DOI: 10.5061/dryad.6djh9w122
title: Dietary composition and fatty acid content of giant salmonflies (Pteronarcys californica) in two Rocky Mountain rivers

description: Many aquatic invertebrates are declining or facing extinction from
 stressors that compromise physiology, resource consumption, reproduction,
 and phenology. However, the influence of these common stressors
 specifically on consumer-resource interactions for aquatic invertebrate
 consumers is only beginning to be understood. We conducted a field study
 to investigate Pteronarcys californica (i.e., the ‘giant salmonfly’), a
 large-bodied insect that is ecologically and culturally significant to
 rivers throughout the western U.S. We sampled gut contents and
 polyunsaturated fatty acid (PUFA) composition of salmonflies to compare
 resource consumption across river (Madison or Gallatin, Montana), sex
 (male or female), and habitat (rock or woody debris). We found that
 allochthonous detritus comprised the majority of salmonfly diets in the
 Gallatin and Madison Rivers, making up 68% of the gut contents on average,
 followed by amorphous detritus, diatoms, and filamentous algae. Diets
 showed little variation across river, sex, or length. Minor differences in
 diets were detected by habitat type, with a higher proportion of diatoms
 in the diets of salmonflies collected from rocky habitat compared to woody
 debris. Fatty acid composition generally supported the results of gut
 content analysis but highlighted the importance of primary producers. The
 presence of eicosapentaenoic acid (EPA; 20:5n-3) and alpha linolenic acid
 (ALA; 18:3n-3) suggest consumption of diatoms and filamentous green algae,
 respectively. Our research underscores the importance of a healthy
 riparian zone that provides allochthonous detritus for invertebrate
 nutrition as well as the role of algae as an important source of fatty
 acids.
description: <i>Study area</i> This study was
 conducted in the Madison and Gallatin Rivers, located in Southwest
 Montana. The Madison and Gallatin Rivers originate in Yellowstone National
 Park, Wyoming, and flow north for 295 and 193 river kilometers,
 respectively. Both rivers join the Jefferson River near Three Forks,
 Montana to form the Missouri River. Study sites on the
 Madison River were located between Hebgen and Ennis Reservoirs, which were
 created by dams constructed in 1914 and 1906, respectively. In this area,
 the Madison River flows through a broad valley, with riparian vegetation
 dominated by willow and grasses. The Madison watershed (6,620
 km<sup>2</sup>) is 39% woodlands and 48% grass and shrublands
 (Anderson et al. 2019). Near our study sties, mean July water temperature
 and discharge were 14.7 ºC and 1,130 cfs, respectively, in 2020 (USGS gage
 06038800). Because the Gallatin River does not have a present-day USGS
 water temperature sensor, we evaluated historical temperature at the same
 location as the USGS gage from NorWeST (Isaak et al. 2017) to compare
 water temperature between rivers. NorWeST reported mean August water
 temperature of 16.4 ºC from 1993-2011. Study sites on
 the Gallatin River were located between the towns of Big Sky and Gallatin
 Gateway. The Gallatin River in the study area flows through both valley
 and canyon topography. The Gallatin River is free flowing, with riparian
 vegetation dominated by willow and conifer forest. The Gallatin watershed
 (4,784 km<sup>2</sup>) is 70% woodlands and 18% grass and
 shrublands (Anderson et al. 2019). Mean July discharge was 975 cfs in 2020
 (USGS gage 06043120). Because the Gallatin River does not have a
 present-day USGS water temperature sensor, we collected historical
 temperature from NorWeST (Isaak et al. 2017) at the same location as the
 USGS gage. NorWeST reported mean August water temperature of 10.7 ºC from
 1993-2011. <i>Salmonfly
 collection</i> Salmonflies were collected from
 three sites each on the Madison and Gallatin Rivers on July 9 – 10, 2020.
 Sites were chosen to span the length of each river known to support
 relatively dense salmonfly populations and were spaced at minimum
 approximately 12.5 river kilometers apart. We collected salmonflies in the
 evening between the hours of 17:00 – 21:00 to increase the likelihood of
 sampling a full gut and to maintain a consistent sampling window across
 all sites. At each site, we collected three male and three female late
 instars from two habitat substrate types: rock surfaces and woody debris
 (n = 12 individuals per site; 36 per river). Individuals were collected
 directly from substrates using forceps. Salmonflies were immediately
 preserved in Kahle’s solution in individual vials and transported to the
 laboratory. <i>Gut content
 analysis</i> Gut contents were identified and
 analyzed in the laboratory following the methods described in
 Rosi-Marshall et al. (2016b). We measured salmonfly body length (mean =
 34.86; s.e. = 0.46 mm) and dissected each individual to remove the gut
 tract. Gut contents from the upper portion of the gut tract were emptied
 into water, the gut wall was removed, and contents were placed in a
 beaker. We placed each beaker in a water bath sonicator for 30 seconds to
 disperse clumps. Gut contents were then filtered onto Metricel membrane
 filters (25-mm diameter, 0.45 µm pore size). Filters were dried at 60ºC
 for ten minutes and then mounted onto slides using standard immersion oil.
