When parasitizing host caterpillars, parasitoids like Cotesia glomerata not only lay their eggs but also inject symbiotic venom and polydnavirus (CgPDV) that enhance offspring survival by suppressing host immune responses. Parasitism also induces changes in caterpillars that are exploited by hyperparasitoids to locate parasitoid larvae, which serve as hosts for their offspring. However, it remains unclear whether these changes in the caterpillar are driven by the injection of CgPDV, venom, or both.
Here, we investigated how CgPDV and venom influence the attractiveness of Pieris brassicae caterpillars to the true hyperparasitoid Baryscapus galactopus. Using a series of choice and non-choice assays, we quantified hyperparasitoid responses to caterpillars injected with CgPDV, and / or venom, compared these with responses to unparasitized caterpillars injected with phosphate-buffered saline (PBS) and naturally parasitized caterpillars.
We found that B. galactopus preferred caterpillars injected with CgPDV alone or in combination with venom compared with caterpillars injected with PBS. However, the injection of venom alone did not increase the attractiveness of caterpillars to hyperparasitoids. Naturally parasitized caterpillars elicited stronger attraction than any injection treatment, indicating that factors beyond polydnavirus and venom contribute to hyperparasitoid attraction. In contrast, neither CgPDV nor venom affected close-range host-foraging behaviour of hyperparasitoids, including time until first contact or duration of contact with caterpillars.
These findings demonstrate that parasitoid-associated polydnavirus contribute to parasitism-induced changes in caterpillar phenotype that are exploited by a hyperparasitoid, and venom only contributes to this when combined with polydnavirus. Additional parasitism-related cues are required to fully explain attraction to naturally parasitized hosts. This study provides evidence that an obligate symbiont can mediate multitrophic interactions by indirectly increasing host vulnerability to higher trophic levels.

Dryad DOI: 10.5061/dryad.79cnp5j9s

Dataset overview

This dataset contains the data and analysis code used to generate Figures 2–6 and Supplementary Figure S1 of the associated article. It includes replicate-level data from two-chamber olfactometer choice assays and individual-level data from non-choice behavioural assays. R Markdown scripts are included for the analyses and plots presented in Figures 2–6 and Figure S1.

The study tested responses of the true hyperparasitoid Baryscapus galactopus to fifth-instar Pieris brassicae caterpillars subjected to treatments involving venom and the Cotesia glomerata polydnavirus (CgPDV), compared with PBS-injected negative controls and naturally parasitized positive controls.

Two experimental approaches were used:

Two-choice olfactometer assays to test the attraction of hyperparasitoids to caterpillars receiving different treatments.
Non-choice behavioural assays to quantify the close-range host-foraging behaviour of hyperparasitoids.

Study system

Herbivore host: Pieris brassicae (Lepidoptera: Pieridae)
Primary parasitoid: Cotesia glomerata (Hymenoptera: Braconidae)
Hyperparasitoid: Baryscapus galactopus (Hymenoptera: Eulophidae)

Treatment abbreviations

NC: Negative control; unparasitized caterpillars injected with phosphate-buffered saline (PBS).
VEN: Unparasitized caterpillars injected with C. glomerata venom.
VIR: Unparasitized caterpillars injected with calyx fluid containing CgPDV particles.
VV: Unparasitized caterpillars injected with both C. glomerata venom and calyx fluid containing CgPDV particles.
PC: Positive control; caterpillars naturally parasitized by C. glomerata and subsequently injected with PBS.

Files and variables

Choice-assay data: Figures 2–4

The following ten CSV files contain the full factorial set of pairwise comparisons among the five treatments:

File	Comparison	Figure
`NC_VEN.csv`	NC versus VEN	Figure 2
`NC_VIR.csv`	NC versus VIR	Figure 2
`NC_VV.csv`	NC versus VV	Figure 2
`NC_PC.csv`	NC versus PC	Figure 2
`VEN_PC.csv`	VEN versus PC	Figure 3
`VIR_PC.csv`	VIR versus PC	Figure 3
`VV_PC.csv`	VV versus PC	Figure 3
`VEN_VIR.csv`	VEN versus VIR	Figure 4
`VEN_VV.csv`	VEN versus VV	Figure 4
`VIR_VV.csv`	VIR versus VV	Figure 4

Variables in the ten pairwise choice-assay files

Variable	Definition
`Treatment`	Treatment represented by the row: NC, VEN, VIR, VV, or PC.
`Group`	Replicate identifier within that pairwise assay.
`Choice`	Number of B. galactopus females choosing the treatment represented by the row.
`NoChoice`	Number of females making no choice in the replicate.
`Total`	Total number of females released in the replicate (five).
`Ratio`	`Choice / Total`.
`RatioNoResp`	`NoChoice / Total`.

Global choice-assay data: Figure 5

FULL_BT_SHEET_FIG123.csv: All ten pairwise choice assays combined in wide format for the global Bradley–Terry analysis.

