DOI: 10.5061/dryad.m905qfv71
title: Temperature affects predation of schistosome-competent snails by a novel invader, the marbled crayfish Procambarus virginalis

description: The human burden of environmentally transmitted infectious diseases can
 depend strongly on ecological factors, including the presence or absence
 of natural enemies. The marbled crayfish (Procambarus virginalis) is a
 novel invasive species that can tolerate a wide range of ecological
 conditions and colonize diverse habitats. Marbled crayfish first appeared
 in Madagascar in 2005 and quickly spread across the country, overlapping
 with the distribution of freshwater snails that serve as the intermediate
 host of schistosomiasis–a parasitic disease of poverty with human
 prevalence ranging up to 94% in Madagascar. It has been hypothesized that
 the marbled crayfish may serve as a predator of schistosome-competent
 snails in areas where native predators cannot and yet no systematic study
 to date has been conducted to estimate its predation rate on snails. Here,
 we experimentally assessed marbled crayfish consumption of uninfected and
 infected schistosome-competent snails (Biomphalaria glabrata and Bulinus
 truncatus) across a range of temperatures, reflective of the habitat range
 of the marbled crayfish in Madagascar. We found that the relationship
 between crayfish consumption and temperature is unimodal with a peak at
 ~27.5°C. Per-capita consumption increased with body size and was not
 affected either by snail species or their infectious status. We detected a
 possible satiation effect, i.e., a small but significant reduction in
 per-capita consumption rate over the 72-hour duration of the predation
 experiment. Our results suggest that ecological parameters, such as
 temperature and crayfish weight, influence rates of consumption and, in
 turn, the potential impact of the marbled crayfish invasion on snail host
 populations.
description: Animal
 husbandry Marbled crayfish were reared in freshwater aquaria
 filled with artificial pond water. Crayfish tanks varied in size (3.72 L,
 11.7 L, or 81.3 L), depending on the age, size, and rearing density of the
 crayfish. Crayfish, prior to becoming subjects in experiments, were
 typically housed with between two and four conspecifics. Juvenile crayfish
 were regularly removed from adult husbandry tanks and either relocated to
 a smaller tank (11.7 L) without adults or euthanized. Once included in the
 experiment, crayfish were housed individually in 11.7-L tanks. Husbandry
 tanks were held at room temperature (~25°C), whereas the temperature of
 experimental tanks was controlled and monitored (see below). All crayfish
 were regularly fed frozen carrots, except during experimental trials.
 Marbled crayfish (<em>Procambarus</em>
 <em>virginalis</em>) were obtained through private sellers on
 Etsy (https://www.etsy.com/)
 and Aquabid (https://www.aquabid.com/). Permission to import and house marbled crayfish for use in this study was provided by the State of Washington’s Department of Fish and Wildlife (Shellfish Import Permit No. 22-3020). Snails were reared in freshwater aquaria (either 3.72- or 11.7-L tanks, depending on the density of snails), filled with artificial pond water [78]. Tanks underwent 100% water changes one to two times per week. Snails were regularly fed romaine lettuce, which was refreshed during the bi-weekly water changes. All <em>Biomphalaria</em> <em>glabrata</em> (M-line, naive and exposed to <em>S. mansoni</em> strain PR-1) and <em>Bulinus</em> <em>truncatus</em> (Egypt, naive and exposed <em>S. haematobium</em> strain Egyptian) snails were provided by the NIAID Schistosomiasis Resource Center of the Biomedical Research Institute (Rockville, MD, USA) through NIH-NIAID Contract HHSN272201700014I for distribution through BRI Resources. Experiments Our methods largely replicated previous experimental predation trials between crustaceans and <em>Schistosoma</em>-competent mollusks. Briefly, one marbled crayfish (<em>Procambarus</em> <em>virginalis</em>) was held in combination with a set density (n =12) of either <em>Bi. glabrata </em>or<em> Bu. truncatus</em> snails in a 11.7-L tank. An "average" size class of snails (6–10 mm shell length for <em>Bi</em>. <em>glabrata</em>; 5–10 mm shell length for <em>Bu. truncatus</em>) was used. Crayfish length and weight were measured prior to the start of each experimental period. Crayfish varied in weight between 1.54 g and 14.44 g with an average ± SE of 6.62 ± 0.117 g. The total duration of each experimental period was 72 hours, with observations and snail replacement taking place every 12 hours. Each experimental period consisted of 7 total time points (0, 12, 24, 36, 48, 60, 72), in which each 12-hour increment constituted a “trial,” for a total of 6 trials per experimental period. At the conclusion of each trial, the number of snails above the water line, the number of snails on the lettuce (described below), the number of snails inside and under the shelter/hiding, and the number of snails in open water were counted and summed to reflect the total number of snails remaining in the tank. Additionally, the total number of empty, intact shells and the total number of dead snails were recorded at the conclusion of each trial. Shattered shell pieces were not included in empty shell counts, as it was too difficult to determine how many broken pieces constituted a singular shell. For each trial, we derived the total number of snails missing and presumably consumed as follows: the initial number of snails at each trial (n =12), minus the number of