不同生物饵料对中国鲎繁殖性能及后代质量的影响

为筛选有效促进中国鲎(Tachypleus tridentatus)亲鲎性腺发育及产卵的生物饵料,选取成年中国鲎15对,于2021年7—8月开展生物饵料实验,以菲律宾蛤仔(A组)、双齿围沙蚕(B组)、近江牡蛎肉(C组)、菲律宾蛤仔+双齿围沙蚕(D组)和近江牡蛎肉+双齿围沙蚕(E组)5组生物饵料投喂亲鲎,采用SPSS 19.0软件统计分析和单因素方差分析,通过比较卵径、受精卵的孵化时间和孵化率以及1龄幼体头胸甲宽,研究不同饵料对中国鲎繁殖性能和后代质量的影响。结果表明:试验D组产卵数量和次数均高于其他试验组,其次是E组,混合试验组产卵数量和质量均高于单独试验组。研究发现,双齿围沙蚕和菲律宾蛤仔组合投喂较适合开发为亲鲎性腺强化培育的生物饵料,对性腺发育及产卵有显著的促进作用。本研究旨在为开发安全、高效、优质的中国鲎性腺促熟饵料提供参考依据。

四种重金属对中国鲎(Tachypleus tridentatus)胚胎发育的影响

分析了不同离子浓度的 Zn、Pb、Cu、Cd对中国鲎胚胎发育的影响。结果表明 :1 4种金属离子浓度≤渔业水域水质标准浓度 4倍时 ,对中国鲎胚胎发育无明显毒性效应 ;≥渔业水域水质标准浓度 8倍时 ,Cd离子和 Cu离子对中国鲎胚胎发育无明显影响 ,而 Zn离子组和 Pb离子组胚体发育随离子浓度提高而卵径变小 ,发育速度下降 ;2浓度为 1 .6mg/L时 ,Zn和 Pb离子组胚胎致畸率分别为 4 4%和 58% ,Pb离子组胚体孵化率仅 2 .67% ;3 4种重金属对中国鲎胚胎发育的毒性效应表现为 Pb>Zn>Cu=Cd;4中国鲎胚胎对重金属有蓄积作用 ,蓄积效应随金属种类不同而异。

盐度对中国鲎(Tachypleus tridentatus)胚胎发育的影响

用雌雄中国鲎进行人工授精育苗，研究盐度对其胚胎发育的影响，并用未授精的卵作对照，观察其授精后 ２～３ ｄ 出现的分裂纹是否可作为受精的标志。结果表明，中国鲎人工授精育苗的可行盐度为１６～４０，适宜盐度为１６～３３。实验中，受精率在可行盐度范围内均达８５％ 以上，但孵化率却较低，授精后所能表现的分裂纹可作为其已受精的标志。卵的成熟度对人工授精后的卵的发育有较大的影响。

中国鲎(Tachypleus tridentatus)人工授精育苗的初步研究

用厦门海滩捕获的成熟雌雄中国鲎进行人工授精育苗试验，并对受精率、孵化率、幼鲎饲养成活率进行测定。结果表明，受精率为95％，孵化率为85％，幼鲎成活率为100％；1996年7月29日人工授精的鲎，至1997年7月11日仍健康生长，并已蜕第一次壳。说明中国鲎用人工授精的方法大量人工育苗是可行的。要提高受精率和育苗效果，应在5～6月为佳。

中国鲎(Tachypleus tridentatus)雌激素相关受体基因的克隆与分析

采用RT-PCR、RACE、Genome Walking等方法从中国鲎克隆得到了雌激素相关受体基因的cDNA全长及基因组DNA序列,命名为ttERR(GenBank登录号为HQ386702),并采用生物信息学方法对其序列和编码的蛋白质的理化性质、分子系统进化关系等进行预测和推断。结果表明,ttERR基因cDNA全长2722bp,包含一个1413bp的开放阅读框、626bp的5’-UTR和683bp的3’-UTR,编码一个由470个氨基酸组成的蛋白质。ttERR基因由10个内含子和11个外显子组成。系统发育分析表明,ttERR属于核受体超家族第三亚族。本文为将来深入研究中国鲎ttERR的功能奠定了基础。

