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CHEN Yue, ZHANG Ruiqi, LI Xixi, HUANG Xiaoting, ZHANG Zhifeng, QIN Zhenkui
2024,31(6):627-639, DOI: 10.12264/JFSC2024-0063
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.
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ZHU Yahua, JIANG Tao, CHEN Xiubao, LIU Hongbo, YANG Jian
2024,31(6):640-651, DOI: 10.12264/JFSC2024-0092
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.
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HUANG Xiaorong, TAN Ru, ZHANG Tao, FENG Guangpeng, GENG Zhi, ZHAO Feng, YANG Gang, ZHUANG Ping
2024,31(6):652-661, DOI: 10.12264/JFSC2023-0357
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.
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HAN Linmei, LIU Mingqiu, LI Chuanwu, XIANG Xing, LI Jiancheng, XIAO Yamei, ZOU Li, TIAN Lu, XIE Zhonggui, LIANG Zhiqiang
2024,31(6):662-673, DOI: 10.12264/JFSC2024-0037
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. -
WENG Zhaohong, CHEN Ruifang, XIE Yangjie, CHEN Zhi, ZHONG Yunmin, ZANG Haonan, WU Siying
2024,31(6):674-683, DOI: 10.12264/JFSC2024-0041
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.
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Developmental characteristics of embryos and early larvae of Carassius carassius in the Irtysh River
HAN Haoxiang, LIAN Jie, JIN Hongyu, MA Bo
2024,31(6):684-692, DOI: 10.12264/JFSC2024-0024
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.
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SU Li, XU Shannan, LI Chunhou, CHEN Zuozhi
2024,31(6):693-703, DOI: 10.12264/JFSC2024-0064
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.
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LI Qingying, CHEN Feng, JIANG Rijin, XU Kaida, DAI Qian, QIAN Weiguo
2024,31(6):704-717, DOI: 10.12264/JFSC2024-0072
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.
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YAO Mengzhu, LIU Jiqiang, CHEN Yi, SHEN Hao, ZHANG Yiming
2024,31(6):718-730, DOI: 10.12264/JFSC2024-0005
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.
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GOU Ziyue, WANG Xiuhua, YANG Bing, ZHU Xinjie, XI Rui, PAN Mingchao, HU Xili
