# Multi-Omic Analysis Identifies Key Genes Driving Testicular Fusion in Spodoptera litura

## TL;DR

This study uses multi-omics to identify genes involved in testicular fusion in the pest Spodoptera litura, revealing the role of a previously unknown gene, Sl3030.

## Contribution

The first multi-omics analysis of testicular fusion in Spodoptera litura, identifying Sl3030 as a key gene in the process.

## Key findings

- Five genes, including Sl3030, were identified as potentially involved in testicular fusion in Spodoptera litura.
- Knocking out Sl3030 disrupted testicular fusion and caused abnormal sperm development and thickened membranes.
- Sl3030 affects testicular fusion by influencing microtubule and cytoskeleton assembly.

## Abstract

The Spodoptera litura, a Lepidopteran pest known for its high fecundity, undergoes a complete metamorphosis, including a distinctive process during which the male testes fuse from two separate organs into a single entity, significantly enhancing its fertility. To elucidate the molecular mechanisms underlying this testicular fusion, this study employed an integrated multi-omics approach to investigate concurrent changes at the transcriptomic and proteomic levels. We identified a series of synchronized alterations on the peritestic larval membrane, including heme binding, peptidase activity, hydrolase activity, metal ion transport, redox reactions, and chitin metabolism, all of which are substantially enriched at specific temporal points during testicular fusion. Nine genes/proteins co-expressed at the mRNA and protein levels were selected for targeted quantitative proteomics (PRM) and quantitative PCR (qPCR) validation, leading to the identification of five genes potentially involved in the testicular fusion process: Sl3030, ARCP, PSLRE, Obstructor-E, and Osris9B. Notably, the gene Sl3030, once knocked out, not only disrupted the normal fusion process but also resulted in reduced testis size, thickened peritestic membranes, and abnormal sperm development. Transcriptomic sequencing of the Sl3030 knockout mutant revealed its primary influence on the fusion process by affecting the assembly of the microtubule system and cytoskeleton. This research, for the first time, provides a multi-omics perspective on the response of key signaling pathways and molecular changes during the testicular fusion of S. litura and validates the role of the previously uncharacterized gene Sl3030 in this process, offering valuable insights into the complex mechanisms of testicular fusion in this species.