, with the 20% group showing the highest values. (2) Egg production and fecundity of female crabs reached their peak in the 20% group, with no significant differences (P＞0.05) in reproductive indices among all groups. (3) The phospholipid content in the 20% group was significantly higher than that in the other groups (P＜0.05), the highest triglyceride (TG) content was found in the 10% group, with an average of (1449.73±101.38) μg/g, significantly higher than other groups (P＜0.05), cholesterol content in the 0% and control groups were significantly higher than that in the 10% and 20% groups (P＜0.05). No significant differences were observed in egg diameter, egg weight, moisture, crude protein, and crude fat between the groups (P＞0.05). (4) The content of C20 : 2 and C20 : 4n6 was highest in the 0% group, with a significant difference compared to the 10% group (P＜0.05). However, no significant differences were found in the total content of saturated fatty acids (SFA), mono-unsaturated fatty acids (MUFA) and poly unsaturated fatty acids (PUFA) among all groups (P＞0.05). The results indicate that 10%~20% and 20% dietary Antarctic krill meal could promote gonadal development of the female E. sinensis and significantly improve reproductive performance and embryo quality, respectively.

Abstract:Onychostoma rara is a fish species distributed only in the Yuan River system in the middle Yangtze River and the Xijiang River system in the Pearl River basin of China. It is locally popular due to its tender meat, delicious taste, and high nutritional value. However, stressors including overfishing and environmental damage have led to a sharp decline in the number of O. rara and it is currently listed as an endangered fish species in the "Red List of Species in China." The artificial breeding and release of endangered fish can aid in protecting them from extinction. To achieve this, wild O. rara were captured from the Yuan River system in Huaihua from 2016–2019. In 2019, numerous artificially reared O. rara reached sexual maturity, and the F1 generation was obtained by artificial cultivation and manual stimulation with oxytocin. Our findings indicate that male F1 individuals attain sexual maturity for the first time at the age of 2 years, whereas female F1 individuals achieve sexual maturity at 3 years. By the age of 4 years, 90% of the F1 population has reached sexual maturity. In 2023, we successfully achieved the controlled reproduction of cultured O. rara. Until Det 2023, there were 100 wild parents, 2000 cultured parents, and 2.02×10⁵ newly hatched larvae. One hundred male and one hundred female parents were sequestered in two separate net cage for the subsequent artificial oxytocin experiment.” Artificial oxytocin was injected with a mixture of luteinizing hormone releasing hormone A₂, chorionic gonadotropin, and carp pituitary gland extract. Each female received two injections, while each male received one that was half the dose of the female's second dose. After the response period was reached, which was approximately 11 h after the second injection, the fertilization was performed using the artificial dry method. The sequence and morphological characteristics of the developing fertilized eggs were observed using a microscope and described in detail. The newly spawned eggs of O. rara were yellow, sinkable, and weakly adhesive, with an average diameter of (2.21±0.06) mm. After absorbing water and approximately 30 min after fertilization, the average diameter of the fertilized eggs expanded to (2.88±0.07) mm and became non-adhesive. When the water temperature was 19.5-25.5 ℃, the time from fertilization to hatching was 45.5 h, and the accumulated temperature from fertilization to hatching was 1024.39 h·℃. The newly hatched larvae showed a light-yellow color, with no other pigments visible throughout the larva body. On the second day, melanosis in the eyes and red blood vessels in the heart and abdomen were clearly visible. When the water temperature was decreased, the time of the embryo hatching from the membrane was extended, and the embryonic development of the prophase stage of incubation was more complete; furthermore, the eye pigments were clearly visible, and red blood flow could be seen in the heart and abdomen. The morphological characteristics of the prophase stage of incubation were basically consistent with those of the larvae on the second day, with higher water temperatures. This suggests that temperature is crucial for the hatching and development of O. rara embryos.
In general, the egg traits of the O. rara are basically the same as those of other fish in the genus Onychostoma, which may be related to their typical habitats such as rivers with strong currents and gravelly bottoms. In such environments, fertilized eggs can be dispersed with water flowing downward into the spaces between rocks to avoid predation and improve survival rates. However, due to the difference between species and environmental factors, the degree and stage of organ development and the time from fertilization to hatching are different. Using artificial breeding and artificial oxytocin technology, this study realized the artificial reproduction of O. rara for the first time and carried out a preliminary study on the development of O. rara embryos and larvae, providing basic data and an important reference for the future implementation of large-scale breeding of this species.

