Publications by authors named "Marce Lorenzen"

33 Publications

Microinjection of Corn Planthopper, Peregrinus maidis, Embryos for CRISPR/Cas9 Genome Editing.

J Vis Exp 2021 03 26(169). Epub 2021 Mar 26.

Department of Entomology and Plant Pathology, North Carolina State University;

The corn planthopper, Peregrinus maidis, is a pest of maize and a vector of several maize viruses. Previously published methods describe the triggering of RNA interference (RNAi) in P. maidis through microinjection of double-stranded RNAs (dsRNAs) into nymphs and adults. Despite the power of RNAi, phenotypes generated via this technique are transient and lack long-term Mendelian inheritance. Therefore, the P. maidis toolbox needs to be expanded to include functional genomic tools that would enable the production of stable mutant strains, opening the door for researchers to bring new control methods to bear on this economically important pest. However, unlike the dsRNAs used for RNAi, the components used in CRISPR/Cas9-based genome editing and germline transformation do not easily cross cell membranes. As a result, plasmid DNAs, RNAs, and/or proteins must be microinjected into embryos before the embryo cellularizes, making the timing of injection a critical factor for success. To that end, an agarose-based egg-lay method was developed to allow embryos to be harvested from P. maidis females at relatively short intervals. Herein are provided detailed protocols for collecting and microinjecting precellular P. maidis embryos with CRISPR components (Cas9 nuclease that has been complexed with guide RNAs), and results of Cas9-based gene knockout of a P. maidis eye-color gene, white, are presented. Although these protocols describe CRISPR/Cas9-genome editing in P. maidis, they can also be used for producing transgenic P. maidis via germline transformation by simply changing the composition of the injection solution.
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http://dx.doi.org/10.3791/62417DOI Listing
March 2021

Characterization, expression patterns, and transcriptional responses of three core RNA interference pathway genes from Ostrinia nubilalis.

J Insect Physiol 2021 Feb-Mar;129:104181. Epub 2020 Dec 25.

Department of Entomology, 123 Waters Hall, Kansas State University, Manhattan, KS 66506, USA. Electronic address:

RNA interference (RNAi) is commonly used in the laboratory to analyze gene function, and RNAi-based pest management strategies are now being employed. Unfortunately, RNAi is hindered by inefficient and highly-variable results when different insects are targeted, especially lepidopterans, such as the European corn borer (ECB), Ostrinia nubilalis (Lepidoptera: Crambidae). Previous efforts to achieve RNAi-mediated gene suppression in ECB revealed low RNAi efficiency with both double-stranded RNA (dsRNA) injection and ingestion. One mechanism that can affect RNAi efficiency in insects is the expression and function of core RNAi pathway genes, such as those encoding Argonaut 2 (Ago2), Dicer 2 (Dcr2), and a dsRNA binding protein (R2D2). To determine if deficiencies in these core RNAi pathway genes contribute to low RNAi efficiency in ECB, full-length complementary DNAs encoding OnAgo2, OnDcr2, and OnR2D2 were cloned, sequenced, and characterized. A comparison of domain architecture suggested that all three predicted proteins contained the necessary domains to function. However, a comparison of evolutionary distances revealed potentially important variations in the first RNase III domain of OnDcr2, the double-stranded RNA binding domains of OnR2D2, and both the PAZ and PIWI domains of OnAgo2, which may indicate functional differences in enzymatic activity between species. Expression analysis indicated that transcripts for all three genes were expressed in all developmental stages and tissues investigated. Interestingly, the introduction of non-target dsRNA into ECB second-instar larvae via microinjection did not affect OnAgo2, OnDcr2, or OnR2D2 expression. In contrast, ingestion of the same dsRNAs resulted in upregulation of OnDcr2 but downregulation of OnR2D2. The unexpected transcriptional responses of the core machinery and the divergence in amino-acid sequence between specific domains in each core RNAi protein may possibly contribute to low RNAi efficiency in ECB. Understanding the contributions of different RNAi pathway components is critical to adapting this technology for use in controlling lepidopteran pests that exhibit low RNAi efficiency.
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http://dx.doi.org/10.1016/j.jinsphys.2020.104181DOI Listing
December 2020

Molecular Characterizations of Double-Stranded RNA Degrading Nuclease Genes from .

Insects 2020 Sep 23;11(10). Epub 2020 Sep 23.

Department of Entomology, 123 Waters Hall, Kansas State University, Manhattan, KS 66506, USA.