2024,31(6):731-743, DOI: 10.12264/JFSC2024-0089
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.
Volume 31,2024 Issue 6
研究论文
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Spatial distribution characteristics of Portunus trituberculatus in offshore water of Zhejiang and their relationship with environmental factors
LI Xiangqian, JIANG Rijin, SHAN Xiaoluan, LI Zhenhua, ZHAO Peng, HAO Huibo, LI Qiqun, ZHOU Yongdong
Abstract:
为探究浙江近海三疣梭子蟹空间分布特征及其影响因素，根据2020—2022年4月(春季)和11月(秋季)浙江近海底拖网渔业资源调查数据，利用全局莫兰指数(Global Moran's I)、热点分析(Getis-Ord G_i^*)和地理加权回归模型(geographically weighted regression, GWR)分析分析三疣梭子蟹资源的空间自相关性及其与环境因子影响的空间异质性。结果表明：(1) 春季空间自相关性从小到大依次为2020年(P>0.1)、2022年(P<0.01)、2021年(P<0.01)；秋季空间自相关性从小到大依次为2022年(P<0.05)、2021年(P<0.01)、2020年(P<0.01)。2021和2022年均表现为春季三疣梭子蟹空间聚集程度高于秋季，而2020年空间聚集程度为春季低于秋季。(2) 局部自相关表明，春季和秋季三疣梭子蟹热点区分布位置分布存在相似性，即春秋季三疣梭子蟹热点区分布存在规律，春季三疣梭子蟹主要聚集在舟山海域西北部，而秋季则聚集在舟山海域东北部。(3) GWR模型表明不同季节的环境因子对三疣梭子蟹资源分布具有空间非平稳效应，春季各环境因子对三疣梭子蟹资源分布影响空间异质性程度大小依次为底层海水溶解氧(SBD)>底层海水温度(SBT)>底层海水盐度(SBS)，秋季空间异质性程度为底层海水温度(SBT)>底层海水溶解氧(SBD)>底层海水盐度(SBS)。三疣梭子蟹的空间自相关性存在一定的年际变化和季节规律，环境因子对三疣梭子蟹资源分布影响存在明显空间异质性。
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Differences in water quality, particle-associated bacteria and surrounding bacterial structure and function in small greenhouse farms of Litopenaeus vannamei under different yield levels
HUANG Yong, WANG Baojie, LIU Mei, JIANG Keyong, ZHONG Chen, XU Kefeng, GAO Yan, WANG Lei
Abstract:
为揭示凡纳滨对虾(Litopenaeus vannamei)小棚养殖模式下影响养殖产量的因素，本研究开展了凡纳滨对虾小棚养殖水质、颗粒相关细菌和非活动自由生活细菌结构和功能差异研究，并基于16s rRNA的高通量测序技术分析了不同产量水平下小棚模式两种细菌结构和潜在功能的差异。其结果显示, 高产小棚的悬浮颗粒浓度显著高于低产小棚(P<0.001)，水体中悬浮颗粒的浓度与对虾产量密切相关；在小棚养殖水体中，PAB的Chao1和Shannon指数均高于NFB，表明PAB的多样性和丰富度均高于NFB；黄杆菌科在颗粒相关细菌中显著富集，表明黄杆菌科细菌是PAB中主要的优势功能微生物；PAB的生物合成、物质和能量代谢功能通路丰度显著高于NFB(P<0.05)，PAB显著上调的代谢通路为硝化反硝化作用，表明PAB对物质合成和营养盐代谢能力更强；实时荧光定量PCR结果表明，高产小棚PAB中氨氧化细菌和亚硝酸盐氧化细菌的主要功能基因丰度均高于低产小棚，表明高产小棚PAB中硝化细菌更为成熟。该研究表明，在对虾小棚模式中，颗粒相关细菌对养殖水体中含氮污染物的去除发挥了重要作用，其通过调控水质而进一步影响对虾产量。本研究结果可为对虾小棚模式水质调控和高效养殖提供重要理论依据。
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Study on the hydrodynamic performance of a semi-submersible aquaculture platform during wet towing transportation
