scholarly journals The use of barcoded Asaia bacteria in mosquito in vivo screens for identification of systemic insecticides and inhibitors of malaria transmission

2021 ◽  
Author(s):  
Sturm Angelika ◽  
Martijn Vos ◽  
Rob Henderson ◽  
Maarten Eldering ◽  
Karin Koolen ◽  
...  
Keyword(s):  
Small Molecules ◽  
Recombinant Dna ◽  
Blood Feeding ◽  
Microtiter Plate ◽  
Parasite Development ◽  
Mosquito Vector ◽  
Vector Borne Diseases ◽  
Anopheles Mosquitoes ◽  
Systemic Insecticides

This work addresses the need for new chemical matter in product development for control of pest insects and vector-borne diseases. We present a barcoding strategy that enables phenotypic screens of blood-feeding insects against small molecules in microtiter plate-based arrays and apply this to discovery of novel systemic insecticides and compounds that block malaria parasite development in the mosquito vector. Encoding of the bloodmeals was achieved through recombinant DNA-tagged Asaia bacteria that successfully colonized Aedes and Anopheles mosquitoes. An arrayed screen of a collection of pesticides showed that chemical classes of avermectins, phenylpyrazoles and neonicotinoids were enriched for compounds with systemic adulticide activity against Anopheles. Using a luminescent Plasmodium falciparum reporter strain, barcoded screens identified 48 drug-like transmission blocking compounds from a 400-compound antimicrobial library. The approach significantly increases the throughput in phenotypic screening campaigns using adult insects, and identifies novel candidate small molecules for disease control.

scholarly journals Barcoded Asaia bacteria enable mosquito in vivo screens and identify novel systemic insecticides and inhibitors of malaria transmission

PLoS Biology ◽  
2021 ◽  
Vol 19 (12) ◽  
pp. e3001426
Author(s):  
Angelika Sturm ◽  
Martijn W. Vos ◽  
Rob Henderson ◽  
Maarten Eldering ◽  
Karin M. J. Koolen ◽  
...  
Keyword(s):  
Small Molecules ◽  
Recombinant Dna ◽  
Blood Feeding ◽  
Microtiter Plate ◽  
Parasite Development ◽  
Mosquito Vector ◽  
Vector Borne Diseases ◽  
Systemic Insecticides ◽  
Vector Borne

This work addresses the need for new chemical matter in product development for control of pest insects and vector-borne diseases. We present a barcoding strategy that enables phenotypic screens of blood-feeding insects against small molecules in microtiter plate-based arrays and apply this to discovery of novel systemic insecticides and compounds that block malaria parasite development in the mosquito vector. Encoding of the blood meals was achieved through recombinant DNA-tagged Asaia bacteria that successfully colonised Aedes and Anopheles mosquitoes. An arrayed screen of a collection of pesticides showed that chemical classes of avermectins, phenylpyrazoles, and neonicotinoids were enriched for compounds with systemic adulticide activity against Anopheles. Using a luminescent Plasmodium falciparum reporter strain, barcoded screens identified 48 drug-like transmission-blocking compounds from a 400-compound antimicrobial library. The approach significantly increases the throughput in phenotypic screening campaigns using adult insects and identifies novel candidate small molecules for disease control.

scholarly journals Inhibition of Plasmodium Liver Infection by Ivermectin

2016 ◽  
Vol 61 (2) ◽  
Author(s):  
António M. Mendes ◽  
Inês S. Albuquerque ◽  
Marta Machado ◽  
Joana Pissarra ◽  
Patrícia Meireles ◽  
...  
Keyword(s):  
Control Strategies ◽  
Parasite Development ◽  
Mammalian Host ◽  
Mosquito Vector ◽  
Content Type ◽  
Vector Borne Diseases ◽  
Host Infection ◽  
Liver Infection

ABSTRACT Avermectins are powerful endectocides with an established potential to reduce the incidence of vector-borne diseases. Here, we show that several avermectins inhibit the hepatic stage of Plasmodium infection in vitro. Notably, ivermectin potently inhibits liver infection in vivo by impairing parasite development inside hepatocytes. This impairment has a clear impact on the ensuing blood stage parasitemia, reducing disease severity and enhancing host survival. Ivermectin has been proposed as a tool to control malaria transmission because of its effects on the mosquito vector. Our study extends the effect of ivermectin to the early stages of mammalian host infection and supports the inclusion of this multipurpose drug in malaria control strategies.

