The fieldwork formed part of a larger bio-surveillance initiative called CamBatRat, which sampled 2,461 bats at 233 sites across Cambodia between December 2016 and February 2020. From this archive, the team selected 1,180 individuals representing 34 species for retrospective screening of blood samples. DNA was extracted and initially tested using a polymerase chain reaction targeting a fragment of the parasites’ mitochondrial cytochrome b gene. Positive samples were then characterized with additional markers spanning the parasites’ three genomes: mitochondrial genes cytb and cox1, an apicoplast marker called clpC, and two nuclear loci, ef2 and pat. This multilocus approach matters because some bat haemosporidians carry highly rearranged mitochondrial genomes that can defeat standard amplification protocols, and because different genes evolve at different rates, offering complementary windows into parasite evolutionary history.

Sequence-confirmed infections turned up in 152 bats belonging to eleven species from four families: Emballonuridae, Hipposideridae, Pteropodidae and Vespertilionidae. Two parasite genera dominated the findings. Polychromophilus, a genus restricted to bats, was confined to a handful of host species, infecting the vespertilionids Scotophilus kuhlii, Scotophilus heathii and Myotis rosseti as well as the hipposiderid Hipposideros diadema. Prevalence differed dramatically between the two Scotophilus species: 54 percent of S. kuhlii were infected, compared with just 4 percent of S. heathii. Hepatocystis, a genus closely related to mammalian Plasmodium, showed a broader footprint, occurring across three bat families and infecting, among others, 42 percent of Cynopterus brachyotis and 23 percent of Hipposideros cf. grandis individuals examined.

The most taxonomically significant discovery came from the emballonurid bat Taphozous melanopogon, in which 18 percent of sampled individuals carried Hepatocystis. This is the first record of the genus in the family Emballonuridae, substantially expanding its known host range. Because emballonurid bats are widely distributed across the Old World yet rarely documented as haemosporidian hosts, the finding suggests that parasite diversity in this family has gone largely unnoticed. Infections were also newly recorded in the fruit bats Megaerops niphanae and Eonycteris spelaea. By contrast, no infections were detected in bats from the families Molossidae, Megadermatidae and Rhinolophidae, although the authors caution that the molossid sample was spatially concentrated and therefore less conclusive.

Phylogenetic analyses revealed two contrasting evolutionary patterns. The Polychromophilus lineage associated with Scotophilus bats proved remarkably genetically conserved: haplotype networks built from cytb, cox1, clpC and ef2 sequences showed identical or near-identical parasites in hosts from Cambodia, Thailand and Madagascar, thousands of kilometres apart. This lineage forms a well-supported monophyletic group that does not cluster with any currently recognized Polychromophilus species, hinting that it may represent a distinct evolutionary lineage with taxonomic significance. Such extreme genetic uniformity across such a vast range could reflect either recent dispersal or long-term evolutionary conservatism, and in either case points to strong host specificity at the level of the host genus.

Hepatocystis told a very different story. Parasites from Cambodian bats fell into several deeply divergent clades, and lineages infecting hipposiderid bats did not even form a single group. Most strikingly, cytb sequences from the Taphozous-associated parasites formed a well-supported clade with Hepatocystis lineages infecting Southeast Asian squirrels of the genus Callosciurus, despite substantial mitochondrial divergence between the host-associated groups. Analyses at additional loci placed the Taphozous lineage basal to other Hepatocystis taxa, though the authors note that interpretation is limited by the scarcity of comparable rodent-derived reference sequences. Taken together, the patterns suggest that the evolutionary history of Hepatocystis may involve multiple historical host transitions among mammalian groups, with Asian bats and rodents playing a more prominent role than previously appreciated.

Within the genus Cynopterus, the parasite picture was equally complex. Hepatocystis from C. brachyotis and C. sphinx did not form a single group but instead split into two deeply divergent clades differing by 30 to 41 nucleotide substitutions, with no clear structure by host species or geography. The clades also included parasites from Megaerops and Eonycteris hosts. This suggests that host specificity in Asian bat Hepatocystis may be looser than classical morphology-based taxonomy implies, or that recognized morphospecies conceal cryptic diversity, a possibility previously raised for African bat parasites. Because no blood smears were collected during the original sampling, the Cambodian parasites could not be confidently assigned to described morphospecies, underscoring the need to integrate morphological and molecular data in future work.

To understand what drives infection, the team fitted a Bayesian generalized linear mixed model to 589 individuals of ten host species from 84 sites, treating host species and sampling site as crossed group-level effects. The result was clear: host species accounted for 26.3 percent of the variance in infection probability and sampling site for 25.3 percent, while the measured covariates added essentially nothing. Knowing a bat’s species and capture location predicted infection as well as any combination of host traits, landscape attributes and climate variables. The strongest single effect was seasonal, with infection probability substantially lower in the wet season than the dry season, a direction that runs counter to a simple vector-abundance expectation, since the presumed Culicoides midge vectors should thrive when wet-season larval habitat is most available.

