DOI: 10.5061/dryad.2fqz6132k
title: Cost of parasite exposure depends on host ontogeny

description: Fear comes at a cost, and exposure to parasites can elicit non-consumptive
 effects (NCEs in hosts that come at such a cost. While NCEs are well
 documented, the influence of host developmental stage on these effects
 remains poorly understood. Using Drosophila nigrospiracula and its
 ectoparasite Macrocheles subbadius, we tested whether early-life exposure
 (egg and pupal stages) to mites altered host development and survival.
 First, we exposed eggs to direct and indirect mite cues to assess effects
 on larval emergence. We then conducted two follow-up experiments to
 evaluate microbial transfer from mites and feeding behaviour. Next, we
 exposed pupae at two distinct developmental stages and recorded both
 emergence success and mite longevity. Mites were highly effective at
 consuming fly eggs, but pupae were resistant to direct parasitism. No NCEs
 were observed from indirect egg exposure; however, host developmental
 state significantly influences pupal NCEs. Less developed pupae had
 reduced emergence when exposed to mites, compared to more developed pupae
 that had detached from the puparium wall. These results indicate that the
 cost of parasite exposure is developmentally dependent, and that
 metamorphosis plays a critical role in shaping host susceptibility to
 non-consumptive parasite effects.
description: # Cost of parasite exposure depends on host ontogeny Dataset DOI:
 [10.5061/dryad.2fqz6132k](https://doi.org/10.5061/dryad.2fqz6132k) ##
 Description of the data and file structure Fear comes at a cost, and
 exposure to parasites can elicit costly non-consumptive effects (NCEs) to
 occur in hosts. While NCEs are well documented, the influence of host
 developmental stage on NCEs remains poorly understood. Using *Drosophila
 nigrospiracula* and its ectoparasite *Macrocheles subbadius*, we tested
 whether early-life exposure (egg and pupal stages) to mites altered host
 development and survival. We exposed eggs to direct and indirect mite cues
 to assess effects on larval emergence. Followed by two experiments to
 evaluate microbial transfer from mites and feeding behaviour. Next, we
 exposed pupae at two distinct developmental stages and recorded both
 emergence success and mite longevity. Mites were highly effective at
 consuming fly eggs, but pupae were resistant to direct parasitism. No NCEs
 were observed from indirect egg exposure. However, host developmental
 state significantly influences pupal NCEs, where less developed pupae had
 reduced emergence when exposed to mites, compared to more developed pupae
 that had detached from the puparium wall. These results indicate that the
 cost of parasite exposure is developmentally dependent, and that
 metamorphosis plays a critical role in shaping host susceptibility to
 non-consumptive parasite effects. ### Files and variables This data set
 contains measurements of fly development success, as well as mite
 longevity and consumptive abilities. There are six data files in total:
 HatchSuccess.csv, HatchSuccessW.csv, Consumption.csv, MiteLongevity.csv,
 PupaeCollection.csv., and FlyWeight.csv. For the egg experiment data files
 “HatchSuccess.csv and HatchSuccessW.csv” each cell represents a single
 measurement out of 100 eggs. However, for the “Consumption.csv” data file
 each cell represents a single measurement out of 25 eggs. For the mite
 longevity and emergence experiment data file “MiteLongevity.csv”, each
 cell represents a single measurement for a single mite. However, for the
 data files “FlyWeight.csv and FlyEmergence.csv”, each cell represents a
 single measurement for a single pupa. #### File: Egg_Exposure.R
 Description: Script for all the egg experiment analysis.  #### File:
 HatchSuccess.csv Description: Egg hatching success was measured by
 transferring the larval medium to a sterile plastic container with a 20%
 sucrose solution for ease of collection. Larvae were collected using fine
 tipped forceps and categorized into the three larval instar stages based
 on size, anterior spiracle morphology (clubbed [L2] vs. branched [L3]),
 and presence of mouth hooks (short [L1] vs. long [L2/L3]) [(Poças,
 Domingos, and Mirth
 2020)](https://www.zotero.org/google-docs/?broken=DjVBW4). Larval
 development was measured as the number of larvae that reached each instar
 stage out of the number of total larvae that successfully hatched in that
 petri dish. In total, there were 2 089 larvae collected from eggs not
 exposed to mites (n = 30), 1 905 from eggs indirectly exposed to mites (n
 = 30), and 266 from eggs directly exposed to mites (n = 30); out of a
 possible 3 000 seeded eggs for each treatment group. Overall,
 approximately 85% of larvae were third instar by day 6, only about 12%
 were second instar, and less than 3% only made it to the first instar.
