Phospholipids from the hepatopancreas of Indian horseshoe crab Carcinoscropius rotundicauda

Total phospholipids were extracted from the heart, hepatopancreas, and hemolymph of the Indian horseshoe crab Carcinoscorpius rotundicauda by the conventional method. Characteristic group reaction and 2-dimensional thin-layer chromatography on silica gel were used for identification of different phospholipids. The phospholipid profile obtained from hemolymph and 2 major organs are comparable and show phosphatidyl choline (PC) and phosphatidyl ethanolamine to be the major phospholipids. A phospholipid has been consistently detected migrating immediately below the PC in the thin-layer chromatogram of lipids extracted from the hepatopancreas. When mixed methyl esters of this slower moving PC are resolved on a silica gel plate ran in hexane ether:acetic acid 80:20:1, with appropriate controls, an additional spot is seen just below the normal methyl ester, indicating a difference between the fatty acid compositions of 2 PC (e.g., regular and slower). The slower mixed methyl esters were found to comprise mainly the 4 saturated fatty acids: lauric, myristic, palmitic, and stearic. The slow moving PC seems to consist mainly of molecular species with the above-mentioned saturated fatty acids at both Sn 1 and Sn 2 positions.     

Estimation of total hemolymph volume in the horseshoe crab Limulus polyphemus

Biomedical companies extract blood from the horseshoe crab, Limulus polyphemus, for the production of Limulus Amebocyte Lysate, used worldwide for detecting endotoxins in injectable solutions and medical devices. Despite the extensive use of horseshoe crabs by the biomedical industry, total hemolymph volume for this species is not known. The hemolymph volume of 60 adult horseshoe crabs was estimated using an inulin dilution technique. Blood volume of the horseshoe crab represented as a percentage of wet body weight was 25?±?2.2% for males and 25?±?5.1% (mean?±?SD) for females. Relationships between hemolymph volume and weight (p?=?0.0026, r ²?=?0.8762), hemolymph volume and prosomal width (p?<?0.0001), and hemolymph volume and inter-ocular width (p?<?0.0001) were observed. No significant differences were observed between males and females. The relationship of animal size and hemolymph volume can be used to predict how much blood can be drawn from horseshoe crabs used by the biomedical industry, and can be of further use in future bleeding mortality studies.     

基于MAXENT模型评估北部湾潮间带中国鲎和圆尾鲎稚鲎的潜在地理分布及种群保育对策

鲎具有极高的经济价值和科研意义。近年来由于过度捕捞和栖息地受损等原因,亚洲鲎种群数量正急剧下降。鲎漫长的生命周期使得鲎资源的保护和增殖迫在眉睫。生态位模型已经广泛应用于物种的潜在地理分布预测。基于实地调研数据和公开发表的北部湾中国海域中国鲎和圆尾鲎地理分布数据,运用MAXENT模型得到中国鲎和圆尾鲎在广西北部湾(中国部分)的栖息地适宜度指数(Habitat suitability index, HSI),确定了这两种稚鲎在北部湾中国海域潜在适生区。模型分析结果表明,潮间带坡度和地形指数是影响中国鲎分布的主要环境因子,而潮间带底质的有机物含量和植被指数是影响圆尾鲎分布的主要环境因子,根据研究结果建议在两种稚鲎适生区建立保护区,进行人工放流稚鲎,加强对海草和红树林的生态建设,进而促进鲎资源种群恢复和发展。     

Microsatellite DNA markers for the study of horseshoe crab (Limulus polyphemus) population structure

Twenty‐two microsatellite DNA loci were identified and characterized for horseshoe crabs (Limulus polyphemus) collected from two Atlantic coast and one Gulf of Mexico site. These markers revealed a high degree of genetic diversity (8–35 alleles per locus), heterozygosity (25.0% to 100.0%), and allelic heterogeneity (69.8% of comparisons). Considerable regional differentiation was observed as genetic distances (chord) ranged between 0.25 and 0.45, and all F_ST values (0.014–0.092) were significant. These preliminary findings are consistent with patterns of regional differentiation observed using allozyme variation and contradictory to findings of limited gene flow reported for sequence variation at the mitochondrial DNA COI region.     

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.     

Bactericidal activity of amebocytes from the horseshoe crab, Limulus polyphemus

The role of amebocytes in the host defense mechanisms of the horseshoe crab, Limulus polyphemus, was examined in a series of in vitro systems. Amebocytes were assayed for their ability to kill one of four bacterial strains in the presence or absence of hemolymph factors. No significant cidal effect was seen with unsupplemented amebocytes during the 1-hr incubation period. Escherichia coli was significantly inactivated when incubated with amebocytes plus either homologous pooled serum or plasma; Aerococcus viridans, Serratia marcescens, and Micrococcus luteus were not. The results are similar to those previously reported for serum from L. polyphemus and suggest an opsonizing activity in the fluid hemolymph.     

