The ontogeny of Limulus polyphemus (Xiphosura s. str., Euchelicerata) revised: looking "under the skin".

In recent years, methods for investigating the exo-morphology of zoological specimens have seen large improvements. Among new approaches, auto-fluorescence imaging offers possibilities to document specimens under high resolution without introducing additional artifacts as, for example, seen in scanning electron microscopy (SEM) imaging. Additionally, while SEM imaging is restricted to the outer morphology of the current instar, auto-fluorescence imaging can be used to document changes of the outer morphology of the next instar underneath the cuticle of the current instar. Thus, reinvestigating seemingly well known species with these methods may lead to interesting new insights. Here we reinvestigate the late embryonic development of the xiphosuran ("sword tail") Limulus polyphemus, which is often treated as a proxy for early eucheliceratan evolution. In addition to entire specimens, the appendages of the embryos were dissected off and documented separately with composite-autofluorescence microscopy. Based on these data, we can distinguish six developmental stages. These stages do not match exactly the formerly described stages, as these were largely based on SEM investigation. Our stages appear to represent earlier or later phases within what has in other studies been identified as one stage. This finer subdivision is visible as we can see the developing cuticle under the outer cuticle. In comparison to data from fossil xiphosurans, our results and those of other studies on the ontogeny of L. polyphemus point to a derived mode of development in this species, which argues against the idea of L. polyphemus as a "living fossil."

Low Oxygen Levels Slow Embryonic Development of Limulus polyphemus.

The American horseshoe crab Limulus polyphemus typically spawns in the upper intertidal zone, where the developing embryos are exposed to large variations in abiotic factors such as temperature, humidity, salinity, and oxygen, which affect the rate of development. It has been shown that embryonic development is slowed at both high and low salinities and temperatures, and that late embryos close to hatching tolerate periodic hypoxia. In this study we investigated the influence of hypoxia on both early and late embryonic development in L. polyphemus under controlled laboratory conditions. Embryos were exposed to four different oxygen levels and their developmental stage was scored every second day. Embryos developed more slowly at both 5% O2 and 10% O2 than at the 21% O2 treatment; late development was arrested when oxygen was reduced to 2%. Our study confims that L. polyphemus not only tolerates pronounced hypoxia in later embryonic developmental stages, but also in earlier, previously unexplored, developmental stages.

Environmental exposure of Atlantic horseshoe crab (Limulus polyphemus) early life stages to essential trace elements.

This study investigated the accumulation Co, Cu, Fe, Mn, Ni, Se, and Zn in Atlantic horseshoe crab (Limulus polyphemus) early life stages (egg, embryo and larvae) and compared the concentrations to the concentration of each element in sediment, pore water and overlying water for 5 sites across Long Island, NY. For the majority of the sites, all essential trace elements accumulated in the embryos and larvae. However, many of the embryos and larvae at specific sites presented different concentration patterns which had no apparent relationship with the local habitat sediment and water values. Generally, Cu, Fe, and Se sequentially increased from egg stage through larval stages for the majority of sites, while Co, Mn, and Ni only did for a few sites. Zinc also showed an increase across sites from embryo to larval stage, however was the only one to show a decrease in concentration from egg to embryo stage at all sites. Interestingly, Mn at Manhasset Bay presented embryo and larval stages to be 50 fold greater than all other sites while the egg stage showed similar values to other sites; this high degree of uptake could be due to a high concentration in the overlying water. All essential trace elements can be accumulated from the environment but greater concentrations may be influenced by abiotic factors and the predominant uptake route (aqueous versus diet) at each life stage. Future laboratory experiments are required to investigate factors that influence essential trace element accumulation and loss in horseshoe crab early life stages.

De Novo Assembly and Characterization of Early Embryonic Transcriptome of the Horseshoe Crab Tachypleus tridentatus.