Global Warming and Vector-borne Infectious Diseases

2008 ◽  
Vol 3 (2) ◽  
pp. 105-112 ◽  
Author(s):  
Mutsuo Kobayashi ◽  
◽  
Osamu Komagata ◽  
Naoko Nihei
Keyword(s):  
Global Warming ◽  
Infectious Diseases ◽  
Heat Waves ◽  
Blood Feeding ◽  
Mosquito Vector ◽  
Vector Borne Diseases ◽  
Extreme Climate ◽  
Ipcc Report ◽  
Vector Borne ◽  
Global Mean Sea Level

Vector-borne diseases result from infections transmitted to humans by blood-feeding arthropods such as mosquitoes, ticks, and fleas. Such cold-blooded animals are influenced by environmental change. A recent IPCC report clearly showed that the emission of greenhouse gases has already changed world climates. Heat waves in Europe, rises in global mean sea level, summer droughts and wild fires, more intense precipitation, and increasing numbers of large cyclones and hurricanes may be typical example of extreme climate phenomena related to global warming. High temperatures may increase survival among arthropods, depending on their vector, behavior, ecology, and valuable factors, and temperate zone warming may accelerate the spread of mosquitoes such asAedes albopictus. The MIROK (K1) Model clearly shows a northern limit forAe.albopictus, particularly in northern Honshu in 2035 and southern and middle Hokkaido Island in 2100 in Japan. The spread of the mosquito vector through global used-tire trading in recent decades to Africa, the Mideast, Europe, and North and South America caused an outbreak of Chikungunya fever in north Italy in 2007. Global warming, extreme climate change, changing physical distribution, and an increase in oversea travel are also expected to influence the epidemiology of vector-borne infectious diseases.

scholarly journals Genetics of Mosquito Vector Competence

2000 ◽  
Vol 64 (1) ◽  
pp. 115-137 ◽  
Author(s):  
Brenda T. Beerntsen ◽  
Anthony A. James ◽  
Bruce M. Christensen
Keyword(s):  
Sindbis Virus ◽  
Molecular Mechanisms ◽  
Germ Line ◽  
Control Strategies ◽  
Vector Competence ◽  
Parasite Development ◽  
Mosquito Vector ◽  
Susceptible Population ◽  
Pathogen Destruction

SUMMARY Mosquito-borne diseases are responsible for significant human morbidity and mortality throughout the world. Efforts to control mosquito-borne diseases have been impeded, in part, by the development of drug-resistant parasites, insecticide-resistant mosquitoes, and environmental concerns over the application of insecticides. Therefore, there is a need to develop novel disease control strategies that can complement or replace existing control methods. One such strategy is to generate pathogen-resistant mosquitoes from those that are susceptible. To this end, efforts have focused on isolating and characterizing genes that influence mosquito vector competence. It has been known for over 70 years that there is a genetic basis for the susceptibility of mosquitoes to parasites, but until the advent of powerful molecular biological tools and protocols, it was difficult to assess the interactions of pathogens with their host tissues within the mosquito at a molecular level. Moreover, it has been only recently that the molecular mechanisms responsible for pathogen destruction, such as melanotic encapsulation and immune peptide production, have been investigated. The molecular characterization of genes that influence vector competence is becoming routine, and with the development of the Sindbis virus transducing system, potential antipathogen genes now can be introduced into the mosquito and their effect on parasite development can be assessed in vivo. With the recent successes in the field of mosquito germ line transformation, it seems likely that the generation of a pathogen-resistant mosquito population from a susceptible population soon will become a reality.