Variations of taurine concentration in the ovaries and hepatopancreas of the female crab Carcinus maenas L. (Decapoda Brachyura) during the different phases of its genital activity

Summary

The concentration of taurine within the ovary remains fairly stable during the different stages of oogenesis. However, there is a marked increase in the taurine level within the hepatopancreas during the terminal phase of vitellogenesis and this can be correlated with the metabolic needs of the oocytes.

In view of the osmoregulatory role of taurine in Crustacea, and of the reduction in hydration of the ovaries that takes place before spawning, at the end of anecdysis, a relation taurine-reproduction-moult is suggested.     

中国鲎保育工作研究进展

近年来中国鲎种群数量呈急剧下降,资源几近枯竭,如何有效开展其保育工作成为研究热点。从中国鲎的地理分布、生活习性、资源价值、保育工作开展现状、保育措施等5个方面对中国鲎资源现状以及其保育工作开展情况进行概括,为完善中国鲎保育工作提供最新资料。     

Histones from spermatozoa of the horseshoe crab

It is shown that histones are the nuclear proteins present in spermatozoa of the horseshoe crab Limmulus polyphemus, an arthropod which is considered a living fossil. They have been characterized and found to be closely related to calf thymus histones. The only difference is the presence of an additional histone in small amounts (2?3% of the whole histones) which has intermediate properties between H1 and H2b.     

Shorebird predation of horseshoe crab eggs in Delaware Bay: species contrasts and availability constraints

1. Functional responses -- the relationship between resource intake rate and resource abundance -- are widely used in explaining predator-prey interactions yet many studies indicate that resource availability is crucial in dictating intake rates. 2. For time-stressed migrant birds refuelling at passage sites, correct decisions concerning patch use are crucial as they determine fattening rates and an individual's future survival and reproduction. Measuring availability alongside abundance is essential if spatial and temporal patterns of foraging are to be explained. 3. A suite of shorebird species stage in Delaware Bay where they consume horseshoe crab Limulus polyphemus eggs. Several factors including spawning activity and weather give rise to marked spatial and temporal variation in the abundance and availability of eggs. We undertook field experiments to determine and contrast the intake rates of shorebird species pecking for surface and probing for buried eggs. 4. Whether eggs were presented on the sand surface or buried, we demonstrate strong aggregative responses and rapid depletion (up to 80%). Depletion was greater at deeper depths when more eggs were present. No consistent give-up densities were found. Type II functional responses were found for surface eggs and buried eggs, with peck success twice as high in the former. Maximum intake rates of surface eggs were up to 83% higher than those of buried eggs. 5. Caution is needed when applying functional responses predicted on the basis of morphology. Our expectation of a positive relationship between body size and intake rate was not fully supported. The smallest species, semipalmated sandpiper, had the lowest intake rate but the largest species, red knot, achieved only the same intake rate as the mid-sized dunlin. 6. These functional responses indicate that probing is rarely more profitable than pecking. Currently, few beaches provide egg densities sufficient for efficient probing. Areas where eggs are deposited on the sand surface are critical for successful foraging and ongoing migration. This may be especially true for red knot, which have higher energetic demands owing to their larger body size yet appear to have depressed intake rates because they consume smaller prey than their body size should permit.     

海洋活化石——鲎的研究进展

海洋活化石——鲎（hòu）是十分宝贵的自然资源,在科研、经济、生态等方面都有着不可估量的价值。鲎作为古老的海洋底栖动物被过度捕捞,现已到了枯竭的程度,2021年2月,我国《国家重点保护野生动物名录》正式更新和发布,我国海域的中国鲎和圆尾鲎被列为国家二级保护动物。为推动鲎研究和有效制定实施鲎资源的保护措施,本文主要从鲎的生物学研究、资源利用和生态保护三个方面入手,瞄准鲎的分类学问题、鲎的生物学前沿问题包括神经系统、视觉系统、消化系统、循环系统、生殖与发育和共附生微生物等、生态保护现状以及存在的问题等方面国内外的研究进展进行综述,明确研究重点和难点的同时,扩大对鲎的认知。     

Sufficiency of horseshoe crab eggs for red knots during spring migration stopover in Delaware Bay USA

Horseshoe crab (Limulus polyphemus) eggs are a dietary staple of the red knot (Calidris canutus) during its spring stopover on the Delaware Bay. Numbers of knots stopping in Delaware Bay declined in the 1990s concurrent with a decline in horseshoe crabs, leading to the hypothesis that reduced horseshoe crab egg abundance limited the red knot population. Management efforts, including a seasonal harvest moratorium in the Delaware Bay, have been instituted to restore crab populations to levels of sustainable use by multiple users, including migratory birds. Our objective was to evaluate the sufficiency of horseshoe crab eggs in Delaware Bay in May–June 2004 and 2005 for knots to refuel for their migratory flight to the Arctic breeding grounds. We examined egg counts to determine if there were fewer high egg-density sites later than earlier in the day and season, as migrating birds might deplete this resource. We studied foraging rates at red knot locations to determine if foraging probes increased with time of day and season as birds depleted surface eggs by pecking, then began probing for subsurface eggs. Finally, we experimentally tested whether red knots and their competitors depleted horseshoe crab eggs. Crab egg numbers at knot foraging sites did not decline throughout the day or season in 2004. In both years, we found no evidence that knots switched from pecking to probing with increases in time since sunrise or start of the stopover. Egg numbers were similar in exclosed and accessible plots on crab nesting depressions and in areas of open intertidal zone, but were significantly lower in accessible than in exclosed plots in the wrack line. Our results indicate that horseshoe crab eggs in Delaware Bay were sufficient to support the refueling of the present-day stopover population of red knots. If an increase in the availability of crab eggs to foraging birds does not result in an increase in knot numbers, managers must prioritize mitigation of limiting factors at other historically important spring stopovers and on the poorly understood breeding and wintering grounds in addition to the Delaware Bay. © 2011 The Wildlife Society.     

