Host-parasite Interactions between a Copepod (Pharodes tortugensis) and Small Reefassociated Gobies (Coryphopterus) in the British Virgin Islands

© 2022 Academia Sinica, Taiwan Open Access Host-parasite Interactions between a Copepod (Pharodes tortugensis) and Small Reefassociated Gobies (Coryphopterus) in the British Virgin Islands Graham E. Forrester1,*,§ and Rachel J. Finley2,§ 1Department of Natural Resources Science, University of Rhode Island, Kingston, Rhode Island 02881, USA. *Correspondence: E-mail: [email protected] (Forrester) 2Department of Natural Resources Science, University of Rhode Island, Kingston, Rhode Island 02881, USA. E-mail: [email protected] (Finley) §GF and RF contributed equally to this work. Received 21 February 2021 / Accepted 4 May 2022 / Published 12 August 2022 Communicated by Benny K.K. Chan The effects of parasitic copepods on free-living hosts are infrequently documented, and the copepod Pharodes tortugensis has remained virtually unstudied since described. For the first time, we document its host range in the British Virgin Islands (BVI), the prevalence and intensity of infections on wild hosts, and its impacts on host morphology and performance. Infections were observed on four benthic gobies in the BVI (Coryphopterus glaucofraenum, C. venezuelae, C. dicrus and C. eidolon) but not on other host species previously reported from other parts of the western Atlantic. Infected gobies were widespread in the BVI (detected at 33 of 52 sites, prevalence from 1–25%) but extremely rare elsewhere in the Caribbean (detected at 2 of 16 sites, prevalence < 0.006%). As is typical of macroparasite infections, P. tortugensis was over-dispersed in BVI host populations (mean intensity = 4.7, range = 1–17). Infections were most common in juvenile and female hosts, and rarely found in larger male hosts. The copepods attach in the branchial chamber of the goby; female copepods show high attachment fidelity to the ventral surface of the chamber, while male copepods attached most often to the first two gill arches and in the branchial chamber adjacent to the female. Infections caused substantial damage to the host’s branchial chamber and gill filaments. Parasitized gobies also had larger livers and smaller gonads than unparasitized individuals of similar length. The changes in organ mass of infected gobies were not sizeable enough to affect total body mass, and host condition (the body-length vs. body-mass relationship) was similar for gobies with and without infections. Parasitized gobies were, however, significantly smaller in body mass at a given age, reflecting slower overall growth. Effects of P. tortugensis on individual hosts were broadly similar to those of other parasitic copepods that infect fish gills and, for unknown reasons, the BVI appears to be a persistent hotspot of infections on these goby hosts. Key words: Coral reef fish, Ectoparasite, Gill pathology, Host-range, Infection intensity, Liver condition, Prevalence, Reproductive output. BACKGROUND The effects of parasites on fish are generally better documented for microparasites (viruses, bacteria and protozoa) than macroparasites (helminthes and arthropods including cymothoid isopods), whose impacts are thought to be chiefly sublethal (Sindermann 1987; Sale 2002). Copepods are the most common and diverse macroparasites of marine fish (Boxshall and Hayes 2019), and most of what is known about Citation: Forrester GE, Finley RJ. 2022. Host-parasite interactions between a copepod (Pharodes tortugensis) and small reef-associated gobies (Coryphopterus) in the British Virgin Islands. Zool Stud 61:32. doi:10.6620/ZS.2022.61-32. Zoological Studies 61:32 (2022) doi:10.6620/ZS.2022.61-32 1 © 2022 Academia Sinica, Taiwan the effects of parasitic copepods on fish hosts is derived from studies of commercial aquaculture. This bias is understandable because parasitic infections in this setting result in potentially severe financial losses (Johnson et al. 2004 2019). Parasitic copepods commonly occupy the gill cavity, oral cavity or skin of their hosts, and their attachment often causes structural damage to the tissues in the area (Kabata 1984). Other pathological consequences of attachment in aquaculture settings include damage to the tongue and sensory organs, whereas parasite feeding can also cause damage to musculature and sometimes atrophy of internal organs (Kabata 1984; Johnson et al. 2019; Kottarathil et al. 2019; Aneesh and Kappalli 2020). Coincident stressrelated physiological responses, including anaemia, and altered immune function are often observed (Kabata 1984). At the individual level, infection can reduce the energy available for growth and reproduction so that host condition (body mass at a given length) and reproductive output is diminished (Johnson et al. 2019). These impacts on individuals can be severe enough to reduce survival, either directly or indirectly by making infected