One landscape variable did stand out: infection probability declined with increasing human population density within a two-kilometre radius of the capture site. The authors interpret this as consistent with reduced availability of vector habitat in built-up areas, since Culicoides midges depend on aquatic and semi-aquatic larval sites and have limited dispersal ranges, making transmission highly sensitive to conditions close to the roost. Habitat fragmentation showed a weaker, statistically ambiguous negative trend, and land cover class at the site showed no association. Among individual host attributes, sex and body condition were unrelated to infection, while reproductive individuals showed a suggestive but inconclusive elevation in infection probability. Notably, residual variation among sites was not distance-structured, meaning that infection hotspots were not clustered spatially in any simple way.

The broader implications reach into both evolutionary biology and surveillance strategy. Because bats host parasites from at least seven genera, collectively representing roughly a third of all described mammalian haemosporidian species, they are a key system for understanding how malaria parasites have diversified across vertebrate hosts. The Cambodian data reinforce the view that diversification within Hepatocystis may have occurred substantially among Asian mammalian hosts, a hypothesis that will require broader sampling of Asian rodents and additional bat families to test. On the ecological side, the finding that infection is structured principally by host identity and fine-scale roost conditions rather than by landscape composition indicates that roost ecology and vector biology are the most promising targets for understanding transmission. Distinguishing among competing explanations for the seasonal pattern, whether vector phenology, temperature or shifts in host demography, will require paired vector sampling and bloodmeal analysis alongside host screening, work the current study design did not include but which the authors identify as the logical next step.

Subject of Research: Diversity, host associations and ecology of haemosporidian malaria parasites infecting bats in Cambodia

Article Title: High diversity and novel host associations of bat haemosporidian parasites in Cambodia: insights from a nationwide bat survey and multilocus analyses

Article References: Schaer, J., Werb, O., Ch´ng, L., Low, D. H. W., Borthwick, S. A., Furey, N., Frias, L., Pimsai, A., Yim, R., Chor, K., Cheang, D., Smith, G. J. D., Hitch, A., & Mendenhall, I. H. (2026). High diversity and novel host associations of bat haemosporidian parasites in Cambodia: insights from a nationwide bat survey and multilocus analyses. Parasites & Vectors, 19(1), Article 436. https://doi.org/10.1186/s13071-026-07732-3

Image Credits: AI Generated

DOI: 10.1186/s13071-026-07732-3

Keywords: bats, haemosporidian parasites, Hepatocystis, Polychromophilus, malaria, Cambodia, Southeast Asia, phylogenetics, host associations, Culicoides, parasite ecology, molecular survey

Avian haemosporidians are a group of apicomplexan parasites belonging chiefly to three genera: Plasmodium, Haemoproteus, and Leucocytozoon. In birds, these organisms can behave much like the malaria parasites that afflict humans, invading blood cells and internal organs, sapping host condition, and in severe cases reducing survival and reproductive success. Their effects ripple outward into population dynamics, migration behavior, and even the structure of whole bird communities. For decades, ecologists have worked to map which insect vectors carry which parasite lineages, because transmission is the hinge on which the entire host-parasite system turns. Mosquitoes, Culicoides biting midges, louse flies, and black flies are the recognized vectors, each genus of parasite showing affinities for particular insect groups and particular bird hosts.

Leptoconops is a genus of haematophagous biting midges in the family Ceratopogonidae, closely related to the better-known Culicoides midges that are established vectors of avian Haemoproteus. Despite this kinship, Leptoconops midges have never been systematically implicated in avian malaria transmission, and no previous study had molecularly screened them for haemosporidian infections. That gap is precisely what motivated the research team, led by Javier Carbonero and Martina Ferraguti of the Estación Biológica de Doñana, a research institute of the Spanish National Research Council (CSIC) in Seville, together with colleagues affiliated with the Centro de Investigación Biomédica en Red de Epidemiología y Salud Pública.

The scale of the fieldwork reflects the difficulty of the question. During 2024, the researchers collected a total of 5,417 female Leptoconops midges across sites in Andalusia, taking advantage of the fact that only female midges take blood meals and can therefore plausibly acquire and transmit blood parasites. Each specimen was morphologically identified to species, a labor-intensive step that matters because vector competence and host preferences can differ sharply even between close relatives. The midges were then grouped into 203 pools for molecular analysis, a pooling strategy that balances laboratory workload against the sensitivity needed to detect low-level infections in wild-caught insects.

The molecular heart of the study is a nested polymerase chain reaction (PCR) protocol targeting the cytochrome b gene, the standard DNA barcode region for avian haemosporidians. Nested PCR uses two successive rounds of amplification with two pairs of primers, dramatically increasing sensitivity and allowing researchers to detect the minute quantities of parasite DNA that may persist in an insect after a blood meal has been digested. Positive amplicons were then sequenced, and the resulting DNA barcodes were compared against reference databases to assign each parasite to a genus and, where possible, to a known lineage with a documented host range. This lineage-level resolution is what gives the study its ecological punch: many haemosporidian lineages have been catalogued from specific bird species, so finding a lineage inside a midge hints at which birds the midge may have bitten.

