# Transcriptomic Analysis of the Cold Resistance Mechanisms During Overwintering in Apis mellifera

## TL;DR

This study explores how Hunchun bees survive winter by analyzing gene activity changes, revealing mechanisms like osmotic balance and antioxidant production.

## Contribution

The study identifies novel gene families and pathways involved in cold resistance during overwintering in Hunchun bees.

## Key findings

- Hunchun bees maintain osmotic balance and reduce fatty acid metabolism to survive winter.
- Genes encoding antifreeze proteins, HSPs, and other cold-resistant proteins are upregulated during overwintering.
- Shared differentially expressed genes across winter months suggest key roles in stress mitigation and colony stability.

## Abstract

The Hunchun bee is an ecotype of Apis mellifera, which is mainly distributed in Hunchun city, Jilin Province, China. It possesses excellent cold resistance, but the molecular mechanisms of its safe overwintering remain unclear. We analyzed the gene expression levels of Hunchun bees during summer breeding and four overwintering intervals, and we found that they mitigated low-temperature-induced stress during winter by maintaining osmotic pressure balance, reducing fatty acid metabolism, and increasing antioxidant capacity. In addition, their resistance to low temperatures was enhanced through the regulation of genes encoding cold-resistant macromolecular proteins, such as antifreeze proteins (AFPs), C2H2 zinc finger proteins (C2H2-ZFPs), Heat Shock Proteins (HSPs), Serine/Threonine Kinase Proteins (STKs), and leucine-rich repeat-containing proteins (LRRCs). This study provides a novel approach to investigating the molecular mechanism of overwintering in the Western honey bee.

Safe overwintering is a challenging issue in rearing management that is inevitably faced by beekeepers in high-latitude regions. Under the combined influence of multiple factors, the overwintering loss rate of Western honey bees has risen continuously, and investigating the molecular mechanisms related to safe overwintering has become key. The Hunchun bee, an Apis mellifera ecotype in Jilin Province, China, exhibits strong overwintering ability during an overwintering period of more than five months. To investigate the molecular mechanisms of its cold resistance, we conducted a comparative transcriptomic analysis between the summer breeding period (July) and different overwintering intervals (November, December, January, and February), and then systematically identified key genes and signaling pathways related to cold resistance. The results showed that the highest number of differentially expressed genes (DEGs) was found between December and July. Compared with July, the upregulated genes in Hunchun bee in December were significantly enriched in several pathways, such as ion transport and neuroactive ligand–receptor interactions, and the downregulated genes were significantly enriched in pathways related to fatty acid metabolism, glutathione metabolism, and the peroxisome. Notably, a total of 378 shared DEGs were obtained from the four comparison groups, and several candidate cold-resistant gene families, such as AFPs, HSPs, C2H2-ZFPs, STKs, and LRRCs, were identified among the shared DEGs of the winter season. Additionally, 749 shared DEGs related to protein modification and metabolic process regulation were identified between the four successive overwintering intervals. Four shared genes, including sensory neuron membrane protein 1 (SNMP1), were revealed by pairwise comparison of the four intervals. The above results collectively indicate that the Hunchun bee attenuates winter-induced stress responses during the overwintering process by maintaining osmotic pressure balance, reducing fatty acid metabolism, increasing antioxidant capacity, and synthesizing cold-resistant macromolecular proteins. It was also found that chemical signal perception may serve a role in maintaining the stability of the overwintering bee colony. The key genes and pathways related to cold resistance identified in this study not only provide a basis for explaining the overwintering molecular mechanism for Apis mellifera of Hunchun bee but also offer key data to improve overwintering management strategies for Western honey bees.