AUTHOR(S): Matthew Boot & Kyle Sozanski

DATE: 5/15/2026

[Access this dataset on Dryad] (https://doi.org/10.5061/dryad.zs7h44jpg )

EXPERIMENTAL CONTEXT

This data repository contains data and codebase from the publication: Boot et al., 2026. Guest-ant social parasites avoid conflict with social hosts using venom signaling. Several datasets and analyses are provided, broken down between 1) behavioral data/analyses and 2) venom data/analyses.

Background & motivation:

As context-dependent mutualists, the two focal species generally cohabit peacefully within shared nests for many years. However, conflict can also occur in these mixed interspecific nests and has been observed at certain points in the host colonies’ lifecycle. Parasite venom is thought to play a role in host colony infiltration and integration as it is toxic and can have communicative properties. Sericomyrmex amabilis hosts lack venom and are stingless but have strong mandibles which they use when in conflict. Megalomyrmex symmetochus social parasites have alkaloid-based venom stings, and these parasites have been observed using conspicuous, indirect venom display behavior—which could play a role in maintaining nest cohesion, even outside of overt conflict. The provided data and code were used to investigate parasite venom use, and if it is used in signaling during host-parasite behavioral conflict. In addition, we also characterized the venom expenditure necessary for parasites to neutralize hosts.

Behavioral interactions:

To analyze host-parasite conflict, we observed staged conflict between naive S. amabilis hosts and M. symmetochus social parasites (5 hosts to a single social parasite) at the group level. When host ants have not lived with a parasite, their initial behaviors are aggressive. By staging these altercations, we are able to induce interspecific conflict. Conflict-related host/parasite behaviors were scored and recorded during video playback of arena trials, along with certain interaction metadata (e.g., video ID, ant colony ID, etc.) for each behavioral observation. Using a generalized linear mixed modeling framework to account for multiple observations at the colony and/or video level, we used these observations to assess: a) a comparison of direct and indirect venom use by parasites, b) the sequence of associated conflict behaviors between hosts and parasites, c) the relationship between parasite venom use and host behavior, d) escalation of aggression between hosts and parasites, e) parasite behavior following host biting, f) parasite venom use relative to conflict escalation, g) host submission following venom use, and h) interaction termination.

Through our analyses, host ants were found to be the primary aggressors, while parasites tended to avoid conflict and conflict escalation. Parasites were observed to dispense alkaloid venom directly through stinging, but more frequently, indirectly via airborne venom dispersal behavior earlier in interactions. We further demonstrated that indirect venom use can alter interaction outcomes if hosts switch to non-aggressive behavioral tactics. Specifically, parasites themselves tended to terminate interactions in which venom was used.

All data for the behavioral aspect of our investigation is contained in the "Behavior" folder. The data is completely contained in a single, large data set, and the analyses are presented in a single code document. Analyses within the behavior code-base are presented in sections dedicated to each major finding/figure presented in the PLOS One publication. Behavioral observations in this dataset are encoded, with associated interaction metadata, line by line. Each row represents a unique record based on a single observed behavior, and includes interaction metadata associated with that observation. Behaviors were categorized for the host as aggressive or submissive. Parasite venom behaviors included venom behavior. Irrelevant behaviors were classified in the "other" category. Cells without recorded data in a given line are not applicable to the current line record, and thus, do not represent missing data. Further information is provided in a dedicated Behavior README document inside the "Behavior" folder.

Venom economy:

To characterize the costs of parasite venom use within the context of interactions, we also assessed limitations to parasite venom usage in terms of 1) parasite anatomical venom storage, and 2) host neutralization capacity. We then used these concepts to define a "venom economy" between hosts and parasites in conflict.

To establish venom storage capacity (in “number of stings”), we calculated the relative ratio in size between the individual droplets discharged by cold-anesthetized parasites directly from their sting, and the total venom liquid stored in the venom sac. To assess the number of hosts a single parasite can neutralize using venom directly (stinging), we performed a combination of mortality assays using both whole venom sting and synthetic venom proxy (stereoisomeric mix of 3-butyl-5-hexylpyrrolizidine). We used the synthetic venom proxy to calibrate a lethal dose-response curve for hosts, which we used as a baseline model against which to compare mortality from whole-venom single stings.

Through our analyses, we showed the costs of parasite venom-use (via stinging) in terms of overall parasite venom reserves (per parasite). Note, however, that we assume that parasite venom supply is not immediately replenishable, that the rate of production is slow relative to the timescale over which host-parasite interactions occur, and that host mortality is not immediate. Additionally note that ‘sting’, in this analysis, refers to the droplet size that is produced by parasites under anesthetized conditions; however, parasite individuals may have finer control over their venom delivery when using venom under normal conditions.

Analyses in the venom codebase are provided in a single code document, but the venom measurement data are split between several datasheets. Data structures within each datasheet vary, but are explained fully in a separate, dedicated venom README document, located inside the "Venom" folder.

Data Navigation

Within the main folder, Boot_et_al_2026_Dryad.zip, the data and code are split between 2 secondary folders (Behavior, Venom), representing the two primary analysis types presented in the associated publication.

Cells without recorded data in a given line are not applicable to the current line record, and thus, do not represent missing data.

FOLDER STRUCTURE:

1) Behavior

2) Venom

*Each folder separately contains both the data and code necessary to reproduce all analyses used in the paper.

The Behavior folder contains:

1 data file (MRB2024_BehaviorData_Submission2.xlsx),

1 R-markdown code document (MRB_SignalingMS_Analysis_Subm2_Behavior.Rmd),

and 1 README file (1_BEHAVIOR_README.ods) explaining the datafile.

The Venom folder contains:

4 data files (KSS_Msymmetochus_venomsize.csv, Venom_sting_summary.xlsx, KSS+MRB_Msymmetochus_Samabilis_StingMort.xlsx, MRB_LD50data.xlsx)

1 R-markdown code document (MRB_SignalingMS_Analysis_Subm2_Venom.Rmd),

and 1 README file (1_VENOM_README.ods) explaining its datafiles.

README DOCS & DATA ENUMERATION:

The README docs within the Behavior and Venom folders are spreadsheets that explain what data is included in each data file, enumerate datatypes (columns), and define the values used in each column. Please refer to the respective READMEs for specific details regarding the datasets in each folder.

CODE:

The codebase uses R-markdown notation to split the code into multiple sections. The code is intended to be viewed in outline form using an IDE such as RStudio (or other similar software) that can parse R-markdown type script since each code document has multiple sections, and the Behavioral analysis in particular is quite long.

In general, each code document has several main sections (in caps), and multiple subsections. For example:

SECTIONS:

LOAD PACKAGES

BEHAVIORAL ANALYSIS
PRIMARY ANALYSES - SET-UP & DESCRIPTIVE ANALYSIS

1) Behavioral summary

2) Behavioral overview

3) ...

Each numbered section contains a set of analyses (incl. sub-analyses) which pertains to a named method/results passage in Boot et. al, 2026. Manuscript figure outputs are demarcated in the detailed outline (R-markdown) above the code chunk which will produce plot outputs used in those figures.

Each code document contains further instructions that pertain to the specifics of that document. In the case that further instructions are provided in the document, line numbers are provided in the document notes header.