Abstract

BACKGROUND

The steroid hormone ecdysone, acting through its nuclear receptor (EcR), is a key regulator of insect development and reproduction and also contributes to immune regulation and gut microbiota homeostasis. In sand flies, both immunity and the gut microbiota are critical determinants for Leishmania establishment in the midgut. Phlebotomus perniciosus is the principal vector of Leishmania infantum in the western Mediterranean basin; however, the mechanisms affecting interactions among parasite, vector immune system, and resident microbiota remain poorly understood in this species.

METHODS

Using RNA interference-mediated silencing of EcR, we assessed how disruption of ecdysone signaling affects vector immunity, gut bacteria, and parasite development. Double-stranded RNA targeting EcR or a control sequence was microinjected into L. infantum-infected females, and gene expression was analyzed by RT-qPCR. Parasite load was quantified via Leishmania actin expression, while gut bacterial relative abundance was assessed using universal 16S rRNA primers.

RESULTS

L. infantum infection induced expression of the sand fly ecdysone signaling-related genes, immune pathway transcription factors, and antimicrobial peptides (AMPs), while significantly reducing gut bacterial load during the late phase of parasite infection. In contrast, EcR silencing suppressed downstream ecdysone-related genes, impaired activation of the immune deficiency (Imd) and JAK/STAT pathways, and disrupted AMPs expression. This was associated with a marked increase in bacterial relative abundance in sugar-fed sand flies and altered microbial and parasite loads in Leishmania-infected sand flies.

DISCUSSION

Collectively, our results indicate that ecdysone signaling regulates the Imd and JAK/STAT transcription factors, thereby modulating P. perniciosus immune responses and suppressing the gut microbiota. Disruption of ecdysone signaling reveals an inverse correlation between parasite burden and bacterial abundance.

CONCLUSION

These findings identify ecdysone signaling as a central regulator coordinating immune responses and maintaining the balance between Leishmania and the gut microbiota in sand flies.

MeSH

Sand, Genes, Actins, Transcription Factors, Immune System Diseases, Immunity, Parasites, Parasite Load, Bacteria, Gastrointestinal Microbiome, Ecdysone, Microbiota, Infections