# The Honeybee Associate Galleria mellonella Can Acquire Arsenophonus apicola Through Oral and Parenteral Infection Routes

## TL;DR

This study shows that the bacterium Arsenophonus apicola can infect wax moths through both oral and injection methods, potentially impacting hive health and offering new research opportunities.

## Contribution

The study demonstrates that Arsenophonus apicola can infect Galleria mellonella through multiple routes, expanding its known host range and suggesting new research directions.

## Key findings

- Arsenophonus apicola can infect Galleria mellonella larvae via injection at low doses.
- Oral infection with A. apicola impacts waxworm development and causes mortality.
- A. apicola crosses from gut to hemocoel in G. mellonella, inducing melanisation.

## Abstract

Members of the genus Arsenophonus are classically considered to be vertically transmitted endosymbiotic associates of invertebrates. Acquisition of Arsenophonus apicola by
Apis mellifera
honeybees through social and environmental pathways raises the possibility that this species can infect a broader range of host species. In this study, we tested whether a natural inhabitant of bee hives, the wax moth Galleria mellonella, was a suitable host for A. apicola. We first demonstrated A. apicola colonised G. mellonella larvae following injection at doses as low as 104 CFU. A similar capacity of A. apicola to infect G. mellonella orally was evidenced, impacting waxworm development and mortality. Microscopy indicated that A. apicola crossed from gut to hemocoel in the G. mellonella crop, inducing melanisation. PCR screening of Galleria individuals in an apiary sample confirmed exposure of Galleria in the hive context. We conclude that A. apicola is capable of infecting and damaging hive associates. These findings raise two onward avenues of research: first, to investigate whether A. apicola's presence could protect hives against Galleria infestations, and second, to utilise model insect G. mellonella for immunity research to uncover the interplay between A. apicola and insect host defences whilst elucidating virulence factors utilised by A. apicola during infection.

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Galleria mellonella larvae were exposed to Arsenophonus apicola orally and via injection, alongside negative control treatments. The exposed larvae were confirmed to be infected by monitoring GFP expression and exhibited increased mortality and melanisation, alongside delayed pupation.