individuals more vulnerable to other agents of mortality, which can translate to populationlevel impacts. Aquaculture settings, however, differ from natural ones is ways that may alter host-parasite dynamics (e.g., crowding, stress and diet) and magnify the impact on hosts (Johnson et al. 2004). Further study of parasitic copepods under natural conditions is thus important to understand whether they have equivalent impacts on wild hosts (Johnson et al. 2019; Sikkel and Welicky 2019; Timi and Poulin 2020). We describe effects on their free-living hosts of the parasitic copepod Pharodes tortugensis (Wilson), a member of the family Chondracanthidae found in the western Atlantic (Milne Edwards) (Ho 1970; Østergaard et al. 2003; Hadfield 2019). Chondracanthids are all highly modified parasites of marine fishes, and relatively little is known of the 193 species in the family aside from morphological descriptions and phylogenetic analysis (Smit et al. 2019). Most Chondracanthids are sexually dimorphic, with dwarf males that are attached to the female. Male Pharodes are distinctive because they attach to the fish host independently from the females and males are larger relative to the size of the female than most other species (Ho 1971a; Østergaard and Boxshall 2004). Evidence for the impact of chondracanthids on their hosts is limited to a few case studies. For example, Chondracanthus goldsmidi (Tang, Andrews and Cobcroft) attaches to the gills, inner operculum and nasal cavities of its hosts (Andrews et al. 2010). Hosts suffer structural damage at the attachment site that includes swelling and tissue necrosis (Andrews et al. 2010), with associated overexpression of inflammatory cytokines (Covello et al. 2009). Pharodes banyulensis (Delamare Deboutteville and NunesRuivo), a close relative of P. tortugensis found in the Mediterranean, also causes substantial physical damage to the gill cavity that compromises the respiratory function of a common host, the Mediterranean blenny Salaria pavo (Risso) (Rousset and Raibaut 1984). Pharodes tortugensis and P. banyulensis are morphologically very similar, differing only slightly in the morphology of the tip of the caudal process of the female copepods; these variations could be due to intraspecific variation among localities (Ho 1971a). We discovered P. tortugensis infecting four species of goby that inhabit mixed reef and coral reef habitats. Three common gobies, Coryphopterus glaucofraenum (Gill), C. venezuelae (Cervigón) and C. dicrus (Böhlke & Robins), are infected, as is a much rarer goby, C. eidolon (Böhlke & Robins). Infected gobies were first noticed in 1993 because their physical appearance differs from that of uninfected gobies: copepods attached in the host branchial chamber cause visible distension of the operculum (Fig. 1). Using this visual criterion, divers can accurately identify unparasitized (92% accuracy) and parasitized (84% accuracy) gobies during underwater surveys (Finley and Forrester 2003). Population-level effects of P. tortugensis on these goby hosts have been documented near Guana Island in the British Virgin Islands. In an observational field study, one of the common goby hosts (C. venezuelae) displayed slower growth, reduced fecundity, and suffered higher mortality when infected (Finley and Forrester 2003). The primary population-level influence of the parasite, however, is to compromise the gobies’ ability to avoid the larger predatory fish that cause most goby deaths. Gobies flee to crevices in the reef when attacked and compete for refuges when they are in short supply (Forrester and Steele 2004; Samhouri et al. 2009; Vance et al. 2010). Infection with P. tortugensis diminished gobies’ effectiveness as competitors for refuges and strongly impacts their abundance (Forrester and Finley 2006; Forrester et al. 2019). Objectives This study had two sets of objectives. (1) We first tested for individual-level effects of P. tortugensis on C. venezuelae hypothesized to underly the populationlevel impacts previously reported. We documented the site(s) of attachment by P. tortugensis and patterns in the intensity of infection, and asked whether attachment damages host tissues at the infection site? We also asked whether infection alters the amount of energy allocated to reproduction (measured as gonad mass relative to body mass, Cole and Shapiro 1990) and to energy stored page 2 of 19Zoological Studies 61:32 (2022) © 2022 Academia Sinica, Taiwan (measured as liver mass relative to body mass, Shulʹman and Love 1999). We also investigated whether infection influenced body condition (using body length-mass relationships and body mass-age relationships, Jakob et al. 1996). (2) Second, we further describe host-parasite dynamics at the population and community levels to assess the scope of their effects over time and over a broader geographic area. We documented the prevalence of P. tortugensis on its common Coryphopterus