Effects of destruxins on free calcium and hydrogen ions in insect hemocytes.

Destruxins, cyclohexadepsipeptidic mycotoxins isolated from the entomopathogenic fungus Metarhizium anisopliae, inhibit innate insect immunity. However, their mechanism of action remains unclear. In this study, the effects of destruxins on changes in free calcium and hydrogen ions in the hemocytes of Exolontha serrulata, Bombyx mori and the Spodoptera litura SL-1 cell line were detected using laser scanning confocal microscopy (LSCM). An instant Ca(2+) influx of hemocytes induced by destruxins A and B (DA and DB) was recorded. The DA/DB-dependent Ca(2+) influx was not influenced by the Ca(2+) channel inhibitors 2-aminoethoxydiphenyl borane (2-APB) and U73122. It also had an apparently different LSCM profile from that of the ionomycin-dependent Ca(2+) influx. However, the instant Ca(2+) influx was not seen in the SL-1 cells; on the contrary, a slow, moderate enhancement of intracellular Ca(2+) was observed. Meanwhile, an instant intracellular free H(+) decrease aroused by DA and DB was found. DB at 20 µmol/L and DA at 690 µmol/L significantly reduced intracellular free H(+) levels. Furthermore, the vacuolar H(+)-ATPase (V-ATPase) inhibitor bafilomycin A1 had obvious effects on the decreases of intracellular free H(+) in hemocytes. These results suggest that the mechanism of DA/DB-dependent Ca(2+) influx is perhaps not related to Ca(2+) channels and ionophores; rather, the intracellular free H(+) decrease might be due to V-ATPase inhibition.

Similarity in venom alkaloid chemistry of alate queens of imported fire ants: implication for hybridization between Solenopsis richteri and S. invicta in the Southern United States.

Both cis- and trans-2-methyl-6-undecylpiperidines, MC11P, have been previously reported as the major components of the venom of alate queens of the imported fire ants, Solenopsis richteri (black) and S. invicta (red). To identify the minor components of venom alkaloids from alate queens and compare the venom alkaloid chemistry of alate queen of their hybrid (S. richteri×S. invicta) with that of the two parental fire ant species (S. richteri and S. invicta), silica-gel short-column chromatography was utilized for separating cis-stereoisomers of venom alkaloids from trans-stereoisomers. GC/MS Analyses of venom-alkaloid chemistry of alate queens demonstrated that fewer alkaloid peaks were detected in the chromatograms of the alate queens compared to those of workers. Three new compounds, 7, 12, and 13, were detected as minor components in the venom of alate queens of all three fire ant species. Alate queens of hybrid fire ants showed cis- and trans-alkaloid patterns similar to those of the parental species. Similarity in venom-alkaloid chemistry of alate queens of S. richteri and S. invicta, and their hybrid may indicate their reproductive compatibility in the hybrid zone in southern United States, where all three species occur sympatrically.

Phthalide derivatives with antifungal activities against the plant pathogens isolated from the liquid culture of Pestalotiopsis photiniae.

Three new phthalide derivatives (1-3) named 5-(3'-methyl-2'-butenyl)-2-hydroxy-3-methoxy-4-methylbenzoic acid (1), 5-(3'-carboxyl-3'-methyl-2E-allyloxy)-3-methoxy-4-methylphthalide (2) and 5-(3',3'-dimethylallyloxy)-2-methoxycarbonyl-3-methoxy-4-methylbenzoic acid (3) together with six known phthalide derivatives named 5-(3',3'-dimethylallyloxy)-3-methoxy-4-methylphthalide (4), zinnimidine (5), 5-(3',3'-dimethylallyloxy)-3-methoxy-4-methylphthalide (6), 5-(3',3'-dimethylallyloxy)-3-methoxy-4-methylphthalic acid (7), zinniol anhydride (8) and porriolide (9) were isolated from the liquid culture of the plant endophytic fungus Pestalotiopsis photiniae isolated from the Chinese Podocarpaceae plant Podocarpus macrophyllus. Their structures were elucidated by extensive spectroscopic analysis. Compounds 1-9 displayed significant antifungal activities against three plant pathogens.

