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Reduced Densities of Ectosymbiotic Worms (Annelida: Branchiobdellida) on Reproducing Female Crayfish
Kaitlin J. Farrell, Robert P. Creed, and Bryan L. Brown

Southeastern Naturalist, Volume 13, Issue 3 (2014): 523–529

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Southeastern Naturalist 523 K.J. Farrell, R.P. Creed, and B.L. Brown 22001144 SOUTHEASTERN NATURALIST 1V3o(3l.) :1532,3 N–5o2. 93 Reduced Densities of Ectosymbiotic Worms (Annelida: Branchiobdellida) on Reproducing Female Crayfish Kaitlin J. Farrell1,3,*, Robert P. Creed1, and Bryan L. Brown2 Abstract - Cleaning symbioses provide net benefits by improving each partner’s fitness. Ectosymbiotic Cambarincola spp. (branchiobdellidans) can increase growth and survival of Cambarus chasmodactylus (New River Crayfish), but the nature of the symbiosis might change with female reproductive state because brooding offspring (eggs, young) and worms inhabit the same surfaces. Here, we present the results of field surveys that examined whether the number and location of branchiobdellidans on New River Crayfish varies as a function of female crayfish reproductive state. Reproducing female New River Crayfish had fewer total worms, an absence of cocoons, and a relatively greater proportion of worms on lateral body surfaces than non-reproducing crayfish. The altered distribution and reduced abundance of worms suggest that the symbiosis changes with female reproductive status, but additional experiments will be needed to identify the mecha nism responsible. Introduction Cleaning symbioses are interspecific interactions in which a cleaner species removes detritus, parasites, or other epibionts from the client organism, and have been considered textbook examples of mutualism (Bshary et al. 2007, Losey 1979, Poulin and Grutter 1996). While much of the literature on cleaning symbioses has focused on interactions between cleaner and client fishes on coral reefs (Cheney and Côté 2003, Limbaugh 1961, Poulin and Grutter 1996), some species of crayfish and branchiobdellidan worms (Annelida, Branchiobdellida) also appear to engage in cleaning symbioses (Brown et al. 2002, 2012; Lee et al. 2009 ). The nature of the crayfish–branchiobdellidan relationship can change depending on the density of branchiobdellidans on a crayfish (Brown et al. 2002, 2012) and environmental conditions (Lee et al. 2009). Cambarus chasmodactylus James (New River Crayfish) hosts an ectosymbiotic branchiobdellidan, Cambarincola ingens Hoffman, which removes particulate matter and fouling epibionts from the crayfish exoskeleton including the gills (Brown et al. 2002). Lab and field experiments have indicated that intermediate densities of worms can have direct positive effects on the crayfish by increasing growth and survival compared to crayfish without C. ingens (Brown et al. 2002, 2012). Lee et al. (2009) also found positive effects of a mixed community of four species of branchiobdellidans on growth rates of a host crayfish, Cambaroides similis Koelbel, in high-fouling environments but no effects in low-fouling environments. The nature of these relationships may also 1Department of Biology, Appalachian State University, Boone, NC 28608. 2Department of Biological Sciences, Virginia Tech, Blacksburg, VA 24061. 3Present address - Odum School of Ecology, University of Georgia, Athens, GA 30602. *Corresponding author - kfarrell@ uga.edu. Manuscript Editor: Nathan Dorn Southeastern Naturalist K.J. Farrell, R.P. Creed, and B.L. Brown 2014 Vol. 13, No. 3 524 change with the reproductive status of female crayfish. Branchio bdellidans inhabit the same abdominal surfaces as brooding offspring (eggs, young), and may affect crayfish offspring and/or be removed by female cleaning behaviors. Altered worm counts and distributions on reproducing female crayfish could indicate that the nature of the symbiosis depends on host reproductive state. To determine whether the relationship is stable through reproductive cycles, we quantified Cambarincola spp. abundance and distribution on reproducing and non-reproducing female crayfish; here, we present the results of our field survey . Methods Field surveys We conducted field surveys of Cambarus chasmodactylus and Cambarincola spp. at 4 locations in the New River watershed in Watauga County, NC (36°12'N, 81°42'W). We collected crayfish and worms during the summer of 2011 from early June to early August in three 3rd-order tributaries of the South Fork of the New River: Meat