Dataset DOI: 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). 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 <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) 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)) 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 > 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.