BackgroundDestruxins (dtxs) are the mycotoxin produced by certain entomopathogenic fungi, such as Metarhizium anisopliae, Aschersonia sp, Alternaria brassicae and Ophiosphaerella herpotrichae. It can affect a wide variety of biological processes in insects, including innate immune, Ca2+ channel in cells, and apoptosis in a dose-dependent manner. Dtxs have been used as biological control agent for a long time, however, their molecular mechanism of action is still unknown.Principal FindingsIn this study, both digital gene expression (DGE) and two-dimensional electrophoresis (2-DE) approaches were adopted to examine the effects of dtx A on Plutella xyllostella (L.) larvae. By using DGE and 2-DE analyses, 1584 genes and 42 protein points were identified as being up- or down regulated at least 2-fold in response to dtx A. Firstly, injection of dtx A to larvae accelerated the increase of peptidoglycan recognition protein (PGRP), which could activate the Toll signal pathway inducing production of antibacterial substances such as cecropin and gloverin. Dtx A also stimulated prophenoloxidase (proPO) system which plays an important role in innate immunity and leads to melanization of external organisms. Secondly, dtx A suppressed the expression of genes related to the Toll pathway, and induced expression of serine proteinase inhibitors (serpins), especially the serpin 2 that blocked process of the proPO system. Finally, other physiological process like xenobiotics detoxification, apoptosis, calcium signaling pathway and insect hormone biosynthesis, were also mediated in response to dtx A toxicity.ConclusionsTranscript and protein profiling analyses will provide an insight into the potential molecular mechanism of action in P. xylostella larvae in response to dtx A.

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Destruxin A is a mycotoxin that is secreted by entomopathogenic fungi which has a broad-spectrum insecticidal effect. Previous transcript and protein profiling analysis showed that destruxin A has significant effects on the expression of serine protease inhibitor genes (serpin-2, 4, 5) in the larvae of Plutella xylostella. In the current study, we aimed to understand the role of serpins under application of destruxin A. We obtained two full-length cDNA sequences of P. xylostella serpins, named serpin-4 and serpin-5, and cloned the serpin-2 gene whose full-length has already been published. Phylogenetic analysis indicated that these two serpin genes were highly clustered with other serpins associated with the immune response in other insects. The temporal and spatial expression of serpin-2, serpin-4 and serpin-5 were determined to be the highest in the fat body and hemolymph of 4th larval stage using qRT-PCR and western blot detection techniques. RNA interference (RNAi) mediated knockdown of P. xylostella serpin genes was carried out by microinjection of double-stranded RNA (dsRNA). The expression levels of serpins decreased significantly after RNAi. Results showed that the depletion of serpins induced cecropins expression, increased phenoloxidase (PO) activity, body melanization and mortality in the larvae of P. xylostella under the same lethal concentration of destruxin A. The superimposed effects of serpins RNAi were similar with the destruxin A treatment upon mortality of P. xylostella larvae. We discovered for the first time that serpins play indispensable role in P. xylostella when challenged by destruxin A and deduced the possible function mechanism of destruxin A. Our findings are conducive to fully understanding the potential insecticidal mechanism of destruxin A and constitute a well-defined potential molecular target for novel insecticides.

Diacetyl is a natural byproduct of fermentation and known to be an important flavor compound in many food products. Because of the potential undesirable effects of diacetyl on health safety and beer flavor, determination of its concentration in beer samples is essential and its analytical methods have attracted close attention recently. The aim of the present work is to develop and validate a novel high-performance liquid chromatography method for the quantification of diacetyl in beer based on the derivatization reaction of diacetyl with 4-nitro-o-phenylenediamine (NPDA). After the derivatization with NPDA in pH 3.0 at 45 °C for 20 min, diacetyl was separated on a kromasil C(18) column at room temperature in the form of the resulting 6-nitro-2,3-dimethylquinoxaline and detected by the ultraviolet detector at 257 nm. The results showed that the correlation coefficient for the method was 0.9992 in the range of 0.0050-10.0 mg L(-1) and the limit of detection was 0.0008 mg L(-1) at a signal-to-noise ratio of 3. The applicability of the proposed method was evaluated in the analysis of beer samples with the recovery range of 94.0-99.0% and relative standard deviation range of 1.20-3.10%. The concentration levels of diacetyl detected in beer samples from 12 brands ranged from 0.034 to 0.110 mg L(-1). The proposed method showed efficient chromatographic separation, excellent linearity, and good repeatability that can be applied to quantification of diacetyl in beer samples.