hosts, and also surveyed other potential hosts for infections, throughout the British Virgin Islands and US Virgin Islands. We also searched for Coryphopterus hosts with visual symptoms of infection throughout the Caribbean and documented changed over time in the prevalence of infections on these hosts near Guana Island. We sampled naturally infected Coryphopterus hosts and correlated parasite presence with host responses, so we could not unambiguously isolate the effects of parasitism per se. We acknowledge that other unmeasured factors, if correlated with parasite presence, might cause the responses we detected. Offsetting the limitations of this approach is the benefit of being able to observe and sample large numbers of infected hosts in a natural setting. MATERIALS AND METHODS Host collection and identification This study includes data collected from 1993–2019, and revisions in the classification of Coryphopterus hosts over this period affected the accuracy with which we could identify hosts. In the early years of our study, Coryphopterus tortugae (Jordan) and C. venezuelae were not considered separate from C. glaucofraenum, but DNA barcoding in the mid 2000s supported the validity of each as distinct species (Victor 2008; Baldwin et al. 2009). Although similar in appearance, these three species can be distinguished morphologically (Baldwin and Robertson 2015; Victor 2015; Robertson and Van Tassell 2019), and we confirmed the identiy of preserved specimens collected prior to 2008. Some host identifications made prior to 2008, such as those made visually by divers, could not be reevaluated, so in each component of the study described below we specify the level of specificity to which hosts are identified. Identification of the copepods as P. tortugensis based on morphology (Petrik-Finley 2005) was corroborated by the author of the species (Ju-shey Ho, University of Fig. 1. Photographs of a goby infected with P. tortugensis (A) and an uninfected goby (B). The arrow in A indicates swelling of the opercular cavity due to infection. page 3 of 19 Zoological Studies 61:32 (2022) © 2022 Academia Sinica, Taiwan California Long Beach, personal communication 2002) and by DNA barcoding (Forrester et al., in review). Attachment locations of P. tortugensis on Coryphopterus hosts To determine if the parasitic copepod showed preference in attachment location on the host, a mix of parasitized C. glaucofraenum and C. venezuelae (n = 74) were collected from Muskmelon Bay and White Bay, near Guana Island in August 2001 and October 2002 (Fig. 2). Goby hosts for this and all other parts of the study were collected individually on SCUBA using hand nets and anaesthetic (Quinaldine). Captured gobies were placed directly into plastic bags and euthanized with an overdose of Quinaldine. No copepods were observed in the bags, suggesting that they remain attached to the hosts after collection. The external body surface, gill arches, branchial chamber and underside of the operculum were carefully searched for P. tortugensis and the attachment location of copepod recorded. To assess whether attachment location differed between male and female copepods, or was affected by body size, copepods were sexed and visually assigned to size classes (Table 5). We divided female copepods into three classes based on their size and morphology: immature transforming, mature non-gravid, and mature gravid. Transforming females are those that are metamorphosing from the typical copepod morphology into the modified fleshy adult female. During the transformation, the lateral processes extend from the trunk, the caudal process elongate, and the head becomes more distinct. Mature females were distinguished from transforming females by their possession of distinct and well-developed heads, and lateral and cephalic processes (Fig. 2). Gravid females were distinguished by their possession of two large egg sacs, which extended from the caudal process (Fig. 3B). Transforming females were assumed to be immature because they never possessed egg sacs. Male copepods were found in several locations, so a Chi2 test was used to test whether the frequency of attachment differed among these locations. Fig. 2. Locations in the United States and British Virgin Islands where Coryphopterus were found infected with Pharodes tortugensis. Numbers correspond to sites listed in table 1. N page 4 of 19Zoological Studies 61:32 (2022) © 2022 Academia Sinica, Taiwan Infection intensity of P. tortugensis on C. venezuelae To describe patterns in the intensity of infections (sensu Bush et al. 1997), a collection of 331 C. venezuelae was made in 2004 (n = 284) and 2018 (n = 47). These gobies were collected at random, without regard for size or infection symptoms, from the White Bay site (Fig. 2). The gobies were measured in standard length (SL, the distance from the tip of the nose to the end of the caudal peduncle) and grouped into four