# Potential impact of marine heatwaves on the survival and transcriptomic signature of free-living third-stage larvae (L3) of Anisakis simplex (Nematoda: Anisakidae)

## TL;DR

This study examines how extreme heat affects the survival and gene activity of Anisakis simplex larvae, showing that high temperatures reduce their viability and alter key biological processes.

## Contribution

The study reveals novel transcriptomic responses of Anisakis simplex larvae to marine heatwaves, linking thermal stress to survival and developmental suppression.

## Key findings

- Larvae survived 60 days at 15°C but only a week at 28°C, indicating severe viability loss under heatwave conditions.
- Transcriptomic analysis showed downregulation of cuticle-related genes and upregulation of peptidases and glycogen metabolism genes under heat stress.
- Neural modulation and neurotransmission-related genes were upregulated, suggesting prioritization of survival processes over development.

## Abstract

This experimental study aims investigates the gene expression plasticity of free-living infective third-stage larvae (L3) of Anisakis simplex in response to extreme thermal stress, potentially linked to the increasing occurrence of climate change-induced marine heatwaves.

Using an RNA-seq approach, the molecular transcriptomic responses of L3 larvae were analyzed under both normal (15 °C) and extreme (28 °C) heating aquarium conditions environmental stress. Data were collected from90 viable L3 in good condition, sampled from viscera of fresh European hake (Merluccius merluccius), and subjected to a seawater survival assay simulating the most extreme ocean warming event currently recorded whitin the Spanish Marine Demarcation. A bioinformatic pipeline involving read alignment (STAR), differential expression (DESeq2), and gene ontology enrichment was applied.

The analysis revealed that larvae remained viable for up to 60 days at 15 °C but only a week at 28 °C. Transcriptomic data showed from 241 to 244 DEGs depending on each comparison, with a progressive downregulation of cuticle-related genes, a strong upregulation of peptidases, and coordinated neural modulation; with reduced ion channel activity and enhanced expression of key neurotransmission-related genes. Additionally, there was strong upregulation of genes involved in glycogen degradation and galactose metabolism, along with consistent downregulation of glycolysis-related genes.

The transcriptomic patterns describe above indicate that short-term marine heatwave conditions (28 °C) severely compromise viability of free L3 of A. simplex, which clearly prioritize survival processes while suppressing developmental pathways. These findings suggest that the more intense, longer-lasting, and widespread marine heatwaves observed along the Spanish marine demarcation over the last 15 years may influence parasite persistence and transmission of infective stages to paratenic fish host. This highlights the One Health significance of such adaptative response, particularly regarding parasite biogeographical dispersion and associated zoonotic risk.