@article{han_klobasa_oliveira_rotenberg_whitfield_lorenzen_2024, title={CRISPR/Cas9-mediated genome editing of Frankliniella occidentalis, the western flower thrips, via embryonic microinjection}, volume={4}, ISSN={["1365-2583"]}, DOI={10.1111/imb.12913}, abstractNote={Abstract The western flower thrips, Frankliniella occidentalis , poses a significant challenge in global agriculture as a notorious pest and a vector of economically significant orthotospoviruses. However, the limited availability of genetic tools for F. occidentalis hampers the advancement of functional genomics and the development of innovative pest control strategies. In this study, we present a robust methodology for generating heritable mutations in F. occidentalis using the CRISPR/Cas9 genome editing system. Two eye‐colour genes, white ( Fo‐w ) and cinnabar ( Fo‐cn ), frequently used to assess Cas9 function in insects were identified in the F. occidentalis genome and targeted for knockout through embryonic microinjection of Cas9 complexed with Fo‐w or Fo‐cn specific guide RNAs. Homozygous Fo‐w and Fo‐cn knockout lines were established by crossing mutant females and males. The Fo‐w knockout line revealed an age‐dependent modification of eye‐colour phenotype. Specifically, while young larvae exhibit orange‐coloured eyes, the colour transitions to bright red as they age. Unexpectedly, loss of Fo‐w function also altered body colour, with Fo‐w mutants having a lighter coloured body than wild type, suggesting a dual role for Fo‐w in thrips. In contrast, individuals from the Fo‐cn knockout line consistently displayed bright red eyes throughout all life stages. Molecular analyses validated precise editing of both target genes. This study offers a powerful tool to investigate thrips gene function and paves the way for the development of genetic technologies for population suppression and/or population replacement as a means of mitigating virus transmission by this vector.}, journal={INSECT MOLECULAR BIOLOGY}, author={Han, Jinlong and Klobasa, William and Oliveira, Lucas and Rotenberg, Dorith and Whitfield, Anna E. and Lorenzen, Marce D.}, year={2024}, month={Apr} }
@article{wang_rivera_klobasa_lorenzen_2024, title={Evaluation of Peregrinus maidis transformer-2 as a target for CRISPR-based control}, volume={19}, ISSN={["1932-6203"]}, DOI={10.1371/journal.pone.0295335}, abstractNote={The corn planthopper, Peregrinus maidis, is an economically important pest of corn and sorghum. Here we report the initial steps towards developing a CRISPR-based control method, precision guided sterile insect technique (pgSIT), for this hemipteran pest. Specifically, we evaluated the potential of transformer-2 (tra-2) as a target for sterilizing insects. First, we identified tra-2 transcripts within our P. maidis transcriptome database and performed RNA interference (RNAi) to confirm functional conservation. RNAi-mediated knockdown of Pmtra-2 in nymphs transformed females into pseudomales with deformed ovipositors resembling male claspers. While males showed no overt difference in appearance, they were indeed sterile. Importantly, the results were similar to those observed in another planthopper, Nilaparvata lugens. We also used CRISPR/Cas9 genome editing to assess the impact of tra-2 knockout in injectees. CRISPR-mediated knockout of Pmtra-2 had lethal effects on embryos, and hence not many injectees reached adulthood. However, mosaic knockout of Pmtra-2 did impact female and male fertility, which supports the use of tra-2 as a target for pgSIT in this hemipteran species.}, number={4}, journal={PLOS ONE}, author={Wang, Yu-Hui and Rivera, Dina Espinoza and Klobasa, William and Lorenzen, Marce D.}, year={2024}, month={Apr} }
@article{wang_mikaelyan_coates_lorenzen_2024, title={The Genome of Arsenophonus sp. and Its Potential Contribution in the Corn Planthopper, Peregrinus maidis}, volume={15}, ISSN={["2075-4450"]}, url={https://www.mdpi.com/2075-4450/15/2/113}, DOI={10.3390/insects15020113}, abstractNote={The co-evolution between symbionts and their insect hosts has led to intricate functional interdependencies. Advances in DNA-sequencing technologies have not only reduced the cost of sequencing but, with the advent of highly accurate long-read methods, have also enabled facile genome assembly even using mixed genomic input, thereby allowing us to more easily assess the contribution of symbionts to their insect hosts. In this study, genomic data recently generated from Peregrinus maidis was used to assemble the genome of a bacterial symbiont, Pm Arsenophonus sp. This ~4.9-Mb assembly is one of the largest Arsenophonus genomes reported to date. The Benchmarking Universal Single-Copy Orthologs (BUSCO) result indicates that this Pm Arsenophonus assembly has a high degree of completeness, with 96% of the single-copy Enterobacterales orthologs found. The identity of the Pm Arsenophonus sp. was further confirmed by phylogenetic analysis. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicates a major contribution by Pm Arsenophonus sp. to the biosynthesis of B vitamins and essential amino acids in P. maidis, where threonine and lysine production is carried out solely by Pm Arsenophonus sp. This study not only provides deeper insights into the evolutionary relationships between symbionts and their insect hosts, but also adds to our understanding of insect biology, potentially guiding the development of novel pest control methods.}, number={2}, journal={INSECTS}, author={Wang, Yu-Hui and Mikaelyan, Aram and Coates, Brad S. and Lorenzen, Marce}, year={2024}, month={Feb} }
@article{coates_walden_lata_vellichirammal_mitchell_andersson_mckay_lorenzen_grubbs_wang_et al._2023, title={A draft Diabrotica virgifera virgifera genome: insights into control and host plant adaption by a major maize pest insect}, volume={24}, ISSN={1471-2164}, url={http://dx.doi.org/10.1186/s12864-022-08990-y}, DOI={10.1186/s12864-022-08990-y}, abstractNote={Abstract}, number={1}, journal={BMC Genomics}, publisher={Springer Science and Business Media LLC}, author={Coates, Brad S. and Walden, Kimberly K. O. and Lata, Dimpal and Vellichirammal, Neetha Nanoth and Mitchell, Robert F. and Andersson, Martin N. and McKay, Rachel and Lorenzen, Marcé D. and Grubbs, Nathaniel and Wang, Yu-Hui and et al.}, year={2023}, month={Jan} }