size categories: < 15, 15–20, 20–25, and 25–30 mm SL, to examine whether the intensity of infection differed among size categories. Each goby was then searched for copepods. To test for over-dispersion, we examined both the variance to mean ratio of the number of copepods per fish and tested if the frequency of infection intensity fit a negative binomial distribution (following the methods described by Krebs 1999). Size-distributions of male and female P. tortugensis To describe the size distribution of P. tortugensis, we took digital photographs of male and female copepods taken from infected C. venezuelae collected in 2004 and measured them using imaging software (ImageJ version 2.10; Schindelin et al. 2012). On male copepods, body length was defined as the combined length of the cephalothorax and genital segment, and body width was the greatest distance across the cephalothorax. For female copepods, body length was the distance from the tip of the head between the antennae to the end of the caudal process, and body width was a linear distance between the tips of the lateral processes (anatomical terminology follows Ho 1971a). We also measured the maximum length and width of egg sacs from gravid female copepods. Gill pathology of parasitized C. venezuelae To describe damage to the gill arches of C. venezuelae by P. tortugensis, we dissected parasitized (n = 94) and unparasitized (n = 190) gobies collected in 2004 and inspected their gill arches. Damage to the gill arches was defined as: compression of the gill filaments, mucus completely covering filaments, and filaments missing from the gill arches and the percent of the arch damaged was quantified visually. Our preliminary impression was that damage was intensified when infections included female copepods, rather than just males and/or juveniles. We therefore divided the gobies into these two groupings and used analysis of variance (ANOVA) to test if they differed in percent of branchial damage (unparasitized gobies were included as a third control group). Prior to this and other analyses using linear models, we used Q-Q plots and plots of the residuals versus predicted values to confirm that the data met the assumptions of the model. For this analysis, data were square root arcsine transformed prior to analysis to meet the assumptions (following Zar 1996). To determine if female copepods enlarge the branchial chamber of parasitized gobies, digital photographs were taken of the right and/or left branchial cavities of some parasitized and unparasitized bridled gobies (n = 8) after the operculum was removed (Fig. 3). Imaging software (ImageJ version 2.10; Schindelin et al. 2012) was used to measure the perimeter of the branchial cavity in gobies parasitized with female copepods and compared to the perimeter of unparasitized gobies (see Fig. 3 for a diagram illustrating the perimeter measured). To adjust for the fact that the branchial chamber size should be a function of the fish size; differences in branchial chamber perimeter were tested using analysis of covariance (ANCOVA) with fish length as a covariate and infection (yes or no) as a categorical variable. Effects of P. tortugensis on host body condition To assess the effect of P. tortugensis on host body condition and energy allocation, a mix of C. glaucofraenum and C. venezuelae were collected from Muskmelon Bay and White Bay near Guana Island in from 2001–2003 (Fig. 2). The gobies were fixed in 70% alcohol, measured (SL) and sexed by examining the genital papilla (Cole and Shapiro 1990 1992). Individuals spanning the size range at which most infections occurred were used in this analysis (n = 163, 12.2–33.7 mm SL). These gobies change sex from female to male (protogynous hermaphroditism), and individuals of this size range comprise mainly juveniles and females (Cole and Shapiro 1990 1992). Excluding larger individuals because they were rarely infected by P. tortugensis thus also excluded most males from the sample. The otoliths (lapilli) were removed from each goby and, after clearing in immersion oil, the postsettlement age (days) was determined by counting the daily growth rings formed after the mark on the otolith that indicates settlement to the reef (Steele and Forrester 2002). The copepods were removed from parasitized gobies, dried at 60°C until a constant mass was achieved (at least 1 h) then weighed in mg to 0.001 mg. The liver and gonads were removed from each goby, and these organs plus the body tissue (minus the alimentary tract) were each dried and weighed. Total goby body mass page 5 of 19Zoological Studies 61:32 (2022) © 2022 Academia Sinica, Taiwan Fig. 3. (A–G). Photographs detailing infection by P. tortugensis in gobies: the size and morphology of male and female copepods, and gill damage in parasitized fish. Scale bars are = 1 mm in all photos. (A) Profile of an infected C. glaucofraenum with operculum removed. Dotted line indicates perimeter