Seasonal dietary shifts enhance parasite transmission to lake salmonids during ice cover

Abstract Changes in abiotic and biotic factors between seasons in subarctic lake systems are often profound, potentially affecting the community structure and population dynamics of parasites over the annual cycle. However, few winter studies exist and interactions between fish hosts and their parasites are typically confined to snapshot studies restricted to the summer season whereas host‐parasite dynamics during the ice‐covered period rarely have been explored. The present study addresses seasonal patterns in the infections of intestinal parasites and their association with the diet of sympatric living Arctic charr (Salvelinus alpinus) and brown trout (Salmo trutta) in Lake Takvatn, a subarctic lake in northern Norway. In total, 354 Arctic charr and 203 brown trout were sampled from the littoral habitat between June 2017 and May 2018. Six trophically transmitted intestinal parasite taxa were identified and quantified, and their seasonal variations were contrasted with dietary information from both stomachs and intestines of the fish. The winter period proved to be an important transmission window for parasites, with increased prevalence and intensity of amphipod‐transmitted parasites in Arctic charr and parasites transmitted through fish prey in brown trout. In Arctic charr, seasonal patterns in parasite infections resulted mainly from temporal changes in diet toward amphipods, whereas host body size and the utilization of fish prey were the main drivers in brown trout. The overall dynamics in the community structure of parasites chiefly mirrored the seasonal dietary shifts of their fish hosts.

summer, as opposed to winter, often correspond with a peak of helminth egg output and increased intensity of infections in wild Red deer populations from temperate regions (Albery et al., 2018). Such seasonal trends in parasite transmission are, however, not universal, as exemplified by the transmission of the nematode Marshallagia marshalli to reindeer in the high Arctic (Svalbard) which occurs throughout the winter months despite extreme cold conditions (Carlsson et al., 2012).
For freshwater fishes, seasonality in parasite transmission is documented from several host and parasite taxa (Chubb, 1979(Chubb, , 1980(Chubb, , 1982. However, few studies exist from lakes at high latitudes, where the changes between seasons are contrasting and profound. These changes are often dictated by the formation of ice cover, which can last for more than 6 months (Thompson, Ventura, & Camarero, 2009;Wrona et al., 2013). Studies of parasites in Finnish boreal lakes and coastal sea areas with similar ice-cover duration have demonstrated the existence of host-parasite dynamics during winter in various fish hosts (Karvonen, Cheng, & Valtonen, 2005;Valtonen & Crompton, 1990;Valtonen, Prost, & Rahkonen, 1990). The winter period has, however, traditionally been viewed as an insignificant season of low ecological importance in ice-bound lakes (Salonen, Leppäranta, Viljanen, & Gulati, 2009), and the majority of host-parasite studies in northern lake systems have addressed the ice-free period (Knudsen, Klemetsen, & Staldvik, 1996;Tedla & Fernando, 1969). This bias toward summer and autumn studies may underestimate the importance of the winter as a transmission window for parasites in high-latitude lakes.
The exposure to trophically transmitted parasites is often related to the abundances of intermediate and final hosts in the environment (Hechinger & Lafferty, 2005;Stutz, Lau, & Bolnick, 2014).
Beside the trophic behavior of the hosts, the availability of potential intermediate prey hosts is often regulated by seasonal changes in environmental factors, which can also affect the viability of free-living parasite stages and thereby alter parasite transmission rates to fish (Chubb, 1982;Pietrock & Marcogliese, 2003). The structure of parasite communities in fish may consequently change seasonally with different prey and parasite species dominating in different seasons (Kennedy, 1997). Accounting for such seasonality in parasite transmission is important when conducting larger ecological studies (e.g., food-web analyses) that include parasites.
In the present study, we address seasonal patterns in the intestinal parasite community of sympatric Arctic charr (Salvelinus alpinus) and brown trout (Salmo trutta) in subarctic Lake Takvatn, northern Norway, which is ice covered for approximately half the year. Lake Takvatn has been the subject of numerous ecological studies, including fish and food-web analyses that comprise parasites (see Amundsen & Knudsen, 2009;Amundsen et al., 2013;, thus constituting a good basis for seasonal studies addressing intestinal parasites. The intestinal parasites of Arctic charr and brown trout are typically transmitted trophically with diet as an important predictor of their community composition (Curtis, Bérubé, & Stenzel, 1995;Knudsen et al., 2008;. Sympatric Arctic charr and brown trout segregate their diets during the ice-free season with the former being the most generalist feeder (Eloranta, Knudsen, & Amundsen, 2013), which seems to drive differences in their parasite communities (Knudsen et al., 2008).