Abstract:The resources of Tachypleus tridentatus face the risk of depletion owing to habitat destruction and overfishing. Implementing artificial cultivation of large-sized juveniles and releasing them into suitable breeding grounds is an effective measure for restoring the population of T. tridentatus. This study used a self-designed cultivation system to rear horseshoe crab larvae and juveniles under controlled laboratory conditions. The impact of four distinct water temperatures (25, 28, 31, and 34 ℃) on molting rates, molting duration, and mortality rates of various instar stages was compared. After 120 days of cultivation, there were no significant differences in the total survival rates of larval and juvenile horseshoe crabs across different temperature groups (P＞0.05). However, higher water temperatures significantly facilitated the molting of larval and juvenile horseshoe crabs. At the end of the experiment, most of the juveniles in the 34 ℃ group had transitioned to the 4th instar stage, with a few individuals having already progressed to the 5th instar stage. In contrast, most individuals in the 31 ℃ group were observed to be at the 4th instar stage. In the 28 ℃ group, a predominant number were observed at the 3rd instar stage, with a few individuals progressing to the 4th instar. Within the 25 ℃ group, most individuals were observed at the 2nd instar stage, with only a few progressing to the 3rd instar. During the experimental period, the mortality rate of the 1st instar larvae ranged from 6.00% to 19.33%. Among them, the mortality rate of the 1st instar larvae in the 25 ℃ temperature group was significantly higher than that in the 31 ℃ and 34 ℃ groups (P＜0.05), while no significant difference was observed among the other temperature groups (P＞0.05). The mortality rates of the 2nd to 4th instar juveniles were not significantly different among the various temperature groups (P＞0.05). Significant differences were observed in the earliest molting times of the 1st instar stages among the various temperature groups (P＜0.05), with a noticeable trend of an earlier onset of molting at higher water temperatures. The development time required for each instar stage during the 2nd and 4th stages was estimated to range between 27 and 43 days, based on the weighted average time and earliest molting time of each instar stage, under temperatures ranging from 25 ℃ to 34 ℃. The influence of temperature on the development time of the 1st instar larvae was greater than that of the 2nd and subsequent instar juveniles. The comprehensive analysis indicated that increasing the temperature within the appropriate range can effectively promote the molting of T. tridentatus, both 31 ℃ and 34 ℃ can achieve excellent cultivation effects. These results suggest that large-scale indoor cultivation of large juvenile horseshoe crabs is feasible. Additionally, it is advisable to rear the larvae at higher temperatures immediately after hatching to facilitate their development into the 2nd and subsequent instars as quickly as possible, thereby preventing them from entering a diapause state at low temperatures.

Abstract:This study was conducted to understand embryonic and larval development of Carassius carassius distributed in the Irtysh River Basin, fill the gap in artificial breeding, and enrich the reproductive biological data of this species. This study collected C. carassius parents from the Irtysh River, obtained fertilized eggs through artificial induction of fertilization, and observed their early developmental characteristics. The results showed that the embryos hatched out of the membrane at a temperature of (19.89±0.64) ℃ for 110 h and 35 min, and a total accumulated temperature of 2274.54 h·℃. The C. carassius embryo started from the fertilized egg, placenta was bulged 50 min after fertilization and developed to a blastocyst in 6 h 40 min. The gastrul embryo developed in 17 h 20 min, nerve developed in 26 h 20 min, sarcomere appeared in 37 h 20 min, heartbeat occurred in 70 h 35 min, and membrane emerged in 110 h 35 min. The embryonic development process went through seven stages (26 periods) of fertilized egg, cleavage, blastocyst, gastrulation, neural embryo, organ formation, and hatching. Lying at the bottom, the total length of the fry hatched after 1 d was (4.45±0.12) mm, and the yolk sac was full; the full length of the fry after 2 d was (5.13±0.18) mm, the pigmentation had increased, and a short swim was observed; the total length of the fry after 3 d was (5.69±0.17) mm, they were sensitive to external stimuli, the yolk sac was absorbed, and anal and caudal fins were clearly visible. This study is the first to report the timing and characteristics of the early development of the Irtysh River C. carassius under artificial induction conditions, thereby providing basic data and an important reference for further large-scale breeding of seedlings.