measurement. Abbreviations are: GA: 1st gill arch, M: placement of males on the gill arch and on perimeter of gill cavity wall, LP: lateral process of gravid female copepod attached to host gill cavity wall, ES: egg sac of gravid female copepod. (B) Two gravid female copepods. (C) Three large male copepods. (D) Size comparison of juvenile (left), small male (center), and large male (right). (E) Gill arch with a male copepod (M) attached. (F) Display of gill arches 4 – 1 (L to R) from an unparasitized goby. (G) Gill arches 1 – 4 (L to R) from a parasitized goby. Damaged and missing filaments are indicated with arrows on the 3rd and 4th arches. Some filaments are also missing, and less severe damage overall is seen in the 1st and 2nd arches. page 6 of 19Zoological Studies 61:32 (2022) © 2022 Academia Sinica, Taiwan was calculated as the sum of liver, gonad, and body tissue mass. To assess the effect of P. tortugensis on body condition, we measured total body mass as a function of body length (SL) (Ogle 2016). To assess the effect of P. tortugensis on investment in energy reserves and reproduction allocation, we measured liver (LM) and gonad (GM) mass respectively as a function of total body mass (BM). To explore whether changes in condition might affect growth we examined body mass as a function of post-settlement age (A). Fish mass-length (BM vs. SL) relationships are non-linear and are typically modeled as a power function of the form BMi = aSLi b eci where a and b are constants and ci is the multiplicative error term for the ith fish (Ogle 2016). This relationship was linearized as follows log(BMi) = log(a) + blog(SLi) + ci and analysis of covariance (ANCOVA) was used to test the effect of parasitic infection (IN), a categorical variable (infected or not), on the transformed relationship as follows log(BMi) = log(a) + blog(SLi) + d(IN) + eIN × log(SLi) + ci Inspection of the data suggested that the other relationships of interest (LM vs. BM, GM vs. BM, and BM vs. A) also resembled power functions rather than linear functions. ANCOVA models of the same form as the BM vs. SL model were thus used to test for effects of parasitism on these other relationships. The effects of some macroparasites are related to the number of parasites per host, so rather than just parasite presence, we tested whether infection abundance (sensu Bush et al. 1997) was a better predictor of host impact than parasite presence. Copepods varied greatly in size, so we used the combined mass of copepods on a host, rather than the number of copepods, as our index of infection abundance. For each of the relationships just described (BM vs. SL, LM vs. BM, GM vs. BM, and BM vs. A), we substituted infection intensity (II) for parasite presence (IN) in the above linear models. Infection abundance (II) is a continuous variable and infection presence (IN) is categorical, so the model became a multiple regression rather than ANCOVA. For each pair of models (II vs. IN), we used Akaike’s Information Criterion (AICc) as a measure of relative model fit, and models differing in AIC by < 6 were judged to have similar support in the data (Richards 2005). Spatial and temporal patterns in the distribution of hosts infected with P. tortugensis To document the spatial distribution of P. tortugensis infections on common Coryphopterus hosts in the British and US Virgin Islands, we performed visual surveys on SCUBA at 52 sites in the area (Table 1; Fig. 2). Individuals were classified as parasitized or not based on visual symptoms of infection. These counts represent the combined abundance of C. glaucofraenum, C. venezuelae and C. tortugae. We made quantitative surveys at 35 sites, in which all gobies within transects (0.5 × 4 m) were inspected and counted. At 17 other sites, we made less rigorous qualitative surveys in which gobies encountered were visually inspected and P. tortugensis was noted as either present or absent at the site. To document changes over time in the prevalence of infection, repeated estimates were made from 1993– 2019 at a BVI site near Guana Island (Harris Ghut, Table 1, and Fig. 2). Coryphopterus venezuelae from this site were classified as parasitized or not based on visual symptoms of infection (Table 2). Some estimates were based on inspection of individuals during underwater surveys, whereas other individuals were captured using hand nets for other experiments and inspected underwater while in the net prior to release back into the field. To document the broader distribution of P. tortugensis infections on three common Coryphopterus hosts (C. glaucofraenum, C. venezuelae and C. tortugae), visual surveys were performed at 16 other sites throughout the Caribbean (Table 3). Hosts were screened visually for symptoms on SCUBA as encountered. Parasitized hosts were counted individually, and the total number of hosts screened per location was recorded to the nearest 10 (n ≈ 4900; Table 3). In