Name: embryo microinjection and knockout mutant identification of crispr/cas9 genome-edited helicoverpa armigera (h&#252;bner)
Uploaded: 2021-07-01T13:00:00
Description: Watch this Scientific Journal Video about Embryo Microinjection and Knockout Mutant Identification of CRISPR/Cas9 Genome-Edited Helicoverpa Armigera HÃ¼bner at JoVE.com

This work was supported by National Natural Science Foundation of China (31725023, 31861133019 to GW, and 31171912 to CY).

1. Design of gene-specific primers and preparation of sgRNA
<ol>
	<li>Verify a conserved genomic region in the gene of interest through PCR amplification and sequencing analyses. Amplify the target gene from the genome DNA of H. armigera and distinguish the exons and introns. 
	NOTE: The sequence specificity of the guide site is necessary to avoid off-target gene editing. Search possible guide sites in the exons are close to the 5&#39; UTR of the gene. Then, it is important to make sure that the gene is c...

<ol>
	<li>Cong, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+genome+engineering+using+CRISPR/Cas+systems.">Multiplex genome engineering using CRISPR/Cas systems.</a> Science. 339 (6121), 819-823 (2013).</li><li>Garneau, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+CRISPR/Cas+bacterial+immune+system+cleaves+bacteriophage+and+plasmid+DNA.">The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA.</a> Nature. 468 (7320), 67-71 (2010).</li><li>Jinek, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+programmable+dual-RNA-guided+DNA+endonuclease+in+adaptive+bacterial+immunity.">A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.</a> Science. 337 (6096), 816-821 (2012).</li><li>Doudna, J. A., Charpentier, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+new+frontier+of+genome+engineering+with+CRISPR-Cas9.">The new frontier of genome engineering with CRISPR-Cas9.</a> Science. 346 (6213), 1258096 (2014).</li><li>Ding, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+efficiency+of+human+pluripotent+stem+cell+genome+editing+through+replacing+TALENs+with+CRISPRs.">Enhanced efficiency of human pluripotent stem cell genome editing through replacing TALENs with CRISPRs.</a> Cell Stem Cell. 12 (4), 393-394 (2013).</li><li>Collins, P. J., Hale, C. M., Xu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Edited+course+of+biomedical+research:+leaping+forward+with+CRISPR.">Edited course of biomedical research: leaping forward with CRISPR.</a> Pharmacological Research. 125, 258-265 (2017).</li><li>Huang, J., Wang, Y., Zhao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR+editing+in+biological+and+biomedical+investigation.">CRISPR editing in biological and biomedical investigation.</a> Journal of Cellular Physiology. 233 (5), 3875-3891 (2018).</li><li>Guan, L., Han, Y., Zhu, S., Lin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+CRISPR-Cas+system+in+gene+therapy:+pre-clinical+progress+in+animal+model.">Application of CRISPR-Cas system in gene therapy: pre-clinical progress in animal model.</a> DNA Repair. 46, 1-8 (2016).</li><li>Wu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+therapy+for+glaucoma+by+ciliary+body+aquaporin+1+disruption+using+CRISPR-Cas9.">Gene therapy for glaucoma by ciliary body aquaporin 1 disruption using CRISPR-Cas9.</a> Molecular Therapy. 28 (3), 820-829 (2020).</li><li>Jiao, R., Gao, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+CRISPR/Cas9+genome+editing+revolution.">The CRISPR/Cas9 genome editing revolution.</a> Journal of Genetics and Genomics. 43, 227-228 (2016).</li><li>Gupta, M., Gerard, M., Padmaja, S. S., Sastry, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trends+of+CRISPR+technology+development+and+deployment+into+Agricultural+Production-Consumption+Systems.">Trends of CRISPR technology development and deployment into Agricultural Production-Consumption Systems.</a> World Patent Information. 60, 101944 (2020).</li><li>Es, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+application+of+the+CRISPR-Cas9+genome+editing+machinery+in+food+and+agricultural+science:+Current+status,+future+perspectives,+and+associated+challenges.">The application of the CRISPR-Cas9 genome editing machinery in food and agricultural science: Current status, future perspectives, and associated challenges.</a> Biotechnology Advances. 37 (3), 410-421 (2019).</li><li>Tarasava, K., Oh, E. J., Eckert, C. A., Gill, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR-enabled+tools+for+engineering+microbial+genomes+and+phenotypes.">CRISPR-enabled tools for engineering microbial genomes and phenotypes.</a> Biotechnology Journal. 13 (9), 1700586 (2018).</li><li>Ma, X., Zhu, Q., Chen, Y., Liu, Y. -. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR/Cas9+platforms+for+genome+editing+in+plants:+developments+and+applications.">CRISPR/Cas9 platforms for genome editing in plants: developments and applications.</a> Molecular Plant. 9 (7), 961-974 (2016).</li><li>Pandey, P. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Versatile+and+multifaceted+CRISPR/Cas+gene+editing+tool+for+plant+research.">Versatile and multifaceted CRISPR/Cas gene editing tool for plant research.</a> Seminars in Cell &amp; Developmental Biology. 96, 107-114 (2019).</li><li>Friedland, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heritable+genome+editing+in+C.+elegans+via+a+CRISPR-Cas9+system.">Heritable genome editing in C. elegans via a CRISPR-Cas9 system.</a> Nature Methods. 10 (8), 741-743 (2013).</li><li>Mojica, F. J., Montoliu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+origin+of+CRISPR-Cas+technology:+from+prokaryotes+to+mammals.">On the origin of CRISPR-Cas technology: from prokaryotes to mammals.</a> Trends in Microbiology. 24 (10), 811-820 (2016).</li><li>Taning, C. N. T., Van Eynde, B., Yu, N., Ma, S., Smagghe, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR/Cas9+in+insects:+Applications,+best+practices+and+biosafety+concerns.">CRISPR/Cas9 in insects: Applications, best practices and biosafety concerns.</a> Journal of Insect Physiology. 98, 245-257 (2017).</li><li>Chang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pheromone+antagonist+regulates+optimal+mating+time+in+the+moth+Helicoverpa+armigera.">A pheromone antagonist regulates optimal mating time in the moth Helicoverpa armigera.</a> Current Biology. 27 (11), 1610-1615 (2017).</li><li>Yan, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+engineered+orco+mutation+produces+aberrant+social+behavior+and+defective+neural+development+in+ants.">An engineered orco mutation produces aberrant social behavior and defective neural development in ants.</a> Cell. 170 (4), 736-747 (2017).</li><li>Koutroumpa, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heritable+genome+editing+with+CRISPR/Cas9+induces+anosmia+in+a+crop+pest+moth.">Heritable genome editing with CRISPR/Cas9 induces anosmia in a crop pest moth.</a> Scientific Reports. 6 (1), 29620 (2016).</li><li>Chen, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR/Cas9-mediated+genome+editing+induces+exon+skipping+by+complete+or+stochastic+altering+splicing+in+the+migratory+locust.">CRISPR/Cas9-mediated genome editing induces exon skipping by complete or stochastic altering splicing in the migratory locust.</a> BMC Biotechnology. 18 (1), 1-9 (2018).</li><li>Fitt, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ecology+of+Heliothis+species+in+relation+to+agroecosystems.">The ecology of Heliothis species in relation to agroecosystems.</a> Annual Review of Entomology. 34 (1), 17-53 (1989).</li><li>Jallow, M. F., Cunningham, J. P., Zalucki, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intra-specific+variation+for+host+plant+use+in+Helicoverpa+armigera+(H&#252;bner)(Lepidoptera:+Noctuidae):+implications+for+management.">Intra-specific variation for host plant use in Helicoverpa armigera (H&#252;bner)(Lepidoptera: Noctuidae): implications for management.</a> Crop Protection. 23 (10), 955-964 (2004).</li><li>Ai, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+cloning,+prokaryotic+expression,+and+biochemical+characterization+of+a+soluble+trehalase+in+Helicoverpa+armigera+H&#252;bner+(Lepidoptera:+Noctuidae).">Gene cloning, prokaryotic expression, and biochemical characterization of a soluble trehalase in Helicoverpa armigera H&#252;bner (Lepidoptera: Noctuidae).</a> Journal of Insect Science. 18 (3), 22 (2018).</li><li>Wan, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chromosome-level+genome+assembly+of+Cydia+pomonella+provides+insights+into+chemical+ecology+and+insecticide+resistance.">A chromosome-level genome assembly of Cydia pomonella provides insights into chemical ecology and insecticide resistance.</a> Nature Communications. 10 (1), 1-14 (2019).</li><li>Cheng, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+adaptation+to+polyphagy+and+insecticides+in+a+major+East+Asian+noctuid+pest.">Genomic adaptation to polyphagy and insecticides in a major East Asian noctuid pest.</a> Nature Ecology &amp; Evolution. 1 (11), 1747-1756 (2017).</li><li>Xu, W., Papanicolaou, A., Zhang, H. J., Anderson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion+of+a+bitter+taste+receptor+family+in+a+polyphagous+insect+herbivore.">Expansion of a bitter taste receptor family in a polyphagous insect herbivore.</a> Science Reports. 6, 23666 (2016).