Leishmaniasis is a parasitic disease transmitted by the bite of infected sand flies. In Thailand, the species responsible belong to the subgenus Mundinia, including Leishmania martiniquensis and Leishmania orientalis, both of which have been implicated in human infections. Yet despite growing recognition of the disease as a local problem, the ecology of its vectors remains poorly understood, particularly in the cave-associated environments of the north where many species shelter and breed. Even less is known about the endosymbionts that live inside these insects and that, in other vector systems, have been shown to manipulate reproduction or interfere with pathogen development.

To close that gap, a team led by Puckavadee Somwang of Mae Fah Luang University and Kanok Preativatanyou of Chulalongkorn University collected female sand flies from two tourist caves and one livestock-associated site in Chiang Rai Province. The haul amounted to 298 specimens representing eight taxa across three genera: Phlebotomus, Sergentomyia, and Idiophlebotomus. Notably, the species composition differed markedly among the three collection sites, a pattern the authors interpret as habitat-associated structuring of sand fly communities. Caves, with their stable humidity and abundant vertebrate hosts, and livestock shelters, with their ready supply of blood meals, evidently support distinct assemblages of these insects.

The molecular screening was thorough. Each fly was tested for Leishmania DNA using quantitative PCR assays targeting the genus-level 18S rRNA gene together with species-specific markers, and positive results were complemented by ITS1-PCR coupled with nanopore sequencing, a portable sequencing technology that allows rapid reading of the parasite’s internal transcribed spacer region. The results were striking: Leishmania DNA was detected in 78 of the 298 flies, an infection rate of 26.2 percent. Both L. martiniquensis and L. orientalis were identified, and in four specimens the two species were detected simultaneously, a co-detection that underscores how multiple parasite species can circulate within the same vector populations.

The endosymbiont picture was even more dramatic. Wolbachia, the world’s most widespread intracellular bacterium in arthropods, was present in 228 flies, or 76.5 percent of the sample. Of these, 124 carried Wolbachia alone while 104 were co-infected with Cardinium, a second endosymbiont best known from mites, spiders, and various insects. Cardinium-only infections, by contrast, were rare, accounting for just 1.3 percent of specimens. This asymmetry suggests that Wolbachia dominates the endosymbiotic landscape of these sand flies, with Cardinium typically riding along as a co-infectant rather than establishing itself independently.

Whether these bacteria actually affect Leishmania presence was the study’s central question. Using Firth’s penalized logistic regression, a statistical method suited to sparse or imbalanced categorical data, the researchers tested the association between endosymbiont status and Leishmania DNA detection. Across all endosymbiont categories the overall association fell short of statistical significance, with a penalized likelihood ratio test yielding P = 0.083. But when the analysis zoomed in on specific categories, a telling pattern emerged: sand flies carrying Wolbachia alone had significantly lower odds of harboring Leishmania DNA than endosymbiont-free flies, with an odds ratio of 0.49 and a 95 percent confidence interval of 0.25 to 0.97, corresponding to P = 0.041. In other words, Wolbachia-positive flies were roughly half as likely to test positive for the parasite.

The authors are careful about interpretation. A lower odds of parasite DNA detection does not prove that Wolbachia blocks Leishmania; it could reflect differences in sand fly species composition, sampling variation, or the biology of particular host-endosymbiont combinations. Still, the finding echoes results from other vector systems, where Wolbachia has been deployed deliberately to reduce the transmission of dengue and other pathogens. If a similar interference operates in sand flies, the implications for leishmaniasis control in Thailand and beyond could be substantial, opening the door to endosymbiont-based strategies that reduce vector competence without relying on insecticides.

Beyond the association analysis, the study delivered a genetic inventory that is itself a first for Thailand. Haplotype-based analyses of the Wolbachia surface protein gene, wsp, revealed 16 distinct haplotypes, while sequencing of the Cardinium 16S rRNA gene uncovered 14 haplotypes. Phylogenetic reconstruction placed the Wolbachia strains within Supergroups A and B, the two lineages most commonly found in insects, and sorted the Cardinium sequences into Groups A and C. Neither the breadth of Wolbachia diversity nor the Cardinium lineages had been documented in Thai sand flies before, making this the first report of their genetic diversity in these vectors from the country.