@article{chu_wu_pinzi_grubbs_cohen_lorenzen_2023, title={An Optimized Small-Scale Rearing System to Support Embryonic Microinjection Protocols for Western Corn Rootworm, Diabrotica virgifera virgifera}, volume={14}, ISSN={2075-4450}, url={http://dx.doi.org/10.3390/insects14080683}, DOI={10.3390/insects14080683}, abstractNote={Western corn rootworm (WCR), a major pest of corn, has been reared in laboratories since the 1960s. While established rearing methods are appropriate for maintaining WCR colonies, they are not optimal for performing germline transformation or CRISPR/Cas9-based genome editing. Here we report the development of an optimized rearing system for use in WCR functional genomics research, specifically the development of a system that facilitates the collection of preblastoderm embryos for microinjection as well as gathering large larvae and pupae for downstream phenotypic screening. Further, transgenic-based experiments require stable and well-defined survival rates and the ability to manipulate insects at every life stage. In our system, the WCR life cycle (egg to adult) takes approximately 42 days, with most individuals eclosing between 41 and 45 days post oviposition. Over the course of one year, our overall survival rate was 67%. We used this data to establish a quality control system for more accurately monitoring colony health. Herein, we also offer detailed descriptions for setting up single-pair crosses and conducting phenotypic screens to identify transgenic progeny. This study provides a model for the development of new rearing systems and the establishment of highly controlled processes for specialized purposes.}, number={8}, journal={Insects}, publisher={MDPI AG}, author={Chu, Fu-Chyun and Wu, Pei-Shan and Pinzi, Sofia and Grubbs, Nathaniel and Cohen, Allen Carson and Lorenzen, Marcé D.}, year={2023}, month={Aug}, pages={683} }
@article{patil_klobasa_espinoza‐rivera_baars_lorenzen_scott_2023, title={Development of transgenic corn planthopper Peregrinus maidis}, volume={32}, ISSN={0962-1075 1365-2583}, url={http://dx.doi.org/10.1111/imb.12836}, DOI={10.1111/imb.12836}, abstractNote={Abstract}, number={4}, journal={Insect Molecular Biology}, publisher={Wiley}, author={Patil, Anandrao A. and Klobasa, William and Espinoza‐Rivera, Dina and Baars, Oliver and Lorenzen, Marcé D. and Scott, Maxwell J.}, year={2023}, month={Mar}, pages={363–375} }
@article{dossey_oppert_chu_lorenzen_scheffler_simpson_koren_johnston_kataoka_ide_2023, title={Genome and Genetic Engineering of the House Cricket (Acheta domesticus): A Resource for Sustainable Agriculture}, volume={13}, ISSN={2218-273X}, url={http://dx.doi.org/10.3390/biom13040589}, DOI={10.3390/biom13040589}, abstractNote={Background: The house cricket, Acheta domesticus, is one of the most farmed insects worldwide and the foundation of an emerging industry using insects as a sustainable food source. Edible insects present a promising alternative for protein production amid a plethora of reports on climate change and biodiversity loss largely driven by agriculture. As with other crops, genetic resources are needed to improve crickets for food and other applications. Methods: We present the first high quality annotated genome assembly of A. domesticus from long read data and scaffolded to chromosome level, providing information needed for genetic manipulation. Results: Gene groups related to immunity were annotated and will be useful for improving value to insect farmers. Metagenome scaffolds in the A. domesticus assembly, including Invertebrate Iridescent Virus 6 (IIV6), were submitted as host-associated sequences. We demonstrate both CRISPR/Cas9-mediated knock-in and knock-out of A. domesticus and discuss implications for the food, pharmaceutical, and other industries. RNAi was demonstrated to disrupt the function of the vermilion eye-color gene producing a useful white-eye biomarker phenotype. Conclusions: We are utilizing these data to develop technologies for downstream commercial applications, including more nutritious and disease-resistant crickets, as well as lines producing valuable bioproducts, such as vaccines and antibiotics.}, number={4}, journal={Biomolecules}, publisher={MDPI AG}, author={Dossey, Aaron T. and Oppert, Brenda and Chu, Fu-Chyun and Lorenzen, Marcé D. and Scheffler, Brian and Simpson, Sheron and Koren, Sergey and Johnston, J. Spencer and Kataoka, Kosuke and Ide, Keigo}, year={2023}, month={Mar}, pages={589} }
@article{wang_klobasa_chu_huot_whitfield_lorenzen_2023, title={Structural and functional insights into the ATP-binding cassette transporter family in the corn planthopper, Peregrinus maidis}, volume={32}, ISSN={0962-1075 1365-2583}, url={http://dx.doi.org/10.1111/imb.12840}, DOI={10.1111/imb.12840}, abstractNote={Abstract}, number={4}, journal={Insect Molecular Biology}, publisher={Wiley}, author={Wang, Yu‐Hui and Klobasa, William and Chu, Fu‐Chyun and Huot, Ordom and Whitfield, Anna E. and Lorenzen, Marcé}, year={2023}, month={Apr}, pages={412–423} }
@article{oppert_muszewska_steczkiewicz_šatović-vukšić_plohl_fabrick_vinokurov_koloniuk_johnston_smith_et al._2022, title={The Genome of Rhyzopertha dominica (Fab.) (Coleoptera: Bostrichidae): Adaptation for Success}, volume={13}, ISSN={2073-4425}, url={http://dx.doi.org/10.3390/genes13030446}, DOI={10.3390/genes13030446}, abstractNote={The lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae), is a major global pest of cereal grains. Infestations are difficult to control as larvae feed inside grain kernels, and many populations are resistant to both contact insecticides and fumigants. We sequenced the genome of R. dominica to identify genes responsible for important biological functions and develop more targeted and efficacious management strategies. The genome was assembled from long read sequencing and long-range scaffolding technologies. The genome assembly is 479.1 Mb, close to the predicted genome size of 480.4 Mb by flow cytometry. This assembly is among the most contiguous beetle assemblies published to date, with 139 scaffolds, an N50 of 53.6 Mb, and L50 of 4, indicating chromosome-scale scaffolds. Predicted genes from biologically relevant groups were manually annotated using transcriptome data from adults and different larval tissues to guide annotation. The expansion of carbohydrase and serine peptidase genes suggest that they combine to enable efficient digestion of cereal proteins. A reduction in the copy number of several detoxification gene families relative to other coleopterans may reflect the low selective pressure on these genes in an insect that spends most of its life feeding internally. Chemoreceptor genes contain elevated numbers of pseudogenes for odorant receptors that also may be related to the recent ontogenetic shift of R. dominica to a diet consisting primarily of stored grains. Analysis of repetitive sequences will further define the evolution of bostrichid beetles compared to other species. The data overall contribute significantly to coleopteran genetic research.