CML8 and GAD4 function in (Z)–3–hexenol–mediated defense by regulating GABA accumulation in Arabidopsis

(Z)–3–hexenol, a small gaseous molecule, is produced in plants under biotic stress and induces defense responses in neighboring plants. However, the research on little is known about how (Z)–3–hexenol induces plant defense–related signaling. In this study, we uncovered how (Z)–3–hexenol treatment enhances insect resistance by increasing γ–aminobutyric acid (GABA) contents in Arabidopsis thaliana leaves. First, (Z)–3–hexenol increases the intracellular content of the signaling molecule calcium in Arabidopsis leaf mesophyll cells. Both intracellular and extracellular calcium stores regulate these changes in calcium content. Then, CML8 and GAD4 are involved in calcium signaling. Yeast two–hybrid assays, firefly luciferase complementation imaging, and GST pull–down assays demonstrated that CML8 interacts with GAD4. Finally, (Z)–3–hexenol treatment increased the GABA contents in Arabidopsis leaves, thus increasing plant resistance to the insect Plutella xylostella. This study revealed the mechanism of activating plant insect defense induced by (Z)–3–hexenol, which is of great significance for the study of volatiles as biological control measures.

Dodecanol, metabolite of entomopathogenic fungus Conidiobolus coronatus, affects fatty acid composition and cellular immunity of Galleria mellonella and Calliphora vicina

One group of promising pest control agents are the entomopathogenic fungi; one such example is Conidiobolus coronatus, which produces a range of metabolites. Our present findings reveal for the first time that C. coronatus also produces dodecanol, a compound widely used to make surfactants and pharmaceuticals, and enhance flavors in food. The main aim of the study was to determine the influence of dodecanol on insect defense systems, i.e. cuticular lipid composition and the condition of insect immunocompetent cells; hence, its effect was examined in detail on two species differing in susceptibility to fungal infection: Galleria mellonella and Calliphora vicina. Dodecanol treatment elicited significant quantitative and qualitative differences in cuticular free fatty acid (FFA) profiles between the species, based on gas chromatography analysis with mass spectrometry (GC/MS), and had a negative effect on G. mellonella and C. vicina hemocytes and a Sf9 cell line in vitro: after 48 h, almost all the cells were completely disintegrated. The metabolite had a negative effect on the insect defense system, suggesting that it could play an important role during C. coronatus infection. Its high insecticidal activity and lack of toxicity towards vertebrates suggest it could be an effective insecticide.

Horizontally transmitted parasitoid killing factor shapes insect defense to parasitoids

Interkingdom competition occurs between hymenopteran parasitoids and insect viruses sharing the same insect hosts. It has been assumed that parasitoid larvae die with the death of the infected host or as result of competition for host resources. Here we describe a gene family, parasitoid killing factor (pkf), that encodes proteins toxic to parasitoids of the Microgastrinae group and determines parasitism success. Pkfs are found in several entomopathogenic DNA virus families and in some lepidopteran genomes. We provide evidence of equivalent and specific toxicity against endoparasites for PKFs found in entomopoxvirus, ascovirus, baculovirus, and Lepidoptera through a mechanism that elicits apoptosis in the cells of susceptible parasitoids. This highlights the evolutionary arms race between parasitoids, viruses, and their insect hosts.

Differential Contributions of MYCs to Insect Defense Reveals Flavonoids Alleviating Growth Inhibition Caused by Wounding in Arabidopsis

In Arabidopsis, basic helix–loop–helix transcription factors (TFs) MYC2, MYC3, and MYC4 are involved in many biological processes, such as defense against insects. We found that despite functional redundancy, MYC-related mutants displayed different resistance to cotton bollworm (Helicoverpa armigera). To screen out the most likely genes involved in defense against insects, we analyzed the correlation of gene expression with cotton bollworm resistance in wild-type (WT) and MYC-related mutants. In total, the expression of 94 genes in untreated plants and 545 genes in wounded plants were strongly correlated with insect resistance, and these genes were defined as MGAIs (MYC-related genes against insects). MYC3 had the greatest impact on the total expression of MGAIs. Gene ontology (GO) analysis revealed that besides the biosynthesis pathway of glucosinolates (GLSs), MGAIs, which are well-known defense compounds, were also enriched in flavonoid biosynthesis. Moreover, MYC3 dominantly affected the gene expression of flavonoid biosynthesis. Weighted gene co-expression network analysis (WGCNA) revealed that AAE18, which is involved in activating auxin precursor 2,4-dichlorophenoxybutyric acid (2,4-DB) and two other auxin response genes, was highly co-expressed with flavonoid biosynthesis genes. With wounding treatment, the WT plants exhibited better growth performance than chalcone synthase (CHS), which was defective in flavonoid biosynthesis. The data demonstrated dominant contributions of MYC3 to cotton bollworm resistance and imply that flavonoids might alleviate the growth inhibition caused by wounding in Arabidopsis.

