Abstract:

Objective: As a member of the pine wood nematode disease system, Bursaphelenchus mucronatus has caused serious ecological and economic losses in China due to its efficient and frequent mating behavior. We therefore investigated Bmu-gpa-1 gene of G protein that is related to mating behaviour, and its expression characteristics and its role in mating behavior in B. mucronatus, which would provide a basis for the study of the molecular mechanisms for mating and reproduction in B. mucronatus. Method: Bmu-gpa-1 gene was cloned by PCR based on the genomic data of B.mucronatus. The sequence alignment, phylogenetic analysis and protein structure prediction were conducted to clarify its evolutional relationship with other species. The RT-qPCR and in situ hybridization were used to analyze the gene expression level and expression location in different development stages to reveal its temporal and spatial dynamic expression characteristics. The gene Bmu-gpa-1 was silenced by RNAi to investigate the effects on mating behavior and reproductive capability. In the meanwhile, RT-qPCR was used to check the efficiency of RNAi. Result: The full-length of Bmu-gpa-1 gene is 1 780 bp, including 1 056 bp CDS, encoding 354 amino acids. Phylogenetic analysis indicated that the gene belonged to the same G protein alpha subunit family as gpa-1 in Caenorhabditis elegans. The results of RT-qPCR and transcriptomes showed that Bmu-gpa-1 was expressed in all developmental stages of B. mucronatus, with the expression level highest in the second and third juvenile stages, followed by embryonic stage, and the expression level in the fourth juvenile stage and the adult stage was the lowest. Meanwhile, we found the gene expression level has sexual dimorphism, showing a higher expression level in the males than that in the females. The result of mRNA in situ hybridization showed that Bmu-gpa-1 was expressed broadly in the nematodes. In the embryonic stage, expression location changed dramatically from sporadic dots to whole embryos. In the second juvenile stages, it was expressed in the whole bodies. In the third and fourth juvenile stages, it was mostly located in the intestinal tracts and gonads. In the adult stage, the expression location had sexual dimorphism, it was seen in the vulvas of females, whereas it was only observed in the spicules and tails in males. In the mating test, the males after silencing Bmu-gpa-1 had more frequent and faster mating behavior events than those of controls, but had fewer offspring. However, there were no significant changes in the mating and reproductive behavior of the RNAi-treated females. It is speculated that Bmu-gpa-1 gene can negatively regulate the mating behavior for males, but not for females. Conclusion: Bmu-gpa-1 is a member of the G protein alpha subunit family. It has sexual dimorphism in the expression level and location. Our result indicate the males treated by RNAi have stronger capacity in signal transduction and massive invalid frequent mating behaviors, but not happened in females. It is suggested that Bmu-gpa-1 plays a negative role in the signal transduction of male nematodes and regulates their mating behaviors and fecundity in B. mucronatus.

Key words: Bursaphelenchus mucronatus, Bmu-gpa-1, mating behavior, fecundity, sexual dimorphism