LIU Changfeng, ZHANG Yu, XIN Yu, YIN Jin, CHEN Changping
Abstract:
为提高深远海浮式网箱平台湿拖运输的安全性，基于三维势流理论和莫里森方程建立半潜式网箱平台拖航过程水动力数值模型，分析有效波高、浪向角、拖航速度、拖缆长度和系缆点位置对网箱平台运动响应及拖缆张力的影响。结果表明, 有效波高由1.25 m增加至4.00 m时，网箱平台垂荡加速度和纵摇角分别提高293%和750%，拖缆张力增加358%；顺浪拖航时网箱平台的纵摇响应比顶浪时更为剧烈，但拖缆张力有所减小；拖航速度由2.0 kn增加至5.0 kn时，垂荡加速度降低56%，拖缆张力提高112%；拖缆长度增加3倍时，纵摇角减小43%，拖缆张力降低31%；拖缆点位置对纵摇运动影响较大。实际工程中，建议网箱平台拖航最高海况限制为4级，拖航速度不超过5.0 kn，避免顺浪拖航，宜采用偏顶浪拖航，通过适当增大拖缆长度来提高拖航稳定性。
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Preliminary study for using otolith 87Sr/86Sr tracking the natal origin and reconstructing the early life history habitats of Coilia nasus from Poyang Lake
Xuan Zhongya, Yang Yanping, Hua Zhong, Ma Fengjiao, Wang Yinping, Liu Kai
Abstract:
目前通过耳石Sr/Ca比值可以确定刀鲚经历淡水、河口及海洋的洄游生活史，然而在追溯刀鲚个体的出生起源及早期发育阶段淡水生境履历转变方面仍存在困难。为探索新的追溯鄱阳湖中刀鲚的孵化场来源及早期生活史中不同淡水栖息地转变的方法，利用激光剥蚀连接多接收电感耦合等离子体质谱(LA-MC-ICP-MS)技术，分析了2023年7-8月采自鄱阳湖都昌水域的10尾刀鲚(Coilia nasus)成鱼个体以及3尾短颌鲚成鱼矢耳石的锶同位素(87Sr/86Sr)组成特征。从耳石核心到边缘的87Sr/86Sr线分析结果显示，3尾短颌鲚的耳石87Sr/86Sr比值变化不大，在0.71448附近波动，反映了其鄱阳湖定居生活；9尾刀鲚核心附近87Sr/86Sr比值接近0.71448，显示其孵化自鄱阳湖，并“回归”至出生栖息地鄱阳湖，其中4尾个体在仔稚鱼阶段迁出鄱阳湖，5尾个体在幼鱼阶段迁出；1尾刀鲚耳石核心附近87Sr/86Sr比值平均值为0.71078，显示为长江干流出生，在上溯洄游的过程中没有回到出生栖息地，而是进入了非出生栖息地鄱阳湖。这些结果反映了耳石87Sr/86Sr比值作为鄱阳湖刀鲚产卵场地理标志和区分鄱阳湖与长江干流生活史方面的客观性、准确性和稳定性，初步显示了其作为刀鲚出生起源和生境自然标签的潜力，也初步表明了鄱阳湖刀鲚早期个体迁出鄱阳湖时机的多样性。
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Effects of phytosterols on growth, digestion, intestinal, liver morphology, antioxidant and immune levels in Procambarus clarkia
YANG Zhigang, WANG Xiao, Ren shengjie, LIU Yongjian, GU Xiaolong, WANG Aiming
Abstract:
本实验旨在探究饲料中添加植物甾醇对克氏原螯虾（Procambarus clarkii）生长消化、抗氧化、免疫及肠肝形态的影响。在基础饲料中分别添加0%、0.10%、0.19%、0.38%、0.76%的植物甾醇，制备5组等氮等脂饲料，分别命名为CON、P1、P2、P3和P4饲料组，对体均重为（9.37 ? 0.02 g）的克氏原螯虾进行6周的养殖实验。结果显示：P1和P2组的增重率、特定生长率显著高于CON组（P < 0.05），且P2组实验虾的生长性能最佳。P3组实验虾肠道蛋白酶活性显著高于CON组，脂肪酶活性显著低于CON组（P < 0.05）。肝胰腺中P1组酸性磷酸酶和碱性磷酸酶活性最高，血淋巴中P2组酸性磷酸酶和碱性磷酸酶活性最高，但与CON差异均不显著（P > 0.05）。随着植物甾醇水平提高，肝胰腺和血淋巴总超氧化物歧化酶活性与过氧化氢酶活性呈上升趋势，丙二醛含量呈下降趋势。相较CON组，P1组肠道结构更加健康完整，植物甾醇水平到0.19%及以上时，克氏原螯虾的肝胰腺与肠道组织形态出现不良影响。随着植物甾醇水平提高，肝胰腺的NF-κB相对表达水平升高。P1组Hsp70相对表达水平显著高于其他组（P < 0.05）。研究表明，本实验条件下添加0.10%?0.19%植物甾醇可以促进克氏原螯虾的生长消化、改善肠肝组织形态，提高克氏原螯虾抗氧化和免疫能力。