Eight of the 203 pools tested positive for haemosporidian DNA. The positive pools corresponded to two of the Leptoconops species examined, Leptoconops communis and Leptoconops nigrithorax, indicating that more than one member of the genus encounters these parasites in the wild. The identified parasites spanned all three target genera. Among the Haemoproteus detections were Haemoproteus noctuae, corresponding to the lineage CIRCUM01, and an unidentified Haemoproteus species of the lineage COLPAL01. The Plasmodium findings included Plasmodium matutinum, the well-characterized lineage LINN1, while Leucocytozoon was represented by the lineages COLIV04 and AEMO02. In addition to these known barcodes, the team uncovered two lineages that had never been recorded before, which they designated Plasmodium P_LEPTO01 and Haemoproteus H_LEPTO01, expanding the global catalogue of avian haemosporidian genetic diversity.

The known host associations of the detected lineages are where the story becomes genuinely suggestive. Haemoproteus noctuae, for example, is a parasite associated with owls and other birds, and lineages such as LINN1 and COLIV04 have been documented from particular avian hosts in previous surveys. By matching the lineages found in the midges to their known bird hosts, the researchers can infer, indirectly, which avian species Leptoconops midges may have fed upon. This approach, sometimes framed within the broader use of insect-derived DNA as a sampling tool, effectively turns each infected midge into a tiny record of both a bird-parasite interaction and a midge feeding choice. It is an indirect inference rather than a direct blood-meal identification, but it provides testable hypotheses about the feeding ecology of a genus whose biting habits remain poorly documented in many regions.

The authors are careful, and appropriately so, about what the results do and do not demonstrate. Detecting parasite DNA in an insect does not establish vector competence, the ability of a vector to support the parasite’s full developmental cycle and transmit it to a new host. Haemosporidian parasites undergo a complex development within a competent vector, including sexual reproduction, invasion of the insect’s gut wall, production of infective sporozoites, and migration to the salivary glands. Parasite DNA could, in principle, simply reflect the digestion of an infected blood meal without any subsequent parasite development. The researchers explicitly state that vector competence has not been demonstrated and that the potential role of Leptoconops in transmission is unknown and merits further investigation. Disentangling genuine biological transmission from mere mechanical carriage of DNA will require experimental infection studies, dissection of midge tissues, and detection of parasite developmental stages rather than DNA alone.

Even with those caveats, the study carries weight for several reasons. First, it establishes a previously unrecorded interaction between a widespread genus of biting midges and avian haemosporidian parasites, adding Leptoconops to the short list of insect groups in which these parasites have been molecularly detected. Second, the diversity of parasites found, spanning Plasmodium, Haemoproteus, and Leucocytozoon, suggests that the encounters are not a rare accident of a single parasite species but a broader pattern involving multiple parasite lineages and at least two midge species. Third, the discovery of two novel lineages underscores how much undocumented parasite diversity circulates in wild bird communities and their vectors, and how vector-focused sampling can surface it. Fourth, the work has practical implications: if Leptoconops midges prove to be competent vectors, current models of avian malaria transmission, which lean heavily on mosquitoes and Culicoides, would need revision, with consequences for understanding disease dynamics in bird populations, the risks faced by vulnerable species, and even the epidemiology of zoonotic or wildlife pathogens in shared landscapes.

The research was conducted by a team based at the Estación Biológica de Doñana in Seville, with funding from projects including ARBOPREVENT, supported by Fundación La Caixa, a national research project financed by Spain’s Ministry of Science and European regional funds, and the Severo Ochoa excellence program. The work formed part of a broader research effort on vector ecology in southern Spain, where wetland habitats concentrate both bird diversity and biting insects. As the authors conclude, the findings suggest real interactions between Leptoconops midges and infected avian hosts, and they call for further studies to determine whether these midges are incidental carriers or genuine players in the transmission of avian malaria. For now, a genus of tiny midges, long noted mainly for the irritating bites of its females, has earned a place on the watch list of avian disease ecology, and the search for its true role in the parasite’s life cycle is only beginning.

Subject of Research: Molecular detection of avian haemosporidian parasites in Leptoconops biting midges

Article Title: First molecular detection of avian haemosporidian parasites in Leptoconops (Diptera: Ceratopogonidae) biting midges

Article References: Carbonero, J., González, M. A., Magallanes, S., Paun-Tanase, D. I., Martínez-de la Puente, J., Figuerola, J., & Ferraguti, M. (2026). First molecular detection of avian haemosporidian parasites in Leptoconops (Diptera: Ceratopogonidae) biting midges. Parasites & Vectors. https://doi.org/10.1186/s13071-026-07713-6

Image Credits: AI Generated

DOI: 10.1186/s13071-026-07713-6

Keywords: avian malaria, haemosporidian parasites, Leptoconops, biting midges, Ceratopogonidae, Plasmodium, Haemoproteus, Leucocytozoon, vector ecology, cytochrome b, nested PCR, Andalusia