Gene expression in Tribolium castaneum life stages: Identifying a species-specific target for pest control applications

The red flour beetle, Tribolium castaneum, is a major agricultural pest of post-harvest products and stored grain. Control of T. castaneum in stored products and grain is primarily by fumigants and sprays, but insecticide resistance is a major problem, and new control strategies are needed. T. castaneum is a genetic model for coleopterans, and the reference genome can be used for discovery of candidate gene targets for molecular-based control, such as RNA interference. Gene targets need to be pest specific, and ideally, they are expressed at low levels for successful control. Therefore, we sequenced the transcriptome of four major life stages of T. castaneum, sorted data into groups based on high or low expression levels, and compared relative gene expression among all life stages. We narrowed our candidate gene list to a cuticle protein gene (CPG) for further analysis. We found that the CPG sequence was unique to T. castaneum and expressed only in the larval stage. RNA interference targeting CPG in newly-emerged larvae caused a significant (p < 0.05) decrease in CPG expression (1,491-fold) compared to control larvae and 64% mortality over 18 d. RNA-Seq of survivors after 18 d identified changes in the expression of other genes as well, including 52 long noncoding RNAs. Expression of three additional cuticle protein genes were increased and two chitinase genes were decreased in response to injection of CPG dsRNA. The data demonstrate that RNA-Seq can identify genes important for insect survival and thus may be used to develop novel biologically-based insect control products

Fitness costs of resistance to \u3ci\u3eBacillus thuringiensis\u3c/i\u3e in the Indianmeal moth, \u3ci\u3ePlodia interpunctella\u3c/i\u3e

Genetic changes in insects that result in insecticide resistance can also affect their fitness. Here, we report measurements of development time and survival of the Indianmeal moth, Plodia interpunctella (Hübner), to compare the relative fitness of Bacillus thuringiensis (Bt)-susceptible and -resistant colonies. Measurements of larval development time and survival indicated that a fitness cost was associated with resistance to Bt in some Bt-resistant colonies but not others. Comparisons of geographically different populations revealed inherent differences in development time and survival. In most cases, Bt-resistant moths suffered no disadvantage when feeding on a Bt-treated diet. In many cases, the development of Bt-resistant moths on Bt-treated diet was slower than the unselected moths on untreated diet, but it is unclear whether these differences would affect the successful mating of susceptible and resistant moths

DiPel-Selected Ostrinia nubilalis Larvae Are Not Resistant to Transgenic Corn Expressing Bacillus thuringiensis Cry1Ab

The survival of KS-SC DiPel-resistant and -susceptible European corn borer, Ostrinia nubilalis (Hübner), was evaluated on different tissues from corn, Zea mays L., hybrids, including a nontransgenic and two transgenic corn plants (events MON810 and Bt11) expressing high doses of Bacillus thuringiensis (Bt) Cry1Ab. The survival of Bt-resistant and -susceptible third instars was similar after a 5-d exposure to transgenic plant tissues. Survivors eventually died when returned to Bt corn tissues, but many were able to continue development when transferred to non-Bt corn tissues. Survival of resistant and susceptible larvae also was evaluated in bioassays with dilutions of leaf extracts from the three corn hybrids incorporated in an artificial diet. In these assays, survival was significantly higher for resistant O. nubilalis neonates at three of the five dilutions compared with the susceptible strain, but the resistance ratio was only 2.2- and 2.4-fold for MON810 and Bt11, respectively. The data demonstrate that Bt-resistant and unselected control O. nubilalis larvae were similar in susceptibility to MON810 and Bt11 event corn hybrids. Although we were unable to evaluate the Cry1Ab protein that larvae were exposed to in the transgenic tissue because of company restrictions, Cry1Ab protoxin produced in Escherichia coli was incubated with extracts from non-Bt corn leaves to simulate the in planta effect on the transgenic protein. Cry1Ab protoxin was hydrolyzed rapidly by enzymes in the corn extract into peptide fragments with molecular masses ranging from 132 to 74 kDa, and eventually 58 kDa. Overall, these data suggest that plant enzymes hydrolyze transgenic toxin to one that is functionally activated. Therefore, resistant insect populations with reduced proteinase activity do not seem to pose a threat to the efficacy of commercial MON810 and Bt11 corn hybrids