scholarly journals Clustered rapid induction of apoptosis limits ZIKV and DENV-2 proliferation in the midguts of Aedes aegypti

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Jasmine B. Ayers ◽  
Heather G. Coatsworth ◽  
Seokyoung Kang ◽  
Rhoel R. Dinglasan ◽  
Lei Zhou
Keyword(s):  
Aedes Aegypti ◽  
Viral Entry ◽  
Ex Vivo ◽  
Systemic Infection ◽  
Blood Feeding ◽  
Mosquito Vector ◽  
Antiviral Responses ◽  
Rapid Induction ◽  
Induction Of Apoptosis

AbstractInter-host transmission of pathogenic arboviruses such as dengue virus (DENV) and Zika virus (ZIKV) requires systemic infection of the mosquito vector. Successful systemic infection requires initial viral entry and proliferation in the midgut cells of the mosquito followed by dissemination to secondary tissues and eventual entry into salivary glands1. Lack of arbovirus proliferation in midgut cells has been observed in several Aedes aegypti strains2, but the midgut antiviral responses underlying this phenomenon are not yet fully understood. We report here that there is a rapid induction of apoptosis (RIA) in the Aedes aegypti midgut epithelium within 2 hours of infection with DENV-2 or ZIKV in both in vivo blood-feeding and ex vivo midgut infection models. Inhibition of RIA led to increased virus proliferation in the midgut, implicating RIA as an innate immune mechanism mediating midgut infection in this mosquito vector.

scholarly journals Clustered Rapid Induction of Apoptosis Limits ZIKV and DENV-2 Proliferation in the Midguts of Aedes aegypti

2020 ◽  
Author(s):  
Jasmine B. Ayers ◽  
Heather G. Coatsworth ◽  
Seokyoung Kang ◽  
Rhoel R. Dinglasan ◽  
Lei Zhou
Keyword(s):  
Aedes Aegypti ◽  
Viral Entry ◽  
Ex Vivo ◽  
Systemic Infection ◽  
Blood Feeding ◽  
Mosquito Vector ◽  
Antiviral Responses ◽  
Rapid Induction ◽  
Induction Of Apoptosis

ABSTRACTInter-host transmission of pathogenic arboviruses such as dengue virus (DENV) and Zika virus (ZIKV) requires systemic infection of the mosquito vector. Successful systemic infection requires initial viral entry and proliferation in the midgut cells of the mosquito followed by dissemination to secondary tissues and eventual entry into salivary glands1. Lack of arbovirus proliferation in midgut cells has been observed in several Aedes aegypti strains2, but the midgut antiviral responses underlying this phenomenon are not yet fully understood. We report here that there is a rapid induction of apoptosis (RIA) in the Aedes aegypti midgut epithelium within 2 hours of infection with DENV-2 or ZIKV in both in vivo blood-feeding and ex vivo midgut infection models. Inhibition of RIA led to increased virus proliferation in the midgut, implicating RIA as an innate immune mechanism mediating midgut infection in this mosquito vector.

scholarly journals Transcriptome of the Aedes aegypti Mosquito in Response to Human Complement Proteins

2020 ◽  
Author(s):  
Gloria I. Giraldo-Calderón ◽  
Arley Calle-Tobon ◽  
Paula Rozo-Lopez ◽  
Tonya M. Colpitts ◽  
Yoonseong Park ◽  
...  
Keyword(s):  
Aedes Aegypti ◽  
Blood Feeding ◽  
Normal Serum ◽  
Effective Control ◽  
Mosquito Vector ◽  
Transcript Expression ◽  
Human Complement ◽  
Complement Proteins