}, number={3}, journal={Genes}, publisher={MDPI AG}, author={Oppert, Brenda and Muszewska, Anna and Steczkiewicz, Kamil and Šatović-Vukšić, Eva and Plohl, Miroslav and Fabrick, Jeffrey and Vinokurov, Konstantin and Koloniuk, Igor and Johnston, J. and Smith, Timothy and et al.}, year={2022}, month={Feb}, pages={446} }
@article{cooper_song_shi_yu_lorenzen_silver_zhang_zhu_2021, title={Characterization, expression patterns, and transcriptional responses of three core RNA interference pathway genes from Ostrinia nubilalis}, volume={129}, ISSN={0022-1910}, url={http://dx.doi.org/10.1016/j.jinsphys.2020.104181}, DOI={10.1016/j.jinsphys.2020.104181}, abstractNote={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.}, journal={Journal of Insect Physiology}, publisher={Elsevier BV}, author={Cooper, Anastasia M.W. and Song, Huifang and Shi, Xuekai and Yu, Zhitao and Lorenzen, Marcé and Silver, Kristopher and Zhang, Jianzhen and Zhu, Kun Yan}, year={2021}, month={Feb}, pages={104181} }
@article{klobasa_chu_huot_grubbs_rotenberg_whitfield_lorenzen_2021, title={Microinjection of Corn Planthopper, Peregrinus maidis, Embryos for CRISPR/Cas9 Genome Editing}, volume={3}, ISSN={1940-087X}, url={http://dx.doi.org/10.3791/62417}, DOI={10.3791/62417}, abstractNote={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.}, number={169}, journal={Journal of Visualized Experiments}, publisher={MyJove Corporation}, author={Klobasa, William and Chu, Fu-Chyun and Huot, Ordom and Grubbs, Nathaniel and Rotenberg, Dorith and Whitfield, Anna E. and Lorenzen, Marcé D.}, year={2021}, month={Mar} }
@article{long_alphey_annas_bloss_campbell_champer_chen_choudhary_church_collins_et al._2020, title={Core commitments for field trials of gene drive organisms}, volume={370}, ISSN={0036-8075 1095-9203}, url={http://dx.doi.org/10.1126/science.abd1908}, DOI={10.1126/science.abd1908}, abstractNote={We must ensure that trials are scientifically, politically, and socially robust, publicly accountable, and widely transparent}, number={6523}, journal={Science}, publisher={American Association for the Advancement of Science (AAAS)}, author={Long, Kanya C. and Alphey, Luke and Annas, George J. and Bloss, Cinnamon S. and Campbell, Karl J. and Champer, Jackson and Chen, Chun-Hong and Choudhary, Amit and Church, George M. and Collins, James P. and et al.}, year={2020}, month={Dec}, pages={1417–1419} }
@article{cooper_song_shi_yu_lorenzen_silver_zhang_zhu_2020, title={Molecular Characterizations of Double-Stranded RNA Degrading Nuclease Genes from Ostrinia nubilalis}, volume={11}, ISSN={2075-4450}, url={http://dx.doi.org/10.3390/insects11100652}, DOI={10.3390/insects11100652}, abstractNote={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), Ostrinia nubilalis, 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 (OndsRNase1, 2, 3, and 4) and one REase (OnREase) 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 OndsRNase2 and OnREase were highly expressed in the larval gut, and OndsRNase1 showed the highest expression in hemolymph, especially in older developmental stages. Transcript level analysis after dsRNA exposure revealed that expression of OnREase rapidly increased upon dsRNA ingestion or injection, whereas OndsRNase4 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.}, number={10}, journal={Insects}, publisher={MDPI AG}, author={Cooper, Anastasia M. W. and Song, Huifang and Shi, Xuekai and Yu, Zhitao and Lorenzen, Marcé and Silver, Kristopher and Zhang, Jianzhen and Zhu, Kun Yan}, year={2020}, month={Sep}, pages={652} }
@article{german_lorenzen_grubbs_whitfield_2020, title={New Technologies for Studying Negative-Strand RNA Viruses in Plant and Arthropod Hosts}, volume={33}, ISSN={0894-0282 1943-7706}, url={http://dx.doi.org/10.1094/MPMI-10-19-0281-FI}, DOI={10.1094/MPMI-10-19-0281-FI}, abstractNote={ The plant viruses in the phylum Negarnaviricota, orders Bunyavirales and Mononegavirales, 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. }, number={3}, journal={Molecular Plant-Microbe Interactions®}, publisher={Scientific Societies}, author={German, Thomas L. and Lorenzen, Marcé D. and Grubbs, Nathaniel and Whitfield, Anna E.}, year={2020}, month={Mar}, pages={382–393} }
@article{oppert_perkin_lorenzen_dossey_2020, title={Transcriptome analysis of life stages of the house cricket, Acheta domesticus, to improve insect crop production}, volume={10}, ISSN={2045-2322}, url={http://dx.doi.org/10.1038/s41598-020-59087-z}, DOI={10.1038/s41598-020-59087-z}, abstractNote={Abstract}, number={1}, journal={Scientific Reports}, publisher={Springer Science and Business Media LLC}, author={Oppert, Brenda and Perkin, Lindsey C. and Lorenzen, Marcé and Dossey, Aaron T.}, year={2020}, month={Feb} }
@article{oppert_chu_reyna_pinzi_adrianos_perkin_lorenzen_2019, title={Effects of targeting eye color in
Tenebrio molitor
through RNA interference of tryptophan 2,3‐dioxygenase (
vermilion
): Implications for insect farming}, volume={101}, ISSN={0739-4462 1520-6327}, url={http://dx.doi.org/10.1002/arch.21546}, DOI={10.1002/arch.21546}, abstractNote={Abstract}, number={1}, journal={Archives of Insect Biochemistry and Physiology}, publisher={Wiley}, author={Oppert, Brenda and Chu, Fu‐Chyun and Reyna, Steven and Pinzi, Sofia and Adrianos, Sherry and Perkin, Lindsey and Lorenzen, Marcé}, year={2019}, month={Mar}, pages={e21546} }