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Effects of two Bacillus probiotics on growth, digestion, immunity and intestinal microbial communities of largemouth bass (Micropterus salmoides)
WANG Shigui, YAN Fajun, ZHANG Minglei, MAO Shuquan, ZHU Yongan, FU Chaosong, SONG Liping, LIU Feng
Abstract:
本文选取热带芽孢杆菌(Bacillus tropicus)和传染病研究所副芽孢杆菌(Metabacillus idriensis)为潜在益生菌，探讨它们对大口黑鲈(Micropterus salmoides)生长、消化、免疫及肠道菌群的影响。将初始体重为(92.95±2.36)g的大口黑鲈随机分为5组，每组设3个重复，分别饲喂基础饲料(对照组)和4种试验饲料[于基础饲料中分别添加104和106 CFU/g的B. tropicus(标记为BT4、BT6)或M. idriensis(MI4、MI6)]，饲喂期为60 d。生长试验结果表明，各菌处理组大口黑鲈的终末体重、增重率及特定生长率均高于对照组，但差异不显著(P>0.05)，而饲料系数均显著低于对照且随菌株添加浓度增加呈显著降低变化(P<0.05)。消化方面结果显示，各菌处理组的肠道淀粉酶和蛋白酶活性均高于对照组，其中BT6和MI6对肠道消化性能具有显著提高效果(P<0.05)。免疫方面结果表明，各菌处理组的血清酸性磷酸酶、碱性磷酸酶及溶菌酶以及肠道溶菌酶、超氧化物歧化酶活性均得到显著的提升(P<0.05)，说明菌株对血清和肠道免疫功能均有显著提高作用，但比较发现，总体以BT6与MI6的免疫增强效果最佳。肠道菌群高通量测序结果显示，支原菌属(Mycoplasma)、邻单胞菌属(Plesiomonas)和鲸杆菌属(Cetobacterium)为大口黑鲈肠道优势菌群；各组比较发现，BT6、MI6与对照之间在OTU数量、多样性指数及菌群结构上差异最明显，BT6可降低主要优势菌支原菌属(P<0.05)和多数劣势菌群的丰度来提高菌群均一性进而提高菌群多样性，而MI6可促进多数劣势类群的生长来提高菌群丰富度和均一性进而提高菌群多样性，这反映了不同菌株对肠道菌群作用方式的差异。本研究表明，热带芽孢杆菌和传染病研究所副芽孢杆菌皆可作为益生菌株应用于大口黑鲈养殖，在本实验条件下最佳使用量均为106 CFU/g，它们具有促进大口黑鲈生长、增强消化和免疫力、促进肠道菌群平衡及提高肠道菌群多样性等作用。本研究为大口黑鲈的益生菌开发与应用提供理论支撑。
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1.3 RT-qPCR
zhanghui, zenglin, xiongyifei, songwei
Abstract:
毒物兴奋效应是指有机体经历低剂量胁迫后,可以提高其对相同或不同高剂量胁迫因子的耐受性。由此探讨盐度驯化改善大黄鱼(Larimichthys crocea)盐度胁迫耐受性的作用机制,实验设置六个盐度梯度(25、25+12、25+40、20、20+12、20+40,分别记为C组、CL组、CH组、A组、AL组、AH组),大黄鱼盐度暴露24 h后取样。结果显示,CL vs C中大黄鱼肝脏的丙二醛(malondialdehyde,MDA)、脂质过氧化物(Lipid oxidation,LPO)、超氧化物歧化酶(superoxide dismutase,SOD)、溶菌酶(lysozyme,LZM)活力显著上升,过氧化氢酶(catalase,CAT)活力显著降低(P<0.05),碱性磷酸酶(alkaline phosphatase,AKP)的活力变化不显著(P>0.05)。CH vs C中MDA和LPO含量显著上升,SOD、 LZM的活力显著上升,CAT和AKP显著降低(P<0.05)。A vs C中MDA和LPO含量显著上升,SOD、LZM和AKP的活力未发生显著变化(P>0.05),CAT活力显著降低(P<0.05)。 AL vs CL与AH vs CH中MDA和LPO含量显著降低,SOD、 CAT 、LZM和AKP的活力显著上升(P<0.05)。铜锌超氧化物歧化酶(Cu/Zn-SOD)、锰超氧化物歧化酶(Mn-SOD)、CAT、非特异性免疫酶［c型溶菌酶(c-type LZM)、g型溶菌酶(g-type LZM)和AKP］基因表达水平与其酶活成正相关；核转录因子NF-E2相关因子2(NF-E2-related factor 2,Nrf2)和核转录因子-κB(nuclear transcription factor-κB,NF-κB)基因表达水平均分别与其目的基因(抗氧化酶基因和非特异性免疫酶基因)表达水平呈正相关(P<0.05),表明Nrf2和NF-κB可能分别参与了抗氧化和非特异性基因表达调控。综上所述,基于毒物兴奋效应原理,盐度驯化可以通过提高大黄鱼抗氧化和非特异性免疫能力来缓解盐度胁迫所带来的氧化损伤,从而改善大黄鱼的盐度耐受性。
