Zum feinbau der cuticula von Limulus polyphemus L. (Chelicerata,Xiphosura)

The surface structure of the fracture in the exuvia of Limulus caused by the shedding of the exoskeleton is compared with the fine-structure of experimentally induced fractures using the electron microscope, Stereoscan.     

Survival and development of horseshoe crab (Limulus polyphemus) embryos and larvae in hypersaline conditions

The horseshoe crab Limulus polyphemus spawns in the mid- to upper intertidal zone where females deposit eggs in nests below the sediment surface. Although adult crabs generally inhabit subtidal regions of estuaries with salinities from 5 to 34 ppt, developing embryos and larvae within nests are often exposed to more extreme conditions of salinity and temperature during summer spawning periods. To test whether these conditions have a negative impact on early development and survival, we determined development time, survival, and molt cycle duration for L. polyphemus embryos and larvae raised at 20 combinations of salinity (range: 30-60 ppt) and temperature (range: 25-40 degrees C). Additionally, the effect of hyperosmotic and hypoosmotic shock on the osmolarity of the perivitelline fluid of embryos was determined at salinities between 5 and 90 ppt. The embryos completed their development and molted at salinities below 60 ppt, yet failed to develop at temperatures of 35 degrees C or higher. Larval survival was high at salinities of 10-70 ppt but declined significantly at more extreme salinities (i.e., 5, 80, and 90 ppt). Perivitelline fluid remained nearly isoosmotic over the range of salinities tested. Results indicate that temperature and salinity influence the rate of crab development, but only the extremes of these conditions have an effect on survival.     

Spermiogenesis in the horseshoe crab,Limulus polyphemus

Groups of spermatids of Limulus polyphemus undergo differentiation in thin-walled cysts within the seminiferous tubules. The nucleus compacts to a spherical shape, but retains a much less condensed nuclear appendage, whose unique pores are each surrounded by a microtubule. The appendage, unmodified mitochondria, glycogen, and coated vesicles, all present in the mature spermatozoon, suggest an unusual degree of metabolic self-sufficiency of the cell. The acrosome is associated with a 50 μ-long acrosomal filament that penetrates the nucleus during spermiogenesis and coils up in the cytoplasm, enveloped by two outer nuclear membranes. The filament, which eventually comes to lie in the circumnuclear cisterna, retains a covering of one membrane during its discharge at the time of the acrosome reaction. The posterior region of the head forms a thin-walled collar with peculiar internal supports around the base of the flagellum. Serverance of intercellular bridges between spermatids, cytoplasm elimination, and rupture of the cyst precede liberation of the immature spermatozoa into the lumen of the seminiferous tubules. Notwithstanding its peculiarities, the Limulus spermatozoon, with its simple shape closely resembling that of annelids and molluscs, represents the most primitive arthropod spermatozoon congruent with the evolutionary stability of the xiphosurans.     

The eyes of Limulus polyphemus (Xiphosura, Chelicerata) and their afferent and efferent projections

The visual system of the American horseshoe crab Limulus polyphemus (L. polyphemus) is an important preparation for studying the photoresponse, the circadian modulation of the photoresponse and visual information processing. Given its unique position in phylogeny the structure of its visual system also informs studies of the relationships among arthropods and the characteristics of eurarthropods. Much has been learned about the organization of the relatively simple L. polyphemus visual system, but much remains to be discovered. This review summarizes current knowledge of the structure of L. polyphemus eyes and the organization of their afferent and efferent projections and points to important unanswered questions.     

Cortical reaction in inseminated eggs of the horseshoe crab,Limulus polyphemus L

The egg cortical reaction in Limulus polyphemus is described in four events. Approximately 10 min after insemination, small “pits” are visible in the cortex of the inseminated Limulus egg. These pits progressively enlarge, eventually coalesce, and finally disappear, leaving a smooth-appearing surface approximately 60–90 min after insemination. Based on these visible changes, the following four events comprise the cortical reaction: (I) uninseminated egg—smooth surface, (II) inseminated egg—appearance and growth of pits, (III) inseminated egg—coalescence of pits, and (IV) inseminated egg—appearance of smooth surface. Preparation of these events for SEM studies demonstrated morphological characteristics and sequential development of the pits. Also numerous microvilli are found on the surface of uninseminated and inseminated eggs during the cortical reaction. An increase in their diameter during the reaction is particularly unique.     

