"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."

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

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.

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

Atlantic horseshoe crabs (Limulus polyphemus) have endured decades of intense harvest pressure. Genetics studies have shown evidence of distinct sub-groups spanning the coast, although few fine-scale studies have been done to delineate these groups on a local level. Massachusetts lies directly between two of these sub-groups. With documented differences in prosomal widths of horseshoe crabs from either side of Cape Cod, it is possible that Cape Cod is a barrier to gene flow and that there are two distinct genetic groups within Massachusetts. Regulations currently consider all horseshoe crabs to be of one stock. I examined 6 microsatellite loci from 193 horseshoe crabs collected from 7 locations across Massachusetts between 5 May and 24 June 2010. I also analyzed the prosomal widths of 324 horseshoe crabs from 8 locations across Massachusetts. Data analysis revealed low divergence with a G′ST of 0.005 (95% CI −0.004–0.013) and a G″ST of 0.015 (95% CI −0.014–0.045). Wellfleet Bay showed evidence of divergence from all other sites except Buzzards Bay. Isolation by distance is apparent via the Atlantic Ocean. Phenotypic variation in the prosomal widths of horseshoe crabs shows greater divergence among sites than neutral markers and indicates the presence of additive genetic effects. Low divergence and high heterozygosity indicate that although documented population declines have occurred, effective population size (Ne) is still large enough to maintain allele frequencies. With isolation by distance, divergence is likely to increase over time if populations remain low. Phenotypic divergence shows the possibility of local adaptation and that the implementation of management units (MUs) to the north and south of Cape Cod would be recommended as a conservative measure.

Horseshoe crabs (Limulus polyphemus) have been harvested along the east coast of the United States since the 1800s, however a Fishery Management Plan (FMP) was only recently created for this species. To date, there have not been any studies that have attempted to identify or quantify bycatch in the horseshoe crab trawl fishery. A horseshoe crab trawl survey was started in 2001 to collect data on the relative abundance, distribution, and population demographics of horseshoe crabs along the Atlantic coast of the United States. In the present study, species composition data were collected at sites sampled by the horseshoe crab trawl survey in 2005 and 2006. Seventy-six different taxa were identified as potential bycatch in the horseshoe crab trawl fishery. Non-metric multidimensional scaling (NMS) was used to cluster sites and identify the spatial distribution of taxa. Sites strongly clustered into distinct groups, suggesting that species composition changes spatially and seasonally. Species composition shifted between northern and southern sites. Location and bottom water temperature explain most of the variation in species composition. These results provide a list of species that are susceptible to this specific trawl gear and describe their distribution during fall months throughout the study area. Identifying these species and describing their distribution is a first step to understanding the ecosystem-level effects of the horseshoe crab trawl fishery. / Master of Science