中国鲎(Tachypleus tridentatus)微卫星标记筛选及种群遗传多样性和遗传结构分析

采用FIASCO方法构建了中国鲎(Tachypleus tridentatus)(CA)_n微卫星富集文库,对214个阳性克隆进行测序,筛选获得60条含有微卫星的序列,其中可设计引物的序列共38条。38对微卫星引物在37个中国鲎个体中PCR检测发现,只有9个位点具有多态性,其等位基因数为5-14个,每个位点平均具有8.1个等位基因。9个位点之间不存在连锁。利用该9个具有多态性微卫星标记分析了中国沿海9个中国鲎地理群体的种群遗传多样性和遗传结构。结果表明:中国沿海的中国鲎种群遗传多态性水平仍较高,9个地方群体间无遗传差异,它们之间未见显著分化,且有着较高的基因流;推测人为的迁移是造成遗传无分化的主要原因。我们建议中国鲎已绝迹的海域可从其他海域引入中国鲎个体来恢复地方群体资源。

中国鲎(Tachypleus tridentatus)基因工程抗菌肽的制备及其抗菌活性

从中国鲎血细胞RT-PCR扩增抗菌肽tachyplesins基因,经改造构建原核和真核表达载体pET-KRN、pPIC9-KRY,并分别在大肠杆菌DE3和毕赤酵母GS115中诱导表达。用Tris-Tricine SDS-PAGE检测并进一步电洗脱纯化表达产物,经体外生物活性测定表明,表达产物KRN对大肠杆菌、金黄色葡萄球菌、枯草杆菌、伤寒沙门菌的抑菌环为25、22、25、16mm;MIC值为3.57、7.14、3.57、14.28mg/L。表达产物KRY对大肠杆菌、金黄色葡萄球菌、枯草杆菌、伤寒沙门菌和李氏杆菌的抑菌环为26、22、25、18、16mm;MIC值为1.57、3.14、1.57、6.28、6.28mg/L,对水霉的抑菌效果尤其显著,抑菌环达35mm。

北部湾中国鲎不同群体及雌雄个体形态差异分析

采用t检验、主成分分析和判别分析等多元统计方法,分析和比较北海市和钦州市中国鲎群体的体质量(Y)和全长(X1)、头胸部长(X2)、头胸部宽(X3)、腹部长(X4)、腹部宽(X5)、剑尾长(X6)等7个可量性状,研究两者形态差异,并对北海群体进行雌雄个体的形态差异分析。t检验结果显示,2组群体仅X6/X1差异极显著(P<0.01),其他指标的差异不显著(P>0.05)。主成分分析构建了1个主成分,其贡献率为50.16%。对北海群体的5项比例性状进行逐步判别分析,筛选出3个比例性状构建的判别方程对该群体的2组群体识别的正确率达73.3%。指出,北海市群体雌性个体体型大,X2和X6均大于雄性,但X4小于雄性。

Impact of Different Diets on Adult Tri-Spine Horseshoe Crab,Tachypleus tridentatus

Effective culture and management of adult tri-spine horseshoe crab,Tachypleus tridentatus can ensure that stock enhancement programs and aquaculture systems are maintained.To explore suitable feed for animals during the breeding season,Pacific oyster(Ostrea gigas)(oyster group;OG)and frozen sharpbelly fish(Hemiculter leucisculus)(frozen fish group;FG)were selected to feed 20 T.tridentatus male and female pairs,respectively.At the end of the experiment,intestinal samples were obtained to measure digestive enzymes activities.The intestinal flora were determined by 16S r DNA sequencing.No eggs were observed in the FG and one T.tridentatus adult died.No animals died in the OG,and 9.7×10;eggs were obtained.These results show that oysters are more suitable for the development and reproduction of adult T.tridentatus than frozen fish.Additionally,the digestive enzyme activity analysis revealed that animals in the OG exhibited higher protein digestibility than those in the FG,but no significant differences in lipid and carbohydrate uptake were observed between the groups.Furthermore,the intestinal flora analysis showed that operational taxonomic units(OTUs)and the Chao1 index were significantly higher in the OG than in the FG,but no significant difference was observed in the Shannon or Simpson indices between the groups.Our data indicate that the oyster diet improved the intestinal microbial diversity of T.tridentatus.We hypothesize that nutrients,such as oyster-based taurine,proteins,and highly unsaturated fatty acids,improve protease activity in the T.tridentatus digestive tract,alter the intestinal floral structure,and improve the reproductive performance of T.tridentatus.