addition to the four species of Coryphopterus we studied, P. tortugensis has been reported from 11 species of fish host at other locations in the western Atlantic (NMNH 2020; WORMS 2020). Most of these other hosts are gobies (Gobiidae Cuvier) or blennies (Blenniidae Rafinesque). We therefore sought to identify potential additional hosts of P. tortugensis from these families in the British Virgin Islands (Table 4). Some potential hosts were collected and their external body surface, gill arches, branchial chamber and underside of the operculum were carefully searched for P. tortugensis. Other hosts were inspected visually on SCUBA for the distended operculum symptomatic of infection. page 7 of 19Zoological Studies 61:32 (2022) © 2022 Academia Sinica, Taiwan Table 1. Geographic pattern of infections of P. tortugensis on Coryphopterus in the British Virgin Islands (BVI) and U. S. Virgin Islands (USVI). Data are prevalence from transect counts (mean % with number of transects in brackets) or presence/absence from visual searches. Dashes (-) indicate no data for a given site and year. Map numbers correspond to sites in figure 2 Island Group Island Site name Latitude (N) Longitude (W) Map # 2001 2002 2003 2004 2008 BVI Guana Island Muskmelon Bay 18°28.9 64°34.78 19% (7) 17% (8) Yes Yes - BVI Guana Island Crab Cove 18°28.79 64°34.70 2 13% (10) 13% (10) Yes Yes - BVI Guana Island White Bay Dock 18°28.54 64°34.65 3 19% (19) Yes Yes Yes - BVI Guana Island Harris Gut 4 21% (9) Yes Yes Yes - BVI Guana Island White Bay 18°28.13 64°34.41 5 21% (19) 12% (15) Yes 16% (5) - BVI Guana Island Monkey Point 18°27.98 64°34.30 6 25% (10) 7% (11) Yes Yes - BVI Guana Island Bigelow Beach 7 1% (20) 5% (8) Yes Yes - BVI Tortola Brewer’s Bay Inside 814% (18) - Yes Yes - BVI Little Camanoe East Bay 18°27.42 64°32.15 10 - 4% (10) - - - BVI Jost van Dyke Great Bay 11 -No --- USVI St John Cinnamon Bay 12 - Yes --- BVI Necker Island Necker Island 18°31.45 64°21.61 13 - 0% (10) - - - BVI Norman Island The Bight 14 - 0% (10) - - - BVI Scrub Island South 18°27.92 64°31.18 15 - 6% (10) - No - BVI Tortola Smuggler’s Cove 16 - - Yes - - BVI Beef Island Long Bay 17 - - Yes Yes - BVI Beef Island Airport Runway Dock 18 - - Yes Yes - BVI Norman Island Money Bay 19 - - No - - BVI Peter Island Deadman Bay 20 - - No - - BVI Peter Island Little Harbour 21 - - No - - BVI Anegada Loblolly Bay 22 - - No - - BVI Great Camanoe Diamond Reef 23 - - No - - BVI Great Dog Coral Gardens 18°28.93 64°27.70 24 - - - 2% (8) - BVI Great Camanoe Northeast Point 25 - - - No - BVI Scrub Island North 18°28.25 64°31.02 26 - - - 0% (5) - BVI Ginger Island North 18°23.32 64°29.14 27 - - - 0% (10) - BVI Ginger Island South 18°23.19 64°28.90 28 - - - 0% (9) - BVI Tortola Brewer's Bay Outside 18°26.90 64°39.21 29 - - - 17% (7) - BVI George Dog Bronco Billy 18°29.49 64°27.52 30 - - - 2% (4) - BVI Mosquito Island South 18°30.57 64°24.05 31 - - - 1% (3) - BVI Virgin Gorda Mountain Point 18°30.06 64°24.94 32 - - - 2% (5) - BVI Dead Chest West 18°22.12 64°33.83 33 - - - 4% (5) - BVI Norman Island North 18°19.42 64°36.65 34 - - - 0% (5) - BVI Pelican Island Reef Check Site 35 - - - 0% (5) - BVI Great Thatch South 18°22.923 64°44.37 36 - - - 2% (9) - BVI Sandy Cay North 18°26.245 64°42.79 37 - - - 0% (5) - BVI Tortola Beaumont Point 18°23.99 64°41.80 38 - - - 10% (10) - BVI Buck Island West Bay 39 - - - No - BVI Beef Island Hans Creek 1 40 - - - No - BVI Beef Island Airport 18°26.22 64°32.76 41 - - - - 8% (5) BVI Dead chest East 18°22.07 64°33.75 42 - - - - 6% (5) BVI Green Cay Green Cay 1 18°27.32 64°42.49 43 - - - - 3% (6) BVI Green Cay Green cay 2 18°45.39 64°70.99 44 - - - - 1% (6) BVI Guana Island Grand Ghut 18°28.79 64°33.70 45 - - - - 6% (5) BVI Beef Island Han’s Creek 2 18°26.21 64°31.90 46 - - - - 2% (5) BVI Guana Island North Bay 18°28.71 64°34.64 47 - - - - 5% (6) BVI Peter Island White Bay 18°21.43 64°35.42 48 - - - - 0% (5) USVI St John Lameshur Bay 49 - Yes -Yes - USVI St John Round Bay 50 - - - Yes - USVI St John Brown Bay 51 - - - Yes - USVI St Croix East End 52 - No -No - page 8 of 19Zoological Studies 61:32 (2022) © 2022 Academia Sinica, Taiwan Table 2. Prevalence of P. tortugensis on C. venezuelae at Harris Ghut, near Guana Island based on visual inspections of hosts for signs of infection Year # hosts inspected # infected Prevalence 1993 99 3 3% 1994 121 3 2% 1995 114 4 4% 1996 95 12 13% 1997 71 18 25% 1998 0 - - 1999 0 - - 2000 138 19 14% 2001 126 27 21% 2002 0 - - 2003 0 - - 2004 237 56 24% 2005 97 12 12% 2006 63 12 19% 2007 88 12 14% 2008 0 - - 2009 19 211% 2010 34 5 15% 2011 74 23 31% 2012 0 - - 2013 0 - - 2014 45 5 11% 2015 101 12 12% 2016 154 21 14% 2017 0 - - 2018 162 29 18% 2019 192 29 15% Table 3. Hosts of P. tortugensis screened for infections at other Caribbean locations. Three hosts (C. glaucofraenum, C. tortugae and C. venezuelae) were inspected visually underwater for symptoms of infection. Hosts were not