Conservation education program for threatened Asian horseshoe crabs: A step towards reducing community apathy to environmental conservation

Long-term and locally-based public education programs are essential in supporting conservation initiatives for horseshoe crab protection, particularly in Asia, where their populations have been largely exploited for food and biomedical applications. However, the implementation of such conservation initiatives can be difficult due to a community’s ingrained apathy towards the environment. In addition, the effectiveness of similar education programs to engage the community in conservation work has rarely been assessed. We described the implementation strategies of a conservation education program in Hong Kong, with the objective to improve the community’s awareness, engagement and environmental attitudes towards the conservation of Asian horseshoe crabs. This study evaluated the program’s outreach, outcomes and impacts between 2009 and 2016. By raising laboratory-cultured juvenile horseshoe crabs at local secondary schools, this long-term conservation education program succeeded in enhancing students’ knowledge on the biology and ecology of horseshoe crabs, as well as promoting positive attitudes and behaviors towards relevant conservation issues. Lessons and recommendations derived from this program can serve as guidance for similar campaigns in other places.     

Complete Mitochondrial Genomes of Carcinoscorpius rotundicauda and Tachypleus tridentatus (Xiphosura,Arthropoda) and Implications for Chelicerate Phylogenetic Studies

Horseshoe crabs (order Xiphosura) are often referred to as an ancient order of marine chelicerates and have been considered as keystone taxa for the understanding of chelicerate evolution. However, the mitochondrial genome of this order is only available from a single species, Limulus polyphemus. In the present study, we analyzed the complete mitochondrial genomes from two Asian horseshoe crabs, Carcinoscorpius rotundicauda and Tachypleus tridentatus to offer novel data for the evolutionary relationship within Xiphosura and their position in the chelicerate phylogeny. The mitochondrial genomes of C. rotundicauda (15,033 bp) and T. tridentatus (15,006 bp) encode 13 protein-coding genes, two ribosomal RNA (rRNA) genes, and 22 transfer RNA (tRNA) genes. Overall sequences and genome structure of two Asian species were highly similar to that of Limulus polyphemus, though clear differences among three were found in the stem-loop structure of the putative control region. In the phylogenetic analysis with complete mitochondrial genomes of 43 chelicerate species, C. rotundicauda and T. tridentatus were recovered as a monophyly, while L. polyphemus solely formed an independent clade. Xiphosuran species were placed at the basal root of the tree, and major other chelicerate taxa were clustered in a single monophyly, clearly confirming that horseshoe crabs composed an ancestral taxon among chelicerates. By contrast, the phylogenetic tree without the information of Asian horseshoe crabs did not support monophyletic clustering of other chelicerates. In conclusion, our analyses may provide more robust and reliable perspective on the study of evolutionary history for chelicerates than earlier analyses with a single Atlantic species.     

A review on fisheries and conservation status of Asian horseshoe crabs

Horseshoe crabs are the only extant xiphosurans and are believed to be morphologically unchanged for more than 200 million years. Of the four extant species namely, Limulus polyphemus, Tachypleus tridentatus, Tapinauchenius gigas and Carcinoscorpius rotundicauda, the latter three are found in Asian waters. Recent evidences showed that Asian horseshoe crabs are facing serious threats such as degradation of their spawning grounds and habitat, environmental pollution, overexploitation as a culinary delicacy and biomedical bleeding practices. Baseline data on the distribution and existing population of the wild horseshoe crabs remain poorly known in several Asian regions. Several studies have clearly revealed that pressure due to over-fishing of wild stock has increased tremendously in the last decade. Due to an increase in demand for Tachypleus Amebocyte Lysate (TAL) analogous to Limulus Amebocyte Lysate (LAL) in the United States, there is an urgent need to comprehensively address their fishing and conservation measures in the Asian region. This review addresses the overall studies on three species of Asian horseshoe crabs in relation to their fishing practices, local exploitation of their wild stock either for human consumption (or) by biomedical industries. The authors have structured the discussion on an international scale to address the existing problems in fishing and conservation of horseshoe crabs. Since no specific regulatory force or legislative protection act or a policy to preserve their natural stock are available to this date, this paper strongly recommends representative countries to include horseshoe crabs under their wildlife protection act to avoid further unsustainable exploitation of their wild populations.     

