# Xenopsylla buxtoni fleas as a dominant species harboring multiple infections of Wolbachia lineages in the ancient plague epicenters of Iran

## TL;DR

This study identifies Xenopsylla buxtoni fleas as a dominant species in Iran with high Wolbachia infection rates, which could help control plague outbreaks.

## Contribution

The study reveals high Wolbachia infection rates in X. buxtoni and multiple Wolbachia supergroups, challenging assumptions about flea-symbiont co-evolution.

## Key findings

- Xenopsylla buxtoni was the most abundant flea species with 62%-75% Wolbachia infection rates.
- Wolbachia sequences from the fleas belonged to supergroups A, B, and F.
- Significant genetic divergence was observed between X. buxtoni and X. nuttalli, suggesting possible cryptic species.

## Abstract

Fleas are permissive and euryxenous ectoparasites capable of transmitting numerous ancient and new pathogens among warm-blooded animals, including humans. Precise identification of flea species involved in disease transmission and understanding the highly specialized morphological characteristics associated with their ectoparasitic lifestyle is essential. Likewise, identifying endosymbionts such as Wolbachia—which have long-lasting and intimate relationships with their hosts—will enhance our knowledge of the epidemiology of flea-borne diseases and their control. Flea sampling was conducted in the western half of Iran, where the highest plague outbreaks have been reported over the past two centuries. A total of 1,439 fleas, comprising 623 males and 816 females, were detached from 223 hosts and were identified as Xenopsylla buxtoni, X. nuttalli, X. astia, Pulex irritans, Nosopsyllus iranus iranus, and Ctenophthalmus rettigi smiti. Also, 116 and 73 nucleotide sequences were analyzed to assess the genetic diversity and phylogenetic position of the fleas, and to determine their infection rate and Wolbachia supergroup. Molecular analysis of the COII and ITS2 genes confirmed the morphological distinctiveness of the six species. Xenopsylla buxtoni, the most abundant taxon, displayed Wolbachia infection rates of 62%-75% (x̄ = 69%). The Wolbachia sequences identified from the fleas were assigned to supergroups A, F, and B. The taxonomic position of X. buxtoni and its closely related species, X. nuttalli, in the conformis group was questioned due to significant genetic divergence. The impact of Wolbachia on flea ecology and its potential impact in controlling flea populations and flea-borne pathogens was highlighted.

Fleas are important vectors of zoonotic diseases, including plague caused by Yersinia pestis. We investigated flea diversity and the presence of Wolbachia endosymbionts in western Iran—a region with a long history of plague outbreaks. Among 1,439 fleas collected from 223 hosts, six species were identified, with Xenopsylla buxtoni being the most common and showing high Wolbachia infection rates (62%–75%). Phylogenetic analysis revealed the presence of multiple Wolbachia supergroups (A, B, F), challenging existing assumptions about flea–symbiont co-evolution. We also observed significant genetic divergence between X. buxtoni and the closely related X. nuttalli, suggesting possible cryptic species that could complicate plague surveillance. Our findings highlight the ecological role of Wolbachia in fleas and its potential for use in targeted biocontrol strategies to reduce disease transmission. This study underscores the need to integrate symbiont-focused research into public health initiatives, particularly in plague-endemic areas.