Variable RNA interference (RNAi) efficiencies limit RNAi-based pest management strategies for many pests. Previous efforts to understand mechanisms contributing to low RNAi efficiency indicate that double-stranded RNA (dsRNA) is degraded in the European corn borer (ECB), due to nuclease activity. To investigate the contribution of dsRNA-degrading endonucleases (dsRNases) and lepidopteran-specific RNAi efficiency-related nucleases (REases) to dsRNA instability and low RNAi efficiency in ECB, five complementary DNAs putatively encoding four dsRNases ( and ) and one REase () were sequenced. Characterization of these transcripts revealed that substrate specificity might vary among the four dsRNases due to different amino acid combinations in the substrate-binding sites. Gene expression analysis indicated that and were highly expressed in the larval gut, and showed the highest expression in hemolymph, especially in older developmental stages. Transcript level analysis after dsRNA exposure revealed that expression of rapidly increased upon dsRNA ingestion or injection, whereas expression only increased after long-term ingestion of dsRNA. While the biological function of these nucleases remains to be verified, our results suggest that OnREase and OndsRNase2, and OndsRNase1 and OndsRNase4 may be responsible for degradation of dsRNAs in the ECB gut and hemolymph, respectively, thereby contributing to low RNAi efficiency.
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http://dx.doi.org/10.3390/insects11100652DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7598268PMC
September 2020

Transcriptome analysis of life stages of the house cricket, Acheta domesticus, to improve insect crop production.

Sci Rep 2020 02 26;10(1):3471. Epub 2020 Feb 26.

All Things Bugs LLC, 755 Research Parkway, Suite 465, Oklahoma City, OK, 73104, USA.

To develop genetic resources for the improvement of insects as food, we sequenced transcripts from embryos, one-day hatchlings, three nymphal stages, and male and female adults of the house cricket, Acheta domesticus. A draft transcriptome was assembled from more than 138 million sequences combined from all life stages and sexes. The draft transcriptome assembly contained 45,866 contigs, and more than half were similar to sequences at NCBI (e value < e). The highest sequence identity was found in sequences from the termites Cryptotermes secundus and Zootermopsis nevadensis. Sequences with identity to Gregarina niphandrodes suggest that these crickets carry the parasite. Among all life stages, there were 5,042 genes with differential expression between life stages (significant at p < 0.05). An enrichment analysis of gene ontology terms from each life stage or sex highlighted genes that were important to biological processes in cricket development. We further characterized genes that may be important in future studies of genetically modified crickets for improved food production, including those involved in RNA interference, and those encoding prolixicin and hexamerins. The data represent an important first step in our efforts to provide genetically improved crickets for human consumption and livestock feed.
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http://dx.doi.org/10.1038/s41598-020-59087-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7044300PMC
February 2020

The impact of local population genetic background on the spread of the selfish element in red flour beetles.

Ecol Evol 2020 Jan 19;10(2):863-874. Epub 2019 Dec 19.

W. M. Keck Center for Behavioral Biology North Carolina State University Raleigh North Carolina.

Selfish genetic elements have been found in the genomes of many species, yet our understanding of their evolutionary dynamics is only partially understood. A number of distinct selfish elements are naturally present in many populations of the red flour beetle (). Although these elements are predicted by models to increase in frequency within populations because any offspring of a -bearing mother that do not inherit at least one allele will die, experiments demonstrating an increase in a naturally occurring element are lacking. Our survey of the specific element M, in the United States showed that it had a patchy geographic distribution. From the survey, it could not be determined if this distribution was caused by a slow process of M colonization of discrete populations or if some populations lacked M because they had genetic factors conferring resistance to the mechanism. We show that populations with naturally low to intermediate M frequencies likely represent transient states during the process of spread. Furthermore, we find no evidence that genetic factors are excluding M from US populations where the element is not presently found. We also show how a known suppressor of can impair the increase of M in populations and discuss the implications of our findings for pest-management applications of elements.
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http://dx.doi.org/10.1002/ece3.5946DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6988536PMC
January 2020

The distribution and spread of naturally occurring selfish genetic elements in the United States.

Ecol Evol 2019 Dec 27;9(24):14407-14416. Epub 2019 Nov 27.

Department of Entomology and Plant Pathology North Carolina State University Raleigh NC USA.

Selfish genetic elements (SGEs) are DNA sequences that are transmitted to viable offspring in greater than Mendelian frequencies. SGEs occur naturally in some populations of red flour beetle () and are expected to increase in frequency within populations and spread among populations. The large-scale U.S. distributions of (M) had been mapped based on samples from 1993 to 1995. We sampled beetles in 2011-2014 and show that the distribution of M in the United States is dynamic and has shifted southward. By using a genetic marker of (M), we found five unique geographic clusters with high and low M frequencies in a pattern not predicted by microsatellite-based analysis of population structure. Our results indicate the absence of rigid barriers to spread in the United States, so assessment of what factors have limited its current distribution requires further investigation. There is great interest in using synthetic SGEs, including synthetic , to alter or suppress pest populations, but there is concern about unpredicted spread of these SGEs and potential for populations to become resistant to them. The finding of patchy distributions of elements suggests that released synthetic SGEs cannot always be expected to spread uniformly, especially in target species with limited dispersal.
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http://dx.doi.org/10.1002/ece3.5876DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6953677PMC
December 2019

New Technologies for Studying Negative-Strand RNA Viruses in Plant and Arthropod Hosts.

Mol Plant Microbe Interact 2020 Mar 8;33(3):382-393. Epub 2020 Jan 8.

Department of Entomology and Plant Pathology, North Carolina State University, Raleigh, NC, U.S.A.