</li><li>Gouin, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+genomes+of+highly+polyphagous+lepidopteran+pests+(Spodoptera+frugiperda,+Noctuidae)+with+different+host-plant+ranges.">Two genomes of highly polyphagous lepidopteran pests (Spodoptera frugiperda, Noctuidae) with different host-plant ranges.</a> Scientific Reports. 7 (1), 1-12 (2017).</li><li>Wang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+validation+of+cadherin+as+a+receptor+of+Bt+toxin+Cry1Ac+in+Helicoverpa+armigera+utilizing+the+CRISPR/Cas9+system.">Functional validation of cadherin as a receptor of Bt toxin Cry1Ac in Helicoverpa armigera utilizing the CRISPR/Cas9 system.</a> Insect Biochemistry and Molecular Biology. 76, 11-17 (2016).</li><li>Wang, J., Wang, H., Liu, S., Liu, L., Wu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR/Cas9+mediated+genome+editing+of+Helicoverpa+armigera+with+mutations+of+an+ABC+transporter+gene+HaABCA2+confers+resistance+to+Bacillus+thuringiensis+Cry2A+toxins.">CRISPR/Cas9 mediated genome editing of Helicoverpa armigera with mutations of an ABC transporter gene HaABCA2 confers resistance to Bacillus thuringiensis Cry2A toxins.</a> Insect Biochemistry and Molecular biology. 87, 147 (2017).</li><li>Wang, J., Ma, H., Zhao, S., Huang, J., Wu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+redundancy+of+two+ABC+transporter+proteins+in+mediating+toxicity+of+Bacillus+thuringiensis+to+cotton+bollworm.">Functional redundancy of two ABC transporter proteins in mediating toxicity of Bacillus thuringiensis to cotton bollworm.</a> PLoS Pathogens. 16 (3), 1008427 (2020).</li><li>Jin, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dominant+point+mutation+in+a+tetraspanin+gene+associated+with+field-evolved+resistance+of+cotton+bollworm+to+transgenic+Bt+cotton.">Dominant point mutation in a tetraspanin gene associated with field-evolved resistance of cotton bollworm to transgenic Bt cotton.</a> Proceedings of the National Academy of Sciences. 115 (46), 11760-11765 (2018).</li><li>Hongtao, Z., Jianan, L., Lian, T., Yang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sexing+of+Helicoverpa+assulta+and+Helicoverpa+armigera+pupae+based+on+machine+vision.">Sexing of Helicoverpa assulta and Helicoverpa armigera pupae based on machine vision.</a> Tobacco Sciene &amp; Technology. 53 (2), 21-26 (2019).</li><li>Wu, K., Gong, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+and+practical+artificial+diet+for+the+cotton+boll-worm.">A new and practical artificial diet for the cotton boll-worm.</a> Insect science. 4 (3), 277-282 (1997).</li><li>Jha, R. K., Chi, H., Li-Cheng, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Comparison+of+Artificial+Diet+and+Hybrid+Sweet+Corn+for+the+Rearing+of+Helicoverpa+armigera+(H&#252;bner)+(Lepidoptera:+Noctuidae)+Based+on+Life+Table+Characteristics.">A Comparison of Artificial Diet and Hybrid Sweet Corn for the Rearing of Helicoverpa armigera (H&#252;bner) (Lepidoptera: Noctuidae) Based on Life Table Characteristics.</a> Environmental Entomology. (1), 30 (2012).</li><li>Bassett, A. R., Tibbit, C., Ponting, C. P., Liu, J. -. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+targeted+mutagenesis+of+Drosophila+with+the+CRISPR/Cas9+system.">Highly efficient targeted mutagenesis of Drosophila with the CRISPR/Cas9 system.</a> Cell Reports. 4 (1), 220-228 (2013).</li><li>Zheng, M. -. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+non-destructive+method+of+genotyping+individual+insects+from+the+exuviate+of+final+instar+larvae,+or+puparia.">A non-destructive method of genotyping individual insects from the exuviate of final instar larvae, or puparia.</a> Chinese Journal of Applied Entomology. (2), 21 (2018).</li><li>Pashley, D. P., Hammond, A. M., Hardy, T. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproductive+isolating+mechanisms+in+fall+armyworm+host+strains+(Lepidoptera:+Noctuidae).">Reproductive isolating mechanisms in fall armyworm host strains (Lepidoptera: Noctuidae).</a> Annals of The Entomological Society of America. 85 (4), 400-405 (1992).</li><li>Kamimura, M., Tatsuki, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diel+rhythms+of+calling+behavior+and+pheromone+production+of+oriental+tobacco+budworm+moth,+Helicoverpa+assulta(Lepidoptera:+Noctuidae).">Diel rhythms of calling behavior and pheromone production of oriental tobacco budworm moth, Helicoverpa assulta(Lepidoptera: Noctuidae).</a> Journal of Chemical Ecology. 19 (12), 2953-2963 (1993).</li><li>Edwards, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity+rhythms+of+lepidopterous+defoliators:+ii.+Halisidota+argentata+pack.+(arctiidae),+and+nepytia+phantasmaria+stkr.+(GEOMETRIDAE).">Activity rhythms of lepidopterous defoliators: ii. Halisidota argentata pack. (arctiidae), and nepytia phantasmaria stkr. (GEOMETRIDAE).</a> Canadian Journal of Zoology. 42 (6), 939-958 (1964).</li></ol>