The horseshoe crab Tachypleus tridentatus is a unique marine species and a potential model for marine invertebrate. Limited genomic and transcriptional data are currently available to understand the molecular mechanisms underlying the embryonic development of T. tridentatus. Here, we reported for the first time the de novo transcriptome assembly for T. tridentatus at embryonic developmental stage using Illumina RNA-seq platform. Approximate 38 million reads were obtained and further assembled into 133,212 unigenes. Sequence homology analysis against public databases revealed that 33,796 unigenes could be annotated with gene descriptions. Of the annotated unigenes, we identified a number of key components of several conserved metazoan signaling pathways (Hedgehog, Wnt, TGF-beta and Notch pathways) and other important regulatory genes involved in embryonic development. Targeted searching of Pax family genes which play critical roles in the formation of tissue and organ during embryonic development identified a complete set of Pax family genes. Moreover, the full length T. tridentatus Pax1/9a (TtPax1/9a) and Pax1/9b (TtPax1/9b) cDNA sequences were determined based on the transcriptome, demonstrating the immediate application of our database. Using quantitative real time PCR, we analyzed the expression patterns of TtPax1/9a and TtPax1/9b in different tissues of horseshoe crab. Taking advantage of Drosophila model, we further found that TtPax1/9b, but not TtPax1/9a, can partly rescue the Drosophila homolog Poxm dysfunction-caused lethality at the larval stage. Our study provides the embryonic transcriptome of T. tridentatus which could be immediately used for gene discovery and characterization, functional genomics studies in T. tridentatus. This transcriptome database will also facilitate the investigations of molecular mechanisms underlying embryonic development of T. tridentatus and other marine arthropods as well.

Multiple-stressor interactions influence embryo development rate in the American horseshoe crab, Limulus polyphemus.

Fertilized eggs of the American horseshoe crab, Limulus polyphemus, are buried in shallow nests above the high tide line, where they are exposed to variations in abiotic conditions during early development. Using a multiple-stressors approach, we examined whether the rate of embryonic development is affected by exposure to combinations of three factors: temperature (25, 30 and 35°C), salinity (5, 15 and 34 ppt) and ambient O2 (5%, 13% and 21% O2). Newly fertilized eggs were incubated under 27 fully factorial stressor combinations for 14 days, then allowed to recover in control conditions (30°C, 34 ppt, 21% O2) for an additional 14 days. Growth rate was measured every 2 days throughout the experiment (N=1289). We found that the effect of isolated stressors (high temperature, low salinity or low O2) reduced developmental success by up to 72% (low salinity), and that stressor combinations showed stronger effects and evidence of complex interactions. For example, low O2 had little effect individually but was lethal in combination with high temperature, and low temperature in isolation slightly decreased the rate of development but reduced the negative effects of low salinity and low O2. Development was delayed under exposure to low O2 but resumed upon return to control conditions after a 10 day lag. These data demonstrate that complex, synergistic interactions among abiotic stressors can substantially alter the development of a coastal invertebrate in ways that may not be predicted from the effects of the stressors in isolation.

A conserved genetic mechanism specifies deutocerebral appendage identity in insects and arachnids.

The segmental architecture of the arthropod head is one of the most controversial topics in the evolutionary developmental biology of arthropods. The deutocerebral (second) segment of the head is putatively homologous across Arthropoda, as inferred from the segmental distribution of the tripartite brain and the absence of Hox gene expression of this anterior-most, appendage-bearing segment. While this homology statement implies a putative common mechanism for differentiation of deutocerebral appendages across arthropods, experimental data for deutocerebral appendage fate specification are limited to winged insects. Mandibulates (hexapods, crustaceans and myriapods) bear a characteristic pair of antennae on the deutocerebral segment, whereas chelicerates (e.g. spiders, scorpions, harvestmen) bear the eponymous chelicerae. In such hexapods as the fruit fly, Drosophila melanogaster, and the cricket, Gryllus bimaculatus, cephalic appendages are differentiated from the thoracic appendages (legs) by the activity of the appendage patterning gene homothorax (hth). Here we show that embryonic RNA interference against hth in the harvestman Phalangium opilio results in homeonotic chelicera-to-leg transformations, and also in some cases pedipalp-to-leg transformations. In more strongly affected embryos, adjacent appendages undergo fusion and/or truncation, and legs display proximal defects, suggesting conservation of additional functions of hth in patterning the antero-posterior and proximo-distal appendage axes. Expression signal of anterior Hox genes labial, proboscipedia and Deformed is diminished, but not absent, in hth RNAi embryos, consistent with results previously obtained with the insect G. bimaculatus. Our results substantiate a deep homology across arthropods of the mechanism whereby cephalic appendages are differentiated from locomotory appendages.

