Analysis of the Bacterial Microbiota in Wild Populations of Prickly Pear Cochineal, <em>Dactylopius opuntiae</em> in Morocco

Dactylopius opuntiae (Cockerell) (Hemiptera: Dactylopiidae), the wild cochineal scale, is a major pest of prickly pear crops worldwide. This study characterized the bacterial community structure of D. opuntiae from four Moroccan regions using targeted PCR and full-length 16S rRNA MinION sequencing. We report the first detection of Wolbachia (16.6% prevalence) in D. opuntiae, with infection rates varying geographically from 0% (Rabat) to 53.3% (Ouazzane). Spiroplasma was detected at a lower prevalence (3.3%) and exclusively in males. Phylogenetic analysis placed the Wolbachia strains in supergroup B and Spiroplasma in the poulsonii-citri complex. MinION sequencing revealed Candidatus Dactylopiibacterium as the dominant taxon (97.7%), consistent with its role as an obligate symbiont. After removing this dominant species, we uncovered a diverse bacterial community, including Flavisolibacter, Pseudomonas, Phyllobacterium, Acinetobacter, and Brevibacillus. Beta diversity analysis showed significant geographic variation (PERMANOVA p&lt;0.008), with distinct communities across regions. Females harbored a more specialized microbiome dominated by Flavisolibacter (except in Agadir), whereas males and nymphs showed Pseudomonas dominance. Core microbiome analysis revealed no universal genera across all groups, with females displaying a more restricted core than males and nymphs did. The detection of reproductive symbionts, combined with geographic and sex-specific microbiome patterns, provides insights into the development of microbiome-based pest management strategies. The complementary use of targeted and untargeted sequencing methods is essential for comprehensive microbiome characterization in this economically important pest.