This study demonstrates that B. candida infections are highly prevalent among adult horseshoe crabs, and that the cocoons of this parasite are capable of occupying a considerable amount of gill real estate (> 10%). To our knowledge, the only previous studies evaluating B. candida infection intensities demonstrated that adult horseshoe crabs had 400–800 (average = 575, n = 4) cocoons across their entire gill area (Pearse 1949) and are capable of having > 50 cocoons within a single gill lamella (Leibovitz and Lewbart 2003). In contrast, the average B. Candida intensity was 267 in this study within our subsamples (10% of the total horseshoe crab gill area), and assuming that B. candida cocoon intensity patterns in the remaining 90% of the gill are homogenous, our results indicate average cocoon intensities could be much higher (average of 2,670 cocoons in entire gill space). Interestingly, cocoon prevalence and intensity were not uniform across life history stages, as only 6.2% (n = 2) of juvenile crabs (instars < 13; 58mm) had cocoons attached to their gills, whereas all adults and sub-adults were observed to have cocoons and adult worms. Moreover, all cocoon intensities were orders of magnitude higher on adult crabs than juveniles (when present). Ontogenetic shifts in parasite infrapopulation characteristics, such as the one we observed here, are not uncommon for a species where both prevalence and infection intensities increase with host body size and age. However, the mechanisms leading to the adult-juvenile dichotomy in B. candida prevalence observed is intriguing, as juvenile (instar groups 8–10) and adult crabs share the same habitat (spawning beaches) during the spring months possibly exposing the juvenile crabs to the parasite. Moreover, the high (100%) prevalence of B. candida in adult crabs suggests that B. candida is well adapted at colonizing a susceptible host with transmission unlikely to be limiting to juveniles; therefore, these age-group prevalence trends may presumably be a result of behavioral, foraging, or physiological differences between the stages.
A possible explanation for the ontogenetic differences in infection are due to the decrease in molting frequency observed in horseshoe crabs as they age. Juvenile horseshoe crabs molt several times a year until reaching instar 10 (49.2mm), after which molting occurs annually until a terminal molt is reached upon sexual maturation (Carmichael et al. 2003, Estes et al. 2015). Following the terminal molt, the accumulation of epibionts on horseshoe crabs (slipper snails, barnacles, macroalgae, etc.) are well documented (Walls et al. 2002) with similar dynamics likely to influence the establishment of B. candida. For example, the antiparasitic effects of molting have been observed in Antarctic krill (Euphausia superba), as recently molted krill had 0% prevalence of ectoparasites as opposed to pre-molt individuals that had a 66% prevalence (Tarling and Cuzin-Roudy 2008). Moreover, this study also found parasite prevalence increased with host age, presumably a result of decreased molting frequency. Further support of molting as a mechanism of parasitic defense was demonstrated in Daphnia magna, as molting was found to limit the adhesion of bacteria and subsequent infection (Duneau and Ebert 2012). Similarly, prevalence and intensity of epizootic shell disease in the American lobster were shown to increase in animals with lower molting frequencies (egg-bearing females) and this was suggested to result from the inability of infected lobsters to eliminate pathogenic microbes during molting (Castro et al. 2012). Although molting seems to be a likely mechanism in B. candida regulation, it is difficult to test, as the most heavily infected cohort (adults) rarely molt and are difficult to maintain in laboratory conditions in sufficient numbers to test such a hypothesis.
Outside of molting, other phenomena such as horseshoe crab behavior, ontogenetic differences in size, physiology and resource use may explain prevalence dynamics across age groups. For instance, it is possible that B. candida is sexually transmitted and initiates infection during the extensive copulatory process observed in horseshoe crabs in which multiple males may attach to one female for days to months each spring (Brockmann and Penn 1992). However, the observation of 100% prevalence infections among the sub-adults (n = 7, instars 16–18) in this study makes this an unlikely scenario, as this age group exhibits different space-use patterns relative to adults, and they do not engage in mating behavior (Rudloe et al 1981). Differences in gill surface area between instars 8–10 and sub-adults/adults may also be a primary factor behind prevalence disparities, as parasite intensity can correlate with increasing body size such is the case with Salmon louse (Lepeophtheirus salmonis) infecting Atlantic salmon (Salmo salar) (Tucker et al. 2002; Drake 2019), presumably a result of increased surface area reducing space competition among conspecific ectoparasites. Additionally, the body size argument may also explain why horseshoe crab sex was a contributing factor in B. candida infection rates because adult females are larger than males (Loveland and Botton 1992) and thus, females have more available surface area or “habitat” for B. candida to reside. However, host size has not been found to be a limiting factor in ectoparasite infection intensities in some organisms; whereas, host whole body metabolism can be a more important determinant of ectoparasite intensities (Hechinger et al. 2019) because host energy can be more constraining to parasite infection loads.
