The production relationship of destruxins and blastospores of Metarhizium anisopliae with virulence against Plutella xylostella

Metarhizium anisopliae as an essential entomopathogenic fungus has been known to produce destruxins （a kind of cyclo-peptidic mycotoxins） and blastospores in submerged culture. Blastospores and destruxins are candidates for in- secticides, but the relations of both productions and the impact factors are unclear yet. In this study, we investigated the effects of inoculums, rotation, dissolved oxygen （DO） on the productions of blastospores and destruxins A and B （DA and DB） in submerged culture of M. anisopliae strain MaQ01. The results indicated that DO levels were regulated by inoculum amounts and rotation speeds, meanwhile, the productions of DA, DB and blastospores were also closely influenced by those factors. Totally, when DO value was more than 40%, the higher productions of destruxins and blastospores were achieved, by contrast, lower than 40% of DO values resulted in lower productions. The regression analysis suggested that the productions of DA, DB and blastospores were positively correlated with the DO levels. Meanwhile, the positive correlations between the productions of DA or DB and blastospores were also found. Briefly, when the rotation is 150 r min-1 and the inoculum is 1.0xl06 spore mL-1, the DA, DB and blastospores achieved the best production of 61.81 mg mL-1, 24.74 mg mL-1 and 5.73x 108 spore mL-1, respectively. In addition, the pathogenicities of blastospores and conidia against Plutella xylostella were bioassayed. The higher mortalities of P. xylostella were totally recorded in blastospore treatments than in conidia treatments, especially in lower dosages and earlier periods. Our research will give some new insights to production of destruxins and blastospores by using M. anisopliae.

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

Destruxins, cyclohexadepsipeptidic mycotoxins isolated from the ento mopathogenic fungus Metarhizium anisopliae, inhibit innate insect immunity. However, their mechanism of action remains unclear. In this study, the effects ofdestruxins on changes in free calcium and hydrogen ions in the hemocytes ofExolontha serrulata, Bombyx mori and the Spodoptera litura SL1 cell line were detected using laser scanning confocal mi croscopy （LSCM）. An instant Ca2＋ influx of hemocytes induced by destruxins A and B （DA and DB） was recorded. The DA/DBdependent Ca2＋ influx was not influenced by the Ca2＋ channel inhibitors 2aminoethoxydiphenyl borane （2APB） and U73122. It also had an apparently different LSCM profile from that of the ionomycindependent Ca2＋ influx. However, the instant Ca2＋ influx was not seen in the SL1 cells; on the contrary, a slow, moderate enhancement of intracellular Ca2＋ was observed. Meanwhile, an instant intracellular free H＋ decrease aroused by DA and DB was found. DB at 20/zmol/L and DA at 690/zmol/L significantly reduced intracellular free H＋ levels. Furthermore, the vacuolar H＋ATPase （VATPase） inhibitor bafilomycin A1 had obvious effects on the decreases ofintracellular free H＋ in hemocytes. These results suggest that the mechanism of DA/DBdependent Ca2＋ influx is perhaps not related to Ca2＋ channels and ionophores; rather, the intracellular free H＋ decrease might be due to VATPase inhibition.

一株真菌所产环缩酚酸肽类化合物的分离和鉴定

对一株真菌ZME及其所产的次级代谢产物进行分离鉴定。通过形态观察和ITS测序对真菌ZME进行菌株鉴定,并对其发酵液上清液和菌丝体分别用乙酸乙酯和甲醇萃取;萃取后的有机试剂粗提物经硅胶柱层析和半制备HPLC分离纯化;纯化后的各组分利用光谱分析进行结构解析。结果表明,真菌ZME初步判断属于Beauveria felina。对这株真菌次级代谢物结构解析,共鉴定5个化合物Isaridin B、Isaridin A、Roseocardin、Roseotoxin B和Destruxin B,这5个化合物均为环缩酚酸肽类化合物,其中Roseocardin具有抗细菌活性。

Effects of Destruxin A on Hemocytes Morphology of Bombyx mori

Destruxin A （DA）, a kind of cyclo-hexadepsipeptide isolated from entomopathogenic fungus, Metarhizium anisopliae, is an inhibitor of insect＇s immunity. But its mechanism has not been clarified yet. In this study, the effects of DA on morphologic changes of in vivo and in vitro hemocytes of silkworm, Bombyx mori, were investigated by means of inverted phase contrast microscopy （IPCM）, fluorescence microscopy （FCM） and environmental scanning electron microscopy （ESEM）. The results indicated that DA was cytotoxic to granulohemocytes （GR） and plasmatocytes （PL）. The LC 50 values of DA against in vitro GR and PL of silkworm were 68.77 and 84.11 μg mL-1, respectively. However, the hemocytes in vivo were more susceptible to DA, although at the extremely low dose of 10 μL of 12.5 μg mL-1 for each insect （i.e., 0.036 μg g-1 body weight, or approximately 0.25 μg mL-1 hemolymph）, DA could induce obviously morphologic alterations of hemocytes in vivo. The results imply that there might be some factors in silkworm＇s hemolymph, which influence the interaction of DA and hemocytes.

Experimental and Computational Analysis of the Solution and Solid-State Conformations of Hexadepsipeptides from Beauveria felina

Chemical investigation of Beouverio felina,a marine-derived entomopathogenic fungus,obtained two new destruxin hexadepsipeptides(1 and 2)containing an/.-Ala^(5)residue instead of the usual A/-Me-L-Ala^(5).Interestingly,the presence of the/.-Ala^(5)residue enabled two slowly-exchanging solution conformers with cis/trons(ILE^(3))-(A/-Me-Val^(4))amide bond for 1 and 2 with 1.5:1 and 1:1 cis/trans ratios,respectively,while only the conformer with c/s amide bond was found in the solid-state.The solution and solid-state conformers were studied by NMR,X-ray diffraction and solution and solid-state ECD measurements,as well as by TDDFT-ECD calculations.Structural(conformational)parameter-activity correlations suggested that the extended destruxin geometry with the preferred c/s(ILE^(3))-(A/-Me-Val^(4))bond probably resulted in improved bioactivity and reduced toxicity for the destruxin class.This was the first report on the interconversion phenomenon of cis/trans amide bonds of destruxin class,which could give insights in the development of bioactive destruxin agents with lower toxicity.