The plant viruses in the phylum , orders and , have common features of single-stranded, negative-sense RNA genomes and replication in the biological vector. Due to the similarities in biology, comparative functional analysis in plant and vector hosts is helpful for understanding host-virus interactions for negative-strand RNA viruses. In this review, we will highlight recent technological advances that are breaking new ground in the study of these recalcitrant virus systems. The development of infectious clones for plant rhabdoviruses and bunyaviruses is enabling unprecedented examination of gene function in plants and these advances are also being transferred to study virus biology in the vector. In addition, genome and transcriptome projects for critical nonmodel arthropods has enabled characterization of insect response to viruses and identification of interacting proteins. Functional analysis of genes using genome editing will provide future pathways for further study of the transmission cycle and new control strategies for these viruses and their vectors.
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http://dx.doi.org/10.1094/MPMI-10-19-0281-FIDOI Listing
March 2020

Structural and functional insights into the Diabrotica virgifera virgifera ATP-binding cassette transporter gene family.

BMC Genomics 2019 Nov 27;20(1):899. Epub 2019 Nov 27.

Department of Entomology and Plant Pathology, North Carolina State University, Box 7613, 1566 Thomas Hall, Raleigh, NC, 27695-7613, USA.

Background: The western corn rootworm, Diabrotica virgifera virgifera, is a pervasive pest of maize in North America and Europe, which has adapted to current pest management strategies. In advance of an assembled and annotated D. v. virgifera genome, we developed transcriptomic resources to use in identifying candidate genes likely to be involved in the evolution of resistance, starting with members of the ATP-binding cassette (ABC) transporter family.

Results: In this study, 65 putative D. v. virgifera ABC (DvvABC) transporters were identified within a combined transcriptome assembly generated from embryonic, larval, adult male, and adult female RNA-sequence libraries. Phylogenetic analysis placed the deduced amino-acid sequences of the DvvABC transporters into eight subfamilies (A to H). To supplement our sequence data with functional analysis, we identified orthologs of Tribolium castaneum ABC genes which had previously been shown to exhibit overt RNA interference (RNAi) phenotypes. We identified eight such D. v. virgifera genes, and found that they were functionally similar to their T. castaneum counterparts. Interestingly, depletion of DvvABCB_39715 and DvvABCG_3712 transcripts in adult females produced detrimental reproductive and developmental phenotypes, demonstrating the potential of these genes as targets for RNAi-mediated insect control tactics.

Conclusions: By combining sequence data from four libraries covering three distinct life stages, we have produced a relatively comprehensive de novo transcriptome assembly for D. v. virgifera. Moreover, we have identified 65 members of the ABC transporter family and provided the first insights into the developmental and physiological roles of ABC transporters in this pest species.
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http://dx.doi.org/10.1186/s12864-019-6218-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6882327PMC
November 2019

Effects of targeting eye color in Tenebrio molitor through RNA interference of tryptophan 2,3-dioxygenase (vermilion): Implications for insect farming.

Arch Insect Biochem Physiol 2019 May 25;101(1):e21546. Epub 2019 Mar 25.

Department of Entomology and Plant Pathology, North Carolina State University, Raleigh, North Carolina.

The gene vermilion encodes tryptophan 2,3-dioxygenase, part of the ommochrome pathway, and is responsible for the dark pigmented eyes in some insects, including beetles. Using RNA interference, we targeted the vermilion gene ortholog in embryos and pupae of the yellow mealworm, Tenebrio molitor, resulting in larvae and adults, respectively, that lacked eye pigment. RNA-Seq was used to analyze the impact of vermilion-specific RNA interference on gene expression. There was a 425-fold reduction in vermilion gene expression (p = 0.0003), as well as significant (p < 0.05) differential expression of 109 other putative genes, most of which were downregulated. Enrichment analysis of Gene Ontology terms found in the differentially expressed data set included genes known to be involved in the ommochrome pathway. However, enrichment analysis also revealed the influence of vermilion expression on genes involved in protein translocation to the endoplasmic reticulum, signal transduction, G-protein-coupled receptor signaling, cell-cycle arrest, mannose biosynthesis, and vitamin transport. These data demonstrate that knockdown of vermilion in T. molitor results in complete loss of eye color (white-eyed phenotype) and identify other interrelated genes in the vermilion metabolic pathway. Therefore, a dominant marker system based on eye color can be developed for the genetic manipulation of T. molitor to increase the value of mealworms as an alternative food source by decreasing negative traits, such as disease susceptibility, and increasing desired traits, such as protein content and vitamin production.
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http://dx.doi.org/10.1002/arch.21546DOI Listing
May 2019

Genome of the small hive beetle (Aethina tumida, Coleoptera: Nitidulidae), a worldwide parasite of social bee colonies, provides insights into detoxification and herbivory.

Gigascience 2018 12 1;7(12). Epub 2018 Dec 1.

Institute of Bee Health, Vetsuisse Faculty, University of Bern, Schwarzenburgstrasse 161, CH-3097, Liebefeld, Switzerland.

Background: The small hive beetle (Aethina tumida; ATUMI) is an invasive parasite of bee colonies. ATUMI feeds on both fruits and bee nest products, facilitating its spread and increasing its impact on honey bees and other pollinators. We have sequenced and annotated the ATUMI genome, providing the first genomic resources for this species and for the Nitidulidae, a beetle family that is closely related to the extraordinarily species-rich clade of beetles known as the Phytophaga. ATUMI thus provides a contrasting view as a neighbor for one of the most successful known animal groups.