The most common intermediate hosts for fish intestinal parasites in
northern lakes include zooplankton (mainly copepods), amphipods, insect larvae, and fish. Typically, Arctic charr have more parasites transmitted via copepods than brown trout, which are more frequently parasitized via the consumption of amphipods, insect larvae, and fish as intermediate hosts (Henriksen et al., 2016;Knudsen et al., 2008;Paterson, Nefjodova, Salis, & Knudsen, 2019). However, under ice cover, both Arctic charr and brown trout feed mainly on benthic macroinvertebrates including amphipods and insect larvae in addition to increased piscivory in brown trout (Amundsen & Knudsen, 2009;. Such seasonal changes in feeding ecology may be crucial for the structuring of parasite communities in salmonid hosts. Here, we explore seasonal patterns in the infections of intestinal parasites and their association with the diet of Arctic charr and brown trout in Lake Takvatn. Firstly, we hypothesized that Arctic charr have a higher parasite diversity, prevalence, and abundance than brown trout throughout all seasons due to a broader dietary niche. We secondly hypothesized that Arctic charr will have increased infections (prevalence and intensity) of amphipod-transmitted parasites while brown trout will aggregate more parasites transmitted via amphipods and fish prey during the winter period.
Thirdly, we hypothesized that diet drives seasonal changes in the intestinal parasite communities. Consequently, the accumulation of parasites during the winter period will be particularly important for the overall differences in parasite community seen between the two fish host species.

| Study site
The study was conducted in Lake Takvatn, a dimictic and oligotrophic subarctic lake located 215 m above mean sea level in Troms county, northern Norway. The lake has a surface area of 15.2 km 2 and a maximum depth of 88 m. Secchi depths range between 14 and 17 m, and phosphorous levels do not exceed five micrograms per liter (Eloranta et al., 2013). The lake is usually ice covered from late November to early June (Amundsen, Knudsen, & Klemetsen, 2007;Klemetsen, Knudsen, Staldvik, & Amundsen, 2003). In the winter of 2017-2018, the lake surface froze during the last week of November and the ice melted at the end of May. The maximum measured ice thickness was 100 cm in March. The temperatures observed in the upper water level (1 m depth) during the field sampling periods ranged between 12.8°C in summer (August) and 1.15°C under ice cover in winter (January). The only fish species present in the lake are brown trout, Arctic charr, and three-spined stickleback (G. aculeatus) (see Amundsen and Knudsen (2009) for further details about the lake).

| Fish sampling and processing
In total, 354 Arctic charr and 203 brown trout were sampled from the littoral habitat (<15 m depth) between June 2017 and May 2018 using multi-meshed gillnets with panels of eight different mesh sizes from 10 to 45 mm, knot to knot ( Table 1). The sampling was carried out monthly during the ice-free season (June to November) and every second month during the ice-covered period (December to May). During the ice-covered period, gill nets were pulled out and retrieved through holes in the ice by means of submerged ropes. The ropes were positioned in the lake in December when the ice thickness was still modest. The nets were left in the lake overnight for approximately 12 hr during the ice-free period and approximately 16 hr during the ice-covered period. In the field, fork length in mm, weight, sex, and gonad maturation of all fish was recorded. Stomachs were opened, and the fullness degree was determined on a scale from 0% to 100%. Prey types were identified, and their contribution to the total stomach contents was calculated according to the method described by Amundsen (1995). The stomach contents were preserved in 96% alcohol, and the intestines were frozen to preserve the content, allowing subsequent parasitological and dietary analyses in the laboratory.

