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Compton A, Sharma A, Hempel M, Aryan A, Biedler JK, Potters MB, Chandrasegaran K, Vinauger C, Tu Z. Differential elimination of marked sex chromosomes enables production of nontransgenic male mosquitoes in a single strain. Proc Natl Acad Sci U S A 2025; 122:e2412149122. [PMID: 40339129 PMCID: PMC12087967 DOI: 10.1073/pnas.2412149122] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/17/2024] [Accepted: 03/25/2025] [Indexed: 05/10/2025] Open
Abstract
Diverse genetic strategies are being pursued to control mosquito-borne infectious diseases. These strategies often rely on the release of nonbiting males to either reduce the target mosquito population or render them resistant to pathogens. Male-only releases are important as any contaminating females can bite and potentially transmit pathogens. Despite significant efforts, it remains a major bottleneck to reliably and efficiently separate males from females, especially when nontransgenic males are preferred. In the yellow fever mosquito Aedes aegypti, sex is determined by a pair of homomorphic sex chromosomes, with the dominant male-determining locus (the M locus) and its counterpart (the m locus) embedded in an M-bearing and an m-bearing chromosome 1, respectively. We utilized both naturally occurring and engineered sex-linked recessive lethal alleles (RLAs) to create sex separation strains for Ae. aegypti on the basis of differential elimination of marked sex chromosomes (DeMark). DeMark strains are self-sustaining and produce nontransgenic males that are readily separated from individuals carrying RLA- and transgene-marked m chromosomes. For example, the marked m chromosome in the heterozygous mother in some strains was only inherited by her female progeny due to RLA-mediated incompatibility with the M-bearing chromosome in the father, producing nontransgenic males and transgenic females, generation after generation. We further explore strategies to conditionally eliminate females that contain marked sex chromosomes. We also discuss DeMark designs that are applicable for efficient sex separation in organisms with well-differentiated X and Y chromosomes, such as the Anopheles mosquitoes.
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Affiliation(s)
- Austin Compton
- Department of Biochemistry, Virginia Tech, Blacksburg, VA24061
- The Fralin Life Sciences Institute, Virginia Tech, Blacksburg, VA24061
| | - Atashi Sharma
- Department of Biochemistry, Virginia Tech, Blacksburg, VA24061
- The Fralin Life Sciences Institute, Virginia Tech, Blacksburg, VA24061
| | - Melanie Hempel
- Department of Biochemistry, Virginia Tech, Blacksburg, VA24061
- The Fralin Life Sciences Institute, Virginia Tech, Blacksburg, VA24061
| | - Azadeh Aryan
- Department of Biochemistry, Virginia Tech, Blacksburg, VA24061
- The Fralin Life Sciences Institute, Virginia Tech, Blacksburg, VA24061
| | - James K. Biedler
- Department of Biochemistry, Virginia Tech, Blacksburg, VA24061
- The Fralin Life Sciences Institute, Virginia Tech, Blacksburg, VA24061
| | - Mark B. Potters
- Department of Biochemistry, Virginia Tech, Blacksburg, VA24061
- The Fralin Life Sciences Institute, Virginia Tech, Blacksburg, VA24061
| | | | - Clément Vinauger
- Department of Biochemistry, Virginia Tech, Blacksburg, VA24061
- The Fralin Life Sciences Institute, Virginia Tech, Blacksburg, VA24061
| | - Zhijian Tu
- Department of Biochemistry, Virginia Tech, Blacksburg, VA24061
- The Fralin Life Sciences Institute, Virginia Tech, Blacksburg, VA24061
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2
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Dong Y, Xu X, Qian L, Kou Z, Andongma AA, Zhou L, Huang Y, Wang Y. Genome-wide identification of yellow gene family in Hermetia illucens and functional analysis of yellow-y by CRISPR/Cas9. INSECT SCIENCE 2025; 32:115-126. [PMID: 38685755 DOI: 10.1111/1744-7917.13371] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/22/2023] [Revised: 07/03/2023] [Accepted: 07/05/2023] [Indexed: 05/02/2024]
Abstract
The yellow gene family plays a crucial role in insect pigmentation. It has potential for use as a visible marker gene in genetic manipulation and transgenic engineering in several model and non-model insects. Sadly, yellow genes have rarely been identified in Stratiomyidae species and the functions of yellow genes are relatively unknown. In the present study, we first manually annotated and curated 10 yellow genes in the black soldier fly (BSF), Hermetia illucens (Stratiomyidae). Then, the conserved amino acids in the major royal jelly proteins (MRJPs) domain, structural architecture and phylogenetic relationship of yellow genes in BSF were analyzed. We found that the BSF yellow-y, yellow-c and yellow-f genes are expressed at all developmental stages, especially in the prepupal stage. Using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, we successfully disrupted yellow-y, yellow-c and yellow-f in the BSF. Consequently, the mutation of yellow-y clearly resulted in a pale-yellow body color in prepupae, pupae and adults, instead of the typical black body color of the wild type. However, the mutation of yellow-c or yellow-f genes did not result in any change in color of the insects, when compared with the wild type. Our study indicates that the BSF yellow-y gene plays a role in body pigmentation, providing an optimal marker gene for the genetic manipulation of BSF.
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Affiliation(s)
- Yongcheng Dong
- Key Laboratory of Biology and Sustainable Management of Plant Diseases and Pests of Anhui Higher Education Institutes, College of Plant Protection, Anhui Agricultural University, Hefei, China
| | - Xiaomiao Xu
- Key Laboratory of Biology and Sustainable Management of Plant Diseases and Pests of Anhui Higher Education Institutes, College of Plant Protection, Anhui Agricultural University, Hefei, China
| | - Lansa Qian
- Chinese Academy of Sciences (CAS) Key Laboratory of Insect Developmental and Evolutionary Biology, CAS Center for Excellence in Molecular Plant Sciences, CAS, Shanghai, China
| | - Zongqing Kou
- Chinese Academy of Sciences (CAS) Key Laboratory of Insect Developmental and Evolutionary Biology, CAS Center for Excellence in Molecular Plant Sciences, CAS, Shanghai, China
| | - Awawing A Andongma
- Insect and Parasite Ecology Group, Lancaster Environment Centre, Lancaster University, Lancaster, UK
| | - Lijun Zhou
- Key Laboratory of Biology and Sustainable Management of Plant Diseases and Pests of Anhui Higher Education Institutes, College of Plant Protection, Anhui Agricultural University, Hefei, China
| | - Yongping Huang
- School of Environmental Science and Engineering, Shanghai Jiao Tong University, Shanghai, China
| | - Yaohui Wang
- Key Laboratory of Biology and Sustainable Management of Plant Diseases and Pests of Anhui Higher Education Institutes, College of Plant Protection, Anhui Agricultural University, Hefei, China
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3
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Chan SW. CRISPR-editing of the virus vector Aedes albopictus cell line C6/36, illustrated by prohibitin 2 gene knockout. MethodsX 2024; 13:102817. [PMID: 39049926 PMCID: PMC11267050 DOI: 10.1016/j.mex.2024.102817] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/13/2024] [Accepted: 06/20/2024] [Indexed: 07/27/2024] Open
Abstract
Aedes mosquitoes are important virus vectors. We provide a toolkit for CRISPR-Cas9-editing of difficult-to-knockdown gene previously shown to be refractory to siRNA silencing in mosquito cells, which is pivotal in understanding vector biology, vector competence, host-pathogen interactions and in gene annotations. Starting from database searches of Ae. albopictus and the C6/36 cell line whole genome shotgun sequences for the prohibitin 2 (PHB2) gene, primers were designed to confirm the gene sequence in our laboratory-passaged C6/36 cell line for the correct design and cloning of CRISPR RNA into an insect plasmid vector to create a single guide RNA for the PHB2 gene target. After transfection of this plasmid vector into the C6/36 cells, cell clones selected by puromycin and/or limiting dilution were analyzed for insertions and deletions (INDELs) using PCR, sequencing and computational sequence decomposition. From this, we have identified mono-allelic and bi-allelic knockout cell clones. Using a mono-allelic knockout cell clone as an example, we characterized its INDELs by molecular cloning and computational analysis. Importantly, mono-allelic knockout was sufficient to reduce >80 % of PHB2 expression, which led to phenotypic switching and the propensity to form foci but was insufficient to affect growth rate or to inhibit Zika virus infection.•We provide a toolkit for CRISPR-Cas9-editing of the virus vector, Aedes albopictus C6/36 cell line•We validate this using a difficult-to-knockdown gene prohibitin 2•This toolkit is pivotal in understanding vector biology, vector competence, host-pathogen interactions and in gene annotations.
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Affiliation(s)
- Shiu-Wan Chan
- Faculty of Biology, Medicine and Health, School of Biological Sciences, The University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, United Kingdom
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4
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Kantor AM, Talyuli OAC, Reid WR, Alvarenga PH, Booker J, Lin J, Franz AWE, Barillas-Mury C. Identification of a dengue 2 virus envelope protein receptor in Aedes aegypti critical for viral midgut infection. Proc Natl Acad Sci U S A 2024; 121:e2417750121. [PMID: 39565309 PMCID: PMC11621822 DOI: 10.1073/pnas.2417750121] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/05/2024] [Accepted: 10/21/2024] [Indexed: 11/21/2024] Open
Abstract
The establishment of a productive dengue virus (DENV) infection in the midgut epithelial cells of Aedes aegypti is critical for the viral transmission cycle. The hypothesis that DENV virions interact directly with specific mosquito midgut proteins was explored. We found that DENV serotype 2 (DENV2) pretreated with trypsin interacted with a single 31 kDa protein, identified as AAEL011180 by protein mass spectrometry. This putative receptor is a highly conserved protein and has orthologs in culicine and anopheline mosquitoes. We confirmed that impairing the expression of AAEL011180 in the midgut of Ae. aegypti females abolished the interaction with DENV2, and the virus also bound to immobilized recombinant purified receptor. Furthermore, recombinant DENV2 surface E glycoprotein bound to recombinant AAEL011180 with high affinity (38.2 nM) in binding kinetic analysis using surface plasmon resonance. The gene for this DENV2 E protein receptor (EPrRec) was disrupted, but since the gene is essential in Ae. aegypti, only heterozygote knockout (ΔEPrRec+/-) females could be recovered. Further reducing EPrRec mRNA expression in the midgut of ΔEPrRec+/- females by systemic dsRNA injection significantly reduced the prevalence of DENV2 midgut infection. EPrRec also interacts with heat shock protein 70 cognate 3 (Hsc70-3), and silencing Hsc70-3 expression in ΔEPrRec females also reduced the prevalence of DENV2 midgut infection.
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Affiliation(s)
- Asher M. Kantor
- Laboratory of Malaria and Vector Research, National Institutes of Allergy and Infectious Diseases, NIH, Rockville, MD20852
| | - Octavio A. C. Talyuli
- Laboratory of Malaria and Vector Research, National Institutes of Allergy and Infectious Diseases, NIH, Rockville, MD20852
| | - William R. Reid
- Department of Veterinary Pathobiology, University of Missouri, Columbia, MO65211
| | - Patricia Hessab Alvarenga
- Laboratory of Malaria and Vector Research, National Institutes of Allergy and Infectious Diseases, NIH, Rockville, MD20852
| | - Jasmine Booker
- Laboratory of Malaria and Vector Research, National Institutes of Allergy and Infectious Diseases, NIH, Rockville, MD20852
| | - Jingyi Lin
- Department of Veterinary Pathobiology, University of Missouri, Columbia, MO65211
| | | | - Carolina Barillas-Mury
- Laboratory of Malaria and Vector Research, National Institutes of Allergy and Infectious Diseases, NIH, Rockville, MD20852
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Yan X, Zhao Z, Feng S, Zhang Y, Wang Z, Li Z. Multi-omics analysis reveal the fall armyworm Spodoptera frugiperda tolerate high temperature by mediating chitin-related genes. INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY 2024; 174:104192. [PMID: 39401552 DOI: 10.1016/j.ibmb.2024.104192] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/29/2024] [Revised: 10/02/2024] [Accepted: 10/11/2024] [Indexed: 10/19/2024]
Abstract
Climate change facilitates the rapid invasion of agricultural pests, threatening global food security. The fall armyworm Spodoptera frugiperda is a highly polyphagous migratory pest tolerant to high temperatures, allowing its proliferation in harsh thermal environments. We aimed to demonstrate mechanisms of its high-temperature tolerance, particularly transcriptional and metabolic regulation, which are poorly understood. To achieve the aim, we examined the impact and mechanism of heat events on S. frugiperda by using multiple approaches: ecological measurements, transcriptomics, metabolomics, RNAi, and CRISPR/Cas9 technology. We observed that several physiological indices (larval survival rate, larval period, pupation rate, pupal weight, eclosion rate, and average fecundity) decreased as the temperature increased, with the 32 °C treatment displaying a significant difference from the control group at 26 °C. Significantly upregulated expression of genes encoding endochitinase and chitin deacetylase was observed in the chitin-binding, extracellular region, and carbohydrate metabolic process GO terms of hemolymph, fat body, and brain, exhibiting a tissue-specific pattern. Significantly enriched pathways (e.g., cutin, suberin, and wax biosynthesis; oxidative phosphorylation and cofactor biosynthesis; diverse amino acid biosynthesis and degradation; carbon metabolism; and energy metabolism), all of which are essential for S. frugiperda larvae to tolerate temperature, were found in metabolites that were expressed differently. Successful RNA interference targeting of the three chitin-related genes reduced gene expression levels and larval survival rate. Knockout of the endochitinase gene by using the CRISPR/Cas9 system significantly reduced the relative gene expression and increased sensitivity to high-temperature exposure. On the basis of our findings, theoretical foundations for understanding the high-temperature tolerance of S. frugiperda populations and latent genetic control strategies were established.
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Affiliation(s)
- Xiaorui Yan
- MARA Key Laboratory of Surveillance and Management for Plant Quarantine Pests, College of Plant Protection, China Agricultural University, Beijing, 100193, China; Sanya Institute of China Agricultural University, Sanya, 572025, China
| | - Zihua Zhao
- MARA Key Laboratory of Surveillance and Management for Plant Quarantine Pests, College of Plant Protection, China Agricultural University, Beijing, 100193, China; Sanya Institute of China Agricultural University, Sanya, 572025, China
| | - Shiqian Feng
- State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, 100193, China
| | - Yongjun Zhang
- State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, 100193, China
| | - Zhenying Wang
- State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, 100193, China
| | - Zhihong Li
- MARA Key Laboratory of Surveillance and Management for Plant Quarantine Pests, College of Plant Protection, China Agricultural University, Beijing, 100193, China; Sanya Institute of China Agricultural University, Sanya, 572025, China.
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6
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Lo IHY, Matthews BJ. Design and Validation of Guide RNAs for CRISPR-Cas9 Genome Editing in Mosquitoes. Cold Spring Harb Protoc 2024; 2024:pdb.top107688. [PMID: 37816601 DOI: 10.1101/pdb.top107688] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/12/2023]
Abstract
CRISPR-Cas9 has revolutionized gene editing for traditional and nontraditional model organisms alike. This tool has opened the door to new mechanistic studies of basic mosquito biology as well as the development of novel vector control strategies based on CRISPR-Cas9, including gene drives that spread genetic elements in the population. Although the promise of the specificity, flexibility, and ease of deployment CRISPR is real, its implementation still requires empirical optimization for each new species of interest, as well as to each genomic target within a given species. Here, we provide an overview of designing and testing single-guide RNAs for the use of CRISPR-based gene editing tools.
