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      Knockdown resistance allele L1014F introduced by CRISPR/Cas9 is not associated with altered vector competence of Anopheles gambiae for o’nyong nyong virus

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          Abstract

          Knockdown resistance ( kdr) alleles conferring resistance to pyrethroid insecticides are widespread amongst vector populations. Previous research has suggested that these alleles are associated with changes in the vector competence of mosquitoes for arboviruses and Plasmodium, however non-target genetic differences between mosquito strains may have had a confounding effect. Here, to minimise genetic differences, the laboratory Anopheles gambiae Kisumu strain was compared to a CRISPR/Cas9 homozygous kdr L1014F mutant Kisumu- kdr line in order to examine associations with vector competence for o’nyong nyong virus (ONNV). Mosquitoes were infected using either blood feeds or intrathoracic microinjections. There were no significant differences in the prevalence of virus in mosquito body parts between kdr mutant and wildtype lines from either oral or intrathoracic injection routes. The ONNV titre was significantly higher in the legs of the wildtype strain at 7dpi following intrathoracic microinjection, but no other significant differences in viral titre were detected. ONNV was not detected in the saliva of mosquitoes from either strain. Our findings from per os infections suggest that the kdr L1014F allele is not associated with altered infection prevalence for ONNV, a key component of vector competence.

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          Off-target Effects in CRISPR/Cas9-mediated Genome Engineering

          Xiao-hui Zhang, Louis Y Tee, Xiao-Gang Wang … (2015)
          CRISPR/Cas9 is a versatile genome-editing technology that is widely used for studying the functionality of genetic elements, creating genetically modified organisms as well as preclinical research of genetic disorders. However, the high frequency of off-target activity (≥50%)—RGEN (RNA-guided endonuclease)-induced mutations at sites other than the intended on-target site—is one major concern, especially for therapeutic and clinical applications. Here, we review the basic mechanisms underlying off-target cutting in the CRISPR/Cas9 system, methods for detecting off-target mutations, and strategies for minimizing off-target cleavage. The improvement off-target specificity in the CRISPR/Cas9 system will provide solid genotype–phenotype correlations, and thus enable faithful interpretation of genome-editing data, which will certainly facilitate the basic and clinical application of this technology.
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            Molecular biology of insect sodium channels and pyrethroid resistance.

            Ke Dong, Yuzhe Du, Frank D Rinkevich … (2014)
            Voltage-gated sodium channels are essential for the initiation and propagation of the action potential in neurons and other excitable cells. Because of their critical roles in electrical signaling, sodium channels are targets of a variety of naturally occurring and synthetic neurotoxins, including several classes of insecticides. This review is intended to provide an update on the molecular biology of insect sodium channels and the molecular mechanism of pyrethroid resistance. Although mammalian and insect sodium channels share fundamental topological and functional properties, most insect species carry only one sodium channel gene, compared to multiple sodium channel genes found in each mammalian species. Recent studies showed that two posttranscriptional mechanisms, alternative splicing and RNA editing, are involved in generating functional diversity of sodium channels in insects. More than 50 sodium channel mutations have been identified to be responsible for or associated with knockdown resistance (kdr) to pyrethroids in various arthropod pests and disease vectors. Elucidation of molecular mechanism of kdr led to the identification of dual receptor sites of pyrethroids on insect sodium channels. Many of the kdr mutations appear to be located within or close to the two receptor sites. The accumulating knowledge of insect sodium channels and their interactions with insecticides provides a foundation for understanding the neurophysiology of sodium channels in vivo and the development of new and safer insecticides for effective control of arthropod pests and human disease vectors.
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              Aedes aegypti vector competence studies: A review

