Thesis (M.S.)--Worcester Polytechnic Institute. / Keywords: juvenile hormone; Limulus; horseshoe crab. Includes bibliographical references (p. 68-79).

Thesis (M.S.)--University of Delaware, 2007. / Principal faculty advisor: Nancy M. Targett and Timothy E. Targett, College of Marine and Earth Studies; Pamela J. Green, Dept. of Plant & Soil Science. Includes bibliographical references.

"Lipid-based hormones known as the juvenile hormones (JH) are ubiquitous among the arthropods, but their presence, functions, and sites of production in the horseshoe crab, Limulus polyphemus, remain unknown. Large size and lack of secondary sex characteristics in adult female horseshoe crabs may indicate continuous growth and molting throughout life, which is the outcome of high JH levels in insects and crustaceans. Here a study was undertaken to detect and localize lipid-based hormones in horseshoe crab hemolymph and tissue. Capillary electrophoresis and RP-HPLC analyses indicate the presence of a JH-like compound in subadult horseshoe crab hemolymph. The compound is present only in much lower amounts in the hemolymph of adult male and adult female horseshoe crabs. Identification of this compound was based on its similar retention time to standard JH, co-migration with added JH, and cross-reactivity with a polyclonal antibody to JH III. In addition, immunohistochemistry was used to localize the production site of this compound. Analysis of neural tissue, the assumed site of production, yielded no reactivity with labeled anti-JH III antiserum. In larval animals, however, reactivity was noted in yolk contained within the digestive tract. Since the larvae are lecithotrophic and feeding only on their yolk reserves, JH in the gut may be maternal, deposited in the egg before laying. Based on these results, we conclude that horseshoe crabs produce a lipid-based, JH-like hormone, with functional similarity to JH III in insects (i.e., maintenance of the juvenile form during growth and molting.) This paper is the first substantiation of such a hormone in horseshoe crabs. Our findings suggest that JH will be found in other chelicerates as well."

Thesis (M.S.) -- Worcester Polytechnic Institute. / Keywords: neuroblastoma; SHSY; Ependymin; Limulus Includes bibliographical references (p. 56-60).

This study examined the effects of blood extraction on the survival of horseshoe crabs and performed a preliminary investigation into amebocyte maintenance in vitro. Hemolymph volume of L. polyphemus was estimated over a representative size range of adults. Hemolymph volume expressed as a percentage of wet body weight was 25 ± 2.2% (mean ± S.D.) for males and 25 ± 5.1% for females. Mortality associated with blood extraction was evaluated for horseshoe crabs bled 0, 10, 20, 30, and 40% of their estimated hemolymph volume (unstressed group, N = 200). Mortality associated with the same bleeding levels was evaluated in horseshoe crabs that underwent simulated transport and handling procedures of the biomedical industry's bleeding process (stressed group, N = 195). Mortality rates of the unbled crabs were not significantly different between the stressed group and unstressed group. Of the bled animals, there was a higher (8.3%) mortality rate in the stressed group, than that (0%) in the unstressed group (P < 0.0001). Within the stressed group, mortality was significantly associated with bleeding (P = 0.0088). Horseshoe crab serum and a variety of standard insect cell culture media were evaluated for their effects on amebocyte morphology and viability after 7 days of maintenance in vitro. Horseshoe crab serum-supplemented cultures had significantly higher cell viability than serum-free cultures (N = 6; P = 0.0147). Significant differences in amebocyte viability were identified among the six insect cell culture media tested (N = 36; P < 0.0001), with the highest amebocyte viability of 77.2 ± 5.1% (mean ± S.D.) in Grace's Insect Medium without serum. Information gained from this study provides guidance on altering biomedical bleeding protocols to decrease horseshoe crab stress and mortality, and advances information on amebocyte culture medium selection, both of which contribute to decreasing the biomedical industry's impact on the horseshoe crab population. / Master of Science