Intraspecific shape variation in horseshoe crabs: The importance of sexual and natural selection for local adaptation

A morphometric analysis of the body shape of three species of horseshoe crabs was undertaken in order to infer the importance of natural and sexual selection. It was expected that natural selection would be most intense, leading to highest regional differentiation, in the American species Limulus polyphemus, which has the largest climatic differences between different populations. Local adaptation driven by sexual selection was expected in males but not females because horseshoe crab mating behaviour leads to competition between males, but not between females. Three hundred fifty-nine horseshoe crabs from nine populations, representing three species, were analyzed using a digitizer to position sixty morphometric landmarks in a three-dimensional space. Discriminant analysis revealed strong regional structuring within a species, which suggests strong philopatry, and showed the existence of geographically-based intraspecific variation. An admixture analysis showed regional intraspecific differentiation for males and females of L. polyphemus and males of the Asian horseshoe crab Carcinoscorpius rotundicauda, but not for females of C. rotundicauda and another Asian horseshoe crab, Tachypleus gigas. Differences in shape variation between sexes were tested with F-tests, which showed lower intrapopulation morphometric variation in males than females. These results indicate a lower degree of local adaptation on body shape in C. rotundicauda and T. gigas than in L. polyphemus and a lower degree of local adaptation in females than in males.     

Position of horseshoe crabs in estuarine food webs: N and C stable isotopic study of foraging ranges and diet composition

To discern the position of horseshoe crabs as a potentially important predator in estuarine food webs, we determined where they foraged and what they ate. We used N and C stable isotopes to link adult horseshoe crabs to their oraging locations and potential food sources in Pleasant Bay, Cape Cod. The δ¹⁵N in tissues of horseshoe crabs and their potential foods suggest crabs were loyal to local foraging sites and did not forage substantially in subestuaries receiving >110 kg N ha⁻¹ year⁻¹. Among locations where crabs foraged, δ¹³C values in potential foods showed that food webs in subestuaries subject to higher N loads were supported by algal producers, while food webs in subestuaries with lower N loads were also supported by Spartina. δ¹³C values in horseshoe crab tissue did not change with load, suggesting they ate a mixed diet, regardless of N load. N and C isotopes in horseshoe crab feces were similar to signatures of estimated diet, suggesting low assimilation efficiency, perhaps due to ingestion of low quality organic matter. Although horseshoe crabs were relatively opportunistic in foraging habits, conservation or culture of horseshoe crabs may require habitats with higher water quality, ample particulate organic matter, and supporting a variety of prey.     

Tachyplesins isolated from hemocytes of Southeast Asian horseshoe crabs (Carcinoscorpius rotundicauda and Tachypleus gigas): identification of a new tachyplesin, tachyplesin III, and a processing intermediate of its precursor

Tachyplesins and their analogs are antimicrobial peptides composed of 17 or 18 amino acid residues present abundantly in acid extracts of hemocyte debris of horseshoe crabs. We purified here tachyplesin isopeptides from hemocytes of two species of Southeast Asian horseshoe crabs, Carcinoscorpius rotundicauda and Tachypleus gigas, and determined their amino acid sequences. The major tachyplesin isolated from both species was identified, respectively, as tachyplesin I, which had previously been found in hemocytes of the Japanese horseshoe crab (Tachypleus tridentatus). The yield from both species was very high (more than 70 mg per 100 g wet weight of hemocytes), i.e., comparable with that from T. tridentatus. In addition to tachyplesin I, a new tachyplesin isopeptide, named tachyplesin III, was also isolated from T. gigas hemocytes, in which an arginine replaced the 15th lysine of tachyplesin I. The carboxyl-terminal residue of the isolated tachyplesins I and III was confirmed, respectively, to be an arginine alpha-amide by chemical analysis. Furthermore, a tachyplesin peptide derivative with a carboxyl-terminal extension of glycine-lysine was newly found in the hemocytes of C. rotundicauda. It appeared to be an intermediate derived from a tachyplesin precursor during processing to the mature form.     

Acrosomal actin bundles of Asian and American horseshoe crab sperm differ in protein composition

Acrosomal actin bundles were isolated from the sperm of horseshoe crabs from four different sources, three from Asia and one from North America, and their protein constituents and structures were compared. The bundle from the American Limulus polyphemus sperm was composed of actin and two associated proteins of MW 95,000 and MW 52,000, as reported previously (Tilney, L. G. (1975) J. Cell Biol. 64, 289-310). However, those from the three Asian species (Tachypleus tridentatus, T. gigas, and Carcinoscorpius rotundicauda) were composed only of actin and the protein of MW 95,000. Electron microscopic and optical diffraction studies indicated that both the helical structures and the interfilament spacing of the actin filaments composing the acrosomal bundle were indistinguishable among the four species. These results suggest that the MW 95,000 protein crosslinks actin filaments in the bundle. Moreover, they support the idea that Limulus and the three Asian species have evolved independently from a common ancestor.     

