First record of mermithid parasitism in adult biting midges, Culicoides huffi (Diptera: Ceratopogonidae), collected from Southern Thailand, with ultrastructural and molecular characterization.

Abstract

Background: Culicoides biting midges are known vectors of several pathogens, including arboviruses, protozoa, and filarial worms. Additionally, mermithid nematodes have been found to parasitize Culicoides midges, causing pathogenic effects that reduce host fitness and often lead to death. Consequently, mermithids have potential as biocontrol agents to reduce midge populations. However, the biology of these entomopathogenic nematodes infecting Culicoides in Thailand remains unknown.

Methods: As part of the leishmaniasis surveillance program in Nakhon Si Thammarat Province, Southern Thailand, we collected Culicoides midges near the residence of a leishmaniasis patient in November 2024. The Culicoides samples were morphologically identified and examined microscopically for parasitic nematodes. Infected midges were dissected to isolate nematodes from each individual. The nematodes were then characterized morphologically using scanning electron microscopy (SEM) and identified molecularly via polymerase chain reaction (PCR) targeting the non-filarial hypervariable region I of the small subunit ribosomal RNA gene (SSU HVR-I) and mermithid small subunit ribosomal RNA (SSU rRNA) gene, followed by nanopore sequencing, phylogenetic analysis, and species delimitation testing.

Results: A total of 263 field-caught adult Culicoides midges were collected, with Culicoides huffi of the Calvipalpis group being the most abundant species (n = 155, 58.9%). Among these, 35 C. huffi samples, including 4 males, 11 females, and 20 intersex males, were infected with nematodes, resulting in an overall infection rate of 13.3%. The parasitized intersex males, presumably genetically male, exhibited a high degree of feminization in their antennae and wings, which likely enhances female-like behaviors such as detecting and flying toward breeding sites, possibly facilitating parasite transmission and reproductive success. The SEM analysis revealed key morphological features consistent with parasitic nematode larvae of the Mermithidae family, including a long, slender body, a stylet, cephalic papillae, amphids, and a trophosome. Basic Local Alignment Search Tool for nucleotide (BLASTn) analysis of non-filarial SSU HVR-I and mermithid-specific SSU rRNA gene sequences identified all nematodes as mermithids, showing 94.2-94.4% similarity to Pheromermis sp. from the hornet and 97.3% similarity to Mermis sp. from Culicoides obsoletus. Phylogenetic analysis and species delimitation suggest that these sequences represent a single putative species distinct from other known mermithids.

Conclusions: This study is the first to report mermithid parasitism in Culicoides midges in Thailand, incorporating both ultrastructural and molecular characterization. The novel morphological and molecular insights provided here contribute to a more comprehensive understanding of the biology of entomopathogenic nematodes. Further research is needed to evaluate the potential of these nematodes for the biological control of Culicoides biting midges.