@article{adedipe_grubbs_coates_wiegmman_lorenzen_2019, title={Structural and functional insights into the Diabrotica virgifera virgifera ATP-binding cassette transporter gene family}, volume={20}, ISSN={1471-2164}, url={http://dx.doi.org/10.1186/s12864-019-6218-8}, DOI={10.1186/s12864-019-6218-8}, abstractNote={Abstract}, number={1}, journal={BMC Genomics}, publisher={Springer Science and Business Media LLC}, author={Adedipe, Folukemi and Grubbs, Nathaniel and Coates, Brad and Wiegmman, Brian and Lorenzen, Marcé}, year={2019}, month={Nov} }
@article{cash_lorenzen_gould_2019, title={The distribution and spread of naturally occurring Medea selfish genetic elements in the United States}, volume={9}, ISSN={2045-7758 2045-7758}, url={http://dx.doi.org/10.1002/ece3.5876}, DOI={10.1002/ece3.5876}, abstractNote={Abstract}, number={24}, journal={Ecology and Evolution}, publisher={Wiley}, author={Cash, Sarah A. and Lorenzen, Marce D. and Gould, Fred}, year={2019}, month={Nov}, pages={14407–14416} }
@article{cash_robert_lorenzen_gould_2019, title={The impact of local population genetic background on the spread of the selfish element Medea‐1 in red flour beetles}, volume={10}, ISSN={2045-7758 2045-7758}, url={http://dx.doi.org/10.1002/ece3.5946}, DOI={10.1002/ece3.5946}, abstractNote={Abstract}, number={2}, journal={Ecology and Evolution}, publisher={Wiley}, author={Cash, Sarah A. and Robert, Michael A. and Lorenzen, Marcé D. and Gould, Fred}, year={2019}, month={Dec}, pages={863–874} }
@misc{grubbs_chu_lorenzen_2019, title={Window to the Fluorescence: TheWhite}, url={http://dx.doi.org/10.1101/552935}, DOI={10.1101/552935}, abstractNote={ABSTRACT}, publisher={Cold Spring Harbor Laboratory}, author={Grubbs, Nathaniel and Chu, Fu-Chyun and Lorenzen, Marcé D.}, year={2019}, month={Feb} }
@article{chu_klobasa_grubbs_lorenzen_2018, title={Cover Image}, volume={97}, ISSN={0739-4462}, url={http://dx.doi.org/10.1002/ARCH.21453}, DOI={10.1002/ARCH.21453}, abstractNote={On the cover: The cover image, by Fu-Chyun Chu et al., is based on the Article Development and use of a piggyBac-based jumpstarter system in Drosophila suzukii, DOI 10.1002/arch.21439.}, number={3}, journal={Archives of Insect Biochemistry and Physiology}, publisher={Wiley}, author={Chu, Fu-Chyun and Klobasa, William and Grubbs, Nathaniel and Lorenzen, Marcé D.}, year={2018}, month={Feb}, pages={e21453} }
@article{evans_mckenna_scully_cook_dainat_egekwu_grubbs_lopez_lorenzen_reyna_et al._2018, title={Genome of the small hive beetle (Aethina tumida, Coleoptera: Nitidulidae), a worldwide parasite of social bee colonies, provides insights into detoxification and herbivory}, volume={7}, ISSN={2047-217X}, url={http://dx.doi.org/10.1093/gigascience/giy138}, DOI={10.1093/gigascience/giy138}, abstractNote={Abstract 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.}, number={12}, journal={GigaScience}, publisher={Oxford University Press (OUP)}, author={Evans, Jay D and McKenna, Duane and Scully, Erin and Cook, Steven C and Dainat, Benjamin and Egekwu, Noble and Grubbs, Nathaniel and Lopez, Dawn and Lorenzen, Marcé D and Reyna, Steven M and et al.}, year={2018}, month={Dec} }
@article{adrianos_lorenzen_oppert_2018, title={Metabolic pathway interruption: CRISPR/Cas9-mediated knockout of tryptophan 2,3-dioxygenase in Tribolium castaneum}, volume={107}, ISSN={["1879-1611"]}, DOI={10.1016/j.jinsphys.2018.03.004}, abstractNote={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, vermilionwhite (vw), 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 vw 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 G0 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 vermilionICE and will be used in future work in addition to or in place of vw.}, journal={JOURNAL OF INSECT PHYSIOLOGY}, author={Adrianos, Sherry and Lorenzen, Marce and Oppert, Brenda}, year={2018}, pages={104–109} }
@article{chu_wu_pinzi_grubbs_lorenzen_2018, title={Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing}, ISSN={1940-087X}, url={http://dx.doi.org/10.3791/57497}, DOI={10.3791/57497}, abstractNote={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.}, number={134}, journal={Journal of Visualized Experiments}, publisher={MyJove Corporation}, author={Chu, Fu-Chyun and Wu, Pei-Shan and Pinzi, Sofia and Grubbs, Nathaniel and Lorenzen, Marcé D.}, year={2018}, month={Apr} }
@article{scott_gould_lorenzen_grubbs_edwards_o’brochta_2017, title={Agricultural production: assessment of the potential use of Cas9-mediated gene drive systems for agricultural pest control}, volume={5}, ISSN={2329-9460 2329-9037}, url={http://dx.doi.org/10.1080/23299460.2017.1410343}, DOI={10.1080/23299460.2017.1410343}, abstractNote={ABSTRACTTo highlight how gene drives could be useful for control of agricultural insect pests, we selected species that are pests of animals (New World screwworm), plants (spotted wing Drosophila, ...}, number={sup1}, journal={Journal of Responsible Innovation}, publisher={Informa UK Limited}, author={Scott, Maxwell J. and Gould, Fred and Lorenzen, Marcé and Grubbs, Nathaniel and Edwards, Owain and O’Brochta, David}, year={2017}, month={Dec}, pages={S98–S120} }
@article{chu_klobasa_grubbs_lorenzen_2017, title={Development and use of a piggyBac
-based jumpstarter system in Drosophila suzukii}, volume={97}, ISSN={0739-4462}, url={http://dx.doi.org/10.1002/arch.21439}, DOI={10.1002/arch.21439}, abstractNote={Abstract}, number={3}, journal={Archives of Insect Biochemistry and Physiology}, publisher={Wiley}, author={Chu, Fu-Chyun and Klobasa, William and Grubbs, Nathaniel and Lorenzen, Marcé D.}, year={2017}, month={Dec}, pages={e21439} }
@article{chu_klobasa_wu_pinzi_grubbs_gorski_cardoza_lorenzen_2017, title={Germline transformation of the western corn rootworm, Diabrotica virgifera virgifera}, volume={26}, ISSN={0962-1075}, url={http://dx.doi.org/10.1111/imb.12305}, DOI={10.1111/imb.12305}, abstractNote={Abstract}, number={4}, journal={Insect Molecular Biology}, publisher={Wiley}, author={Chu, F. and Klobasa, W. and Wu, P. and Pinzi, S. and Grubbs, N. and Gorski, S. and Cardoza, Y. and Lorenzen, M. D.}, year={2017}, month={Apr}, pages={440–452} }
