Characterization of the role of alpha-arylphorin in the \u3ci\u3eHeliothis virescens\u3c/i\u3e midgut response to Cry1Ac toxin from \u3ci\u3eBacillus thuringiensis\u3c/i\u3e

Homeostasis of the intestinal epithelium in Heliothis virescens is mediated by the proliferation and differentiation of multipotent intestinal stem cells (ISCs) that lie adjacent to the basal lamina. In response to extrinsic and intrinsic signals, ISC proliferation and differentiation promotes epithelial growth and regeneration following the loss of integrity. We tested the in vivo effects of the ISC mitogen, a [alpha]-arylphorin, on ISC proliferation and the morphological changes of the midgut during larval development. Additionally, we examined how these changes affected the intestinal epithelium response to Cry1Ac toxin from Bacillus thuringiensis. Histological and in vitro evidence supported two distinct mechanisms whereby ingestion of -arylphorin reduced Cry1Ac toxicity in H. virescens larvae. To characterize the role of alkaline phosphatase as a Cry toxin receptor in the H. virescens larval midgut, we used mechanistically distinct double-stranded RNA (dsRNA) delivery strategies to activate RNA-interference (RNAi) and induce transient silencing of midgut alkaline phosphatase (HvmALP) prior to exposing larvae to Cry1Ac toxin. Quantitative and qualitative analyses of gene and protein expression supported that hemocoelic microinjections resulted in the largest reduction in HvmALP protein expression and, furthermore, that bypassing physiological barriers such as the intestinal epithelium may enhance the efficiency of target gene silencing

Reduced Levels of Membrane-Bound Alkaline Phosphatase Are Common to Lepidopteran Strains Resistant to Cry Toxins from Bacillus thuringiensis

Development of insect resistance is one of the main concerns with the use of transgenic crops expressing Cry toxins from the bacterium Bacillus thuringiensis. Identification of biomarkers would assist in the development of sensitive DNA-based methods to monitor evolution of resistance to Bt toxins in natural populations. We report on the proteomic and genomic detection of reduced levels of midgut membrane-bound alkaline phosphatase (mALP) as a common feature in strains of Cry-resistant Heliothis virescens, Helicoverpa armigera and Spodoptera frugiperda when compared to susceptible larvae. Reduced levels of H. virescens mALP protein (HvmALP) were detected by two dimensional differential in-gel electrophoresis (2D-DIGE) analysis in Cry-resistant compared to susceptible larvae, further supported by alkaline phosphatase activity assays and Western blotting. Through quantitative real-time polymerase chain reaction (qRT-PCR) we demonstrate that the reduction in HvmALP protein levels in resistant larvae are the result of reduced transcript amounts. Similar reductions in ALP activity and mALP transcript levels were also detected for a Cry1Ac-resistant strain of H. armigera and field-derived strains of S. frugiperda resistant to Cry1Fa. Considering the unique resistance and cross-resistance phenotypes of the insect strains used in this work, our data suggest that reduced mALP expression should be targeted for development of effective biomarkers for resistance to Cry toxins in lepidopteran pests

Enterococcus faecalis Colonization of the Helicoverpa zea Midgut Epithelium

In recent years, there has been a surge in interdisciplinary efforts to elucidate the nutrient acquisition mechanisms that contribute to the success of a bacterium to not only survive in the gastrointestinal tract (G.I.) of Lepidopteran insects, but rather to thrive in it. However, bacterial colonization of Lepidoptera, one of the largest insect orders containing over 180,000 species is, to this day, poorly understood. Most of the works describing intestinal colonization have employed methods that inoculate larvae with an excessive number of bacteria relative to their natural abundance in the G.I. tract and/or disregarded key principles laid forth in Freter’s nutrient niche hypothesis which asserts that nutrient availability drives successful colonization. We developed a feeding bioassay using diet amended with rifampin to non-invasively introduce Enterococcus faecalis OG1RF into the Helicoverpa zea (corn earworm) G.I. tract in a single dose. We quantified E. faecalis populations using fecal plate counting. Preliminary results suggest that the continuous administration of artificial diet amended with 50 μg/mL rifampin opens a nutrient niche for E. faecalis to colonize at approximately 106 CFU/g frass (feces), whereas E. faecalis populations in solvent and negative control groups steadily declined. Collectively, this data supports a role for rifampin in suppressing susceptible endogenous bacteria and, subsequently, permitting stable colonization of E. faecalis OG1RF. Future works will continue optimizing this feeding protocol and characterize the role of metabolic pathways in colonization of isogenic E. faecalis mutants

Characterization of the Intestinal Mucin Role in Colonization of the Spodoptera frugiperda Midgut

The animal intestinal microbiome is a complex multispecies community which influences host development, physiology, and response to invasive pathogens. In lepidopteran (caterpillars) insects, intestinal mucins (IMs) are densely O-glycosylated proteins found in the midgut epithelium as either membrane-bound or gel-forming variants and possess chitin-binding domains which facilitate attachment to the protective mucus layer, the peritrophic matrix (PM) (Wang and Granados 1997). IMs are analogous to MUC2 the vertebrate mucin and major constituent in the mucosal layer of the mammalian gastrointestinal (G.I.) tract. In mice, it has been demonstrated that anaerobes secrete hydrolases that degrade mucin polysaccharides to mono- and disaccharides utilized by other intestinal residents (Mahowald et al. 2009). However, this mechanism of nutrient acquisition by stably colonized intestinal residents has not been elucidated. Using Spodoptera frugiperda (Fall armyworm) larvae, we attempted to clone and recombinantly express IMs with the goal of then using the purified mucins to supplement minimal growth media to test enteric bacteria for the ability to metabolize IM polysaccharides in vitro. Second, we attempted to quantify IM gene expression in each developmental instar by quantitative PCR to determine the optimal stage to induce gene silencing by RNA interference (RNAi). We anticipate that RNAi-mediated silencing of IM would significantly reduce the nutrient content in the mucosal layer, leading to a significant reduction in the number of stably colonized resident microorganisms. We have designed multiple primer sets that specifically target coding regions from S. frugiperda mucin transcripts (Legeai et al. 2014) and Aequorea victoria GFP to use as a control. We have also made complementary DNA (cDNA) from extracted RNAs of midgut epithelial tissue. With primers designed to specifically target S. frugiperda midgut IMs, we are closer to cloning the full-length transcript. Gradient polymerase chain reaction will be used in the next series of experiments to identify optimal amplification temperatures of S. Frugiperda intestinal mucin