Perhaps the most technically intriguing result came from structural analysis of the Wolbachia surface protein, or WSP, a protein embedded in the bacterium’s outer membrane and exposed to the host cell environment. One Wolbachia variant recovered from the sand fly species Sergentomyia barraudi carried a nine-amino-acid insertion, with the motif KDSTKQVTD, located within the HVR4 hypervariable region of the protein. Three-dimensional structural modeling predicted that this insertion produces localized conformational variation while leaving the conserved beta-barrel core of the protein intact. Crucially, the insertion sits within a surface-exposed loop region, precisely the kind of structural feature that can mediate interactions between the symbiont and its host. A closely related reference sequence, PQ606075, carries a nearly identical motif differing by a single substitution from arginine to lysine, suggesting this insertion is not a one-off anomaly but a recurring variant in the region’s Wolbachia populations.

Why does a surface protein insertion matter? WSP is a prime candidate for host-symbiont recognition, and variation in its exposed loops has been linked to differences in cytoplasmic incompatibility, the reproductive manipulation that allows Wolbachia to spread through insect populations. If certain WSP variants confer different interaction profiles with sand fly tissues, they might also differ in how they affect Leishmania development inside the insect. The study’s structural evidence therefore provides a concrete molecular handle for future work: researchers can now ask whether flies carrying the HVR4-insertion variant show different parasite loads than those with canonical WSP sequences.

The research also carries practical value for disease surveillance. Because sand flies pick up Leishmania from infected hosts, screening them offers a form of xenomonitoring, a way to track parasite circulation in an area without directly sampling every human or animal resident. The high infection rate recorded in Chiang Rai, more than one in four flies, signals active parasite transmission in these environments and justifies closer surveillance in cave and livestock settings frequented by both local communities and tourists. The finding of two Mundinia species, including co-detections, further complicates the epidemiological picture, since different species can cause distinct clinical forms and may involve different reservoir hosts.

Funded by Thailand’s National Science, Research and Innovation Fund through Mae Fah Luang University and partially supported by the Asahi Glass Foundation, the study was approved by the animal research ethics committees of both Mae Fah Luang University and Chulalongkorn University. The authors, drawn from the School of Medicine at Mae Fah Luang University and the Center of Excellence in Vector Biology and Vector-Borne Disease at Chulalongkorn University, stress that their work is a starting point rather than a conclusion. The statistical signal linking Wolbachia to reduced parasite detection is suggestive but modest, and the cross-sectional design cannot establish causation.

What the study does establish is that the endosymbiotic world of Thai sand flies is far richer than previously appreciated, and that its diversity is structured, heritable, and potentially consequential for disease transmission. The next steps are clear from the data: experimental infections comparing Wolbachia-positive and Wolbachia-free sand fly lines, functional studies of the WSP insertion variant, and broader sampling across Thailand’s endemic regions to test whether the inverse association between Wolbachia and Leishmania holds at larger scales. If those experiments confirm a blocking effect, the humble bacteria living inside cave-dwelling sand flies could become unexpected allies in the fight against a neglected tropical disease.

Subject of Research: Endosymbiont–parasite associations between Leishmania (Mundinia), Wolbachia, and Cardinium in phlebotomine sand flies from Northern Thailand

Article Title: Molecular co-occurrence of Leishmania (Mundinia) spp., Wolbachia, and Cardinium in cave-associated phlebotomine sand flies from Northern Thailand: genetic diversity and endosymbiont–parasite associations

Article References: Somwang, P., Khositharattanakool, P., Sunantaraporn, S., Promrangsee, C., Yuanlae, S., Tepboonrueng, P., Siriyasatien, P., & Preativatanyou, K. (2026). Molecular co-occurrence of Leishmania (Mundinia) spp., Wolbachia, and Cardinium in cave-associated phlebotomine sand flies from Northern Thailand: genetic diversity and endosymbiont–parasite associations. Parasites & Vectors. https://doi.org/10.1186/s13071-026-07727-0