Identification and distribution of gonadotropin-releasing hormone-like peptides in the brain of horseshoe crab Tachypleus tridentatus

Gonadotropin-releasing hormone （GnRH） is a crucial peptide for the regulation of reproduction. Using immunological techniques, we investigated the presence of GnRH in horseshoe crab Tachypleus tridentatus. Octopus GnRH-like immunoreactivity, tunicate GnRH-like immunoreactivity, and lamprey GnRH-I-like immunoreactivity were detected in the neurons and fibers of the protocerebrum. However, no mammal GnRH-like immunoreactivity or lamprey GnRH-LII-like immunoreactivity was observed. Our results suggest that a GnRH-like factor, an ancient peptide, existed in the brain of T. tridentatus and may be involved in the reproductive endocrine system.

中华鲎(Tachypleus tridentatus)稚鲎对不同比例泥沙类型选择行为特征

中华鲎(Tachypleus tridentatus)是保障我国医疗检测安全的重要战略生物资源,国内外中华鲎行为生态学相关研究极少。在实验室内模拟砾石底(NS)、细砂(SS)、中砂(MS)、粗砂(CS)、泥(AR)、泥沙1(AA)、泥沙2(AB)和沙泥(SA)8种不同比例的泥沙类型对中华鲎行为特征的影响。结果表明,稚鲎较多时间潜藏于泥沙中,较少时间裸露于泥沙表面活动(如爬行、游泳等)。稚鲎偏好在含淤泥较多的泥沙类型中潜沙,且在泥质和泥沙质类型中埋栖时间较在沙质中埋栖时间长;在裸露于泥沙表面时,稚鲎相对偏好栖息于沙质底。泥沙环境下稚鲎用于游泳的时间比例高于砾石底。在凌晨、上午、中午、下午、夜晚5个时间段中,稚鲎在夜晚和凌晨活动较活跃。通过揭示不同比例的泥沙类型底质对中华鲎稚鲎行为特征的影响,为野外增殖放流计划的设计与实施、放流适宜地点的选择以及稚鲎人工养殖环境的优化提供科学理论依据。

Effect of Intertidal Elevation at Tsuyazaki Cove,Fukuoka,Japan on Survival Rate of Horseshoe Crab Tachypleus tridentatus Eggs

Physical factors affecting the survival of Tachypleus tridentatus eggs were investigated by translocating their eggs between the high intertidal zone and the low intertidal zone of a known spawning site.The mean egg survival rates per day were highest in the mid intertidal zone(45.1%±25.4%)and the lowest in the low intertidal zone(13.3%±27.6%).Differences in the elevation,air exposure time,and water content of the spawning ground were significant factors determining the egg survival rates.Excessive or insufficient air exposure time resulted in inadequate water content at higher and lower intertidal zones and could reduce egg survival.On the other hand,moderate saturation and dehydration were repeated with each tidal movement in the mid intertidal zone.This dynamic is considered as one of the crucial factors for the survival of eggs and is considered optimal for spawning.Therefore,the protection of the mid intertidal zone is imperative for maximizing the egg survival rate in Tsuyazaki Cove where almost all suitable nesting sites have disappeared due to coastal development.By protecting these optimal sites for spawning and recovering other optimal sites on suitable beaches,a positive contribution can be made to future management and conservation.The study also suggests that translocating eggs from marginal to optimal spawning sites might be a recovery strategy for this globally endangered species.

北部湾中华鲎(Tachypleus tridentatus)幼鲎生态位及种间竞争分析

中华鲎(Tachypleus tridentatus)属于国家二级重点保护野生动物,近年来数量锐减已成为濒危物种。该研究基于北部湾沿岸滨海湿地中华鲎种群调查,利用中华鲎幼鲎栖息地主要大型底栖动物资源数据,采用生态位宽度和生态位重叠指数分析探讨中华鲎幼鲎对资源利用情况及其与各物种之间竞争强弱,进一步采用种间联结性探究中华鲎幼鲎与各物种竞争情况。结果表明:(1)中华鲎幼鲎在底栖群落中属于广生态位物种,其生态位宽度最大(4.2522);(2)中华鲎幼鲎与各底栖生物均有一定程度生态位重叠,其中与短指和尚蟹(Mictyris brevidactylus)生态位重叠高达0.8375;(3)中华鲎幼鲎与短指和尚蟹种群动态模拟结果显示,二者种群规模在西背岭和三娘湾地区呈此消彼长状态,自然补充量减少是限制种群增长的重要因素。生态位分析表明,中华鲎幼鲎可作为滨海湿地生态系统健康程度指示物种,建议以中华鲎为旗舰物种开展滨海湿地生物多样性保护和管理宣传。