The amino acid sequence of coagulogen isolated from southeast Asian horseshoe crab, Tachypleus gigas

The amino acid sequence of coagulogen isolated from Southeast Asian horseshoe crab (Tachypleus gigas) has been determined. The NH2-terminal sequence of the first 51 residues was obtained by automated Edman degradation. The intact protein was then treated with a Tachypleus clotting enzyme, to form a gel and to remove an internal peptide C (28 residues) located near the NH2-terminal portion. The gel protein, which consisted of A chain (18 residues) and B chain (129 residues), was S-alkylated and the resulting two chains were separated by acetone precipitation. Among these segments, A chain and peptide C were assigned to the NH2-terminal portion of whole coagulogen, as judged from their amino acid compositions. On the other hand, the covalent structure of B chain was determined by sequencing the peptides obtained from its tryptic digest. The alignments of the tryptic peptides were deduced from the sequence homology in comparison with the previously established B chain sequence of Japanese horseshoe crab (T. tridentatus) coagulogen. T. gigas coagulogen had a total of 175 amino acids and a calculated molecular weight of 19,770. When the sequence was compared with those of Japanese and American horseshoe crab (Limulus polyphemus) coagulogens, extensive structural homology was found: T. tridentatus/T. gigas, 87% and L. polyphemus/T. gigas, 67%. This comparison suggests that Japanese and Southeast Asian horseshoe crabs have a crab, based on amino acid sequence data.     

Chelicerate Skeletal Neuromuscular Systems

In the past decade several laboratories began investigatingthe neuromuscular physiology of the arthropod subphylum, Chelicerata.This paper concerning chelicerate skeletal neuromuscular systemsconcentrates on three major areas: fine structural featuresof the skeletal muscles; fine structure of synaptic areas; andinnervation patterns and neuromuscular physiology. For all cheliceratesinvestigated, the fine structural features of the muscle fibersappear to be similar with the exception that the sarcomere lengthsfall into two general size categories (ca. 4 µ. and 7µ.) . Two types of synaptic areas are found in the chelicerates.In horseshoe crabs the synaptic region is formed in a largeevagination of the sarcoplasm, and evaginations from more thanone muscle fiber may combine into one synaptic region. In thearachnids the synaptic area is formed in a slight furrow inthe muscle fiber or along the surface of the fiber. Studiesof innervalion patterns reveal that horseshoe crabs have a muchgreater number of motor axons innervating the skeletal musculaturethan do other chelicerates. The motor axons appear to be similarin function in horseshoe crabs, but in scorpions there are atleast two functional types. In only one investigation has peripheralinhibition been demonstrated in Chelicerata.     

Cell lineage analysis of neural induction: origins of cells forming the induced nervous system

Horseradish peroxidase (HRP) was used as an intracellular lineage tracer in two experiments designed to reveal the sites of origin of cells that formed the duplicate embryo which developed in relation to an organizer grafted in the ventral marginal zone (VMZ) of Xenopus laevis embryos. In the first experiment a dorsal blastoporal lip fully labeled with HRP was grafted in the VMZ of an unlabeled embryo at the beginning of gastrulation. This resulted in development of a second embryo in which labeled cells, of graft origin, formed the notochord, and parts of the somites, endoderm, and neural tube. The second experiment was designed to show the sites of origin of the host's cells that formed parts of the induced embryo. HRP was injected into individual blastomeres in a series of Xenopus embryos at the 32-cell stage and each embryo received an unlabeled organizer graft in the VMZ at the beginning of gastrulation. In these embryos the lineages that contributed to the host's primary neural tube did not contribute any cells to the induced neural tube. All the cells in the induced neural tube which originated from the host were descendants of ventral blastomeres that did not contribute to the neural tube normally. This shows that the second neural tube is formed as a result of the action of the organizer on cells in its immediate vicinity which would not normally have entered neural pathways of differentiation.     

