DOI: 10.5061/dryad.w6m905qs1
title: Effects of mycotoxin treatment on fly survival, development time, thorax length, fecundity, and longevity in four mycophagous Drosophila species

description: Many mycophagous Drosophila species have adapted to tolerate high
 concentrations of mycotoxins, an ability not reported in any other
 eukaryotes. Although an association between mycophagy and mycotoxin
 tolerance has been established in many Drosophila species, the genetic
 mechanisms of the tolerance are unknown. This study presents the inter-
 and intraspecific variation in the mycotoxin tolerance trait. We studied
 the mycotoxin tolerance in four Drosophila species from four separate
 clades within the immigrans-tripunctata radiation from two distinct
 locations. The effect of mycotoxin treatment on 20 isofemale lines per
 species was studied using seven gross phenotypes: survival to pupation,
 survival to eclosion, development time to pupation and eclosion, thorax
 length, fecundity, and longevity. We observed interspecific variation
 among four species, with D. falleni being the most tolerant, followed by
 D. recens, D. neotestacea, and D. tripunctata, in that order. The results
 also revealed geographical variation and intraspecific genetic variation
 in mycotoxin tolerance. This report provides the foundation for further
 delineating the genetic mechanisms of the mycotoxin tolerance trait.
description: Fly isofemale lines Four species were included
 in this study: D. falleni, D. recens, D. neotestacea, and D. tripunctata.
 Adult flies were collected by net sweeping on fermented banana baits,
 tomato baits, and mushroom baits over the summer months of 2017-2019 from
 two distant locations: Great Smoky Mountain National Park near Gatlinburg,
 TN (hereafter referred to as GSM) and Little Bay de Noc in Escanaba in The
 Upper Peninsula of Michigan (hereafter referred to as ESC). These two
 sites are approximately 1400 km apart. Multiple sites were used for fly
 collection within each location spanning over 3-5 square kilometers. The
 species and the sex of the captured flies were identified, and isofemale
 lines were set up by adding one wild-caught female with one wild-caught
 male from the same species and location and collecting their progeny
 (David 2005). The established isofemale lines were maintained on a diet of
 Carolina Biological Formula 4- 24 Instant Drosophila Medium supplemented
 with finely ground, freeze-dried Agaricus bisporus mushrooms (Oregon
 mushrooms, OR) at a ratio of 33.28:1 w/w, and a dental roll was added to
 the food vial as a pupation site. The standard conditions for maintenance
 and experiments were 22°C and a 14 h:10 h (L:D) photoperiod at 60%
 humidity. The authors note here that the isofemale lines were maintained
 in the laboratory for at least over a year before the experiments were
 conducted. 2.2 Mycotoxin tolerance assays
 Basic food was prepared by mixing 28.3 g freeze-dried A. bisporus
 mushrooms (Oregon mushrooms, Oregon) with 941.9 g Carolina 4-24 Instant
 Drosophila Medium and grinding them together into a fine powder. For
 mycotoxin tolerance assays, clean glass vials were filled with 250 mg of
 basic food. The natural-toxin mix was provided by Dr. Clare Scott-Chialvo
 (Scott Chialvo et al. 2020), which contained methanol as eluent. To
 account for this methanol, one mL of 0.56% methanol solution was added to
 the control vials containing 250 mg of basic food. The mycotoxin vials
 were prepared by adding 1 mL of the natural-toxin mix (100 µg/mL of known
 amatoxins) to the vials containing 250 mg of basic food. Both control and
 mycotoxin vials were weighed and subjected to vacuum evaporation for 96
 hours at room temperature to remove methanol from the vials. The loss in
 weight (in grams) in the vials was replenished with the appropriate amount
 (in mL) of sterile distilled water. The optimal duration of vacuum
 evaporation was identified using preliminary studies and 96 hours of
 vacuum evaporation showed survivorship that was comparable to vials
 without methanol. Water agar plates were prepared using
 15 g Bacto Agar (Sigma Aldrich) in 500 mL of distilled water and adding
 Tegosept to a final concentration of 0.1% and poured into 30 mm
 Petri-plates. These plates snugly fit the plastic bottles that were used
 to make egg- laying chambers. Tiny holes were punched into these plastic
 bottles for aeration. Equal amounts of dry yeast and freeze-dried mushroom
 powder were mixed together with autoclaved distilled water to prepare a
 paste (prepared fresh daily). A drop of this paste was applied to the
 water agar plate. Recently eclosed males and females of each isofemale
 line were transferred to egg-lay chambers and allowed to oviposit at 22°C
 and a 14 h:10 h (L:D) photoperiod at 60% humidity. The next day, the
 plates were replaced with fresh plates, and the water agar plates with
 oviposited eggs were allowed to hatch at 22°C and a 14 h:10 h (L:D)
 photoperiod at 60% humidity. The hatched first-instar larvae were used for
 the experiments. Pilot studies were performed to identify the optimal
 larval density for each species. As a result, fifteen first-instar larvae
 were added to each vial in the case of D. falleni, D. recens, and D.
 tripunctata, whereas 20 first-instar larvae were added to each vial for D.
 neotestacea. The experiments were conducted in triplicates. Each
 experiment was conducted on consecutive days to generate three replicates
 for each of the ten isofemale lines/location/species for two treatments
 (control and mycotoxin). 2.2.1 Development time, thorax
 length measurements, and survival The vials were
 checked daily to record the time to pupation, survival to pupation, time
 to eclosion, and survival to eclosion. The eclosed flies were collected
 within 24 hours by light CO 2 anesthesia, sexed, and placed laterally to
 measure the thorax length. The thorax’s anterior margin length to the
 scutellum&#39;s posterior tip was measured and recorded as the
 thorax length. The thorax length of the eclosed flies was measured to the
 nearest 0.025 mm with an Olympus SZX16 dissection microscope fitted with
 an Olympus DP72 camera, using the ImageScan software (Hasson et al. 1992).
