scholarly journals Programmable CRISPR interference for gene silencing using Cas13a in mosquitoes

2019 ◽  
Author(s):  
Aditi Kulkarni ◽  
Wanqin Yu ◽  
Alex Moon ◽  
Ashmita Pandey ◽  
Kathryn A. Hanley ◽  
...  
Keyword(s):  
Aedes Aegypti ◽  
Gene Silencing ◽  
Anopheles Gambiae ◽  
Blood Meal ◽  
Egg Production ◽  
Mosquito Species ◽  
Single Strand ◽  
Crispr Interference ◽  
Target Mrna

AbstractIn the CRISPR-Cas systems, Cas13a is an RNA-guided RNA nuclease specifically targeting single strand RNA. We developed a Cas13a mediated CRISPR interference tool to target mRNA for gene silencing in mosquitoes. The machinery was tested in two mosquito species. A Cas13a expressing plasmid was delivered to mosquitoes by intrathoracic injection, and Cas13a transcripts were detectable at least10 days post-delivery. In Anopheles gambiae, vitellogenin gene was silenced by Vg-crRNA injection two hours post-blood meal, which was accompanied by a significant reduction in egg production. In Aedes aegypti, the α- and δ-subunits of COPI genes were silenced by a post-blood meal crRNA injection, which resulted in mortality and fragile midguts, reproducing a phenotype reported previously. Co-silencing genes simultaneously is achievable when a cocktail of target crRNAs is given. No detectable collateral cleavages of non-target transcripts were observed in the study. This study adds a programmable CRISPR tool to manipulate RNA in mosquitoes.

scholarly journals Serine hydroxymethyltransferase controls blood-meal digestion in the midgut of Aedes aegypti mosquitoes

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Xuemei Li ◽  
Jinyu Yang ◽  
Qian Pu ◽  
Xinyue Peng ◽  
Lili Xu ◽  
...  
Keyword(s):  
Aedes Aegypti ◽  
Blood Meal ◽  
Digestive Enzymes ◽  
Egg Production ◽  
Sequence Similarity ◽  
Mrna Level ◽  
Serine Hydroxymethyltransferase ◽  
Blood Protein ◽  
Transcriptional Expression ◽  
Blood Meal Digestion

Abstract Background Female Aedes aegypti mosquitoes are vectors of arboviruses that cause diverse diseases of public health significance. Blood protein digestion by midgut proteases provides anautogenous mosquitoes with the nutrients essential for oocyte maturation and egg production. Midgut-specific miR-1174 affects the functions of the midgut through its target gene serine hydroxymethyltransferase (SHMT). However, less is known about SHMT-regulated processes in blood digestion by mosquitoes. Methods RNAi of SHMT was realized by injection of the double-stranded RNA at 16 h post-eclosion. The expression of SHMT at mRNA level and protein level was assayed by real-time PCR and Western blotting, respectively. Statistical analyses were performed with GraphPad7 using Student’s t-test. Results Here, we confirmed that digestion of blood was inhibited in SHMT RNAi-silenced female A. aegypti mosquitoes. Evidence is also presented that all SHMT-depleted female mosquitoes lost their flight ability and died within 48 h of a blood meal. Furthermore, most examined digestive enzymes responded differently in their transcriptional expression to RNAi depletion of SHMT, with some downregulated, some upregulated and some remaining stable. Phylogenetic analysis showed that transcriptional expression responses to SHMT silence were largely unrelated to the sequence similarity between these enzymes. Conclusions Overall, this research shows that SHMT was expressed at a low level in the midgut of Aedes aegypti mosquitoes, but blood-meal digestion was inhibited when SHMT was silenced. Transcriptional expressions of different digestive enzymes were affected in response to SHMT depletion, suggesting that SHMT is required for the blood-meal digestion in the midgut and targeting SHMT could provide an effective strategy for vector mosquito population control.

scholarly journals 20-Hydroxyecdysone Primes Innate Immune Responses That Limit Bacterial and Malarial Parasite Survival in Anopheles gambiae

mSphere ◽  
2020 ◽  
Vol 5 (2) ◽  
Author(s):  
Rebekah A. Reynolds ◽  
Hyeogsun Kwon ◽  
Ryan C. Smith
Keyword(s):  
Immune Function ◽  
Anopheles Gambiae ◽  
Blood Meal ◽  
Egg Production ◽  
Blood Feeding ◽  
Innate Immune ◽  
Content Type ◽  
Innate Immune Function ◽  
Cellular Immune Function ◽  
Cellular Immune