| Parasite sampling
The intestinal parasites were sampled by cutting the intestines open and sieving the contents including that of the pyloric caeca under running water with a 120-micron mesh size nylon net. The collected material was then placed into a Petri dish with a physiological saltwater solution (9% NaCl). We found 5 taxa: Crepidostomum spp., Cyathocephalus truncatus, Eubothrium salvelini, E. crassum, and Proteocephalus sp., which use Arctic charr or brown trout as their final host. (Table 2, Figure 1). At least four potentially different species belonging to the genus Crepidostomum are found in Lake Takvatn (Soldánová et al., 2017), here grouped as Crepidostomum spp. as they are only distinguishable via genetic analysis. The only representative of the genus Proteocephalus is here described as Proteocephalus sp.
since the exact species is not known. Additionally, the larval stage (plerocercoids) of two different species of Dibothriocephalus (formerly Diphyllobothrium (Waeschenbach, Brabec, Scholz, Littlewood, & Kuchta, 2017)) were also recorded in the intestines of both Arctic charr and brown trout and are here grouped together as Dibothriocephalus spp. (Figure 1). The Dibothriocephalus spp. plerocercoids analyzed in this study include only the unencysted larvae found in the intestine, not those encysted in the viscera. The presence of unencysted plerocercoids in the intestine has previously been considered accidental. However, a high correlation between the number of unencysted plerocercoids and the degree of piscivory (see later), particularly in brown trout, strongly indicates that their presence was the result of recent ingestion of infected fish prey, and the Dibothriocephalus spp. plerocercoids were therefore taken into consideration for the analyses.

| Prey types in the gastrointestinal tract
Only amphipods, insect larvae, zooplankton, and fish were consid-
Mean number of taxa is defined as the mean number of parasite taxa per host individual. Mean number of parasite taxa was used instead of observed parasite species richness, as no seasonal differences between Arctic charr and brown trout were detected using the Jackknife method (Zelmer & Esch, 1999) as an estimator of parasite richness. The mean number of parasite taxa was compared between Arctic charr and brown trout using the Mann-Whitney U test. Abundance is the number of parasite individuals of a particular species in a single host species (infected and uninfected). Prevalence is defined as the proportion of host individuals infected by a particular parasite among the examined sample of a specific host species, usually expressed in percentage. Prevalence was compared between host species using a χ 2 test with Yates correction for each parasite taxa separately. Intensity is the number of parasite individuals of a particular species in a single infected host species. Mean intensity represents the average number of parasite individuals belonging to a particular species found in all hosts infected by that parasite (uninfected hosts excluded). To test for differences in mean intensity of each parasite taxa separately a nonparametric maximum test that combines Brunner-Munzel and Welch U tests was used as suggested by Welz, Ruxton, and Neuhäuser (2018).
To analyze seasonal variations in the infections of intestinal parasites, monthly data were merged into four seasonal periods to cope with the low winter sample size (Table 1). As the length of both Arctic charr and brown trout significantly differed among sampling seasons (One-way ANOVA, F(3) = 3.844, p = .01 and F(3) = 21.78, p < .001, respectively), any size effect on the seasonal variation in parasite infections was also tested using a negative binomial generalized linear model (GLM) with length as a covariate.
Negative binomial GLM is best suited to model the overdispersion of parasites distributions among hosts which is typically aggregated (Lindén & Mäntyniemi, 2011;Paterson & Lello, 2003;Rózsa, Reiczigel, & Majoros, 2000;Wilson & Grenfell, 1997). The model included parasite counts of infected hosts (i.e., intensity) as the response variable with seasons and fish length as predictors. The use of sex as a covariate did not produce significant results, and age was excluded from the analysis because part of the age data

| The intestinal parasite communities of Arctic charr and brown trout
Of all Arctic charr examined, 98% were infected with at least one intestinal parasite taxon and the mean number of parasite taxa per fish was 2.3 (±0.95 SD, Figure 2a). In total, five intestinal parasites taxa were recorded in Arctic charr (Figure 2b). Of these, the most common were E. salvelini with 92% and Crepidostomum spp.
Of all brown trout examined, 88% were infected with at least one intestinal parasite taxon and the mean number of parasite taxa per fish was 1.5 (±0.91 SD), which was significantly less than in Arctic charr (Mann-Whitney U test, W = 52,280, p < .01; Figure 2a). Overall, five intestinal parasite taxa were found. The most common parasite was Crepidostomum spp. with a prevalence of 71%, whereas E.

| Seasonal variations of intestinal parasites in Arctic charr and brown trout
Both Arctic charr and brown trout displayed significant seasonal variations in the prevalence and intensity of several intestinal parasites (Table 3a,b, Figure 3). Parasite infections in the two salmonids were strongly influenced by both seasons and fish length (

| Seasonal variations in diet
Insect larvae, zooplankton, and amphipods were the most common overall prey for Arctic charr (Figure 5a). Insect larvae were the most common prey during late winter and summer, whereas zooplankton was the most common prey during autumn and amphipods during early winter. Fish prey had in contrast just a minor contribution to the diet of Arctic charr in all seasons. In brown trout, insect larvae and fish (mainly three-spined stickleback) were the most important prey groups (Figure 5b). Insect larvae were the dominant prey from summer to early winter, while fish were the main prey in late winter.
The occurrence of amphipods in the brown trout diet was relatively modest throughout all seasons, whereas zooplankton was recorded only in autumn and early winter.