Effect of Probiotics on Juvenile Tachypleus tridentatus Gut Microbiota

The population of tri-spine horseshoe crabs(HSCs)Tachypleus tridentatus has sharply decreased in recent decades.Domestication and manual releasing are now used to recover it.In order to accelerate the growth of T.tridentatus in indoor environment,in this study,gut microbiota of 2nd instar T.tridentatus was supposed to be improved by putting probiotics in water environment and thus promoted the health and growth of the host.Using 16S rRNA sequencing of gut samples,we examined the intestinal microbiome of juvenile HSCs and studied the effect of probiotics.Results showed that there was no significant difference between groups fed with high and low concentrations(1×10^(7),1×10^(12)CFU mL^(−1))of Bacillus subtilis and high concentration(1×10^(12)CFU mL^(−1))of Lactobacillus,and no significant difference compared with the control group,but the diversity of Lactobacillus group with low concentration(1×10^(7)CFU mL^(−1))was significantly higher than those of other groups.Microbial-mediated functions predicted by PICRUSt showed that‘Cellular Processes’,‘Metabolism’and‘Organismal Systems’pathways significantly increased in groups of Bacillus subtilis and Lactobacillus group with low concentration.The results showed that two types of probiotics with different concentrations have other effects on the structure,richness and function of intestinal microbes.By searching the literature and conducting pre-experiments,we forecasted the short time of breeding under the probiotic environment may not have much effect.The present study for the first time investigated the impact of probiotics on gut microbiota of juvenile HSCs and found them may benefit for the growth and health of HSCs.

Indiscriminate Dietary Compositions of Two Asian Horseshoe Crabs,Tachypleus tridentatus and Carcinoscorpius rotundicauda:Evidence from Hemolymph Stable Isotopes

Knowledge of dietary compositions is essential to the recovery of the dwindling populations of horseshoe crabs in China.The feeding habits of Tachypleus tridentatus and Carcinoscorpis rotundicauda of Pearl Bay in southwestern China were studied by stable isotope analysis.Hemolymph samples of the two species were obtained from two age groups living in different habitats(inner,central,and outer Bay).In addition,their potential food source samples were collected in May 2019.Results showed that the mean values ofδ^(13)C andδ^(15)N in C.rotundicauda individuals ranged from−19.01‰to−16.47‰and from 10.49‰to 13.5‰,respectively,and those of T.tridentatus ranged from−19.12‰to−14.96‰and from 8.78‰to 13.48‰,respectively.These values indicated that the horseshoe crabs have a wide variety of food sources and therefore are highly omnivorous.No remarkably correlations were found betweenδ^(13)C andδ^(15)N values and individual widths in the two species.Thirteen potential food sources were selected,in which Sipunculus nudus,Batillaria zonalisi,and Ulva lactuca contributed largely to the dietary compositions of the two species of horseshoe crabs.No significant correlation was found between the feeding habits and habitat geochemical characteristics of the horseshoe crabs.All these results possibly indicate the high diversity of their food sources and the indiscriminate dietary compositions of the two Asian horseshoe crabs.

AFLP analysis of genetic variation among three natural populations of horseshoe crab Tachypleus tridentatus along Chinese coast

The AFLP(amplified fragment length polymorphism) technique was used to analyze and compare the genetic diversity of Tachypleus tridentatus from three south-eastern coastal sites of China(Pingtan,Hong Kong and Beihai).Eight pairs of primers generated 361 loci,including 285 polymorphic loci.The ratio of polymorphic loci was 96.97%.Nei's genetic diversity index was 0.420 8 and the Shannon information index was 0.607 5,both of which were higher than that reported for many other arthropods.These results show that the genetic diversity detected was mainly caused by individual differences within a population.Genetic distance showed that the rational division of the three geographic populations of T.tridentatus along the south-eastern coast of China was not significant,in which the genetic distance was not proportional to the geographic distance.All three horseshoe crab populations may belong to a large group,and had a high degree of genetic similarity.The high level of genetic diversity obtained from the present AFLP analysis may be due to the large effective population size of the species in Chinese waters.

