Lindsey Perkin

Functional analysis of C1 family cysteine peptidases in the larval gut of Тenebrio molitor and Tribolium castaneum.

BackgroundLarvae of the tenebrionids Tenebrio molitor and Tribolium castaneum have highly compartmentalized guts, with primarily cysteine peptidases in the acidic anterior midgut that contribute to the early stages of protein digestion.ResultsHigh throughput sequencing was used to quantify and characterize transcripts encoding cysteine peptidases from the C1 papain family in the gut of tenebrionid larvae. For T. castaneum, 25 genes and one questionable pseudogene encoding cysteine peptidases were identified, including 11 cathepsin L or L-like, 11 cathepsin B or B-like, and one each F, K, and O. The majority of transcript expression was from two cathepsin L genes on chromosome 10 (LOC659441 and LOC659502). For cathepsin B, the major expression was from genes on chromosome 3 (LOC663145 and LOC663117). Some transcripts were expressed at lower levels or not at all in the larval gut, including cathepsins F, K, and O. For T. molitor, there were 29 predicted cysteine peptidase genes, including 14 cathepsin L or L-like, 13 cathepsin B or B-like, and one each cathepsin O and F. One cathepsin L and one cathepsin B were also highly expressed, orthologous to those in T. castaneum. Peptidases lacking conservation in active site residues were identified in both insects, and sequence analysis of orthologs indicated that changes in these residues occurred prior to evolutionary divergence. Sequences from both insects have a high degree of variability in the substrate binding regions, consistent with the ability of these enzymes to degrade a variety of cereal seed storage proteins and inhibitors. Predicted cathepsin B peptidases from both insects included some with a shortened occluding loop without active site residues in the middle, apparently lacking exopeptidase activity and unique to tenebrionid insects. Docking of specific substrates with models of T. molitor cysteine peptidases indicated that some insect cathepsins B and L bind substrates with affinities similar to human cathepsin L, while others do not and have presumably different substrate specificity.ConclusionsThese studies have refined our model of protein digestion in the larval gut of tenebrionid insects, and suggest genes that may be targeted by inhibitors or RNA interference for the control of cereal pests in storage areas.

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.

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.

Expression patterns of cysteine peptidase genes across the Tribolium castaneum life cycle provide clues to biological function.

The red flour beetle, Tribolium castaneum, is a major agricultural pest responsible for considerable loss of stored grain and cereal products worldwide. T. castaneum larvae have a highly compartmentalized gut, with cysteine peptidases mostly in the acidic anterior part of the midgut that are critical to the early stages of food digestion. In previous studies, we described 26 putative cysteine peptidase genes in T. castaneum (types B, L, O, F, and K) located mostly on chromosomes 3, 7, 8, and 10. In the present study, we hypothesized that specific cysteine peptidase genes could be associated with digestive functions for food processing based on comparison of gene expression profiles in different developmental stages, feeding and non-feeding. RNA-Seq was used to determine the relative expression of cysteine peptidase genes among four major developmental stages (egg, larvae, pupae, and adult) of T. castaneum. We also compared cysteine peptidase genes in T. castaneum to those in other model insects and coleopteran pests. By combining transcriptome expression, phylogenetic comparisons, response to dietary inhibitors, and other existing data, we identified key cysteine peptidases that T. castaneum larvae and adults use for food digestion, and thus new potential targets for biologically-based control products.