Camp Creek, Howard’s Creek, and the Middle Fork of the New River. New River Crayfish is a dominant crayfish in these tributaries (Fortino and Creed 2007). We also collected crayfish and worms in the South Fork of the New River, a 4th-order stream, where adult New River Crayfish are co-dominant with Orconectes cristavarius Taylor (Spiny Stream Crayfish) in the sampled areas (Fortino and Creed 2007, Helms and Creed 2005). Reaches surveyed in each stream were short (~100 m), and the predominant substrate in all reaches was cobb le/boulder. We captured crayfish by lifting boulders from the stream bed and washing crayfish into dip nets; exposed individuals on the stream bed were also captured with dip nets. After capture, we placed crayfish in individual, covered plastic containers filled with stream water to prevent possible transfer of worms between crayfish. We examined the exterior of each female crayfish on site by holding the crayfish in a shallow dish of water and visually examining ventral, lateral, and dorsal body surfaces using a 10x OptiVisor binocular headband magnifier (Donegan Optical Company, Lenexa, KS). Cambarincola spp. attached to the exterior carapace are easily located on submerged crayfish by the swaying of the anterior portion of their bodies (Brown and Creed 2004). For each crayfish, we recorded total carapace length (CL, from the tip of the rostrum to the posterior margin of the cephalothorax), the location (dorsal, lateral, or ventral crayfish surfaces) and number of large, potentially reproductive (≥6 mm; Brown et al. 2002, 2012) and small (<6 mm) Cambarincola spp., and the location and number of branchiobdellidan cocoons. Other researchers have found that most visible branchiobdellidans on the external surfaces of New River Crayfish in these tributaries are C. ingens; thus, we are confident that many of the field-identified worms were C. ingens, though a few may have been Cambarincola philadelphicus Leidy. For this reason, we will refer to the worms as Cambarincola. The only other branchiobdellidan species observed on the external surfaces of New River Crayfish during these surveys was Pterodrilus alcicornis Moore, which we recovered from a few crayfish in Howard’s Creek but did not include in this analysis. We also examined female crayfish to determine if there were Southeastern Naturalist 525 K.J. Farrell, R.P. Creed, and B.L. Brown 2014 Vol. 13, No. 3 attached eggs, i.e., they were ovigerous, or if they were carrying recently hatched young; crayfish were then released. Data analysis We compared the number and location of large, small, and total Cambarincola as well as the number of Cambarincola cocoons found on ovigerous, young-bearing, and non-reproducing females. We excluded surveyed female crayfish from this analysis if their carapace length was less than that of the smallest reproducing female (32 mm CL) to control for reduced worm numbers on smaller crayfish (Brown and Creed 2004). In addition, we excluded recently molted crayfish (identified by incomplete hardening of the carapace) from this analysis because these individuals usually have fewer worms and cocoons than intermolt individuals (R.P. Creed, unpubl. data). The analysis thus included 18 females without eggs or attached young, 7 ovigerous females, and 3 bearing recently hatched young crayfish. The carapace lengths of these crayfish ranged from 32 mm to 45 mm. When assessing differences in worm number between reproducing and nonreproducing crayfish, we used general linear models fitted to negative binomial distributions because these models fit our overdispersed count data better than models fitted to a Poisson distribution (Chatterjee and Simonoff 2013). We also tested for effects of crayfish size (CL) on worm counts through a model that included crayfish CL as a covariate. We used a chi-square goodness-of-fit test to assess whether branchiobdellidans were distributed on crayfish exoskeletons in proportion to the available area (sensu Brown and Creed 2004). We used Fisher’s exact test to compare the proportion of worms found on each body surface (dorsal, lateral, ventral) between reproducing and non-reproducing female crayfish. We used R v3.0.2 (R Core Team 2013) to conduct our analyses. General linear models were fitted to a negative binomial distribution using the MASS package (Venables and Ripley 2002). Results We captured a total of 18 non-reproducing female crayfish (32–38 mm CL), 7 ovigerous females (CL 32–45 mm), and 3 carrying recently hatched young (34–35 mm CL). There