ABSTRACT Blood feeding is an integral behavior of mosquitoes to acquire nutritional resources needed for reproduction. This requirement also enables mosquitoes to serve as efficient vectors to acquire and potentially transmit a multitude of mosquito-borne diseases, most notably malaria. Recent studies suggest that mosquito immunity is stimulated following a blood meal, independent of infection status. Since blood feeding promotes production of the hormone 20-hydroxyecdysone (20E), we hypothesized that 20E plays an important role in priming the immune response for pathogen challenge. Here, we examine the immunological effects of priming Anopheles gambiae with 20E prior to pathogen infection, demonstrating a significant reduction in bacteria and Plasmodium berghei survival in the mosquito host. Transcriptome sequencing (RNA-seq) analysis following 20E treatment identifies several known 20E-regulated genes, as well as several immune genes with previously reported function in antipathogen defense. Together, these data demonstrate that 20E influences cellular immune function and antipathogen immunity following mosquito blood feeding, arguing the importance of hormones in the regulation of mosquito innate immune function. IMPORTANCE Blood feeding is required to provide nutrients for mosquito egg production and serves as a mechanism to acquire and transmit pathogens. Shortly after a blood meal is taken, there is a peak in the production of 20-hydroxyecdysone (20E), a mosquito hormone that initiates physiological changes, including yolk protein production and mating refractoriness. Here, we examine additional roles of 20E in the regulation of mosquito immunity, demonstrating that priming the immune system with 20E increases mosquito resistance to pathogens. We identify differentially expressed genes in response to 20E treatment, including several involved in innate immune function as well as lipid metabolism and transport. Together, these data argue that 20E stimulates mosquito cellular immune function and innate immunity shortly after blood feeding.

scholarly journals Effectiveness of Three Fruit Seed Extracts as Larvicide against Three Major Mosquito Vectors Aedes aegypti Linnaeus, Culex quinquefasciatus Say and Anopheles gambiae Giles (Diptera: Culicidae)

2020 ◽  
pp. 16-29
Author(s):  
Lame Younoussa ◽  
Kary Mallam Oumarou ◽  
Theodora Kopa Kowa ◽  
Serge Eteme Enama ◽  
Gabriel Agbor Agbor ◽  
...  
Keyword(s):  
Aedes Aegypti ◽  
Anopheles Gambiae ◽  
Culex Quinquefasciatus ◽  
Tap Water ◽  
Mosquito Species ◽  
Mangifera Indica ◽  
Larval Mortality ◽  
Persea Americana ◽  
Plant Seed ◽  
Seed Extracts

The CH2Cl2-MeOH (30:70 v/v) extracts of the seeds of Mangifera indica (Mango), Persea americana (Avocado) and Dacryodes edulis (African plum) were evaluated for potential mosquito larvicidal activity against 3rd and 4th instar larvae of Aedes aegypti, Culex quinquefasciatus and Anopheles gambiae. Extracts were diluted with 1 mL of methanol and concentrations ranging from 1000 to 125 mg/L in 4 replicates each, were prepared in the volume of 100 mL in the plastic cups (250 mL). A volume of 1 mL of methanol added to 99 mL of tap water was prepared as negative control and Bi-one (1000 mg/L) constituted a positive control. In each test solution, 25 larvae of each mosquito species were separately transferred and larval mortality was recorded after 24 h post-treatment. As results, the three plant seed extracts applied at 1000 mg/L caused for at least 79% mortality of each mosquito species larvae assessed. The seed extract of P. americana (LC50 of 98.31, 129.24 and 136.26 mg/L, respectively against An. gambiae, Ae. aegypti and Cx. quiquefasciatus larvae) was the most potent followed by D. edulis (LC50 of 176.87 mg/L for An. gambiae, 198.68 mg/L for Ae. aegypti and 201.70 mg/L for Cx. quinquefasciatus) and M. indica (LC50 of 258.98 mg/L for An. gambiae, 297.35 mg/L for Ae. aegypti and 435.45 mg/L for Cx. quinquefasciatus).  Globally, all the seed extracts were more toxic against An. gambiae larvae compared to other mosquito species and need further exploration for the development of a new botanical larvicide to reduce mosquito densities.

scholarly journals The impact of the age of first blood meal and Zika virus infection on Aedes aegypti egg production and longevity

PLoS ONE ◽  
2018 ◽  
Vol 13 (7) ◽  
pp. e0200766 ◽  
Author(s):  
Martha Thieme Petersen ◽  
Isabella Dias da Silveira ◽  
Aline Tátila-Ferreira ◽  
Mariana Rocha David ◽  
Thais Chouin-Carneiro ◽  
...  
Keyword(s):  
Virus Infection ◽  
Aedes Aegypti ◽  
Blood Meal ◽  
Zika Virus ◽  
Egg Production ◽  
Zika Virus Infection ◽  
The Impact