The effects of naphthalene on the ultrastructure of the hepatopancreas of the fiddler crab, Uca minax

The hepatopancreas of the red-jointed fiddler crab, Uca minax, is a bilateral evagination of the midgut, composed of numerous blind-ending tubules. Groups of these tubules empty into collecting ducts which join to form the main hepatopancreatic duct. Ultrastructural examination of tubules from the hepatopancreas of adult fiddler crabs revealed four major cell types, designated as E, R, B, and F cells. The E cells were found at the apex of the tubule and were assumed to serve as meristematic tissue. The R cells were most numerous and were scattered along the length of the tubule. Characterized by extensive smooth and rough endoplasmic reticulum and abundant lipid and glycogen reserves, the R cell was assumed to function in absorption and storage of the organic products of digestion. The B cells were recognized by the presence of a single, large apical vacuole that likely functioned in the secretion of digestive enzymes into the lumen of the hepatopancreas. The F cells, which contained extensive amounts of rough endoplasmic reticulum, were believed to be responsible for the synthesis of digestive enzymes. Electron microscopy of the hepatopancreas of crabs exposed to naphthalene for 5 days revealed that those cells with abundant membrane lipids (F cells) and abundant storage lipids (R cells) were most altered while those cells having little membrane or storage lipids (B and E cells) were only slightly altered. Furthermore, alterations in the F and R cells were not uniform along the length of the tubule, but increased in severity toward the proximal end.     

Incorporation of ω6 polyunsaturated fatty acids into phospholipids of the crab Carcinus maenas

The incorporation in vivo of ¹⁴C-18:2 ω6 and ³H-20:4 ω6 fatty acids in phospholipids isolated from gills, hepatopancreas and hemolymph of the crab Carcinus maenas was analysed. PC was the most heavily labelled phospholipid from these ω6-unsaturated fatty acids and appeared to play an important part in the phospholipids metabolism in Crustaceans. The pathway of fatty acids synthesis in phospholipids of C. maenas seems to be similar to those described for mammals. It is at the level of tissue Pl of C. maenas that the renewal of the 20:4 ω6 fatty acid is the most important. It is suggested that the rapid reorganization of phospholipid molecular species composition in the crab is checked by deacylation—reacylation cycle.     

Methyl farnesoate binding proteins in tissues of the spider crab, Libinia emarginata

Methyl farnesoate (MF) binding proteins (MFBPs) were found in the ovaries, testes, accessory glands, and hemolymph of the spider crab Libinia emarginata, by photoaffinity labeling the tissues in vitro with tritiated farnesyl diazomethyl ketone ([3H]-FDK). Specificity was demonstrated by competitive displacement of [3H]-FDK with MF. SDS-PAGE followed by fluorography revealed several labeled proteins in the hemolymph and testes with molecular masses ranging from 29 to 116 kDa, and two in the ovary that were 97 and 70 kDa. Tissues from reproductive animals bound twice as much label per gram weight compared to those that were from non-reproductive crabs.     

Endogenous rhythms of locomotion in the American horseshoe crab, Limulus polyphemus

American horseshoe crabs (Limulus polyphemus) exhibit clear circadian rhythms of visual sensitivity in the laboratory and in the field they exhibit seasonal patterns of mating behavior that are closely associated with the tides. Recent reports suggest that Limulus locomotor activity may be controlled by endogenous circadian and/or circatidal clocks and that light:dark (LD) cycles may affect the rhythmic output of both of these clocks. In this study, we examined locomotor behavior in the laboratory to determine the extent of this endogenous activity and to examine the influence of LD cycles on these rhythms. Thirty-three L. polyphemus were captured during the breeding season and their activity was monitored with activity boxes and “running wheels” in seawater kept at constant temperature and salinity. Activity patterns were analyzed using visual inspection of actograms and Chi-square and Lomb-Scargle periodograms. Overall, 36% of the animals was significantly more active during L, while only 12% was more active during D (52% showed no preference). Circatidal rhythms were observed in LD in 67% of the horseshoe crabs. Surprisingly, LD cycles appeared to synchronize these rhythms at times. In DD, the majority of animals tested (63%) exhibited circatidal rhythms that persisted for at least seven days. Overall, the results demonstrate that an endogenously controlled tidal rhythm of locomotion operates during, and significantly after, the breeding season in this species. In addition, the present results are consistent with the presence of circalunidian oscillators controlling these rhythms.     

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