identified to species and data are pooled; the number of hosts screened is given to the nearest 10 Location Years # hosts inspected Prevalence Barbados 2007, 2014 110 0 Bahamas 1995–2006 2200 0.005 Belize 2006, 2016 340 0 Bonaire 2006, 2008, 2014–2019 480 0 Curacao 2008 120 0 Dominica 2008–2009 160 0 Grenada 2010 120 0 Honduras 2019 40 0 Jamaica 1994–1999, 2002 330 0.006 Mexico 2002 140 0 Puerto Rico 2012 70 0 Saba 2008, 2010 160 0 St. Eustatius 2008 150 0 St. Lucia 2007–2008 200 0 St. Vincent 2007 140 0 Tobago 2008, 2010 110 0 page 9 of 19Zoological Studies 61:32 (2022) © 2022 Academia Sinica, Taiwan common responses observed in fish with macroparasitic infections (Pennycuick 1971; Gordon and Rau 1982; Lemly and Esch 1984; Collyer and Stockwell 2004; Johnson et al. 2004; Katakura et al. 2004). Unlike several other fish hosts infected with parasitic copepods, Coryphopterus hosts showed no reduction in body condition associated with infection (reviewed by Johnson et al. 2019). The mass-age relationship, however, clearly indicated that infected gobies grew in mass more slowly than uninfected ones, as did previous mark-recapture data (Finley and Forrester 2003). In addition to reduced overall growth, infected Coryphopterus had enlarged livers and smaller gonads than uninfected hosts. Whereas the enlargement of the liver was relatively slight and consistent at all body sizes, the reduced investment in reproduction increased with body size and was most severe for larger female Coryphopterus. Enlarged livers have been observed in some parasitized fish and are usually associated with a pathological response, stress, or parasites encysting in the liver itself (Takashima et al. 1972; Tierney et al. 1996; Francis 1997; Malek 2001). We found no parasites in the livers of Coryphopterus and so can exclude this possibility. A pathological or stress response to initial infection would be expected to be greatest in young fish and decrease with age, as observed when sticklebacks are infected with cestodes (Tierney et al. 1996), and so is not consistent with our data. Consistent liver enlargement may thus reflect prolonged stress leading to fatty degeneration and impaired liver function (Hilton and Dixon 1982; Shulʹman and Love 1999). Biochemical analysis of lipid levels in the liver would resolve physiological and metabolic differences in parasitized and unparasitized fish and determine the cause and consequence of differences in liver size. An additional possible contributor to increased liver size in parasitized individuals is a trade-off in resource allocation to energy storage and reproductive output. Reduced gonad mass is perhaps the most common reproductive impact of parasitic copepods and is argued to result from general host debility (Kabata 1984). In Coryphopterus hosts, reductions in gonad size associated with parasitism were experienced primarily by females. This sex-related bias is partly related to protogynous hermaphroditism; these gobies maturing first as female at around 55 mg (24 mm SL) and then changing sex to male around 120 mg (35 mm SL) (Cole and Shapiro 1990 1992). It is also partly because very few large Coryphopterus > 120 mg are infected with P. tortugensis. Effects of parasitism on reproduction in sex changing fish are rarely studied, but these two findings suggest the potential for infected juveniles and small females to allocate energy to lipid storage and growth rather than reproduction as a life-history response to infection. If growing to a larger large size facilitates shedding infection or reduces its impact, then deferring female function may increase reproductive value via the dual benefits of improved survival and future male reproductive function (Warner 1988). In aquaculture settings, where infection intensities can be very high, it is common for the severity of parasite impacts on individual hosts to increase with the burden of infection (reviewed by Johnson et al. 2019). For free-living Coryphopterus, we found no evidence that a greater mass of copepods was associated with stronger impacts on growth or liver mass, but decreases in gonad mass were more severe for Coryphopterus with high-intensity infections (see also Katakura et al. 2004). We cannot say for certain why infection intensity only affected reproductive allocation. One possibility is limited variability in infection intensity. Like most macroparasites, P. tortugensis is aggregated among individual Coryphopterus hosts, but the degree of overdispersion is at the low end of the range observed for other macroparasites of similar mean infection intensity (see Fig. 1 in Poulin 1998), including parasitic crustaceans (Tavares-Dias et al. 2015). This modest degree of overdispersion may be related to the small size of Coryphopterus hosts relative to P. tortugensis. Limited space within the branchial cavity could simply restrict the maximum number and size of copepods that can infect a host. Consistent