were no significant differences between the numbers of Cambarincola nor worm cocoons present on ovigerous female crayfish and those bearing recently hatched young (large worms: Z = -0.612, P = 0.540; small worms: Z = -1.115, P = 0.265; cocoons: not present on any individuals), so we pooled these 2 groups for subsequent analyses and hereafter, we refer to them as reproducing females. Worm number did not differ as a function of crayfish size because both CL and CL × reproductive status were non-significant factors (P-values > 0.10) in the models; CL was removed from subsequent analyses. There were significantly more total (large and small) worms on non-reproducing females (6.72 ± 0.87) than on reproducing females (3.10 ± 0.66; Z = -3.161, P = 0.002). Although the number of small worms did not differ between reproducing and non-reproducing females (Fig. 1, Z = -1.440, P = 0.150), the number of large worms was significantly lower on reproducing female crayfish, with Southeastern Naturalist K.J. Farrell, R.P. Creed, and B.L. Brown 2014 Vol. 13, No. 3 526 non-reproducing females hosting over two times as many large worms as reproducing females (Fig. 1; Z = -2.976, P = 0.003). Visible large worms were not distributed evenly on the external body surfaces on either reproducing or non-reproducing crayfish. We found more large worms and Cambarincola cocoons on the ventral surfaces of non-reproducing crayfish than would be expected given an area-weighted distribution (χ2 2 df = 17.52, P = 0.0002 and χ2 2 df = 72.04, P < 0.0001, respectively). In contrast, reproducing crayfish had fewer large worms than expected on the ventral surfaces (χ2 2 df = 7.21, P = 0.027) and lacked any attached Cambarincola cocoons. Small worms on reproducing crayfish were not evenly distributed, with more small worms found on the lateral surfaces than would be expected (χ2 2 df = 6.57, P = 0.023). Small worms were distributed evenly on non-reproducing crayfish. Figure 1. Counts (bars) and spatial distribution (pie charts) of Cambarincola spp. and worm cocoons on reproducing (n = 10) and non-reproducing (n = 18) female New River Crayfish. Pie charts show mean proportion of worms and cocoons on lateral (white), ventral (medium gray), and dorsal (dark gray) surfaces of female crayfish. Bars represent mean counts ± 1 SE. Statistical results reported on the figure are for negative binomial general linear models comparing counts of worms on reproducing and non-reproducing females. Two asterisks (**) denotes P < 0.01. Southeastern Naturalist 527 K.J. Farrell, R.P. Creed, and B.L. Brown 2014 Vol. 13, No. 3 When we compared worm distributions between crayfish groups, non-reproducing females had a greater proportion of visible large worms on the ventral surfaces (0.52 ± 0.08) than did reproducing females (0.08 ± 0.06), where large worms were most often found on lateral surfaces (Fisher’s exact test: two-sided P = 0.0005; Fig. 1). Distributions of small worms also differed between crayfish groups, with small worms found most often on the lateral surfaces of reproducing crayfish; their distribution was more even on non-reproducing crayfish (Fig. 1, Fisher’s exact test: two-sided P = 0.002). Discussion Cambarincola worms and cocoons on reproducing female New River Crayfish were significantly reduced compared to the number on non-reproducing female crayfish. The difference was mainly due to a significant reduction in the number of large worms. Worms on reproducing crayfish were also located on different surfaces of the exoskeleton than on non-reproducing crayfish, with large worms found predominantly on the lateral surfaces of reproducing females, versus on the ventral surfaces of non-reproducing females. The data suggest that the nature of the symbiosis between female New River Crayfish and Cambarincola spp. may vary depending on the reproductive state of the host, a condition that warrants experimentation to pinpoint potential mechanisms driving the dif ferences we observed. Several mechanisms could explain the reduced worm numbers we documented on reproducing female crayfish. Crayfish grooming behaviors prior to egg extrusion may contribute to the reduction in large branchiobdellidans and their cocoons. Previous studies on the natural history of various crayfish taxa reported that females thoroughly clean the ventral surfaces of their exoskeletons 4–5 days prior to egg extrusion (Andrews 1904, Tack 1941). It is possible that such intensive grooming behaviors could dislodge branchiobdellidans and their cocoons from