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.

The Q biotype of Bemisia tabaci (Gennadius), which has been described from the Mediterranean/North African region, was identified for the first time infesting ornamental crop species in several locations in China. Identification and partial distributions of the exotic B biotype and the recently introduced Q biotype in China were established by using the mitochondrial cytochrome oxidase I gene (mtCOI) as a molecular marker. Collections of B. tabaci were made from representative geographical locations and plant hosts in different provinces of China. MtCOI sequence analysis revealed that collections from Beijing [AY582872, AY589499], Yunnan [AY518189, AY587516], and Henan [AY587514] shared >99.6% sequence identity with the Q biotype from Spain [AY587513, AY562216, AY596950]. The Q type from China shared 98.9-99.4% nucleotide sequence identity with Q-like relatives of B. tabaci described from Israel [AY518191, AY582869]. Phylogenetic analyses indicated that certain B. tabaci populations that are pres... Resumen El biotipo Q de Bemisia tabaci (Genn.), el cual fue descrito de la región Mediterráneo/Norte de Africa, fue identificado por primera vez infestando especies de cultivos ornamentales en varios lugares de China. La identificación y la distribución parcial del exótico biotipo B y el recién introducido biotipo Q en China fueron determinados usando el gen citocromo oxidase I mitocondrial (mtCOI) como un marcador molecular. Se realizaron colecciones de Bemisia tabaci de lugares geográficos representativos y hospederos de plantas en diferentes provincias de China. El análisis de la secuencia de mtCOI reveló que las colecciones de Beijing [AY582872, AY589499], Yunnan [AY518189, AY587516] y Henan [AY587514] compartieron >99.6% de la identidad de la secuencia con el biotipo Q de España. [AY587513, AY562216, AY596950]. El tipo Q de China compartieron 98.9-99.4% de la identidad de la secuencia de nucleótidos con los relacionados de clase como del tipo Q de B. tabaci descritos de Israel [AY518191, AY582869]. E...

Quantitative real-time PCR (qRT-PCR), a primary tool in gene expression analysis, requires an appropriate normalization strategy to control for variation among samples. The best option is to compare the mRNA level of a target gene with that of reference gene(s) whose expression level is stable across various experimental conditions. In this study, expression profiles of eight candidate reference genes from the diamondback moth, Plutella xylostella, were evaluated under diverse experimental conditions. RefFinder, a web-based analysis tool, integrates four major computational programs including geNorm, Normfinder, BestKeeper, and the comparative ΔCt method to comprehensively rank the tested candidate genes. Elongation factor 1 (EF1) was the most suited reference gene for the biotic factors (development stage, tissue, and strain). In contrast, although appropriate reference gene(s) do exist for several abiotic factors (temperature, photoperiod, insecticide, and mechanical injury), we were not able to identify a single universal reference gene. Nevertheless, a suite of candidate reference genes were specifically recommended for selected experimental conditions. Our finding is the first step toward establishing a standardized qRT-PCR analysis of this agriculturally important insect pest.

Accurate evaluation of gene expression requires normalization relative to the expression of reliable reference genes. Expression levels of “classical” reference genes can differ, however, across experimental conditions. Although quantitative real-time PCR (qRT-PCR) has been used extensively to decipher gene function in the sweetpotato whitefly Bemisia tabaci, a world-wide pest in many agricultural systems, the stability of its reference genes has rarely been validated. In this study, 15 candidate reference genes from B. tabaci were evaluated using two Excel-based algorithms geNorm and Normfinder under a diverse set of biotic and abiotic conditions. At least two reference genes were selected to normalize gene expressions in B. tabaci under experimental conditions. Specifically, for biotic conditions including host plant, acquisition of a plant virus, developmental stage, tissue (body region of the adult), and whitefly biotype, ribosomal protein L29 was the most stable reference gene. In contrast, the expression of elongation factor 1 alpha, peptidylprolyl isomerase A, NADH dehydrogenase, succinate dehydrogenase complex subunit A and heat shock protein 40 were consistently stable across various abiotic conditions including photoperiod, temperature, and insecticide susceptibility. Our finding is the first step toward establishing a standardized quantitative real-time PCR procedure following the MIQE (Minimum Information for publication of Quantitative real time PCR Experiments) guideline in an agriculturally important insect pest, and provides a solid foundation for future RNA interference based functional study in B. tabaci.

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.