Activity and Expression of Midgut Proteases from Mexican and US Trichoplusia ni (Hübner) Strains Exposed to Bacillus thuringiensis

Abstract Proteases in the insect midgut have been associated with differences in susceptibility to Bacillus thuringiensis (Bt) insecticidal toxins. Therefore, we evaluated the protease activity of laboratory versus field strains of Trichoplusia ni that were previously characterized by their susceptibility to Bt Cry toxins or protoxins and antimicrobial peptide production (Tamez-Guerra et al. 2006, 2008). In addition, because aminopeptidases may be involved in Bt resistance, the expression of T. ni aminopeptidase N1 transcript (tnapn1) was analyzed by RT-PCR. These strains included Mexican (NL) and American (US) laboratory strains, as well as a field collected strain (GTO), and strains obtained by XenTari® selection from the laboratory strains (NLX and USX) or field strain (GTOX). The activity of proteases in midgut extracts were evaluated by in vitro assays or in-gel activity with specific substrates. The results indicated that the ratio of midgut protease activity (trypsin:chymotrypsin) was higher in the laboratory NL and US strains, but the differences did not correlate with Bt resistance. The ratio was slightly increased in the Xentari-selected field strain GTOX, and zymograms with Xentari or Cry1Ac as substrates suggested that both high and low molecular mass protease activities were increased in GTOX. Selection also resulted in 87% and 300% higher tnapn1 amplification in Xentari-selected NLX and USX, respectively. The relationship between Bt susceptibility, proteases and tnapn1 expression and activity alterations is discussed. Key Words: trypsin; chymotrypsin; Bt protoxins and toxins activation; aminopeptidase N1; enzymatic alterations Resumen Diferencias en la susceptibilidad de insectos a Bacillus thuringiensis (Bt) se han asociado a la actividad de proteasas intestinales del insecto. El presente estudio se realizó para evaluar la actividad de proteasas del intestino medio de cepas de Trichoplusia ni de laboratorio y campo en las que anteriormente se había caracterizado su susceptibilidad a protoxinas y toxinas Cry de Bt y producción de péptidos antimicrobiales (Tamez-Guerra et al. 2006, 2008). Debido a que la aminopeptidasa N1 se ha relacionado con la baja de susceptibilidad a Bt, también se analizó la amplificación de este transcrito (tnapn1) por RT-PCR entre las cepas de T. ni. Las cepas evaluadas fueron de laboratorio de México (NL) y Estados Unidos (US), así como las cepas colectadas de campo (GTO) o después de sobreexponer las larvas a XenTari® (NLX, USX y GTOX). La actividad de proteasas presentes en el extracto del intestino medio se evaluó en ensayos in vitro o en gel usando sustratos específicos. Los resultados mostraron que el rango entre la relación de tripsina:quimotripsina era mayor en las cepas de laboratorio NL y US, pero estas diferencias no mostraron correlación con la resistencia a Bt. El rango se incrementó ligeramente al seleccionar la cepa de campo GTOX con XenTari, mientras que los zimogramas usando Cry1Ac o XenTari como sustrato sugieren que se incrementó la actividad de proteasas de alta y baja masa molecular, especialmente en la cepa GTOX. También se observó un incremento del 87% y 300% en la expresión del tnapn1 en las cepas NLX y USX, respectivamente, seleccionadas con XenTari. Se discute la relación entre la susceptibilidad a Bt con la alteración de la expresión de tnapn1 y la actividad de proteasas. Palabras Clave: tripsina; quimiotripsina; activación de protoxinas y toxinas de Bt; aminopeptidasa N1; alteraciones enzimática

Transcriptome Profiling of the Intoxication Response of Tenebrio molitor Larvae to Bacillus thuringiensis Cry3Aa Protoxin