Aedes aegypti is the primary mosquito vector of several human arboviruses including dengue virus (DENV). Vector control is the principal intervention to decrease the transmission of these viruses. The characterization of molecules involved in the mosquito physiological responses to blood-feeding may help to identify novel targets useful in the design of effective control strategies. In this study, we evaluated the in vivo effect of feeding adult female mosquitoes with human blood containing either heat-inactivated (IB), normal serum (NB), and RNA-seq based transcript expression was compared against sugar-fed (SF) mosquitoes. In the in vitro experiments, we treated Aag2 cells with a recombinant version of the complement proteins (hC3 or hC5a) and compared transcript expression to untreated control cells after 24h. The transcript expression analysis revealed that human complement proteins modulate approximately 2,300 transcripts involved in multiple biological functions, including the immune system. We also found 161 up-regulated and 168 down-regulated transcripts differentially expressed when hC3 and hC5a were compared against the control untreated cells. We conclude that active human complement induces significant changes in the transcriptome of Ae. aegypti mosquitoes, which can influence the infective capacity of pathogens ingested during blood meals.

scholarly journals Transcriptome of the Aedes aegypti Mosquito in Response to Human Complement Proteins

2020 ◽  
Vol 21 (18) ◽  
pp. 6584
Author(s):  
Gloria I. Giraldo-Calderón ◽  
Arley Calle-Tobón ◽  
Paula Rozo-López ◽  
Tonya M. Colpitts ◽  
Yoonseong Park ◽  
...  
Keyword(s):  
Aedes Aegypti ◽  
Blood Feeding ◽  
Effective Control ◽  
Mosquito Vector ◽  
Transcript Expression ◽  
Human Complement ◽  
Complement Protein ◽  
Complement Proteins

Aedes aegypti is the primary mosquito vector of several human arboviruses, including the dengue virus (DENV). Vector control is the principal intervention to decrease the transmission of these viruses. The characterization of molecules involved in the mosquito physiological responses to blood-feeding may help identify novel targets useful in designing effective control strategies. In this study, we evaluated the in vivo effect of feeding adult female mosquitoes with human red blood cells reconstituted with either heat-inactivated (IB) or normal plasma (NB). The RNA-seq based transcript expression of IB and NB mosquitoes was compared against sugar-fed (SF) mosquitoes. In in vitro experiments, we treated Aag2 cells with a recombinant version of complement proteins (hC3 or hC5a) and compared transcript expression to untreated control cells after 24 h. The transcript expression analysis revealed that human complement proteins modulate approximately 2300 transcripts involved in multiple biological functions, including immunity. We also found 161 upregulated and 168 downregulated transcripts differentially expressed when human complement protein C3 (hC3) and human complement protein C5a (hC5a) treated cells were compared to the control untreated cells. We conclude that active human complement induces significant changes to the transcriptome of Ae. aegypti mosquitoes, which may influence the physiology of these arthropods.

scholarly journals Activation of the autophagy pathway affects Dengue virus infection in Aedes aegypti

2021 ◽  
Author(s):  
Tse-Yu Chen ◽  
Chelsea T. Smartt
Keyword(s):  
Aedes Aegypti ◽  
Dengue Virus ◽  
Small Molecules ◽  
Virus Titer ◽  
Denv Infection ◽  
Control Group ◽  
Mosquito Vector ◽  
Autophagy Pathway