Results: We present a robust genome assembly and a gene set possessing 97.5% of the core proteins known from the holometabolous insects. The ATUMI genome encodes fewer enzymes for plant digestion than the genomes of wood-feeding beetles but nonetheless shows signs of broad metabolic plasticity. Gustatory receptors are few in number compared to other beetles, especially receptors with known sensitivity (in other beetles) to bitter substances. In contrast, several gene families implicated in detoxification of insecticides and adaptation to diverse dietary resources show increased copy numbers. The presence and diversity of homologs involved in detoxification differ substantially from the bee hosts of ATUMI.

Conclusions: Our results provide new insights into the genomic basis for local adaption and invasiveness in ATUMI and a blueprint for control strategies that target this pest without harming their honey bee hosts. A minimal set of gustatory receptors is consistent with the observation that, once a host colony is invaded, food resources are predictable. Unique detoxification pathways and pathway members can help identify which treatments might control this species even in the presence of honey bees, which are notoriously sensitive to pesticides.
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http://dx.doi.org/10.1093/gigascience/giy138DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6302959PMC
December 2018

Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing.

J Vis Exp 2018 04 27(134). Epub 2018 Apr 27.

Department of Entomology and Plant Pathology, North Carolina State University;

The western corn rootworm (WCR) is an important pest of corn and is well known for its ability to rapidly adapt to pest management strategies. Although RNA interference (RNAi) has proved to be a powerful tool for studying WCR biology, it has its limitations. Specifically, RNAi itself is transient (i.e. does not result in long-term Mendelian inheritance of the associated phenotype), and it requires knowing the DNA sequence of the target gene. The latter can be limiting if the phenotype of interest is controlled by poorly conserved, or even novel genes, because identifying useful targets would be challenging, if not impossible. Therefore, the number of tools in WCR's genomic toolbox should be expanded by the development of methods that could be used to create stable mutant strains and enable sequence-independent surveys of the WCR genome. Herein, we detail the methods used to collect and microinject precellular WCR embryos with nucleic acids. While the protocols described herein are aimed at the creation of transgenic WCR, CRISPR/Cas9-genome editing could also be performed using the same protocols, with the only difference being the composition of the solution injected into the embryos.
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http://dx.doi.org/10.3791/57497DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6101034PMC
April 2018

Metabolic pathway interruption: CRISPR/Cas9-mediated knockout of tryptophan 2,3-dioxygenase in Tribolium castaneum.

J Insect Physiol 2018 May - Jun;107:104-109. Epub 2018 Mar 15.

USDA, Agricultural Research Service, Center for Grain and Animal Health Research, Manhattan, KS 66502, United States. Electronic address:

The Tribolium castaneum vermilion gene encodes tryptophan 2,3-dioxygenase, a pivotal enzyme in the ommochrome pathway that is required for proper pigmentation of the eye. A white-eyed mutant strain of T. castaneum, vermilion (v), lacks eye pigmentation due to a deletion of unknown size that removes all but the 3'-end of the vermilion gene. To create a more defined mutation in vermilion, the CRISPR/Cas9-nuclease system was used to target wild type vermilion in preblastoderm T. castaneum embryos. As adults, all injected beetles had wild type (black) eye pigmentation; however, when outcrossed to v mates, one cross produced 19% white-eyed offspring. When the vermilion locus of these offspring was analyzed by target-site sequencing, it was determined that white-eyed individuals had a 2 bp deletion that resulted in a frame-shift mutation, presumably producing a nonfunctional enzyme. Interestingly, some of their black-eyed siblings also had a small deletion of 6 bp, but the resultant loss of two amino acids had no apparent impact on enzyme function. To establish a mutant strain homozygous for the CRISPR-induced knock-out allele, a CRISPR positive G male was crossed to wild type females. Their progeny were self-crossed, and white-eyed progeny were used to establish the new strain. This mutant strain is herein named vermilion and will be used in future work in addition to or in place of v.
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http://dx.doi.org/10.1016/j.jinsphys.2018.03.004DOI Listing
September 2019

Development and use of a piggyBac-based jumpstarter system in Drosophila suzukii.

Arch Insect Biochem Physiol 2018 Mar 1;97(3). Epub 2017 Dec 1.

Department of Entomology and Plant Pathology, North Carolina State University, Raleigh, NC, USA.

