Entomopathogenic fungi infect insects via penetration through the cuticle, which varies remarkably in chemical composition across species and life stages. Fungal infection involves the production of enzymes that hydrolyse cuticular proteins, chitin and lipids. Host specificity is associated with fungus-cuticle interactions related to substrate utilization and resistance to host-specific inhibitors. The soil fungus Conidiobolus coronatus (Constantin) (Entomophthorales: Ancylistaceae) shows virulence against susceptible species. The larvae and pupae of Calliphora vicina (Robineau-Desvoidy) (Diptera: Calliphoridae), Calliphora vomitoria (Linnaeus), Lucilia sericata (Meigen) (Diptera: Calliphoridae) and Musca domestica (Linnaeus) (Diptera: Muscidae) are resistant, but adults exposed to C. coronatus quickly perish. Fungus was cultivated for 3 weeks in a minimal medium. Cell-free filtrate, for which activity of elastase, N-acetylglucosaminidase, chitobiosidase and lipase was determined, was used for in vitro hydrolysis of the cuticle from larvae, puparia and adults. Amounts of amino acids, N-glucosamine and fatty acids released were measured after 8 h of incubation. The effectiveness of fungal enzymes was correlated with concentrations of compounds detected in the cuticles of tested insects. Positive correlations suggest compounds used by the fungus as nutrients, whereas negative correlations may indicate compounds responsible for insect resistance. Adult deaths result from the ingestion of conidia or fungal excretions.

Coronatin-2, a 14.5 kDa protein, was isolated from culture filtrates of the entomopathogenic fungus Conidiobolus coronatus (Costantin) Batko (Entomophthoramycota: Entomophthorales). After LC-MS/MS (liquid chromatography tandem mass spectrometry) analysis of the tryptic peptide digest of coronatin-2 and a mass spectra database search no orthologs of this protein could be found in fungi. The highest homology was observed to the partial translation elongation factor 1a from Sphaerosporium equinum (protein sequence coverage, 21%), with only one peptide sequence, suggesting that coronatin-2 is a novel fungal protein that has not yet been described. In contrast to coronatin-1, an insecticidal 36 kDa protein, which shows both elastolytic and chitinolytic activity, coronatin-2 showed no enzymatic activity. Addition of coronatin-2 into cultures of hemocytes taken from larvae of Galleria mellonella Linnaeus (Lepidoptera: Pyralidae), resulted in progressive disintegration of nets formed by granulocytes and plasmatocytes due to rapid degranulation of granulocytes, extensive vacuolization of plasmatocytes accompanied by cytoplasm expulsion, and cell disintegration. Spherulocytes remained intact, while oenocytes rapidly disintegrated. Coronatin-2 produced 80% mortality when injected into G. mellonella at 5 µg larva-1. Further study is warranted to determine the relevance of the acute toxicity of coronatin-2 and its effects on hemocytes in vitro to virulence of C. coronatus against its hosts.

The most effective and important strategy in the insect immune response is based on cellular reactions incorporating haemocytes. The present study uses Galleria mellonella (Lepidoptera: Pyralidae) as a host to study the pathogenesis caused by the entomopthoralean fungus Conidiobolus coronatus (Entomophthorales). Five types of haemocytes with different morphologies and behaviour are observed in the haemolymph of G. mellonella: granulocytes (GRs), plasmatocytes (PLs), spherulocytes (SPs), oenocytes (OEs) and prohaemocytes (PRs). During in vitro cultivation, three morphological subtypes of PLs are distinguished: flattened PLs, sun‐like PLs and oval PLs. In fresh smears of haemolymph observed under phase‐contrast microscopy, only flattened PLs are identified. No morphological changes are observed between fresh smears and in vitro cultures for GR, OE, SP and PR. Haemocytes cultured in vitro form a cellular network composed of PLs and GRs. Changes in the numbers, morphology and behaviour of haemocytes induced by fungal infection are compared with those observed in normally‐developing untreated larvae. Infection results in a significant drop in the number of haemocyte types. Fresh smears of haemocytes from mycosed larvae reveal malformed OEs, vacuolized PLs and GRs, as well as PLs with apoptotic blebs. Haemocytes from mycosed larvae incubated in vitro look similar, with degranulated GRs and vacuolized PLs forming microaggregations, as well as deformed OEs; only the SPs remain unharmed. Fungal infection impairs the ability of haemocytes to attach and spread on the culture dish. The actin cytoskeleton of haemocytes from mycosed larvae appear disorganized.