The research team, led by Khajid Ullah Khan and Muhammad Tariq Zahid of Government College University, Lahore, together with colleagues in Pakistan and South Korea, collected 126 scorpions, 85 females and 41 males, from rocky field sites during June and July, the peak of the animals’ annual activity window. Specimens were located by day by overturning stones and by night using ultraviolet light, which causes scorpion cuticles to fluoresce. Morphological examination under a stereomicroscope documented the diagnostic features of the species, including its dark brown coloration, densely granulated carapace keels, a triangular sternum characteristic of the family Buthidae, and a bulbous reddish telson bearing a curved, dark-tipped aculeus. Females were consistently larger and thicker-bodied than males, with mean total lengths of roughly 72.8 millimeters compared with 62.9 millimeters in males, and males carried significantly more pectinal teeth, averaging about 30 to 32 per comb versus 26 in females.

To confirm species identity beyond morphology, the researchers amplified and Sanger-sequenced approximately 650 base pairs of the mitochondrial cytochrome c oxidase subunit I gene, the standard animal DNA barcode. BLAST comparison against the NCBI GenBank database returned nucleotide identities of 96.95 and 96.81 percent with existing H. tamulus sequences, and maximum-likelihood phylogenetic analysis in MEGA11 clustered the new sequences firmly within the species. The roughly three percent divergence from the nearest reference strain, which originated from a commercial insect market in China with undocumented provenance, suggests notable geographic structuring within the species, a pattern consistent with the deep intraspecific lineages documented in other scorpion genera such as Scorpio in the Levant and Alpiscorpius in the European Alps.

The centerpiece of the study was its metataxonomic analysis of the gut contents of six adult scorpions, three males and three females, dissected aseptically after surface sterilization. Genomic DNA was extracted with a bead-beating kit and sequenced on an Oxford Nanopore MinION flow cell using primers targeting the full V1 through V9 hypervariable regions of the 16S rRNA gene. The run produced 355,264 high-quality reads, averaging about 71,000 per sample with a mean length of approximately 1,390 nucleotides, effectively covering the entire gene. Because nanopore sequencing carries higher raw error rates than short-read platforms, the team clustered sequences de novo into operational taxonomic units at a deliberately relaxed 85 percent identity threshold using VSEARCH, then classified them against the SILVA reference database within the QIIME 2 pipeline, removing putative contaminants flagged in negative controls.

The results reveal a gut community dominated overwhelmingly by two bacterial phyla. Firmicutes accounted for between 79.5 and 90.4 percent of sequences across individuals, followed by Proteobacteria at 9.5 to 20.5 percent, while Actinobacteriota and Bacteroidota were barely detectable. At the class level, Bacilli made up roughly 73 percent of the community, with Gammaproteobacteria and Mollicutes contributing about 13 and 7 percent respectively. Four genera stood out as the core microbiota: Bacillus, Mycoplasma, Pantoea, and Enterobacter together comprised approximately 84 percent of all recovered 16S sequences. Bacillus alone represented 17 to 30 percent of the gut community, echoing recent findings in the black scorpion Heterometrus longimanus, where the same genus also dominates. The authors suggest Bacillus may function as a key endosymbiont, supporting antioxidant defenses, gut barrier integrity, and the synthesis of vitamins and amino acids for the host.

Perhaps the most evolutionarily intriguing member of the community is Mycoplasma, a genus of wall-less bacteria that has repeatedly surfaced in scorpion microbiome surveys. Previous work by Bolaños and colleagues documented Mycoplasma lineages in 15 scorpion morphospecies across Mexico, with cophylogenetic analysis suggesting cospeciation between the bacteria and their hosts. The new Pakistani data reinforce the idea that Mycoplasma is an indigenous, long-term associate of scorpions rather than a transient dietary contaminant, a conclusion paralleled by reports of Mycoplasma comprising up to 40 percent of the respiratory microbiota in some snake families. In the present study, Mycoplasma was roughly twice as abundant in females, averaging 10.5 percent relative abundance, as in males at 5.3 percent.