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Affiliation(s)
- Ivan Hok Yin Lo
- Department of Zoology, The University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada
| | - Benjamin J Matthews
- Department of Zoology, The University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada
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7
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Cui Y, Megawati D, Lin J, Rehard DG, Grant DG, Liu P, Jurkevich A, Reid WR, Mooney BP, Franz AW. Cytoskeleton-associated gelsolin responds to the midgut distention process in saline meal-fed Aedes aegypti and affects arbovirus dissemination from the midgut. FASEB J 2024; 38:e23764. [PMID: 39042395 PMCID: PMC11268798 DOI: 10.1096/fj.202302684rr] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/27/2023] [Revised: 06/06/2024] [Accepted: 06/13/2024] [Indexed: 07/24/2024]
Abstract
The mosquito, Aedes aegypti, is the principal vector for several arboviruses. The mosquito midgut is the initial tissue that gets infected with an arbovirus acquired along with a blood meal from a vertebrate host. Blood meal ingestion leads to midgut tissue distention thereby increasing the pore size of the surrounding basal lamina. This allows newly synthesized virions to exit the midgut by traversing the distended basal lamina to infect secondary tissues of the mosquito. We conducted a quantitative label-free proteomic time course analysis with saline meal-fed Ae. aegypti females to identify host factors involved in midgut tissue distention. Around 2000 proteins were detected during each of the seven sampling time points and 164 of those were uniquely expressed. Forty-five of 97 differentially expressed proteins were upregulated during the 96-h time course and most of those were involved in cytoskeleton modulation, metabolic activity, and vesicle/vacuole formation. The F-actin-modulating Ae. aegypti (Aa)-gelsolin was selected for further functional studies. Stable knockout of Aa-gelsolin resulted in a mosquito line, which showed distorted actin filaments in midgut-associated tissues likely due to diminished F-actin processing by gelsolin. Zika virus dissemination from the midgut of these mosquitoes was diminished and delayed. The loss of Aa-gelsolin function was associated with an increased induction of apoptosis in midgut tissue indicating an involvement of Aa-gelsolin in apoptotic signaling in mosquitoes. Here, we used proteomics to discover a novel host factor, Aa-gelsolin, which affects the midgut escape barrier for arboviruses in mosquitoes and apoptotic signaling in the midgut.
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Affiliation(s)
- Yingjun Cui
- Department of Veterinary Pathobiology, University of Missouri, Columbia, Missouri, USA
| | - Dewi Megawati
- Department of Veterinary Pathobiology, University of Missouri, Columbia, Missouri, USA
- Department of Microbiology and Parasitology, School of Medicine, Warmadewa University, Bali, Indonesia
| | - Jingyi Lin
- Department of Veterinary Pathobiology, University of Missouri, Columbia, Missouri, USA
| | - David G. Rehard
- Department of Veterinary Pathobiology, University of Missouri, Columbia, Missouri, USA
| | - DeAna G. Grant
- Electron Microscopy Core, University of Missouri, Columbia, Missouri, USA
| | - Pei Liu
- Charles W. Gehrke Proteomics Center, University of Missouri, Columbia, Missouri, USA
| | - Alexander Jurkevich
- Advanced Light Microscopy Core, University of Missouri, Columbia, Missouri, USA
| | - William R. Reid
- Department of Veterinary Pathobiology, University of Missouri, Columbia, Missouri, USA
| | - Brian P. Mooney
- Charles W. Gehrke Proteomics Center, University of Missouri, Columbia, Missouri, USA
| | - Alexander W.E. Franz
- Department of Veterinary Pathobiology, University of Missouri, Columbia, Missouri, USA
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8
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Li M, Zhou Y, Cheng J, Wang Y, Lan C, Shen Y. Response of the mosquito immune system and symbiotic bacteria to pathogen infection. Parasit Vectors 2024; 17:69. [PMID: 38368353 PMCID: PMC10874582 DOI: 10.1186/s13071-024-06161-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2023] [Accepted: 01/24/2024] [Indexed: 02/19/2024] Open
Abstract
Mosquitoes are the deadliest animal in the word, transmitting a variety of insect-borne infectious diseases, such as malaria, dengue fever, yellow fever, and Zika, causing more deaths than any other vector-borne pathogen. Moreover, in the absence of effective drugs and vaccines to prevent and treat insect-borne diseases, mosquito control is particularly important as the primary measure. In recent decades, due to the gradual increase in mosquito resistance, increasing attention has fallen on the mechanisms and effects associated with pathogen infection. This review provides an overview of mosquito innate immune mechanisms in terms of physical and physiological barriers, pattern recognition receptors, signalling pathways, and cellular and humoral immunity, as well as the antipathogenic effects of mosquito symbiotic bacteria. This review contributes to an in-depth understanding of the interaction process between mosquitoes and pathogens and provides a theoretical basis for biological defence strategies against mosquito-borne infectious diseases.
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Affiliation(s)
- Manjin Li
- The Affiliated Wuxi Center for Disease Control and Prevention of Nanjing Medical University, Wuxi Center for Disease Control and Prevention, Wuxi, 214023, China
| | - Yang Zhou
- Nanjing Medical University, Nanjing, 211166, China
| | - Jin Cheng
- The Affiliated Wuxi Center for Disease Control and Prevention of Nanjing Medical University, Wuxi Center for Disease Control and Prevention, Wuxi, 214023, China
| | - Yiqing Wang
- The Affiliated Wuxi Center for Disease Control and Prevention of Nanjing Medical University, Wuxi Center for Disease Control and Prevention, Wuxi, 214023, China
| | - Cejie Lan
- The Affiliated Wuxi Center for Disease Control and Prevention of Nanjing Medical University, Wuxi Center for Disease Control and Prevention, Wuxi, 214023, China.
| | - Yuan Shen
- The Affiliated Wuxi Center for Disease Control and Prevention of Nanjing Medical University, Wuxi Center for Disease Control and Prevention, Wuxi, 214023, China.
- Nanjing Medical University, Nanjing, 211166, China.
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Anderson MAE, Gonzalez E, Edgington MP, Ang JXD, Purusothaman DK, Shackleford L, Nevard K, Verkuijl SAN, Harvey-Samuel T, Leftwich PT, Esvelt K, Alphey L. A multiplexed, confinable CRISPR/Cas9 gene drive can propagate in caged Aedes aegypti populations. Nat Commun 2024; 15:729. [PMID: 38272895 PMCID: PMC10810878 DOI: 10.1038/s41467-024-44956-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2023] [Accepted: 01/11/2024] [Indexed: 01/27/2024] Open
Abstract
Aedes aegypti is the main vector of several major pathogens including dengue, Zika and chikungunya viruses. Classical mosquito control strategies utilizing insecticides are threatened by rising resistance. This has stimulated interest in new genetic systems such as gene drivesHere, we test the regulatory sequences from the Ae. aegypti benign gonial cell neoplasm (bgcn) homolog to express Cas9 and a separate multiplexing sgRNA-expressing cassette inserted into the Ae. aegypti kynurenine 3-monooxygenase (kmo) gene. When combined, these two elements provide highly effective germline cutting at the kmo locus and act as a gene drive. Our target genetic element drives through a cage trial population such that carrier frequency of the element increases from 50% to up to 89% of the population despite significant fitness costs to kmo insertions. Deep sequencing suggests that the multiplexing design could mitigate resistance allele formation in our gene drive system.
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Affiliation(s)
- Michelle A E Anderson
- Arthropod Genetics, The Pirbright Institute, Ash Road, Pirbright, GU24 0HN, UK
- The Department of Biology, University of York, Wentworth Way, York, YO10 5DD, UK
| | - Estela Gonzalez
- Arthropod Genetics, The Pirbright Institute, Ash Road, Pirbright, GU24 0HN, UK
- Animal and Plant Health Agency, Woodham Lane, Addlestone, Surrey, KT15 3NB, UK
| | - Matthew P Edgington
- Arthropod Genetics, The Pirbright Institute, Ash Road, Pirbright, GU24 0HN, UK
- The Department of Biology, University of York, Wentworth Way, York, YO10 5DD, UK
| | - Joshua X D Ang
- Arthropod Genetics, The Pirbright Institute, Ash Road, Pirbright, GU24 0HN, UK
- The Department of Biology, University of York, Wentworth Way, York, YO10 5DD, UK
| | - Deepak-Kumar Purusothaman
- Arthropod Genetics, The Pirbright Institute, Ash Road, Pirbright, GU24 0HN, UK
- MRC-University of Glasgow Centre for Virus Research, Henry Wellcome Building, 464 Bearsden Road, Glasgow, G61 1QH, UK
| | - Lewis Shackleford
- Arthropod Genetics, The Pirbright Institute, Ash Road, Pirbright, GU24 0HN, UK
- The Department of Biology, University of York, Wentworth Way, York, YO10 5DD, UK
| | - Katherine Nevard
- Arthropod Genetics, The Pirbright Institute, Ash Road, Pirbright, GU24 0HN, UK
| | - Sebald A N Verkuijl
- Arthropod Genetics, The Pirbright Institute, Ash Road, Pirbright, GU24 0HN, UK
- Department of Biology, University of Oxford, 11a Mansfield Road, Oxford, OX1 3SZ, UK
| | | | - Philip T Leftwich
- Arthropod Genetics, The Pirbright Institute, Ash Road, Pirbright, GU24 0HN, UK
- School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich, Norfolk, NR4 7TJ, UK
| | - Kevin Esvelt
- Media Laboratory, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA
| | - Luke Alphey
- Arthropod Genetics, The Pirbright Institute, Ash Road, Pirbright, GU24 0HN, UK.
- The Department of Biology, University of York, Wentworth Way, York, YO10 5DD, UK.
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10
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Martin-Martin I, Kojin BB, Aryan A, Williams AE, Molina-Cruz A, Valenzuela-Leon PC, Shrivastava G, Botello K, Minai M, Adelman ZN, Calvo E. Aedes aegypti D7 long salivary proteins modulate blood feeding and parasite infection. mBio 2023; 14:e0228923. [PMID: 37909749 PMCID: PMC10746281 DOI: 10.1128/mbio.02289-23] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/25/2023] [Accepted: 09/27/2023] [Indexed: 11/03/2023] Open
Abstract
IMPORTANCE During blood feeding, mosquitoes inject saliva into the host skin, preventing hemostasis and inflammatory responses. D7 proteins are among the most abundant components of the saliva of blood-feeding arthropods. Aedes aegypti, the vector of yellow fever and dengue, expresses two D7 long-form salivary proteins: D7L1 and D7L2. These proteins bind and counteract hemostatic agonists such as biogenic amines and leukotrienes. D7L1 and D7L2 knockout mosquitoes showed prolonged probing times and carried significantly less Plasmodium gallinaceum oocysts per midgut than wild-type mosquitoes. We hypothesize that reingested D7s play a vital role in the midgut microenvironment with important consequences for pathogen infection and transmission.
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Affiliation(s)
- Ines Martin-Martin
- Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, USA
- Laboratory of Medical Entomology, National Center for Microbiology, Instituto de Salud Carlos III, Madrid, Spain
| | | | - Azadeh Aryan
- Department of Entomology, Fralin Life Science Institute, Virginia Tech, Blacksburg, Virginia, USA
| | - Adeline E. Williams
- Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, USA
| | - Alvaro Molina-Cruz
- Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, USA
| | - Paola Carolina Valenzuela-Leon
- Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, USA
| | - Gaurav Shrivastava
- Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, USA
| | - Karina Botello
- Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, USA
| | - Mahnaz Minai
- Infectious Disease Pathogenesis Section, Comparative Medicine Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, USA
| | - Zach N. Adelman
- Department of Entomology, Texas A&M University, College Station, Texas, USA
- Department of Entomology, Fralin Life Science Institute, Virginia Tech, Blacksburg, Virginia, USA
| | - Eric Calvo
- Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, USA
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11
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Gouda MNR, Jeevan H, Shashank HG. CRISPR/Cas9: a cutting-edge solution for combatting the fall armyworm, Spodoptera frugiperda. Mol Biol Rep 2023; 51:13. [PMID: 38085335 DOI: 10.1007/s11033-023-08986-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2023] [Accepted: 10/13/2023] [Indexed: 12/18/2023]
Abstract
The utilization of CRISPR/Cas9 in Spodoptera frugiperda, commonly known as fall armyworm, presents a groundbreaking avenue for pest management. With its ability to precisely modify the insect's genome, CRISPR/Cas9 offers innovative strategies to combat this destructive pest. The application of CRISPR/Cas9 in S. frugiperda holds immense potential. It enables the identification and functional analysis of key genes associated with its behavior, development, and insecticide resistance. This knowledge can unveil novel target sites for more effective and specific insecticides. Additionally, CRISPR/Cas9 can facilitate the development of population control methods by disrupting vital genes essential for survival. However, challenges such as off-target effects and the efficient delivery of CRISPR/Cas9 components remain. Addressing these obstacles is vital to ensure accurate and reliable results. Furthermore, ethical considerations, biosafety protocols, and regulatory frameworks must be integral to the adoption of this technology. Looking forward, CRISPR/Cas9-based gene drive systems hold the potential to promulgate desirable genetic traits within S. frugiperda populations, offering a sustainable and eco-friendly approach. This could curtail their reproductive capabilities or make them more susceptible to certain interventions. In conclusion, CRISPR/Cas9 presents a transformative platform for precise and targeted pest management in S. frugiperda. By deciphering the insect's genetic makeup and developing innovative strategies, we can mitigate the devastating impact of fall armyworm on agriculture while ensuring environmental sustainability.
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Affiliation(s)
- M N Rudra Gouda
- Division of Entomology, Indian Agricultural Research Institute, New Delhi, 110012, India.
| | - H Jeevan
- Division of Nematology, Indian Agricultural Research Institute, New Delhi, 110012, India
| | - H G Shashank
- Division of Plant Genetic Resources, Indian Agricultural Research Institute, New Delhi, 110012, India
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12
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Carabajal Paladino LZ, Wilson R, Tng PYL, Dhokiya V, Keen E, Cuber P, Larner W, Rooney S, Nicholls M, Uglow A, Williams L, Anderson MAE, Basu S, Leftwich PT, Alphey L. Optimizing CRE and PhiC31 mediated recombination in Aedes aegypti. Front Bioeng Biotechnol 2023; 11:1254863. [PMID: 37811374 PMCID: PMC10557486 DOI: 10.3389/fbioe.2023.1254863] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2023] [Accepted: 08/31/2023] [Indexed: 10/10/2023] Open
Abstract
Introduction: Genetic manipulation of Aedes aegypti is key to developing a deeper understanding of this insects' biology, vector-virus interactions and makes future genetic control strategies possible. Despite some advances, this process remains laborious and requires highly skilled researchers and specialist equipment. Methods: Here we present two improved methods for genetic manipulation in this species. Use of transgenic lines which express Cre recombinase and a plasmid-based method for expressing PhiC31 when injected into early embryos. Results: Use of transgenic lines which express Cre recombinase allowed, by simple crossing schemes, germline or somatic recombination of transgenes, which could be utilized for numerous genetic manipulations. PhiC31 integrase based methods for site-specific integration of genetic elements was also improved, by developing a plasmid which expresses PhiC31 when injected into early embryos, eliminating the need to use costly and unstable mRNA as is the current standard. Discussion: Here we have expanded the toolbox for synthetic biology in Ae. aegypti. These methods can be easily transferred into other mosquito and even insect species by identifying appropriate promoter sequences. This advances the ability to manipulate these insects for fundamental studies, and for more applied approaches for pest control.