The difference in infection intensities among age groups may partially reflect contrasting foraging behaviors among life history stages and may make the juvenile cohort (< 12 instars) unsuitable for B. candida establishment. For example, B. candida is believed to indirectly consume food particle remnants from horseshoe crab feeding activities (Jennings 1977). Juvenile crabs (instars < 10) predominantly rely on sedimentary organic matter and meiofauna adjacent to salt-marsh habitats (Botton et al. 2003b); in contrast, older juvenile and adult crabs predominantly forage on larger-bodied prey, such as bivalves and polychaetes (i.e. Neries spp.) (Botton and Ropes 1987; Gaines et al. 2002). Therefore, the nutritional resources on juvenile horseshoe crabs may not be sufficient or optimal for B. candida’s nutritional requirements. However, the theory of B. candida foraging on remnants of horseshoe crab prey items remains controversial due to chemical analysis indicating that B. candida may obtain some nutritional energy directly from horseshoe crabs (Lauer and Fried 1977). The application of modern techniques to assess resource-use, such as stable isotope analysis (bulk or compound-specific), could be used to resolve this controversy, as it could identify the nutritional resources adult B. candida predominantly relies on.
Given that this study emphasized one population of horseshoe crab hosts, we cannot state these infection intensity patterns apply to other populations, as other factors such as biogeographic differences in reproduction ratios, environmental conditions, migration patterns, abundance, and size may result in varying B. candida. For example, host population density is often positively correlated with ectoparasite intensity as a result of increased probability of direct transmission (e.g. contact, breeding, etc.) among conspecifics within a population after controlling for other covariates (Arneberg et al. 1998). However, controversy surrounds the contribution of population density to increased parasite infection intensities. Bagge et al. (2004) noted that the primary determinant behind infection rate variability for multiple Monogenean species’ in crucian carp (Carrasius carassius) was host population size, presumably due to a required infection density threshold for effective transmission, and thus numbers of hosts were the limiting transmission factor. For horseshoe crabs, size-at-maturation and population densities are the largest in Mid-Atlantic populations (Delaware and Chesapeake Bays) and can be 2-400 times greater than their northern counterparts (Shuster 1955; James-Pirri 2005; Smith et al. 2009, 2017 ). In turn, B. candida intensity may contrast between host populations due to both disparate population densities, abundances, and body size differences of L. polyphemus. Previous studies have demonstrated that ectoparasite intensities increase with host body size in a variety of animal species, including chigger parasites on the Spiny lizard (Sceloporus clarkii,) (Watkins and Blouin-Demers 2019), multiple species of Woodpeckers (Galloway and Lamb 2017), and ectoparasitic copepods in Brook trout (Salvelinus fontinalis) (Poulin et al. 1991). Therefore, assessing B. candida population dynamics between horseshoe crab populations with different characteristics (e.g. abundance, density, etc.) may be beneficial for elucidating mechanisms that regulate ectoparasite- host relationships, particularly in such an ancient and stable host species.