Image Credits: AI Generated

DOI: 10.1186/s13071-026-07727-0

Keywords: Leishmania, Mundinia, sand flies, Wolbachia, Cardinium, endosymbionts, genetic diversity, leishmaniasis, Thailand, vector biology, xenomonitoring, phlebotomine

L. martiniquensis has attracted growing concern in Southeast Asia as an emerging cause of visceral leishmaniasis, a disease that can be fatal particularly among patients with advanced HIV infection whose immune systems cannot contain the parasite. Amphotericin B remains a mainstay of therapy, yet clinicians in Thailand have documented relapse and treatment failure in patients infected with this species, raising the possibility that some parasite populations carry or acquire reduced susceptibility to the drug. Until now, the molecular machinery underlying such reduced susceptibility in L. martiniquensis has remained largely unexplored, making paired isolates drawn from the same patient at different clinical time points an unusually valuable window into how the parasite changes under drug pressure.

The research team, led by investigators at Chulalongkorn University in Bangkok in collaboration with Hatyai Hospital and King Chulalongkorn Memorial Hospital, obtained one isolate before treatment, designated CULE7.2, and a second, CULE8, after the patient relapsed. They first confirmed the biological difference between the two isolates using in vitro susceptibility assays: CULE8 displayed significantly higher half-maximal and 90 percent inhibitory concentrations of amphotericin B than CULE7.2, with the dose-response curve shifted markedly to the right. Transmission electron microscopy of CULE7.2 promastigotes exposed acutely to the drug revealed the expected cellular damage, including prominent cytoplasmic vacuolization, lipid-like inclusions, mitochondrial swelling, and focal disruption of the nuclear membrane, consistent with amphotericin B’s mechanism of binding membrane sterols and destabilizing cellular membranes.

To explore the transcriptional landscape underlying these phenotypic differences, the researchers profiled both isolates across two developmental stages of the parasite, promastigotes and axenic amastigotes, using RNA sequencing. Importantly, the two isolates were cultured under different conditions: CULE8 was maintained under continuous exposure to a sublethal 0.3 micromolar concentration of amphotericin B, while CULE7.2 was cultured in parallel without the drug. Differential expression analysis, hierarchical clustering, and functional enrichment were performed independently within each stage. This design choice means the observed patterns likely reflect a combination of intrinsic isolate-associated characteristics and the transcriptional response to ongoing drug exposure, an important caveat the authors emphasize in interpreting their results.

In the promastigote stage, the post-relapse isolate CULE8 showed predominant downregulation of genes involved in sterol and lipid metabolism, membrane transport, protein synthesis, motility, and broader metabolic and regulatory processes. Among the affected genes were sterol C24 reductase, an enzyme central to the parasite’s membrane sterol composition and a known determinant of amphotericin B activity, together with upstream enzymes of the mevalonate pathway. At the same time, CULE8 promastigotes displayed increased expression of genes associated with thiol-based redox homeostasis, suggesting a shift toward enhanced management of oxidative stress, a plausible countermeasure to drug-induced membrane and mitochondrial damage.

The axenic amastigote stage, the form that proliferates inside the mammalian host, told a different story. Here, CULE8 showed increased expression of genes linked to lipid metabolism, redox homeostasis, protein synthesis, and proteostasis, alongside broader downregulation of transport genes and genes involved in genome maintenance. Despite the predominantly stage-specific patterns, a small subset of genes showed concordant regulation across both life stages, including genes involved in lipid metabolism and transport. Genes tied to thiol-based redox homeostasis stood out for their elevated expression in both stages, marking this antioxidant capacity as a consistent candidate feature of the relapse-associated parasite and a potential target for functional validation studies.

