Regulation of soldier caste differentiation by microRNAs in Formosan subterranean termite (Coptotermes formosanus Shiraki).

The soldier caste is one of the most distinguished castes inside the termite colony. The mechanism of soldier caste differentiation has mainly been studied at the transcriptional level, but the function of microRNAs (miRNAs) in soldier caste differentiation is seldom studied. In this study, the workers of Coptotermes formosanus Shiraki were treated with methoprene, a juvenile hormone analog which can induce workers to transform into soldiers. The miRNomes of the methoprene-treated workers and the controls were sequenced. Then, the differentially expressed miRNAs (DEmiRs) were corrected with the differentially expressed genes DEGs to construct the DEmiR-DEG regulatory network. Afterwards, the DEmiR-regulated DEGs were subjected to GO enrichment and KEGG enrichment analysis. A total of 1,324 miRNAs were identified, among which 116 miRNAs were screened as DEmiRs between the methoprene-treated group and the control group. A total of 4,433 DEmiR-DEG pairs were obtained. No GO term was recognized as significant in the cellular component, molecular function, or biological process categories. The KEGG enrichment analysis of the DEmiR-regulated DEGs showed that the ribosome biogenesis in eukaryotes and circadian rhythm-fly pathways were enriched. This study demonstrates that DEmiRs and DEGs form a complex network regulating soldier caste differentiation in termites.

Screening of reference genes for microRNA analysis in the study of solider caste differentiation of Formosan subterranean termite Coptotermes formosanus Shiraki.

The soldier caste differentiation is a complex process that is governed by the transcriptional regulation and post-transcriptional regulation. microRNAs (miRNAs) are noncoding RNAs that control a wide range of activities. However, their roles in solider caste differentiation are barely studied. RT-qPCR is a powerful tool to study the function of genes. A reference gene is required for normalization for the the relative quantification method. However, no reference gene is available for miRNA quantification in the study of solider caste differentiation of Coptotermes formosanus Shiraki. In this research, in order to screen the suitable reference genes for the study of the roles of miRNAs in solider caste differentiation, the expression levels of 8 candidate miRNA genes were quantified in the head and thorax + abdomen during soldier differentiation. The qPCR data were analyzed using geNorm, NormFinder, BestKeeper, ΔCt method and RefFinder. The normalization effect of the reference genes was evaluated using the let-7-3p. Our study showed that novel-m0649-3p was the most stable reference gene, while U6 was the least stable reference gene. Our study has selected the most stable reference gene, and has paved the way for functional analysis of miRNAs in solider caste differentiation.

Methoprene-Induced Genes in Workers of Formosan Subterranean Termites (Coptotermes formosanus Shiraki).

Termites have a distinct polyphenism controlled by concise hormonal and molecular mechanisms. Workers undergo double molts to transform into soldiers (worker-presoldier-soldier). Juvenile hormone analogs, such as methoprene, can induce workers to transform into presoldiers. However, the molecular mechanism underlying the worker-to-presoldier transformation in Coptotermes formosanus Shiraki is still not clear. We sequenced the transcriptome of workers four days after they had fed on methoprene-treated filter paper and control group workers, which fed on acetone-treated filter paper. The transcriptome of C. formosanus was assembled using the de novo assembly method. Expression levels of unigenes in the methoprene-treated group and the control group were compared. The differentially expressed genes were further analyzed by Gene Ontology (GO) term enrichment analysis and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis. Tetrapyrrole binding, oxidoreductase activity, and metal ion binding were the only three enriched GO terms. Juvenile hormone synthesis was the first ranked enriched pathway. Carbohydrate, amino acid, and lipid metabolism pathways were also enriched. These three pathways may be related to fat body development, which is critical for presoldier formation. Our results have demonstrated the significance of JH synthesis pathways, and pathways related to fat body development in the artificial induction of presoldiers.