Spotted wing drosophila, Drosophila suzukii, is an invasive pest that primarily attacks fresh, soft-skinned fruit. Although others have reported successful integration of marked piggyBac elements into the D. suzukii genome, with a very respectable transgenesis rate of ∼16%, here we take this work a step further by creating D. suzukii jumpstarter strains. These were generated through integration of a fluorescent-marked Minos element carrying a heat shock protein 70-driven piggyBac transposase gene. We demonstrate that there is a dramatic increase in transformation rates when germline transformation is performed in a transposase-expressing background. For example, we achieved transformation rates as high as 80% when microinjecting piggyBac-based plasmids into embryos derived from one of these D. suzukii jumpstarter strains. We also investigate the effect of insert size on transformation efficiency by testing the ability of the most efficient jumpstarter strain to catalyze integration of differently-sized piggyBac elements. Finally, we demonstrate the ability of a jumpstarter strain to remobilize an already-integrated piggyBac element to a new location, demonstrating that our jumpstarter strains could be used in conjunction with a piggyBac-based donor strain for genome-wide mutagenesis of D. suzukii.
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http://dx.doi.org/10.1002/arch.21439DOI Listing
March 2018

Multifaceted biological insights from a draft genome sequence of the tobacco hornworm moth, Manduca sexta.

Insect Biochem Mol Biol 2016 09 12;76:118-147. Epub 2016 Aug 12.

Centre of Systems Biology, Biomedical Research Foundation, Academy of Athens, Athens, Greece.

Manduca sexta, known as the tobacco hornworm or Carolina sphinx moth, is a lepidopteran insect that is used extensively as a model system for research in insect biochemistry, physiology, neurobiology, development, and immunity. One important benefit of this species as an experimental model is its extremely large size, reaching more than 10 g in the larval stage. M. sexta larvae feed on solanaceous plants and thus must tolerate a substantial challenge from plant allelochemicals, including nicotine. We report the sequence and annotation of the M. sexta genome, and a survey of gene expression in various tissues and developmental stages. The Msex_1.0 genome assembly resulted in a total genome size of 419.4 Mbp. Repetitive sequences accounted for 25.8% of the assembled genome. The official gene set is comprised of 15,451 protein-coding genes, of which 2498 were manually curated. Extensive RNA-seq data from many tissues and developmental stages were used to improve gene models and for insights into gene expression patterns. Genome wide synteny analysis indicated a high level of macrosynteny in the Lepidoptera. Annotation and analyses were carried out for gene families involved in a wide spectrum of biological processes, including apoptosis, vacuole sorting, growth and development, structures of exoskeleton, egg shells, and muscle, vision, chemosensation, ion channels, signal transduction, neuropeptide signaling, neurotransmitter synthesis and transport, nicotine tolerance, lipid metabolism, and immunity. This genome sequence, annotation, and analysis provide an important new resource from a well-studied model insect species and will facilitate further biochemical and mechanistic experimental studies of many biological systems in insects.
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http://dx.doi.org/10.1016/j.ibmb.2016.07.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5010457PMC
September 2016

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

BMC Genomics 2015 Nov 18;16:968. Epub 2015 Nov 18.

USDA Agricultural Research Service, Center for Grain and Animal Health Research, Manhattan, KS, USA.

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.
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http://dx.doi.org/10.1186/s12864-015-2121-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4650509PMC
November 2015

The ABCs of eye color in Tribolium castaneum: orthologs of the Drosophila white, scarlet, and brown Genes.

Genetics 2015 Mar 2;199(3):749-59. Epub 2015 Jan 2.

Department of Entomology, North Carolina State University, Raleigh, North Carolina 27695

In Drosophila melanogaster, each of the three paralogous ABC transporters, White, Scarlet and Brown, is required for normal pigmentation of the compound eye. We have cloned the three orthologous genes from the beetle Tribolium castaneum. Conceptual translations of Tribolium white (Tcw), scarlet (Tcst), and brown (Tcbw) are 51, 48, and 32% identical to their respective Drosophila counterparts. We have identified loss-of-eye-pigment strains that bear mutations in Tcw and Tcst: the Tcw gene in the ivory (i) strain carries a single-base transversion, which leads to an E → D amino-acid substitution in the highly conserved Walker B motif, while the Tcst gene in the pearl (p) strain has a deletion resulting in incorporation of a premature stop codon. In light of these findings, the mutant strains i and p are herein renamed white(ivory) (w(i)) and scarlet(pearl) (st(p)), respectively. In addition, RNA inhibition of Tcw and Tcst recapitulates the mutant phenotypes, confirming the roles of these genes in normal eye pigmentation, while RNA interference of Tcbw provides further evidence that it has no role in eye pigmentation in Tribolium. We also consider the evolutionary implications of our findings.
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http://dx.doi.org/10.1534/genetics.114.173971DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4349069PMC
March 2015

Functional analysis of the ATP-binding cassette (ABC) transporter gene family of Tribolium castaneum.

BMC Genomics 2013 Jan 16;14. Epub 2013 Jan 16.

Department of Biology/Chemistry, Animal Physiology, University of Osnabrück, Osnabrück 49069, Germany.

Background: The ATP-binding cassette (ABC) transporters belong to a large superfamily of proteins that have important physiological functions in all living organisms. Most are integral membrane proteins that transport a broad spectrum of substrates across lipid membranes. In insects, ABC transporters are of special interest because of their role in insecticide resistance.