@article{kanost_arrese_cao_chen_chellapilla_goldsmith_grosse-wilde_heckel_herndon_jiang_et al._2016, title={Multifaceted biological insights from a draft genome sequence of the tobacco hornworm moth, Manduca sexta}, volume={76}, ISSN={0965-1748}, url={http://dx.doi.org/10.1016/J.IBMB.2016.07.005}, DOI={10.1016/J.IBMB.2016.07.005}, abstractNote={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.}, journal={Insect Biochemistry and Molecular Biology}, publisher={Elsevier BV}, author={Kanost, Michael R. and Arrese, Estela L. and Cao, Xiaolong and Chen, Yun-Ru and Chellapilla, Sanjay and Goldsmith, Marian R. and Grosse-Wilde, Ewald and Heckel, David G. and Herndon, Nicolae and Jiang, Haobo and et al.}, year={2016}, month={Sep}, pages={118–147} }
@article{kanost_arrese_cao_chen_chellapilla_goldsmith_grosse-wilde_heckel_herndon_jiang_et al._2016, title={Multifaceted biological insights from a draft genome sequence of the tobacco hornworm moth, Manduca sexta}, volume={76}, journal={Insect Biochemistry and Molecular Biology}, author={Kanost, M. R. and Arrese, E. L. and Cao, X. L. and Chen, Y. R. and Chellapilla, S. and Goldsmith, M. R. and Grosse-Wilde, E. and Heckel, D. G. and Herndon, N. and Jiang, H. B. and et al.}, year={2016}, pages={118–147} }
@article{oppert_guedes_aikins_perkin_chen_phillips_zhu_opit_hoon_sun_et al._2015, title={Genes related to mitochondrial functions are differentially expressed in phosphine-resistant and -susceptible Tribolium castaneum}, volume={16}, ISSN={["1471-2164"]}, DOI={10.1186/s12864-015-2121-0}, abstractNote={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.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.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.}, journal={BMC GENOMICS}, author={Oppert, Brenda and Guedes, Raul N. C. and Aikins, Michael J. and Perkin, Lindsey and Chen, Zhaorigetu and Phillips, Thomas W. and Zhu, Kun Yan and Opit, George P. and Hoon, Kelly and Sun, Yongming and et al.}, year={2015}, month={Nov} }
@article{grubbs_haas_beeman_lorenzen_2015, title={The ABCs of Eye Color in Tribolium castaneum: Orthologs of the Drosophila white, scarlet, and brown Genes}, volume={199}, ISSN={["1943-2631"]}, DOI={10.1534/genetics.114.173971}, abstractNote={Abstract}, number={3}, journal={GENETICS}, author={Grubbs, Nathaniel and Haas, Sue and Beeman, Richard W. and Lorenzen, Marce D.}, year={2015}, month={Mar}, pages={749-+} }
@book{hodgson_roe_mailman_chambers_2014, place={Amsterdam}, edition={3rd}, title={Dictionary of Toxicology}, ISBN={9780124202412 9780124201699 9780124201699 9781322192246}, publisher={Academic Press}, author={Hodgson, E. and Roe, M. and Mailman, R. and Chambers, J.}, year={2014} }
@inproceedings{chu_lorenzen_klobasa_2014, title={Genetics and Genomics of Tribolium Medea Elements}, author={Chu, F and Lorenzen, MD and Klobasa, W.A.}, year={2014} }
@article{broehan_kroeger_lorenzen_merzendorfer_2013, title={Functional analysis of the ATP-binding cassette (ABC) transporter gene family of Tribolium castaneum}, volume={14}, ISSN={["1471-2164"]}, DOI={10.1186/1471-2164-14-6}, abstractNote={Abstract}, journal={BMC GENOMICS}, author={Broehan, Gunnar and Kroeger, Tobias and Lorenzen, Marce and Merzendorfer, Hans}, year={2013}, month={Jan} }
@article{semeao_campbell_beeman_lorenzen_whitworth_sloderbeck_2012, title={Genetic Structure of Tribolium castaneum (Coleoptera: Tenebrionidae) Populations in Mills}, volume={41}, ISSN={0046-225X 1938-2936}, url={http://dx.doi.org/10.1603/en11207}, DOI={10.1603/en11207}, abstractNote={ABSTRACT
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 (FST 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.}, number={1}, journal={Environmental Entomology}, publisher={Oxford University Press (OUP)}, author={Semeao, Altair A. and Campbell, James F. and Beeman, Richard W. and Lorenzen, Marcé D. and Whitworth, R. Jeff and Sloderbeck, Phillip E.}, year={2012}, month={Feb}, pages={188–199} }
@inproceedings{semeao_campbell_beeman_whitworth_sloderbeck_lorenzen_2012, title={Genetic structure of Tribolium castaneum (Coleoptera: Tenebrionidae) populations in mills}, author={Semeao, A.A. and Campbell, J.F. and Beeman, R.W. and Whitworth, R.J. and Sloderbeck, P.E. and Lorenzen, M.D.}, year={2012} }
@article{oppert_dowd_bouffard_li_conesa_lorenzen_toutges_marshall_huestis_fabrick_et al._2012, title={Transcriptome Profiling of the Intoxication Response of Tenebrio molitor Larvae to Bacillus thuringiensis Cry3Aa Protoxin}, volume={7}, ISSN={1932-6203}, url={http://dx.doi.org/10.1371/journal.pone.0034624}, DOI={10.1371/journal.pone.0034624}, abstractNote={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.}, number={4}, journal={PLoS ONE}, publisher={Public Library of Science (PLoS)}, author={Oppert, Brenda and Dowd, Scot E. and Bouffard, Pascal and Li, Lewyn and Conesa, Ana and Lorenzen, Marcé D. and Toutges, Michelle and Marshall, Jeremy and Huestis, Diana L. and Fabrick, Jeff and et al.}, editor={Palli, Subba ReddyEditor}, year={2012}, month={Apr}, pages={e34624} }
@inbook{aronstein_oppert_lorenzen_2011, title={RNAi in Agriculturally-Important Arthropods}, ISBN={9789533075570}, url={https://www.intechopen.com/books/rna-processing/rnai-in-agriculturally-important-arthropods}, DOI={10.5772/19768}, abstractNote={This chapter was inspired by rapid developments in the field of RNA interference (RNAi), an evolutionarily-conserved cellular mechanism that directs protection against nucleic-acid invaders (e.g., viruses and repetitive DNA sequences including transposable elements) in plants, animals,protozoans and fungi. It is now evident that prokaryotes also possess an RNA-based defense system, though completely distinct from that in eukaryotes. The recent discovery of post-transcriptional gene silencing (PTGS) has generated tremendous interest in basic and applied research, including the development of in vitro and in vivo therapeutic approaches to reduce expression of disease-associated genes. This cutting-edge technology has already been successfully used in genomic manipulations of insect disease vectors, such as development of Dengue-resistant mosquitoes (Blair et al., 2006; Franz et al., 2006 ; Mathur et al., 2010). Our aim in this chapter is to provide an overview of the profound knowledge accumulated in recent years from invertebrate RNAi studies, but with a focus on agriculturally important arthropods. We start with a brief discussion of the RNAi mechanism to introduce readers to key concepts that