Book gill development in embryos and first and second instars of the horseshoe crab Limulus polyphemus L. (Chelicerata, Xiphosura).

The scanning electron microscope (SEM) was used to study the development of the opisthosomal appendages and book gills of the horseshoe crab, Limulus polyphemus. Later embryonic stages were examined as well as the first and second instars. The observations are compared with a much earlier light microscopic description of book gill development in the horseshoe crab and with book lung development in scorpion embryos and first and second instars in a recent study with SEM. After the third embryonic molt in the horseshoe crab, the opisthosomal appendages are of sufficient size so they could be fractured or dissected open so internal cells and other structures could be examined. The opisthosomal appendages and book gill lamellae of first and second instars were also opened. The observations support the earlier histological report that the gill lamellae are a hypodermal outgrowth from the posterior surface of the preceding branchial appendages. The genital operculum, branchial appendages and gill lamellae are very thin and consist of external cuticle, hypodermis and space holders. The latter help hold the cuticle walls in place so hemolymph can flow through the narrow channels. The space holders are formed from cell processes that extend into the lumen from the hypodermis just inside the external cuticle. In the recent SEM study in scorpion embryos and in some histological investigations in spider embryos, the book lung lamellae are formed by alignment of cells from an invaginated sac or mass of cells. This clearly differs from the mode of formation of gill lamellae as observed in this and earlier investigations. These reports of differences in embryology refine but do not preclude hypotheses about book gill/book lung homology since addition, deletion or modification of ancestral features often occur for the benefit of the embryos and larvae.

Enhancement of vertebrate cardiogenesis by a lectin from perivitelline fluid of horseshoe crab embryo.

Cardiac myocytes are the first cells to differentiate during the development of a vertebrate embryo. A wide variety of molecules take part in various steps in this process. While exploring biologically active molecules from marine sources, we found that a constituent of perivitelline fluid from embryos of the Indian horseshoe crab can enhance growth and differentiation of chick embryonic heart. We have purified the factor and identified the cardiac promoting molecule to be a novel lectin. We show that this molecule influences cardiac development by increasing the number of cells constituting the heart and by modulating the expression of several cardiac development regulatory genes in chick embryos. Using mouse embryonic stem cells we show that the cardiac myocyte-enhancing capacity of this molecule extends to mammals and its effects can be blocked using methylated sugars. This molecule may prove to be an important tool in the study of cardiomyocyte differentiation.

Isolation and expression of Pax6 and atonal homologues in the American horseshoe crab, Limulus polyphemus.

Pax6 regulates eye development in many animals. In addition, Pax6 activates atonal transcription factors in both invertebrate and vertebrate eyes. Here, we investigate the roles of Pax6 and atonal during embryonic development of Limulus polyphemus rudimentary lateral, medial and ventral eyes, and the initiation of lateral ommatidial eye and medial ocelli formation. Limulus eye development is of particular interest because these animals hold a unique position in arthropod phylogeny and possess multiple eye types. Furthermore, the molecular underpinnings of eye development have yet to be investigated in chelicerates. We characterized a Limulus Pax6 gene, with multiple splice products and predicted protein isoforms, and one atonal homologue. Unexpectedly, neither gene is expressed in the developing eye types examined, although both genes are present in the lateral sense organ, a structure of unknown function.

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.

Evolution of arthropod visual systems: development of the eyes and central visual pathways in the horseshoe crab Limulus polyphemus Linnaeus, 1758 (Chelicerata, Xiphosura).

