The conserved role of miR-2 and novel miR-109 in the increase in fecundity of Diaphorina citri induced by symbiotic bacteria and pathogenic fungi.

Infection with pathogens can increase the fecundity and other fitness-related traits of insect vectors for their own advantage. Our previous research has reported the pivotal role of DcKr-h1 in the fecundity improvement of Diaphorina citri induced by the bacterium, "Candidatus Liberibacter asiaticus" (CLas), and the fungus, Cordyceps fumosorosea (Cf). However, the posttranscriptional regulation of this process remains poorly understood. Given the significance of miRNAs in gene regulation, we delved into their roles in shaping phenotypes and their underlying molecular mechanisms. Our results indicated that two miRNAs, miR-2 and novel-miR-109, jointly inhibited DcKr-h1 expression by binding to its 3' untranslated region (UTR). In the D. citri-CLas interaction, the expression levels of miR-2 and novel-miR-109 in the ovaries of CLas-positive psyllids were lower compared to CLas-negative individuals. Overexpression of miR-2 or novel-miR-109 significantly decreased fecundity and CLas titer in ovaries and caused reproductive defects reminiscent of DcKr-h1 knockdown. Similarly, in the D. citri-Cf interaction, the levels of miR-2 and novel-miR-109 markedly decreased in the ovaries. Upregulation of miR-2 or novel-miR-109 also resulted in reduced fecundity and ovary defects similar to those caused by DcKr-h1 silencing. Moreover, feeding antagomir-2 or antagomir-109 partially rescued the defective phenotypes caused by DcKr-h1 silencing in both model systems, and miR-2 and novel-miR-109 were repressed by juvenile hormone (JH) and regulated the genes associated with egg development. This study shows a conserved regulatory mechanism, whereby JH suppresses the expression of miR-2 and novel-miR-109 which, together with JH-induced transcription of DcKr-h1, increases female fecundity induced by both symbiotic bacteria and pathogenic fungi.

Importance: Infection with pathogens can increase the fecundity and other fitness-related traits of insect vectors for their own advantage. Our previous research has reported that DcKr-h1 plays a critical role in the increase in fecundity of Diaphorina citri induced by the bacterium, "Candidatus Liberibacter asiaticus" (CLas) and the fungus, Cordyceps fumosorosea (Cf). However, the posttranscriptional regulation of this process remains poorly understood. Given the significance of miRNAs in gene regulation, we delved into their roles in shaping phenotypes and their underlying molecular mechanisms. Our results indicated that two miRNAs, miR-2 and novel-miR-109, jointly inhibited DcKr-h1 expression by binding to its 3' untranslated region (UTR). In both D. citri-CLas and D. citri-Cf interactions, the increased juvenile hormone (JH) titer and reduced abundance of miR-2 and novel-miR-109 ensure high levels of DcKr-h1 expression, consequently stimulating ovarian development and enhancing fecundity. These observations provide evidence that miR-2 and miR-109 are crucial players in the JH-dependent increase in fecundity in psyllids induced by infection with different pathogens.