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

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

Variants in the mitochondrial genome sequence of \u3ci\u3eRhyzopertha dominica\u3c/i\u3e (Fabricius) (Coleoptera: Bostrycidae)

The lesser grain borer, Rhyzopertha dominica, is a coleopteran pest of stored grains and is mainly controlled by phosphine fumigation, but the increase in phosphine-resistant populations threatens efficacy. Some phosphine-resistant insects have reduced respiration, and thus studying the mitochondrial genome may provide additional information regarding resistance. Genomic DNA from an inbred laboratory strain of R. dominica was extracted and sequenced with both short (Illumina) and long (Pacific Biosciences) read technologies for whole genome sequence assembly and annotation. Short read sequences were assembled and annotated by open software to identify mitochondrial sequences, and the assembled sequence was manually annotated and verified by long read sequences. The mitochondrial genome sequence for R. dominica had a total length of 15,724 bp and encoded 22 trna genes, 2 rRNA genes, 13 protein coding genes (7 nad subunits, 3 cox, 2 atp, and 1 cytB), flanked by a long control region. We compared our predicted mitochondrial genome to that of another from a R. dominica strain from Jingziguan (China). While there was mostly agreement between the two assemblies, key differences will be further examined to determine if mutations in populations are related to insecticide control pressure, mainly that of phosphine. Differences in sequence data, assembly, and annotation also may result in different genome interpretations

Gene Disruption Technologies Have the Potential to Transform Stored Product Insect Pest Control

Stored product insects feed on grains and processed commodities manufactured from grain post-harvest, reducing the nutritional value and contaminating food. Currently, the main defense against stored product insect pests is the pesticide fumigant phosphine. Phosphine is highly toxic to all animals, but is the most effective and economical control method, and thus is used extensively worldwide. However, many insect populations have become resistant to phosphine, in some cases to very high levels. New, environmentally benign and more effective control strategies are needed for stored product pests. RNA interference (RNAi) may overcome pesticide resistance by targeting the expression of genes that contribute to resistance in insects. Most data on RNAi in stored product insects is from the coleopteran genetic model, Tribolium castaneum, since it has a strong RNAi response via injection of double stranded RNA (dsRNA) in any life stage. Additionally, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology has been suggested as a potential resource for new pest control strategies. In this review we discuss background information on both gene disruption technologies and summarize the advances made in terms of molecular pest management in stored product insects, mainly T. castaneum, as well as complications and future needs

Variants in the Mitochondrial Genome Sequence of Rhyzopertha dominica (Fabricius) (Coleoptera: Bostrycidae)

The lesser grain borer, Rhyzopertha dominica, is a coleopteran pest of stored grains and is mainly controlled by phosphine fumigation, but the increase in phosphine-resistant populations threatens efficacy. Some phosphine-resistant insects have reduced respiration, and thus studying the mitochondrial genome may provide additional information regarding resistance. Genomic DNA from an inbred laboratory strain of R. dominica was extracted and sequenced with both short (Illumina) and long (Pacific Biosciences) read technologies for whole genome sequence assembly and annotation. Short read sequences were assembled and annotated by open software to identify mitochondrial sequences, and the assembled sequence was manually annotated and verified by long read sequences. The mitochondrial genome sequence for R. dominica had a total length of 15,724 bp and encoded 22 trna genes, 2 rRNA genes, 13 protein coding genes (7 nad subunits, 3 cox, 2 atp, and 1 cytB), flanked by a long control region. We compared our predicted mitochondrial genome to that of another from a R. dominica strain from Jingziguan (China). While there was mostly agreement between the two assemblies, key differences will be further examined to determine if mutations in populations are related to insecticide control pressure, mainly that of phosphine. Differences in sequence data, assembly, and annotation also may result in different genome interpretations

The Transcriptomic Response of the Boll Weevil, <i>Anthonomus grandis grandis</i> Boheman (Coleoptera: Curculionidae), following Exposure to the Organophosphate Insecticide Malathion