 The eclosed females were used for the fecundity assays, and the eclosed
 males were used for the longevity assays. The experiments were terminated
 after ensuring that no new flies had emerged for four consecutive
 days. 2.2.2 Fecundity assays Only
 female flies were used for the fecundity assays. Female flies eclosed from
 the mycotoxin tolerance assay vials were labeled appropriately and
 maintained individually in food vials for three days as virgins. They were
 then transferred individually into a fresh food vial with three 3-day-old
 virgin males from the laboratory stocks of the same isofemale line. These
 parent flies were transferred to a new vial every three days. After 15
 days, the adult flies were removed. The offspring of the females that
 survived the full 15 days were counted to provide an estimate of
 fecundity. We note that this assay cannot be used to evaluate egg-to-adult
 survival (Dyer and Hall 2019). 2.2.3 Longevity
 assays Only male flies were used for the longevity
 study. Male flies eclosed from the mycotoxin tolerance assay vials were
 maintained individually in tiny 5-mL glass vials containing approximately
 250 mg of the basic food used to create the mycotoxin tolerance assay
 vials. The vials were checked every alternate day to record any dead
 flies, and the remaining flies were transferred to fresh food vials every
 2-3 days. 2.3 Statistical Analyses
 All statistical analyses were done using R version 3.6.1 (https://www.r-
 project.org/foundation/) and R Studio version 2021.09.2+382 (https://www.rstudio.com/). We used the linear-mixed model (LMM) and the generalized linear-mixed effects model (GLMM), implemented in R package &#39;lme4&#39; (Bates et al. 2015), to determine the independent variables that can explain the variation in survival, development time, body size, fecundity, and longevity. We modeled pupal and survival to eclosion using a binomial linear-mixed model with the logistic link function. We used the linear-mixed model to model development time, fecundity, longevity, and thorax lengths. The development time and thorax lengths were analyzed for each sex separately. To detect whether mycotoxin tolerance shows interspecific variation, we first fitted a GLMM that includes the main effects, the two-way interactions, and the three-way interaction of the species, the treatment, and the location as the fixed effects. The likelihood test (LRT) was used to test if the three interaction was significant. The final model includes the main effects and the two-way interactions of the species, the treatment, and the three-way interaction only if the three-way interaction is significant (in other words, the p-value from the LRT for the three-way interaction is less than 0.05). In all models, the isofemale lines and the replicate vials were included as the random effects. To check the sufficiency of the model, the scatter plots of the deviance residuals against the predicted values were generated, and the dispersion parameter was estimated based on the ratio of the sum of squared deviance residuals and the degrees of freedom of the model if the binomial linear-mixed model was used. To evaluate the effect of toxin treatment, we fitted either a binomial linear-mixed or linear-mixed model to assess whether the treatment affects the seven gross phenotypes. In all models, the main effect of the treatment was the only fixed effect, and the isofemale lines and the replicate vials were included as the random effects. The analysis was conducted for each of four species and seven gross phenotypes separately. Among seven gross phenotypes, the development time of eclosion and the thorax length of eclosion were analyzed for the males and the females separately. Therefore, 36 p-values were obtained. To account for the multiple testing adjustment, both the p-value adjusted using the Bonferroni correction and the false discovery rate (FDR) calculated with the Benjamini-Hochberg method (Benjamini and Hochberg, 1995) were presented. The FDR &lt; 0.05 was used as a cut-off for significant results. To identify the extent of tolerance for each isofemale line, the binomial linear-mixed model was performed on each isofemale line with the replicate vial as a random effect, and the lines were segregated based on their p-values. High-tolerance lines were identified as those in which no significant difference in survival between the control and the mycotoxin treatments was observed or where the survival was significantly higher in the mycotoxin treatment. Isofemale lines with significantly low survivorship on the mycotoxin treatment (p-value &lt; 0.05) were categorized as low-tolerance lines. For the scope of this study, high tolerance is defined as the ability of an isofemale line to survive in the presence of the natural-toxin mix (100 µg/mL of known amatoxins). For intraspecific variation, before model fitting, we pruned the data to exclude isofemale lines where only one data point was observed per treatment. This exclusion allowed us to eliminate data that could not estimate variation within an isofemale line. We assessed whether the main effects: a) isofemale line, b) treatment (presence or absence of mycotoxin), c) location, and d) interactions between the main effects affect the seven gross phenotypes (survival to pupation, survival to eclosion, development time to pupation and eclosion, thorax length, fecundity, and longevity) in each species. The isofemale lines and the replicate vials were included in the analysis as random effects.
description: R version 3.6.1 (https://www.r-project.org/foundation/)
 R Studio version 2021.09.2+382
 (https://www.rstudio.com/). lme4 (Bates et al.
 2015)