Development of single-locus DNA microsatellite markers using 5′anchored ISSR-PCR method for the mangrove horseshoe crab, <Emphasis Type="Italic">Carcinoscorpius rotundicauda</Emphasis> (Latreille, 1802) in Peninsular Malaysia

Horseshoe crabs are said to be declining worldwide. However, there is still no published report on the status of horseshoe crabs in Malaysia. Thus, we report here eight informative microsatellite markers that were developed using the 5′-anchored ISSR-PCR enrichment procedure to diagnose the population genetic structure of the mangrove horseshoe crab, Carcinoscorpius rotundicauda from Peninsular Malaysia. This set of markers was tested on 127 samples and showed polymorphism in this species. Hence they should be useful in future essential population genetic studies of these living fossils in the Southeast Asian region.     

Rhythms of locomotion expressed by Limulus polyphemus, the American horseshoe crab: II. Relationship to circadian rhythms of visual sensitivity

In the laboratory, horseshoe crabs express a circadian rhythm of visual sensitivity as well as daily and circatidal rhythms of locomotion. The major goal of this investigation was to determine whether the circadian clock underlying changes in visual sensitivity also modulates locomotion. To address this question, we developed a method for simultaneously recording changes in visual sensitivity and locomotion. Although every animal (24) expressed consistent circadian rhythms of visual sensitivity, rhythms of locomotion were more variable: 44% expressed a tidal rhythm, 28% were most active at night, and the rest lacked statistically significant rhythms. When exposed to artificial tides, 8 of 16 animals expressed circatidal rhythms of locomotion that continued after tidal cycles were stopped. However, rhythms of visual sensitivity remained stable and showed no tendency to be influenced by the imposed tides or locomotor activity. These results indicate that horseshoe crabs possess at least two biological clocks: one circadian clock primarily used for modulating visual sensitivity, and one or more clocks that control patterns of locomotion. This arrangement allows horseshoe crabs to see quite well while mating during both daytime and nighttime high tides.     

Activin-treated ectoderm has complete organizing center activity in Cynops embryos

The differentiation and organizer activity of newt ectoderm treated with activin A was studied in explantation and transplantation experiments. In the explantation experiments, ectoderm dissected from late morulae–early gastrulae stage embryos treated with a high concentration of activin A (100 ng/mL) formed only yolk-rich endodermal cells. Mesodermal tissues, such as notochord and muscle, were seldom found in these explants. When they were transplanted into the blastocoele of other early gastrulae, they formed part of the endoderm of the host embryo and induced a secondary axis with only posterior characters (including axial mesoderm and neural tissues). In contrast, whole secondary axes were induced when activin-treated ectoderm was transplanted into the ventral marginal zone (VMZ) of early blastulae. The transplanted pieces invaginated by themselves and differentiated into foregut structures including pharynx, stomach, and liver. These phenomena were also observed in experiments in which presumptive foregut was used instead of activin-treated ectoderm. These findings show that activin-treated ectoderm can act as the complete organizing center in Cynops .     

中国鲎和圆尾鲎血淋巴细胞分类和特征的比较研究

为了更好地了解中国鲎(Tachpleus tridentatus)和圆尾鲎(Carcinoscorpius rotundicauda)血淋巴细胞的种类组成和特征差异,综合运用光学显微镜、扫描电镜和粒度仪,较为系统地对两种鲎的血淋巴细胞进行了分类和特征研究,从而为两种鲎的血淋巴细胞和分子生物学研究提供基础资料。根据血淋巴细胞大小、核质比、细胞着色特点、细胞中颗粒存在与否、颗粒的密集程度等,中国鲎和圆尾鲎的血淋巴细胞均可分为大颗粒细胞、小颗粒细胞和透明细胞三种主要类型,且两种鲎的血淋巴细胞均以颗粒细胞为主,透明细胞在血淋巴细胞中所占比例最小,但具有高核质比。两种鲎的同类血淋巴细胞在染色和形态上无显著性差异,但在同一种鲎中,血淋巴细胞密度存在显著的雌雄差异。       

Entrainment of juvenile horseshoe crab activity to artificial tides

Juvenile American horseshoe crabs, Limulus polyphemus, express both daily and tidal rhythms. To determine if, and how, tidal cues influence the expression of these rhythms, we exposed 25 animals to artificial tides, and 17 to artificial tides with inundation, both with a 12:12 LD cycle. In the first experiment, 24% expressed daily rhythms of activity, 24% tidal rhythms, 12% a combination of the two, and the rest were arrhythmic. Under subsequent atidal conditions some expressed daily rhythms, but more were circatidal. In the second experiment, 6% expressed daily rhythms, 71% tidal, 12% a combination, and 12% were arrhythmic. Those expressing tidal rhythms were more active during flood/high tide, while daily animals tended to be nocturnal. Under subsequent constant conditions, the majority exhibited circatidal activity, with some expressing one activity bout per day. We conclude that juvenile horseshoe crabs entrain to artificial tides, with inundation cycles providing stronger cues than water depth changes.     