ABSTRACT This thesis was divided into two parts. The purpose of part I was to clone and sequence the full-length ependymin gene from the invertebrate Limulus polyphemus, or portions of the gene, and to use RT-PCR to determine whether expression of this gene increases during leg regeneration. PCR was chosen as the method for obtaining the gene due to the success our lab had previously characterizing several ependymin genes using this approach. Three sets of primers were designed based on the conserved domains between teleost fish and three invertebrate ependymin sequences. “Sea primers" were designed based on the nucleotide sequence of the sea cucumber H. glaberrima for each conserved domain, and these primers produced all four of the expected size amplicons with Limulus DNA, but surprisingly only one such band with the sea cucumber Sclerodactyla briareus. The consensus primers (con-primers) were designed based on the most conserved nucleotide among all known ependymin species at each particular position in the conserved domains. Primers designated“5-11 primers" were designed based on the absolutely conserved domains among the three known invertebrate ependymins. Neither con-primers nor 5-11 primers produced any bands of the expected size; this was true for both species of DNA. One very strong band was produced using“5-11" primer pair 6/10 with both species. One of the bands from this reaction from Limulus was cloned and sequenced, and showed a very strong homology (88% over 292 bp) with mouse FGF-14, a neurotrophic factor involved in mouse neurogenesis. The expression of this gene during leg regeneration will be tested in future experiments. Limulus GAPDH was also cloned and sequenced, and a genomic intron was identified for the first time in this study. This Limulus housekeeping gene will be used in future studies for gene expression comparisons. The purpose of part two of this thesis was to study the up-regulation of growth-related genes induced by treatment of a human neuroblastoma SH-SY5Y cell line with a human ependymin peptide mimetic (hEPN-1), in an attempt to help provide a basis for using human EPN mimetics as therapeutics in stroke and neurodegenerative diseases. The sequence of this mimetic is derived from an area of human MERP-1 analogous to goldfish mimetic CMX-8933. The human mimetic was previously found to up-regulate growth related genes L-19, EF-2 and ATP Synthase in the mouse neuroblastoma cell line Nb2a (Saif, 2004). The expression levels of genes encoding ribosomal proteins and ribosomal RNA were studied using RT-PCR as hallmarks of proliferating cells. hEPN-1 was found to increase the expression of the nuclear-encoded ribosomal proteins S-19 and S-12, an average of 2.76 fold and 1.74 fold, with statistically significant p-values of 0.031 and 0.015 (<0.05), respectively. The expression levels of nuclear-encoded 5.8S ribosomal RNA (p = 0.018) and the mitochondrial-encoded 16S RNA (p = 0.046) were found to be increased an average of 14.04 fold and 3.91 fold, respectively. Thus, human ependymin mimetic hEPN-1 appears to stimulate growth-related genes, a property which can be useful to regenerate neuronal tissue after injury.

Information regarding spawning site fidelity and movement patterns of the American horseshoe crab (Limulus polyphemus) is crucial for developing effective conservation and management strategies on the correct spatial scale. To investigate the spatial ecology of American horseshoe crabs, 75 adult animals were tracked off the coast of Chatham, Cape Cod, MA from June 2010 to November 2011 using acoustic telemetry. Two groups of horseshoe crabs were tagged in spawning habitats (separated by ~2.0 km) with differing commercial harvesting pressure: one group inside Stage Harbor, where harvesting is permitted and the other within a Marine Protected Area (MPA) where harvesting is prohibited. Network analysis revealed that horseshoe crabs exhibited fidelity to spawning habitat, but not necessarily to the habitat where they were initially tagged. Fifty-nine percent of horseshoe crabs tagged inside Stage Harbor were detected in the MPA and 13% of horseshoe crabs tagged in the MPA were detected inside Stage Harbor. Although horseshoe crabs were utilizing both spawning habitats, predictive modeling revealed little temporal overlap, suggesting that horseshoe crabs from the two spawning habitats represent local populations. Isolated and local populations are more susceptible to overexploitation than are larger populations with many migrants. To protect against overharvest and extinction of isolated and local populations, the correct identification of management units (MUs) must be a priority of fisheries managers. Horseshoe crab populations around Cape Cod, Massachusetts and New England behave differently, requiring the collection of more information so that conservation tools such as MUs or MPAs can be used most effectively.

