# Development of Antisense Tools to Study Bodo saltans and Its Intracellular Symbiont

## TL;DR

This study shows that antisense inhibition cannot be used to study gene functions in the symbiotic relationship between Bodo saltans and its bacterial partner.

## Contribution

The paper demonstrates the infeasibility of antisense inhibition in this aquatic symbiosis and provides alternative protocols for study.

## Key findings

- Antisense inhibition is not feasible in Bodo saltans and its symbiont due to the holobiont feeding on the antisense molecules.
- The study developed protocols to investigate the biology of Bodo saltans and its microbial associates.
- The symbiosis increases in numbers when treated with antisense constructs, making the method ineffective.

## Abstract

Obligate symbioses are common in nature and present a particular challenge for functional genetic analysis. In many cases, the host is a non‐model species with poor tools for genetic manipulation, and the symbiont cannot be cultured or its gene expression manipulated to investigate function. Here, we investigated the potential for using antisense inhibition to analyze host and symbiont gene function within an obligate aquatic symbiosis. We focused on the kinetoplastid host Bodo saltans and its bacterial symbiont, Candidatus Bodocaedibacter vickermanii, a member of Rickettsiales. We conclude that antisense inhibition is not feasible in the Bodo saltans and its symbiont, as the holobiont feeds on the antisense molecules—and increases in numbers—upon treatment with the antisense construct. Although our approach has proven unsuccessful, we have developed an array of protocols that can be used to study the biology of this microeukaryote and its microbial associates.

Obligate symbioses present challenges for functional genetic analysis. This study shows that antisense inhibition cannot be used to investigate gene functions in the aquatic symbiosis between Bodo saltans and its bacterial symbiont. Our work stresses the importance of the development of alternative protocols to study the biology of both the microeukaryote and the associated bacterium.