Results: We have identified 73 ABC transporter genes in the genome of T. castaneum, which group into eight subfamilies (ABCA-H). This coleopteran ABC family is significantly larger than those reported for insects in other taxonomic groups. Phylogenetic analysis revealed that this increase is due to gene expansion within a single clade of subfamily ABCC. We performed an RNA interference (RNAi) screen to study the function of ABC transporters during development. In ten cases, injection of double-stranded RNA (dsRNA) into larvae caused developmental phenotypes, which included growth arrest and localized melanization, eye pigmentation defects, abnormal cuticle formation, egg-laying and egg-hatching defects, and mortality due to abortive molting and desiccation. Some of the ABC transporters we studied in closer detail to examine their role in lipid, ecdysteroid and eye pigment transport.

Conclusions: The results from our study provide new insights into the physiological function of ABC transporters in T. castaneum, and may help to establish new target sites for insect control.
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http://dx.doi.org/10.1186/1471-2164-14-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3560195PMC
January 2013

Transcriptome profiling of the intoxication response of Tenebrio molitor larvae to Bacillus thuringiensis Cry3Aa protoxin.

PLoS One 2012 25;7(4):e34624. Epub 2012 Apr 25.

USDA Agricultural Research Service, Center for Grain and Animal Health Research, Manhattan, Kansas, United States of America.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0034624PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3338813PMC
September 2012

Genetic structure of Tribolium castaneum (Coleoptera: Tenebrionidae) populations in mills.

Environ Entomol 2012 Feb;41(1):188-99

Department of Entomology, Kansas State University, Manhattan, KS 66506, USA.

The red flour beetle, Tribolium castaneum (Herbst), is primarily found associated with human structures such as wheat and rice mills. Such structures are predicted to be spatially isolated resource patches with frequent population bottlenecks that should influence their genetic structure. Genetic diversity and differentiation among nine populations of T. castaneum collected from wheat and rice mills (ranging from <1-5,700 km apart) were investigated using eight polymorphic loci (microsatellites and other insertion-deletion polymorphisms, each with 3-14 alleles). Seventy-two locus-by-population combinations were evaluated, of which 31 deviated significantly from Hardy-Weinberg equilibrium, all because of a deficiency of heterozygotes. AMOVA analysis indicated significant differences among populations, with 8.3% of the variation in allele frequency resulting from comparisons among populations, and commodity type and geographic region not significant factors. Although there were significant differences in genetic differentiation among populations (F(ST) values = 0.018-0.149), genetic distance was not significantly correlated with geographic distance. Correct assignment to the source population was successful for only 56% of individuals collected. Further analyses confirmed the occurrence of recent genetic bottlenecks in five out of nine populations. These results provide evidence that populations of T. castaneum collected from mills show spatial genetic structure, but the poor ability to assign individuals to source populations and lack of isolation by distance suggest greater levels of gene flow than predicted originally.
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http://dx.doi.org/10.1603/EN11207DOI Listing
February 2012

RNA interference as a method for target-site screening in the Western corn rootworm, Diabrotica virgifera virgifera.

J Insect Sci 2010 ;10:162

Department of Entomology, University of Nebraska - Lincoln, Lincoln, NE 68583-0816, USA.

To test the efficacy of RNA interference (RNAi) as a method for target-site screening in Diabrotica virgifera virgifera LeConte (Coleptera: Chrysomelidae) larvae, genes were identified and tested for which clear RNAi phenotypes had been identified in the Coleopteran model, Tribolium castaneum. Here the cloning of the D. v. vergifera orthologs of laccase 2 (DvvLac2) and chitin synthase 2 (DvvCHS2) is reported. Injection of DvvLac2-specific double-stranded RNA resulted in prevention of post-molt cuticular tanning, while injection of DvvCHS2-specific dsRNA reduced chitin levels in midguts. Silencing of both DvvLac2 and DvvCHS2 was confirmed by RT-PCR and quantitative RT-PCR. As in T. castaneum, RNAi-mediated gene silencing is systemic in Diabrotica. The results indicate that RNAi-induced silencing of D. v. vergifera genes provides a powerful tool for identifying potential insecticide targets.
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http://dx.doi.org/10.1673/031.010.14122DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3395163PMC
February 2011

Large-scale insertional mutagenesis of a coleopteran stored grain pest, the red flour beetle Tribolium castaneum, identifies embryonic lethal mutations and enhancer traps.

BMC Biol 2009 Nov 5;7:73. Epub 2009 Nov 5.

1Department of Biology, Developmental Biology, Friedrich-Alexander-University Erlangen, Erlangen, Germany.

Background: Given its sequenced genome and efficient systemic RNA interference response, the red flour beetle Tribolium castaneum is a model organism well suited for reverse genetics. Even so, there is a pressing need for forward genetic analysis to escape the bias inherent in candidate gene approaches.

Results: To produce easy-to-maintain insertional mutations and to obtain fluorescent marker lines to aid phenotypic analysis, we undertook a large-scale transposon mutagenesis screen. In this screen, we produced more than 6,500 new piggyBac insertions. Of these, 421 proved to be recessive lethal, 75 were semi-lethal, and eight indicated recessive sterility, while 505 showed new enhancer-trap patterns. Insertion junctions were determined for 403 lines and often appeared to be located within transcription units. Insertion sites appeared to be randomly distributed throughout the genome, with the exception of a preference for reinsertion near the donor site.