Patterning of avian craniofacial muscles

Vertebrate voluntary muscles are composed of myotubes and connective tissue cells. These two cell types have different embryonic origins: myogenic cells arise from paraxial mesoderm, while in the head many of the connective tissues are formed by neural crest cells. The objective of this research was to study interactions between heterotopically transplanted trunk myotomal cells and presumptive connective tissue-forming cephalic neural crest mesenchyme. Presumptive or newly formed cervical somites from quail embryos were implanted lateral to the midbrain of chick hosts prior to the onset of neural crest emigration. Hosts were sacrificed between 7 and 12 days of incubation, and sections examined for the presence of quail cells. Some grafted tissues differentiated in situ, forming ectopic skeletal, connective, and muscle tissues. However, many myotomal cells broke away from the implant, became integrated into adjacent neural crest mesenchyme, and subsequently formed normal extrinsic ocular or jaw muscles. In these muscles it was evident that only the myogenic populations were derived from grafted trunk cells. Ancillary findings were that grafted trunk paraxial mesoderm frequently interfered with the movement of neural crest cells which form the corneal posterior epithelial and stromal tissues, and that some grafted cells formed ectopic intramembranous bones adjacent to the eye. These results verify that presumptive connective tissue-forming mesenchyme derived from the neural crest imparts spatial patterning information upon myogenic cells that invade it. Moreover, interactions between myotomal cells and both lateral plate somatic mesoderm in the trunk and neural crest mesenchyme in the head appear to operate according to similar mechanisms.     

Induction of multiple embryos with NaHCO3 or calcium free sea water in the horseshoe crab

Summary When horseshoe crab embryos were treated with NaHCO₃ at the developmental stage when the germ disc appears, multiple embryos were formed. NaHCO₃ may effect the formation of multiple embryos by binding Ca²⁺ ions of the embryo since multiple embryos were also formed by treatment with Ca²⁺ free sea water.The treatment caused the blastoderm layer to tear. When the embryos were returned to normal sea water after the treatment, the blastoderm recovered. Some cell masses, probably derived from the germ disc or its prospective cells, formed during the process of the recovery. Each cell mass developed into an embryo.Contributions from the Shimoda Marine Research Center, Nor. 348     

An anatomical study of secondary embryogenesis inCamellia reticulata

Summary An anatomical study was carried out during the sequences of events which lead to the differentiation of secondary embryos ofCamellia reticulata cv ‘Mouchang’. Secondary embryogenesis can be induced by culturing somatic embryos on a modified Murashige and Skoog medium supplemented with 0.5 mg·liter⁻¹ 6-benzylaminopurine and 0.1 mg·liter⁻¹ indole-3-butyric acid. After about 12 days of culture, globular-shaped secondary embryos became apparent, and by 18 to 20 days of culture cotyledonary stages were formed. Embryos developed mainly on the hypocotyl of primary embryos without an intermediate callus. Histologic monitoring revealed that secondary embryos apparently had a multicellular origin from embryogenic areas originating in both epidermal and subepidermal layers of the hypocotyl region. This morphogenetic competence is related to the presence, at the time of culture, of relatively undifferentiated cells in superfical layers of the primary embryo hypocotyl. Microcomputer image analysis was applied for quantifying cytological events associated with somatic embryogenesis. This method showed an increasing gradient in the nucleus-to-cell area ratio from differentiated cells passing through preembryogenic cells to embryogenic cells. The formation of embryogenic areas was preceded by accumulation of starch in the surrounding cortical cells. The cells underlying globular secondary embryos still contained abundant starch, but it declined as the secondary embryos developed.     

鲎的分布及生活习性

鲎是一种古老的节肢动物,现存的鲎分为二亚科三属四种,本文对鲎在世界上的分布及其生活习性做了概述。