Hemocyanins (Hcs) and phenoloxidases (POs) are both members of the type-3 copper protein family, possessing di-cupric active sites which facilitate the binding of dioxygen. While Hcs and POs share a high degree of sequence homology, Hcs have been associated traditionally with oxygen transport whereas POs are catalytic proteins with a role in innate immunity. Evidence gathered in recent years details numerous immune functions for Hc, including an inducible PO activity. Unlike the pro-phenoloxidase activation cascade in arthropods, the endogenous mechanism(s) involved in the conversion of Hc into an immune enzyme is lacking in detail. The overall aim of this research was to characterise the physiological circumstances in which Hc is converted into a PO-like enzyme during immune challenge. A series of biochemical, biophysical and cellular techniques were used to assess the ability of phospholipid liposomes to mimic the well-characterised induction of PO activity in Hc by SDS micelles. Incubation of Hc purified from Limulus polyphemus, in the presence of phosphatidylserine (PS) liposomes, yielded ~ 90% of the PO activity observed upon incubation of Hc with the non-physiological activator, SDS. Phospholipid–induced PO activity in Hc was accompanied by secondary and tertiary structural changes similar to those observed in the presence of SDS. Subsequent analysis revealed that electrostatic interactions appear to be important in the PS-Hc activation complex. In vivo, PS-Hc interactions are assumed to be limited in quiescent cells. However, amebocytes undergoing apoptosis redistribute PS onto the outer leaflet of the plasma membrane, resulting in the potential for increased Hc-PS interactions. In the absence of a reliable culturing technique for L. polyphemus amebocytes, in vitro conditions were optimised for the short term maintenance of this labile cell type. Amebocytes retained viability and functionality in a medium that mimicked most-closely, the biochemical properties of L. polyphemus hemolymph. When presented with a fungal, bacterial or synthetic challenge, ~9% of amebocytes in vitro were found to be phagocytically active. Target internalisation was confirmed via the use of fluorescent quenchers and membrane probes. Within 4 hours of target internalisation, amebocytes underwent apoptosis, characterised by the loss of plasma and mitochondrial membrane potential, increased caspase-3 activity and extracellularisation of PS. Phagocytosis-induced cell death led to a proportional increase in the level of Hc-derived PO activity, suggesting that Hc may be interacting with PS present on terminal amebocyte membranes. The PO activity of Hc was investigated further in order to address an economically important issue; hyperpigmentation in commercial shellfish. While PO enzymes are thought to be the cause of hyperpigmentation in Nephrops norvegicus, evidence presented here suggests that cellular PO is inactivated after freeze-thawing, while extracellular Hc retains stability and displays a heightened level of inducible PO activity under similar treatments. Known PO inhibitors were used successfully to reduce Hc-derived PO activity, with inhibitors assumed to bind Hc in a manner similar to PO-inhibitor complexes. Structural and functional studies of hemocyanins and immune cells presented here provide new insights into the interactions of hemocyanin-activator complexes in invertebrates.

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.