Reduction of venom alkaloids in Solenopsis richteri×Solenopsis invicta hybrid: an attempt to identify new alkaloidal components.

The alkaloid chemistry of the venom of hybrid fire ant, Solenopsis richteri × Solenopsis invicta, was investigated using silica gel chromatography and GC-MS techniques. In addition to most cis alkaloids of parental species, S. richteri Forel and S. invicta Buren, the cis alkaloid fraction of the body extract of hybrid fire ants also contains five significant new alkaloids. Hydrogenation of the cis alkaloid fraction yielded only five piperidines, 4', 12', 12, 20', and 20. Sodium borohydride and lithium aluminum hydride selectively reduced C═N double bond in piperideine alkaloids to give a mixture of cis and trans piperidines. However, reduction of the five new components yielded several new peaks with much longer retention times and increasing molecular weights over 30. It is evident that the chemical identities of the five new peaks are quite different from those known piperidines or piperideines found in Solenopsis fire ants.

Toxicities of destruxins against Bemisia tabaci and its natural enemy, Serangium japonicum.

The bioactivities of destruxins against whitefly, Bemisia tabaci and its natural enemy, ladybird beetle Serangium japonicum were evaluated. Destruxins A and B (DA and DB) showed insignificant ovicidal, oviposition deterrent and systemic insecticidal activities to B. tabaci; however, DA and DB had certain contact virulence to its nymphs. The LC(50) values of DA at 120h to 2nd, 3rd and 4th instars were 89.8 (95% confidence interval as 85.4-94.4), 199.3 (187.7-211.5) and 270.7 (251.5-291.5)mg/L, while the LC(50)s of DB at 120h were 96.5 (92.0-101.2), 216.7 (203.0-231.2), 359.4 (326.6-395.4)mg/L, respectively. In addition, DA exhibited moderate acute contact toxicities towards S. japonicum, the LC(50)s at 48h were 165.4 (132.3-229.4) and 192.5 (148.1-289.2)mg/L for 4th instar larvae and adults. Furthermore, the results from experiments of residual toxicities of DA towards mortalities of 4th instar larvae and adults, pupation rate, emergence rate, average number of egg/female and hatching rate suggested that DA had minimal effects to the ladybird beetle. Generally, the toxicity decreased about 50% from 1st to 3rd-5th day of post-treatment. Specially, the residual toxicity at 50mg/L and the 7th day post-treatment was down to a value not differing significantly from the control.

Investigation of destruxin A and B from 80 Metarhizium strains in China, and the optimization of cultural conditions for the strain MaQ10.

The production levels of destruxin A and B (DA and DB) of 80 Metarhizium strains from China were investigated. The average yields of DA and DB were 14.87+/-2.30 and 3.65+/-0.58 microg/mL, respectively. The strains isolated from soil had significantly (p<0.05) lower production of DA and DB than the strains isolated from insect cadavers. Also, there was a positive correlation in the yields of DA and DB, and the regressive equations were established. Of the 80 strains, MaQ10 had the highest yields of DA and DB, amounted to 106.78+/-9.41 and 29.52+/-2.63 microg/mL, respectively, at 10 days fermentation period. Followed by the strains MaQ05, MaQ07 and MaQ12, their yields of DA were 79.72+/-5.36, 77.69+/-8.54 and 70.04+/-10.1 microg/mL, respectively, and DB were 16.81+/-1.51, 13.38+/-0.41 and 16.88+/-1.15 microg/mL, respectively. Furthermore, the cultural conditions of MaQ10 were optimized to produce DA and DB. The optimal inoculum, initial pH, temperature and rotary speed were 8%, pH 9.0, 27 degrees C and 240 r/min for DA, and 8%, pH 9.0, 25 degrees C and 220 r/min for DB. Under these optimal culture conditions, the predicted production of DA and DB was 193.87 and 39.85 microg/mL, and the actual production was 189.13 and 43.35 microg/mL, respectively.