snails remaining, minus the number of snails dead but not consumed. The number of snails consumed and the number of dead snails were totaled to determine the total number of snails to be replaced/added to the experimental tank. All counts were repeated and confirmed by a second observer. At the conclusion of each trial, dead snails and empty, intact shells were removed, snail density (n =12) was reset, and the number of snails replaced/added was recorded. At the conclusion of the 72-hour experimental period, any remaining snails were removed from experiment tanks and returned to temperature-acclimated holding tanks. Crayfish remained in their tanks, allowing us to control for individual crayfish identity in analyses. Crayfish and snails were provided with food throughout the duration of the experimental period. Specifically, at the beginning of the experimental period (time point “0”), a piece of romaine lettuce was added to each experimental tank to serve as a food source for snails. Additionally, one invertebrate pellet was placed into each of the tanks, including control tanks, to serve as an alternative source of food for crayfish. This reflected our assumption that crayfish are omnivorous, and are not limited to eating only snails in their natural habitats. Each experimental tank also contained a piece of PVC pipe, which served as a shelter for the crayfish and snails. We observed crayfish consumption rate across five temperature conditions – 15, 20, 25, 30, and 35°C. This range reflects the diverse temperatures at which marbled crayfish have been found in Madagascar (20°C to 37°C). Though marbled crayfish can survive in temperatures as low as 5°C for extended periods of time, previous experiments have suggested that consumption ceases below 10°C. However, consumption has been observed at 15°C, and therefore, this may reflect the lower thermal limit of crayfish feeding behavior. Animals underwent a temperature acclimation period, in which the water temperature changed 1–1.5 °C/day until the desired temperature was reached. Animals were then held at the experimental temperature for at least 12 hours prior to the start of the experiment. Given that 15°C and 35°C would near the thermal limits of both the crayfish and snails, we included control tanks, from which crayfish were absent, to exclude the effect of temperature-associated snail death and ensure that snail mortality accurately reflected crayfish consumption.  We were interested in the influence of snail infection status on crayfish consumption rates and, therefore, varied snail infection status between experimental tanks. Each individual crayfish was held either with all “exposed” or “naive” (hereafter, “uninfected”) snails of one of two species included in the present study: <em>Biomphalaria glabrata </em>and<em> Bulinus truncatus</em>. Exposed snails were held at room temperature (~25°C) for ~30 days post-exposure (exposure date provided by the reagent provider, BRI) to allow infections to adequately mature before being used in experiments. Following the post-exposure period, exposed snails were assumed to be infected and will be referred to as such hereafter. We were limited by the availability of infected <em>Bu. truncatus </em>and<em> Bi. glabrata </em>snails (table 1, figure 1). As such, the first round of experiments included only uninfected snails. Round 1 of experiments began on 14 June 2021 and concluded on 13 August 2021. In Round 1 of experiments, we conducted seven (72-hour long) predation experiments for each set temperature (15, 20, 25, 30, and 35°C) for a total of 42 (12-hour long) trials (observations) per temperature, with one crayfish individual held in combination with either uninfected <em>Bi. glabrata </em>or uninfected <em>Bu. truncatus</em> snails. Round 1 also included two (72-hour long) experiments in control tanks with snails and no crayfish for each set temperature for uninfected <em>Bi. glabrata </em>and<em> Bu. truncatus</em>, for a total of 14 (12-hour long) trials for each temperature for each species in control conditions Round 2 of experiments, which included both uninfected and infected snails, began on 25 October 2021 and concluded on 17 December 2021. In Round 2 of experiments, two 72-hour experiments, for a total of 14 (12-hour long) trials for each set temperature (15, 20, 25, 30, and 35°C) were conducted in both the experimental and control conditions for both uninfected and infected <em>Bi. glabrata </em>and<em> Bu. truncatus</em> snails. Crayfish individuals used in Round 1 of experiments were also used in Round 2, barring mortality.
description: # Temperature affects predation of schistosome-competent snails by a novel
 invader, the marbled crayfish Procambarus virginalis --- These are the
 data underlying the following manuscript: Faiad SM et al. 2023.
 Temperature affects predation of schistosome-competent snails by a novel
 invader, the marbled crayfish Procambarus virginalis. PLoS One. ##
 Description of the data and file structure For a complete description of
 how these data were collected, please see the corresponding manuscript. In
 brief, the data include two csv files used in this analysis:
 `all_trials_crayfish_digital.csv`, which contains data on each crayfish
 taken at the start of each experimental run, and
 `all_trials_snail_digital.csv` which contains data on snail abundance,
 mortality, and behavior at the trial level within experimental runs.
 Variable descriptions are provided in `metadata.csv`. ## Sharing/access
 information Data are available in this repository, and are also
 permanently archived in GitHub at
 .