Modeling the complex relationships between habitat characteristics and a species' habitat preferences pose many difficult problems for ecological researchers. These problems are complicated further when information is collected over a range of time or space. Additionally, the variety of factors affecting these choices is difficult to understand and even more difficult to accurately collect information about. In light of these concerns, we evaluate the performance of current standard habitat preference models that are based on Bayesian methods and then present some extensions and supplements to those methods that prove to be very useful. More specifically, we demonstrate the value of extending the standard Bayesian hierarchical model using finite mixture model methods. Additionally, we demonstrate that an extension of the Bayesian hierarchical changepoint model to allow for estimating multiple changepoints simultaneously can be very informative when applied to data about multiple habitat locations or species. These models allow the researcher to compare the sites or species with respect to a very specific ecological question and consequently provide definitive answers that are often not available with more commonly used models containing many explanatory factors. Throughout our work we use a complex data set containing information about horseshoe crab spawning habitat preferences in the Delaware Bay over a five-year period. These data epitomize some of the difficult issues inherent to studying habitat preferences. The data are collected over time at many sites, have missing observations, and include explanatory variables that, at best, only provide surrogate information for what researchers feel is important in explaining spawning preferences throughout the bay. We also looked at a smaller data set of freshwater mussel habitat selection preferences in relation to bridge construction on the Kennerdell River in Western Pennsylvania. Together, these two data sets provided us with insight in developing and refining the methods we present. They also help illustrate the strengths and weaknesses of the methods we discuss by assessing their performance in real situations where data are inevitably complex and relationships are difficult to discern. / Ph. D.

In conjunction with Cambrex, a biomedical company that utilizes horseshoe crabs for the production of Limulus Amoebocyte Lysate (LAL), a study was conducted to investigate movement patterns and population distributions of horseshoe crabs to increase understanding of mid-Atlantic horseshoe crab populations. In addition, areas of the shoreline of Tom's Cove, Assateague Island, Virginia were investigated as possible locations for annual spawning surveys. Twelve thousand five hundred horseshoe crabs were tagged and released in Chincoteague, Virginia and Ocean City, Maryland as part of a movement study; 431 (3.45%) were reported as resights. The mean distance between site of release and site of recapture for all resighted crabs was 68.3 km; maximum distance moved was 493.7 km. During 1999-2004, demographic data were collected from horseshoe crabs harvested in Chincoteague, VA and Ocean City, MD. The proportion of females (p < 0.0001) and juveniles (p < 0.0001) sampled varied from year-to-year, but no trends were observed. This study also showed that a greater proportion of females were observed in the juvenile cohort sampled compared to the adults sampled (p < 0.0001). The spawning survey revealed that spawning activity in Tom's Cove varied between years. On May 30th, 2003, 1,192 horseshoe crabs were observed spawning on the northern shoreline of Tom's Cove. The maximum number of horseshoe crabs observed spawning in the same area in 2004 was 94. This study provides no evidence for isolated subpopulations in Chincoteague, VA or Ocean City, MD. It also shows that horseshoe crab sex and age ratios fluctuate annually, therefore requiring a long time series of data to detect trends. / Master of Science

C-reactive protein (CRP) is an acute phase protein of the innate immune system that has been evolutionarily conserved. Human CRP is known to exist in two different pentameric conformations; native CRP and non-native CRP that possess differential ligand recognition functions. The structure of CRP evolved from arthropods to humans, in terms of subunit composition, disulfide bonds, and glycosylation pattern. Along with change in structure, the gene expression pattern of CRP also evolved from a constitutive protein in lower invertebrates to an acute phase protein in humans. The objective of this study was to determine the function of a non-native pentameric CRP, that binds to atherogenic LDL, in atherosclerosis and compare the ligand recognition functions of human pentameric CRP with an evolutionary distant CRP for understanding the evolution of the structure of CRP. Additionally, in vitro reporter gene assays were used to gain further insight into the regulation of human CRP gene expression by an IL-6 inducible transcription factor, STAT3. We observed that CRP, in its non-native pentameric conformation, binds to atherogenic LDL and slows the progression of atherosclerosis in a site-specific manner in high fat diet fed LDLR-/- mice. Further, we observed that the ligand recognition function of CRP from an evolutionary conserved species, Limulus polyphemus, is different than that of native pentameric human CRP, but overlaps that of non-native pentameric human CRP. Lastly, we screened the proximal 300 bp region of the CRP promoter and identified a novel STAT3 binding site at position -134 located upstream of the previously identified, transcriptionally active STAT3 site at -108. In conclusion, non-native pentameric human CRP is an atheroprotective molecule whose ligand recognition functions exhibit similarity with CRP from an evolutionarily distant species. IL-6 mediated transcriptional regulation of human CRP is modulated, in part, by STAT3 binding to two distinct positions on the CRP promoter.