Ukauwa virus proliferation in mosquitoes

1968 ◽  
Vol 14 (2) ◽  
pp. 125-129 ◽  
Author(s):  
Oladeinde Ogunbi
Keyword(s):  
Aedes Aegypti ◽  
Salivary Glands ◽  
North American ◽  
Blood Meal ◽  
Mosquito Species ◽  
Infective Virus ◽  
Biological Criteria ◽  
Eclipse Phase ◽  
Initial Recovery ◽  
Intrathoracic Inoculation

Ukauwa virus, a Bunyamwera group arbovirus endemic in the Ethiopian zoogeographic region, multiplied in the tissues of North American mosquito species Aedes canadensis (Theobald) and Aedes triseriatus (Say) after intrathoracic inoculation. This virus also multiplied in laboratory-bred Aedes aegypti (L.) after both feeding and injection. Ukauwa virus now fulfills the biological criteria of an arbovirus. In these three mosquito species, after initial detection of infective virus immediately after inoculation, an eclipse phase with lack of detection of infectivity was observed 6 hours later, and was followed by initial recovery of virus (1.0 log mouse LD50) at 12 hours and thereafter steady virus increments in thorax, salivary glands, gut, and legs which reached maximum titers of 5.4 to 6.3 log mouse LD50 in 4 days. In A. aegypti fed Ukauwa virus no infectivity was detected during the first 8 days after the meal. After initial detection of infective virus (3.0 log mouse LD50) on day 10, maximum titers of 4.3 to 5.3 log mouse LD50 were reached 4 days later.A. aegypti transmitted Ukauwa virus to weaned mice on the 12th and 14th days after an infective blood meal, when virus titers in the salivary glands exceeded 4.3 log mouse LD50. The infection threshold of Ukauwa virus for A. aegypti by feeding was 4.7 log mouse LD50 per 0.002 ml.

scholarly journals The impact of mating and sugar feeding on blood-feeding physiology and behavior in the arbovirus vector mosquito Aedes aegypti

2021 ◽  
Vol 15 (9) ◽  
pp. e0009815
Author(s):  
Garrett P. League ◽  
Ethan C. Degner ◽  
Sylvie A. Pitcher ◽  
Yassi Hafezi ◽  
Erica Tennant ◽  
...  
Keyword(s):  
Aedes Aegypti ◽  
Blood Meal ◽  
Egg Production ◽  
Control Strategies ◽  
Blood Feeding ◽  
Female Behavior ◽  
Sugar Feeding ◽  
And Behavior ◽  
The Impact ◽  
Initial Blood

Background Aedes aegypti mosquitoes are globally distributed vectors of viruses that impact the health of hundreds of millions of people annually. Mating and blood feeding represent fundamental aspects of mosquito life history that carry important implications for vectorial capacity and for control strategies. Females transmit pathogens to vertebrate hosts and obtain essential nutrients for eggs during blood feeding. Further, because host-seeking Ae. aegypti females mate with males swarming near hosts, biological crosstalk between these behaviors could be important. Although mating influences nutritional intake in other insects, prior studies examining mating effects on mosquito blood feeding have yielded conflicting results. Methodology/Principal findings To resolve these discrepancies, we examined blood-feeding physiology and behavior in virgin and mated females and in virgins injected with male accessory gland extracts (MAG), which induce post-mating changes in female behavior. We controlled adult nutritional status prior to blood feeding by using water- and sugar-fed controls. Our data show that neither mating nor injection with MAG affect Ae. aegypti blood intake, digestion, or feeding avidity for an initial blood meal. However, sugar feeding, a common supplement in laboratory settings but relatively rare in nature, significantly affected all aspects of feeding and may have contributed to conflicting results among previous studies. Further, mating, MAG injection, and sugar intake induced declines in subsequent feedings after an initial blood meal, correlating with egg production and laying. Taking our evaluation to the field, virgin and mated mosquitoes collected in Colombia were equally likely to contain blood at the time of collection. Conclusions/Significance Mating, MAG, and sugar feeding impact a mosquito’s estimated ability to transmit pathogens through both direct and indirect effects on multiple aspects of mosquito biology. Our results highlight the need to consider natural mosquito ecology, including diet, when assessing their physiology and behavior in the laboratory.

scholarly journals Video Recording Can Conveniently Assay Mosquito Locomotor Activity

2020 ◽  
Author(s):  
Maisa da Silva Araujo ◽  
Fang Guo ◽  
Michael Rosbash
Keyword(s):  
Locomotor Activity ◽  
Aedes Aegypti ◽  
Anopheles Gambiae ◽  
Tracking System ◽  
Mosquito Species ◽  
Activity Patterns ◽  
Video Recording ◽  
Constant Darkness ◽  
Daily Rhythms ◽  
Wide Range