Abstract:To provide reference for the protection and management of the ecological environment in the sea area around Qilianyu Island, the characteristics of phytoplankton communities and environmental conditions in this area were analyzed based on survey data of four voyages from 2018 to 2021. The results showed that 341 species of phytoplankton were identified from 111 genera of five phyla, with Bacillariophyta (69.21%) and Pyrrophyta (23.75%) as the major species. Phytoplankton abundance fluctuated in the range of (2.34–2841.16)×10³ cells/m³ , with the highest mean value in autumn 2018 (797.47×10³ cells/m³ ), followed by summer 2018 (675.40×10³ cells/m³ ), with the lowest value found in summer 2020 (91.39×10³ cells/m³ ). Phytoplankton abundance was mainly composed of Bacillariophyta (48.67%) and Cyanophyta (47.22%). Significant differences were observed in phytoplankton communities between the four periods (P=0.001). The diversity and evenness index of phytoplankton first increased and then decreased. The RDA analysis results showed that the main environmental factors affecting phytoplankton communities in Qilianyu Island were transparency, water depth, ammonia nitrogen, DIN, and N∶P with dominant factors changing in different periods. The nutrient factor limiting phytoplankton growth in this area changed from nitrogen to phosphorus. The decrease in phytoplankton diversity may be related to a nitrogen-phosphorus ratio imbalance caused by a surge of nitrogen content in the sea area. Therefore, the discharge of nitrogen-containing wastewater must be reduced and the protection and management of the ecological environment in the sea area must be strengthened.

Abstract:This study investigated the niche and interspecific associations of shellfish, as well as the impact of environmental factors on the niche differentiation of dominant species in shellfish, based on four quarters of benthic shellfish biomass and marine environment survey data conducted in the coastal waters of Zhejiang from August 2020 to April 2021. The results showed that a total of 41 species of shellfish were surveyed and collected, of which 16 were the main shellfish, divided into three ecological groups. The niche width was 1.1–3.7, with an average of 2.4. Among the shellfish, Bursa rana had the largest niche width of 3.7, and Phalium bisulcatum had the lowest of 1.1. The niche overlap value between species was 0–0.66, with the maximum niche overlap value between species of Fusinus longicaudus and Hemifusus tuba being 0.66. The niche overlap between 58 species was 0, and the degree of niche overlap was relatively low. The overall correlation between the main shellfish species was not significant (VR＞1), and 89.7% of the species showed no significant correlation (P＞0.05), indicating that the overall competition for resources among the major shellfish species in the sea area was relatively low. However, some species such as Turritella terebra bacillum, Glossaulax didyma, Tonna olearium, and Bursa rana also had strong competitive relationships. The environmental factors that affected the niche differentiation of dominant species of shellfish in different seasons were slightly different; however, the major factors affecting the niche differentiation of nearshore shellfish are habitat water depth and salinity (0.2≤r＜0.4, P＜0.01). These results can provide a scientific basis for the assessment of ecological stability and resource conservation of nearshore shellfish.

Abstract:Microplastics are widely present in the atmosphere, soil, and water and can be transferred, enriched, and bioaccumulated through the food chain. The choice of digestion solution depends on test sample characteristics and the digestion time varies from a few hours to several days, which may have detrimental effects on the original plastic by causing dissolution, fracture, and degradation, thereby potentially interfering with experimental data. Each existing detection technology has its advantages and limitations. To establish an efficient pre-separation treatment scheme for shellfish microplastics based on fluorescence staining technology, this study focused on the Manila clam (Ruditapes philippinarum) because they are one of the main indicators of microplastic pollution in shellfish in China. We investigated digestion efficiency, membrane obstruction rate, microplastic recovery rate, as well as changes in the infrared spectrum under two digestion systems: 10% potassium hydroxide (KOH) and protease K. Digestion conditions were determined and optimized accordingly. Furthermore, we combined the optimization scheme with Nile red fluorescence staining to validate the unknown fluorescent particles using a confocal micro-Raman spectrometer to confirm the applicability of the proposed scheme. The results showed that under a 10% KOH digestion system, there was an extremely significant difference in digestion efficiency among the three groups (P˂0.01). The highest digestion efficiency was observed in the 4-h group at (99.46±0.49)% with a blocking rate of (126.17±53.30)%. For the protease K digestion system, a significant difference was observed among the three groups (P˂0.05), with the average digestion efficiency being highest in the 16-h group at (99.66±0.08)%. No statistically significant differences were observed in recovery rates of standard microplastic particles among the experimental groups (P＞0.05). The major characteristic peaks observed between the experimental and quality control groups were consistent; however, differences existed in peak intensity, position, spectral quality, and other aspects. Based on the aforementioned comparative results, it is evident that under all experimental conditions, the most effective digestion occurred with 10% KOH at 50 ℃ for 4 h and protease K at 50 ℃ for 16 h. To enhance vacuum filtration time, we optimized the two digestion schemes by incorporating a pH adjustment using a 5% dilute hydrochloric acid solution after the initial treatment with 10% KOH at 50 ℃ for 4 h. Additionally, sodium laurylsulfonate (SDS) was added followed by ultrasound treatment for an additional duration of 30 min after digestion with protease K for 16 h. The experimental data demonstrated that the optimized KOH digestion scheme improved digestion efficiency to (99.80±0.06)% while reducing the membrane blockage rate to (95.78±6.51)%. The recovery rate of plastic standard samples with particle sizes ranging from 3 to 5 mm and 400 to 600 μm was (97.92±3.61)% and (89.58±4.14)%, respectively. For the optimized protease K digestion scheme, digestion efficiency improved and was found to be (99.97±0.02)% while the membrane blockage rate decreased to (94.37±9.85)%. The recovery rates for both plastic standards (PP, PS, PET and LDPE) were over 95% and 85%, respectively. The infrared spectrum changes observed in plastics following each treatment scheme were not significant. Therefore, the optimized method presented here is suitable for microplastic extraction in samples. After staining with Nile red solution, SDS ions possess adsorption activity on the surface of Nile red molecules, resulting in solubilization and formation of original micelles. This phenomenon leads to numerous false positive fluorescent bright spots within the optimized protease K group which may interfere with the micro-Raman measurement. However, the KOH optimization group did not exhibit the same occurrence, and microplastics detected in the sample were 0.99 g (wet weight). These included fibrous polyethylene terephthalate with a length of (971.23±22.01) μm and particle-like acrylonitrile butadiene styrene with a particle size of (26.88±1.69) μm. In conclusion, a 10% KOH solution was utilized as the digestion agent, followed by ultrasound treatment for 10 min. Subsequently, the solution was dissolved in a water bath at 50 ℃ and agitated at 150 rpm for 4 h. The pH was adjusted using a 5% dilute hydrochloric acid solution to achieve an optimal pre-treatment scheme. This approach was combined with Nile red fluorescence staining observation and confocal micro-Raman characterization, providing a simple, cost-effective, and efficient method for detecting and analyzing bivalve shellfish microplastics.

