DOI: 10.5061/dryad.v9s4mw6s6
title: Data from: Fluid preservation causes minimal reduction of parasite detectability in fish specimens: a new approach for reconstructing parasite communities of the past?

description: Long-term datasets are needed to evaluate temporal patterns in wildlife
 disease burdens, but historical data on parasite abundance are extremely
 rare. For more than a century, natural history collections have been
 accumulating fluid-preserved specimens, which should contain the parasites
 infecting the host at the time of its preservation. However, before this
 unique data source can be exploited, we must identify the artefacts that
 are introduced by the preservation process. Here, we experimentally
 address whether the preservation process alters the degree to which
 metazoan parasites are detectable in fluid-preserved fish specimens when
 using visual parasite detection techniques. We randomly assigned fish of
 three species (Gadus chalcogrammus, Thaleichthys pacificus, Parophrys
 vetulus) to two treatments. In the first treatment, fish were preserved
 according to the standard procedures used in ichthyological collections.
 Immediately after the fluid-preservation process was complete, we
 performed parasitological dissection on those specimens. The second
 treatment was a control, in which fish were dissected without being
 subjected to the fluid-preservation process. We compared parasite
 abundance between the two treatments. Across 298 fish individuals and 59
 host–parasite pairs, we found few differences between treatments, with 24
 of 27 host–parasite pairs equally abundant between the two treatments. Of
 these, one pair was significantly more abundant in the preservation
 treatment than in the control group, and two pairs were significantly less
 abundant in the preservation treatment than in the control group. Our data
 suggest that the fluid-preservation process does not have a substantial
 effect on the detectability of metazoan parasites. This study addresses
 only the effects of the fixation and preservation process; long-term
 experiments are needed to address whether parasite detectability remains
 unchanged in the months, years, and decades of storage following
 preservation. If so, ecologists will be able to reconstruct novel,
 long-term datasets on parasite diversity and abundance over the past
 century or more using fluid-preserved specimens from natural history
 collections.
description: <i>Study
 species</i> We used three species of marine
 fish for our experiment. Two species, Walleye Pollock <i>Gadus
 chalcogrammus</i> and Eulachon <i>Thaleichthys
 pacificus</i>, were provided to us by the University of Washington
 Burke Museum Ichthyology Collection (UWFC). Both fish species were
 collected by National Oceanic and Atmospheric Administration research
 cruises in Alaska, frozen, and shipped to UWFC, where they were stored
 frozen as they awaited cataloging. The Walleye Pollock were collected from
 the Bering Sea and Gulf of Alaska between 2000 and 2002 and the Eulachon
 were collected from Shelikof Strait in the Gulf of Alaska in March of
 2002. On 11 and 12 May 2018, we collected English sole <i>Parophrys
 vetulus</i> by otter trawl in Port Madison, WA, in
 conjunction with the Fisheries Ecology course offered by the School of
 Aquatic and Fisheries Sciences at the University of Washington. English
 sole were euthanized, placed on ice, and frozen within 6 hours of
 collection. These three species of fish represent a variety of trophic
 strategies (suspension feeder, benthic predator, and pelagic predator,
 respectively), life history (i.e., short lived [Eulachon] and long lived
 [English Sole and Walleye Pollock]), habitats (benthic [English Sole] and
 pelagic [Walleye Pollock and Eulachon]), as well as body plans (i.e., flat
 fish [English Sole] and fusiform [Walleye Pollock and Eulachon]). Fish
 traits might influence the parasites that the fish can be infected with or
 the effects of preservative on fish tissues, and thus having a diversity
 of fish traits allows more robust conclusions regarding the effects of
 preservation on parasite detectability. In total, we collected 99 English
 Sole, 109 Walleye Pollock, and 70 Eulachon. <i>Experimental test of the
 effect of preservation on parasite detectability</i> We took an experimental approach
 to assess whether and how the preservation process affects the
 detectability of parasites. For each of the three fish species,
 individuals were randomly assigned to one of two treatments using a
 stratified design. Prior to randomly assigning fish to a treatment, fish
 were visually paired according to length within each species. Then, an
 individual from each pair was randomly assigned to the preservation
 treatment by a coin toss. This stratified random design equalized host
 size, a potential driver of parasite abundance, between treatments, and
 ensured that the mean, median, and range of host body sizes were similar
 between the two treatments. Two treatments were included in
 this experiment. In the control group, frozen fish were thawed and
 dissected, and their parasites identified. In the treatment group, frozen
 fish were thawed and preserved according to methods used at the University
 of Washington Fish Collection (UWFC). We followed the preservation
 protocol used by the UWFC, by first placing completely thawed fish in 10%
 buffered formalin solution until the fish is fixed (absolute amount of
 time is variable due to variation in fish body size, range = 9–16 days). A
 fish was considered to be fixed when its tissues had become firm
 (particularly around the abdominal cavity) but retained some pliability.
 Over-fixation and decalcification are possible if fish are left in
 formalin beyond the time required to fix all the tissues. To prevent this
 from happening, fish were monitored during the preservation process at
 least every three days. Once the fish were fully fixed, they underwent two
 consecutive freshwater rinses, each lasting 24 hours. After this, the fish
 were placed directly in 70% ethanol, where they remained for at least
 three days prior to dissection. Across the entire preservation process,
 care was taken to ignore parasites that could be dislodged during the
 preservation process, to mimic actual preservation conditions in
 collections. After preservation was complete, fish were dissected, and
 their parasites identified. <i>Parasitological
 dissection</i> To avoid confounding the effects
 of preservation and dissection method, we used a consistent dissection
 protocol across both treatments. This could result in lower overall
 detectability (across both treatments) for parasites found in muscle,
 since candling was used rather than a muscle squash to avoid excessive
 destruction of the musculature of specimens – a variation in dissection
 protocol that is often used for museum specimens to preserve external
 morphology. Methods for all other tissues follow standard parasitological
 techniques (see below). We compared parasite abundance between the two
 treatments to assess the effects of preservation on detectability for each
 parasite taxon detected. For each specimen, we made a
 ventral incision from the anus to the gill isthmus. We then removed all
 viscera for examination. The viscera were separated by organ and
 individually squashed between two glass plates and examined for parasites.
 Gills from the right or blind side of the fish were removed, placed in a
 vial with artificial seawater (for control group) or 70% ethanol (for
 preservation treatment), shaken to free parasites from the gills, and
 examined under a dissecting microscope. External surfaces and the buccal
 cavity of the fish were also examined for ectoparasites. The specimen was
 then spread open along its ventral incision and placed over a strong light
 to examine the flesh and skin for parasites. All examinations were
 conducted under a stereomicroscope to best capture the entire parasite
 burden and not simply the large-bodied parasite taxa and all parasites
 were identified to the lowest possible taxonomic level (hereafter,
 morphotypes) using standard parasite identification keys.
description: This deposition includes several datasets: one raw dataset on the
 abundance of various parasite taxa in Walleye Pollock (Gadus
 chalcogrammus; PollockParasites.csv), one raw dataset on the abundance of
 various parasite taxa in Eulachon (Thaleichthys pacificus;
 EulachonParasites.csv), one raw dataset on the abundance of various
 parasite taxa in English sole (Parophrys vetulus; EngSoleParasites.csv),
 and a dataset containing meta-data collected on each host fish individual
 sampled (HostData.csv). Data are available in this
 repository, and are also permanently archived in
 GitHub: https://github.com/wood-lab/Fiorenza_et_al_2020_Ecol_Evol