Coronatin-2 from the entomopathogenic fungus Conidiobolus coronatus kills Galleria mellonella larvae and incapacitates hemocytes.

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.

Cuticular and internal n-alkane composition of Lucilia sericata larvae, pupae, male and female imagines: application of HPLC-LLSD and GC/MS-SIM.

The composition of cuticular and internal n-alkanes in Lucilia sericata larvae, pupae, and male and female imagines were studied. The cuticular and internal lipid extracts were separated by HPLC-LLSD, after which the hydrocarbon fraction was identified by GC/MS in selected ion monitoring (SIM) and total ion current (TIC) modes. The cuticular lipids of the larvae contained seven n-alkanes from C23 to C31. The major n-alkane in L. sericata larvae was C29 (42.1%). The total cuticular n-alkane content in the cuticular lipids was 31.46 µg g-1 of the insect body. The internal lipids of L. sericata larvae contained five n-alkanes ranged from C25 to C31. The most abundant compound was C27 (61.71 µg g-1 of the insect body). Eighteen n-alkanes from C14 to C31 were identified in the cuticular lipids of the pupae. The most abundant n-alkanes ranged from C25 to C31; those with odd-numbered carbon chains were particularly abundant, the major one being C29:0 (59.5%). Traces of eight cuticular n-alkanes were present. The internal lipids of L. sericata pupae contained five n-alkanes, ranging from C25 to C31. The cuticular lipids of female imagines contained 17 n-alkanes from C12 to C30. Among the cuticular n-alkanes of females, C27 (47.5%) was the most abundant compound. Four n-alkanes, with only odd-numbered carbon chains, were identified in the internal lipids of females. The lipids from both sexes of L. sericata had similar n-alkane profiles. The cuticular lipids of adult males contained 16 n-alkanes ranging from C13 to C31. C27 (47.9%) was the most abundant cuticular n-alkanes in males. The same n-alkanes only with odd-numbered carbon chains and in smaller quantities of C27 (0.1%) were also identified in the internal lipids of males. The highest amounts of total cuticular n-alkanes were detected in males and females of L. sericata (330.4 and 158.93 µg g-1 of the insect body, respectively). The quantities of total cuticular alcohols in larvae and pupae were smaller (31.46 µg g-1 and 42.08 µg g-1, respectively). The internal n-alkane contents of larvae, pupae, and male and female imagines were significantly higher than the cuticular n-alkane contents (153.53, 99.60, 360.06 and 838.76 µg g-1 of the insect body, respectively).