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13
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Dong S, Dimopoulos G. Aedes aegypti Argonaute 2 controls arbovirus infection and host mortality. Nat Commun 2023; 14:5773. [PMID: 37723154 PMCID: PMC10507101 DOI: 10.1038/s41467-023-41370-y] [Citation(s) in RCA: 12] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/07/2023] [Accepted: 08/30/2023] [Indexed: 09/20/2023] Open
Abstract
Ae. aegypti mosquitoes transmit some of the most important human viral diseases that are responsible for a significant public health burden worldwide. The small interfering RNA (siRNA) pathway is considered the major antiviral defense system in insects. Here we show that siRNA pathway disruption by CRISPR/Cas9-based Ago2 knockout impaired the mosquitoes' ability to degrade arbovirus RNA leading to hyper-infection accompanied by cell lysis and tissue damage. Ago2 disruption impaired DNA repair mechanisms and the autophagy pathway by altering histone abundance. This compromised DNA repair and removal of damaged cellular organelles and dysfunctional aggregates promoted mosquito death. We also report that hyper-infection of Ago2 knockout mosquitoes stimulated a broad-spectrum antiviral immunity, including apoptosis, which may counteract infection. Taken together, our studies reveal novel roles for Ago2 in protecting mosquitoes from arbovirus infection and associated death.
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Affiliation(s)
- Shengzhang Dong
- W. Harry Feinstone Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, 615 N. Wolfe Street, Baltimore, MD, 21205-2179, USA
| | - George Dimopoulos
- W. Harry Feinstone Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, 615 N. Wolfe Street, Baltimore, MD, 21205-2179, USA.
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14
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Sun R, Raban R, Akbari OS. CRISPR-Cas9 Methods and Key Considerations in the Production of Aedes aegypti Mutant Strains. Cold Spring Harb Protoc 2023; 2023:607-613. [PMID: 36931732 PMCID: PMC10901255 DOI: 10.1101/pdb.top107693] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/19/2023]
Abstract
Since the characterization of the CRISPR-Cas9 system in prokaryotes, it has become the prime choice in gene editing because of its exceptional flexibility, ease of use, high efficiency, and superior specificity. As a result, CRISPR-Cas9-mediated gene-editing technologies have enabled researchers not only to engineer transgenic animal strains with site-directed insertions more efficiently but also to generate desired mutants for previously intractable species. One such species is the invasive yellow fever mosquito, Aedes aegypti, which is notorious for its ability to transmit many blood-borne human pathogens. Methods for developing new transgenic strains of the yellow fever mosquito may aid in the effort to control its populations and provide significant benefits for the public. Here, we provide an overview of injection and noninjection methods for generating transgenic mosquitoes and also highlight important experimental design features.
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Affiliation(s)
- Ruichen Sun
- Division of Biological Sciences, Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, California 92093, USA
| | - Robyn Raban
- Division of Biological Sciences, Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, California 92093, USA
| | - Omar S Akbari
- Division of Biological Sciences, Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, California 92093, USA
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15
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Merkling SH, Crist AB, Henrion-Lacritick A, Frangeul L, Couderc E, Gausson V, Blanc H, Bergman A, Baidaliuk A, Romoli O, Saleh MC, Lambrechts L. Multifaceted contributions of Dicer2 to arbovirus transmission by Aedes aegypti. Cell Rep 2023; 42:112977. [PMID: 37573505 DOI: 10.1016/j.celrep.2023.112977] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2022] [Revised: 06/20/2023] [Accepted: 07/28/2023] [Indexed: 08/15/2023] Open
Abstract
Arthropod-borne viruses (arboviruses) transmitted by Aedes aegypti mosquitoes are an increasing threat to global health. The small interfering RNA (siRNA) pathway is considered the main antiviral immune pathway of insects, but its effective impact on arbovirus transmission is surprisingly poorly understood. Here, we use CRISPR-Cas9-mediated gene editing in vivo to mutate Dicer2, a gene encoding the RNA sensor and key component of the siRNA pathway. The loss of Dicer2 enhances early viral replication and systemic viral dissemination of four medically significant arboviruses (chikungunya, Mayaro, dengue, and Zika viruses) representing two viral families. However, Dicer2 mutants and wild-type mosquitoes display overall similar levels of vector competence. In addition, Dicer2 mutants undergo significant virus-induced mortality during infection with chikungunya virus. Together, our results define a multifaceted role for Dicer2 in the transmission of arboviruses by Ae. aegypti mosquitoes and pave the way for further mechanistic investigations.
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Affiliation(s)
- Sarah Hélène Merkling
- Institut Pasteur, Université Paris Cité, CNRS UMR2000, Insect-Virus Interactions Unit, 75015 Paris, France
| | - Anna Beth Crist
- Institut Pasteur, Université Paris Cité, CNRS UMR2000, Insect-Virus Interactions Unit, 75015 Paris, France
| | - Annabelle Henrion-Lacritick
- Institut Pasteur, Université Paris Cité, CNRS UMR3569, Viruses and RNA Interference Unit, 75015 Paris, France
| | - Lionel Frangeul
- Institut Pasteur, Université Paris Cité, CNRS UMR3569, Viruses and RNA Interference Unit, 75015 Paris, France
| | - Elodie Couderc
- Institut Pasteur, Université Paris Cité, CNRS UMR2000, Insect-Virus Interactions Unit, 75015 Paris, France; Sorbonne Université, Collège Doctoral, 75005 Paris, France
| | - Valérie Gausson
- Institut Pasteur, Université Paris Cité, CNRS UMR3569, Viruses and RNA Interference Unit, 75015 Paris, France
| | - Hervé Blanc
- Institut Pasteur, Université Paris Cité, CNRS UMR3569, Viruses and RNA Interference Unit, 75015 Paris, France
| | - Alexander Bergman
- Institut Pasteur, Université Paris Cité, CNRS UMR2000, Insect-Virus Interactions Unit, 75015 Paris, France
| | - Artem Baidaliuk
- Institut Pasteur, Université Paris Cité, CNRS UMR2000, Insect-Virus Interactions Unit, 75015 Paris, France; Sorbonne Université, Collège Doctoral, 75005 Paris, France
| | - Ottavia Romoli
- Institut Pasteur, Université Paris Cité, CNRS UMR3569, Viruses and RNA Interference Unit, 75015 Paris, France
| | - Maria-Carla Saleh
- Institut Pasteur, Université Paris Cité, CNRS UMR3569, Viruses and RNA Interference Unit, 75015 Paris, France.
| | - Louis Lambrechts
- Institut Pasteur, Université Paris Cité, CNRS UMR2000, Insect-Virus Interactions Unit, 75015 Paris, France.
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16
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Chae K, Overcash JM, Dawson C, Valentin C, Tsujimoto H, Myles KM, Adelman ZN. CRISPR-based gene editing of non-homologous end joining factors biases DNA repair pathway choice toward single-strand annealing in Aedes aegypti. CURRENT RESEARCH IN BIOTECHNOLOGY 2023; 5:100133. [PMID: 37475832 PMCID: PMC10357993 DOI: 10.1016/j.crbiot.2023.100133] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 07/22/2023] Open
Abstract
To maintain genome stability, eukaryotic cells orchestrate DNA repair pathways to process DNA double-strand breaks (DSBs) that result from diverse developmental or environmental stimuli. Bias in the selection of DSB repair pathways, either non-homologous end joining (NHEJ) or homology-directed repair (HDR), is also critical for efficient gene editing and for homing-based gene drive approaches developed for the control of disease-transmitting vector mosquitoes. However, little is understood about DNA repair homeostasis in the mosquito genome. Here, we utilized CRISPR/Cas9 to generate indel mutant strains for core NHEJ factors ku80, DNA ligase IV (lig4), and DNA-PKcs in the mosquito Aedes aegypti and evaluated the corresponding effects on DNA repair. In a plasmid-based assay, disruption of ku80 or lig4, but not DNA-PKcs, reduced both NHEJ and SSA. However, a transgenic reporter strain-based test revealed that those mutations significantly biased DNA repair events toward SSA. Interestingly, ku80 mutation also significantly increased the end joining rate by a yet-characterized mechanism in males. Our study provides evidence that the core NHEJ factors have an antagonistic effect on SSA-based DSB repair of the Ae. aegypti genome. Down-modulating the NHEJ pathway can enhance the efficiency of nuclease-based genetic control approaches, as most of those operate by homology-based repair processes along with extensive DNA end resection that is antagonized by NHEJ.
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Affiliation(s)
- Keun Chae
- Department of Entomology, Texas A&M University, College Station, TX 77843, United States
| | - Justin M. Overcash
- U.S. Department of Agriculture-Animal and Plant Health Inspection Service (USDA-APHIS), Biotechnology Regulatory Services, Riverdale, MD 20737, United States
| | - Chanell Dawson
- Department of Entomology, Texas A&M University, College Station, TX 77843, United States
| | - Collin Valentin
- Department of Entomology, Texas A&M University, College Station, TX 77843, United States
| | - Hitoshi Tsujimoto
- Department of Entomology, Texas A&M University, College Station, TX 77843, United States
| | - Kevin M. Myles
- Department of Entomology, Texas A&M University, College Station, TX 77843, United States
| | - Zach N. Adelman
- Department of Entomology, Texas A&M University, College Station, TX 77843, United States
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17
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Samuel GH, Pohlenz T, Dong Y, Coskun N, Adelman ZN, Dimopoulos G, Myles KM. RNA interference is essential to modulating the pathogenesis of mosquito-borne viruses in the yellow fever mosquito Aedes aegypti. Proc Natl Acad Sci U S A 2023; 120:e2213701120. [PMID: 36893279 PMCID: PMC10089172 DOI: 10.1073/pnas.2213701120] [Citation(s) in RCA: 12] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2022] [Accepted: 12/15/2022] [Indexed: 03/11/2023] Open
Abstract
While it has long been known that the transmission of mosquito-borne viruses depends on the establishment of persistent and nonlethal infections in the invertebrate host, specific roles for the insects' antiviral immune pathways in modulating the pathogenesis of viral infections is the subject of speculation and debate. Here, we show that a loss-of-function mutation in the Aedes aegypti Dicer-2 (Dcr-2) gene renders the insect acutely susceptible to a disease phenotype upon infection with pathogens in multiple virus families associated with important human diseases. Additional interrogation of the disease phenotype demonstrated that the virus-induced pathology is controlled through a canonical RNA interference (RNAi) pathway, which functions as a resistance mechanism. These results suggest comparatively modest contributions of proposed tolerance mechanisms to the fitness of A. aegypti infected with these pathogens. Similarly, the production of virus-derived piwi-interacting RNAs (vpiRNAs) was not sufficient to prevent the pathology associated with viral infections in Dcr-2 null mutants, also suggesting a less critical, or potentially secondary, role for vpiRNAs in antiviral immunity. These findings have important implications for understanding the ecological and evolutionary interactions occurring between A. aegypti and the pathogens they transmit to human and animal hosts.
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Affiliation(s)
- Glady Hazitha Samuel
- Department of Entomology, Minnie Belle Heep Center, Texas A & M University, College Station, TX77843-2475
| | - Tyler Pohlenz
- Department of Entomology, Minnie Belle Heep Center, Texas A & M University, College Station, TX77843-2475
| | - Yuemei Dong
- W. Harry Feinstone Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD21205-2179
| | - Nese Coskun
- Department of Entomology, Minnie Belle Heep Center, Texas A & M University, College Station, TX77843-2475
| | - Zach N. Adelman
- Department of Entomology, Minnie Belle Heep Center, Texas A & M University, College Station, TX77843-2475
| | - George Dimopoulos
- W. Harry Feinstone Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD21205-2179
| | - Kevin M. Myles
- Department of Entomology, Minnie Belle Heep Center, Texas A & M University, College Station, TX77843-2475
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18
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Asad M, Liu D, Chen J, Yang G. Applications of gene drive systems for population suppression of insect pests. BULLETIN OF ENTOMOLOGICAL RESEARCH 2022; 112:724-733. [PMID: 36043456 DOI: 10.1017/s0007485322000268] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/15/2023]
Abstract
Population suppression is an effective way for controlling insect pests and disease vectors, which cause significant damage to crop and spread contagious diseases to plants, animals and humans. Gene drive systems provide innovative opportunities for the insect pests population suppression by driving genes that impart fitness costs on populations of pests or disease vectors. Different gene-drive systems have been developed in insects and applied for their population suppression. Here, different categories of gene drives such as meiotic drive (MD), under-dominance (UD), homing endonuclease-based gene drive (HEGD) and especially the CRISPR/Cas9-based gene drive (CCGD) were reviewed, including the history, types, process and mechanisms. Furthermore, the advantages and limitations of applying different gene-drive systems to suppress the insect population were also summarized. This review provides a foundation for developing a specific gene-drive system for insect population suppression.
