Differences between uncapping and removal behaviors in Apis cerana from the perspective of long non-coding RNAs

Abstract

Background: Hygienic behavior, a specialized form of immune response evolved in social insects, plays a crucial role in safeguarding colonies from disease spread. In honeybee colonies, such behavior typically entails the dual steps of uncapping and removal of unhealthy and deceased brood. Although in recent years, numerous studies have examined the development of hygienic behavior, the mechanisms underlying the division in the performance of uncapping and removal have yet to be sufficiently elucidated. In this regard, long non-coding RNAs (lncRNAs) have been evidenced to be engaged in regulating the physiological activities of honeybees; however, whether lncRNAs are likewise involved in the uncapping and removal tasks has not been clarified.

Results: In this study, the strong hygienic Apis cerana worker bees were used and the processes of uncapping and removal behaviors in three colonies were assayed with freeze-killed brood in the field. We then sequenced the antennal RNAs of honeybees to identify differentially expressed lncRNAs and performed lncRNA-mRNA association analysis to establish the differences between uncapping and removal. We detected 1,323 differentially expressed lncRNAs in the antennae, and the findings of lncRNA-mRNA association analyses revealed that the target genes of differentially expressed lncRNAs between uncapping and removal worker bees were predominantly linked to response to stimulus, receptor activity, and synapse. Notably, among the lncRNAs enriched in cellular response to stimulus, XR_001766094.2 was exclusively expressed in the uncapping worker bees. Based on these findings, we hypothesize that XR_001766094.2 plays a key role in distinguishing uncapping from removal behaviors by responding to external stimulus, thereby suggesting that the division of hygienic behaviors is governed by differential thresholds of responsiveness to environmental cues.

Conclusion: We characterized differences in the uncapping and removal behaviors of worker bees from a perspective of lncRNAs. Uncapping bees may be equipped with a more rapid stimulatory response and more acute olfactory sensitivity, contributing to the rapid hygienic behavior in honeybee colonies. Our results thus establish a foundation for potential lncRNA-mediated gene expression regulation in hygienic behavior.

Keywords: Apis cerana; Antenna; Honeybee; Hygienic behavior; Removal; Uncapping; lncRNA.

Fig. 1

The process of uncapping and subsequent removal of freeze-killed broods over time in three different Apis cerana colonies. Kaplan-Meier plots are shown for cell uncapping and brood removal in colonies C01 (a), C02 (b), and C03 (c). The percentage values indicate the proportion of uncapping and removal behaviors in each of the three colonies. Throughout the 72-h observation period, the increase in the proportions of uncapping and removal behaviors is represented by line segments connecting the time points at 3, 6, 24, 27, 30, 48, 51, 54, and 72 h. Log-rank P: the P value obtained from the log-rank test

Fig. 2

The number of differentially expressed transcripts between honeybee workers performing uncapping (u) and removal (r) behaviors in the three Apis. cerana colonies C01, C02, and C03, respectively

Fig. 3

Venn diagram showing commonly differentially expressed lncRNAs between honeybees performing uncapping (u) and removal (r) behaviors. Thirty-eight lncRNAs were differentially expressed in all the three comparison groups (C01-u vs. C01-r, C02-u vs. C02-r, and C03-u vs. C03-r)

Fig. 4

GO enrichment analysis of differentially expressed lncRNAs (DElncRNAs). Enrichment is shown for genes regulated by DElncRNAs in C01-u vs. C01-r, C02-u vs. C02-r, and C03-u vs. C03-r comparisons via antisense regulatory mechanisms

Fig. 5

KEGG enrichment analysis of differentially expressed lncRNAs (DElncRNAs). Enrichment is shown for genes regulated by DElncRNAs via antisense regulatory mechanisms in C01-u vs. C01-r, C02-u vs. C02-r, and C03-u vs. C03-r comparisons

Fig. 6

GO enrichment analysis of differentially expressed lncRNAs (DElncRNAs). Enrichment is shown for genes regulated by DElncRNAs via cis regulatory mechanisms in C01-u vs. C01-r, C02-u vs. C02-r, and C03-u vs. C03-r comparisons

Fig. 7

GO enrichment analysis of target genes regulated by differentially expressed lncRNAs (DElncRNAs) based on colony intersection. Enrichment is shown for DElncRNAs-regulated genes via cis regulatory mechanisms for intersections among C01-u vs. C01-r, C02-u vs. C02-r, and C03-u vs. C03-r

Fig. 8

Top 20 KEGG terms from enrichment analysis of target genes regulated by differentially expressed lncRNAs (DElncRNAs). Enrichment is shown for DElncRNAs-regulated genes via cis regulatory mechanisms for the intersections among C01-u vs. C01-r, C02-u vs. C02-r, and C03-u vs. C03-r