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.

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.

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.

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.

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.

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.

Ontogenetic resource use and trophic dynamics of endangered juvenile Tachypleus tridentatus among diversified nursery habitats in the northern Beibu Gulf,China

Horseshoe crabs,the most well-known example of“living fossils”,are iconic and ecologically important macroinvertebrates in coastal and estuarine ecosystems.Their blood is a crucial resource for manufacturing Limulus or Tachypleus amebocyte lysate to detect bacterial endotoxins or fungal contamination in drug and medical devices.An enhanced understanding of their ecological roles and trophic interactions in the food webs is fundamental to facilitate resource management for the declining populations in Asia.Foraging information of the Asian species,however,is mainly derived from preliminary,scattered reports from a limited number of study locations.In this study,resource utilization,trophic niche dynamics,and trophic interaction of the juvenile tri-spine horseshoe crab,Tachypleus tridentatus(instars 1–12,approximately 0.5–8 years old)across ontogeny was assessed in diversified nursery habitats along the northern Beibu Gulf,China,using carbon and nitrogen stable isotopes.Our results suggest that:(i)T.tridentatus are ecological generalists given the vast range of carbon isotopic values and trophic niche width estimates exhibited between multiple instar groups;(ii)juvenile T.tridentatus across most habitat types predominantly assimilated energy from a variety of basal production sources in the food web,but primarily depended on sedimentary organic matter and seagrass resource pools;(iii)ontogenetic shifts in juvenile dietary proportions were evident,with decreased reliance on sedimentary organic matter,coupled with increased reliance on benthic macroinvertebrate grazers,detritivores,and omnivores with age;and(iv)nearly all juvenile instars occupied similar trophic positions in the food web with slight shifts in trophic position present with increasing size.Our findings indicate that resource availability and ontogenetic diet shifts strongly influence horseshoe crab trophic dynamics,and age should be accounted when formulating habitat conservation measures based on resource use for Asian horseshoe crabs.

中国鲎变形细胞超微结构的进一步观察

中国鲎的变形细胞可在血液中发育成熟,根据其超微结构,可分为原始变形细胞、幼变形细胞和变形细胞三个阶段。 变形细胞受到外环境或内毒素的刺激后,第一型颗粒出现一系列变化,最终把内容物释放到细胞外。与此同时,细胞表面形成若干胞质性突起,边缘带分散断裂,质膜下和胞质性突起中出现微丝。第二型颗粒无变化,提示它们和血液凝胶化过程无直接关系。

三种离子通道在中国鲎脑神经节的分布

应用组织切片和免疫组织化学的方法在中国鲎脑神经中定位了3种离子通道的分布。研究检测到Na+通道和K+通道免疫阳性细胞和阳性纤维,但没有检测到Ca2+通道的免疫阳性。Na+通道主要分布在中间体神经纤维网和背面中央细胞群,K+通道分布于背面侧后方第二细胞群和背面中央细胞群。

Spatiotemporal Distribution of Asian Horseshoe Crab Eggs Are Highly Intermingled with Anthropogenic Structures in Northern Beibu Gulf,China

Identification,protection and restoration of spawning habitats are vital for protecting the depleted species.Asian horseshoe crabs are ecologically important macroinvertebrates in coastal and estuarine ecosystems.However,their spawning habitat studies were limited to several reports in tropical regions,possibly due to the lack of modified survey methods,particularly in habitats with a lower density of spawning adults,and/or intermingled with anthropogenic structures.In this study,the year-round egg distribution and spawning habitat baselines of Carcinoscorpius rotundicauda and Tachypleus tridentatus were determined in the northern Beibu Gulf,China.Our findings demonstrated that the peak spawning occurred in June–July and ceased in November–January when the average water temperature dropped below 20℃.Egg aggregations were found<10 cm beneath the sediment surface with regular tidal inundation,regardless of seasonal changes,in the vicinity of natural and artificial structures with elevated,mildly sloping substratum within the high tide zones.The nests were characterized by medium-sized sediment grains(0.5–0.9 mm),high temperatures(31–34℃),low water contents(0.8%–0.9%),and total organic carbon contents(0.5%–0.7%),which might maximize the hatching success.The identified nesting beaches were close to nursery habitats for juveniles,and tidal creeks were present as the possible corridor connecting these two important habitats through the dominant mangrove forests.The findings provide valuable insights in the scope of spawning behavior and nest-site selection of Asian horseshoe crabs under a mixture of natural and artificial structures,which could benefit future management efforts for the exploited spawning populations.

Asian Horseshoe Crab Bycatch in Intertidal Zones of the Northern Beibu Gulf:Suggestions for Conservation Management

Bycatch poses a severe challenge to global fishery management.Although studies have focused on marine megafauna bycatch,research on relevant issues regarding invertebrates is limited,particularly for the threatened horseshoe crabs.In this study,the distribution of fishing gears and the bycatch intensity of Asian horseshoe crabs in the intertidal zones of the northern Beibu Gulf that harbors the most abundant juvenile Asian horseshoe crabs populations in China were evaluated.Seven intertidal nursery habitats for Tachypleus tridentatus and Carcinoscorpius rotundicauda were surveyed from May to July in 2021.A transect that crossed the tidal creeks and tidal flats between the high tide embankment/vegetation and low tide line of a given habitat was surveyed during the ebb tides.The type,number,and GPS of fishing gears were recorded when sighted,and the number and prosomal width of each horseshoe crab species were measured.Bycatch intensities differed among habitats,ranging from 0.3 to 18.4 and 1.2 to 22.7 individuals per kilometer of transect for T.tridentatus and C.rotundicauda,respectively.Among the three identified fishing gear types,ground cages and stick net sets caused a stronger bycatch pressure on these two species.Most T.tridentatus bycatch and C.rotundicauda bycatch were large individuals in late juvenile and adult stages.Therefore,the ground cages and stick net sets along/near the tidal creeks,mangrove fringe,and low tide line should be regulated and removed to ensure the functionality of the intertidal zone as the spawning corridor and nursery habitat of Asian horseshoe crabs.

Activity Recording of Free-Ranging Tri-spine Horseshoe Crabs in the Southeastern Coast of Sabah,Borneo

In Inderasabah(southeast Sabah),tri-spine horseshoe crab Tachypleus tridentatus was observed for their locomotion activity using data loggers from September to November in 2015.A female with acceleration and depth-temperature loggers and five males with acceleration loggers were recaptured between 10 and 49 days after their release.From the record of 194 activity days that involve all six T.tridentatus,four horseshoe crabs,including the female,were active throughout the 24.0 h cycle,whereas the activity of the remaining two males was consistent with the 12.4 h cycle.Using the 40-day recording,three horseshoe crabs,including the female,were primarily active around the new moon and full moon,but they were dormant around the first and third quarter moon days.The female spent much time in shallow shores(depth<0.3 m)during the new moon and full moon.This result indicated that the female attempted to spawn in a minimum of three spring tide periods while lingering in the vicinity.Meanwhile,after spawning,the female spent time foraging in shallow water(depth 0.3–18 m).As for the two male individuals,their activity was consistent with semi-lunar periodicity.Therefore,both of them were in amplexus.In addition,a solitary male individual was active only during the first and third quarter moon days.Through activity recording,all the T.tridentatus in Inderasabah was active during daytime and nighttime.This result was contrary to T.tridentatus activity cycles in western Japan,where the species was found to be primarily nocturnal.Perhaps,the regional differences in activity cycles for T.tridentatus were related to their population adaptation toward water temperature,depth,and prey-searching periods.