Analysis of the Bacterial Microbiota in Wild Populations of Prickly Pear Cochineal, Dactylopius opuntiae in Morocco

<i>Dactylopius opuntiae</i> (Cockerell) (Hemiptera: Dactylopiidae), the wild cochineal scale, is a major pest of prickly pear crops worldwide. This study characterized the bacterial community structure of <i>D. opuntiae</i> from four Moroccan regions using targeted PCR and full-length 16S rRNA MinION sequencing. We report the first detection of <i>Wolbachia</i> (16.6% prevalence) in <i>D. opuntiae</i>, with infection rates varying geographically from 0% (Rabat) to 53.3% (Ouazzane). <i>Spiroplasma</i> was detected at a lower prevalence (3.3%) and exclusively in males. Phylogenetic analysis showed that <i>Wolbachia</i> sequences likely belong to supergroup B, based on their similarity to reference sequences, while <i>Spiroplasma</i> sequences were placed within the poulsonii-citri complex. MinION sequencing revealed <i>Candidatus</i> 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 <i>Flavisolibacter</i>, <i>Pseudomonas</i>, <i>Phyllobacterium</i>, <i>Acinetobacter</i>, and <i>Brevibacillus</i>. Beta diversity analysis showed significant geographic variation (PERMANOVA <i>p</i> < 0.008), with distinct communities across regions. Females harbored a more specialized microbiome dominated by <i>Flavisolibacter</i> (except in Agadir), whereas males and nymphs showed <i>Pseudomonas</i> 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.