Abstract:Clamworm is a biological bait with high nutritional value, widely used in promoting gonadal maturation of shrimp brood stock. However, as most clamworm occur in wild environments, they are prone to carrying pathogenic bacteria and viruses, posing a potential disease risk for farmed shrimp. To eliminate the pathogens carried by clamworm, antibiotics are often used by immersion. Due to the disadvantages of using antibiotics, novel safe and effective alternative products must be developed urgently. Sorbic acid, malic acid, citric acid, and other organic acids and their salts are commonly used as preservatives and fungicides in food and have potential development value for biological sterilization of aquatic bait. To research the profile of Vibrio pathogens carried by bait clamworm and establish effective pathogen elimination technology, the pathogen species and Vibrio load in 10 coastal areas of China were investigated. Sodium citric acid, povidone iodine, potassium sorbate, sodium dehydroacetate, malic acid, potassium dimethyl acid, propionic acid, and a Vibrio-killing product (VKP)—a commercial product containing citric acid and acid calcium sulfate—were employed to test the bactericidal effect in vitro in this study. The lowest and safest bactericidal concentrations of citric acid, povidone iodine, and VKP were tested in vitro for the elimination effect of Vibrios in clamworm. Additionally, protein, fat, cholesterol, and vitamin E content changes in clamworm after soaking in citric acid, povidone iodine, and VKP for 3 h were analyzed. The results displayed that the sample clamworm carried Vibrio alginolyticus, V. harveyi, V. owensii, V. parahaemolyticus, and V. parahaemolyticus with detection rates of 86.7%, 20.0%, 13.3%, 13.3%, and 6.7%, respectively. The highest and lowest pathogenic Vibrio loads in the sample were 7.0×10⁴ and 2.0×10³ cfu/g, respectively. The minimum bactericidal concentrations of the nine drugs in vitro from low to high were povidone-iodine (10.0×10^–3 g/L), formic acid (0.5 g/L), VKP (1.0 g/L), citric acid/malic acid/propionic acid (1.2 g/L), potassium dicarboxylate (1.6 g/L), sodium dehydroacetate (12.0 g/L), and potassium sorbate (30.0 g/L). The inactivation rates of Vibrio in vivo were 96.2%, 66.4%, 99.9% and 97.7%, 98.0% 100.0%, respectively, when the clamworm were soaked in the citric acid, povidone-iodine and VKP solutions at the minimum effective bactericidal concentration for 0.5 and 3 h. Safe concentrations of the three drugs were 48.9, 120.3 and 963.0 mg/L, respectively, meanwhile the contents of protein, fat, cholesterol, and vitamin E in the clamworm were significantly reduced compared with those in the control group (P＜0.05) after the 3 h treatment. The results suggest that the clamworm has a great biosafety risk as biological bait, and the soaking treatment in organic acids can reduce the pathogenic bacterial load. The results of this study provide a basis for the comprehensive prevention and control of shrimp diseases.