Bacillus thuringiensis (Bt) crystal (Cry) proteins are effective against a select number of insect pests, but improvements are needed to increase efficacy and decrease time to mortality for coleopteran pests. To gain insight into the Bt intoxication process in Coleoptera, we performed RNA-Seq on cDNA generated from the guts of Tenebrio molitor larvae that consumed either a control diet or a diet containing Cry3Aa protoxin. Approximately 134,090 and 124,287 sequence reads from the control and Cry3Aa-treated groups were assembled into 1,318 and 1,140 contigs, respectively. Enrichment analyses indicated that functions associated with mitochondrial respiration, signalling, maintenance of cell structure, membrane integrity, protein recycling/synthesis, and glycosyl hydrolases were significantly increased in Cry3Aa-treated larvae, whereas functions associated with many metabolic processes were reduced, especially glycolysis, tricarboxylic acid cycle, and fatty acid synthesis. Microarray analysis was used to evaluate temporal changes in gene expression after 6, 12 or 24 h of Cry3Aa exposure. Overall, microarray analysis indicated that transcripts related to allergens, chitin-binding proteins, glycosyl hydrolases, and tubulins were induced, and those related to immunity and metabolism were repressed in Cry3Aa-intoxicated larvae. The 24 h microarray data validated most of the RNA-Seq data. Of the three intoxication intervals, larvae demonstrated more differential expression of transcripts after 12 h exposure to Cry3Aa. Gene expression examined by three different methods in control vs. Cry3Aa-treated larvae at the 24 h time point indicated that transcripts encoding proteins with chitin-binding domain 3 were the most differentially expressed in Cry3Aa-intoxicated larvae. Overall, the data suggest that T. molitor larvae mount a complex response to Cry3Aa during the initial 24 h of intoxication. Data from this study represent the largest genetic sequence dataset for T. molitor to date. Furthermore, the methods in this study are useful for comparative analyses in organisms lacking a sequenced genome

Comparative Evaluation of Phenoloxidase Activity in Different Larval Stages of Four Lepidopteran Pests After Exposure toBacillus thuringiensis

Microbial entomopathogen–based bioinsecticides are recognized as alternatives to synthetic pesticides. Insects defend themselves against microbial pathogens by innate mechanisms, including increased phenoloxidase (PO) activity, but its relationship with microbial bioinsecticides efficacy is little known. This study evaluated the differences in PO activity at different developmental stages of the tobacco budworm Heliothis virescens Fabricius (Lepidoptera: Noctuidae), Indian meal moth Plodia interpunctella (Hübner) (Pyralidae), beet armyworm Spodoptera exigua (Hübner) (Noctuidae), and cabbage looper Trichoplusia ni (Hübner) (Noctuidae). Additionally, 2nd- and 4th-instars were exposed to the LC50 value of the commercial Bacillus thuringiensis (Bt) spray, Biobit®. The percentage of insecticidal activity (IA%) on 2nd-instar Biobit–exposed larvae was approximately the predicted 50 % mortality for all species except S. exigua. With all 4th instar Biobit–exposed larvae, mortality was not significantly different from that of unexposed larvae. Unexposed insects had a significantly higher PO activity in pre–pupae and pupae than early–instar larvae and adults, whereas PO activity was higher in adult females than in males. Correlation analysis between IA% and PO activity revealed significant r–values (p < 0.01) in 2nd instar H. virescens (r = 0.979) and P. interpunctella (r = 0.930). Second instar Biobit–exposed P. interpunctella had 10 times more PO activity than unexposed larvae. Similarly, the amount of total protein was lower in 4th instar Biobit–exposed H. virescens and higher in S. exigua. Therefore, the results indicated a relationship between Biobit susceptibility and PO activity in some cases. This information may be useful if the Biobit application period is timed for a developmental stage with low PO activity. However, more studies are needed to determine the correlation of each insect with a particular bioinsecticide

Active and covert infections of cricket Iridovirus and Acheta domesticus Densovirus in reared Gryllodes sigillatus crickets