Transcriptome Analysis of the Digestive Tract of Tachypleus tridentatus and Carcinoscorpius rotundicauda

Transcriptome assemblies for the stomach,midgut,and fecal mucosa of Tachypleus tridentatus and Carcinoscorpius rotundicauda are reported for the first time in this study.Genome assembly resulted in 373069 unigenes with an N50 of 1314,while transcriptome assembly resulted in 736378 unigenes and an N50 of 2121.The annotated unigenes showed the highest similarity to fishes,amphibians,and reptiles,with most unigenes closely related to metabolism,translation,biogenesis,signal transduction,energy production,immune response,and secretion.Unigenes(4768)were mapped to KEGG pathways,and the most enriched pathways were involved in translation,environmental information signal processing,metabolism,endocrine system,immune system,nervous system,and varying metabolism.Totally 65889,2001,and 7162 differentially expressed genes(DEGs)were identified in the stomach,midgut,and fecal mucosa between the two horseshoe crab species,respectively.Compared with C.rotundicauda,99.95%of the DEGs in the T.tridentatus stomach were down-regulated,while 99.97%of the DEGs of T.tridentatus fecal mucosa and 55.42%of the DEGs of the T.tridentatus midgut were up-regulated.Most midgut DEGs were involved in hydrolase activity,protein metabolism,and cell cytoplasm,while most stomach and fecal mucosa DEGs were involved in catalytic activity and primary metabolic processes.Most stomach DEGs were assigned to the cellular component in cellular macromolecular complexes,while most fecal mucosa DEGs were assigned to the cellular component in cell and cell parts.These results will benefit the investigation of the molecular mechanisms of the digestive tract related to the feeding habits and environmental traces of horseshoe crabs.

Genetic difference of Chinese horseshoe crab(Tachypleus tridentatus) in southeast coast of China based on mitochondrial COI gene analysis

Population genetic structure and historical demography of Chinese horseshoe crab (T.tridentatus)along southeast coast of China were inferred from the sequence data of mitochondrial cytochrome c oxidase subunit Ⅰ (COI) fragment.The sequence analysis for 964 bp COI fragment was conducted in 28 individuals collected from five localities:Ninghai in Zhejiang Province,Meizhou and Zhangpu in Fujian Province,Beihai of Guangxi Zhuang Autonomous Region and Danzhou of Hainan Province.Sequence variation was relatively low with a total of seven transitions observed.In all localities,Haplotype H3 was the dominant type observed among eight haplotypes defined previously,and was at the center of radiation in Median-Joining network.The prolonged star-like network suggests a signature of population expansions.The level of diversity was low in total,with haplotype diversity ( Hd) being equal to 0.765 and nucleotide diversity (π) being equal to 0.00118,respectively.The genetic structure analysis revealed the significant genetic difference between Ninghai and Danzhou populations.Both mismatch distribution analysis and Fu's Fs test provided consistent inference of historic population expansion.The low genetic diversity of horseshoe crab observed along China coast indicated that urgent measures should be taken to protect this rare marine animal.

雄性中国鲎不同组织重金属含量分析和比较

采用原子吸收分光光度计法测定天然海区野生平均体质量(1.23±0.12)kg成年中国鲎(Tachypleus tridentatus)结缔组织、血液、书鳃和肌肉中Pb、Cd、Cu和Zn的含量,旨在揭示海洋典型重金属类污染物在雄性中国鲎体内组织特异性富集特征。结果表明:肌肉中Pb、Cu和Zn的蓄积最高,其次是书鳃;Cd在书鳃中蓄积最高,其次是肌肉、血液和结缔组织。中国鲎不同组织的均值污染指数(PI)由大到小排序为:书鳃>肌肉>血液>结缔组织,其中结缔组织属于微污染级别,血液属于中污染级别,书鳃和肌肉达到严重污染级别,因此,食用中国鲎存在一定的健康风险。本研究结果可为濒危物种中国鲎种群数量恢复提供技术参考。

中国鲎人工驯养繁殖技术初步研究

为恢复濒危物种中国鲎(Tachypleus tridentatus)种群数量,2021—2022年在福建省罗源县开展中国鲎人工驯养繁殖技术研究。基于野外自然产卵场及幼体生境观测结果,构建人工模拟产卵环境,诱导野生亲本在仿生态环境中自然受精、产卵繁殖,并开展了人工孵化和苗种培育研究。研究组初步掌握了中国鲎成体对底质的栖息及繁殖偏好习性和生态需求,形成了适宜福建沿海中国鲎的人工驯养繁殖技术,对中国鲎人工驯养和自然繁殖有重要意义,可为福建沿海中国鲎人工育苗及野外种群资源恢复提供理论基础资料。