Insecticide tolerance and resistance have evolved countless times in insect systems. Molecular drivers of resistance include mutations in the insecticide target site and/or gene duplication, and increased gene expression of detoxification enzymes. The boll weevil, Anthonomus grandis grandis Boheman (Coleoptera: Curculionidae), is a pest of commercial cotton and has developed resistance in the field to several insecticides; however, the current organophosphate insecticide, malathion, used by USA eradication programs remains effective despite its long-term use. Here, we present findings from an RNA-seq experiment documenting gene expression post-exposure to field-relevant concentrations of malathion, which was used to provide insight on the boll weevil’s continued susceptibility to this insecticide. Additionally, we incorporated a large collection of boll weevil whole-genome resequencing data from nearly 200 individuals collected from three geographically distinct areas to determine SNP allele frequency of the malathion target site, as a proxy for directional selection in response to malathion exposure. No evidence was found in the gene expression data or SNP data consistent with a mechanism of enhanced tolerance or resistance adaptation to malathion in the boll weevil. Although this suggests continued effectiveness of malathion in the field, we identified important temporal and qualitative differences in gene expression between weevils exposed to two different concentrations of malathion. We also identified several tandem isoforms of the detoxifying esterase B1 and glutathione S-transferases, which are putatively associated with organophosphate resistance

A New qPCR Assay for the Rapid Diagnosis of <i>Anthonomus grandis</i> Subspecies

Rapid and accurate identification of Anthonomus grandis subspecies is crucial for effective management and eradication. Current diagnostic methods have limitations in terms of time to diagnosis (up to seven days) and can yield ambiguous results. Here, we present the validation of a custom TaqMan SNP Genotyping Assay for the rapid and accurate identification of A. grandis grandis (boll weevil) and A. g. thurberiae (thurberia weevil) subspecies. To validate the assay, we conducted three main experiments: (1) a sensitivity test to determine the DNA concentration range at which the assay performs, (2) a non-target specificity test to ensure no amplification in non-target weevils (false positives), and (3) an accuracy test comparing the results of the new assay to previously established methods. These experiments were carried out in parallel at three independent facilities to confirm the robustness of the assay to variations in equipment and personnel. We used DNA samples from various sources, including field-collected specimens, museum specimens, and previously isolated DNA. The assay demonstrated high sensitivity (PCR success with ≥0.05 ng/µL DNA template), specificity (0.02 false positive rate), and accuracy (97.7%) in diagnosing boll weevil and thurberia weevil subspecies. The entire workflow, including DNA extraction, assay preparation, PCR run time, and data analysis, can be completed within a single workday (7–9 h) by a single technician. The deployment of this assay as a diagnostic tool could benefit boll weevil management and eradication programs by enabling same-day diagnosis of trap-captured or intercepted weevil specimens. Furthermore, it offers a more reliable method for identifying unknown specimens, contributing to the overall effectiveness of boll weevil research and control efforts

A model species for agricultural pest genomics: the genome of the Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae)

The Colorado potato beetle is one of the most challenging agricultural pests to manage. It has shown a spectacular ability to adapt to a variety of solanaceaeous plants and variable climates during its global invasion, and, notably, to rapidly evolve insecticide resistance. To examine evidence of rapid evolutionary change, and to understand the genetic basis of herbivory and insecticide resistance, we tested for structural and functional genomic changes relative to other arthropod species using genome sequencing, transcriptomics, and community annotation. Two factors that might facilitate rapid evolutionary change include transposable elements, which comprise at least 17% of the genome and are rapidly evolving compared to other Coleoptera, and high levels of nucleotide diversity in rapidly growing pest populations. Adaptations to plant feeding are evident in gene expansions and differential expression of digestive enzymes in gut tissues, as well as expansions of gustatory receptors for bitter tasting. Surprisingly, the suite of genes involved in insecticide resistance is similar to other beetles. Finally, duplications in the RNAi pathway might explain why Leptinotarsa decemlineata has high sensitivity to dsRNA. The L. decemlineata genome provides opportunities to investigate a broad range of phenotypes and to develop sustainable methods to control this widely successful pest