Despite ongoing interest into the architecture, biochemistry, and physiology of the visual systems of the xiphosuran Limulus polyphemus, their ontogenetic aspects have received little attention. Thus, we explored the development of the lateral eyes and associated neuropils in late embryos and larvae of these animals. The first external evidence of the lateral eyes was the appearance of white pigment spots-guanophores associated with the rudimentary photoreceptors-on the dorsolateral side of the late embryos, suggesting that these embryos can perceive light. The first brown pigment emerges in the eyes during the last (third) embryonic molt to the trilobite stage. However, ommatidia develop from this field of pigment toward the end of the larval trilobite stage so that the young larvae at hatching do not have object recognition. Double staining with the proliferation marker bromodeoxyuridine (BrdU) and an antibody against L. polyphemus myosin III, which is concentrated in photoreceptors of this species, confirmed previous reports that, in the trilobite larvae, new cellular material is added to the eye field from an anteriorly located proliferation zone. Pulse-chase experiments indicated that these new cells differentiate into new ommatidia. Examining larval eyes labeled for opsin showed that the new ommatidia become organized into irregular rows that give the eye field a triangular appearance. Within the eye field, the ommatidia are arranged in an imperfect hexagonal array. Myosin III immunoreactivity in trilobite larvae also revealed the architecture of the central visual pathways associated with the median eye complex and the lateral eyes. Double labeling with myosin III and BrdU showed that neurogenesis persists in the larval brain and suggested that new neurons of both the lamina and the medulla originate from a single common proliferation zone. These data are compared with eye development in Drosophila melanogaster and are discussed with regard to new ideas on eye evolution in the Euarthropoda.

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.

Development of the nervous system in the "head" of Limulus polyphemus (Chelicerata: Xiphosura): morphological evidence for a correspondence between the segments of the chelicerae and of the (first) antennae of Mandibulata.

We investigated brain development in the horseshoe crab Limulus polyphemus and several other arthropods via immunocytochemical methods, i.e. antibody stainings against acetylated alpha-tubulin and synapsin. According to the traditional view, the first appendage-bearing segment in chelicerates (the chelicerae) is not homologous to the first appendage-bearing segment of mandibulates (first antenna, deutocerebrum) but to the segment of the second antenna (tritocerebrum) or the intercalary segment in hexapods and myriapods. Accordingly, the segment of the deutocerebrum in chelicerates would be completely reduced. The main arguments for this view are: (1) the postoral origin of the cheliceral ganglion, (2) a poststomodaeal commissure, and (3) a connection of the cheliceral ganglion to the stomatogastric system. Our data show that these arguments are not convincing. During the development of horseshoe crabs there is no evidence for a former additional segment in front of the chelicerae. Instead, comparison of the brain structure (neuropil ring) between chelicerates, crustaceans and insects shows remarkable similarities. Furthermore, the cheliceral commissure in horseshoe crabs runs mainly praestomodaeal, which would be unique for a tritocerebral commissure. An unbiased view of the developing nervous system in the "head" of chelicerates, crustaceans and insects leads to a homologisation of the cheliceral segment and that of the (first) antenna (= deutocerebrum) of mandibulates that is also congruous to the interpretation of the Hox gene expression patterns. Thus, our data provide morphological evidence for the existence of a chelicerate deutocerebrum.

Amebocyte production begins at stage 18 during embryogenesis in Limulus polyphemus, the American horseshoe crab.

Limulus polyphemus, the American horseshoe crab, has a single type of circulating blood cell, the granular amebocyte, which is the horseshoe crab's primary cellular defense against microbial infection. On exposure to gram-negative bacteria or their endotoxins, the amebocytes degranulate, releasing the clotting protein coagulogen and a number of proteases. The protease cascade converts the soluble coagulogen to insoluble coagulin, which forms fibrous clots that seal off the site of infection. The first description of this clotting reaction in the 1950s initiated development of Limulus amebocyte lysate and spurred an intensive study of the amebocytes. However, the site or sites and timing of amebocyte production have yet to be determined. We report here that during embryonic development in Limulus polyphemus, amebocyte production begins at stage 18. The first amebocytes detected are found in developing hemocoel cavities, and the cells may derive from previously undifferentiated yolk nuclei.

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.

Conservation and variation in Ubx expression among chelicerates.