              Jayme Souza-Neto, Jeffrey R. Powell, Mariangela Bonizzoni (2018)
              Aedes aegypti is the primary transmitter of the four viruses that have had the greatest impact on human health, the viruses causing yellow fever, dengue fever, chikungunya, and Zika fever. Because this mosquito is easy to rear in the laboratory and these viruses grow in laboratory tissue culture cells, many studies have been performed testing the relative competence of different populations of the mosquito to transmit many different strains of viruses. We review here this large literature including studies on the effect of the mosquito microbiota on competence. Because of the heterogeneity of both mosquito populations and virus strains used, as well as methods measuring potential to transmit, it is very difficult to perform detailed meta-analysis of the studies. However, a few conclusions can be drawn: (1) almost no population of Ae. aegypti is 100% naturally refractory to virus infection. Complete susceptibility to infection has been observed for Zika (ZIKV), dengue (DENV) and chikungunya (CHIKV), but not yellow fever viruses (YFV); (2) the dose of virus used is directly correlated to the rate of infection; (3) Brazilian populations of mosquito are particularly susceptible to DENV-2 infections; (4) the Asian lineage of ZIKV is less infective to Ae. aegypti populations from the American continent than is the African ZIKV lineage; (5) virus adaptation to different species of mosquitoes has been demonstrated with CHIKV; (6) co-infection with more than one virus sometimes causes displacement while in other cases has little effect; (7) the microbiota in the mosquito also has important effects on level of susceptibility to arboviral infection; (8) resistance to virus infection due to the microbiota may be direct (e.g., bacteria producing antiviral proteins) or indirect in activating the mosquito host innate immune system; (9) non-pathogenic insect specific viruses (ISVs) are also common in mosquitoes including genome insertions. These too have been shown to have an impact on the susceptibility of mosquitoes to pathogenic viruses. One clear conclusion is that it would be a great advance in this type of research to implement standardized procedures in order to obtain comparable and reproducible results.
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                Author and article information

                Contributors
                Grant A. Kay: Role: ConceptualizationRole: Formal analysisRole: InvestigationRole: Writing – original draft
                Edward I. Patterson: Role: Methodology
                Grant L. Hughes:
                ORCID: https://orcid.org/0000-0002-7567-7185
                Role: ResourcesRole: Writing – review & editing
                Jennifer S. Lord: Role: Formal analysisRole: Writing – review & editing
                Lisa J. Reimer:
                ORCID: https://orcid.org/0000-0002-9711-4981
                Role: ConceptualizationRole: ResourcesRole: SupervisionRole: Writing – review & editing
                Pierre Roques: Role: Editor
                Journal
                Journal ID (nlm-ta): PLoS One
                Journal ID (iso-abbrev): PLoS One
                Journal ID (publisher-id): plos
                Title: PLOS ONE
                Publisher: Public Library of Science (San Francisco, CA USA )
                ISSN (Electronic): 1932-6203
                Publication date (Electronic): 10 August 2023
                Publication date Collection: 2023
                Volume: 18
                Issue: 8
                Electronic Location Identifier: e0288994
                Affiliations
                [1 ] Department of Vector Biology, Liverpool School of Tropical Medicine, Liverpool, United Kingdom
                [2 ] Biological Sciences, Brock University, St. Catharines, Ontario, Canada
                CEA, FRANCE
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Author information
                https://orcid.org/0000-0002-7567-7185
                https://orcid.org/0000-0002-9711-4981
                Article
                Publisher ID: PONE-D-23-03395
                DOI: 10.1371/journal.pone.0288994
                PMC ID: 10414658
                SO-VID: 2db61431-5044-4c48-bce4-07be61ee587e
                Copyright © © 2023 Kay et al
                License:

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                Date received : 6 February 2023
                Date accepted : 9 July 2023
                Page count
                Figures: 3, Tables: 0, Pages: 12
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100000265, Medical Research Council;
                Award ID: N013514
                Award Recipient :
                GK was supported by the Medical Research Council Doctoral Training Programme at LSTM (grant number N013514/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Subject: Research Article
                Subject: Medicine and Health Sciences
                Subject: Medical Conditions
                Subject: Infectious Diseases
                Subject: Disease Vectors
                Subject: Insect Vectors
                Subject: Mosquitoes
                Subject: Biology and Life Sciences
                Subject: Species Interactions
                Subject: Disease Vectors
                Subject: Insect Vectors
                Subject: Mosquitoes
                Subject: Biology and Life Sciences
                Subject: Zoology
                Subject: Entomology
                Subject: Insects
                Subject: Mosquitoes
                Subject: Biology and Life Sciences
                Subject: Organisms
                Subject: Eukaryota
                Subject: Animals
                Subject: Invertebrates
                Subject: Arthropoda
                Subject: Insects
                Subject: Mosquitoes
                Subject: Biology and Life Sciences
                Subject: Zoology
                Subject: Animals
                Subject: Invertebrates
                Subject: Arthropoda
                Subject: Insects
                Subject: Mosquitoes
                Subject: Biology and Life Sciences
                Subject: Agriculture
                Subject: Agrochemicals
                Subject: Insecticides
                Subject: Biology and life sciences
                Subject: Organisms
                Subject: Viruses
                Subject: RNA viruses
                Subject: Togaviruses
                Subject: Alphaviruses
                Subject: O'Nyong'Nyong Virus
                Subject: Biology and Life Sciences
                Subject: Microbiology
                Subject: Medical Microbiology
                Subject: Microbial Pathogens
                Subject: Viral Pathogens
                Subject: Togaviruses
                Subject: Alphaviruses
                Subject: O'Nyong'Nyong Virus
                Subject: Medicine and Health Sciences
                Subject: Pathology and Laboratory Medicine
                Subject: Pathogens
                Subject: Microbial Pathogens
                Subject: Viral Pathogens
                Subject: Togaviruses
                Subject: Alphaviruses
                Subject: O'Nyong'Nyong Virus
                Subject: Biology and Life Sciences
                Subject: Organisms
                Subject: Viruses
                Subject: Viral Pathogens
                Subject: Togaviruses
                Subject: Alphaviruses
                Subject: O'Nyong'Nyong Virus
                Subject: Biology and Life Sciences
                Subject: Anatomy
                Subject: Body Fluids
                Subject: Saliva
                Subject: Medicine and Health Sciences
                Subject: Anatomy
                Subject: Body Fluids
                Subject: Saliva
                Subject: Biology and Life Sciences
                Subject: Physiology
                Subject: Body Fluids
                Subject: Saliva
                Subject: Medicine and Health Sciences
                Subject: Medical Conditions
                Subject: Infectious Diseases
                Subject: Viral Diseases
                Subject: Arboviral Infections
                Subject: Biology and Life Sciences
                Subject: Anatomy
                Subject: Body Fluids
                Subject: Blood
                Subject: Medicine and Health Sciences
                Subject: Anatomy
                Subject: Body Fluids
                Subject: Blood
                Subject: Biology and Life Sciences
                Subject: Physiology
                Subject: Body Fluids
                Subject: Blood
                Subject: Medicine and Health Sciences
                Subject: Medical Conditions
                Subject: Infectious Diseases
                Subject: Vector-Borne Diseases
                Subject: Medicine and Health Sciences
                Subject: Medical Conditions
                Subject: Infectious Diseases
                Subject: Disease Vectors
                Subject: Insect Vectors
                Subject: Mosquitoes
                Subject: Anopheles Gambiae
                Subject: Biology and Life Sciences
                Subject: Species Interactions
                Subject: Disease Vectors
                Subject: Insect Vectors
                Subject: Mosquitoes
                Subject: Anopheles Gambiae
                Subject: Biology and Life Sciences
                Subject: Zoology
                Subject: Entomology
                Subject: Insects
                Subject: Mosquitoes
                Subject: Anopheles Gambiae
                Subject: Biology and Life Sciences
                Subject: Organisms
                Subject: Eukaryota
                Subject: Animals
                Subject: Invertebrates
                Subject: Arthropoda
                Subject: Insects
                Subject: Mosquitoes
                Subject: Anopheles Gambiae
                Subject: Biology and Life Sciences
                Subject: Zoology
                Subject: Animals
                Subject: Invertebrates
                Subject: Arthropoda
                Subject: Insects
                Subject: Mosquitoes
                Subject: Anopheles Gambiae
                Custom metadata
                Data Availability Data are available in Open Science Framework: https://doi.org/10.17605/OSF.IO/3NFR7.

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