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Affiliation(s)
- Muhammad Asad
- State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, Institute of Applied Ecology, Fujian Agriculture and Forestry University, Fuzhou 350002, China
- Joint International Research Laboratory of Ecological Pest Control, Ministry of Education, Fuzhou 350002, China
- Key Laboratory of Integrated Pest Management for Fujian-Taiwan Crops, Ministry of Agriculture, Fuzhou 350002, China
- Key Laboratory of Green Pest Control, Fujian Province University, Fuzhou 350002, China
| | - Dan Liu
- State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, Institute of Applied Ecology, Fujian Agriculture and Forestry University, Fuzhou 350002, China
- Joint International Research Laboratory of Ecological Pest Control, Ministry of Education, Fuzhou 350002, China
- Key Laboratory of Integrated Pest Management for Fujian-Taiwan Crops, Ministry of Agriculture, Fuzhou 350002, China
- Key Laboratory of Green Pest Control, Fujian Province University, Fuzhou 350002, China
| | - Jing Chen
- State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, Institute of Applied Ecology, Fujian Agriculture and Forestry University, Fuzhou 350002, China
- Joint International Research Laboratory of Ecological Pest Control, Ministry of Education, Fuzhou 350002, China
- Key Laboratory of Integrated Pest Management for Fujian-Taiwan Crops, Ministry of Agriculture, Fuzhou 350002, China
- Key Laboratory of Green Pest Control, Fujian Province University, Fuzhou 350002, China
| | - Guang Yang
- State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, Institute of Applied Ecology, Fujian Agriculture and Forestry University, Fuzhou 350002, China
- Joint International Research Laboratory of Ecological Pest Control, Ministry of Education, Fuzhou 350002, China
- Key Laboratory of Integrated Pest Management for Fujian-Taiwan Crops, Ministry of Agriculture, Fuzhou 350002, China
- Key Laboratory of Green Pest Control, Fujian Province University, Fuzhou 350002, China
- Ministerial and Provincial Joint Innovation Centre for Safety Production of Cross-Strait Crops, Fujian Agriculture and Forestry University, Fuzhou 350002, China
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19
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Zhu GH, Gaddelapati SC, Jiao Y, Koo J, Palli SR. CRISPR-Cas9 Genome Editing Uncovers the Mode of Action of Methoprene in the Yellow Fever Mosquito, Aedes aegypti. CRISPR J 2022; 5:813-824. [PMID: 36374965 PMCID: PMC9805843 DOI: 10.1089/crispr.2022.0066] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022] Open
Abstract
Methoprene, a juvenile hormone (JH) analog, is widely used for insect control, but its mode of action is not known. To study methoprene action in the yellow fever mosquito, Aedes aegypti, the E93 (ecdysone-induced transcription factor) was knocked out using the CRISPR-Cas9 system. The E93 mutant pupae retained larval tissues similar to methoprene-treated insects. These insects completed pupal ecdysis and died as pupa. In addition, the expression of transcription factors, broad complex and Krüppel homolog 1 (Kr-h1), increased and that of programmed cell death (PCD) and autophagy genes decreased in E93 mutants. These data suggest that methoprene functions through JH receptor, methoprene-tolerant, and induces the expression of Kr-h1, which suppresses the expression of E93, resulting in a block in PCD and autophagy of larval tissues. Failure in the elimination of larval tissues and the formation of adult structures results in their death. These results answered long-standing questions on the mode of action of methoprene.
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Affiliation(s)
- Guan-Heng Zhu
- Department of Entomology, College of Agriculture, Food and Environment, University of Kentucky, Lexington, Kentucky, USA
| | - Sharath Chandra Gaddelapati
- Department of Entomology, College of Agriculture, Food and Environment, University of Kentucky, Lexington, Kentucky, USA
| | - Yaoyu Jiao
- Department of Entomology, College of Agriculture, Food and Environment, University of Kentucky, Lexington, Kentucky, USA
| | - Jinmo Koo
- Department of Entomology, College of Agriculture, Food and Environment, University of Kentucky, Lexington, Kentucky, USA
| | - Subba Reddy Palli
- Department of Entomology, College of Agriculture, Food and Environment, University of Kentucky, Lexington, Kentucky, USA.,Address correspondence to: Subba Reddy Palli, Department of Entomology, College of Agriculture, Food and Environment, University of Kentucky, Lexington, KY 40546, USA.
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20
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Reid W, Williams AE, Sanchez-Vargas I, Lin J, Juncu R, Olson KE, Franz AWE. Assessing single-locus CRISPR/Cas9-based gene drive variants in the mosquito Aedes aegypti via single-generation crosses and modeling. G3 (BETHESDA, MD.) 2022; 12:jkac280. [PMID: 36250791 PMCID: PMC9713460 DOI: 10.1093/g3journal/jkac280] [Citation(s) in RCA: 13] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/09/2022] [Accepted: 10/09/2022] [Indexed: 07/29/2023]
Abstract
The yellow fever mosquito Aedes aegypti is a major vector of arthropod-borne viruses, including dengue, chikungunya, and Zika viruses. A novel approach to mitigate arboviral infections is to generate mosquitoes refractory to infection by overexpressing antiviral effector molecules. Such an approach requires a mechanism to spread these antiviral effectors through a population, for example, by using CRISPR/Cas9-based gene drive systems. Critical to the design of a single-locus autonomous gene drive is that the selected genomic locus is amenable to both gene drive and appropriate expression of the antiviral effector. In our study, we used reverse engineering to target 2 intergenic genomic loci, which had previously shown to be highly permissive for antiviral effector gene expression, and we further investigated the use of 3 promoters (nanos, β2-tubulin, or zpg) for Cas9 expression. We then quantified the accrual of insertions or deletions (indels) after single-generation crossings, measured maternal effects, and assessed fitness costs associated with various transgenic lines to model the rate of gene drive fixation. Overall, MGDrivE modeling suggested that when an autonomous gene drive is placed into an intergenic locus, the gene drive system will eventually be blocked by the accrual of gene drive blocking resistance alleles and ultimately be lost in the population. Moreover, while genomic locus and promoter selection were critically important for the initial establishment of the autonomous gene drive, it was the fitness of the gene drive line that most strongly influenced the persistence of the gene drive in the simulated population. As such, we propose that when autonomous CRISPR/Cas9-based gene drive systems are anchored in an intergenic locus, they temporarily result in a strong population replacement effect, but as gene drive-blocking indels accrue, the gene drive becomes exhausted due to the fixation of CRISPR resistance alleles.
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Affiliation(s)
- William Reid
- Department of Veterinary Pathobiology, University of Missouri, Columbia, MO 65211, USA
| | - Adeline E Williams
- Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO 80523, USA
| | - Irma Sanchez-Vargas
- Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO 80523, USA
| | - Jingyi Lin
- Department of Veterinary Pathobiology, University of Missouri, Columbia, MO 65211, USA
| | - Rucsanda Juncu
- Department of Veterinary Pathobiology, University of Missouri, Columbia, MO 65211, USA
| | - Ken E Olson
- Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO 80523, USA
| | - Alexander W E Franz
- Department of Veterinary Pathobiology, University of Missouri, Columbia, MO 65211, USA
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21
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Chen C, Compton A, Nikolouli K, Wang A, Aryan A, Sharma A, Qi Y, Dellinger C, Hempel M, Potters M, Augustinos A, Severson DW, Bourtzis K, Tu Z. Marker-assisted mapping enables forward genetic analysis in Aedes aegypti, an arboviral vector with vast recombination deserts. Genetics 2022; 222:iyac140. [PMID: 36083009 PMCID: PMC9630976 DOI: 10.1093/genetics/iyac140] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2022] [Accepted: 08/29/2022] [Indexed: 11/14/2022] Open
Abstract
Aedes aegypti is a major vector of arboviruses that cause dengue, chikungunya, yellow fever, and Zika. Although recent success in reverse genetics has facilitated rapid progress in basic and applied research, integration of forward genetics with modern technologies remains challenging in this important species, as up to 47% of its chromosome is refractory to genetic mapping due to extremely low rate of recombination. Here, we report the development of a marker-assisted mapping strategy to readily screen for and genotype only the rare but informative recombinants, drastically increasing both the resolution and signal-to-noise ratio. Using marker-assisted mapping, we mapped a transgene that was inserted in a >100-Mb recombination desert and a sex-linked spontaneous red-eye (re) mutation just outside the region. We subsequently determined, by CRISPR/Cas9-mediated knockout, that cardinal is the causal gene of re, which is the first forward genetic identification of a causal gene in Ae. aegypti. The identification of the causal gene of the sex-linked re mutation provides the molecular foundation for using gene editing to develop versatile and stable genetic sexing methods. To facilitate genome-wide forward genetics in Ae. aegypti, we generated and compiled a number of lines with markers throughout the genome. Thus, by overcoming the challenges presented by the vast recombination deserts and the scarcity of markers, we have shown that effective forward genetic analysis is increasingly feasible in this important arboviral vector species.
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Affiliation(s)
- Chujia Chen
- Genetics Bioinformatics and Computational Biology Program, Virginia Tech, Blacksburg, VA 24061, USA
- Fralin Life Sciences Institute, Virginia Tech, Blacksburg, VA 24061, USA
| | - Austin Compton
- Fralin Life Sciences Institute, Virginia Tech, Blacksburg, VA 24061, USA
- Department of Biochemistry, Virginia Tech, Blacksburg, VA 24061, USA
| | - Katerina Nikolouli
- Insect Pest Control Laboratory, Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, IAEA Laboratories, 2444 Seibersdorf, Austria
| | - Aihua Wang
- Fralin Life Sciences Institute, Virginia Tech, Blacksburg, VA 24061, USA
- Department of Biochemistry, Virginia Tech, Blacksburg, VA 24061, USA
| | - Azadeh Aryan
- Fralin Life Sciences Institute, Virginia Tech, Blacksburg, VA 24061, USA
- Department of Biochemistry, Virginia Tech, Blacksburg, VA 24061, USA
| | - Atashi Sharma
- Fralin Life Sciences Institute, Virginia Tech, Blacksburg, VA 24061, USA
- Department of Biochemistry, Virginia Tech, Blacksburg, VA 24061, USA
| | - Yumin Qi
- Fralin Life Sciences Institute, Virginia Tech, Blacksburg, VA 24061, USA
- Department of Biochemistry, Virginia Tech, Blacksburg, VA 24061, USA
| | - Camden Dellinger
- Fralin Life Sciences Institute, Virginia Tech, Blacksburg, VA 24061, USA
- Department of Biochemistry, Virginia Tech, Blacksburg, VA 24061, USA
| | - Melanie Hempel
- Fralin Life Sciences Institute, Virginia Tech, Blacksburg, VA 24061, USA
- Department of Biochemistry, Virginia Tech, Blacksburg, VA 24061, USA
| | - Mark Potters
- Fralin Life Sciences Institute, Virginia Tech, Blacksburg, VA 24061, USA
- Department of Biochemistry, Virginia Tech, Blacksburg, VA 24061, USA
| | - Antonios Augustinos
- Insect Pest Control Laboratory, Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, IAEA Laboratories, 2444 Seibersdorf, Austria
| | - David W Severson
- Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556, USA
| | - Kostas Bourtzis
- Insect Pest Control Laboratory, Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, IAEA Laboratories, 2444 Seibersdorf, Austria
| | - Zhijian Tu
- Genetics Bioinformatics and Computational Biology Program, Virginia Tech, Blacksburg, VA 24061, USA
- Fralin Life Sciences Institute, Virginia Tech, Blacksburg, VA 24061, USA
- Department of Biochemistry, Virginia Tech, Blacksburg, VA 24061, USA
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22
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Agarwal A, Sarma DK, Chaurasia D, Maan HS. Novel molecular approaches to combat vectors and vector-borne viruses: Special focus on RNA interference (RNAi) mechanisms. Acta Trop 2022; 233:106539. [PMID: 35623398 DOI: 10.1016/j.actatropica.2022.106539] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/19/2022] [Revised: 05/21/2022] [Accepted: 05/23/2022] [Indexed: 11/16/2022]
Abstract
Vector-borne diseases, such as dengue, chikungunya, zika, yellow fever etc pose significant burden among the infectious diseases globally, especially in tropical and sub-tropical regions. Globalization, deforestation, urbanization, climate change, uncontrolled population growth, inadequate waste management and poor vector-management infrastructure have all contributed to the expansion of vector habitats and subsequent increase in vector-borne diseases throughout the world. Conventional vector control methods, such as use of insecticides, have significant negative environmental repercussions in addition to developing resistance in vectors. Till date, a very few vaccines or antiviral therapies have been approved for the treatment of vector borne diseases. In this review, we have discussed emerging molecular approaches like CRISPR (clustered regularly interspaced short palindromic repeats)/Cas-9, sterile insect technique (SIT), release of insects carrying a dominant lethal (RIDL), Wolbachia (virus transmission blocking) and RNA interference (RNAi) to combat vector and vector-borne viruses. Due to the extensive advancements in RNAi research, a special focus has been given on its types, biogenesis, mechanism of action, delivery and experimental studies evaluating their application as anti-mosquito and anti-viral agent. These technologies appear to be highly promising in terms of contributing to vector control and antiviral drug development, and hence can be used to reduce global vector and vector-borne disease burden.
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Affiliation(s)
- Ankita Agarwal
- State Virology Laboratory, Department of Microbiology, Gandhi Medical College, Bhopal 462001, Madhya Pradesh, India.
| | - Devojit Kumar Sarma
- ICMR-National Institute for Research in Environmental Health, Bhopal 462030, Madhya Pradesh, India
| | - Deepti Chaurasia
- State Virology Laboratory, Department of Microbiology, Gandhi Medical College, Bhopal 462001, Madhya Pradesh, India
| | - Harjeet Singh Maan
- State Virology Laboratory, Department of Microbiology, Gandhi Medical College, Bhopal 462001, Madhya Pradesh, India
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23
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Martin-Martin I, Valenzuela Leon PC, Amo L, Shrivastava G, Iniguez E, Aryan A, Brooks S, Kojin BB, Williams AE, Bolland S, Ackerman H, Adelman ZN, Calvo E. Aedes aegypti sialokinin facilitates mosquito blood feeding and modulates host immunity and vascular biology. Cell Rep 2022; 39:110648. [PMID: 35417706 PMCID: PMC9082008 DOI: 10.1016/j.celrep.2022.110648] [Citation(s) in RCA: 15] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2021] [Revised: 02/01/2022] [Accepted: 03/17/2022] [Indexed: 11/28/2022] Open
Abstract
Saliva from mosquitoes contains vasodilators that antagonize vasoconstrictors produced at the bite site. Sialokinin is a vasodilator present in the saliva of Aedes aegypti. Here, we investigate its function and describe its mechanism of action during blood feeding. Sialokinin induces nitric oxide release similar to substance P. Sialokinin-KO mosquitoes produce lower blood perfusion than parental mosquitoes at the bite site during probing and have significantly longer probing times, which result in lower blood feeding success. In contrast, there is no difference in feeding between KO and parental mosquitoes when using artificial membrane feeders or mice that are treated with a substance P receptor antagonist, confirming that sialokinin interferes with host hemostasis via NK1R signaling. While sialokinin-KO saliva does not affect virus infection in vitro, it stimulates macrophages and inhibits leukocyte recruitment in vivo. This work highlights the biological functionality of salivary proteins in blood feeding.
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Affiliation(s)
- Ines Martin-Martin
- Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852, USA.
| | - Paola Carolina Valenzuela Leon
- Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852, USA
| | - Laura Amo
- Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852, USA
| | - Gaurav Shrivastava
- Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852, USA
| | - Eva Iniguez
- Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852, USA
| | - Azadeh Aryan
- Department of Entomology and Fralin Life Science Institute, Virginia Polytechnic Institute and State University, Blacksburg, VA 24060, USA
| | - Steven Brooks
- Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852, USA
| | - Bianca B Kojin
- Department of Entomology, Texas A&M University, College Station, TX 77843, USA
| | - Adeline E Williams
- Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852, USA; Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins 80523, CO, USA
| | - Silvia Bolland
- Department of Entomology and Fralin Life Science Institute, Virginia Polytechnic Institute and State University, Blacksburg, VA 24060, USA
| | - Hans Ackerman
- Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852, USA
| | - Zach N Adelman
- Department of Entomology and Fralin Life Science Institute, Virginia Polytechnic Institute and State University, Blacksburg, VA 24060, USA; Department of Entomology, Texas A&M University, College Station, TX 77843, USA
| | - Eric Calvo
- Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852, USA.