AbstractMosquito-borne Dengue virus (DENV) has caused major disease worldwide, impacting 50 to 100 million people every year, and is spread by the major mosquito vector Aedes aegypti. Understanding mosquito physiology and developing new control strategies becomes an important issue to eliminate DENV. We focused on autophagy, a pathway suggested as having a positive influence on virus replication in humans, as a potential anti-viral target in the mosquito. To understand the role played by autophagy in Ae. aegypti, we examined the expression of the pathway in vitro (Aag-2 cell) and in vivo (Ae. aegypti). The results indicated that DENV infection in Aag-2 cells caused the microtubule-associated protein light chain 3-phosphatidylethanolamine conjugate (LC3-II) protein levels to increase which indicated the activation of the autophagy pathway. Rapamycin and 3-Methyladenine were used to activate or suppress the autophagy pathway, respectively. Rapamycin treatment decreased the virus titer in the Aag-2 cells, but the 3-Methyladenine treatment did not affect DENV titer. In Ae. aegypti, microinjected rapamycin increased the DENV titer after one-day infection and was significantly different compared to the control group titer. Two ATG genes, ATG4 and ATG12, were expressed differentially under the rapamycin treatments. Although the results differed between in vitro and in vivo studies, findings from both support the interaction between autophagy and DENV. Our studies revealed the activation of the autophagy pathway through rapamycin could be related to DENV infection in the mosquito. The possibility of autophagy being associated with different antiviral mechanisms at different extrinsic incubation times and tissues in Ae. aegypti is discussed.Author SummaryDengue virus (DENV) has been a great threat to public health and has not developed an efficient method to stop the transmission. To understand the complex interaction between virus and mosquito, we investigate the autophagy pathway and its role during the infection process. We noticed the induction of autophagy pathways from DENV infection in Aag-2 cells and blood meal from Ae. aegypti. Moreover, activation of the autophagy pathway from rapamycin could alter the DENV titer. Our results indicated the autophagy pathway is associated with DENV and could be crucial during the DENV infection. Furthermore, we proved the practicality of small molecules in altering the autophagy pathway in mosquitoes, and thus the usage of small molecules as possible mosquito pathogen vaccines should be evaluated.

A Sensitive and Facile Assay for the Measurement of Activated Protein C Activity Levels in Vivo

1993 ◽  
Vol 69 (05) ◽  
pp. 441-447 ◽  
Author(s):  
Carolyn L Orthner ◽  
Billy Kolen ◽  
William N Drohan
Keyword(s):  
Protein C ◽  
Plasma Concentrations ◽  
Limited Proteolysis ◽  
Activated Protein C ◽  
Microtiter Plate ◽  
Reversible Inhibitor ◽  
Activity Levels ◽  
Standard Curve ◽  
Agarose Beads

SummaryActivated protein C (APC) is a serine protease which plays an important role as a naturally occurring antithrombotic enzyme. APC, which is formed by thrombin-catalyzed limited proteolysis of the zymogen protein C, functions as an anticoagulant by proteolytic inactivation of the coagulation cofactors VIIIa and Va. APC is inhibited by several members of the serpin family as well a by α2-macroglobulin. APC is being developed as a therapeutic for the prevention and treatment of thrombosis. We have developed an assay to quantify circulating levels of enzymatically active APC during its administration to patients, in healthy individuals, and in various disease states. This assay utilizes an EDTA-dependent anti-protein C monoclonal antibody (Mab) 7D7B10 to capture both APC and protein C from plasma, prepared from blood collected in an anticoagulant supplemented with the reversible inhibitor p-aminobenzamidine. Mab 7D7B10-derivatized agarose beads are added to the wells of a 96-well filtration plate, equilibrated with Tris-buffered saline, and incubated for 10 min with 200 μl of plasma. After washing, APC and protein C are eluted from the immunosorbent beads with a calcium-containing buffer into the wells of a 96-well microtiter plate containing antithrombin III (ATIII) and heparin. The amidolytic activity of APC is then measured on a kinetic plate reader following the addition of L-pyroglutamyl-L-prolyl-L-arginine-p-nitroanilide (S-2366) substrate.The rate of substrate hydrolysis was proportional to APC concentration over a 200-fold concentration range (5.0 to 1,000 ng/ml) when measured continuously over a 15 to 30 min time period. The coefficient of variation was 5.9% at 35 ng/ml and 8.8% at 350 ng/ml APC. The sensitivity of the assay could be increased by measuring the amount of color produced after longer incubation times in the endpoint mode. The measured APC activity levels were little affected by varying protein C or prothrombin over the extremes of 0 to 150% of normal plasma concentrations. By constructing the standard curve in protein C-deficient plasma, the concentration of APC activity in normal pooled plasma was determined to be 2.8 ng/ml (45 pM), which represents 0.08% of the protein C concentration. The assay was approximately 50-fold more sensitive than the identical assay, but using Mab-coated microtiter wells rather than immunosorbent beads as the capture step.

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