Insecticidal crystal toxins derived from the soil bacterium Bacillus thuringiensis (Bt) are widely used as biopesticide sprays or expressed in transgenic crops to control insect pests. However, large-scale use of Bt has led to field-evolved resistance in several lepidopteran pests. Resistance to Bt Cry1Ac toxin in the diamondback moth, Plutella xylostella (L.), was previously mapped to a multigenic resistance locus (BtR-1). Here, we assembled the 3.15 Mb BtR-1 locus and found high-level resistance to Cry1Ac and Bt biopesticide in four independent P. xylostella strains were all associated with differential expression of a midgut membrane-bound alkaline phosphatase (ALP) outside this locus and a suite of ATP-binding cassette transporter subfamily C (ABCC) genes inside this locus. The interplay between these resistance genes is controlled by a previously uncharacterized trans-regulatory mechanism via the mitogen-activated protein kinase (MAPK) signaling pathway. Molecular, biochemical, and functional analyses have established ALP as a functional Cry1Ac receptor. Phenotypic association experiments revealed that the recessive Cry1Ac resistance was tightly linked to down-regulation of ALP, ABCC2 and ABCC3, whereas it was not linked to up-regulation of ABCC1. Silencing of ABCC2 and ABCC3 in susceptible larvae reduced their susceptibility to Cry1Ac but did not affect the expression of ALP, whereas suppression of MAP4K4, a constitutively transcriptionally-activated MAPK upstream gene within the BtR-1 locus, led to a transient recovery of gene expression thereby restoring the susceptibility in resistant larvae. These results highlight a crucial role for ALP and ABCC genes in field-evolved resistance to Cry1Ac and reveal a novel trans-regulatory signaling mechanism responsible for modulating the expression of these pivotal genes in P. xylostella. Biopesticide and transgenic crops based on Bacillus thuringiensis (Bt) Cry toxins are widely used worldwide, yet the development of field resistance seriously threatens their sustainability. Unraveling these resistance mechanisms are of great importance for delaying insect field resistance evolution. The diamondback moth was the first insect to evolve field resistance to Bt biopesticides and it is an excellent model for the study of Bt resistance mechanisms. In this work, we present strong empirical evidence supporting that (1) field-evolved resistance to Bt in P. xylostella is tightly associated with differential expression of a membrane-bound alkaline phosphatase (ALP) and a suite of ATP-binding cassette transporter subfamily C (ABCC) genes, and (2) a constitutively transcriptionally-activated upstream gene (MAP4K4) in the MAPK signaling pathway is responsible for this trans-regulatory signaling mechanism. These findings identify key resistance genes and provide the first comprehensive mechanistic description responsible for the field-evolved Bt resistance in P. xylostella. Given that expression alterations of multiple receptor genes result in Bt resistance in many other insects, it can now be tested to determine whether the previously unidentified trans-regulatory mechanism characterized in this study is also involved in these cases.

Significance: Most studies of pesticide resistance have focused on the identification and functional analysis of resistance genes, but few studies have considered the signaling pathways involved in their regulation. In this work, we discovered that overexpression of a P450 that confers resistance to neonicotinoid insecticides in whitefly is trans -regulated by the transcription factor CREB . Further studies demonstrated that exposure to neonicotinoid insecticides activates a key pathway involved in the cellular response to extracellular signals, the MAPK signaling pathway, that activates CREB by phosphorylation. CREB then binds to a specific site on the promoter of CYP6CM1 resulting in its increased expression. These findings reveal mechanisms underlying the regulation of P450-mediated pesticide resistance and also provide a potential target for pest control.

Clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-associated gene Cas9 represent an invaluable system for the precise editing of genes in diverse species. The CRISPR/Cas9 system is an adaptive mechanism that enables bacteria and archaeal species to resist invading viruses and phages or plasmids. Compared with zinc finger nucleases and transcription activator-like effector nucleases, the CRISPR/Cas9 system has the advantage of requiring less time and effort. This efficient technology has been used in many species, including diverse arthropods that are relevant to agriculture, forestry, fisheries and public health; however, there is no review that systematically summarizes its successful application in the editing of both insect and non-insect arthropod genomes. Thus, this paper seeks to provide a comprehensive and impartial overview of the progress of the CRISPR/Cas9 system in different arthropods, reviewing not only fundamental studies related to gene function exploration and experimental optimization but also applied studies in areas such as insect modification and pest control. In addition, we also describe the latest research advances regarding two novel CRISPR/Cas systems (CRISPR/Cpf1 and CRISPR/C2c2) and discuss their future prospects for becoming crucial technologies in arthropods.

The arms race between entomopathogenic bacteria and their insect hosts is an excellent model for decoding the intricate coevolutionary processes of host-pathogen interaction. Here, we demonstrate that the MAPK signaling pathway is a general switch to trans-regulate differential expression of aminopeptidase N and other midgut genes in an insect host, diamondback moth (Plutella xylostella), thereby countering the virulence effect of Bacillus thuringiensis (Bt) toxins. Moreover, the MAPK cascade is activated and fine-tuned by the crosstalk between two major insect hormones, 20-hydroxyecdysone (20E) and juvenile hormone (JH) to elicit an important physiological response (i.e. Bt resistance) without incurring the significant fitness costs often associated with pathogen resistance. Hormones are well known to orchestrate physiological trade-offs in a wide variety of organisms, and our work decodes a hitherto undescribed function of these classic hormones and suggests that hormonal signaling plasticity is a general cross-kingdom strategy to fend off pathogens.