# Combining in vivo and in vitro approaches to better understand host‐pathogen nutritional interactions

## TL;DR

This study shows that the outcome of insect-bacteria infections is mainly influenced by nutrition, not the host's immune system, using a combination of lab and real-world experiments.

## Contribution

The first study combining in vitro and in vivo methods to distinguish between top-down and bottom-up effects of host nutrition on host-pathogen interactions.

## Key findings

- Low-protein diets in larvae led to higher mortality and faster bacterial growth when infected.
- In vitro bacterial growth mirrored in vivo results, indicating nutrition, not immunity, drives the interaction.
- Nutrient availability has broader implications for host-pathogen dynamics across species.

## Abstract

Nutrition often shapes the outcome of host–parasite interactions, however understanding the mechanisms by which this occurs is often confounded by the intimate nature of the association and by the fact that the host and parasite may compete for the same limiting nutrients. One way of disentangling this interaction is to combine in vivo and in vitro approaches.Here, we explore the role of host nutrition in determining the outcome of infections using a model insect‐bacterium system: the cotton leafworm Spodoptera littoralis and the blood‐borne bacterium Xenorhabdus nematophila.
Spodoptera littoralis larvae were reared on one of a series of 20 chemically‐defined diets ranging in their protein: carbohydrate (P:C) ratio and caloric density. They were then challenged with either a fixed dose of X. nematophila cells (live or dead) or were sham‐injected. Survivorship of larvae challenged with live bacterial cells was strongly dependent on the protein levels of the diet, with mortality being highest on low‐protein diets. This trend was reflected in the bacterial growth rate in vivo, which peaked in larvae fed low‐protein diets.To determine whether in vivo bacterial growth rates were driven by the direct effects of blood nutrients or by the indirect effects of the host immune response, we used 20 synthetic haemolymphs (‘nutribloods’) that mimicked the nutritional content of host blood. In vitro bacterial growth rate was negatively impacted by the protein content of the nutribloods, replicating the patterns seen in vivo and suggesting that nutrient availability and not host immunity was driving the interaction.By comparing standardized bacterial growth rates in vivo and in vitro, we conclude that the outcome of this host–parasite interaction is largely driven by the ‘bottom‐up’ effects of nutrients on bacterial growth, rather than by the ‘top‐down’ effects of nutrients on host‐mediated immune responses. The outcome of host–parasite interactions is typically assumed to be strongly determined by the host immune response. The direct effects of nutrition have been underexplored and may have broad consequences for host–parasite interactions across taxa.

Nutrition often shapes the outcome of host–parasite interactions, however understanding the mechanisms by which this occurs is often confounded by the intimate nature of the association and by the fact that the host and parasite may compete for the same limiting nutrients. One way of disentangling this interaction is to combine in vivo and in vitro approaches.

Here, we explore the role of host nutrition in determining the outcome of infections using a model insect‐bacterium system: the cotton leafworm Spodoptera littoralis and the blood‐borne bacterium Xenorhabdus nematophila.

Spodoptera littoralis larvae were reared on one of a series of 20 chemically‐defined diets ranging in their protein: carbohydrate (P:C) ratio and caloric density. They were then challenged with either a fixed dose of X. nematophila cells (live or dead) or were sham‐injected. Survivorship of larvae challenged with live bacterial cells was strongly dependent on the protein levels of the diet, with mortality being highest on low‐protein diets. This trend was reflected in the bacterial growth rate in vivo, which peaked in larvae fed low‐protein diets.

To determine whether in vivo bacterial growth rates were driven by the direct effects of blood nutrients or by the indirect effects of the host immune response, we used 20 synthetic haemolymphs (‘nutribloods’) that mimicked the nutritional content of host blood. In vitro bacterial growth rate was negatively impacted by the protein content of the nutribloods, replicating the patterns seen in vivo and suggesting that nutrient availability and not host immunity was driving the interaction.

By comparing standardized bacterial growth rates in vivo and in vitro, we conclude that the outcome of this host–parasite interaction is largely driven by the ‘bottom‐up’ effects of nutrients on bacterial growth, rather than by the ‘top‐down’ effects of nutrients on host‐mediated immune responses. The outcome of host–parasite interactions is typically assumed to be strongly determined by the host immune response. The direct effects of nutrition have been underexplored and may have broad consequences for host–parasite interactions across taxa.

This is one of the first studies that use a combination of in vitro and in vivo approaches to tease apart the importance of the ‘top‐down’ and ‘bottom‐up’ effects of host nutrition in determining the outcome of host‐pathogen interactions.