Alpha-arylphorin is a mitogen in the Heliothis virescens midgut cell secretome upon Cry1Ac intoxication

Insecticidal crystal (Cry) proteins produced by the bacterium Bacillus thuringiensis (Bt) target cells in the midgut epithelium of susceptible larvae. While the mode of action of Cry toxins has been extensively investigated, the midgut response to Cry intoxication and its regulation are not well characterized. In this work, we describe the secreted proteome (secretome) of primary mature midgut cell cultures from Heliothis virescens larvae after exposure to Cry1Ac toxin compared to control buffer treatment. The Cry1Ac-induced secretome caused higher proliferation and differentiation and an overall reduction in total cell mortality over time in primary H. virescens midgut stem cell cultures when compared to treatment with control buffer secretome. Differential proteomics identified four proteins with significant differences in abundance comparing Cry1Ac-treated and control secretomes. The most significant difference detected in the Cry1Ac secretome was an arylphorin subunit alpha protein not detected in the control secretome. Feeding of purified alpha-arylphorin to H. virescens larvae resulted in midgut hyperplasia and significantly reduced susceptibility to Cry1Ac toxin compared to controls. These data identify alpha-arylphorin as a protein with a new putative role in the midgut regeneration process in response to Cry1Ac intoxication and possibly pathogen/abiotic stress, identifying alpha-arylphorin as a potential gene to target with insecticidal gene silencing for pest control

Reduced levels of membrane-bound alkaline phosphatase are common to lepidopteran strains resistant to cry toxins in bacillus thuringiensis

Development of insect resistance is one of the main concerns with the use of transgenic crops expressing Cry toxins from the bacterium Bacillus thuringiensis. Identification of biomarkers would assist in the development of sensitive DNA-based methods to monitor evolution of resistance to Bt toxins in natural populations. We report on the proteomic and genomic detection of reduced levels of midgut membrane-bound alkaline phosphatase (mALP) as a common feature in strains of Cry-resistant Heliothis virescens, Helicoverpa armigera and Spodoptera frugiperda when compared to susceptible larvae. Reduced levels of H. virescens mALP protein (HvmALP) were detected by two dimensional differential in-gel electrophoresis (2D-DIGE) analysis in Cry-resistant compared to susceptible larvae, further supported by alkaline phosphatase activity assays and Western blotting. Through quantitative real-time polymerase chain reaction (qRT-PCR) we demonstrate that the reduction in HvmALP protein levels in resistant larvae are the result of reduced transcript amounts. Similar reductions in ALP activity and mALP transcript levels were also detected for a Cry1Ac-resistant strain of H. armigera and field-derived strains of S. frugiperda resistant to Cry1Fa. Considering the unique resistance and cross-resistance phenotypes of the insect strains used in this work, our data suggest that reduced mALP expression should be targeted for development of effective biomarkers for resistance to Cry toxins in lepidopteran pests. DOI: 10.1371/journal.pone.001760

Reduced ALP activity correlates with reduced levels of mALP in BBMV from Cry1Fa-resistant <i>S. frugiperda</i> larvae.

<p>BBMV proteins from susceptible (Mon and Ben) and Cry1Fa-resistant (456 and 512) strains of <i>S. frugiperda</i> were used in specific ALP (A) or APN (B) activity assays. Different letters indicate statistically significant differences (p<0.05; Holm-Sidak method) among the samples. C) Detection of mALP in BBMV from susceptible (Mon and Ben) and resistant (456 and 512) strains of <i>S. frugiperda</i> using Western blotting. Lane 1, Mon, lane 2, 456, lane 3, 512, lane 4, Ben. Arrow points to ALP protein band.</p

BBMV from Cry1Ac-resistant <i>H. virescens</i> larvae display reduced ALP levels.

<p>(A) BBMV proteins from <i>H. virescens</i> strains as indicated were used in specific ALP or APN activity assays. Different letters indicate statistically significant differences (p<0.05; Holm-Sidak method) among the samples. (B) Western blot analysis of HvmALP, APN, or HevCaLP in BBMV proteins from <i>H. virescens</i> strains: Lane 1, YDK; lane 2, YHD2-B; lane 3, CXC; lane 4, KCBhyb.</p

Reduced levels of HvmALP1 and HvmALP2 transcripts in Cry1Ac-resistant <i>H. virescens</i> larvae relative to YDK as detected by qRT-PCR.

<p>Data shown are the mean transcript quantity relative to the YDK sample with the highest transcript amounts from three independent biological replicates for each HvmALP isoform and strain. All reactions were performed with triplicate technical replicates. Bars denote standard error of the mean; different letters on each bar indicate significant differences (P<0.05; Holm-Sidak method).</p