The cAMP levels were measured with cAMP Enzyme Immunoassay Kit (Sigma, USA), buy Momelotinib according to the manufacturer’s instructions. In total, each assay was repeated three times independently with three biological replicates for every strain. To test whether Go6983 in vivo exogenous cAMP could restore the growth of RNAi mutant, the cAMP analog, 8-Br-cAMP (Sigma, USA) was added to PDA at a final concentration of 5 mM. 8-Br-cAMP (a membrane permeable variant of cAMP) has

been extensively used in various studies to artificially cause the enhancement of endogenous cAMP levels [27–29]. Biomass assay and fungal growth in the haemolymph of locust in vivo and in vitro The virulence of the RNAi mutant and the wild type was tested by topical inoculation and injection into Locusta migratoria adults reared under crowded conditions as previously described by He et al. [30]. The

Locusta migratoria used were all male adult 3 days post-molt. Wild type and RNAi mutants were incubated at 28°C on 1/4 SDAY plates for 15 d. Aliquots of 5 μL solution of 107 conidia/mL Fedratinib price of either wild type M. acridum or RNAi mutant in cottonseed oil were inoculated on the pronotum. Aliquots of 5 μL suspensions (2 × 106 conidia/mL) in sterile water were injected into the hemocoel. Both experiments were repeated five times with 30 insects per replicate. Mortality was recorded every 12 h after topical inoculation and injection. Mortality was then recorded daily, and lethal time

values for 50% mortality (LT50) values were used to estimate the infectivity of M. acridum by DPS software [31]. The growth of M. acridum in the host locust was quantified by the detection of fungal rDNA in the infected locust using real-time PCR [32]. After the extraction of M. acridum DNA and fungal DNA from the infected locust, fungal DNA was detected by an Icycler iQ Quantitative PCR was performed using specific primers of M. acridum: CQMaP-F1: 5′-TGGCATCTTCTGAGTGGTG-3′and CQMaP-R1: 5′-CCCGTTGCGAGTGAGTTA- 3′. To test the fungal growth in the haemolymph of locust in vitro, 50 μL of a conidial suspension (1 × 107 conidia/mL) was inoculated into 950 μL of locust haemolymph, and the growth of the wild type and mutant was detected 24 h post inoculation. Germination and appressoria formation against insect cuticles The percentage of germination Monoiodotyrosine of wild type and RNAi mutant were measured as described by Liu et al.[18]. The appressorium formation rates were determined from 300 conidia after an 18 h induction on locust hind wings according to He and Xia [33]. The assay was replicated at least three times. Oxidative stress, osmotic stress, heat shock and UV-B treatment test Growth characterization of the wild type and RNAi mutants were carried out on 1/4 SDAY supplemented with H2O2 (6 mM) or KCl (1 M). Samples of conidial suspensions (2 μL; 5 × 105 conidia/mL) were spotted on each Petri dish and the plates were incubated at 28°C for 10 d.