underlie the practical application of RNAi discussed in the remainder of the chapter. Our discussion will include a number of important issues that should be carefully considered when working with multi-cellular organisms, including bidirectional transport of silencing signals, processing of dsRNA, homology-driven mRNA degradation and subsequent gene silencing. We will look at examples of gene silencing in different arthropod systems to illustrate commonalities and differences found in their silencing machinery. Arthropods are a diverse group of organisms, including ticks, mites, spiders, crabs, and insects, many of which are economically significant pests of agricultural crops, parasites of commercially managed pollinators and vectors of livestock diseases. Considering the huge economic impact of honey bees on crop production, and particularly on pollination of specialty crops, a substantial part of this chapter will be dedicated to honey bee research. Recently, catastrophic losses of honey bee colonies drew national media attention and ignited a renewed interest in basic and applied bee research (Aronstein et al., 2006; Campbell et al., 2010; Dearden et al., 2009; Maori et al., 2009a, 2009b; Nelson et al., 2007). The latest achievements in basic honey bee research provide a better understanding of bee physiology and behavior. Analysis of gene function by selective gene silencing has been}, booktitle={RNA Processing}, publisher={InTechOpen}, author={Aronstein, Katherine and Oppert, Brenda and Lorenzen, Marce}, editor={Grabowski, PaulaEditor}, year={2011}, month={Aug}, pages={157–180} }
@article{alves_lorenzen_beeman_foster_siegfried_2010, title={RNA Interference as a Method for Target-Site Screening in the Western Corn Rootworm, Diabrotica Virgifera Virgifera}, volume={10}, ISSN={1536-2442}, url={http://dx.doi.org/10.1673/031.010.14122}, DOI={10.1673/031.010.14122}, abstractNote={Abstract 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.}, number={162}, journal={Journal of Insect Science}, publisher={Oxford University Press (OUP)}, author={Alves, Analiza P. and Lorenzen, Marcé D. and Beeman, Richard W. and Foster, John E. and Siegfried, Blair D.}, year={2010}, month={Sep}, pages={1–16} }
@article{fabrick_oppert_lorenzen_morris_oppert_jurat-fuentes_2009, title={A NovelTenebrio molitorCadherin Is a Functional Receptor forBacillus thuringiensisCry3Aa Toxin}, volume={284}, ISSN={0021-9258 1083-351X}, url={http://dx.doi.org/10.1074/jbc.M109.001651}, DOI={10.1074/jbc.M109.001651}, abstractNote={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.}, number={27}, journal={Journal of Biological Chemistry}, publisher={American Society for Biochemistry & Molecular Biology (ASBMB)}, author={Fabrick, Jeff and Oppert, Cris and Lorenzen, Marcé D. and Morris, Kaley and Oppert, Brenda and Jurat-Fuentes, Juan Luis}, year={2009}, month={May}, pages={18401–18410} }
@article{kim_murphy_xia_caragea_park_beeman_lorenzen_butcher_manak_brown_2009, title={BeetleBase in 2010: revisions to provide comprehensive genomic information for Tribolium castaneum}, volume={38}, ISSN={0305-1048 1362-4962}, url={http://dx.doi.org/10.1093/nar/gkp807}, DOI={10.1093/nar/gkp807}, abstractNote={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.}, number={suppl_1}, journal={Nucleic Acids Research}, publisher={Oxford University Press (OUP)}, author={Kim, Hee Shin and Murphy, Terence and Xia, Jing and Caragea, Doina and Park, Yoonseong and Beeman, Richard W. and Lorenzen, Marcé D. and Butcher, Stephen and Manak, J. Robert and Brown, Susan J.}, year={2009}, month={Oct}, pages={D437–D442} }
@article{trauner_schinko_lorenzen_shippy_wimmer_beeman_klingler_bucher_brown_2009, title={Large-scale insertional mutagenesis of a coleopteran stored grain pest, the red flour beetle Tribolium castaneum, identifies embryonic lethal mutations and enhancer traps}, volume={7}, ISSN={1741-7007}, url={http://dx.doi.org/10.1186/1741-7007-7-73}, DOI={10.1186/1741-7007-7-73}, abstractNote={Abstract
}, number={1}, journal={BMC Biology}, publisher={Springer Science and Business Media LLC}, author={Trauner, Jochen and Schinko, Johannes and Lorenzen, Marcé D and Shippy, Teresa D and Wimmer, Ernst A and Beeman, Richard W and Klingler, Martin and Bucher, Gregor and Brown, Susan J}, year={2009}, pages={73} }
@article{brown_shippy_miller_bolognesi_beeman_lorenzen_bucher_wimmer_klingler_2009, title={The Red Flour Beetle, Tribolium castaneum (Coleoptera): A Model for Studies of Development and Pest Biology}, volume={2009}, ISSN={1559-6095}, url={http://dx.doi.org/10.1101/pdb.emo126}, DOI={10.1101/pdb.emo126}, abstractNote={INTRODUCTION}, number={8}, journal={Cold Spring Harbor Protocols}, publisher={Cold Spring Harbor Laboratory}, author={Brown, S. J. and Shippy, T. D. and Miller, S. and Bolognesi, R. and Beeman, R. W. and Lorenzen, M. D. and Bucher, G. and Wimmer, E. A. and Klingler, M.}, year={2009}, month={Aug}, pages={pdb.emo126-pdb.emo126} }
@article{morris_lorenzen marcé d._hiromasa_tomich_oppert_elpidina_vinokurov_jurat-fuentes_fabrick_oppert_2009, title={Tribolium castaneum Larval Gut Transcriptome and Proteome: A Resource for the Study of the Coleopteran Gut}, volume={8}, ISSN={1535-3893 1535-3907}, url={http://dx.doi.org/10.1021/pr900168z}, DOI={10.1021/pr900168z}, abstractNote={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.}, number={8}, journal={Journal of Proteome Research}, publisher={American Chemical Society (ACS)}, author={Morris, Kaley and Lorenzen Marcé D. and Hiromasa, Yasuaki and Tomich, John M. and Oppert, Cris and Elpidina, Elena N. and Vinokurov, Konstantin and Jurat-Fuentes, Juan Luis and Fabrick, Jeff and Oppert, Brenda}, year={2009}, month={Aug}, pages={3889–3898} }
@article{wang_lorenzen_beeman_brown_2008, title={Analysis of repetitive DNA distribution patterns in the Tribolium castaneum genome}, volume={9}, ISSN={1465-6906}, url={http://dx.doi.org/10.1186/gb-2008-9-3-r61}, DOI={10.1186/gb-2008-9-3-r61}, abstractNote={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.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.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.}, number={3}, journal={Genome Biology}, publisher={Springer Science and Business Media LLC}, author={Wang, Suzhi and Lorenzen, Marce D and Beeman, Richard W and Brown, Susan J}, year={2008}, pages={R61} }