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Development of vertebral column and appendicular skeleton in Collichthys lucidus larvae and juveniles
WU Chenyan, TANG Pengyan, ZHONG Junsheng, WANG Xiaodong, HUANG Jieli
Abstract:
本研究基于2014年6-7月在杭州湾北部水域用大型仔稚鱼网（口径1.3m、网目0.5mm）进行表层拖网所采集的棘头梅童鱼（Collichthys luncidus）仔稚鱼样本，采用软骨-硬骨双染色技术，探讨了棘头梅童鱼仔稚鱼（体长范围为3.3-12.7mm）脊柱及附肢骨骼的早期发育和骨化特征。研究结果表明：脊柱骨化之前不分节，而是由前向后逐渐长出的一个个骨环把脊柱分节，硬骨化出现的顺序大体上为由前向后。髓弓在脊柱骨化之前完全形成，相对于一些低等鱼类髓弓等的生成方向是从中间向头尾和从后向前进行，棘头梅童鱼的髓弓由前向后生长，属于相对高等鱼类的生长类型。胸鳍支鳍骨是最先发育的附肢骨骼，为能够自由地游泳摄食和从内源性营养转变为外源性营养提供了保障。后颞骨的形状十分特殊，具有四枚向不同方向突起的棘；背鳍支鳍骨由体中部开始向前后生长，尾鳍支鳍骨的骨化晚于其他附肢骨骼，侧尾下骨与其相邻的两枚尾下骨的基部融合，为其游泳提供了强劲的动力。该研究结果不仅可以为早期形态学分类提供骨骼发育学方面的科学依据，对棘头梅童鱼人工繁殖过程中结合骨骼发育进行技术上的契合也具有重要的参考价值。
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Detection of Nile tilapia specific antibody secreting cells induced by human IgG by ELISPOT assay
Huang Yu, Pang Bijian, Li Qi, Wu Zaohe, Jian Jichang
Abstract:
通过酶联免疫斑点法(ELISPOT)检测了经人IgG免疫后尼罗罗非鱼(Oreochromis niloticus)头肾、外周血和脾脏中的特异性抗体分泌细胞(antibody secreting cell,ASC)数量。结果表明：首次免疫1天后可在头肾中检测出ASC,而外周血和脾脏第3天才能检测出ASC；头肾、外周血以及脾脏中的ASC均在第12天达到峰值,随后头肾,外周血中的ASC数量显著减少,而在脾脏中ASC数量减少不显著。二次免疫1天后在头肾、外周血和脾脏中均可检测到ASC,ASC数量均在第9天达到峰值,时间早于首次免疫,且在首次免疫和二次免疫中,头肾组织的ASC数量均是三个组织中最高的。酶联免疫吸附试验(ELISA)分别对首次免疫和二次免疫后血清中的抗体水平检测发现,其变化趋势与ASC数量变化规律相同。研究结果表明,尼罗罗非鱼在初次免疫后产生了免疫记忆,在二次免疫过程中产生了更多的ASC和抗体,头肾是ASC的主要来源组织。该结果为鱼类的免疫记忆研究提供重要的科学依据。
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Effects of Cu acclimation on oxidative damage in the large yellow croaker under cold stress based on transcriptome analysis
Zeng Lin, Wang Yong Hong, songwei
Abstract:
为探讨铜驯化对低温胁迫下大黄鱼氧化损伤和基因表达水平的影响,本实验将体质量为(48.92±3.62) g的大黄鱼暴露在铜浓度为0和10 μg L?1的水体中14 d,再暴露在温度为8°C的水体中24 h。结果显示,低温胁迫显著增加了活性氧(ROS)和脂质过氧化物(LPO)含量。尽管铜驯化对ROS和LPO含量不产生影响,但铜驯化显著增加了低温胁迫下大黄鱼ROS和LPO含量,表明铜驯化加剧了低温胁迫对大黄鱼的氧化损伤。从铜驯化vs.对照组、低温胁迫vs.对照组和铜驯化+低温胁迫vs.低温胁迫中分别筛选到2 288个、1 425个和1 382个差异基因。GO和KEGG分析发现差异基因主要富集在与脂肪酸代谢、糖类有氧代谢、谷胱甘肽代谢、内质网应激、自噬和凋亡等相关的通路中。聚类分析表明,低温胁迫上调了不饱和脂肪酸合成、内质网应激、自噬和凋亡等相关通路中的大部分基因表达,而铜驯化则对低温胁迫下大黄鱼的这些基因表达调控产生了拮抗效应,表明铜驯化通过抑制不饱和脂肪酸合成、内质网应激、自噬和凋亡来降低大黄鱼的低温胁迫耐受性。研究结果为深入研究铜污染物对大黄鱼低温胁迫耐受性的影响及其分子机制提供科学依据。
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