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

Background The Colorado potato beetle, Leptinotarsa decemlineata Say, is one of the most challenging agricultural pests to manage. It has shown a spectacular ability to not only rapidly adapt to a broad range of solanaceaeous plants and variable climates during its global invasion, but, most notably, to rapidly evolve resistance to insecticides (over 50 different compounds in all major classes, in some cases within the first year of use). 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 whole-genome sequencing, transcriptome sequencing, and a large community-driven annotation effort. Results We present a 140x coverage whole genome sequence from a single female L. decemlineata, with a reference gene set of 24,740 genes. Transposable elements comprise at least 17% of the genome, and are heavily represented in an analysis of rapidly evolving gene families compared to other Coleoptera. Population genetic analyses provide evidence of high levels of nucleotide diversity, local geographic structure, and recent population growth in pest populations, pointing to the availability of considerable standing genetic variation. These factors may play an important role in rapid evolutionary change. Adaptations to plant feeding are evident in gene expansions and differential expression of digestive enzymes (e.g. cysteine peptidase genes) in gut tissues, as well as expansions of the gustatory receptors for bitter tasting plants in the nightshade family, Solanaceae. Despite its notoriety for adapting to insecticides, L. decemlineata has a similar suite of genes involved in resistance (metabolic detoxification and cuticle penetration) compared to other beetles, although expansions in specific cytochrome P450 subfamilies are known to be associated with insecticide resistance. Finally, this beetle has interesting duplications in RNAi genes that might be linked to its high sensitivity to RNAi and could be important in the future development of gene targeted pesticides. Conclusions As a representative of one of the most evolutionarily diverse lineages, the L. decemlineata genome will undoubtedly provide new opportunities for deeper understanding on the ecology, evolution, and management of this species, as well as new opportunities to leverage genomic technologies to understand the basis of a broad range of phenotypes and to develop sustainable methods to control this widely successful pest.

RNA-Seq Validation of RNAi Identifies Additional Gene Connectivity in Tribolium castaneum (Coleoptera: Tenebrionidae)

Abstract RNA interference (RNAi) is a functional genomics tool to correlate genotype and phenotype by delivering targeted, gene-specific, and complementary dsRNA into a host via injection, feeding, or other means in order to reduce gene expression. In the red flour beetle, Tribolium castaneum, RNAi has been successful via injected dsRNA at all life stages. Traditionally, successful transcript knockdown has been quantified by qPCR on a gene-by-gene basis, where only expression of the target gene and normalization genes are evaluated. In this study, RNA-Seq was used to quantify transcript expression in larvae injected with dsRNA for aspartate 1-decarboxylase (ADC), which gives a reliable phenotype of an adult with a black cuticle instead of the wild-type red-brown. ANOVA of control, mock-injected, and ADC-dsRNA injected larvae indicated that target gene expression was significantly (P = 0.002) reduced 4-fold, and the black phenotype was achieved in all adults injected with ADC-dsRNA as larvae. In a pairwise analysis, significant (P < 0.05) differential expression of other genes in ADC-injected larvae suggested connections between gene pathways. One gene, dopamine receptor 2, was increased in expression 227-fold (P = 0.025), presumably connected to previous data that showed a reduction in expression of ADC results in increased levels of dopamine. To evaluate the hypothesis that increased dopamine levels can affect mobility, T. castaneum adults injected with ADC-dsRNA as larvae were significantly impaired in movement tests compared to controls, similar to black mutants in Drosophila melanogaster. The data demonstrate that RNA-Seq can reveal gene connectivity and provide more complete data validation and analysis compared to qPCR.

Cross-species comparison of the gut: Differential gene expression sheds light on biological differences in closely related tenebrionids

The gut is one of the primary interfaces between an insect and its environment. Understanding gene expression profiles in the insect gut can provide insight into interactions with the environment as well as identify potential control methods for pests. We compared the expression profiles of transcripts from the gut of larval stages of two coleopteran insects, Tenebrio molitor and Tribolium castaneum. These tenebrionids have different life cycles, varying in the duration and number of larval instars. T. castaneum has a sequenced genome and has been a model for coleopterans, and we recently obtained a draft genome for T. molitor. We assembled gut transcriptome reads from each insect to their respective genomes and filtered mapped reads to RPKM>1, yielding 11,521 and 17,871 genes in the T. castaneum and T. molitor datasets, respectively. There were identical GO terms in each dataset, and enrichment analyses also identified shared GO terms. From these datasets, we compiled an ortholog list of 6907 genes; 45% of the total assembled reads from T. castaneum were found in the top 25 orthologs, but only 27% of assembled reads were found in the top 25 T. molitor orthologs. There were 2281 genes unique to T. castaneum, and 2088 predicted genes unique to T. molitor, although improvements to the T. molitor genome will likely reduce these numbers as more orthologs are identified. We highlight a few unique genes in T. castaneum or T. molitor that may relate to distinct biological functions. A large number of putative genes expressed in the larval gut with uncharacterized functions (36 and 68% from T. castaneum and T. molitor, respectively) support the need for further research. These data are the first step in building a comprehensive understanding of the physiology of the gut in tenebrionid insects, illustrating commonalities and differences that may be related to speciation and environmental adaptation.