Laboratory culture and maintenance of the horseshoe crab (Limulus polyphemus)

Often referred to as a living fossil, the American horseshoe crab, Limulus polyphemus, is one of the most-studied invertebrate animals in the world. It has served as a model in Nobel Prize-winning eye research, and researchers use a component of its blood to detect bacterial contamination in medical devices and drugs. The authors review the conditions necessary for housing these animals in the laboratory.     

The morphology of the horseshoe crab (Limulus polyphemus) visual system

Summary Efferent fibers to the compound eye of the horseshoe crab, Limulus polyphemus, not only innervate the various pigment cells, but also invade the eccentric cell dendrite and the retinula cells. This finding provides a structural basis for the coupling of circadian rhythm between the efferents and the receptor cells.This short communication is Publication No. 1130 of the Oregon Regional Primate Research Center. The research was supported by Grants RR-00163 and EY-00392 from the National Institutes of Health     

Distal-less expression in embryos of Limulus polyphemus (Chelicerata, Xiphosura) and Lepisma saccharina (Insecta, Zygentoma) suggests a role in the development of mechanoreceptors, chemoreceptors, and the CNS

The homeobox gene Distal-less (Dll) is well known for its participation in the development of arthropod limbs and their derivatives. Dll activity has been described for all groups of arthropods, but also for molluscs, echinoderms and vertebrates. Generally, Dll participates in the establishment of the proximo-distal-axis and differentiation along this axis. During our investigation of the expression pattern in the silverfish Lepisma saccharina and the horseshoe crab Limulus polyphemus, we found several expressions in late stages which cannot be explained with the "normal" limb-specific function. The antenna, cerci and terminal filament of the silverfish show a striped expression; single cells on the labrum, mandibles, maxillary palps and anal valves are also strongly stained by the Dll antibody. In addition to cell groups in the developing ganglia of the CNS, in the coxal endites and several nerve cells in femur and the trochanter of the prosomal limbs, the whole prosomal shield of Limulus polyphemus is surrounded by Dll-positive cell clusters. Furthermore, the lateral processes of the opisthosoma and the edges of the opisthosomal appendages are Dll positive. To get an indication of the cell fate of these regions, we examined hatched larvae and juvenile stages of both species with the SEM. We found a striking correlation of these Dll-positive areas and different sense organs, especially mechanoreceptors. Since many sense organs in arthropods are situated on the limbs, interpretation of the Dll expression in limbs is problematical. This has critical implications for comparative analysis of Dll expression patterns between arthropods and for the claim of homology between limb-like structures. Furthermore, we discuss the possibility of convergent appendage evolution in various bilaterian groups based on the improvement of spatial sensory resolution.     

Lipids from nerve tissues of the horseshoe crab, Limulus polyphemus.

1. The predominant lipids of nerve cords, ganglion and brain from horseshoe crabs were cholesterol (11% of lipid) and phospholipid (81% of lipid). 2. Major phospholipids were phosphatidyl ethanolamine and phosphatidyl choline with lesser amounts of phosphatidyl serine and phosphatidyl inositol and sphingomyelin. 3. The phospholipid fraction was characterized by a high content of plasmalogen, i.e. alk-1-enyl acyl phosphatides, so that 42% of the ethanolamine phosphatides were the plasmalogen, phosphatidal ethanolamine. 4. Phosphatidyl choline and phosphatidyl ethanolamine were high in polyunsaturation with 20:4 and 20:5 major fatty acids. Sphingomyelin had predominantly long chain saturated fatty acids. 5. Cerebrosides and gangliosides, which are associated with vertebrate nerve tissues, were absent from nerves of horseshoe crabs.     