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24
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Bottino-Rojas V, Ferreira-Almeida I, Nunes RD, Feng X, Pham TB, Kelsey A, Carballar-Lejarazú R, Gantz V, Oliveira PL, James AA. Beyond the eye: Kynurenine pathway impairment causes midgut homeostasis dysfunction and survival and reproductive costs in blood-feeding mosquitoes. INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY 2022; 142:103720. [PMID: 34999199 PMCID: PMC11055609 DOI: 10.1016/j.ibmb.2022.103720] [Citation(s) in RCA: 12] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/24/2021] [Revised: 01/03/2022] [Accepted: 01/04/2022] [Indexed: 06/14/2023]
Abstract
Insect ommochrome biosynthesis pathways metabolize tryptophan to generate eye-color pigments and wild-type alleles of pathway genes are useful phenotypic markers in transgenesis studies. Pleiotropic effects of mutations in some genes exert a load on both survival and reproductive success in blood-feeding species. Here, we investigated the challenges imposed on mosquitoes by the increase of tryptophan metabolites resulting from blood meal digestion and the impact of disruptions of the ommochrome biosynthesis pathway. Female mosquitoes with spontaneous and induced mutations in the orthologs of the genes encoding kynurenine hydroxylase in Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus exhibited impaired survival and reproductive phenotypes that varied in type and severity among the species. A compromised midgut permeability barrier function was also observed in An. stephensi. Surprisingly, mutant mosquitoes displayed an increase in microbiota compared to controls that was not accompanied by a general induction of immune genes. Antibiotic treatment rescued some deleterious traits implicating a role for the kynurenine pathway (KP) in midgut homeostasis. Supplemental xanthurenic acid, a KP end-product, rescued lethality and limited microbiota proliferation in Ae. aegypti. These data implicate the KP in the regulation of the host/microbiota interface. These pleiotropic effects on mosquito physiology are important in the development of genetic strategies targeting vector mosquitoes.
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Affiliation(s)
- Vanessa Bottino-Rojas
- Department of Microbiology & Molecular Genetics, University of California, Irvine, CA, USA
| | - Igor Ferreira-Almeida
- Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
| | - Rodrigo D Nunes
- Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
| | - Xuechun Feng
- Section of Cell and Developmental Biology, University of California San Diego, La Jolla, CA, USA
| | - Thai Binh Pham
- Department of Microbiology & Molecular Genetics, University of California, Irvine, CA, USA
| | - Adam Kelsey
- Department of Microbiology & Molecular Genetics, University of California, Irvine, CA, USA
| | | | - Valentino Gantz
- Section of Cell and Developmental Biology, University of California San Diego, La Jolla, CA, USA
| | - Pedro L Oliveira
- Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil; Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular, Rio de Janeiro, RJ, Brazil.
| | - Anthony A James
- Department of Microbiology & Molecular Genetics, University of California, Irvine, CA, USA; Department of Molecular Biology & Biochemistry, University of California, Irvine, CA, USA.
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25
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Ang JXD, Nevard K, Ireland R, Purusothaman DK, Verkuijl SAN, Shackleford L, Gonzalez E, Anderson MAE, Alphey L. Considerations for homology-based DNA repair in mosquitoes: Impact of sequence heterology and donor template source. PLoS Genet 2022; 18:e1010060. [PMID: 35180218 PMCID: PMC8893643 DOI: 10.1371/journal.pgen.1010060] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2021] [Revised: 03/03/2022] [Accepted: 01/28/2022] [Indexed: 12/23/2022] Open
Abstract
The increasing prevalence of insecticide resistance and the ongoing global burden of vector-borne diseases have encouraged new efforts in mosquito control. For Aedes aegypti, the most important arboviral vector, integration rates achieved in Cas9-based knock-ins so far have been rather low, highlighting the need to understand gene conversion patterns and other factors that influence homology-directed repair (HDR) events in this species. In this study, we report the effects of sequence mismatches or donor template forms on integration rates. We found that modest sequence differences between construct homology arms [DNA sequence in the donor template which resembles the region flanking the target cut] and genomic target comprising 1.2% nucleotide dissimilarity (heterology) significantly reduced integration rates. While most integrations (59-88%) from plasmid templates were the result of canonical [on target, perfect repair] HDR events, no canonical events were identified from other donor types (i.e. ssDNA, biotinylated ds/ssDNA). Sequencing of the transgene flanking region in 69 individuals with canonical integrations revealed 60% of conversion tracts to be unidirectional and extend up to 220 bp proximal to the break, though in three individuals bidirectional conversion of up to 725 bp was observed.
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Affiliation(s)
| | | | | | | | - Sebald A. N. Verkuijl
- The Pirbright Institute, Pirbright, Woking, United Kingdom
- Mathematical Ecology Research Group, Department of Zoology, University of Oxford, Oxford, United Kingdom
| | | | | | | | - Luke Alphey
- The Pirbright Institute, Pirbright, Woking, United Kingdom
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26
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Abstract
CRISPR-mediated genome engineering technologies have been adapted to a wide variety of organisms with high efficiency and specificity. The yellow fever mosquito, Aedes aegypti , is one such organism. It is also responsible for transmitting a wide variety of deadly viruses including Dengue, Zika, Yellow fever, and Chikungunya. The key to successful CRISPR-mediated gene editing applications is the delivery of both Cas9 ribonuclease and single-guide RNA (sgRNA ) to the nucleus of desired cells. Various methods have been developed for supplying the Cas9 endonuclease, sgRNA , and donor DNA to Ae. aegypti. In this chapter, we focus on methods of direct embryo delivery of editing components, presenting detailed step-by-step CRISPR/Cas9-based genome-editing protocols for inducing desired heritable edits in mosquitoes as well as insights into successful application of these protocols. We also highlight potential opportunities for customizing these protocols to manipulate the mosquito genome for innovative in vivo gene function studies.
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Affiliation(s)
- Ruichen Sun
- Division of Biological Sciences, Section of Cell and Developmental Biology, University of California San Diego, La Jolla, CA, USA
| | - Ming Li
- Division of Biological Sciences, Section of Cell and Developmental Biology, University of California San Diego, La Jolla, CA, USA
| | - Conor J McMeniman
- W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Malaria Research Institute, Johns Hopkins Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA
- The Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD, USA
| | - Omar S Akbari
- Division of Biological Sciences, Section of Cell and Developmental Biology, University of California San Diego, La Jolla, CA, USA.
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27
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Zhu GH, Albishi NM, Chen X, Brown RL, Palli SR. Expanding the Toolkit for Genome Editing in a Disease Vector, Aedes aegypti: Transgenic Lines Expressing Cas9 and Single Guide RNA Induce Efficient Mutagenesis. CRISPR J 2021; 4:846-853. [PMID: 33450159 PMCID: PMC8742270 DOI: 10.1089/crispr.2020.0052] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023] Open
Abstract
CRISPR-Cas9 mediated genome editing methods are being used for the analysis of gene function. However, it is hard to identify gene knockout mutants for genes whose knockout does not cause distinct phenotypes. To overcome this issue in the disease vector, Aedes aegypti, a transgenic Cas9/single guide RNA (sgRNA) method, was used to knock out the eye marker gene, kynurenine 3-monooxygenase (kmo), and the juvenile hormone receptor, Methoprene-tolerant (Met). PiggyBac transformation vectors were prepared to express sgRNAs targeting kmo and Met under the control of the U6 promoter. Transgenic Ae. aegypti expressing kmo-sgRNA or Met-sgRNA under the control of the U6 promoter and enhanced green fluorescent protein (eGFP) under the control of the hr5ie1 promoter were produced. The U6-sgRNA adults were mated with AAEL010097-Cas9 adults. The progeny were screened, and the insects expressing eGFP and DsRed were selected and evaluated for mutations in target genes. About 77% and 78% of the progeny that were positive for both eGFP and DsRed in kmo-sgRNA and Met-sgRNA groups, respectively, showed mutations in their target genes.
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Affiliation(s)
- Guan-Heng Zhu
- Department of Entomology, College of Agriculture, Food and Environment, University of Kentucky, Lexington, Kentucky, USA
| | - Najla M. Albishi
- Department of Entomology, College of Agriculture, Food and Environment, University of Kentucky, Lexington, Kentucky, USA
| | - Xien Chen
- Department of Entomology, College of Agriculture, Food and Environment, University of Kentucky, Lexington, Kentucky, USA
| | - Rachel L. Brown
- Department of Entomology, College of Agriculture, Food and Environment, University of Kentucky, Lexington, Kentucky, USA
| | - Subba Reddy Palli
- Department of Entomology, College of Agriculture, Food and Environment, University of Kentucky, Lexington, Kentucky, USA
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28
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Han W, Tang F, Zhong Y, Zhang J, Liu Z. Identification of yellow gene family and functional analysis of Spodoptera frugiperda yellow-y by CRISPR/Cas9. PESTICIDE BIOCHEMISTRY AND PHYSIOLOGY 2021; 178:104937. [PMID: 34446204 DOI: 10.1016/j.pestbp.2021.104937] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/16/2020] [Revised: 06/06/2021] [Accepted: 07/14/2021] [Indexed: 06/13/2023]
Abstract
For a devastating agricultural pest, functional genomics promotes the finding of novel technology to control Spodoptera frugiperda, such as the genetics-based strategies. In the present study, 11 yellow genes were identified in Spodoptera frugiperda. The transcriptome analysis showed the tissue-specific expression of part yellow genes, which suggested the importance of yellow genes in some biological processes in S. frugiperda, such as pigmentation. Among these yellow genes, the expression profiles of yellow-y gene showed that it was expressed in all life stages. In order to realize the further study of yellow-y, we employed CRISPR/Cas9 system to knock out this gene. Following knock out, diverse phenotypes were observed, such as color changes in both larvae and adults. Different from the wild-type larvae and adults, G0 mutants were yellowed since hatching. However, no color difference was observed with the pupal cuticle between the wild-type and mutant pupae before the 8th day. On the basis of the single-pair strategy of G0 generation, the yellow-y gene was proved to be a recessive gene. The G1 yellowish larvae with biallelic mutations displayed a relatively longer development period than wild-type, and often generated abnormal pupae and moths. The deletion of yellow-y also resulted in a decline in the fecundity. The results revealed that yellow-y gene was important for S. frugiperda pigmentation, as well as in its development and reproduction. Besides, the present study set up a standard procedure to knock out genes in S. frugiperda, which could be helpful for our understanding some key molecular processes, such as functional roles of detoxification genes as insecticide resistance mechanisms or modes of action of insecticides to facilitate the management of this insect pest.
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Affiliation(s)
- Weikang Han
- Key Laboratory of Integrated Management of Crop Diseases and Pests (Ministry of Education), College of Plant Protection, Nanjing Agricultural University, Weigang 1, Nanjing 210095, China
| | - Fengxian Tang
- Key Laboratory of Integrated Management of Crop Diseases and Pests (Ministry of Education), College of Plant Protection, Nanjing Agricultural University, Weigang 1, Nanjing 210095, China
| | - Yanni Zhong
- Key Laboratory of Integrated Management of Crop Diseases and Pests (Ministry of Education), College of Plant Protection, Nanjing Agricultural University, Weigang 1, Nanjing 210095, China
| | - Junteng Zhang
- Key Laboratory of Integrated Management of Crop Diseases and Pests (Ministry of Education), College of Plant Protection, Nanjing Agricultural University, Weigang 1, Nanjing 210095, China
| | - Zewen Liu
- Key Laboratory of Integrated Management of Crop Diseases and Pests (Ministry of Education), College of Plant Protection, Nanjing Agricultural University, Weigang 1, Nanjing 210095, China.
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29
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Caragata EP, Dong S, Dong Y, Simões ML, Tikhe CV, Dimopoulos G. Prospects and Pitfalls: Next-Generation Tools to Control Mosquito-Transmitted Disease. Annu Rev Microbiol 2021; 74:455-475. [PMID: 32905752 DOI: 10.1146/annurev-micro-011320-025557] [Citation(s) in RCA: 29] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Mosquito-transmitted diseases, including malaria and dengue, are a major threat to human health around the globe, affecting millions each year. A diverse array of next-generation tools has been designed to eliminate mosquito populations or to replace them with mosquitoes that are less capable of transmitting key pathogens. Many of these new approaches have been built on recent advances in CRISPR/Cas9-based genome editing. These initiatives have driven the development of pathogen-resistant lines, new genetics-based sexing methods, and new methods of driving desirable genetic traits into mosquito populations. Many other emerging tools involve microorganisms, including two strategies involving Wolbachia that are achieving great success in the field. At the same time, other mosquito-associated bacteria, fungi, and even viruses represent untapped sources of new mosquitocidal or antipathogen compounds. Although there are still hurdles to be overcome, the prospect that such approaches will reduce the impact of these diseases is highly encouraging.
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Affiliation(s)
- E P Caragata
- Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland 21205, USA; , , , , ,
| | - S Dong
- Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland 21205, USA; , , , , ,
| | - Y Dong
- Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland 21205, USA; , , , , ,
| | - M L Simões
- Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland 21205, USA; , , , , ,
| | - C V Tikhe
- Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland 21205, USA; , , , , ,
| | - G Dimopoulos
- Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland 21205, USA; , , , , ,
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30
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Rosendo Machado S, van der Most T, Miesen P. Genetic determinants of antiviral immunity in dipteran insects - Compiling the experimental evidence. DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY 2021; 119:104010. [PMID: 33476667 DOI: 10.1016/j.dci.2021.104010] [Citation(s) in RCA: 18] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/30/2020] [Revised: 01/08/2021] [Accepted: 01/09/2021] [Indexed: 06/12/2023]
Abstract
The genetic basis of antiviral immunity in dipteran insects is extensively studied in Drosophila melanogaster and advanced technologies for genetic manipulation allow a better characterization of immune responses also in non-model insect species. Especially, immunity in vector mosquitoes is recently in the spotlight, due to the medical impact that these insects have by transmitting viruses and other pathogens. Here, we review the current state of experimental evidence that supports antiviral functions for immune genes acting in different cellular pathways. We discuss the well-characterized RNA interference mechanism along with the less well-defined JAK-STAT, Toll, and IMD signaling pathways. Furthermore, we highlight the initial evidence for antiviral activity observed for the autophagy pathway, transcriptional pausing, as well as piRNA production from endogenous viral elements. We focus our review on studies from Drosophila and mosquito species from the lineages Aedes, Culex, and Anopheles, which contain major vector species responsible for virus transmission.
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Affiliation(s)
- Samara Rosendo Machado
- Department of Medical Microbiology, Radboud Institute for Molecular Life Sciences, Radboud University Nijmegen Medical Center, P.O. Box 9101, 6500 HB, Nijmegen, the Netherlands
| | - Tom van der Most
- Department of Medical Microbiology, Radboud Institute for Molecular Life Sciences, Radboud University Nijmegen Medical Center, P.O. Box 9101, 6500 HB, Nijmegen, the Netherlands
| | - Pascal Miesen
- Department of Medical Microbiology, Radboud Institute for Molecular Life Sciences, Radboud University Nijmegen Medical Center, P.O. Box 9101, 6500 HB, Nijmegen, the Netherlands.
