Studies on the pathogenicity of the entomopathogenic fungus Metarhizium spp. for the desert locust Schistocerca gregaria

The entomopathogenic fungi Metarhizium anisopliae and M. flavoviride are being developed as mycoinsecticides for the control of the desert locust Schistocerca gregaria. An important part of this development is to identify possible virulence determinants. This study has investigated in detail steps in the penetration of host insect cuticle, and the production of toxins by the fungus. Excised locust wings were used in a semi-in vivo technique to investigate germination, appressoria formation, penetration and protease production of 19 M anisopliae and M. flavoviride isolates. Germination started at between 6 and 8 h after inoculation for all isolates, but 7 of them had not reached 50% germination by 12 h. Generally, the more pathogenic isolates initially produced only short germ tubes prior to appressoria formation, with the less pathogenic isolates producing longer, branched hyphae. Penetration of the wing occurred from the appressoria. A great range of protease production was seen for the isolates at the time of initial penetration. A significant correlation was found between the rate of germination and the median lethal time for the 19 isolates. No other significant correlations were found between the other parameters measured and virulence. Destruxins are cyclic peptide lactone toxins isolated from M. anisopliae. A previous study has suggested a link between destruxin production and isolate virulence against the desert locust. The role of destruxins in fungal pathogenesis was investigated in particular with regard to their effect on the locust's excretory system. Destruxins A, A2 , B and E all inhibited fluid secretion in a dose dependent manner in vitro by Malpighian tubules of the desert locust S. gregaria. Experiments were conducted with ion channel blockers, inhibitors of cAMP phosphodiesterase and in calcium free conditions. From the results it is suggested that the inhibition by destruxin A of desert locust Malpighian tubule fluid secretion involves a cellular mechanism beyond the level of control by calcium or cAMP. Destruxin A (25|ig) inhibited the initial rate of removal of an injected dye, amaranth, from the haemolymph of freshly fed adult locusts indicating that destruxin A inhibits Malpighian tubule function in vivo in S. gregaria as well as in vitro. Locusts on day 4 of infection with M anisopliae isolate Mel had a slower, reduced haemolymph circulation as indicated by the speed of spread of an injected dose of amaranth. Mycosed locusts were difficult to bleed and appeared to contain less haemolymph than controls. However, there was no significant difference in wet weight or the rate of water loss between control and infected locusts. This finding is discussed with regard to the involvement of destruxins in mycosis. Destruxin A altered the ultrastructure of S. gregaria Malpighian tubules in the in vitro preparation used in the physiological experiments. Both control and destruxin treated tubules had changes in ultrastructure, that is vacuolisation and swelling of the organelles, consistent with slight anoxia due to the methods employed. Minor differences between control and destruxin-treated cells, however, were still apparent. It is concluded that destruxin inhibition of S. gregaria Malpighian tubule fluid secretion in vivo and in vitro is not the result of gross cellular damage. These observations suggest that destruxins act on the regulatory apparatus of the cell. Haemolymph from locusts on day 4 of infection with M. anisopliae isolate Mel was processed and analysed using an analytical RP-HPLC. No peaks were found to correspond with the 16 min retention time of the destruxin A standard. However, one or more substances present in fractions 15 and 16 (corresponding to retention times of 15 and 16 min) accelerated the heart rate of a semi-isolated Manduca larval heart preparation with an increase of 24% each. This translates to a level of 14-32 pg/pl of blood of presumptive destruxin A. This concentration of destruxin in the blood of mycosed locusts is a much lower level than that found by Samuels et al. (1988b) in M. sexta larvae. The reasons for this are discussed. ABBREVIATIONS cAMP adenosine 3',5'-cyclic monophosphate DMF dimethyl formamide DTNB 5-5'-dithiobis-(2nitrobenzoic acid) E D 50 the 50% effective dose EGTA ethylene glycol-bis(l3-aminoethyl ether)N,N,N',N'-tetraacetic acid EPPS N-(2-hydroxyethyl) piperazine-N'-(3-propane-sulfonic acid) HEPES N-2-hydroxyethyl piperazine-N’-2-ethanesulphonic acid IBMX 3-isobutyl-1 -methylxanthine IC50 concentration required to give 50% inhibition o a dose required to give 50% kill l t 50 time to 50% mortality OD optical density PMSF phenylmethylsulphonyl fluoride ppm parts per million RH relative humidity RP-HPLC reverse phase high performance liquid chromatography SITS 4-acetamido-4'-isothio-cyanatostilbene-2,2’-disulphonic acid spp. species ULV ultra low volume UV ultra-violet w/v weight for volume