Infection with an acanthocephalan helminth reduces anxiety-like behaviour in crustacean host

Organisms Once a month from October 2021 to February 2022, we collected <em>P. tereticollis</em>-infected and uninfected <em>G. fossarum</em> individuals in the river Chiron in Burgundy, France (47° 27' 25.7"N, 5° 18' 21.2"E). They were maintained in large tanks filled with a mix of oxygenated dechlorinated ultraviolet-treated water (conditioned water; CW) and water from their river, in a room at 16°C with a 12L:12D photoperiod. Rocks were available as substrate and refuge, and gammarids were fed with dry elm leaves and chironomid larvae. Water was partly renewed every week. Gammarids were acclimatized to the room conditions for at least three weeks before taking part in the experiment. Treatments: acute and chronic stress using a semi-automated electric shock device Gammarids were placed in plastic containers (150 x 105mm, height: 60mm) with 400ml of CW by batches of 15 to 20 individuals, separated by treatments and according to the presence or absence of cystacanths. The cystacanths and old acanthellae of <em>P. tereticollis</em> form orange spots visible through <em>G. fossarum</em> exoskeleton, which allows straightforward diagnosis in living hosts; younger acanthellae have a lighter colouration and their presence can be assessed after host dissection. They were provided a small stone and half a terracotta saucer as refuges, and an elm leaf as food. Exposure to ES was done using a semi-automatic ES device (ESD) made of a rectangular piece of 3D-printed polymer containing two electrodes connected to an electronic driver 30, that fitted inside the above-mentionned plastic containers. In the “low intensity” (LI) stress treatment, once a day for 6 days at varying hours, the “chronically stressed” (CS) gammarid groups were placed in the ESD that delivered an ES program consisting in ten 9V pulses of 2sec administered every minute for 10min. Water was daily renewed at the time of the ES treatment. “Chronic controls” (CC) were not placed in ESDs but their water was renewed at the same time. Chronic treatment ended the day before behavioural tests (see below). On the seventh day, the “acutely stressed” (AS) gammarids were placed individually in plastic containers holding 200ml of CW, inside an ESD. They were given 5min to acclimatize before the ES program started. “Acute controls” (AC) were in the same conditions, except that they were not placed in ESDs, and their refuge was moved to the centre of their container. When the ES program ended, we removed the ESDs and placed the refuges back to the top edge of each container. In the “high intensity” (HI) stress treatment, the same protocol was applied to another set of gammarids except that a higher number of pulses and/or voltage was used. The HI CS treatment consisted in twenty 9V pulses of 2sec administered every 30sec for 10min once a day for the three first days, and then ten 15V pulses of 2sec every minute for 10min once a day for the last three days. On the seventh day, the AS gammarids received the 10x15V ES program before the refuge use test. Finally, to differentiate between a truly chronic effect of the CS treatment and a persistent effect of the last ES session, we exposed uninfected gammarids to a 10x15V ES session and conducted refuge use tests 24 hs later; controls were kept in the same conditions, but they did not receive ES. Although we configured the ESD to administer 9V or 15V pulses, effective voltage inside the device was lower and peaked at around 4V and 9V, respectively (measured with Voltcraft Digital Multimeter VC830). Refuge use test Refuge use tests were conducted on a bench receiving light between 90 and 130 lux. The choice of dimmed light (around sunset/sunrise values) was motivated by the decreased photophobia of uninfected gammarids at low illuminance 27, thus allowing the detection of a possible increase in photophobia in stressed gammarids. Five minutes after the end of the AS ES program (acclimation time), we started recording gammarids’ position every thirty seconds for ten minutes (focal and time sampling). Each gammarid’s score, thereafter abbreviated “RUs” was calculated as the sum of observations during which the gammarid was inside the refuge (from 0 to 20). Refuge use tests were generally conducted in blocks of 12 gammarids in individual plastic containers, each of the 8 treatments being represented by at least one individual in each block (with a few exceptions). After each refuge use test, gammarids were sedated in a 0.6g/l MS222 solution until they stopped moving 30, weighted and dissected to count the number of cystacanth(s) and acanthella(e).