the ventral surface of the crayfish abdomen prior to egg-extrusion. Indeed, laboratory observations of crayfish grooming have demonstrated that crayfish can dislodge attached branchiobdellidans (Farrell et al. 2014, Skelton et al. 2014). Crayfish behaviors following egg extrusion may also limit worm re-colonization of the ventral surface of the abdomen. After the eggs are extruded and attached, females beat the pleopods to ensure water circulation around the eggs and, after hatching, the young crayfish, which prevents embryo fouling and death (Bauer 1989). Such frequent disturbances could contribute to the continued reduction in large worms and cocoons found on the ventral abdomens of female crayfish carrying recently hatche d young. Emigration could also contribute to the observed reduction of large worms on reproducing females. Branchiobdellidans are known to disperse between crayfish when the hosts come into direct contact (Young 1966), thus, large worms could leave reproducing females if the crayfish come into contact with other crayfish. Our observed pattern of large worm reductions only on reproducing females suggests that the worms emigrate prior to, or soon after, egg extrusion. Further investigation is needed to determine why large worms remaining on reproducing crayfish were not laying cocoons because cocoons were absent from the dorsal and lateral surfaces on reproducing female crayfish. Southeastern Naturalist K.J. Farrell, R.P. Creed, and B.L. Brown 2014 Vol. 13, No. 3 528 The number of small worms did not differ between reproducing and non-reproducing females, though the distribution of small worms was different between crayfish groups. We predict that when small worms mature into large worms, they recolonize the ventral surfaces after the crayfish reproductive period is over, though more extensive investigations are needed to evaluate this hypothesis, as well as assess other patterns of small-worm distribution, such as the lack of small worms on the dorsal surfaces of reproducing crayfish. The observed differences in worm location between crayfish groups suggest that worms may be moving from the ventral surfaces to other locations on the crayfish exoskeleton. Previous surveys in this system found that nearly 50% of C. ingens were located on the ventral surface of the abdomen or on the underside of the cephalothorax (Brown et al. 2002). Similarly, more than 80% of Cambarincola cocoons are located on the ventral surface of the abdomen (R.P. Creed, unpubl. data). In our surveys, this pattern held true for non-reproducing females, but on reproducing females, the majority of large and small worms were found on the lateral surfaces, specifically the margins of the carapace. Worms may relocate when the host extrudes eggs to avoid reductions in their own fitness due to smothering. However, such relocation alone would not result in a reduction in worm number as was observed in our surveys unless some worms move into the gill chamber. Because we only quantified worms on the exterior of the crayfish, additional investigations are needed to test these hypotheses. While it is possible that our counts failed to account for branchiobdellidans in the gill chamber, extensive observations of Cambarincola in lab and field experiments suggest that few worms, if any, spend significant amounts of time in the gill chambers. Large worms tend to be found in the same places on the external surface of the crayfish for extended periods (Thomas et al. 2013). Therefore, we doubt that our counts missed a significant proportion of the worms on these crayfish. If some large worms moved from the ventral surface to the gill chamber, it is immaterial to the distributional result (reduced density on ventral surfaces) . The results of our field surveys suggest that reproduction by female crayfish may alter the nature of the symbiosis between New River Crayfish and Cambarincola spp. such that the mutual benefits temporarily wane for one or both partners. Further experiments will be necessary to assess the mechanisms responsible and determine whether branchiobdellidans have measurable direct effects on crayfish reproduction. Acknowledgments We are grateful to Michael J. Thomas and April L. Meeks for field assistance. Comments from Nathan Dorn and two reviewers improved this manuscript. Funding was provided by National Science Foundation grants to R.P. Creed (DEB-0949823) and B.L. Brown (DEB-0949780). Literature Cited Andrews, E.A. 1904. 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