Isolation of a novel Pythium mosquito larval pathogen, P. themoculicivorax, and Trichoderma sp. from natural enzootic mosquito larval infections

Abstract

Only a handful of microbial mosquito larval pathogens have been described to date. Sampling of several natural enzootic infections of mosquito larvae in southwestern Florida indicated the presence of microbial pathogens capable of extensive larval mortality. Microscopic analysis of one sample site revealed extensive apparent growth of a Pythium-like microbe on mosquito lar-vae, with highest infection observed on the siphon and head regions. Structures consistent with sporangia were seen on infected insects after lactophenol blue staining, and higher resolution scanning electron micrographs (SEM) showed sporangia and encysted zoospores targeting the head and siphon regions. The isolate was single colony purified and molecular identification targeting the ITS and COX1 loci coupled to phylogenetic reconstruction indicated that the isolate belonged to the Pythium genus, but was distinct from its closest characterized species, P. angusta-tum. Morphological features were characterized, with the isolate showing rapid growth on all mycological media tested and relatively high thermotolerance, capable of robust growth at 37oC, and hence was designated, P. thermoculicivorax. Sampling from a second series of natural infec-tions of mosquito larvae resulted in the molecular identification of three Trichoderma isolates, one with high similarity to T. strigosum, and the other two clustering with, but distinct from, T. asperellum. These data highlight the occurrence of natural enzootic infections of mosquito larvae, potentially as a resource for the identification of new mosquito pathogens.