 ##### Variables * Block: Grouping of replicates completed together *
 Treatment: Designates groups of 100 eggs that had either “Direct mite”
 exposure with free-roaming mites, “Caged mite” exposure with mites in a
 connected dish, or “No mites”. * L1: First instar * L2: Second instar *
 L3: Third instar * Total: Sum count of the three larval instar stages ####
 File: HatchSuccessW.csv Description: Each group of 100 eggs were placed on
 media in a 6 cm petri dish and left to develop as larvae for 6 days. The
 developed larvae were then collected and categorized into the 3 larval
 instar stages. Egg success was measured following the same protocol as the
 first egg exposure experiment. In total, there were 414 larvae collected
 from eggs exposed to mite-wash (n = 10), and 372 larvae from eggs exposed
 to distilled water (n = 10); out of a possible 1,000 seeded eggs for each
 treatment group. On average, more than 60% of larvae were third instar by
 day 6, less than 40% were second instar, and less than 1% made it to the
 first instar. ##### Variables * Block: Grouping of replicates completed
 together * Treatment: Designates groups of 100 eggs that had either 1mL of
 “No mites” in distilled water, or 1mL of distilled water that had been
 soaked with “Mites” for 48 hours * L1: First instar * L2: Second instar *
 L3: Third instar * Total: sum count of the three larval instar stages ####
 File: Consumption.csv Description: The fly eggs were left for 24 hours and
 then observed for deformities, discolouration, and trachea development.
 Each column: “Deformed”, “Discolouration”, and “Trachea.Present”, is the
 number of eggs out of 25 with the associated trait. In total, there were 7
 deformed, 2 discoloured, and 96 eggs with trachea development from the the
 eggs directly exposed to mites (n = 4), 74 deformed, 36 discoloured, and
 53 eggs with trachea development from the eggs not exposed to mites (n =
 4). ##### Variables * Treatment: Designates groups of 25 eggs that had
 either been directly exposed to free-roaming “Mites” or “No mites”. ####
 File: Pupae_Experiment2.R Description: Script for all the mite-pupae
 analysis.  #### File: PupaeCollection.csv Description: All exposed pupae
 were retained from the mite arenas and observed for fly emergence. The
 number of pupae for each sample mite was dependent on the longevity of the
 mite. Therefore, “Emergence” was measured as the proportion of pupae that
 successfully emerged as adults out of the total possible pupae for that
 sample. Pupae from exposure vials were retained in petri dishes and
 monitored daily for successful emergence of adult flies (7 days
 post-pharate and 9 days pre-pharate; no emergence was observed after day
 5). In total, there were 67 pre-pharate pupae retained (*n*= 23), of which
 40 emerged as adults, and 116 post-pharate pupae retained (*n= 23*), with
 93 emerging as adults. ##### Variables * Sample: identifier for each
 individual mite arena, which would produce multiple pupae.  * Block:
 grouping of when replicates were completed together  *
 Treatment: designates whether the pupa was from the pre- (“Young”) or
 post-pharate (“Pharate”) treatment group * Pupae: total number of pupae
 collected from that specific sample * Emergence: total number of
 successful emergences from possible pupae for that sample * Proportion:
 the proportion of successful adult emergences out of possible pupae for
 that sample.  #### File: MiteLongevity.csv Description: Mites were
 individually placed in small vial arenas and were offered various
 resources. Mite longevity was observed by checking mites daily for
 response to stimulus (light/touch) as well as curled legs. If no response
 occurred and legs were curled, the mites were removed from the vial and
 sexed. Mites were sexed based on colouration (females are darker than
 males), the ventral plate morphology (shape, continuity), as well as leg
 thickness and morphology (Özbek, Bal, and Doğan 2015). Overall, there were
 4 treatment groups which included: no food source (negative control, *n=
 19*), an immobilized adult fly (positive control, *n= 22*), a pre-pharate
 pupae (*n*= 23), or post-pharate pupae (*n= 23*). ##### Variables *
 Sample: Identifier for each individual mite arena, which would produce
 multiple pupae. * Block: Grouping of when replicates were completed
 together  * Treatment: Designates mites that were provisioned with a pupa
 in early development (“Young”), pupa in late development that is
 considered “Pharate”, an immobilized “Fly”, or “Water”. * Days Alive:
 Number of days a mite was alive since they were placed in the vial arena *
 Sex: Mite sex * Status: Designated if the mite was alive “1” or dead “2”.
 #### File: FlyWeight.csv Description: Successfully emerged adult flies
 from the petri dishes were frozen and weighed to the nearest 0.01mg
 (XP105DR, Mettler Toledo, Columbus). In total, 35 adults from pre-pharate
 pupae (*n*= 23), and 84 adults from post-pharate pupae (*n= 23*) were
 weighed. ##### Variables * Sample: Designates the specific treatment arena
 each fly was from, as each sample had a varied number of pupae * Block:
 Denotes the batch of flies weighed on each measurement day * Treatment:
 Designates whether the pupa were from the “Pre-Pharate” or “Post-Pharate”
 treatment group * Pupa: identifier for individual pupae that were retained
 from each arena * Weight: Fly weight in mg's * Sex: Flies sexed
 as either female or male ## Code/software #### File: Egg_Exposure.R
 *(Hatch Success)* A generalized linear model (GLM) with a negative
 binomial distribution was used to analyze egg hatching success, using the
 glm.nb() function from the MASS package in R (Venables and Ripley, 2002).