| Associations between parasites and diet
In Arctic charr, host length and the frequency of occurrence of am- of the CCA accounted for 21.6% of the total variation (Figure 6a).
Dimension 1 was mostly correlated with the explanatory variables fish and zooplankton prey, which accounted for 18.1% of the total variation in the parasite abundance data. Dimension 2 was mostly correlated with amphipods prey and accounted for 3.5% of the total variation ( Figure 6a). In brown trout, host length and the frequency of occurrence of fish prey and amphipods in the diet were significantly associated with the variation in parasite abundance data (CCA; permutation test, all p < .05), whereas the frequencies of insect larvae and zooplankton were not significant. The first two dimensions explained 19.9% of the total variation (Figure 6b). Dimension 1, which accounted for 17.4% of the total variation, was mostly correlated with fish prey in the diet and host length, while dimension 2 was driven mainly by predation on zooplankton and explained 2.5% of the total variation (Figure 6b). Overall, the parasite communities of Arctic charr and brown trout were segregated (Figure 7). In Arctic charr, the elevated abundance of C. truncatus and Crepidostomum spp. was associated with amphipod consumption, while Proteocephalus sp. was associated with fish and zooplankton prey groups. Brown trout consumed more fish and consequently had a higher abundance of Dibothriocephalus spp. communities. The more opportunistic feeding behavior observed in Arctic charr apparently increased its exposure to trophically transferred parasites. In accordance with our first hypothesis, Arctic charr not only hosted more parasite taxa but also had an overall higher abundance and prevalence of helminths compared with brown trout.