@article{park_aikins_wang_beeman_oppert_lord_brown_lorenzen_richards_weinstock_et al._2008, title={Analysis of transcriptome data in the red flour beetle, Tribolium castaneum}, volume={38}, ISSN={0965-1748}, url={http://dx.doi.org/10.1016/j.ibmb.2007.09.008}, DOI={10.1016/j.ibmb.2007.09.008}, abstractNote={The whole genome sequence of Tribolium castaneum, a worldwide coleopteran pest of stored products, has recently been determined. In order to facilitate accurate annotation and detailed functional analysis of this genome, we have compiled and analyzed all available expressed sequence tag (EST) data. The raw data consist of 61,228 ESTs, including 10,704 obtained from NCBI and an additional 50,524 derived from 32,544 clones generated in our laboratories. These sequences were amassed from cDNA libraries representing six different tissues or stages, namely: whole embryos, whole larvae, larval hindguts and Malpighian tubules, larval fat bodies and carcasses, adult ovaries, and adult heads. Assembly of the 61,228 sequences collapsed into 12,269 clusters (groups of overlapping ESTs representing single genes), of which 10,134 mapped onto 6,463 (39%) of the 16,422 GLEAN gene models (i.e. official Tribolium gene list). Approximately 1,600 clusters (13% of the total) lack corresponding GLEAN models, despite high matches to the genome, suggesting that a considerable number of transcribed sequences were missed by the gene prediction programs or were removed by GLEAN. We conservatively estimate that the current EST set represents more than 7,500 transcription units.}, number={4}, journal={Insect Biochemistry and Molecular Biology}, publisher={Elsevier BV}, author={Park, Yoonseong and Aikins, Jamie and Wang, L.J. and Beeman, Richard W. and Oppert, Brenda and Lord, Jeffrey C. and Brown, Susan J. and Lorenzen, Marcé D. and Richards, Stephen and Weinstock, George M. and et al.}, year={2008}, month={Apr}, pages={380–386} }
@article{richards_gibbs_weinstock_brown_denell_beeman_bucher_friedrich_grimmelikhuijzen_klingler_et al._2008, title={The genome of the model beetle and pest Tribolium castaneum}, volume={452}, ISSN={0028-0836 1476-4687}, url={http://dx.doi.org/10.1038/nature06784}, DOI={10.1038/nature06784}, abstractNote={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.}, number={7190}, journal={Nature}, publisher={Springer Science and Business Media LLC}, author={Richards, Stephen and Gibbs, Richard A. and Weinstock, George M. and Brown, Susan J. and Denell, Robin and Beeman, Richard W. and Bucher, Gregor and Friedrich, Markus and Grimmelikhuijzen, Cornelis J. P. and Klingler, Martin and et al.}, year={2008}, month={Mar}, pages={949–955} }
@article{lorenzen_gnirke_margolis_garnes_campbell_stuart_aggarwal_richards_park_beeman_2008, title={The maternal-effect, selfish genetic element Medea is associated with a composite Tc1 transposon}, volume={105}, ISSN={0027-8424 1091-6490}, url={http://dx.doi.org/10.1073/pnas.0800444105}, DOI={10.1073/pnas.0800444105}, abstractNote={
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.
}, number={29}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Lorenzen, M. D. and Gnirke, A. and Margolis, J. and Garnes, J. and Campbell, M. and Stuart, J. J. and Aggarwal, R. and Richards, S. and Park, Y. and Beeman, R. W.}, year={2008}, month={Jul}, pages={10085–10089} }
@article{siebert_lorenzen_brown_park_beeman_2008, title={Tubulin superfamily genes in Tribolium castaneum and the use of a Tubulin promoter to drive transgene expression}, volume={38}, ISSN={0965-1748}, url={http://dx.doi.org/10.1016/j.ibmb.2008.04.007}, DOI={10.1016/j.ibmb.2008.04.007}, abstractNote={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 α-Tubulin1 (TcαTub1) 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 TcαTub1 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.}, number={8}, journal={Insect Biochemistry and Molecular Biology}, publisher={Elsevier BV}, author={Siebert, Kendra S. and Lorenzen, Marcé D. and Brown, Susan J. and Park, Yoonseong and Beeman, Richard W.}, year={2008}, month={Aug}, pages={749–755} }
@article{lorenzen_kimzey_shippy_brown_denell_beeman_2007, title={piggyBac-based insertional mutagenesis in Tribolium castaneum using donor/helper hybrids}, volume={16}, ISSN={0962-1075 1365-2583}, url={http://dx.doi.org/10.1111/j.1365-2583.2007.00727.x}, DOI={10.1111/j.1365-2583.2007.00727.x}, abstractNote={Abstract}, number={3}, journal={Insect Molecular Biology}, publisher={Wiley}, author={Lorenzen, M. D. and Kimzey, T. and Shippy, T. D. and Brown, S. J. and Denell, R. E. and Beeman, R. W.}, year={2007}, month={Jun}, pages={265–275} }
@article{insights into social insects from the genome of the honeybee apis mellifera_2006, volume={443}, ISSN={0028-0836 1476-4687}, url={http://dx.doi.org/10.1038/nature05260}, DOI={10.1038/nature05260}, abstractNote={Here we report the genome sequence of the honeybee Apis mellifera, a key model for social behaviour and essential to global ecology through pollination. Compared with other sequenced insect genomes, the A. mellifera genome has high A+T and CpG contents, lacks major transposon families, evolves more slowly, and is more similar to vertebrates for circadian rhythm, RNA interference and DNA methylation genes, among others. Furthermore, A. mellifera has fewer genes for innate immunity, detoxification enzymes, cuticle-forming proteins and gustatory receptors, more genes for odorant receptors, and novel genes for nectar and pollen utilization, consistent with its ecology and social organization. Compared to Drosophila, genes in early developmental pathways differ in Apis, whereas similarities exist for functions that differ markedly, such as sex determination, brain function and behaviour. Population genetics suggests a novel African origin for the species A. mellifera and insights into whether Africanized bees spread throughout the New World via hybridization or displacement.}, number={7114}, journal={Nature}, publisher={Springer Science and Business Media LLC}, year={2006}, month={Oct}, pages={931–949} }