AbstractAnopheles gambiae and Aedes aegypti are perhaps the best studied mosquito species and important carriers of human malaria and arbovirus, respectively. Mosquitoes have daily rhythms in behaviors and show a wide range of activity patterns. Although Anopheles is known to be principally nocturnal and Aedes principally diurnal, details of mosquito activity are not easily assayed in the laboratory. We recently described FlyBox, a simple tracking system for assaying Drosophila locomotor activity rhythms and thought that it might also be applicable to monitoring mosquito activity. Indeed, we show here that FlyBox can easily, conveniently, affordably and accurately measure the activity of Anopheles as well as Aedes over several days. The resulting profiles under light-dark as well as constant darkness conditions are compatible with results in the literature, indicating that this or similar systems will be useful in the future for more detailed studies on a range of insect species and under more diverse laboratory conditions.

scholarly journals Heterogeneity of midgut cells and their differential responses to blood meal ingestion by the mosquito, Aedes aegypti

2020 ◽  
Author(s):  
Yingjun Cui ◽  
Alexander W.E. Franz
Keyword(s):  
Aedes Aegypti ◽  
Blood Meal ◽  
Mosquito Species ◽  
Cell Types ◽  
Blood Feeding ◽  
Nutrient Absorption ◽  
Cell Type ◽  
Blood Digestion ◽  
Mosquito Midgut ◽  
Meal Ingestion

AbstractMosquitoes are the most notorious hematophagous insects and due to their blood feeding behavior and genetic compatibility, numerous mosquito species are highly efficient vectors for certain human pathogenic parasites and viruses. The mosquito midgut is the principal organ of blood meal digestion and nutrient absorption. It is also the initial site of infection with blood meal acquired parasites and viruses. We conducted an analysis based on single-nucleus RNA sequencing (snRNA-Seq) to assess the cellular diversity of the midgut and how individual cells respond to blood meal ingestion to facilitate its digestion. Our study revealed the presence of 20 distinguishable cell-type clusters in the female midgut of Aedes aegypti. The identified cell types included intestinal stem cell (ISC), enteroblasts (EB), differentiating EB (dEB), enteroendocrine cells (EE), enterocytes (EC), EC-like cells, cardia cells, and visceral muscle (VM) cells. Blood meal ingestion dramatically changed the overall midgut cell type composition, profoundly increasing the proportions of ISC and three EC/EC like clusters. In addition, transcriptional profiles of all cell types were strongly affected while genes involved in various metabolic processes were significantly upregulated. Our study provides a basis for further physiological and molecular studies on blood digestion, nutrient absorption, and cellular homeostasis in the mosquito midgut.

A Simple PCR Assay for Discrimination of Dengue Vectors from Nsukka LGA, Nigeria

2020 ◽  
Vol 10 (1) ◽  
pp. 67-77
Author(s):  
Amos Watentena ◽  
Ikem Chris Okoye ◽  
Ikechukwu Eugene Onah ◽  
Onwude Cosmas Ogbonnaya ◽  
Emmanuel Ogudu
Keyword(s):  
Aedes Aegypti ◽  
Aedes Albopictus ◽  
Mosquito Species ◽  
Public Health Problem ◽  
Major Public Health Problem ◽  
Mosquito Vector ◽  
Aedes Mosquitoes ◽  
Allele Specific ◽  
Arboviral Diseases ◽  
Dengue Vectors

Mosquitoes of Aedes species are vectors of several arboviral diseases which continue to be a major public health problem in Nigeria. This study among other things, morphologically identified Aedes mosquitoes collected from Nsukka LGA and used an allele specific PCR amplification for discrimination of dengue vectors. Larval sampling, BG-sentinel traps and modified human landing catches were used for mosquito sampling in two selected autonomous communities of Nsukka LGA (Nsukka and Obimo). A total of 124 Aedes mosquitoes consisting of five (5) different species were collected from April to June, 2019 in a cross-sectional study that covered 126 households, under 76 distinct geographical coordinates. Larvae was mainly collected from plastic containers 73% (n=224), metallic containers 14% (n=43), earthen pots 9% (n=29) and used car tyres 3% (n=9), reared to adult stage 69.35% (n=86), and all mosquitoes were identified using standard morphological keys. Five (5) Aedes mosquito species were captured; Aedes aegypti 83(66.94%), Aedes albopictus 33(26.61%), Aedes simpsoni (4.48%), Aedes luteocephalus (≤1%) and Aedes vittatus (≤1%). Nsukka autonomous community had higher species diversity than Obimo. Allele specific amplification confirmed dengue vectors, Aedes aegypti and Aedes albopictus species on a 2% agarose gel. Since the most recent re-emergence of arboviral diseases is closely associated with Aedes species, findings of this study, therefore, give further evidence about the presence of potential arboviral vectors in Nigeria and describe the role of a simple PCR in discriminating some. Further entomological studies should integrate PCR assays in mosquito vector surveillance.

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