DOI: 10.5061/dryad.dr7sqvb1q
title: Implication of the myo-inositol pathway in behavioral alterations of infected threespine sticklebacks

description: Threespine stickleback (Gasterosteus aculeatus) infected with the tapeworm
 Schistocephalus solidus display impairments in their anti-predator
 responses. They also have increased expression of the gene encoding the
 IMPase 1 enzyme in their brains, which is part of a key step in
 myo-inositol synthesis. IMPase 1 and myo-inositol levels are the targets
 of lithium treatment in patients with bipolar disorder. Although promising
 candidates, we do not know if IMPase 1 and myo-inositol are directly
 implicated in the changes in risky behaviors measured in
 Schistocephalus-infected fish. Understanding the molecular mechanisms
 directly or indirectly involved in these behavioral alterations is crucial
 to understand the evolution of host-parasite interactions. Here, we
 increased myo-inositol levels of uninfected fish and inhibited IMPase 1
 activity in infected fish to test the prediction that it would decrease
 and increase their anti-predator behaviour, respectively. We found that
 uninfected fish with increased myo-inositol levels (by injecting exogenous
 myo-inositol or by inducing endogenous production using an osmotic
 challenge) did not decrease their anti-predator responses. However,
 infected fish treated with lithium chloride had some of their
 anti-predator behaviors restored, but not all. They spent less time
 swimming close to the surface, swam a shorter distance, had a higher
 latency to feed, and spent more time frozen after a predator attack. Our
 results suggest that the target of lithium treatment is implicated in the
 risky behaviors of infected fish and supports the idea that the
 parasite-associated alteration in behavior has a multifactorial nature.
description: SAMPLING: Using a beach seine,
 we caught threespine sticklebacks from the wild population of
 Lac-Témiscouata (47°48'37.1"N 68°51'56.6"W, Québec,
 Canada) in June 2018 (n = 250). We brought all fish to the “Laboratoire
 Aquatique de Recherche en Sciences Environnementales et Médicales” (LARSA)
 at Université Laval. During transportation, we kept fish in coolers filled
 with the lake water and kept it aerated with air stones. In the animal
 facility, we held all fish in three 1000 L water tanks under a Light: Dark
 photoperiod of 13 h: 11 h and a temperature of 12 °C, similar to their
 natural conditions. We fed fish daily, twice a day, with a mix of artemia
 (Hikari Bio-Pur) and flakes (Nutrafin-Basix).
 EXPERIMENTAL GROUPS: For the uninfected fish
 experiment, we created four groups. Uninfected control for the stress of
 the injection (UNI CTRL, n = 20); uninfected control for the injection of
 phosphate-buffered saline (PBS), which was used as the vehicle (UNI PBS, n
 = 20); uninfected fish exposed to exogenous
 <em>myo</em>-inositol (UNI INO, n = 20); and uninfected
 exposed to an osmotic challenge (UNI OSC, n = 20). For the infected fish
 experiment, we had two groups. The infected control group (INF CTRL, n =
 20) and infected treated with lithium group (INF LIT, n =
 20). TREATMENTS:
 We exposed uninfected sticklebacks to exogenous
 <em>myo</em>-inositol (CAS #87-89-8, Sigma-Aldrich,
 ≥ 99% purity) at a dose of 90 mM dissolved in
 buffered saline (PBS, pH 7.4; Life Technologies, Carlsbad, CA, USA) by
 intraperitoneal injection (with a total volume of 100 µL) during three
 days, once per day. In another treatment group,
 we performed
 an intraperitoneal injection of a saline solution at 25
 ppt in uninfected sticklebacks, by dissolving Instant Ocean
 salt mix (Spectrum Brands, Blacksburg, VA, USA) in PBS in a final volume
 of 100 µL. We exposed fish infected
 with <em>Schistocephalus</em> <em>solidus</em> for
 three days to lithium chloride (CAS #7447-41-8, Sigma-Aldrich) using
 static exposures, by dissolving lithium in the aquarium water
 at a concentration of 12.5 mM (in a total volume of 2.7 L).
 BEHAVIOUR QUANTIFICATION:
 During the control week, fish were submitted to the handling of the
 experiment for three days but without receiving any treatment. We changed
 the water of the aquarium completely 48h after the beginning of the
 experiment and from this moment they did not receive any food until the
 behavioral tests. On the fourth day, each fish was transferred to the
 behavioral tanks for testing, which lasted one hour in total. After the
 behavioral tests, each fish returned to its home tank where they could
 rest for three days. After this period, the second week (treatment)
 started and we treated all fish, except those from the control group, and
 then we submitted fish to the behavior tests, as in the first
 week. We measured time spent in the upper part of the
 aquarium, time spent in the center of aquarium, total distance travelled,
 latency to feed, latency to freeze and time spent frozen after a predator
 attack. We recorded all behavioral experiments using a digital camera
 (Super Circuits PC212XS) placed in the front or at the top of the
 behavioral tanks. Videos were analyzed using Ethovision software
 (Ethovision XT 11.5, Noldus Information Technology, Wageningen, The
 Netherlands, Noldus et al., 2001). Water depth preference, distance
 traveled and total time spent in the center were measured with an
 automatic tracking module of Ethovision and latency to feed, latency to
 freeze, and time spent frozen were measured using a manual behavior
 setting module of the software. All tests and analyses were done by
 V.A.A., while blind for the fish experimental group.
 FINAL SAMPLING: At the end of the behavioral tests, we
 anesthetized each fish with an overdose of MS-222 (75 mg/L, pH 7.5). We
 quickly measured the standard length of the fish, their mass and rapidly
 dissected their whole brains. Brains were weighed and placed individually
 in 0.5 mL sample tubes and snap-frozen in liquid nitrogen. Brains were
 then kept at -80 °C until analysis. We verified every fish infection
 status (parasitized or not by <em>S</em>.
 <em>solidus</em>) upon dissection. We calculated the parasite index as
 the proportion of infected
 fish mass that is contributed by parasite tissue.