Chelicerates are an ancient arthropod group with a distinct body plan composed of an anterior (prosoma) and a posterior portion (opisthosoma). The expression of the Hox gene Ultrabithorax (Ubx) has been examined in a single representative of the chelicerates, the spider Cupiennius salei. In spiders, Ubx expression starts in the second opisthosomal segment (O2). Because the first opisthosomal segment (O1) in spiders is greatly reduced relative to other chelicerates, we hypothesized that the observed Ubx expression pattern might be secondarily modified. Shifts in the anterior boundary of the expression of Ubx have been correlated with functional shifts in morphology within malacostracan crustaceans. Thus, the boundary of Ubx expression between chelicerates with different morphologies in their anterior opisthosoma could also be variable. To test this prediction, we examined the expression patterns of Ubx and abdominal-A (collectively referred to as UbdA) in two basal chelicerate lineages, scorpions and xiphosurans (horseshoe crabs), which exhibit variation in the morphology of their anterior opisthosoma. In the scorpion Paruroctonus mesaensis, the anterior border of early expression of UbdA is in a few cells in the medial, posterior region of the O2 segment, with a predominant expression in O3 and posterior. Expression later spreads to encompass the whole O2 segment and a ventral, posterior portion of the O1 segment. In the xiphosuran Limulus polyphemus, early expression of UbdA has an anterior boundary in the segment. Later in development, the anterior boundary moves forward one segment to the chilarial (O1) segment. Thus, the earliest expression boundary of UbdA lies within the second opisthosomal segment in all the chelicerates examined. These results suggest that rather than being derived, the spider UbdA expression in O2 likely reflects the ancestral expression boundary. Changes in the morphology of the first opisthosomal segment are either not associated with changes in UbdA expression or correlate with late developmental changes in UbdA expression.

Purification, characterization, and amino acid sequence of an embryonic lectin in perivitelline fluid of the horseshoe crab.

Hemagglutinating activity in perivitelline fluid of the horseshoe crab embryo dramatically increases during the third and fourth molt of the embryo. A 27-kDa lectin, which we named tachylectin-P (TL-P), was newly identified in perivitelline fluid of the horseshoe crab Tachypleus tridentatus. TL-P preferentially agglutinated human A-type erythrocytes, and the activity was inhibited by N-acetyl group-containing monosaccharides. The amino acid sequence analysis indicated that TL-P is almost structurally the same as a hemocyte-derived lectin with no hemagglutinating activity, tachylectin-1 (TL-1), and that 218 out of 221 amino acid residues in total were conserved between the two lectins. Despite the high sequence similarity, biological and biochemical characteristics of TL-P differed from those of TL-1: (i) unlike TL-1, TL-P agglutinates several animal-derived erythrocytes; (ii) unlike TL-1, TL-P has no significant affinity for bacterial lipopolysaccharides or antibacterial activity; (iii) Based on apparent molecular masses determined by gel filtration, TL-P forms a dimer in solution, while TL-1 is present as a monomer; (iv) and TL-P interacts with endogenous proteins of 13 and 14 kDa present in the perivitelline fluid; however, neither has any affinity for TL-1. We propose that TL-P may have an important role in completing embryonic development by interacting with endogenous glycoproteins or N-acetylhexosamines.

Studies on perivitelline fluid of horseshoe crab embryo. I. Purification and properties of agglutinin from the perivitelline fluid of Tachypleus gigas embryo.

Agglutinin in the perivitelline fluid (PVF) of Tachypleus gigas, horseshoe crab, embryo was isolated and purified by a combination of affinity column chromatography on Sepharose 4B coupled with bovine submaxillary gland mucin and gel-filtration of Fractogel TSK (Toyopearl) HW-60 in Tris-NaCl-CaCl2 (0.05 M Tris-HCl, pH 7.5, containing 0.5 M NaCl and 0.1 M CaCl2) buffer, containing 1 M urea. The specific activity of the purified protein was increased about 1,300 times in comparison with that of the starting material. The active protein was present in highly polymerized forms which were multimers of an identical subunit with a molecular weight of approximately 40,000 as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This agglutinin was shown to have multimeric activity towards different kinds of erythrocytes and its hemagglutinating activity was inhibited by N-acetylamino sugars and bovine submaxillary gland mucin containing sialic acid. Urea and guanidine-HCl inhibited the agglutinating activity but the activity recovered after dilution or dialysis, whereas the effect of HCl, NaOH, or 2-mercaptoethanol was irreversible.