Vesicle involvement in the egg cortical reaction of the horseshoe crab,Limulus polyphemus L

Ultrastructural observations (TEM) of the cortical reaction in Limulus polyphemus have been difficult to obtain due to the relative impermeability of the transparent egg envelope to standard fixatives. With the application of trialdehyde fixation techniques [Kalt, M. R., and Tandler, B. (1971). J. Ultrastruct. Res.36, 633–645], the cortical reaction has now been examined and the role of cortical vesicles has been determined. The size of these vesicles in uninseminated eggs is heterogeneous, with small vesicles (0.5 μm) being apposed to the plasmalemma and with large vesicles (4 μm) located in a lower layer of the egg cortex. The contents of the small vesicles are translucent under the electron beam. With the onset of egg activation these vesicles fuse with the overlying plasmalemma. The contents of the large vesicles appear electron dense and exhibit distinctly different morphologies. Shortly after insemination these large vesicles begin to enlarge by fusing together. By 9 min after insemination some enlarged vesicles fuse with the plasmalemma to form pits on the egg surface. The remaining enlarged vesicles continue to fuse with the plasmalemma until approximately 60 min after insemination when few vesicles are remaining.     

The chemorepellent semaphorin is expressed in the horseshoe crab, Limulus polyphemus.

Semaphorins are a family of soluble and membrane-bound proteins that play a critical role in axonal guidance and other processes of neuronal development. Currently, more than twenty semaphorins have been identified, all of which share a conserved 500 amino acid domain near the amino terminus. Semaphorins are divided into eight classes according to species of origin and structural similarities. Classes 1 and 2 are found in invertebrates, classes 3 through 7 are present in vertebrates and viruses encode class V semaphorin. Microarray analysis of Limulus CNS RNA revealed the presence of a semaphorin-like gene in Limulus polyphemus. Based on these data, we aligned 31 different sequences and designed degenerate primers for the consensus domains (WTT/SFLKA) and (DPY/VCA/GW). RT-PCR products were generated using 6 forward primers and 4 reverse primers. The expected size PCR products (750 bp) was obtained and then ligated with pCR II TOPO vector and transferred into E. coli Top 10. Five partial semaphorin cDNAs were found in Limulus: semaphorins 1a, 1b, 2a, 2b and F (now known as 5) were partially cloned. Subsequent Northern blot analyses using these Limulus specific-probes revealed hybridization with total RNAs purified from six different tissues.     

Presence of microorganisms in hemolymph of the horseshoe crab Limulus polyphemus.

Hemolymph samples obtained from Limulus polyphemus at the time of collection and after a 1-week holding period exhibited a significant increase in bacterial levels. No differences were observed in the ability of amoebocyte lysate, prepared from these same samples, to gel in the presence of lipopolysaccharide.     

Green algal infection of American horseshoe crab (Limulus polyphemus) exoskeletal structures

Degenerative lesions in the dorsum of the horseshoe crab (Limulus polyphemus) exoskeleton, eyes, arthrodial membrane, and base of the telson were documented in a population of wild caught laboratory animals. The disease can lead to loss of tissue structure and function, deformed shells, abnormal molting, loss of ocular structures, erosion of interskeletal membranes, and cardiac hemorrhage. Microscopy, histopathology, and in vitro culture confirmed the causative agent to be a green algae of the family Ulvaceae. Further research may explain how green algae overcome horseshoe crab innate immunity leading to external and internal damage.     

Presence of microorganisms in hemolymph of the horseshoe crab Limulus polyphemus

Hemolymph samples obtained from Limulus polyphemus at the time of collection and after a 1-week holding period exhibited a significant increase in bacterial levels. No differences were observed in the ability of amoebocyte lysate, prepared from these same samples, to gel in the presence of lipopolysaccharide.     

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.     

N epsilon(gamma-glutamyl)lysine crosslinks in the blood clot of the horseshoe crab, Limulus polyphemus.

Clots were allowed to form in samples of whole blood taken from the American horseshoe crab, Limulus polyphemus, in the absence and presence of dansylcadaverine (16), and were analyzed for their contents of N epsilon(gamma-glutamyl)lysine and gamma-glutamyl-dansylcadaverine. Clots obtained without dansylcadaverine yielded significant amounts of N epsilon(gamma-glutamyl)lysine product. Clots formed in the presence of dansylcadaverine yielded only gamma-glutamyl-dansylcadaverine. Formation of these products reflects on the activity of transglutaminase released from the blood cells during coagulation.