Conclusion: A large collection of enhancer-trap and embryonic lethal beetle lines has been made available to the research community and will foster investigations into diverse fields of insect biology, pest control, and evolution. Because the genetic elements used in this screen are species-nonspecific, and because the crossing scheme does not depend on balancer chromosomes, the methods presented herein should be broadly applicable for many insect species.
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http://dx.doi.org/10.1186/1741-7007-7-73DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2779179PMC
November 2009

BeetleBase in 2010: revisions to provide comprehensive genomic information for Tribolium castaneum.

Nucleic Acids Res 2010 Jan 9;38(Database issue):D437-42. Epub 2009 Oct 9.

KSU Bioinformatics Center, Division of Biology, Kansas State University, Manhattan, KS 66506, USA.

BeetleBase (http://www.beetlebase.org) has been updated to provide more comprehensive genomic information for the red flour beetle Tribolium castaneum. The database contains genomic sequence scaffolds mapped to 10 linkage groups (genome assembly release Tcas_3.0), genetic linkage maps, the official gene set, Reference Sequences from NCBI (RefSeq), predicted gene models, ESTs and whole-genome tiling array data representing several developmental stages. The database was reconstructed using the upgraded Generic Model Organism Database (GMOD) modules. The genomic data is stored in a PostgreSQL relatational database using the Chado schema and visualized as tracks in GBrowse. The updated genetic map is visualized using the comparative genetic map viewer CMAP. To enhance the database search capabilities, the BLAST and BLAT search tools have been integrated with the GMOD tools. BeetleBase serves as a long-term repository for Tribolium genomic data, and is compatible with other model organism databases.
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http://dx.doi.org/10.1093/nar/gkp807DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2808946PMC
January 2010

Tribolium castaneum larval gut transcriptome and proteome: A resource for the study of the coleopteran gut.

J Proteome Res 2009 Aug;8(8):3889-98

Department of Biochemistry, Kansas State University, Manhattan, 66506, USA.

Tribolium castaneum is an important agricultural pest and an advanced genetic model for coleopteran insects. We have taken advantage of the recently acquired T. castaneum genome to identify T. castaneum genes and proteins in one of the more critical environmental interfaces of the insect, the larval alimentary tract. Genetic transcripts isolated from the T. castaneum larval gut were labeled and hybridized to a custom array containing oligonucleotides from predicted genes in the T. castaneum genome. Through a ranking procedure based on relative labeling intensity, we found that approximately 17.6% of the genes represented in the array were predicted to be highly expressed in gut tissue. Several genes were selected to compare relative expression levels in larval gut, head, or carcass tissues using quantitative real-time PCR, and expression levels were, with few exceptions, consistent with the gut rankings. In parallel with the microarrays, proteins extracted from the T. castaneum larval gut were subjected to proteomic analysis. Two-dimensional electrophoretic analysis combined with MALDI-TOF resulted in the identification of 37 of 88 selected protein samples. As an alternative strategy, one-dimensional electrophoretic separation of T. castaneum larval gut proteins followed by two-dimensional nano-HPLC and ESI-MS/MS resulted in the identification of 98 proteins. A comparison of the proteomic studies indicated that 16 proteins were commonly identified in both, whereas 80 proteins from the proteomic analyses corresponded to genes with gut rankings indicative of high expression in the microarray analysis. These data serve as a resource of T. castaneum transcripts and proteins in the larval gut and provide the basis for comparative transcriptomic and proteomic studies related to the gut of coleopteran insects.
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http://dx.doi.org/10.1021/pr900168zDOI Listing
August 2009

A novel Tenebrio molitor cadherin is a functional receptor for Bacillus thuringiensis Cry3Aa toxin.

J Biol Chem 2009 Jul 5;284(27):18401-10. Epub 2009 May 5.

United States Department of Agriculture Agricultural Research Service United States Arid-Land Agricultural Research Center, Maricopa, Arizona 85238, USA.

Cry toxins produced by the bacterium Bacillus thuringiensis are effective biological insecticides. Cadherin-like proteins have been reported as functional Cry1A toxin receptors in Lepidoptera. Here we present data that demonstrate that a coleopteran cadherin is a functional Cry3Aa toxin receptor. The Cry3Aa receptor cadherin was cloned from Tenebrio molitor larval midgut mRNA, and the predicted protein, TmCad1, has domain structure and a putative toxin binding region similar to those in lepidopteran cadherin B. thuringiensis receptors. A peptide containing the putative toxin binding region from TmCad1 bound specifically to Cry3Aa and promoted the formation of Cry3Aa toxin oligomers, proposed to be mediators of toxicity in lepidopterans. Injection of TmCad1-specific double-stranded RNA into T. molitor larvae resulted in knockdown of the TmCad1 transcript and conferred resistance to Cry3Aa toxicity. These data demonstrate the functional role of TmCad1 as a Cry3Aa receptor in T. molitor and reveal similarities between the mode of action of Cry toxins in Lepidoptera and Coleoptera.
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http://dx.doi.org/10.1074/jbc.M109.001651DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2709378PMC
July 2009

Tubulin superfamily genes in Tribolium castaneum and the use of a Tubulin promoter to drive transgene expression.