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31
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Değirmenci L, Geiger D, Rogé Ferreira FL, Keller A, Krischke B, Beye M, Steffan-Dewenter I, Scheiner R. CRISPR/Cas 9-Mediated Mutations as a New Tool for Studying Taste in Honeybees. Chem Senses 2021; 45:655-666. [PMID: 32968780 DOI: 10.1093/chemse/bjaa063] [Citation(s) in RCA: 20] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Honeybees rely on nectar as their main source of carbohydrates. Sucrose, glucose, and fructose are the main components of plant nectars. Intriguingly, honeybees express only 3 putative sugar receptors (AmGr1, AmGr2, and AmGr3), which is in stark contrast to many other insects and vertebrates. The sugar receptors are only partially characterized. AmGr1 detects different sugars including sucrose and glucose. AmGr2 is assumed to act as a co-receptor only, while AmGr3 is assumedly a fructose receptor. We show that honeybee gustatory receptor AmGr3 is highly specialized for fructose perception when expressed in Xenopus oocytes. When we introduced nonsense mutations to the respective AmGr3 gene using CRISPR/Cas9 in eggs of female workers, the resulting mutants displayed almost a complete loss of responsiveness to fructose. In contrast, responses to sucrose were normal. Nonsense mutations introduced by CRISPR/Cas9 in honeybees can thus induce a measurable behavioral change and serve to characterize the function of taste receptors in vivo. CRISPR/Cas9 is an excellent novel tool for characterizing honeybee taste receptors in vivo. Biophysical receptor characterization in Xenopus oocytes and nonsense mutation of AmGr3 in honeybees unequivocally demonstrate that this receptor is highly specific for fructose.
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Affiliation(s)
- Laura Değirmenci
- Behavioral Physiology and Sociobiology, University of Würzburg, Biocenter, Am Hubland, Würzburg, Germany
| | - Dietmar Geiger
- Julius-von-Sachs-Institute, Molecular Plant Physiology and Biophysics, University of Würzburg, Biocenter, Würzburg, Germany
| | - Fábio Luiz Rogé Ferreira
- Julius-von-Sachs-Institute, Molecular Plant Physiology and Biophysics, University of Würzburg, Biocenter, Würzburg, Germany
| | - Alexander Keller
- Department of Bioinformatics, Biocenter, Am Hubland, Würzburg, Germany
| | - Beate Krischke
- Animal Ecology and Tropical Biology, University of Würzburg, Biocenter, Am Hubland, Würzburg, Germany
| | - Martin Beye
- Evolutionary Genetics, Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany
| | - Ingolf Steffan-Dewenter
- Animal Ecology and Tropical Biology, University of Würzburg, Biocenter, Am Hubland, Würzburg, Germany
| | - Ricarda Scheiner
- Behavioral Physiology and Sociobiology, University of Würzburg, Biocenter, Am Hubland, Würzburg, Germany
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32
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Li X, Xu Y, Zhang H, Yin H, Zhou D, Sun Y, Ma L, Shen B, Zhu C. ReMOT Control Delivery of CRISPR-Cas9 Ribonucleoprotein Complex to Induce Germline Mutagenesis in the Disease Vector Mosquitoes Culex pipiens pallens (Diptera: Culicidae). JOURNAL OF MEDICAL ENTOMOLOGY 2021; 58:1202-1209. [PMID: 33590868 DOI: 10.1093/jme/tjab016] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/13/2020] [Indexed: 06/12/2023]
Abstract
The wide distribution of Culex (Cx.) pipiens complex mosquitoes makes it difficult to prevent the transmission of mosquito-borne diseases in humans. Gene editing using CRISPR/Cas9 is an effective technique with the potential to solve the growing problem of mosquito-borne diseases. This study uses the ReMOT Control technique in Culex pipiens pallens (L.) to produce genetically modified mosquitoes. A microinjection system was established by injecting 60 adult female mosquitoes-14 µl injection mixture was required, and no precipitation occurred with ≤1 µl of endosomal release reagents (chloroquine or saponin). The efficiency of delivery of the P2C-enhanced green fluorescent protein-Cas9 (P2C-EGFP-Cas9) ribonucleoprotein complex into the ovary was 100% when injected at 24 h post-bloodmeal (the peak of vitellogenesis). Using this method for KMO knockout, we found that gene editing in the ovary could also occur when P2C-Cas9 RNP complex was injected into the hemolymph of adult Cx. pipiens pallens by ReMOT Control. In the chloroquine group, of the 2,251 G0 progeny screened, 9 individuals showed with white and mosaic eye phenotypes. In the saponin group, of the 2,462 G0 progeny screened, 8 mutant individuals were observed. Sequencing results showed 13 bp deletions, further confirming the fact that gene editing occurred. In conclusion, the successful application of ReMOT Control in Cx. pipiens pallens not only provides the basic parameters (injection parameters and injection time) for this method but also facilitates the study of mosquito biology and control.
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Affiliation(s)
- Xixi Li
- Department of Pathogen Biology, Nanjing Medical University, Nanjing, Jiangsu, PR China
| | - Yang Xu
- Department of Pathogen Biology, Nanjing Medical University, Nanjing, Jiangsu, PR China
| | - Hongbo Zhang
- Department of Pathogen Biology, Nanjing Medical University, Nanjing, Jiangsu, PR China
| | - Haitao Yin
- Department of Pathogen Biology, Nanjing Medical University, Nanjing, Jiangsu, PR China
| | - Dan Zhou
- Department of Pathogen Biology, Nanjing Medical University, Nanjing, Jiangsu, PR China
| | - Yan Sun
- Department of Pathogen Biology, Nanjing Medical University, Nanjing, Jiangsu, PR China
| | - Lei Ma
- Department of Pathogen Biology, Nanjing Medical University, Nanjing, Jiangsu, PR China
| | - Bo Shen
- Department of Pathogen Biology, Nanjing Medical University, Nanjing, Jiangsu, PR China
| | - Changliang Zhu
- Department of Pathogen Biology, Nanjing Medical University, Nanjing, Jiangsu, PR China
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Hillary VE, Ceasar SA. Genome engineering in insects for the control of vector borne diseases. PROGRESS IN MOLECULAR BIOLOGY AND TRANSLATIONAL SCIENCE 2021; 179:197-223. [PMID: 33785177 DOI: 10.1016/bs.pmbts.2020.12.017] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
Insects cause many vector-borne infectious diseases and have become a major threat to human health. Although many control measures are undertaken, some insects are resistant to it, exacerbated by environmental changes which is a major challenge for control measures. Genetic studies by targeting the genomes of insects may offer an alternative strategy. Developments with novel genome engineering technologies have stretched our ability to target and modify any genomic sequence in Eukaryotes including insects. Genome engineering tools such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and most recently discovered, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) systems hold the potential to control the vector-borne diseases. In this chapter, we review the vector control strategy undertaken by employing three major genome engineering tools (ZFNs, TALENs, and CRISPR/Cas9) and discuss the future prospects of this system to control insect vectors. Finally, we also discuss the CRISPR-based gene drive system and its concerns due to ecological impacts.
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Affiliation(s)
- V Edwin Hillary
- Division of Biotechnology, Entomology Research Institute, Loyola College, University of Madras, Chennai, Tamil Nadu, India
| | - S Antony Ceasar
- Division of Biotechnology, Entomology Research Institute, Loyola College, University of Madras, Chennai, Tamil Nadu, India; Division of Plant Molecular Biology and Biotechnology, Department of Biosciences, Rajagiri College of Social Sciences, Kalamassery, Kochi, India.
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Kwarteng A, Sylverken A, Asiedu E, Ahuno ST. Genome editing as control tool for filarial infections. Biomed Pharmacother 2021; 137:111292. [PMID: 33581654 DOI: 10.1016/j.biopha.2021.111292] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2020] [Revised: 01/11/2021] [Accepted: 01/13/2021] [Indexed: 12/26/2022] Open
Abstract
Human filarial infections are vector-borne nematode infections, which include lymphatic filariasis, onchocerciasis, loiasis, and mansonella filariasis. With a high prevalence in developing countries, filarial infections are responsible for some of the most debilitating morbidities and a vicious cycle of poverty and disease. Global initiatives set to eradicate these infections include community mass treatments, vector control, provision of care for morbidity, and search for vaccines. However, there are growing challenges associated with mass treatments, vector control, and antifilarial vaccine development. With the emergence of genome editing tools and successful applications in other infectious diseases, the integration of genetic editing techniques in future control strategies for filarial infections would offer the best option for eliminating filarial infections. In this review, we briefly discuss the mechanisms of the three main genetic editing techniques and explore the potential applications of these powerful tools to control filarial infections.
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Affiliation(s)
- Alexander Kwarteng
- Department of Biochemistry and Biotechnology, Kwame Nkrumah University of Science and Technology, KNUST, Kumasi, Ghana; Kumasi Centre for Collaborative Research in Tropical Medicine, Kwame Nkrumah University of Science and Technology, KNUST, Kumasi, Ghana.
| | - Augustina Sylverken
- Kumasi Centre for Collaborative Research in Tropical Medicine, Kwame Nkrumah University of Science and Technology, KNUST, Kumasi, Ghana; Department of Theoretical and Applied Biology, Kwame Nkrumah University of Science and Technology, KNUST, Kumasi, Ghana
| | - Ebenezer Asiedu
- Kumasi Centre for Collaborative Research in Tropical Medicine, Kwame Nkrumah University of Science and Technology, KNUST, Kumasi, Ghana
| | - Samuel Terkper Ahuno
- Department of Biochemistry and Biotechnology, Kwame Nkrumah University of Science and Technology, KNUST, Kumasi, Ghana; Kumasi Centre for Collaborative Research in Tropical Medicine, Kwame Nkrumah University of Science and Technology, KNUST, Kumasi, Ghana
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Macias VM, Palatini U, Bonizzoni M, Rasgon JL. Leaning Into the Bite: The piRNA Pathway as an Exemplar for the Genetic Engineering Need in Mosquitoes. Front Cell Infect Microbiol 2021; 10:614342. [PMID: 33520739 PMCID: PMC7840538 DOI: 10.3389/fcimb.2020.614342] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2020] [Accepted: 11/30/2020] [Indexed: 11/16/2022] Open
Abstract
The piRNA pathway is a specialized small RNA interference that in mosquitoes is mechanistically distant from analogous biology in the Drosophila model. Current genetic engineering methods, such as targeted genome manipulation, have a high potential to tease out the functional complexity of this intricate molecular pathway. However, progress in utilizing these methods in arthropod vectors has been geared mostly toward the development of new vector control strategies rather than to study cellular functions. Herein we propose that genetic engineering methods will be essential to uncover the full functionality of PIWI/piRNA biology in mosquitoes and that extending the applications of genetic engineering on other aspects of mosquito biology will grant access to a much larger pool of knowledge in disease vectors that is just out of reach. We discuss motivations for and impediments to expanding the utility of genetic engineering to study the underlying biology and disease transmission and describe specific areas where efforts can be placed to achieve the full potential for genetic engineering in basic biology in mosquito vectors. Such efforts will generate a refreshed intellectual source of novel approaches to disease control and strong support for the effective use of approaches currently in development.
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Affiliation(s)
- Vanessa M. Macias
- Department of Entomology, The Pennsylvania State University, University Park, PA, United States
| | - Umberto Palatini
- Department of Biology and Biotechnology, University of Pavia, Pavia, Italy
| | | | - Jason L. Rasgon
- Department of Entomology, The Pennsylvania State University, University Park, PA, United States
- Huck Institutes of the Life Sciences, The Pennsylvania State University, University Park, PA, United States
- Center for Infectious Disease Dynamics, The Pennsylvania State University, University Park, PA, United States
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Shults P, Cohnstaedt LW, Adelman ZN, Brelsfoard C. Next-generation tools to control biting midge populations and reduce pathogen transmission. Parasit Vectors 2021; 14:31. [PMID: 33413518 PMCID: PMC7788963 DOI: 10.1186/s13071-020-04524-1] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/07/2020] [Accepted: 12/05/2020] [Indexed: 02/06/2023] Open
Abstract
Biting midges of the genus Culicoides transmit disease-causing agents resulting in a significant economic impact on livestock industries in many parts of the world. Localized control efforts, such as removal of larval habitat or pesticide application, can be logistically difficult, expensive and ineffective if not instituted and maintained properly. With these limitations, a population-level approach to the management of Culicoides midges should be investigated as a means to replace or supplement existing control strategies. Next-generation control methods such as Wolbachia- and genetic-based population suppression and replacement are being investigated in several vector species. Here we assess the feasibility and applicability of these approaches for use against biting midges. We also discuss the technical and logistical hurdles needing to be addressed for each method to be successful, as well as emphasize the importance of addressing community engagement and involving stakeholders in the investigation and development of these approaches.
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Affiliation(s)
- Phillip Shults
- Texas A&M University, 370 Olsen Blvd, College Station, TX, 77843, USA.
| | - Lee W Cohnstaedt
- USDA-ARS Arthropod Borne Animal Disease Research Unit, 1515 College Ave, Manhattan, KS, 66502, USA
| | - Zach N Adelman
- Texas A&M University, 370 Olsen Blvd, College Station, TX, 77843, USA
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Kojin BB, Martin-Martin I, Araújo HRC, Bonilla B, Molina-Cruz A, Calvo E, Capurro ML, Adelman ZN. Aedes aegypti SGS1 is critical for Plasmodium gallinaceum infection of both the mosquito midgut and salivary glands. Malar J 2021; 20:11. [PMID: 33407511 PMCID: PMC7787129 DOI: 10.1186/s12936-020-03537-6] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2020] [Accepted: 12/07/2020] [Indexed: 12/13/2022] Open
Abstract
BACKGROUND The invasion of the mosquito salivary glands by Plasmodium sporozoites is a critical step that defines the success of malaria transmission and a detailed understanding of the molecules responsible for salivary gland invasion could be leveraged towards control of vector-borne pathogens. Antibodies directed against the mosquito salivary gland protein SGS1 have been shown to reduce Plasmodium gallinaceum sporozoite invasion of Aedes aegypti salivary glands, but the specific role of this protein in sporozoite invasion and in other stages of the Plasmodium life cycle remains unknown. METHODS RNA interference and CRISPR/Cas9 were used to evaluate the role of A. aegypti SGS1 in the P. gallinaceum life cycle. RESULTS Knockdown and knockout of SGS1 disrupted sporozoite invasion of the salivary gland. Interestingly, mosquitoes lacking SGS1 also displayed fewer oocysts. Proteomic analyses confirmed the abolishment of SGS1 in the salivary gland of SGS1 knockout mosquitoes and revealed that the C-terminus of the protein is absent in the salivary gland of control mosquitoes. In silico analyses indicated that SGS1 contains two potential internal cleavage sites and thus might generate three proteins. CONCLUSION SGS1 facilitates, but is not essential for, invasion of A. aegypti salivary glands by P. gallinaceum and has a dual role as a facilitator of parasite development in the mosquito midgut. SGS1 could, therefore, be part of a strategy to decrease malaria transmission by the mosquito vector, for example in a transgenic mosquito that blocks its interaction with the parasite.