The American horseshoe crab (HSC), Limulus polyphemus, is one of four species of horseshoe crabs found throughout the world, and the only one found in North America. It is an economically and ecologically important species throughout its native range from Maine to the Yucatan Peninsula. Harvested for fertilizer and livestock feed in the 19th century, the species is now harvested as bait for whelk and eel fisheries, and for their blood by the biomedical industry. The Atlantic States Marine Fisheries Commission (ASMFC) started to formally manage HSC in 1998 with its Interstate Fisheries Management Plan (IFMP). Unique emphasis and harvest limits have been placed on the Delaware Bay stock, as it is commercially exploited and a critical food source for the threatened red knot, Calidris canutus rufa. Previously, estimates of relative and total abundance of HSC in the Delaware Bay area were based on a design-based approach using a stratified random sampling design. In Chapter 1 of this work, I developed hurdle models for each of the six HSC demographic groups to standardize catch-per-unit-effort (CPUE) and estimate relative abundance using a model-based approach. It was determined that while the two approaches resulted in mostly convergent estimates of relative abundance, external factors such as month, time-of-day, and average depth have major effects on the observed CPUE of all demographic groups. Chapter 2 involved the development of hurdle models for the three species of bycatch frequently caught in our trawls, channeled whelk (Busysotypus canaliculatus), knobbed whelk (Busycon carica), and summer flounder (Paralichthys dentatus). It was found that channeled whelk relative abundance has been at a historical low since 2016, while summer flounder has been at a consistent high. Recent estimates of knobbed whelk relative abundance have been less variable than previously seen, with estimates since 2016 being similar to those seen before 2012. These results provide the first estimates for whelk population trends in the mid-Atlantic region and add to the growing knowledge of summer flounder relative abundance in the area. In Chapter 3, I applied the hurdle models developed in Chapter 1 to estimate the total abundance of HSC in the Delaware Bay area. For this work, I developed two spatio-temporal variograms to estimate bottom temperature and bottom salinity at unmeasured cells per month in the time series. The results showed that night estimates of total abundance were consistently higher than daytime estimates, and estimates from September or November resulted in the highest estimated catch for all demographic groups. The results suggest that when comparing September model-based estimates at night to those of the design-based approach, nearly a third of all previous design-based estimates significantly underestimated the total abundance of HSC in the Delaware Bay area. This result suggests that the ASMFC can recommend increased harvest limits for mature individuals if that action aligns with the goals of their adaptive resource management (ARM) framework. / Master of Science / The American horseshoe crab (HSC), Limulus polyphemus, is one of four species of horseshoe crabs found throughout the world, and the only one found in North America. It is an economically and ecologically important species throughout its native range from Maine to the Yucatan Peninsula. Harvested by the millions in the 19th century, the species is still harvested as bait for whelk and eel fisheries, and for their blood by the biomedical industry, on the order of hundreds of thousands. Formal management of HSC by the Atlantic States Marine Fisheries Commission (ASMFC) began in 1998, and allowable catch and landings have decreased since 1999. A strong focus has been placed on the Delaware Bay population, as it is the center of HSC abundance and provides a critical food source for the threatened red knot, Calidris canutus rufa, while also being commercially exploited. To effectively manage the species, it is important to accurately estimate relative and total abundance so that proper harvest limits can be set. Previously, estimates of relative and total abundance of HSC in the Delaware Bay area were based on a design-based approach using a stratified random sampling design. In Chapter 1 of this work, I developed hurdle models (a generalized linear model that models the probability of observations and the observed positive counts using two separate regression models that are then combined) for each of the six HSC demographic groups to remove the effect of external factors (year, latitude, longitude, depth [inshore/offshore], topography, average trawl depth, time-of-day, month, bottom temperature, bottom salinity, and distance from shore) on our observed catch-per-unit-effort (CPUE) and estimate relative abundance using a model-based approach. It was determined that while the two approaches resulted in mostly convergent estimates of relative abundance, factors like month, time-of-day, and average depth had major effects on the observed CPUE of all demographic groups. Chapter 2 involved developing similar hurdle models for three species of bycatch frequently caught in our trawls, i.e., channeled whelk (Busysotypus canaliculatus), knobbed whelk (Busycon carica), and summer flounder (Paralichthys dentatus). It was found that channeled whelk relative abundance has been at a historical low since 2016, while summer flounder has been at a consistent high. Recent estimates of knobbed whelk relative abundance have been less variable than previously seen, with estimates since 2016 being similar to those seen before 2012. These results provide the first estimates for whelk population trends in the mid-Atlantic region and add to the growing knowledge of summer flounder relative abundance in the area. In Chapter 3, I applied the hurdle models developed in Chapter 1 to estimate the total abundance of HSC in the Delaware Bay area. To do this, the bottom temperature and salinity had to be estimated for each geographic cell. This was accomplished by developing two spatio-temporal variograms which allowed me to estimate either variable at an unmeasured point and time based on its spatial and temporal distance from a measured value in a process known as spatio-temporal kriging. The results showed that night estimates of total abundance were consistently higher than daytime estimates and that estimates from September or November resulted in the highest estimated total abundance for all demographic groups. The results suggest that when using September model-based estimates at night to compare against the design-based approach, nearly a third of all previous design-based estimates significantly underestimated the total abundance of HSC in the Delaware Bay area. This outcome could justify ASMFC increasing recommended harvest limits for mature individuals if that action aligns with the goals of their adaptive resource management (ARM) framework.