The application of the CRISPR/Cas9 system has provided powerful technical support for the analysis of gene function and interaction among various genes. The detailed protocol we present here demonstrates the generation of a homozygote mutant in H. armigera via CRISPR/Cas9 genome editing. This reliable procedure provides a straightforward way for directed gene mutagenesis in H. armigera.
The choice of CRISPR target sites could affect the mutagenesis efficiency<sup class="xref"...

The application of genome editing technology provides an efficient tool to achieve target-gene mutants in diverse species. The emergence of the clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 (Cas9) system provides a novel method to manipulate genomes1. The CRISPR/Cas9 system consists of a guide RNA (gRNA) and the Cas9 endonuclease2,3, while the gRNA can be further divided into two parts, a target complementary CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA). The gRNA integrates with Cas9 endonuclease and forms a ribonucleoprotein (RNP). With the gRNA, Cas9 endonuclease can be directed to a specific site of the genome via base complementation. The RuvC and HNH domains of the Cas9 cleave the target site of the genome three bases before the protospacer-adjacent motif (PAM) sequence and create a double-strand break (DSB). The DNA cleavage can then be repaired through two mechanisms, non-homologous end joining (NHEJ) or homology-directed repair (HDR)4. Repair of the DSB introduces insertions or deletions as a way to inactivate the targeted gene, potentially causing a complete loss of gene function. Hence, the hereditable and specificity of the CRISPR/Cas9 system make it a robust method to characterize gene functions in vivo and analyze gene interactions5.
With numerous merits, the CRISPR/Cas9 system has been applied to various fields, including biomedicine6,7, gene therapy8,9, and agriculture10,11,12, and has been used for various biological systems including microorganisms13, plants14,15, nematodes16 , and mammals17. In invertebrates, many insect species have been subjected to CRISPR/Cas9 genome editing, such as the fruit fly Drosophila melanogaster and beyond18,19,20,21,22.
Helicoverpa armigera is one of the most destructive pests worldwide23, and damages numerous crops, including cotton, soybean, and sorghum24,25. With the development of sequencing technology, the genome of H. armigera, as well as that of a range of Lepidoptera insect species, have been sequenced completely26,27,28,29. A large number of resistance and olfactory receptor genes have been identified and characterized from these insects in recent years19,27,28,29. Some resistance-related genes have been identified in H. armigera, such as the genes encoding for cadherin30, an ATP-binding cassette transporter31,32, as well as HaTSPAN133. Knockout of these genes using CRISPR/Cas9 technology results in a high level of resistance to Bacillus thuringiensis (BT) toxin in susceptible strains. Also, Chang et al. (2017) knocked out a pheromone receptor, which validated its significant function in mating time regulation19. These reports suggest that CRISPR/Cas9 can act as an effective tool to study gene function in vivo in insect systems. However, a detailed procedure for CRISPR/Cas9 modification in insect systems remains incomplete, which limits its application range in insect functional genomics.
Here, we present a protocol for knocking out a functional gene in H. armigera using the CRISPR/Cas9 system. A detailed step-by-step protocol is provided, including the design and preparation of gene-specific primers for gRNA production, embryo collection, microinjection, insect rearing, and mutant identification. This protocol serves as a valuable reference to manipulate any functional genes in H. armigera and can be extended to other Lepidoptera species.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2kb DNA ladder</td>
			<td>TransGen Biotech</td>
			<td>BM101</td>
			<td></td>
		</tr>
		<tr>
			<td>Capillary Glass</td>
			<td>World Precision Instrucments</td>
			<td>504949</td>
			<td>referred to as &#34;capillary glass&#34; in the protocol</td>
		</tr>
		<tr>
			<td>Double Sided Tape</td>
			<td>Minnesota Mining and Manufacturing Corporation</td>
			<td>665</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf FemtoJet 4i Microinjector</td>
			<td>Eppendorf Corporate</td>
			<td>E5252000021</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf InjectMan 4 micromanipulator</td>
			<td>Eppendorf Corporate</td>
			<td>5192000051</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Microloader Pipette Tips</td>
			<td>Eppendorf Corporate</td>
			<td>G2835241</td>
			<td></td>
		</tr>
		<tr>
			<td>GeneArt Precision gRNA Synthesis Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>A29377</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Slide</td>
			<td>Sail Brand</td>
			<td>7105</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus Microscope</td>
			<td>Olympus Corporation</td>
			<td>SZX16</td>
			<td></td>
		</tr>
		<tr>
			<td>PrimeSTAR HS (Premix)</td>
			<td>Takara Biomedical Technology</td>
			<td>R040</td>
			<td>used for mutant detection</td>
		</tr>
		<tr>
			<td>Sutter Micropipette Puller</td>
			<td>Sutter Instrument Company</td>
			<td>P-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>TIANamp Genomic DNA Kit</td>
			<td>TIANGEN Corporate</td>
			<td>DP304-03</td>
			<td></td>
		</tr>
		<tr>
			<td>TrueCut Cas9 Protein v2</td>
			<td>Thermo Fisher Scientific</td>
			<td>A36499</td>
			<td></td>
		</tr>
	</tbody>
</table>

embryo microinjection and knockout mutant identification of crispr/cas9 genome-edited helicoverpa armigera (h&#252;bner)

The cotton bollworm, Helicoverpa armigera, is one of the most destructive pests in the world. A combination of molecular genetics, physiology, functional genomics, and behavioral studies has made H. armigera a model species in Lepidoptera Noctuidae. To study the in vivo functions of and interactions between different genes, clustered regularly interspaced short palindromic repeats (CRISPR)/ associated protein 9 (Cas9) genome editing technology is a convenient and effective method used for performing functional genomic studies. In this study, we provide a step-by-step systematic method to complete gene knockout in H. armigera using the CRISPR/Cas9 system. The design and synthesis of guide RNA (gRNA) are described in detail. Then, the subsequent steps consisting of gene-specific primer design for guide RNA (gRNA) creation, embryo collection, microinjection, insect rearing, and mutant detection are summarized. Finally, troubleshooting advice and notes are provided to improve the efficiency of gene editing. Our method will serve as a reference for the application of CRISPR/Cas9 genome editing in H. armigera as well as other Lepidopteran moths.

This protocol provides detailed steps for obtaining gene knock-out lines of H. armigera using CRISPR/Cas9 technology. The representative results obtained by this protocol are summarized for gDNA selection, embryo collection and injection, insect rearing, and mutant detection.
In this study, the target site of our gene of interest was located in its second exon (Figure 2A). This site was hig...

Watch this Scientific Journal Video about Embryo Microinjection and Knockout Mutant Identification of CRISPR/Cas9 Genome-Edited Helicoverpa Armigera HÃ¼bner at JoVE.com

Embryo Microinjection and Knockout Mutant Identification of CRISPR/Cas9 Genome-Edited Helicoverpa Armigera H&amp;#252;bner

Presented here is a protocol of Helicoverpa armigera (H&#252;bner) embryo microinjection and knockout mutant identification created by CRISPR/Cas9 genome editing. Mutant insects enable further research of gene function and interaction among different genes in vivo.

