Cas9-mediated gene-editing in the malaria mosquito Anopheles stephensi by ReMOT Control

AbstractInnovative tool development is essential for continued advancement in malaria control and depends on a deeper understanding of the molecular mechanisms that govern transmission of malaria parasites by Anopheles mosquitoes. Targeted disruption of genes in mosquito vectors is a powerful method to uncover the underlying biology of vector-pathogen interactions, and genome manipulation technologies can themselves form the basis of mosquito and pathogen control strategies. However, the embryo injection methods used to genetically manipulate mosquitoes, and in particular Anopheles species, are difficult and inefficient, particularly for non-specialist laboratories. We have adapted a strategy called ReMOT Control (Receptor-mediated Ovary Transduction of Cargo) to deliver the Cas9 ribonucleoprotein complex to adult mosquito ovaries and generate targeted and heritable mutations in the malaria vector Anopheles stephensi. We found that gene editing by ReMOT Control in Anopheles mosquitoes was comparable to the technique in Ae. aegypti and as efficient in editing as standard embryo injections. The adaptation of this technology to Anopheles mosquitoes opens up the power of reverse genetics to malaria vector labs that do not have the equipment or technical expertise to perform embryo injections and establishes the flexibility of ReMOT Control for gene-editing in non-Aedes species.

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Cas9-Mediated Gene-Editing in the Malaria Mosquito Anopheles stephensi by ReMOT Control

G3 Genes|Genome|Genetics ◽

10.1534/g3.120.401133 ◽

2020 ◽

Vol 10 (4) ◽

pp. 1353-1360 ◽

Cited By ~ 7

Author(s):

Vanessa M. Macias ◽

Sage McKeand ◽

Duverney Chaverra-Rodriguez ◽

Grant L. Hughes ◽

Aniko Fazekas ◽

...

Keyword(s):

Gene Editing ◽

Molecular Mechanisms ◽

Anopheles Stephensi ◽

Mosquito Control ◽

Control Strategies ◽

Mosquito Species ◽

Adult Mosquito ◽

Malaria Mosquito ◽

Powerful Method ◽

Injection Methods

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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Inhibition of Asaia in Adult Mosquitoes Causes Male-Specific Mortality and Diverse Transcriptome Changes

Pathogens ◽

10.3390/pathogens9050380 ◽

2020 ◽

Vol 9 (5) ◽

pp. 380 ◽

Cited By ~ 1

Author(s):

Maria Vittoria Mancini ◽

Claudia Damiani ◽

Sarah M. Short ◽

Alessia Cappelli ◽

Ulisse Ulissi ◽

...

Keyword(s):

Mosquito Control ◽

Control Strategies ◽

Mosquito Species ◽

Acetic Acid Bacteria ◽

Adult Mosquito ◽

Great Expectations ◽

Innovative Tool ◽

Male Specific

Mosquitoes can transmit many infectious diseases, such as malaria, dengue, Zika, yellow fever, and lymphatic filariasis. Current mosquito control strategies are failing to reduce the severity of outbreaks that still cause high human morbidity and mortality worldwide. Great expectations have been placed on genetic control methods. Among other methods, genetic modification of the bacteria colonizing different mosquito species and expressing anti-pathogen molecules may represent an innovative tool to combat mosquito-borne diseases. Nevertheless, this emerging approach, known as paratransgenesis, requires a detailed understanding of the mosquito microbiota and an accurate characterization of selected bacteria candidates. The acetic acid bacteria Asaia is a promising candidate for paratransgenic approaches. We have previously reported that Asaia symbionts play a beneficial role in the normal development of Anopheles mosquito larvae, but no study has yet investigated the role(s) of Asaia in adult mosquito biology. Here we report evidence on how treatment with a highly specific anti-Asaia monoclonal antibody impacts the survival and physiology of adult Anopheles stephensi mosquitoes. Our findings offer useful insight on the role of Asaia in several physiological systems of adult mosquitoes, where the influence differs between males and females.

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Plant evolution driven by interactions with symbiotic and pathogenic microbes

Science ◽

10.1126/science.aba6605 ◽

2021 ◽

Vol 371 (6531) ◽

pp. eaba6605 ◽

Cited By ~ 4

Author(s):

Pierre-Marc Delaux ◽

Sebastian Schornack

Keyword(s):

Cell Biology ◽

Genetic Basis ◽

Reverse Genetics ◽

Molecular Mechanisms ◽

Plant Evolution ◽

Symbiotic Microorganisms ◽

Protection Mechanisms ◽

Evolutionary Trajectories ◽

Microbe Interactions ◽

Pathogenic Microbes

During 450 million years of diversification on land, plants and microbes have evolved together. This is reflected in today’s continuum of associations, ranging from parasitism to mutualism. Through phylogenetics, cell biology, and reverse genetics extending beyond flowering plants into bryophytes, scientists have started to unravel the genetic basis and evolutionary trajectories of plant-microbe associations. Protection against pathogens and support of beneficial, symbiotic, microorganisms are sustained by a blend of conserved and clade-specific plant mechanisms evolving at different speeds. We propose that symbiosis consistently emerges from the co-option of protection mechanisms and general cell biology principles. Exploring and harnessing the diversity of molecular mechanisms used in nonflowering plant-microbe interactions may extend the possibilities for engineering symbiosis-competent and pathogen-resilient crops.