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Background Entomogenous fungi are important factors in biological control, but innate immunity of insects restricts the efficiency of fungus infection. 20‐hydroxyecdysone (20E) is involved in regulating the immune response of insects. Our previous studies have revealed that 20E enhances the expression of antibacterial peptides in the worldwide pest Locusta migratoria. However, the mechanism by which 20E controls innate immunity against entomogenous fungi is still unknown. Results In the present study, based on the transcriptome of L. migratoria fat bodies challenged by 20E, immulectin‐1 (LmIML‐1) was screened and identified to be involved in modulating antifungal immunity. Spatio‐temporal expression analysis showed LmIML‐1 was highly expressed in the fifth instar nymph stage, and mainly distributed in the fat bodies and hemolymph. Both exogenous and endogenous 20E could increase the transcription of LmIML‐1. In contrast, transcription of LmIML‐1 did not increase when the 20E signal was blocked by RNAi of LmEcR (ecdysone receptor). The expressed recombinant protein rLmIML‐1 possessed agglutination activity and promoted the encapsulation. RNA interference of LmIML‐1 reduced the encapsulation of hemocytes, decreased the antifungal activity of plasma against Metarhizium anisopliae and accelerated the death of nymphs under the stress of entomogenous fungus. Meanwhile, 20E did not increase the antifungal activity with silence of LmIML‐1 in L. migratoria. Conclusion 20E enhances antifungal immunity by activating immulectin‐1 in L. migratoria. Our findings indicate a potential mechanism of 20E systematically regulating innate immune response to resist pathogens and provide a well‐defined molecular target for improving biological control. © 2019 Society of Chemical Industry

Destruxin A (DA), a cyclodepsipeptidic secondary metabolite of the entomopathogenic fungus, Metarhizium anisopliae, is an important anti-immunity agent against insect hemocytes. To understand the mechanism of the molecular responses to DA, fifth-instar larvae of the silkworm, Bombyx mori, were injected with 2 μg of DA. The proteomics of hemocytes were then investigated using two-dimensional electrophoresis and mass spectrometry, and validated qPCR. As a result, a total of 47 differently expressed protein spots were detected and 22 proteins in 26 spots were identified. There are eight immunity-related proteins, including three downregulated proteins (antitrypsin isoform 3, p50 protein, and calreticulin precursor) and five upregulated proteins (C-type lectin 10 precursor, serine proteinase-like protein, paralytic peptide, PPO-1, and PPO-2). Four resistance- and/or stress-related proteins (arginine kinase, carboxylesterase clade H, member 1, aminoacylase, and thiol peroxiredoxin) were upregulated. Ten proteins with other or unknown functions were also recorded. Five selected proteins were verified with qPCR. These results provide new insights into the molecular mechanism of host immune response to DA challenge.

BACKGROUNDPPO (prophenoloxidase) cascade plays an important role in resisting invasion of entomogenous fungus. The 20‐hydroxyecdysone (20E) exerts potent effect on the innate immunity in many insects. However, whether 20E controls the PPO cascade system against fungi and the regulatory mechanism in insects remains unclear.RESULTSIn this study, both the proteome and transcriptome of Locusta migratoria were determined followed by the induction of 20E. Pattern recognition receptor GNBP‐2 (Gram‐negative binding proteins) has been identified that responded to 20E at both messenger RNA (mRNA) and protein levels. The PPO gene expression in fat body and PO (phenoloxidase) activity in plasma was found significantly induced after 20E injection and during the high‐20E developmental stage. However, when 20E signal was blocked by RNA interference (RNAi) of ecdysone receptor, the expression level of PPO and PO activity failed to be increased by 20E. Thus, 20E could not significantly induce the expression of PPO gene and PO activity after RNAi of GNBP‐2. Furthermore, 20E treatment notably enhanced the resistance of L. migratoria against Metarhizium anisopliae. Followed by of GNBP‐2 silencing, the mortality of nymphs was significantly increased under the stress of Metarhizium anisopliae, and 20E injection could not increase the resistance.CONCLUSIONThe 20E regulates the PPO system to resist fungal invasion via regulating GNBP‐2 in worldwide pest L. migratoria. Our results provide insight into the mechanism of how 20E enhances the antimicrobial immunity, and will be beneficial for modification of entomogenous fungi targeting on hormones and the immune system. © 2020 Society of Chemical Industry