Variables in `FULL_BT_SHEET_FIG123.csv`

Variable	Definition
`Comb`	Identifier for the treatment pairing.
`Group`	Replicate identifier within that pairwise assay.
`Treatment_left`	Treatment presented in the left chamber.
`Treatment_right`	Treatment presented in the right chamber.
`Choice_left`	Number of females choosing the left chamber.
`NoChoice`	Number of females making no choice.
`Choice_right`	Number of females choosing the right chamber.

Non-choice assay: Figure 6 and Figure S1

NoChoice_Data_Combined.csv: Individual-level behavioural observations from the non-choice assays.

Variables in `NoChoice_Data_Combined.csv`

Variable	Definition	Unit
`Treatment`	Caterpillar treatment: NC, VEN, VIR, VV, or PC.	Categorical
`Contact`	Time from the start of observation until the female first contacted the caterpillar.	Seconds
`TotalTime`	Total time the female spent on the caterpillar.	Seconds
`TotalOviNum`	Total number of oviposition attempts.	Count
`TotalOviPrepT`	Total time spent preparing for oviposition, including antennal drumming.	Seconds
`OviPrepTRatio`	Proportion of combined oviposition-related time allocated to oviposition preparation.	Proportion
`TotalOviT`	Total time spent in oviposition behaviour.	Seconds
`OviTRatio`	Proportion of combined oviposition-related time allocated to oviposition.	Proportion
`AveOviPrepT`	Mean duration of an oviposition-preparation event.	Seconds
`AveOviT`	Mean duration of an oviposition attempt.	Seconds

Figure 1 illustrates the experimental setups and has no underlying numerical dataset.

Analysis code

Liu_EcolEnto_2026_Fig_2_NC.Rmd: R Markdown script for the analysis and visualization of two-choice olfactometer data used to generate Figure 2.
Liu_EcolEnto_2026_Fig_3_PC.Rmd: R Markdown script for the analysis and visualization of two-choice olfactometer data used to generate Figure 3.
Liu_EcolEnto_2026_Fig_4_sideways.Rmd: R Markdown script for the analysis and visualization of two-choice olfactometer data used to generate Figure 4.
Liu_EcolEnto_2026_Fig_5_BT.R: R script for the global ranking analysis with a Bradley–Terry model used to generate Figure 5.
Liu_EcolEnto_2026_Fig_6_Fig_S1_NoChoice.Rmd: R Markdown script that performs the Kruskal–Wallis tests and creates the plots for Figure 6 and Figure S1.

Statistical analyses and reproducibility

All analyses were conducted in R version 4.4.3.

Choice-assay data were analysed using binomial generalized linear models (GLMs) with a logit link, specifying the response as cbind(Choice, Total - Choice).

A global Bradley–Terry model incorporating all pairwise comparisons was used to obtain the overall treatment ranking in the two-chamber olfactometer choice assays.

Non-choice behavioural data were analysed using separate Kruskal–Wallis rank-sum tests for each behavioural response.

Figures were generated using the ggplot2 package, with individual data points displayed using ggbeeswarm.

Running the provided R and R Markdown scripts reproduces the statistical analyses and figures reported in the manuscript.

File formats and missing data

All data files are comma-delimited CSV files. Missing values in the non-choice dataset are represented by NA. Treatment labels are case-sensitive. No confidential, personal, or sensitive information is contained in the dataset.

Reuse notes

Users should consult the associated article for full experimental details, including insect rearing, the preparation and microinjection of venom and CgPDV treatments, behavioural procedures, and interpretation of the results. Please cite both the published article and the Dryad dataset when reusing these data.

Contact

Questions about the data or analyses should be directed to the corresponding author named in the published article.

Data and code from: Maternal factors from a parasitoid wasp mediate hyperparasitoid attraction to parasitized caterpillars

Data files

Abstract

Dataset overview

Study system

Treatment abbreviations

Files and variables

Choice-assay data: Figures 2–4

Variables in the ten pairwise choice-assay files

Global choice-assay data: Figure 5

Variables in `FULL_BT_SHEET_FIG123.csv`

Non-choice assay: Figure 6 and Figure S1

Variables in `NoChoice_Data_Combined.csv`

Analysis code

Statistical analyses and reproducibility

File formats and missing data

Reuse notes

Contact

Data and code from: Maternal factors from a parasitoid wasp mediate hyperparasitoid attraction to parasitized caterpillars

Data files

Abstract

README: Data and code from: Maternal factors from a parasitoid wasp mediate hyperparasitoid attraction to parasitized caterpillars

Dataset overview

Study system

Treatment abbreviations

Files and variables

Choice-assay data: Figures 2–4

Variables in the ten pairwise choice-assay files

Global choice-assay data: Figure 5

Variables in FULL_BT_SHEET_FIG123.csv

Non-choice assay: Figure 6 and Figure S1

Variables in NoChoice_Data_Combined.csv

Analysis code

Statistical analyses and reproducibility

File formats and missing data

Reuse notes

Contact

Variables in `FULL_BT_SHEET_FIG123.csv`

Variables in `NoChoice_Data_Combined.csv`