The virological dimension of the study may prove equally consequential. De novo assembly of RNA-seq reads that failed to map to the parasite genome recovered complete L and S genomic segments of a genetically distinct leishbuvirus from CULE8. This represents, to the authors’ knowledge, the first molecular characterization of a leishbuvirus in a Southeast Asian L. martiniquensis isolate, expanding the known geographic and host range of these viruses. Comparative structural modeling using predicted aligned error matrices supported conservation of the RNA-dependent RNA polymerase core and its Motif C between the CULE8 virus and a reference variant, while negative-stain transmission electron microscopy revealed structures consistent with virus-like particles. Viral RNA abundance was higher in CULE8 than in CULE7.2 across both developmental stages.

The significance of leishbuviruses remains a subject of active debate in the field. Related viruses, including Leishmania RNA virus 1, have been associated in some studies with heightened virulence and treatment failure in other Leishmania species, particularly through modulation of the host inflammatory response, although such associations are not universal and their mechanisms are incompletely understood. The authors are careful to note that the higher leishbuvirus RNA abundance observed in the post-relapse isolate has no established causal relationship with reduced amphotericin B susceptibility or with the patient’s clinical relapse. The finding is best viewed as an observation warranting further investigation in independent isolates rather than as evidence of a viral driver of drug resistance.

Indeed, the authors frame the entire study as hypothesis-generating. Because the paired isolates were examined under different drug-exposure conditions, the transcriptional differences they report cannot be attributed cleanly to either intrinsic isolate biology or adaptive response to amphotericin B, and the findings will require validation in independent isolates under matched experimental conditions. Nevertheless, the breadth of the observed changes, encompassing sterol biosynthesis, lipid handling, membrane transport, redox metabolism, protein homeostasis, and genome maintenance, broadens the molecular landscape associated with reduced amphotericin B susceptibility in this species and identifies multiple candidate pathways for future functional studies, including gene knockout or overexpression experiments that could disentangle correlation from causation.

For a disease that disproportionately strikes immunocompromised patients in resource-limited settings, the stakes of understanding amphotericin B failure are high. Visceral leishmaniasis caused by L. martiniquensis in Thailand is emerging in HIV-coinfected populations, and the therapeutic arsenal is narrow. By pairing rigorous susceptibility testing and ultrastructural imaging with stage-resolved transcriptomics and sensitive viral discovery, this study demonstrates how a single patient’s clinical course can illuminate molecular processes that no laboratory strain could fully recapitulate. The identified candidate pathways, from mevalonate-dependent sterol metabolism to thiol-based antioxidant defenses, now provide concrete starting points for the functional experiments and epidemiological surveillance needed to determine how widespread reduced drug susceptibility might be, and whether hidden viral passengers within these parasites play any role in the clinical outcomes that matter most to patients.

Subject of Research: Transcriptomic analysis of paired Leishmania martiniquensis clinical isolates to identify molecular pathways associated with reduced amphotericin B susceptibility

Article Title: Transcriptomic profiling of paired clinical Leishmania (Mundinia) martiniquensis isolates reveals candidate pathways associated with reduced amphotericin B susceptibility and identifies a novel leishbuvirus variant

Article References: Phadungsaksawasdi, K., Pongpanich, M., Yuanlae, S., Lerona, P. G. E., Sricharoensuk, C., Songumpai, N., Siriyasatien, P., Asawanonda, P., Shotelersuk, V., & Preativatanyou, K. (2026). Transcriptomic profiling of paired clinical Leishmania (Mundinia) martiniquensis isolates reveals candidate pathways associated with reduced amphotericin B susceptibility and identifies a novel leishbuvirus variant. Parasites & Vectors. https://doi.org/10.1186/s13071-026-07628-2

Image Credits: AI Generated

DOI: 10.1186/s13071-026-07628-2

Keywords: Leishmania martiniquensis, Mundinia, amphotericin B, drug susceptibility, transcriptomics, leishbuvirus, visceral leishmaniasis, Thailand, RNA sequencing, parasite virome, sterol metabolism, redox homeostasis