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Reduced susceptibility to selected synthetic pyrethroids in urban malaria vector Anopheles stephensi: a case study in Mangalore city, South India

Malaria Journal ◽

10.1186/1475-2875-9-179 ◽

2010 ◽

Vol 9 (1) ◽

pp. 179 ◽

Cited By ~ 22

Author(s):

Satyanarayan Tiwari ◽

Susanta K Ghosh ◽

Vijay P Ojha ◽

Aditya P Dash ◽

Kamaraju Raghavendra

Keyword(s):

Malaria Vector ◽

South India ◽

Anopheles Stephensi ◽

Synthetic Pyrethroids ◽

Urban Malaria

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Molecular evidence for a kdr-like pyrethroid resistance mechanism in the malaria vector mosquito Anopheles stephensi

Medical and Veterinary Entomology ◽

10.1046/j.1365-2915.2003.00418.x ◽

2003 ◽

Vol 17 (2) ◽

pp. 138-144 ◽

Cited By ~ 81

Author(s):

A. A. Enayati ◽

H. Vatandoost ◽

H. Ladonni ◽

H. Townson ◽

J. Hemingway

Keyword(s):

Malaria Vector ◽

Anopheles Stephensi ◽

Pyrethroid Resistance ◽

Resistance Mechanism ◽

Molecular Evidence ◽

Vector Mosquito

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Astrodaucus persicus as a new source of bioinsectisides against malaria vector, Anopheles stephensi

Asian Pacific Journal of Tropical Medicine ◽

10.1016/j.apjtm.2017.08.007 ◽

2017 ◽

Vol 10 (9) ◽

pp. 896-899 ◽

Cited By ~ 2

Author(s):

Saied Goodarzi ◽

Hassan Vatandoost ◽

Mohammad Reza Abai ◽

Saeed Tavakoli ◽

Amir Hatamian ◽

...

Keyword(s):

Malaria Vector ◽

Anopheles Stephensi

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Does the roof type of a house influence the presence of adult Anopheles stephensi, urban malaria vector? – evidence from a few slum settings in Chennai, India

Parasitology Research ◽

10.1007/s00436-021-07376-5 ◽

2021 ◽

Author(s):

Sangamithra Ravishankaran ◽

Aswin Asokan ◽

N. A. Johnson Amala Justin ◽

Shalu Thomas ◽

Vasna Joshua ◽

...

Keyword(s):

Malaria Vector ◽

Anopheles Stephensi ◽

Urban Malaria

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MicroRNA-8 targets the Wingless signaling pathway in the female mosquito fat body to regulate reproductive processes

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1424408112 ◽

2015 ◽

Vol 112 (5) ◽

pp. 1440-1445 ◽

Cited By ~ 50

Author(s):

Keira J. Lucas ◽

Sourav Roy ◽

Jisu Ha ◽

Amanda L. Gervaise ◽

Vladimir A. Kokoza ◽

...

Keyword(s):

Blood Meal ◽

Molecular Mechanisms ◽

Fat Body ◽

Control Strategies ◽

Functional Characterization ◽

Target Prediction ◽

Underlying Mechanism ◽

Female Mosquito ◽

Reproductive Events

Female mosquitoes require a blood meal for reproduction, and this blood meal provides the underlying mechanism for the spread of many important vector-borne diseases in humans. A deeper understanding of the molecular mechanisms linked to mosquito blood meal processes and reproductive events is of particular importance for devising innovative vector control strategies. We found that the conserved microRNA miR-8 is an essential regulator of mosquito reproductive events. Two strategies to inhibit miR-8 function in vivo were used for functional characterization: systemic antagomir depletion and spatiotemporal inhibition using the miRNA sponge transgenic method in combination with the yeast transcriptional activator gal4 protein/upstream activating sequence system. Depletion of miR-8 in the female mosquito results in defects related to egg development and deposition. We used a multialgorithm approach for miRNA target prediction in mosquito 3′ UTRs and experimentally verified secreted wingless-interacting molecule (swim) as an authentic target of miR-8. Our findings demonstrate that miR-8 controls the activity of the long-range Wingless (Wg) signaling by regulating Swim expression in the female fat body. We discovered that the miR-8/Wg axis is critical for the proper secretion of lipophorin and vitellogenin by the fat body and subsequent accumulation of these yolk protein precursors by developing oocytes.

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