Analysis of the Bacterial Microbiota in Wild Populations of Prickly Pear Cochineal, Dactylopius opuntiae 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 showed that Wolbachia sequences likely belong to supergroup B, based on their similarity to reference sequences, while Spiroplasma sequences were placed within 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 < 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. The detection of reproductive symbionts, combined with geographic and sex-specific microbiome patterns, provides valuable insights into the potential roles of these bacteria in host adaptation and their implications for 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.