Embryo Microinjection and Knockout Mutant Identification of CRISPR/Cas9 Genome-Edited Helicoverpa Armigera (H&#252;bner)

The cotton bollworm, Helicoverpa armigera, is one of the most destructive pests in the world. A combination of molecular genetics, physiology, functional ...

Cotton bollworm is one of the most destructive pests worldwide. Genome editing with the CRISPR/Cas9 technology enables further research of gene function and interaction among different genes in this organism. The two main advantages of this technique are that it has high specificity and is hereditable.In order to choose the sgRNA targets, go to the CRISPR online website to search for possible guide sites and the exons close to the five prime untranslated region of the gene. Input the exon sequence into the text box and select the target genome to Helicoverpa armigera. Choose the PAM option for 20 BPNGG and leave the other settings on the default parameters, according to the user manuals of the website.Compare the predicted guide sequences from the software and choose a guide sequence of 20 base pairs containing one or two guanines near the five prime untranslated region with the highest predicted efficiency and fewest mismatches to improve the editing efficiency and reduce off-target editing. For embryo preparation, select 50 healthy individuals from three-day-old males and two-day-old females, and mix them in a clean net cage. Place a piece of moist cotton containing 10%sugar solution in the cage.Cover the net cages with gauze and fix the gauze with a rubber band. Spray water onto the gauze to keep it moist. Cut a black cloth into an appropriate size and replace the moist gauze with this black cloth to enable free ova-position for 30 minutes.Paste double-sided tape onto a microscope slide. Using forceps, paste the patches with eggs in a row on the surface of the double-sided tape. Press the margin of each patch to make sure they stick firmly to the tape.Collect 50 to 100 eggs per microscope slide. Before microinjection, keep the microscope slide on ice to delay the development of embryos. Prepare the needle by pulling a capillary glass using a micro pipette puller as described in the text manuscript.Grind the needle tip using a micro grinder to a sharp-edged tip. Prepare injection solutions by adding two microliters of commercialized Cas9 protein and sgRNA to RNace-free water in a PCR tube to obtain 10 microliter volume mixture. Mix well by pipetting and put it on ice.Set the parameters of the electronic microinjector. Load two microliters of the mixture into a needle using a microloader pipette tip, exhausting as much residual air in the tip of the needle as possible. Connect the injection needle to a micromanipulator and ensure a tight connection between the two parts.Place a slide in a Petri dish and put it on the stage of the microscope. Carefully insert the needle tip into the top hemisphere of an embryo at a 45 degree angle. Press the pedal to deliver the mixture into the embryo, leading to a slight expansion of the embryo.Retract the needle immediately from the embryo and move the Petri dish with one hand until the next embryo is in proximity to the needle. Inject at least 300 embryos using the same procedure to ensure a sufficient hatching amount. Cover the lid of the Petri dishes after injection.When the surface color of embryos has darkened, put artificial diet in the Petri dish around the microscope slide. Prepare 24-well culture plates and fill each well to 1/3 of its volumetric capacity with artificial diet. Pick out hatching larva using a small paint brush and transfer them to the 24-well culture plate, such that one well has one larva.When larva grow to the third instar stage, transfer them to a new glass ductile ethrae. Transfer the newly enclosed G-zero male adults and wild-type female adults into a fresh net cage at an equal ratio. Supply them with 10%sugar solution dropped in cotton balls.Rear insects using routine methods until the pupation of G-one. Use forceps to carefully remove a hind leg and put each leg in a Lysing Matrix tube. Homogenize the hind leg using a tissue homogenizer.Extract the genomic DNA of the homogenized sample using a commercial gDNA extraction kit according to the manufacturer's instructions. Amplify the gene segment using genotyping primers and the PCR reaction conditions from the text manuscript. Confirm the genotype with a gene-sequencing service.Put G-one individuals of the same genotype in one net cage, self cross the G-one progenys and continue to screen using the same methods. The target site of the gene of interest was located in its second exon. This site was highly conserved and the target band fragment of synthesized sgRNA was confirmed using agarose gel electrophoresis.The mutant detection of a single sgRNA target was performed by sequencing the PCR products from G-one parental units. The effectiveness of using non-overlapping sgRNA pairs across different exons was demonstrated as the large deletion of the mutants was easily distinguished from the wild-type pans. After obtaining a certain number of mutant individuals, electrophysiological or behavioral experiments can be performed to clarify gene function and interaction among different genes.

embryo-microinjection-knockout-mutant-identification-crisprcas9

Research

JoVE Journal

Behavior

Stereotaxic Microinjection of Viral Vectors Expressing Cre Recombinase to Study the Role of Target Genes in Cocaine Conditioned Place Preference

Microinjecting recombinant adenoassociated viral (rAAV) vectors expressing Cre recombinase into distinct mouse brain regions to selectively knockout genes of interest allows for enhanced temporally- and regionally-specific control of gene deletion, compared to existing methods. While conditional deletion can also be achieved by mating mice that express Cre recombinase under the control of specific gene promoters with mice carrying a floxed gene, stereotaxic microinjection allows for targeting of discrete brain areas at experimenter-determined time points of interest. In the context of cocaine conditioned place preference, and other cocaine behavioral paradigms such as self-administration or psychomotor sensitization that can involve withdrawal, extinction and/or reinstatement phases, this technique is particularly useful in exploring the unique contribution of target genes to these distinct phases of behavioral models of cocaine-induced plasticity. Specifically, this technique allows for selective ablation of target genes during discrete phases of a behavior to test their contribution to the behavior across time. Ultimately, this understanding allows for more targeted therapeutics that are best able to address the most potent risk factors that present themselves during each phase of addictive behavior.