@article{lorenzen_doyungan_savard_snow_crumly_shippy_stuart_brown_beeman_2005, title={Genetic Linkage Maps of the Red Flour Beetle, Tribolium castaneum, Based on Bacterial Artificial Chromosomes and Expressed Sequence Tags}, volume={170}, ISSN={0016-6731 1943-2631}, url={http://dx.doi.org/10.1534/genetics.104.032227}, DOI={10.1534/genetics.104.032227}, abstractNote={Abstract}, number={2}, journal={Genetics}, publisher={Genetics Society of America}, author={Lorenzen, Marcé D. and Doyungan, Zaldy and Savard, Joel and Snow, Kathy and Crumly, Lindsey R. and Shippy, Teresa D. and Stuart, Jeffrey J. and Brown, Susan J. and Beeman, Richard W.}, year={2005}, month={Apr}, pages={741–747} }
@article{arakane_muthukrishnan_kramer_specht_tomoyasu_lorenzen_kanost_beeman_2005, title={The Tribolium chitin synthase genes TcCHS1 and TcCHS2 are specialized for synthesis of epidermal cuticle and midgut peritrophic matrix}, volume={14}, ISSN={0962-1075 1365-2583}, url={http://dx.doi.org/10.1111/j.1365-2583.2005.00576.x}, DOI={10.1111/j.1365-2583.2005.00576.x}, abstractNote={Abstract}, number={5}, journal={Insect Molecular Biology}, publisher={Wiley}, author={Arakane, Y. and Muthukrishnan, S. and Kramer, K. J. and Specht, C. A. and Tomoyasu, Y. and Lorenzen, M. D. and Kanost, M. and Beeman, R. W.}, year={2005}, month={Oct}, pages={453–463} }
@article{goodwin_poulter_lorenzen_beeman_2004, title={DIRS retroelements in arthropods: identification of the recently active TcDirs1 element in the red flour beetle Tribolium castaneum}, volume={272}, ISSN={1617-4615 1617-4623}, url={http://dx.doi.org/10.1007/s00438-004-1028-2}, DOI={10.1007/s00438-004-1028-2}, abstractNote={Members of the DIRS family of retrotransposons differ from most other known retrotransposons in that they encode a tyrosine recombinase (YR), a type of enzyme frequently involved in site-specific recombination. This enzyme is believed to insert the extrachromosomal DNA intermediate of DIRS element retrotransposition into the host genome. DIRS elements have been found in plants, a slime mold, fungi, and a variety of animals including vertebrates, echinoderms and nematodes. They have a somewhat patchy distribution, however, apparently being absent from a number of model organisms such as Saccharomyces cerevisiae, Arabidopsis thaliana and Drosophila melanogaster. In this report we describe the first DIRS retroelement to be identified in an arthropod. This element, TcDirs1, was found in the red flour beetle Tribolium castaneum (Coleoptera). It is generally similar in sequence and structure to several previously described members of the DIRS group: it is bordered by inverted terminal repeats and it has a similar set of protein-coding domains (Gag, reverse transcriptase/ribonuclease H, and the YR), although these are arranged in a novel fashion. TcDirs1 elements exhibit several features indicative of recent activity, such as intact coding regions, a high level of sequence similarity between distinct elements and polymorphic insertion sites. Given their presence in an experimentally tractable host, these potentially active elements might serve as useful models for the study of DIRS element retrotransposition. An element closely related to TcDirs1 was also detected in sequences from a second arthropod, the honey bee Apis mellifera (Hymenoptera), suggesting that these retrotransposons are long-term residents of arthropod genomes.}, number={1}, journal={Molecular Genetics and Genomics}, publisher={Springer Science and Business Media LLC}, author={Goodwin, T. J. D. and Poulter, R. T. M. and Lorenzen, M. D. and Beeman, R. W.}, year={2004}, month={Jun}, pages={47–56} }
@article{lorenzen_berghammer_brown_denell_klingler_beeman_2003, title={piggyBac-mediated germline transformation in the beetle Tribolium castaneum}, volume={12}, ISSN={0962-1075 1365-2583}, url={http://dx.doi.org/10.1046/j.1365-2583.2003.00427.x}, DOI={10.1046/j.1365-2583.2003.00427.x}, abstractNote={Abstract}, number={5}, journal={Insect Molecular Biology}, publisher={Wiley}, author={Lorenzen, M. D. and Berghammer, A. J. and Brown, S. J. and Denell, R. E. and Klingler, M. and Beeman, R. W.}, year={2003}, month={Oct}, pages={433–440} }
@article{lorenzen_brown_denell_beeman_2002, title={Cloning and characterization of the Tribolium castaneum eye-color genes encoding tryptophan oxygenase and kynurenine 3-monooxygenase}, volume={160}, DOI={10.1093/genetics/160.1.225}, abstractNote={Abstract}, number={1}, journal={Genetics}, author={Lorenzen, M.D. and Brown, S.J. and Denell, R.E. and Beeman, R.W.}, year={2002}, pages={225–234} }
@article{lorenzen_brown_denell_beeman_2002, title={Transgene expression from the Tribolium castaneum Polyubiquitin promoter}, volume={11}, ISSN={0962-1075 1365-2583}, url={http://dx.doi.org/10.1046/j.1365-2583.2002.00349.x}, DOI={10.1046/j.1365-2583.2002.00349.x}, abstractNote={Abstract}, number={5}, journal={Insect Molecular Biology}, publisher={Wiley}, author={Lorenzen, M. D. and Brown, S. J. and Denell, R. E. and Beeman, R. W.}, year={2002}, month={Oct}, pages={399–407} }
@article{brown_mahaffey_lorenzen_denell_mahaffey_1999, title={Using RNAi to investigate orthologous homeotic gene function during development of distantly related insects}, volume={1}, ISSN={1520-541X 1525-142X}, url={http://dx.doi.org/10.1046/j.1525-142x.1999.99013.x}, DOI={10.1046/j.1525-142x.1999.99013.x}, abstractNote={Gene product distribution is often used to infer developmental similarities and differences in animals with evolutionarily diverse body plans. However, to address commonalties of developmental mechanisms, what is really needed is a method to assess and compare gene function in divergent organisms. This requires mutations eliminating gene function. Such mutations are often difficult to obtain, even in organisms amenable to genetic analysis. To address this issue we have investigated the use of double‐stranded RNA interference to phenocopy null mutations. We show that RNA interference can be used to phenocopy mutations of the Deformed orthologues in Drosophila and Tribolium. We discuss the possible use of this technique for comparisons of developmental mechanisms in organisms with differing ontogenies.}, number={1}, journal={Evolution and Development}, publisher={Wiley}, author={Brown, Susan J. and Mahaffey, James P. and Lorenzen, Marce D. and Denell, Robin E. and Mahaffey, James W.}, year={1999}, month={Jul}, pages={11–15} }