 We photographed particles at 100x to 400x magnification depending on the size of item and
 the resolution required to accurately identify the item type.
 We identified and measured the area of up to 150 particles per slide using
 NIS-Elements imaging software (Nikon, 2019) and estimated the proportion
 of area comprised of each diet item type as our response variable. Diet
 item types identified included wood and leaf debris combined, amorphous
 detritus, animal material, fungi, diatoms, and filamentous algae (Figure
 2). <i>Fatty acid analysis</i>
 Late-instar larval salmonflies were collected from two sites on
 the Madison River, Palisades and Varney Bridge, on November 27, 2019. We
 collected three females and three males from each site for fatty acid
 composition analyses. Salmonflies were frozen, shipped overnight, and
 stored at -80ºC upon arrival at the processing facility. We freeze-dried
 and then weighed salmonflies prior to extracting fatty acid methyl esters
 (FAMEs) for composition analyses. Freeze-dried salmonflies were deposited
 in 7 mL dichloromethane:methanol (2:1, v:v), crushed using a glass rod,
 vigorously sonicated, and stored over night at −20°C. Total lipids were
 extracted three times from tissues with dichloromethane:methanol (2:1,
 v:v). Pooled cell-free lipid extracts were evaporated to dryness under
 N<sub>2</sub>-atmosphere and transesterified with methanolic
 HCl (3 mol L<sup>-1</sup>, 60 °C, 15 min, Sigma-Aldrich
 33050-U). FAMEs were extracted three times with isohexane (2 mL). Pooled
 FAME-containing fractions were evaporated to dryness under
 N<sub>2</sub> and resuspended in 50 µL of isohexane. FAMEs
 were analyzed by gas chromatography (GC) using a HP 6890 gas chromatograph
 (Agilent Technologies) equipped with a flame ionization detector (FID) and
 a DB-225 (J&W Scientific, 30 m × 0.25 mm inner diameter (id) ×
 0.25 μm film) capillary column. Configuration details are given elsewhere
 (Martin-Creuzburg et al., 2010; Martin-Creuzburg et al., 2017). FAMEs were
 quantified by comparison to internal standards (C17:0, C23:0 ME) of known
 concentrations (adapted to dry mass in the sample), using multipoint
 calibration curves generated using FAME standards (Sigma). FAMEs were
 identified by their retention times and their mass spectra, which were
 recorded with a quadrupole gas chromatograph-mass spectrometer (GC-MS;
 Agilent Technologies, 5975C inert MSD) equipped with a DB-225MS
 fused-silica capillary column (J&W Scientific, 30 m × 0.25 mm id ×
 0.25 μm film); gas chromatographic settings as for FID. Mass spectra were
 recorded between 50 and 600 m/z in the electron ionization (EI) mode. The
 limit of quantitation was 10 ng of fatty acid.
 <i>Data analysis</i> We used both
 multivariate and univariate approaches to analyze diet proportions for
 differences by sex, habitat, and river. We used non-metric
 multidimensional scaling (NMDS) based on Bray-Curtis dissimilarities to
 visualize how salmonfly gut contents differed by sex, habitat, and river.
 To test whether the multivariate position of diets differed by these
 variables, we used a mixed effects PERMANOVA including sex, habitat, and
 river as fixed effects and site as a random effect. Testing for
 differences in multivariate position provides information on the
 composition of the diet items. We used a PERMDISP to test whether
 multivariate dispersion of diets differed across the same three
 covariates. Testing for differences in multivariate dispersion, or spread,
 provides information about how variable the diet items were (Anderson
 2006). Multivariate analysis was conducted using the vegan package
 (Oksanen et al. 2019). We used a univariate approach to
 determine if the proportions either of wood and leaf material or of
 diatoms, two of the most abundant diet groups, differed by length, sex,
 habitat, and river. We constructed linear mixed effects models with either
 proportion of wood and leaf or proportion of diatoms as the response
 variable. We included site as a random effect and each combination of
 length, river, habitat, and sex as fixed effects. We chose not to include
 any interaction terms as we had no <i>a priori</i> reason to
 do so. We constructed a total of 16 models for each response variable,
 ranging from a model including all four fixed effects to a null model
 which included only the random effect. We used Akaike’s Information
 Criterion adjusted for small sample sizes (AICc) for model selection, and
 we selected the models with the lowest AICc scores for each response
 variable as our final models. Linear mixed effects models were fit using
 the lme4 package (Bates et al. 2015), and we used the MuMIn package for
 AICc model selection (Barton 2020). We used two-sided Student’s t tests to
 determine if the percent composition of fatty acids differed by site. All
 statistical analysis was completed in R version 4.0.4 (R Core Team
 2019).