This article describes how to microinject viral vectors into mouse brain and then test in a conditioned place preference paradigm that includes an acquisition, extinction and reinstatement phase.

Microinjecting recombinant adenoassociated viral (rAAV) vectors expressing Cre recombinase into distinct mouse brain regions to selectively knockout genes ...

Measurement of Neurophysiological Signals of Ignoring and Attending Processes in Attention Control

Attention control is the ability to selectively attend to some sensory signals while ignoring others. This ability is thought to involve two processes: enhancement of sensory signals that are to be attended and the attenuation of sensory signals that are to be ignored. The overall strength of attentional modulation is often measured by comparing the amplitude of a sensory neural response to an external input when attended versus when ignored. This method is robust for detecting attentional modulation, but precludes the ability to assess the separate dynamics of attending and ignoring processes. Here, we describe methodology to measure independently the neurophysiological signals of attending and ignoring using the intermodal attention task (IMAT). This task, when combined with electroencephalography, isolates neurophysiological sensory responses in auditory and visual modalities, when either attending or ignoring, with respect to a passive control. As a result, independent dynamics of attending and of a ignoring can be assessed in either modality. Our results using this task indicate that the timing and cortical sources of attending and ignoring effects differ, as do their contributions to the attention modulation effect, pointing to unique neural trajectories and demonstrating sample utility of measuring them separately.

Attention control comprises enhancement of target signals and attenuation of distractor signals. We describe an approach to measure separately but concurrently, the neurophysiology of attending and ignoring in sustained intermodal attention, utilizing a passive control condition during which neither process is continuously engaged.

Attention control is the ability to selectively attend to some sensory signals while ignoring others. This ability is thought to involve two processes: ...

Rodent Brain Microinjection to Study Molecular Substrates of Motivated Behavior

Brain microinjection can aid elucidation of the molecular substrates of complex behaviors, such as motivation. For this purpose rodents can serve as appropriate models, partly because the response to behaviorally relevant stimuli and the circuitry parsing stimulus-action outcomes is astonishingly similar between humans and rodents. In studying molecular substrates of complex behaviors, the microinjection of reagents that modify, augment, or silence specific systems is an invaluable technique. However, it is crucial that the microinjection site is precisely targeted in order to aid interpretation of the results. We present a method for the manufacture of surgical implements and microinjection needles that enables accurate microinjection and unlimited customizability with minimal cost. Importantly, this technique can be successfully completed in awake rodents if conducted in conjunction with other JoVE articles that covered requisite surgical procedures. Additionally, there are many behavioral paradigms that are well suited for measuring motivation. The progressive ratio is a commonly used method that quantifies the efficacy of a reinforcer to maintain responding despite an (often exponentially) increasing work requirement. This assay is sensitive to reinforcer magnitude and pharmacological manipulations, which allows reinforcing efficacy and/ or motivation to be determined. We also present a straightforward approach to program operant software to accommodate a progressive ratio reinforcement schedule.

Rodents are an appropriate model to investigate the molecular substrates of behavior and complex psychiatric disorders. Brain microinjection in awake rodents can be used to elucidate disease substrates. An efficient and customizable brain microinjection method as well as the execution of an operant paradigm that quantifies motivation is presented.

Brain microinjection can aid elucidation of the molecular substrates of complex behaviors, such as motivation. For this purpose rodents can serve as ...

Holistic Facial Composite Creation and Subsequent Video Line-up Eyewitness Identification Paradigm

The paradigm detailed in this manuscript describes an applied experimental method based on real police investigations during which an eyewitness or victim to a crime may create from memory a holistic facial composite of the culprit with the assistance of a police operator. The aim is that the composite is recognized by someone who believes that they know the culprit. For this paradigm, participants view a culprit actor on video and following a delay, participant-witnesses construct a holistic system facial composite. Controls do not construct a composite. From a series of arrays of computer-generated, but realistic faces, the holistic system construction method primarily requires participant-witnesses to select the facial images most closely meeting their memory of the culprit. Variation between faces in successive arrays is reduced until ideally the final image possesses a close likeness to the culprit. Participant-witness directed tools can also alter facial features, configurations between features and holistic properties (e.g., age, distinctiveness, skin tone), all within a whole face context. The procedure is designed to closely match the holistic manner by which humans&#8217; process faces. On completion, based on their memory of the culprit, ratings of composite-culprit similarity are collected from the participant-witnesses. Similar ratings are collected from culprit-acquaintance assessors, as a marker of composite recognition likelihood. Following a further delay, all participants &#8212; including the controls&#160;&#8212; attempt to identify the culprit in either a culprit-present or culprit-absent video line-up, to replicate circumstances in which the police have located the correct culprit, or an innocent suspect. Data of control and participant-witness line-up outcomes are presented, demonstrating the positive influence of holistic composite construction on identification accuracy. Correlational analyses are conducted to measure the relationship between assessor and participant-witness composite-culprit similarity ratings, delay, identification accuracy, and confidence to examine which factors influence video line-up outcomes.

