@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{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={AbstractThe 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.}, 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{chu_wu_pinzi_grubbs_lorenzen_2018, title={Microinjection of Western Corn Rootworm, <em>Diabrotica virgifera virgifera</em>, 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{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={AbstractThe western corn rootworm (WCR), a major pest of maize, is notorious for rapidly adapting biochemically, behaviourally and developmentally to a variety of control methods. Despite much effort, the genetic basis of WCR adaptation remains a mystery. Since transformation‐based applications such as transposon tagging and enhancer trapping have facilitated genetic dissection of model species such as Drosophila melanogaster, we developed a germline‐transformation system for WCR in an effort to gain a greater understanding of the basic biology of this economically important insect. Here we report the use of a fluorescent‐marked Minos element to create transgenic WCR. We demonstrate that the transgenic strains express both an eye‐specific fluorescent marker and piggyBac transposase. We identified insertion‐site junction sequences via inverse PCR and assessed insertion copy number using digital droplet PCR (ddPCR). Interestingly, most WCR identified as transgenic via visual screening for DsRed fluorescence proved to carry multiple Minos insertions when tested via ddPCR. A total of eight unique insertion strains were created by outcrossing the initial transgenic strains to nontransgenic WCR mates. Establishing transgenic technologies for this beetle is the first step towards bringing a wide range of transformation‐based tools to bear on understanding WCR biology.}, 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} }