Interest in developing food, feed, and other useful products from farmed insects has gained remarkable momentum in the past decade. Crickets are an especially popular group of farmed insects due to their nutritional quality, ease of rearing, and utility. However, production of crickets as an emerging commodity has been severely impacted by entomopathogenic infections, about which we know little. Here, we identified and characterized an unknown entomopathogen causing mass mortality in a lab-reared population of Gryllodes sigillatus crickets, a species used as an alternative to the popular Acheta domesticus due to its claimed tolerance to prevalent entomopathogenic viruses. Microdissection of sick and healthy crickets coupled with metagenomics-based identification and real-time qPCR viral quantification indicated high levels of cricket iridovirus (CrIV) in a symptomatic population, and evidence of covert CrIV infections in a healthy population. Our study also identified covert infections of Acheta domesticus densovirus (AdDNV) in both populations of G. sigillatus. These results add to the foundational research needed to better understand the pathology of mass-reared insects and ultimately develop the prevention, mitigation, and intervention strategies needed for economical production of insects as a commodity

Genes related to mitochondrial functions are differentially expressed in phosphine-resistant and -susceptible Tribolium castaneum

Background: Phosphine is a valuable fumigant to control pest populations in stored grains and grain products. However, recent studies indicate a substantial increase in phosphine resistance in stored product pests worldwide.Results: To understand the molecular bases of phosphine resistance in insects, we used RNA-Seq to compare gene expression in phosphine-resistant and susceptible laboratory populations of the red flour beetle, Tribolium castaneum. Each population was evaluated as either phosphine-exposed or no phosphine (untreated controls) in triplicate biological replicates (12 samples total). Pairwise analysis indicated there were eight genes differentially expressed between susceptible and resistant insects not exposed to phosphine (i.e., basal expression) or those exposed to phopshine (>8-fold expression and 90 % C.I.). However, 214 genes were differentially expressed among all four treatment groups at a statistically significant level (ANOVA, p < 0.05). Increased expression of 44 cytochrome P450 genes was found in resistant vs. susceptible insects, and phosphine exposure resulted in additional increases of 21 of these genes, five of which were significant among all treatment groups (p < 0.05). Expression of two genes encoding anti-diruetic peptide was 2- to 8-fold reduced in phosphine-resistant insects, and when exposed to phosphine, expression was further reduced 36- to 500-fold compared to susceptible. Phosphine-resistant insects also displayed differential expression of cuticle, carbohydrate, protease, transporter, and many mitochondrial genes, among others. Gene ontology terms associated with mitochondrial functions (oxidation biological processes, monooxygenase and catalytic molecular functions, and iron, heme, and tetrapyyrole binding) were enriched in the significantly differentially expressed dataset. Sequence polymorphism was found in transcripts encoding a known phosphine resistance gene, dihydrolipoamide dehydrogenase, in both susceptible and resistant insects. Phosphine-resistant adults also were resistant to knockdown by the pyrethroid deltamethrin, likely due to the increased cytochrome P450 expression.Conclusions: Overall, genes associated with the mitochondria were differentially expressed in resistant insects, and these differences may contribute to a reduction in overall metabolism and energy production and/or compensation in resistant insects. These data provide the first gene expression data on the response of phosphine-resistant and -susceptible insects to phosphine exposure, and demonstrate that RNA-Seq is a valuable tool to examine differences in insects that respond differentially to environmental stimuli.Peer reviewedEntomology and Plant Patholog

Brown marmorated stink bug, Halyomorpha halys (Stål), genome: putative underpinnings of polyphagy, insecticide resistance potential and biology of a top worldwide pest

Background Halyomorpha halys (Stål), the brown marmorated stink bug, is a highly invasive insect species due in part to its exceptionally high levels of polyphagy. This species is also a nuisance due to overwintering in human-made structures. It has caused significant agricultural losses in recent years along the Atlantic seaboard of North America and in continental Europe. Genomic resources will assist with determining the molecular basis for this species’ feeding and habitat traits, defining potential targets for pest management strategies. Results Analysis of the 1.15-Gb draft genome assembly has identified a wide variety of genetic elements underpinning the biological characteristics of this formidable pest species, encompassing the roles of sensory functions, digestion, immunity, detoxification and development, all of which likely support H. halys’ capacity for invasiveness. Many of the genes identified herein have potential for biomolecular pesticide applications. Conclusions Availability of the H. halys genome sequence will be useful for the development of environmentally friendly biomolecular pesticides to be applied in concert with more traditional, synthetic chemical-based controls