This applied experimental paradigm replicates circumstances by which an eyewitness to a real crime may create a holistic facial composite of the culprit from memory, and then attempt to identify the culprit from a video line-up containing the culprit or one in which he or she is not present.

The paradigm detailed in this manuscript describes an applied experimental method based on real police investigations during which an eyewitness or victim ...

Implantation and Recording of Wireless Electroretinogram and Visual Evoked Potential in Conscious Rats

The full-field electroretinogram (ERG) and visual evoked potential (VEP) are useful tools to assess retinal and visual pathway integrity in both laboratory and clinical settings. Currently, preclinical ERG and VEP measurements are performed with anesthesia to ensure stable electrode placements. However, the very presence of anesthesia has been shown to contaminate normal physiological responses. To overcome these anesthesia confounds, we develop a novel platform to assay ERG and VEP in conscious rats. Electrodes are surgically implanted sub-conjunctivally on the eye to assay the ERG and epidurally over the visual cortex to measure the VEP. A range of amplitude and sensitivity/timing parameters are assayed for both the ERG and VEP at increasing luminous energies. The ERG and VEP signals are shown to be stable and repeatable for at least 4 weeks post surgical implantation. This ability to record ERG and VEP signals without anesthesia confounds in the preclinical setting should provide superior translation to clinical data.

We show surgical implantation and Recording procedures to measure visual electrophysiological signals from the eye (electroretinogram) and brain (visual evoked potential) in conscious rats, which is more analogous to the human condition where recordings are conducted without anesthesia confounds.

The full-field electroretinogram (ERG) and visual evoked potential (VEP) are useful tools to assess retinal and visual pathway integrity in both ...

Manipulation of Epileptiform Electrocorticograms (ECoGs) and Sleep in Rats and Mice by Acupuncture

Ancient Chinese literature has documented that acupuncture possesses efficient therapeutic effects on epilepsy and insomnia. There is, however, little research to reveal the possible mechanisms behind these effects. To investigate the effect of acupuncture on epilepsy and sleep, several issues need to be addressed. The first is to identify the acupoints, which correspond between humans, rats, and mice. Furthermore, the depth of insertion of the acupuncture needle, the degree of needle twist in manual needle acupuncture, and the stimulation parameters for electroacupuncture (EA) need to be determined. To evaluate the effects of acupuncture on epilepsy and sleep, a feasible model of epilepsy in rodents is required. We administer pilocarpine into the left central nucleus of the amygdala (CeA) to simulate focal temporal lobe epilepsy (TLE) in rats. Intraperitoneal (IP) injection of pilocarpine induces generalized epilepsy and status epilepticus (SE) in rats. Five IP injections of pentylenetetrazol (PTZ) with a one-day interval between each injection successfully induces spontaneous generalized epilepsy in mice. Recordings of electrocorticograms (ECoGs), electromyograms (EMGs), brain temperature, and locomotor activity are used for sleep analysis in rats, while ECoGs, EMGs, and locomotor activity are employed for sleep analysis in mice. ECoG electrodes are implanted into the frontal, parietal, and contralateral occipital cortices, and a thermistor is implanted above the cerebral cortex by stereotactic surgery. EMG electrodes are implanted into the neck muscles, and an infrared detector determines locomotor activity. The criteria for categorizing vigilance stages, including wakefulness, rapid eye movement (REM) sleep, and non-REM (NREM) sleep are based on information from ECoGs, EMGs, brain temperature, and locomotor activity. Detailed classification criteria are stated in the text.

Manipulation of Epileptiform Electrocorticograms ECoGs and Sleep in Rats and Mice by Acupuncture

This paper demonstrates the performance of acupuncture, epilepsy models, and the analysis of sleep in rodents. The acupuncture procedure and the identification of acupoints are described. Pilocarpine or pentylenetetrazol (PTZ) is used to induce epilepsy. Electrocorticogram (ECoG), electromyogram (EMG), brain temperature, and locomotor activity recordings are employed for sleep analysis.

Ancient Chinese literature has documented that acupuncture possesses efficient therapeutic effects on epilepsy and insomnia. There is, however, little ...

Task Interruption and Resumption Paradigm for Testing the Activation and Pursuit of an Abstract Thinking Goal

This protocol is based on the task interruption and resumption paradigm, the premise of which is that active goals lead to persistent behavior and thus a higher resumption rate after a period of delay or interruption. The task interruption and resumption protocol described in this research is tailored to test the activation of cognitive goals (e.g., a goal to think more abstractly). Cognitive goals may be pursued even during the interruption period; thus, to prevent this, the protocol involves cognitive distraction. The protocol consists of several stages. Specifically, the initial stage includes the goal activation process, where the treatment (versus control) condition receives a manipulation to activate the cognitive goal being tested by the researcher. In the next stage, participants are presented with the introduction of a task that is perceived to either satisfy or not satisfy the cognitive goal of interest. Importantly, this task is interrupted a few seconds after it begins. The task interruption forces a delay period and introduces a cognitive distraction to prevent the automatic pursuit and fulfillment of the cognitive goal. After the interruption period, participants are given a choice between resuming the interrupted task and abandoning the interrupted task to complete an alternative task instead. Among participants whose cognitive goals had been activated at the earlier stage, the task resumption rate should be higher if the task was perceived as an opportunity to satisfy (versus not satisfy) the goal. Such a finding would provide empirical evidence that the cognitive goal has been activated and pursued. In previous research, this protocol has been used to test whether causal uncertainty activates an abstract thinking goal. Adapting the protocol to test the activation of other cognitive goals is also discussed.