Sex emerged as a consistent theme throughout the diversity analyses. Alpha diversity metrics, including observed species, Chao1, Shannon, and Simpson indices, showed that female scorpions harbored markedly richer and more evenly distributed bacterial communities, whereas male guts were dominated by a smaller set of taxa. Two intestinal fermenters, Enterococcus and Romboutsia, were significantly enriched in females. Both genera are associated with carbohydrate and bile acid metabolism in nutrient-rich gut environments, and the authors speculate that the reproductive energy demands of females may create a more favorable niche for these bacteria, potentially influencing host energy harvest from food. Comparable sex-dependent microbiome patterns have been reported in ticks, where males and females of species such as Amblyomma americanum carry distinct communities, and in the wolf spider Pardosa astrigera, where seasonal and physiological state modulate sex-specific diversity.

To move beyond taxonomy, the researchers applied PICRUSt2, a bioinformatic tool that infers metagenome functions from 16S profiles by matching marker genes against reference genomes. The analysis predicted 23 enriched MetaCyc metabolic pathways spanning energy metabolism, amino acid and lipid biosynthesis, nucleotide metabolism, and the biodegradation of xenobiotic compounds. Male samples appeared enriched for energy metabolism and biosynthetic routes, while female samples showed greater representation of lipid metabolism and fatty acid biosynthesis pathways, hints that the sex-specific microbial differences may extend into function. The authors are careful to stress, however, that PICRUSt2 output is indicative rather than definitive: it does not measure actual gene expression, and the predictions require validation through shotgun metagenomics or transcriptomics, particularly given the study’s small sample of six individuals.

Situated in a broader ecological context, the findings portray scorpions as hosting a moderately diverse microbiome that sits above the extremely depauperate communities of social spiders, which average a Shannon index of just 0.66, yet below the hundreds to thousands of operational taxonomic units recoverable from soil or lepidopteran eggs. The overlap between the H. tamulus core genera and those documented in wolf spiders, crab spiders, and moths suggests a partially conserved gut microbial blueprint across predatory arthropods. The authors also raise an untested but plausible transmission hypothesis: because scorpions exhibit extensive brood care, including mothers provisioning enzymatically treated prey to offspring, maternal transfer could explain how these symbionts persist across generations, a mechanism previously documented in brown widow spiders.

As a first baseline for a medically iconic species in a previously unsampled region of Pakistan, the study opens concrete research avenues. Bacillus and Mycoplasma emerge as prime candidates for integrated follow-up work combining metagenomics, transcriptomics, and analysis of the venom apparatus microbiome, where Proteobacteria have been shown in other species to exceed half of the bacterial load and may contribute to nutrient cycling within the telson. Whether the gut community actively underpins H. tamulus adaptation to arid, resource-limited habitats, as microbiome-mediated stress tolerance studies in spiders and flies suggest it might in other arthropods, remains a compelling but unproven hypothesis. The authors are explicit that their six-individual sample cannot define a species-wide core microbiome; larger geographic surveys and controlled experiments will be needed before the hidden microbial partners of the world’s deadliest scorpion can be fully credited with any of its remarkable ecological success.

Subject of Research: Gut bacterial microbiome diversity and functional potential of the Indian red scorpion Hottentotta tamulus in Pakistan

Article Title: Gut Microbiome Diversity, Functional Potential, and Ecological Relevance of Hottentotta tamulus

Article References: Khan, K. U., Zahid, M. T., Mustafa, G., Tanpure, R. S., Tahir, H. M., Kumar, R., Kim, D.-W., Park, H.-K., & Jeon, B.-H. (2026). Gut Microbiome Diversity, Functional Potential, and Ecological Relevance of Hottentotta tamulus. MicrobiologyOpen, 15(5), Article e70373. https://doi.org/10.1002/mbo3.70373

Image Credits: AI Generated

DOI: 10.1002/mbo3.70373

Keywords: Hottentotta tamulus, Indian red scorpion, gut microbiome, 16S rRNA sequencing, Oxford Nanopore, Firmicutes, Bacillus, Mycoplasma, PICRUSt2, sexual dimorphism, scorpion venom, Pakistan