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Affiliation(s)
- Bianca B Kojin
- Department of Entomology and Agrilife Research, Texas A&M University, College Station, TX, USA
| | - Ines Martin-Martin
- Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD, 20852, USA
| | - Helena R C Araújo
- Departamento de Parasitologia, Laboratório de Mosquitos Geneticamente Modificados, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP, 05508-000, Brazil
| | - Brian Bonilla
- Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD, 20852, USA
| | - Alvaro Molina-Cruz
- Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD, 20852, USA
| | - Eric Calvo
- Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD, 20852, USA
| | - Margareth L Capurro
- Departamento de Parasitologia, Laboratório de Mosquitos Geneticamente Modificados, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP, 05508-000, Brazil
| | - Zach N Adelman
- Department of Entomology and Agrilife Research, Texas A&M University, College Station, TX, USA.
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Choo A, Fung E, Chen IY, Saint R, Crisp P, Baxter SW. Precise single base substitution in the shibire gene by CRISPR/Cas9-mediated homology directed repair in Bactrocera tryoni. BMC Genet 2020; 21:127. [PMID: 33339510 PMCID: PMC7747451 DOI: 10.1186/s12863-020-00934-3] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
Background Pest eradication using the Sterile Insect Technique (SIT) involves high-density releases of sterilized males that mate with wild females and ultimately suppress the population. Sterilized females are not required for SIT and their removal or separation from males prior to release remains challenging. In order to develop genetic sexing strains (GSS), conditional traits such as temperature sensitive lethality are required. Results Here we introduce a known Drosophila melanogaster temperature sensitive embryonic lethal mutation into Bactrocera tryoni, a serious horticultural pest in Australia. A non-synonymous point mutation in the D. melanogaster gene shibire causes embryonic lethality at 29 °C and we successfully used CRISPR/Cas9 technology to recreate the orthologous shibire temperature sensitive-1 (shits1) mutation in B. tryoni. Genotypic analyses over three generations revealed that a high fitness cost was associated with the shits1 mutant allele and shits1 homozygotes were not viable at 21 °C, which is a more severe phenotype than that documented in D. melanogaster. Conclusions We have demonstrated the first successful use of CRISPR/Cas9 to introduce precise single base substitutions in an endogenous gene via homology-directed repair in an agricultural pest insect and this technology can be used to trial other conditional mutations for the ultimate aim of generating genetic sexing strains for SIT. Supplementary Information The online version contains supplementary material available at 10.1186/s12863-020-00934-3.
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Affiliation(s)
- Amanda Choo
- School of Biological Sciences, University of Adelaide, Adelaide, SA, Australia.
| | - Elisabeth Fung
- South Australian Research and Development Institute (SARDI), Adelaide, SA, Australia
| | - Isabel Y Chen
- School of Biological Sciences, University of Adelaide, Adelaide, SA, Australia
| | | | - Peter Crisp
- South Australian Research and Development Institute (SARDI), Adelaide, SA, Australia.,School of Agriculture, Food and Wine, University of Adelaide, Adelaide, SA, Australia
| | - Simon W Baxter
- School of BioSciences, University of Melbourne, Melbourne, Australia
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O’Leary S, Adelman ZN. CRISPR/Cas9 knockout of female-biased genes AeAct-4 or myo-fem in Ae. aegypti results in a flightless phenotype in female, but not male mosquitoes. PLoS Negl Trop Dis 2020; 14:e0008971. [PMID: 33338046 PMCID: PMC7781531 DOI: 10.1371/journal.pntd.0008971] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/06/2020] [Revised: 01/04/2021] [Accepted: 11/11/2020] [Indexed: 02/04/2023] Open
Abstract
Aedes aegypti is a vector of dengue, chikungunya, and Zika viruses. Current vector control strategies such as community engagement, source reduction, and insecticides have not been sufficient to prevent viral outbreaks. Thus, interest in novel strategies involving genetic engineering is growing. Female mosquitoes rely on flight to mate with males and obtain a bloodmeal from a host. We hypothesized that knockout of genes specifically expressed in female mosquitoes associated with the indirect flight muscles would result in a flightless female mosquito. Using CRISPR-Cas9 we generated loss-of-function mutations in several genes hypothesized to control flight in mosquitoes, including actin (AeAct-4) and myosin (myo-fem) genes expressed specifically in the female flight muscle. Genetic knockout of these genes resulted in 100% flightless females, with homozygous males able to fly, mate, and produce offspring, albeit at a reduced rate when compared to wild type males. Interestingly, we found that while AeAct-4 was haplosufficient, with most heterozygous individuals capable of flight, this was not the case for myo-fem, where about half of individuals carrying only one intact copy could not fly. These findings lay the groundwork for developing novel mechanisms of controlling Ae. aegypti populations, and our results suggest that this mechanism could be applicable to other vector species of mosquito.
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Affiliation(s)
- Sarah O’Leary
- Department of Entomology, Texas A&M University, College Station, Texas, United States of America
- Interdisciplinary Program in Genetics, Texas A&M University, College Station, Texas, United States of America
| | - Zach N. Adelman
- Department of Entomology, Texas A&M University, College Station, Texas, United States of America
- Interdisciplinary Program in Genetics, Texas A&M University, College Station, Texas, United States of America
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EFSA Panel on Genetically Modified Organisms (GMO), Naegeli H, Bresson J, Dalmay T, Dewhurst IC, Epstein MM, Guerche P, Hejatko J, Moreno FJ, Mullins E, Nogué F, Rostoks N, Sánchez Serrano JJ, Savoini G, Veromann E, Veronesi F, Bonsall MB, Mumford J, Wimmer EA, Devos Y, Paraskevopoulos K, Firbank LG. Adequacy and sufficiency evaluation of existing EFSA guidelines for the molecular characterisation, environmental risk assessment and post-market environmental monitoring of genetically modified insects containing engineered gene drives. EFSA J 2020; 18:e06297. [PMID: 33209154 PMCID: PMC7658669 DOI: 10.2903/j.efsa.2020.6297] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
Advances in molecular and synthetic biology are enabling the engineering of gene drives in insects for disease vector/pest control. Engineered gene drives (that bias their own inheritance) can be designed either to suppress interbreeding target populations or modify them with a new genotype. Depending on the engineered gene drive system, theoretically, a genetic modification of interest could spread through target populations and persist indefinitely, or be restricted in its spread or persistence. While research on engineered gene drives and their applications in insects is advancing at a fast pace, it will take several years for technological developments to move to practical applications for deliberate release into the environment. Some gene drive modified insects (GDMIs) have been tested experimentally in the laboratory, but none has been assessed in small-scale confined field trials or in open release trials as yet. There is concern that the deliberate release of GDMIs in the environment may have possible irreversible and unintended consequences. As a proactive measure, the European Food Safety Authority (EFSA) has been requested by the European Commission to review whether its previously published guidelines for the risk assessment of genetically modified animals (EFSA, 2012 and 2013), including insects (GMIs), are adequate and sufficient for GDMIs, primarily disease vectors, agricultural pests and invasive species, for deliberate release into the environment. Under this mandate, EFSA was not requested to develop risk assessment guidelines for GDMIs. In this Scientific Opinion, the Panel on Genetically Modified Organisms (GMO) concludes that EFSA's guidelines are adequate, but insufficient for the molecular characterisation (MC), environmental risk assessment (ERA) and post-market environmental monitoring (PMEM) of GDMIs. While the MC,ERA and PMEM of GDMIs can build on the existing risk assessment framework for GMIs that do not contain engineered gene drives, there are specific areas where further guidance is needed for GDMIs.
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The Antiviral Small-Interfering RNA Pathway Induces Zika Virus Resistance in Transgenic Aedes aegypti. Viruses 2020; 12:v12111231. [PMID: 33142991 PMCID: PMC7692394 DOI: 10.3390/v12111231] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2020] [Revised: 10/27/2020] [Accepted: 10/28/2020] [Indexed: 01/21/2023] Open
Abstract
The resurgence of arbovirus outbreaks across the globe, including the recent Zika virus (ZIKV) epidemic in 2015–2016, emphasizes the need for innovative vector control methods. In this study, we investigated ZIKV susceptibility to transgenic Aedes aegypti engineered to target the virus by means of the antiviral small-interfering RNA (siRNA) pathway. The robustness of antiviral effector expression in transgenic mosquitoes is strongly influenced by the genomic insertion locus and transgene copy number; we therefore used CRISPR/Cas9 to re-target a previously characterized locus (Chr2:321382225) and engineered mosquitoes expressing an inverted repeat (IR) dsRNA against the NS3/4A region of the ZIKV genome. Small RNA analysis revealed that the IR effector triggered the mosquito’s siRNA antiviral pathway in bloodfed females. Nearly complete (90%) inhibition of ZIKV replication was found in vivo in both midguts and carcasses at 7 or 14 days post-infection (dpi). Furthermore, significantly fewer transgenic mosquitoes contained ZIKV in their salivary glands (p = 0.001), which led to a reduction in the number of ZIKV-containing saliva samples as measured by transmission assay. Our work shows that Ae. aegypti innate immunity can be co-opted to engineer mosquitoes resistant to ZIKV.
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Nix alone is sufficient to convert female Aedes aegypti into fertile males and myo-sex is needed for male flight. Proc Natl Acad Sci U S A 2020; 117:17702-17709. [PMID: 32661163 PMCID: PMC7395513 DOI: 10.1073/pnas.2001132117] [Citation(s) in RCA: 37] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/02/2022] Open
Abstract
The presence of a dominant male-determining locus (M-locus) in one of a pair of autosomes establishes the male sex in the dengue fever mosquito Aedes aegypti. The Ae. aegypti M-locus contains 30 genes, including Nix, a previously reported male-determining factor. Here we show that the Nix transgene alone was sufficient to convert females into fertile males, which continued to produce sex-converted progeny. We also show that a second M-locus gene named myo-sex was needed for male flight. Nix-mediated sex conversion was 100% penetrant, heritable, and stable, indicating great potential for developing mosquito-control strategies to reduce vector populations by female-to-male conversion. This work also sheds lights into the molecular basis of the function of the M-locus. A dominant male-determining locus (M-locus) establishes the male sex (M/m) in the yellow fever mosquito, Aedes aegypti. Nix, a gene in the M-locus, was shown to be a male-determining factor (M factor) as somatic knockout of Nix led to feminized males (M/m) while transient expression of Nix resulted in partially masculinized females (m/m), with male reproductive organs but retained female antennae. It was not clear whether any of the other 29 genes in the 1.3-Mb M-locus are also needed for complete sex-conversion. Here, we report the generation of multiple transgenic lines that express Nix under the control of its own promoter. Genetic and molecular analyses of these lines provided insights unattainable from previous transient experiments. We show that the Nix transgene alone, in the absence of the M-locus, was sufficient to convert females into males with all male-specific sexually dimorphic features and male-like gene expression. The converted m/m males are flightless, unable to perform the nuptial flight required for mating. However, they were able to father sex-converted progeny when presented with cold-anesthetized wild-type females. We show that myo-sex, a myosin heavy-chain gene also in the M-locus, was required for male flight as knockout of myo-sex rendered wild-type males flightless. We also show that Nix-mediated female-to-male conversion was 100% penetrant and stable over many generations. Therefore, Nix has great potential for developing mosquito control strategies to reduce vector populations by female-to-male sex conversion, or to aid in a sterile insect technique that requires releasing only non-biting males.
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Cas9-Mediated Gene-Editing in the Malaria Mosquito Anopheles stephensi by ReMOT Control. G3-GENES GENOMES GENETICS 2020; 10:1353-1360. [PMID: 32122959 PMCID: PMC7144067 DOI: 10.1534/g3.120.401133] [Citation(s) in RCA: 45] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 01/14/2023]
Abstract
Innovative tools are essential for advancing malaria control and depend on an understanding of molecular mechanisms governing transmission of malaria parasites by Anopheles mosquitoes. CRISPR/Cas9-based gene disruption is a powerful method to uncover underlying biology of vector-pathogen interactions and can itself form the basis of mosquito control strategies. However, embryo injection methods used to genetically manipulate mosquitoes (especially Anopheles) are difficult and inefficient, particularly for non-specialist laboratories. Here, we adapted the ReMOT Control (Receptor-mediated Ovary Transduction of Cargo) technique to deliver Cas9 ribonucleoprotein complex to adult mosquito ovaries, generating targeted and heritable mutations in the malaria vector Anopheles stephensi without injecting embryos. In Anopheles, ReMOT Control gene editing was as efficient as standard embryo injections. The application of ReMOT Control to Anopheles opens the power of CRISPR/Cas9 methods to malaria laboratories that lack the equipment or expertise to perform embryo injections and establishes the flexibility of ReMOT Control for diverse mosquito species.
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Wang Y, Gao B, Zhang G, Qi X, Cao S, Akami M, Huang Y, Niu C. Mutation of Bdpaired induces embryo lethality in the oriental fruit fly, Bactrocera dorsalis. PEST MANAGEMENT SCIENCE 2020; 76:944-951. [PMID: 31461218 DOI: 10.1002/ps.5602] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/03/2019] [Revised: 06/10/2019] [Accepted: 08/27/2019] [Indexed: 06/10/2023]
Abstract
BACKGROUND Pair-rule genes were identified and named for their role in segmentation in animal embryos. Paired, a homolog of mammalian PAX3, acts as one of several pair-rule genes and is key in defining the boundaries of future parasegments and segments during insect embryogenesis. RESULTS We cloned the paired gene from the oriental fruit fly, Bactrocera dorsalis, and then applied CRISPR/Cas9-mediated genome editing to investigate its physiological function in the embryonic stage of this pest. We identified one transcript for a paired homolog in B. dorsalis, which encodes a protein containing a Paired Box domain and a Homeobox domain. Phylogenetic analysis confirmed that the paired gene is highly conserved and the gene was highly expressed at the 12-14 h-old embryonic stage. Knock-out of Bdpaired led to lack of segment boundaries, cuticular deficiency, and embryonic lethality. Sequence analysis of the CRISPR/Cas9 mutants exhibited different insertion and deletions in the Bdpaired locus. In addition, the relative expression of Wingless (Wg) and Abdominal A (Abd-A) genes were significantly down-regulated in the Bdpaired mutant embryos. CONCLUSION These results indicate that Bdpaired gene is critical for the embryonic development of B. dorsalis, and could be a novel molecular target for genetic-based pest management practices to combat this serious invasive pest. © 2019 Society of Chemical Industry.
