A survey of Cambarus crayfish and their symbiotic branchiobdellidans, Sinking Creek, Newport, VA, USA 2017-2018

Crayfish throughout the holarctic are found in association with an order of worms known as branchiobdellidans. This relationship has been confirmed as a cleaning symbiosis in several species. The Conhaway crayfish, Cambarus appalachiensis, is a species of crayfish endemic to the New River Basin in Virginia and West Virginia. We studied a population of C. appalachiensis in Sinking Creek in Newport, VA from March 2017 until February 2018. We collected morphological data and quantified the branchiobdellidan communities on 986 individuals, and kept note of egg brooding and young of year throughout the study period. The life cycle of C. appalachiensis was found to be similar to other large-bodied species of Cambarus crayfish. Molting occurred throughout the year, peaking in the months of April and September. This molting served as a disturbance effect to the symbiotic branchiobdellidan community and reset community assembly. The worm communities on larger, recently molted crayfish more closely resembled the less diverse communities on smaller crayfish. Most worms on recently molted crayfish were ones that we know are early colonizers. This survey provides the first life history information on a newly described species of Cambarus crayfish and provides both seasonal data on its branchiobdellidan associates and one of the first empirical examples of host ontogeny acting as a disturbance on a symbiotic community.

Methods
We sampled the crayfish Cambarus appalachiensis, previously C. sciotensis (Loughman et al. 2017), bimonthly in Sinking Creek at the Newport Recreation Area in Newport, VA (37.303096°N, 80.485522°W) from March 2017 until February 2018. The stream reach we chose to sample had multiple riffles and pools, and covered a broad range of crayfish habitats. We sampled crayfish from these varied microhabitats across the stream reach using dip nets, totaling 986 crayfish captures during our study. Our monthly sample sizes ranged from 53 to 104 crayfish. After measuring the physical properties of the crayfish and symbiont identification (detailed below), crayfish were returned to the stream and deposited downstream of the collection site to reduce the chance of resampling the same crayfish.

Crayfish were separated from each other at the stream to prevent symbiont dispersal between sampled crayfish, transported from the stream to a laboratory, and stored live in the refrigerator separated from each other in repurposed hardware parts organizers, termed “crayfish hotels”. We measured physical attributes of the crayfish, including the carapace length (the distance from the rostrum to the separation between the cephalothorax and the abdomen of the crayfish), the chelae length, the palm width (the width of the chelae at the widest point), the palm length, (length of the chelae up to the joint), the blotted wet mass, the sex, and the reproductive form. We measured total carapace length (a standard measure of crayfish size) of sampled crayfish, and determined carapace hardness using manual palpations to assess exoskeleton elasticity and a visual assessment of carapace fouling. This assessment of carapace hardness (exoskeleton hardening stage, or EHS) was used as a proxy for time since last molt, with a carapace graded as EHS 0/1 (soft exoskeleton), EHS 2 (hard exoskeleton, but with some elasticity and limited fouling), or EHS 3 (very hard and fouled exoskeleton, no elasticity) (Castillo-Escrivà et al. 2013). A score of EHS 0/1 indicated that the crayfish had molted very recently, a score of EHS 2 indicated the crayfish had molted somewhat recently, and a score of EHS 3 indicated that it had been some time since the crayfish molted.

We counted branchiobdellidan symbionts on the exterior of living crayfish using a low-magnification dissecting microscope. Symbiont counts were performed by a single observer throughout the study to prevent interobserver error. Counting symbionts on living crayfish had the advantage of allowing us to see the symbiont location on the host, and how the distribution of symbionts on the crayfish changed with the makeup of the symbiont community. We identified 13466 symbionts on the exoskeletons of the sampled crayfish according to a key developed for the crayfish and symbionts in southwestern Virginia (Hobbs et al. 1967). The branchiobdellidan symbiont community present on the exoskeleton of the crayfish in this population consisted of four species: Cambarincola ingens (Hoffman 1963), Cambarincola fallax (Hoffman 1963), Ankyrodrilus koronaeus (Holt 1965), and Pterodrilus alcicornus (Moore 1894). The location of each branchiobdellidan symbiont on the host crayfish was also noted using a classification based on the one used by Brown et al. (2002).

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