Within an individual host or even an organ, parasites are known to aggregate in regions that provide the best niche for them and in turn higher fitness, this was observed in the cocoons of B. candida in this study as cocoons of were significantly more prevalent in the dorsal most quartile of gill lamellae (Fig. 6). The ventral most gills were preferentially used for cocoon placement, possibly due to the larger size of these lamellae as larger lamellae size not only provides more habitat, but also allows cocoon location to be away further from edge of the lamellae sheltering the cocoons from excessive flow, a critical concern for ectoparasites (Wootten 1974). Additionally, larger lamellae can pump more water, which may be necessary to meet the oxygen demand of the flatworm cocoons which require oxygen to sclerotize (Huggins and Waite 1993). Unsurprisingly, the realized niche of a parasite is frequently smaller than the potential niche (Sukhdeo and Croll 1981), resulting from constraints on attachment, competition and nutrient acquisition, factors that can lead to hyper specialization within a larger organ. For example, the gills of fish are often segregated between parasites such as Monogenean flatworms or parasitic copepods which will localize to particular gill arches in fish (Arme and Halton 1972; Teemer et al. 2020). Similar results were observed in this study as B. candida cocoons were infrequently placed in the CMRA’s, a specialized zone of the lamellae important for nitrogenous waste excretion (Henry et al. 1996; Hans et al. 2018). However, we postulate that cocoon placement is fairly random across the CMRA vs. PMPA sections of horseshoe crab gills because the CMRA region in this study comprised an average area of 0.30 ± 0.60 SE across the total gill surface while the average proportion of cocoons was 0.21 ± 0.02 SE, indicating that the placement of B. candida cocoons is nearly proportional to the CMRA. It is important to note that, the CMRA cocoon placement was not entirely avoided in this study and the likelihood of CMRA cocoon placement appeared to slightly increase with cocoon intensity, albeit, the beta regression results indicated a weak relationship between overall cocoon intensity and the proportion of cocoons in the CMRA region. Therefore, in other horseshoe crab populations it is important to determine if the spatial arrangement (random or clustered) of cocoons on gill lamella varies across B. candida intensity levels.
The extent of the deleterious impacts imposed by B. candida infections remains uncertain; however, horseshoe crab fitness could be affected from the combination of anthropogenic and ambient environmental stressors coupled with B. candida infection. This study revealed no more than 15% of gill surface area in any adult was covered with B. candida cocoons, however this estimate is likely conservative as our analysis was unable to detect the cocoon cemented regions (Fig. 5C, D). Regardless of this potential underestimation, light infection intensities on gills from ectoparasites may have adverse impacts on horseshoe crab fitness. For example, ectoparasite coverage on gills appears to be directly proportional to reduction on the velocity of oxygen uptake in aquatic organisms (Duthie and Hughes 1987) and may potentially affect horseshoe crab fitness by reducing respiration efficiency, especially in hypoxic conditions. Hypoxic conditions are expected to become more chronic and frequent in coastal marine environments in the coming decades (Diaz and Rosenberg 2008), and the combined stress of B. candida and hypoxia may adversely impact horseshoe crab physiology. For example, when exposed to hypoxic conditions (< 2.0 O2 mgL− 1) and parasitic nematode (Anguillicola crassus) infections over 4 days, eels with low swim bladder degenerative indexes (0–1) and the highest infection loads exhibited shorter time until death (10–25 hours shorter on average) than their uninfected counterparts (Lefebvre et al. 2007). Additionally, horseshoe crabs face a unique and direct anthropogenic stressor, in the form of blood extraction for biomedical purposes, that may make individuals with intense infections of B. candida more susceptible to sublethal effects (i.e. reduced oxygen uptake, increased respiration energy expenditure, etc.) or mortality (Smith et al. 2017; Owings et al. 2019, 2020). The sublethal impacts of biomedical blood extraction on horseshoe crab survivors (~ 70% survival) are numerous, such as a significant reduction in hemocyanin concentrations following typical blood volume extractions (30% blood volume) and reduced spawning frequency (Owings et al. 2019, 2020), and it can take up to 4 months for amebocytes to fully recover to baseline levels (Novitsky 1984). Hemocyanin is essential for maintaining oxygen transport (Mangum 1980), immune response (Coates et al. 2011), wound repair and molting (Adachi et al. 2005) and can be altered by environmental conditions (Coates et al. 2012). Additionally, these amebocytes are involved in the immunological response to B. candida cocoons (Fig. 5), so the impairment of these cells could reduce the crabs’ ability to respond to immunological insults and/or increase baseline level of stress of these animals and potentially increasing susceptibility to stressors. Therefore, understanding the simultaneous impacts of projected intensifying environmental conditions (i.e. temperature, ocean acidification, and hypoxia), biomedical blood harvest on hemocyanin levels, and B. candida infections are required, as their combined effect may engender more severe consequences for horseshoe crabs than when these effects are isolated.