Insect Biochem Mol Biol 2008 Aug 16;38(8):749-55. Epub 2008 May 16.

Department of Entomology, Kansas State University, Manhattan, KS 66506, USA.

The use of native promoters to drive transgene expression has facilitated overexpression studies in Drosophila and other insects. We identified 12 Tubulin family members from the genome sequence of the red flour beetle, Tribolium castaneum, and used the promoter from one of these to drive constitutive expression of a transgene. The activity of the T. castaneum alpha-Tubulin1 (TcalphaTub1) putative promoter was pre-tested in conjunction with an eye-color gene, T. castaneum vermilion (Tcv), by transient expression in Tcv-deficient embryos. Such embryos showed complete rescue of larval eyespot pigmentation. We also examined the TcalphaTub1 expression pattern in germline transformants using the enhanced green fluorescent protein (EGFP) reporter. Beetles transformed with this piggyBac-based reporter ubiquitously expressed EGFP at all stages.
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http://dx.doi.org/10.1016/j.ibmb.2008.04.007DOI Listing
August 2008

The maternal-effect, selfish genetic element Medea is associated with a composite Tc1 transposon.

Proc Natl Acad Sci U S A 2008 Jul 11;105(29):10085-9. Epub 2008 Jul 11.

United States Department of Agriculture, Grain Marketing and Production Research Center, Agricultural Research Service, Manhattan, KS 66502, USA.

Maternal-Effect Dominant Embryonic Arrest ("Medea") factors are selfish nuclear elements that combine maternal-lethal and zygotic-rescue activities to gain a postzygotic survival advantage. We show that Medea(1) activity in Tribolium castaneum is associated with a composite Tc1 transposon inserted just downstream of the neurotransmitter reuptake symporter bloated tubules (blot), whose Drosophila ortholog has both maternal and zygotic functions. The 21.5-kb insertion contains defective copies of elongation initiation factor-3, ATP synthase subunit C, and an RNaseD-related gene, as well as a potentially intact copy of a prokaryotic DUF1703 gene. Sequence comparisons suggest that the current distribution of Medea(1) reflects global emanation after a single transpositional event in recent evolutionary time. The Medea system in Tribolium represents an unusual type of intragenomic conflict and could provide a useful vehicle for driving desirable genes into populations.
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http://dx.doi.org/10.1073/pnas.0800444105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2481321PMC
July 2008

Analysis of repetitive DNA distribution patterns in the Tribolium castaneum genome.

Genome Biol 2008 26;9(3):R61. Epub 2008 Mar 26.

Department of Biology, Kansas State University, Manhattan, KS 66506, USA.

Background: Insect genomes vary widely in size, a large fraction of which is often devoted to repetitive DNA. Re-association kinetics indicate that up to 42% of the genome of the red flour beetle, Tribolium castaneum, is repetitive. Analysis of the abundance and distribution of repetitive DNA in the recently sequenced genome of T. castaneum is important for understanding the structure and function of its genome.

Results: Using TRF, TEpipe and RepeatScout we found that approximately 30% of the T. castaneum assembled genome is composed of repetitive DNA. Of this, 17% is found in tandem arrays and the remaining 83% is dispersed, including transposable elements, which in themselves constitute 5-6% of the genome. RepeatScout identified 31 highly repetitive DNA elements with repeat units longer than 100 bp, which constitute 7% of the genome; 65% of these highly repetitive elements and 74% of transposable elements accumulate in regions representing 40% of the assembled genome that is anchored to chromosomes. These regions tend to occur near one end of each chromosome, similar to previously described blocks of pericentric heterochromatin. They contain fewer genes with longer introns, and often correspond with regions of low recombination in the genetic map.

Conclusion: Our study found that transposable elements and other repetitive DNA accumulate in certain regions in the assembled T. castaneum genome. Several lines of evidence suggest these regions are derived from the large blocks of pericentric heterochromatin in T. castaneum chromosomes.
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http://dx.doi.org/10.1186/gb-2008-9-3-r61DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2397513PMC
June 2008

The genome of the model beetle and pest Tribolium castaneum.

Nature 2008 Apr 23;452(7190):949-55. Epub 2008 Mar 23.

Human Genome Sequencing Center, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030, USA.

Tribolium castaneum is a member of the most species-rich eukaryotic order, a powerful model organism for the study of generalized insect development, and an important pest of stored agricultural products. We describe its genome sequence here. This omnivorous beetle has evolved the ability to interact with a diverse chemical environment, as shown by large expansions in odorant and gustatory receptors, as well as P450 and other detoxification enzymes. Development in Tribolium is more representative of other insects than is Drosophila, a fact reflected in gene content and function. For example, Tribolium has retained more ancestral genes involved in cell-cell communication than Drosophila, some being expressed in the growth zone crucial for axial elongation in short-germ development. Systemic RNA interference in T. castaneum functions differently from that in Caenorhabditis elegans, but nevertheless offers similar power for the elucidation of gene function and identification of targets for selective insect control.
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http://dx.doi.org/10.1038/nature06784DOI Listing
April 2008