Characterization of PGRP-LB and immune deficiency in the white-backed planthopper Sogatella furcifera (Hemiptera: Delphacidae)

Peptidoglycan recognition proteins (PGRPs) participate in insect defense against bacterial pathogens by recognizing bacterial cell wall peptidoglycans (PGNs). Here, we identified the PGRP-LB gene in the white-backed planthopper Sogatella furcifera (SfPGRP-LB). SfPGRP-LB is a secreted protein with a typical PGN-binding domain and five conserved amino acid (aa) residues required for amidase activity. Expression analysis showed that the SfPGRP-LB transcript levels were significantly higher in the midgut than in other tissues. Silencing SfPGRP-LB with dsRNA significantly downregulated the expression of Toll pathway genes Toll and Dorsal and Imd pathway genes Imd and Relish after Escherichia coli challenge. However, only Toll and Dorsal expressions were downregulated after Staphylococcus aureus challenge. E. coli and S. aureus challenges rapidly and strongly upregulated SfPGRP-LB expression. Recombinantly expressed SfPGRP-LB (rSfPGRP-LB) had strong affinities for E. coli Dap-type PGN and S. aureus Lys-type PGN and agglutinated the bacteria. However, rSfPGRP-LB inhibited S. aureus but not E. coli growth. Furthermore, rSfPGRP-LB had amidase activity, degraded Lys-type PGN, and destroyed S. aureus cell walls but had no such effects on E. coli Dap-type PGN. Thus, SfPGRP-LB recognizes and binds various bacterial PGNs but only has amidase activity against Lys-type PGN.

Cloning, expression and characterization of arcelin and its impact on digestive enzymes of the stored product insect pest, Callosobruchus maculatus (F.)

Key message The defensive role of arcelin from Phaseolus lunatus for its protection against insect pest Callosobruchus maculatus was elucidated. The potent insecticidal property of arcelin and its impact on pest molecular physiology has identified the adaptive strategies. Abstract The pulse beetle Callosobruchus maculatus causes potential damage to legume crops by infesting the seeds and causes a reduction in the content of total protein. Arcelin found in the wild accessions of the common bean, is an insecticidal protein that has the potency to hamper the metabolism of the bruchid beetle. The arcelin gene from the wild accession of Phaseolus lunatus was isolated and the ORF encoding 158 amino acids was cloned in pET-45b (+) vector. The recombinant was transformed into BL21 STAR (DE3) pLysS cells, and the expressed arcelin was purified using Ni-NTA column. The recombinant protein was used in preparing artificial diet and the insecticidal activity was elucidated against the bruchid pest C. maculatus. Adult emergence and seed damage were drastically reduced in the treated groups. The response towards ingested diet by digested enzymes was elucidated through quantitative gene expression. Maximum expression was observed in the aminopeptidase, followed by upregulation of alpha-amylase, glycoside hydrolase family 31 and cathepsin D-like aspartic protease, and downregulation of cathepsin L-like cysteine protease. These results showed the anti-metabolic nature of the recombinant arcelin. The changes in digestive enzymes to counteract the anti-nutritional nature of the protein were the strategies of the insect defense mechanism.

Insects defend against fungal infection by employing microRNAs to silence virulence-related genes

Chemical insecticides remain the main strategy to combat mosquito-borne diseases, but the growing threat of insecticide resistance prompts the urgent need to develop alternative, ecofriendly, and sustainable vector control tools. Entomopathogenic fungi can overcome insecticide resistance and represent promising biocontrol tools for the control of mosquitoes. However, insects have evolved robust defense mechanisms against infection. Better understanding of mosquito defenses against fungal infection is critical for improvement of fungal efficacy. Here, we show that as the pathogenic fungus Beauveria bassiana penetrates into the host hemocoel, mosquitoes increase expression of the let-7 and miR-100 microRNAs (miRNAs). Both miRNAs translocate into fungal hyphae to specifically silence the virulence-related genes sec2p and C6TF, encoding a Rab guanine nucleotide exchange factor and a Zn(II)2Cys6 transcription factor, respectively. Inversely, expression of a let-7 sponge (anti–let-7) or a miR-100 sponge (anti–miR-100) in the fungus efficiently sequesters the corresponding translocated host miRNA. Notably, B. bassiana strains expressing anti–let-7 and anti–miR-100 are markedly more virulent to mosquitoes. Our findings reveal an insect defense strategy that employs miRNAs to induce cross-kingdom silencing of pathogen virulence-related genes, conferring resistance to infection.

Survival Sounds in Insects: Diversity, Function, and Evolution

Insect defense sounds have been reported for centuries. Yet, aside from the well-studied anti-bat sounds of tiger moths, little is understood about the occurrence, function, and evolution of these sounds. We define a defense sound as an acoustic signal (air- or solid-borne vibration) produced in response to attack or threat of attack by a predator or parasitoid and that promotes survival. Defense sounds have been described in 12 insect orders, across different developmental stages, and between sexes. The mechanisms of defensive sound production include stridulation, percussion, tymbalation, tremulation, and forced air. Signal characteristics vary between species, and we discuss how morphology, the intended receiver, and specific functions of the sounds could explain this variation. Sounds can be directed at predators or non-predators, and proposed functions include startle, aposematism, jamming, and alarm, although experimental evidence for these hypotheses remains scant for many insects. The evolutionary origins of defense sounds in insects have not been rigorously investigated using phylogenetic methodology, but in most cases it is hypothesized that they evolved from incidental sounds associated with non-signaling behaviors such as flight or ventilatory movements. Compared to our understanding of visual defenses in insects, sonic defenses are poorly understood. We recommend that future investigations focus on testing hypotheses explaining the functions and evolution of these survival sounds using predator-prey experiments and comparative phylogenetics.