Virulence of three Aspergillus species to the model insect Galleria mellonella and the contribution of ergot alkaloids to the pathogenic potential of Aspergillus leporis

Larvae of the model insect Galleria mellonella were purchased from Josh’s Frogs (Owosso, MI) and injected with conidia (20 µL at 3,000 conidia/µL, or in one experiment 250 conidia/µL) of Aspergillus leporis, A. hancockii, and A. homomorphus with the aid of a 29-gauge insulin syringe. Control groups of 12 to 15 larvae per trial were injected with 20 µL of PBS solution (phosphate buffered saline) lacking Aspergillus spores. Injected larvae were incubated at room temperature (or in one trial at 37 °C) and observed for one to two weeks following inoculation; any dead larvae were removed throughout this period. Insects also were inoculated topically by placing larvae on sporulating cultures on sucrose-yeast extract agar (20 g sucrose, 10 g yeast extract, 1 g magnesium sulfate heptahydrate, and 15 g agar per liter) for 24 hours before removing them to empty Petri dishes. Insects inoculated through this topical, or natural, approach were observed for 16 days following inoculation. Alkaloids were extracted from individual larvae eight days post-inoculation by bead-beating in 1 mL of methanol. Alkaloids also were extracted from fungi cultured on sucrose-yeast extract agar medium for at least six days at room temperature, 30°C, or 37°C. Samples of ~400 µL volume (containing the hyphae and spores of the fungus and the supporting agar medium) were combined with 400 µL of methanol, rotated end-over-end (~40 rpm) for 30 minutes, and then centrifuged for 10 minutes for clarification. Extracts (20 µL) were analyzed by high performance liquid chromatography (HPLC) with fluorescence detection. Fluorescence of lysergic acid derivatives was detected by excitation at 310 nm and measuring the emission at 410 nm; chanoclavine-I, which has a different double bond structure in its ring system, was detected with fluorescence settings of 272 nm/372 nm. Concentrations of lysergic acid amides were estimated by comparison of areas under HPLC peaks to a standard curve prepared from dilutions of ergonovine, which contains the same fluorophore as the other lysergic acid amides. Because of this comparison to ergonovine, the concentrations of LAH and ergine should be considered as ‘relative to ergonovine’ rather than as absolute.

模式昆虫大蜡螟（Galleria mellonella）的幼虫购自Josh’s Frogs（位于美国密歇根州奥沃索市），借助29号胰岛素注射器，注射兔形曲霉（Aspergillus leporis）、汉考克曲霉（A. hancockii）及同形曲霉（A. homomorphus）的分生孢子：注射体积为20 µL，孢子浓度为3000个孢子/µL，其中一组实验采用250个孢子/µL的浓度。每组试验设置12~15只对照幼虫，注射20 µL不含曲霉孢子的磷酸盐缓冲液（phosphate buffered saline，PBS）。注射后的幼虫置于室温（其中一组试验置于37 ℃）下培养，接种后观察1~2周，试验期间随时移除死亡幼虫。此外，研究还采用体表接种法：将幼虫置于蔗糖-酵母提取物琼脂培养基（每升培养基含蔗糖20 g、酵母提取物10 g、七水硫酸镁1 g、琼脂15 g）的产孢培养物上放置24小时，随后转移至空的培养皿中。采用该体表（即天然）接种方式的幼虫，需在接种后观察16天。接种后第8天，通过研磨珠破碎法，在1 mL甲醇中提取单只幼虫体内的生物碱。同时，将真菌接种于蔗糖-酵母提取物琼脂培养基上，分别置于室温、30 ℃或37 ℃下培养至少6天后，提取其生物碱：取约400 µL样品（含真菌菌丝、孢子及配套琼脂培养基），加入400 µL甲醇，以约40 rpm的转速滚转混匀30分钟，随后离心10分钟以澄清样品。取20 µL提取物，采用高效液相色谱（high performance liquid chromatography，HPLC）结合荧光检测法进行分析。其中，麦角酸类衍生物的荧光检测参数为：激发波长310 nm，发射波长410 nm；而查诺克拉文-I（chanoclavine-I）因其环系双键结构不同，采用272 nm/372 nm的荧光检测参数。通过将色谱峰面积与麦角新碱（ergonovine）的稀释标准曲线进行比对，估算麦角酰胺类化合物的浓度。由于麦角新碱与其他麦角酰胺类化合物拥有相同的荧光基团，因此本次检测中LAH及麦角碱（ergine）的浓度均为相对于麦角新碱的相对浓度，而非绝对浓度。