| D ISCUSS I ON
Hence, the host with the broadest niche (Arctic charr) harbored the richest community of food-transmitted parasites, which is also in agreement with the expectations proposed by Kennedy, Bush, and Aho (1986) and Holmes (1990). Similar patterns have been observed in terrestrial animals. A study conducted on six sympatric species of lemurs in Kirindy Forest (Madagascar) showed that the mouse lemur (Microcebus murinus), which possessed the widest trophic niche, also had the highest burden of gastrointestinal parasites (Springer & Kappeler, 2016).
In accordance with our second hypothesis, that Arctic charr will have increased infections of amphipod-transmitted parasites during the ice-covered period, the prevalence and intensity of C. truncatus and Crepidostomum spp. reached a peak in Arctic charr in early winter. Their elevated infections are explained by an early winter peak in predation on their intermediate host, the amphipod G. lacustris, which is also in agreement with findings from a nearby lake (Amundsen & Knudsen, 2009;Amundsen, Knudsen, Kuris, & Kristoffersen, 2003;Knudsen et al., 2008). Furthermore, the establishment success and residence time of C. truncatus in the fish host seem to increase with temperatures lower than 10°C Awachie, 1968). Hence, the low water temperatures in the ice-bound period likely prolong the residence time in the fish host , resulting in increased infection levels.
In brown trout, on the other hand, a distinct peak in prevalence of Dibothriocephalus spp. and E. crassum was observed in late winter coinciding with a profound rise in piscivory. The copepod-transmitted helminths Dibothriocephalus spp. and E. crassum do have the ability to re-establish in piscivorous fish (Von Bonsdorff & Bylund, 1982;Bylund, 1969;Halvorsen, 1970;Vik, 1963;Williams & Jones, 1994).
Given the low frequency of occurrence of zooplankton in the diet of brown trout, piscivory appears to be important for transmission of these parasite taxa through reinfection processes, in particular by eating three-spined sticklebacks. This assumption is further supported by the fact that Dibothriocephalus spp. was solely present as unencysted plerocercoids in the intestines of both Arctic charr and brown trout; and not as procercoids as should have been the case if they had been transmitted through ingestion of copepods. Previous studies conducted in Lake Takvatn, have highlighted the importance of stickleback as paratenic hosts in transmitting parasite to brown trout Henriksen et al., 2016;Klemetsen et al., 2002;Kuhn, Amundsen, Kristoffersen, Frainer, & Knudsen, 2016). Similarly, a study of the brackish water of the Bothnian Bay, which included the winter period, found that the predatory burbot (Lota lota) was infected by Diphyllobotrium ditremum and Eubothrium rugosum through fish consumption (Valtonen & Julkunen, 1995). The present study shows that a substantial portion of this transmission probably occurs under ice cover in winter, which is also seen in other host-parasites systems (Karvonen et al., 2005;Valtonen & Crompton, 1990;. In accordance with the third hypothesis, the accumulation of parasites through the winter period proved to be important for the differences in the overall parasite community seen between Arctic charr and brown trout. This pattern was to a great extent explained by seasonal changes in resource availability and feeding behavior between the two host species. Seasonal changes in parasite trans- In Arctic charr, the prevalence of the copepod-transmitted parasite Proteocephalus sp. peaked in autumn and strongly declined during winter while the prevalence of amphipod-transmitted parasites, notably C. truncatus, was high during the ice-covered period. These temporal changes in the helminth communities of Arctic charr were well reflected by a corresponding dietary shift from zooplankton to benthic invertebrate, as most cladocerans enter the winter egg diapause and zooplankton availability thus decreases (Amundsen & Knudsen, 2009;Klemetsen et al., 2002;Klemetsen, Knudsen, et al., 2003). A reduction in zooplankton feeding during winter will consequently diminish the exposure to Proteocephalus sp., whereas and increase in consuming larger and more abundant zoobenthos favors the transmission of helminths like Crepidostomum spp. and C. truncatus that use amphipods and insect larvae as intermediate hosts.
Reduced infections of Proteocephalus longicollis in common whitefish (Coregonus lavaretus) during the winter period following a decrease in zooplankton availability were also observed in a French perialpine lake by Hanzelová and Gerdeaux (2003). Increased infections of Crepidostomum metoecus and C. truncatus due to amphipod predation were also previously observed in Dolly Varden (Salvelinus malma)  Marcogliese, Goater, & Esch, 1990), these may have higher prevalence during summer when the feeding on insect prey is high, while the prevalence of those transmitted through amphipods may increase toward winter, consequently explaining the lack of seasonality in Crepidostomum ssp. harbored by Arctic charr.
The stability in the infections of E. salvelini suggests that the prevalence of this parasite is less affected by seasonal changes in diet due to a life span of more than 1 year in the fish host (Hanzelová, Scholz, Gerdeaux, & Kuchta, 2002;Hernandez & Muzzall, 1998). The low and stable prevalence of Dibothriocephalus spp. coincided with a low inclusion of fish prey in the diet of Arctic charr.
Similar to Arctic charr, distinct parasite-diet relationships were also found in brown trout. The prevalence of parasites potentially transmitted by fish prey (i.e., Dibothriocephalus spp. and E. crassum) increased through the winter in correspondence with a transition from an insect-dominated diet during summer to a more fish-dominated diet in late winter. This shift in diet also explains a rise in the overall diversity of the intestinal parasite community of brown trout.
The high degree of piscivory observed in trout during the winter period might partially relate to increased competition for benthic resources with Arctic charr (Amundsen & Knudsen, 2009 (Curtis et al., 1995;Knudsen, Kristoffersen, & Amundsen, 1997;Kristmundsson & Richter, 2009;Thomas, 1958). Overall, the intestinal parasite communities of both Arctic charr and brown trout changed distinctly throughout the year and were associated with seasonal dietary shifts, with the winter period playing an important role for parasite transmissions. Interspecific interactions among the intestinal parasites might also have influenced the observed patterns, but such interactions have not been reported as important structuring forces for these taxa .
In conclusion, our study reveals that the long-lasting ice-covered period represents an important transmission window for parasites to fish in high-latitude lakes. Enhanced infections of amphipod-transmitted parasites in Arctic charr and piscivore-related parasites in brown trout during the ice-cover period are chiefly explained by their winter diet. In essence, our findings document that the intestinal parasite communities of Arctic charr and brown trout are highly influenced by their seasonal dietary changes.

ACK N OWLED G M ENTS
We thank Laina Dalsbø, Karin Strand Johannessen, Cesilie Bye, and Runar Kjaer for assistance in the field sampling and laboratory work.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

E TH I C A L A PPROVA L
All applicable institutional and/or national guidelines for the care and use of animals were followed. Per-Arne Amundsen https://orcid.org/0000-0002-2203-8216