This protocol was designed to test the activation and pursuit of cognitive goals (e.g., an abstract thinking goal) using the task interruption and resumption paradigm. The protocol is suitable for cognitive goals that are automatically pursued once activated, as the distraction procedure prevents goal pursuit during the interruption period.

This protocol is based on the task interruption and resumption paradigm, the premise of which is that active goals lead to persistent behavior and thus a ...

Methods of Pairing and Pair Maintenance of New Zealand White Rabbits (Oryctolagus Cuniculus) Via Behavioral Ethogram, Monitoring, and Interventions

New Zealand White (NZW) laboratory rabbits (Oryctolagus cuniculus), as well as their ancestors the European Rabbit, are a social species that exhibit numerous benefits to being housed accordingly. Although these rabbits are innately gregarious, certain behaviors can still arise when kept in captivity, which if left unchecked, can confound research results or lead to wounding, which in extreme cases can be severe. To prevent these issues, there must be a well-structured plan for the monitoring and maintenance of paired laboratory rabbits. The purpose of this protocol is to present effective procedures for establishing newly paired NZW rabbits as well as methods for successful maintenance. Multiple methods have been tested for the creation of newly paired female rabbits from the vendor, but the most efficacious technique emphasizes capitalizing on the stress bonding from transport, urine marking, pairing in a neutral cage with no forced sharing of resources and a system of monitoring and intervention. To determine the best method of housing paired rabbits in a standard caging environment, data were collected to generate a behavioral ethogram. Behaviors were then quantified as positive, neutral or negative and were tracked across the lifespan of the pair to determine which behaviors indicated pair success or failure. With the newfound knowledge of socially housed laboratory NZW rabbit behavior, enrichment intervention was applied to alleviate aggression and prevent wounding, thus resulting in a higher percentage of successful pairs. Through several years of trialing different pairing methods, the development of the ethogram and the resulting enrichment interventions, understanding of the highly complex social constructs that dominate pair housed rabbit behavior has dramatically increased and allowed for the provision of more species-specific care and increased standards of welfare.

Methods of Pairing and Pair Maintenance of New Zealand White Rabbits Oryctolagus Cuniculus Via Behavioral Ethogram, Monitoring, and Interventions

Though European rabbits are a social species, socially housing them can be challenging. Therefore, there must be a thorough understanding of behaviors and social structures of pair-housed laboratory rabbits. Here we present a protocol to identify pairing methods, species-typical hierarchy establishment behaviors and behaviors that warrant appropriate intervention.

New Zealand White (NZW) laboratory rabbits (Oryctolagus cuniculus), as well as their ancestors the European Rabbit, are a social species that exhibit ...

Continuous Noninvasive Measuring of Crayfish Cardiac and Behavioral Activities

A crayfish is a pivotal aquatic organism that serves both as a practical biological model for behavioral and physiological studies of invertebrates and as a useful biological indicator of water quality. Even though crayfish cannot directly specify the substances that cause water quality deterioration, they can immediately (within a few seconds) warn humans of water quality deterioration via acute changes in their cardiac and behavioral activities.
In this study, we present a noninvasive method that is simple enough to be implemented under various conditions due to a combination of simplicity and reliability in one model.
This approach, in which the biological organisms are implemented into environmental evaluation processes, provides a reliable and timely alarm for warning of and preventing acute water deterioration in an ambient environment. Therefore, this noninvasive system based on crayfish physiological and ethological parameter recordings was investigated for the detection of changes in an aquatic environment. This system is now applied at a local brewery for controlling&#160;quality of the water used for beverage production, but it can be used at any water treatment and supply facility for continuous, real-time water quality evaluation and for regular laboratory investigations of crayfish cardiac physiology and behavior.

This article presents a noninvasive biomonitoring system for the continuous recording and analyses of crayfish cardiac and locomotor activities. This system consists of a near-infrared optical sensor, a video-tracking module, and software for evaluating crayfish heartbeats that reflects its physiological condition and characterizes crayfish behavior during heartbeat fluctuations.

A crayfish is a pivotal aquatic organism that serves both as a practical biological model for behavioral and physiological studies of invertebrates and as ...

Behavioral Tracking and Neuromast Imaging of Mexican Cavefish

Cave-dwelling animals have evolved a series of morphological and behavioral traits to adapt to their perpetually dark and food-sparse environments. Among these traits, foraging behavior is one of the useful windows into functional advantages of behavioral trait evolution. Presented herein are updated methods for analyzing vibration attraction behavior (VAB: an adaptive foraging behavior) and imaging of associated mechanosensors of cave-adapted tetra, Astyanax mexicanus. In addition, methods are presented for high-throughput tracking of a series of additional cavefish behaviors including hyperactivity and sleep-loss. Cavefish also show asociality, repetitive behavior and higher anxiety. Therefore, cavefish serve as an animal model for evolved behaviors. These methods use free-software and custom-made scripts that can be applied to other types of behavior. These methods provide practical and cost-effective alternatives to commercially available tracking software.

Here, we present methods for high-throughput study of a series of the Mexican cavefish behaviors and vital staining of a mechanosensory system. These methods use free-software and custom-made scripts, providing a practical and cost-effective method for the studies of behaviors.

Cave-dwelling animals have evolved a series of morphological and behavioral traits to adapt to their perpetually dark and food-sparse environments. Among ...