with this hypothesis, we rarely observed more than one adult female P. tortugensis per opercular cavity, and the same was true for P. clinii infecting a similarly sized blenny C. argentatus (Chabanaud 1951). In contrast two or three P. banyulensis females were routinely observed in each opercular cavity of a larger blenny host (S. pavo) (Rousset and Raibaut 1984). The scope of populationand community-level impacts of P. tortugensis The number and extent of disease outbreaks in marine organisms is argued to be increasing (Lafferty et al. 2004), but most data comes from overt outbreaks and a lack of baseline data makes reliable estimates difficult (Ward and Lafferty 2004). Pharodes tortugensis was previously reported on 11 fish species, mostly blennies and gobies in the Western Atlantic (NMNH 2020; WORMS 2020), but these reports come from museum specimens and so lack ecological context. Our field surveys identified what appears to be a persistent outbreak of P. tortugensis infections in the BVI that is limited to a subset of its known hosts (four Coryphopterus species). One of the few baseline surveys in marine fish, also showed that visible symptoms of disease in Dab (Limanda linnaeus) were clustered in page 16 of 19Zoological Studies 61:32 (2022) © 2022 Academia Sinica, Taiwan space and stable over several years (Stentiford et al. 2009). Further work to assess the spatial and temporal consistency of outbreaks would thus be informative. Many parasites infect multiple host species and are widely distributed across heterogeneous seaand landscapes (Johnson et al. 2015). In this setting, just one or a few host species can maintain a high prevalence of infection that allows the parasite to persist in the area, but the reasons why certain hosts and sites act as the primary reservoir for infections are not well understood (Wilber et al. 2020). We cannot explain why the outbreak of P. tortugensis was limited to just four of its fifteen known hosts and nor can we explain why the BVI was a hotspot for infections. One possibility, unusual crowding or aggregation of hosts, can be eliminated because ecological studies of Coryphopterus elsewhere, show that the BVI is unremarkable in these respects (e.g., Forrester and Steele 2004). Interestingly, one of our BVI sites (White Bay, Guana Island) was also a site of high prevalence for infections of isopods on French grunts (Welicky and Sikkel 2014) and of monogeneans on surgeon fish (Sikkel et al. 2009). If further work confirms that certain sites are hotspots for multiple sets of host-parasite interactions, then environmental factors, such as climate or pollution, may be the underlying cause (Behringer et al. 2020). CONCLUSIONS Coupled with past population-level analyses, this study provides a comprehensive assessment of impacts of P. tortugensis on four of its hosts, which appear to be plausibly interrelated from the individual-level to community-level. Like many other copepod parasites, P. tortugensis damaged the gills and branchial chamber of Coryphopterus hosts, which seems to compromise respiratory function and possibly feeding. This damage appears to slow the growth of hosts and alter their energy allocation to lipid storage and reproduction, providing a rare example of strong impacts on individual hosts in nature. These debilitating impacts on individual Coryphopterus credibly explain the main populationlevel impact of P. tortugensis, which is to diminish the gobies’ effectiveness as competitors for refuges and so increase their vulnerability to predators. We show that this ecologically significant host-parasite interaction appears to be limited to these hosts in the BVI, even though the parasite is a widespread generalist. Acknowledgments: We thank C. D. Tran, B. Finley, L. Forrester, and J. Messineo for field and laboratory assistance, plus L. Jarecki, the Guana Island staff and dive BVI for logistical support. Financial support to G.F. came from the US National Science Foundation (OCE 0096061) and the Falconwood Foundation. R.F. was supported by an award from the International Women’s Fishing Association, a Sigma Xi Grant in Aid of Research, and a URI Graduate Fellowship. We thank D. Bengtson, F. Golet, L. Gonzalez, P. Paton, S. Twombly, and S. McWilliams for comments on earlier versions of the paper. Authors’ contributions: Both authors contributed to all aspects of the study. GF acquired most funding, RF and GF conceived and designed the study, RF performed most field and lab work, GF performed most data analysis, RF wrote a draft of some elements of the paper in thesis form and GF rewrote it for publication. Competing interests: The authors declare that they have no competing interests. Availability of data and materials: The data that support the findings of this study are available on Dryad: doi:10.5061/dryad.sbcc2fr8p. Consent for publication: Not applicable. 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