 The dependent variable was the number of larvae that emerged in each
 replicate dish, and exposure treatment was the independent variable. A
 negative binomial distribution was selected to account for overdispersion
 in the count data, which violated the assumptions of the Poisson
 distribution (dispersion ratio = 6.73, p-value &lt;0.001). Akaike’s
 Information Criterion (AIC; Akaike, 1974) values further supported the use
 of a negative binomial model (∆AIC= 371.54). Further model selection was
 also computed using the AIC() function. Post hoc pairwise comparisons
 between treatment groups were then conducted using estimated marginal
 means with the emmeans package (Lenth, 2025). We used a
 Dirichlet-multinomial regression model to evaluate the distribution of
 larvae collected from the sucrose solution. The brm() function from the
 brms package [(Bürkner 2021)](https://www.zotero.org/google-docs/?S9Ogae)
 was used to jointly model the three instar stages against the total number
 of successful larvae within a treatment group (trial(Total)). Four Markov
 chains were run with 4000 iterations each (including a 1000 warm-up), and
 the adapt_delta parameter set to 0.95 to ensure proper convergence. The
 Pareto-smoothed importance sampling Leave-One-Out cross-validation (LOO;
 [(Vehtari et al. 2017)](https://www.zotero.org/google-docs/?gOap5B)) was
 used to estimate the expected log predictive density (ELPD) for each
 model. Odds ratios (OR) and 95% credible intervals (CrI) were obtained
 from a binomial model comparing third instar larvae to the pooled first
 and second instars, representing the relative probability of larvae
 reaching the third instar stage across treatments. *(Hatch SuccessW)* A
 glm.nb() function was used to analyze egg hatching success in the
 mite-wash experiment. The dependent variable was the number of larvae that
 emerged in each replicate dish, and exposure treatment was the independent
 variable. A negative binomial distribution was selected to account for
 overdispersion in the count data, which violated the assumptions of the
 Poisson distribution (dispersion ratio = 2.20, p-value = 0.002). We used a
 Dirichlet-multinomial regression model to evaluate the distribution of
 larvae collected from the sucrose solution. The brm() function was used to
 jointly model the three instar stages against the total number of
 successful larvae within a treatment group (trial(Total)). Four Markov
 chains were run with 4000 iterations each (including a 1000 warm-up), and
 the adapt_delta parameter set to 0.95 to ensure proper convergence. The
 Pareto-smoothed importance sampling LOO was used to estimate the expected
 log predictive density (ELPD) for each model. OR and CrIs were obtained
 using a binomial model. *(Consumption)* A glm() function with Poisson
 distribution was considered and checked for overdispersion, which was not
 found (dispersion ratio = 1.18, p-value = 0.31; dispersion ratio = 1.26,
 p-value = 0.27; dispersion ratio = 0.44, p-value = 0.85); further model
 selection was conducted using the AIC() function.  #### File:
 Pupae_Experiment2.R *(Mite Longevity)* The survival package (Therneau,
 2022) was used for the following analysis. A Cox-Proportional hazards
 regression model was fitted using the coxph() function to assess the
 effects of resource type and mite sex on mite survival (number of days
 alive). Model selection was performed using AIC values. The proportional
 hazards assumption was evaluated using Schoenfeld residuals (cox.zph), and
 no violations were detected (Treatment: *p* = 0.79; Sex: *p* = 0.38;
 Global: *p* = 0.81). Pairwise post-hoc contrasts were computed from the
 Cox proportional hazards model using the emmeans package. P-values were
 adjusted for multiple testing using the Benjamini-Hochberg (BH) method.
 Hazard ratios (HR) and 95% confidence intervals (CI) were obtained by
 exponentiating the model estimates. HRs &gt; 1 indicate higher hazard
 (lower survival) relative to the fly reference group. 
 *(PupaeCollection)* A generalized linear model (GLM) with binomial
 distribution was used to evaluate pupation success (number of successfully
 emerged adults out of total pupae). This model predicted the successes and
 failures (cbind(Emergence, Pupae – Emergence)) as a function of the pupal
 development stage. The dispersion ratio of 0.87 indicated adequate model
 fit. Model selection was then performed using AIC(). *(FlyWeight)* Emerged
 adult fly weight was analyzed using a linear model (LM) with treatment
 (pupation development stage) and fly sex as fixed effects. Model
 assumptions were checked by inspecting diagnostic plots and conducting a
 Shapiro-Wilk test for normality of residuals. Model selection was
 completed by checking AIC values.