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Affiliation(s)
- Yaohui Wang
- Hubei Key Laboratory of Insect Resource Application and Sustainable Pest Control, College of Plant Science & Technology, Huazhong Agricultural University, Wuhan, China
| | - Bingli Gao
- Hubei Key Laboratory of Insect Resource Application and Sustainable Pest Control, College of Plant Science & Technology, Huazhong Agricultural University, Wuhan, China
| | - Guijian Zhang
- Hubei Key Laboratory of Insect Resource Application and Sustainable Pest Control, College of Plant Science & Technology, Huazhong Agricultural University, Wuhan, China
| | - Xuewei Qi
- Hubei Key Laboratory of Insect Resource Application and Sustainable Pest Control, College of Plant Science & Technology, Huazhong Agricultural University, Wuhan, China
| | - Shuai Cao
- Hubei Key Laboratory of Insect Resource Application and Sustainable Pest Control, College of Plant Science & Technology, Huazhong Agricultural University, Wuhan, China
| | - Mazarin Akami
- Hubei Key Laboratory of Insect Resource Application and Sustainable Pest Control, College of Plant Science & Technology, Huazhong Agricultural University, Wuhan, China
| | - Yongping Huang
- CAS Key Laboratory of Insect Developmental and Evolutionary Biology, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Shanghai, China
| | - Changying Niu
- Hubei Key Laboratory of Insect Resource Application and Sustainable Pest Control, College of Plant Science & Technology, Huazhong Agricultural University, Wuhan, China
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Matthews BJ, Vosshall LB. How to turn an organism into a model organism in 10 'easy' steps. ACTA ACUST UNITED AC 2020; 223:223/Suppl_1/jeb218198. [PMID: 32034051 DOI: 10.1242/jeb.218198] [Citation(s) in RCA: 68] [Impact Index Per Article: 13.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023]
Abstract
Many of the major biological discoveries of the 20th century were made using just six species: Escherichia coli bacteria, Saccharomyces cerevisiae and Schizosaccharomyces pombe yeast, Caenorhabditis elegans nematodes, Drosophila melanogaster flies and Mus musculus mice. Our molecular understanding of the cell division cycle, embryonic development, biological clocks and metabolism were all obtained through genetic analysis using these species. Yet the 'big 6' did not start out as genetic model organisms (hereafter 'model organisms'), so how did they mature into such powerful systems? First, these model organisms are abundant human commensals: they are the bacteria in our gut, the yeast in our beer and bread, the nematodes in our compost pile, the flies in our kitchen and the mice in our walls. Because of this, they are cheaply, easily and rapidly bred in the laboratory and in addition were amenable to genetic analysis. How and why should we add additional species to this roster? We argue that specialist species will reveal new secrets in important areas of biology and that with modern technological innovations like next-generation sequencing and CRISPR-Cas9 genome editing, the time is ripe to move beyond the big 6. In this review, we chart a 10-step path to this goal, using our own experience with the Aedes aegypti mosquito, which we built into a model organism for neurobiology in one decade. Insights into the biology of this deadly disease vector require that we work with the mosquito itself rather than modeling its biology in another species.
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Affiliation(s)
- Benjamin J Matthews
- Department of Zoology, The University of British Columbia, Vancouver, BC, Canada, V6T 1Z4
| | - Leslie B Vosshall
- Laboratory of Neurogenetics and Behavior, The Rockefeller University, New York, NY 10065, USA.,Howard Hughes Medical Institute, New York, NY 10065, USA.,Kavli Neural Systems Institute, New York, NY 10065, USA
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A mosquito juvenile hormone binding protein (mJHBP) regulates the activation of innate immune defenses and hemocyte development. PLoS Pathog 2020; 16:e1008288. [PMID: 31961911 PMCID: PMC6994123 DOI: 10.1371/journal.ppat.1008288] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/25/2019] [Revised: 01/31/2020] [Accepted: 12/17/2019] [Indexed: 11/29/2022] Open
Abstract
Insects rely on the innate immune system for defense against pathogens, some aspects of which are under hormonal control. Here we provide direct experimental evidence showing that the juvenile hormone-binding protein (mJHBP) of Aedes aegypti is required for the regulation of innate immune responses and the development of mosquito blood cells (hemocytes). Using an mJHBP-deficient mosquito line generated by means of CRISPR-Cas9 gene editing technology we uncovered a mutant phenotype characterized by immunosuppression at the humoral and cellular levels, which profoundly affected susceptibility to bacterial infection. Bacteria-challenged mosquitoes exhibited significantly higher levels of septicemia and mortality relative to the wild type (WT) strain, delayed expression of antimicrobial peptides (AMPs), severe developmental dysregulation of embryonic and larval hemocytes (reduction in the total number of hemocytes) and increased differentiation of the granulocyte lineage. Interestingly, injection of recombinant wild type mJHBP protein into adult females three-days before infection was sufficient to restore normal immune function. Similarly, injection of mJHBP into fourth-instar larvae fully restored normal larval/pupal hemocyte populations in emerging adults. More importantly, the recovery of normal immuno-activation and hemocyte development requires the capability of mJHBP to bind JH III. These results strongly suggest that JH III functions in mosquito immunity and hemocyte development in a manner that is perhaps independent of canonical JH signaling, given the lack of developmental and reproductive abnormalities. Because of the prominent role of hemocytes as regulators of mosquito immunity, this novel discovery may have broader implications for the understanding of vector endocrinology, hemocyte development, vector competence and disease transmission. There are many unanswered questions concerning the nature of immune responses of mosquitoes to bacteria, viruses and parasites. This is important because a variety of human pathogens are transmitted by mosquitoes during the process of consuming blood. Much of mosquito physiology is under the control of hormones and we aim to understand a potential role for an important hormone known as juvenile hormone in anti-bacterial immunity. We have produced a strain of the mosquito, Aedes aegypti, that is deficient in the production of a protein that circulates in the blood while carrying juvenile hormone. This strain is shown to have less ability to control bacterial infection, to have lower levels of proteins involved in immunity and to have smaller numbers of blood cells that are known to be important in the mosquito immune response. If the protein is administered to the deficient strain by injection, the immune response and blood cell numbers return to near-normal levels. Other results suggest that the association of the protein with juvenile hormone is important for its ability to function in the immune system. Overall, this study describes an important new protein regulator of mosquito immunity and a potential role of juvenile hormone in this process.
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Antiviral Effectors and Gene Drive Strategies for Mosquito Population Suppression or Replacement to Mitigate Arbovirus Transmission by Aedes aegypti. INSECTS 2020; 11:insects11010052. [PMID: 31940960 PMCID: PMC7023000 DOI: 10.3390/insects11010052] [Citation(s) in RCA: 17] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 12/12/2019] [Revised: 01/07/2020] [Accepted: 01/09/2020] [Indexed: 12/11/2022]
Abstract
The mosquito vector Aedes aegypti transmits arthropod-borne viruses (arboviruses) of medical importance, including Zika, dengue, and yellow fever viruses. Controlling mosquito populations remains the method of choice to prevent disease transmission. Novel mosquito control strategies based on genetically manipulating mosquitoes are being developed as additional tools to combat arbovirus transmission. Genetic control of mosquitoes includes two basic strategies: population suppression and population replacement. The former aims to eliminate mosquito populations while the latter aims to replace wild populations with engineered, pathogen-resistant mosquitoes. In this review, we outline suppression strategies being applied in the field, as well as current antiviral effector genes that have been characterized and expressed in transgenic Ae. aegypti for population replacement. We discuss cutting-edge gene drive technologies that can be used to enhance the inheritance of effector genes, while highlighting the challenges and opportunities associated with gene drives. Finally, we present currently available models that can estimate mosquito release numbers and time to transgene fixation for several gene drive systems. Based on the recent advances in genetic engineering, we anticipate that antiviral transgenic Ae. aegypti exhibiting gene drive will soon emerge; however, close monitoring in simulated field conditions will be required to demonstrate the efficacy and utility of such transgenic mosquitoes.
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Xu X, Wang Y, Bi H, Xu J, Liu Z, Niu C, He L, James AA, Li K, Huang Y. Mutation of the seminal protease gene, serine protease 2, results in male sterility in diverse lepidopterans. INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY 2020; 116:103243. [PMID: 31541694 DOI: 10.1016/j.ibmb.2019.103243] [Citation(s) in RCA: 25] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/28/2019] [Revised: 09/04/2019] [Accepted: 09/14/2019] [Indexed: 06/10/2023]
Abstract
Sterile insect technology (SIT) is an environmentally friendly method for pest control. As part of our efforts to develop a strategy that results in engineered male-sterile strains with minimum effects on viability and mating competition, we used CRISPR/Cas9 technology to disrupt Ser2, which encodes a seminal fluid protein, in the model lepidopteran insect, Bombyx mori, and an important agricultural pest, Plutella xylostella. Disruption of Ser2 resulted in dominant heritable male sterility. Wild-type females mated with Ser2-deficient males laid eggs normally, but the eggs did not hatch. We detected no differences in other reproductive behaviors in the mutant males. These results support the conclusion that Ser2 gene is necessary for male reproductive success in diverse lepidopterans. Targeting Ser2 gene has the potential to form the basis for a new strategy for pest control.
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Affiliation(s)
- Xia Xu
- School of Life Science, East China Normal University, 200241, Shanghai, China; Key Laboratory of Insect Developmental and Evolutionary Biology, Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, 200032, Shanghai, China
| | - Yaohui Wang
- Key Laboratory of Insect Developmental and Evolutionary Biology, Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, 200032, Shanghai, China; Hubei Key Laboratory of Insect Resource Application and Sustainable Pest Control, College of Plant Science & Technology, Huazhong Agricultural University, 430070, Wuhan, China
| | - Honglun Bi
- School of Life Science, East China Normal University, 200241, Shanghai, China; Key Laboratory of Insect Developmental and Evolutionary Biology, Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, 200032, Shanghai, China
| | - Jun Xu
- Key Laboratory of Insect Developmental and Evolutionary Biology, Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, 200032, Shanghai, China
| | - Zulian Liu
- Key Laboratory of Insect Developmental and Evolutionary Biology, Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, 200032, Shanghai, China
| | - Changying Niu
- Hubei Key Laboratory of Insect Resource Application and Sustainable Pest Control, College of Plant Science & Technology, Huazhong Agricultural University, 430070, Wuhan, China
| | - Lin He
- School of Life Science, East China Normal University, 200241, Shanghai, China.
| | - Anthony A James
- Department of Microbiology & Molecular Genetics and Molecular Biology & Biochemistry, University of California, Irvine, CA, 92697-3900, USA
| | - Kai Li
- School of Life Science, East China Normal University, 200241, Shanghai, China.
| | - Yongping Huang
- Key Laboratory of Insect Developmental and Evolutionary Biology, Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, 200032, Shanghai, China.
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Liu T, Yang WQ, Xie YG, Liu PW, Xie LH, Lin F, Li CY, Gu JB, Wu K, Yan GY, Chen XG. Construction of an efficient genomic editing system with CRISPR/Cas9 in the vector mosquito Aedes albopictus. INSECT SCIENCE 2019; 26:1045-1054. [PMID: 30311353 DOI: 10.1111/1744-7917.12645] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/31/2018] [Revised: 09/04/2018] [Accepted: 09/17/2018] [Indexed: 06/08/2023]
Abstract
Aedes (Stegomyia) albopictus, also known as the Asian tiger mosquito, is a mosquito which originated in Asia. In recent years, it has become increasingly rampant throughout the world. This mosquito can transmit several arboviruses, including dengue, Zika and chikungunya viruses, and is considered a public health threat. Despite the urgent need of genome engineering to analyze specific gene functions, progress in genetical manipulation of Ae. albopictus has been slow due to a lack of efficient methods and genetic markers. In the present study, we established targeted disruptions in two genes, kynurenine hydroxylase (kh) and dopachrome conversion enzyme (yellow), to analyze the feasibility of generating visible phenotypes with genome editing by the clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated protein 9 (Cas9) system in Ae. albopictus. Following Cas9 single guide RNA ribonucleoprotein injection into the posterior end of pre-blastoderm embryos, 30%-50% of fertile survivors produced alleles that failed to complement existing kh and yellow mutations. Complete eye and body pigmentation defects were readily observed in G1 pupae and adults, indicating successful generation of highly heritable mutations. We conclude that the CRISPR/Cas9-mediated gene editing system can be used in Ae. albopictus and that it can be adopted as an efficient tool for genome-scale analysis and biological study.
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Affiliation(s)
- Tong Liu
- Department of Pathogen Biology, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health, Southern Medical University, Guangzhou, China
| | - Wen-Qiang Yang
- Department of Pathogen Biology, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health, Southern Medical University, Guangzhou, China
| | - Yu-Gu Xie
- Department of Pathogen Biology, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health, Southern Medical University, Guangzhou, China
| | - Pei-Wen Liu
- Department of Pathogen Biology, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health, Southern Medical University, Guangzhou, China
| | - Li-Hua Xie
- Department of Pathogen Biology, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health, Southern Medical University, Guangzhou, China
| | - Feng Lin
- Department of Pathogen Biology, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health, Southern Medical University, Guangzhou, China
| | - Chen-Ying Li
- Department of Pathogen Biology, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health, Southern Medical University, Guangzhou, China
| | - Jin-Bao Gu
- Department of Pathogen Biology, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health, Southern Medical University, Guangzhou, China
| | - Kun Wu
- Department of Pathogen Biology, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health, Southern Medical University, Guangzhou, China
| | - Gui-Yun Yan
- Department of Pathogen Biology, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health, Southern Medical University, Guangzhou, China
| | - Xiao-Guang Chen
- Department of Pathogen Biology, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health, Southern Medical University, Guangzhou, China
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Mota MBS, Carvalho MA, Monteiro ANA, Mesquita RD. DNA damage response and repair in perspective: Aedes aegypti, Drosophila melanogaster and Homo sapiens. Parasit Vectors 2019; 12:533. [PMID: 31711518 PMCID: PMC6849265 DOI: 10.1186/s13071-019-3792-1] [Citation(s) in RCA: 29] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/17/2019] [Accepted: 11/05/2019] [Indexed: 01/18/2023] Open
Abstract
Background The maintenance of genomic integrity is the responsibility of a complex network, denominated the DNA damage response (DDR), which controls the lesion detection and DNA repair. The main repair pathways are base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), homologous recombination repair (HR) and non-homologous end joining repair (NHEJ). They correct double-strand breaks (DSB), single-strand breaks, mismatches and others, or when the damage is quite extensive and repair insufficient, apoptosis is activated. Methods In this study we used the BLAST reciprocal best-hit methodology to search for DDR orthologs proteins in Aedes aegypti. We also provided a comparison between Ae. aegypti, D. melanogaster and human DDR network. Results Our analysis revealed the presence of ATR and ATM signaling, including the H2AX ortholog, in Ae. aegypti. Key DDR proteins (orthologs to RAD51, Ku and MRN complexes, XP-components, MutS and MutL) were also identified in this insect. Other proteins were not identified in both Ae. aegypti and D. melanogaster, including BRCA1 and its partners from BRCA1-A complex, TP53BP1, PALB2, POLk, CSA, CSB and POLβ. In humans, their absence affects DSB signaling, HR and sub-pathways of NER and BER. Seven orthologs not known in D. melanogaster were found in Ae. aegypti (RNF168, RIF1, WRN, RAD54B, RMI1, DNAPKcs, ARTEMIS). Conclusions The presence of key DDR proteins in Ae. aegypti suggests that the main DDR pathways are functional in this insect, and the identification of proteins not known in D. melanogaster can help fill gaps in the DDR network. The mapping of the DDR network in Ae. aegypti can support mosquito biology studies and inform genetic manipulation approaches applied to this vector.
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Affiliation(s)
- Maria Beatriz S Mota
- Departamento de Bioquímica, Instituto de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
| | - Marcelo Alex Carvalho
- Instituto Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil.,Instituto Nacional de Câncer, Divisão de Pesquisa Clínica, Rio de Janeiro, RJ, Brazil
| | - Alvaro N A Monteiro
- Cancer Epidemiology Program, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA
| | - Rafael D Mesquita
- Departamento de Bioquímica, Instituto de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil. .,Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil.
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