Molecular Identification, Phylogenetic Characterisation and Conidial Formulation of Emericella nidulans for Mosquito-larvicidal Bioassay

Abstract

This study confirmed the molecular identity and phylogenetic placement of an entomopathogenic fungal isolate previously identified morphologically as Emericella nidulans and standardised its conidial suspension for subsequent larvicidal evaluation against Anopheles gambiae. Genomic DNA was extracted using the CTAB method and amplified with ITS1/ITS4 primers targeting the ITS region. The amplification produced a single 526 bp band, and the extracted DNA showed a concentration of 52 ng µL⁻¹ with an A₂₆₀/A₂₈₀ ratio of 1.83, indicating purity suitable for PCR. Bidirectional sequencing followed by BLAST analysis showed 98.07% nucleotide similarity to E. nidulans strain IHB F 1876 (GenBank accession MN045781), thereby supporting the morphological identification. Neighbour-joining phylogenetic analysis placed the isolate within the Emericella clade with 99% bootstrap support, and the genetic distance between the isolate and the closest reference strain was 0.021 substitutions per site. Field-collected fourth-instar mosquito larvae were identified morphologically as Anopheles gambiae based on a horizontal resting posture, absence of a respiratory siphon and the presence of palmate hairs on abdominal segments II–VII. Four conidial suspensions were prepared at 9.2 × 10⁴, 9.2 × 10⁵, 9.2 × 10⁶ and 9.2 × 10⁷ conidia mL⁻¹. Viability after 24 hours ranged from 91% to 96%, and mean germination time decreased from 20 to 14 hours as conidial concentration increased. These standardised preparations were therefore suitable for use in planned larvicidal bioassays, although larval mortality was not evaluated in the present work. The findings provide an authenticated molecular, phylogenetic and biological baseline for future mosquito-larvicidal bioassays using E. nidulans metabolites.