scholarly journals Knockout of Anopheles stephensi immune gene LRIM1 by CRISPR-Cas9 reveals its unexpected role in reproduction and vector competence

2021 ◽  
Vol 17 (11) ◽  
pp. e1009770
Author(s):  
Ehud Inbar ◽  
Abraham G. Eappen ◽  
Robert T. Alford ◽  
William Reid ◽  
Robert A. Harrell ◽  
...  
Keyword(s):  
Anopheles Stephensi ◽  
Vector Competence ◽  
Bacterial Load ◽  
Acetic Acid Bacteria ◽  
Reproductive Capacity ◽  
Immune Gene ◽  
Microbiome Composition ◽  
Embryonic Genome ◽  
Mosquito Mortality ◽  
Knockout Line

PfSPZ Vaccine against malaria is composed of Plasmodium falciparum (Pf) sporozoites (SPZ) manufactured using aseptically reared Anopheles stephensi mosquitoes. Immune response genes of Anopheles mosquitoes such as Leucin-Rich protein (LRIM1), inhibit Plasmodium SPZ development (sporogony) in mosquitoes by supporting melanization and phagocytosis of ookinetes. With the aim of increasing PfSPZ infection intensities, we generated an A. stephensi LRIM1 knockout line, Δaslrim1, by embryonic genome editing using CRISPR-Cas9. Δaslrim1 mosquitoes had a significantly increased midgut bacterial load and an altered microbiome composition, including elimination of commensal acetic acid bacteria. The alterations in the microbiome caused increased mosquito mortality and unexpectedly, significantly reduced sporogony. The survival rate of Δaslrim1 and their ability to support PfSPZ development, were partially restored by antibiotic treatment of the mosquitoes, and fully restored to baseline when Δaslrim1 mosquitoes were produced aseptically. Deletion of LRIM1 also affected reproductive capacity: oviposition, fecundity and male fertility were significantly compromised. Attenuation in fecundity was not associated with the altered microbiome. This work demonstrates that LRIM1’s regulation of the microbiome has a major impact on vector competence and longevity of A. stephensi. Additionally, LRIM1 deletion identified an unexpected role for this gene in fecundity and reduction of sperm transfer by males.

scholarly journals Knockout of Anopheles stephensi immune gene LRIM1 by CRISPR-Cas9 reveals its unexpected role in reproduction and vector competence

2021 ◽  
Author(s):  
Ehud Inbar ◽  
Abraham Eappen ◽  
Robert T Alford ◽  
William Reid ◽  
Robert A Harrell ◽  
...  
Keyword(s):  
Anopheles Stephensi ◽  
Vector Competence ◽  
Bacterial Load ◽  
Acetic Acid Bacteria ◽  
Reproductive Capacity ◽  
Immune Gene ◽  
Microbiome Composition ◽  
Anopheles Mosquitoes ◽  
Embryonic Genome ◽  
Mosquito Mortality

PfSPZ Vaccine against malaria is composed of Plasmodium falciparum (Pf) sporozoites (SPZ) manufactured using aseptically reared Anopheles stephensi mosquitoes. Immune response genes of Anopheles mosquitoes such as Leucin-Rich protein (LRIM1), inhibit Plasmodium SPZ development (sporogony) in mosquitoes by supporting melanization and phagocytosis of ookinetes. With the aim of increasing PfSPZ infection intensities, we generated an A. stephensi LRIM1 knockout line , Δaslrim1 , by embryonic genome editing using CRISPR-Cas9. Δaslrim1 mosquitoes had a significantly increased midgut bacterial load and an altered microbiome composition, including elimination of commensal acetic acid bacteria.  The alterations in the microbiome caused increased mosquito mortality and unexpectedly, significantly reduced sporogony. The survival rate of Δaslrim1 and their ability to support PfSPZ development, were partially restored by antibiotic treatment of the mosquitoes, and fully restored to baseline when Δaslrim1 mosquitoes were produced aseptically. Deletion of LRIM1 also affected reproductive capacity: oviposition, fecundity and male fertility were significantly compromised. Attenuation in fecundity was not associated with the altered microbiome. This work demonstrates that LRIM1’s regulation of the microbiome has a major impact on vector competence and longevity of A. stephensi. Additionally, LRIM1 deletion identified an unexpected role for this gene in fecundity and reduction of sperm transfer by males.

scholarly journals “Limiting access to iron decreases infection of Atlantic salmon SHK-1 cells with bacterium Piscirickettsia salmonis”

2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Rodrigo Díaz ◽  
José Troncoso ◽  
Eva Jakob ◽  
Stanko Skugor
Keyword(s):  
Gene Expression ◽  
Iron Deficiency ◽  
Atlantic Salmon ◽  
Bacterial Load ◽  
Host Cells ◽  
Immune Gene ◽  
Immune Signaling ◽  
Immune Effector ◽  
Infected Cells ◽  
Piscirickettsia Salmonis

Abstract Background Vertebrate hosts limit the availability of iron to microbial pathogens in order to nutritionally starve the invaders. The impact of iron deficiency induced by the iron chelator deferoxamine mesylate (DFO) was investigated in Atlantic salmon SHK-1 cells infected with the facultative intracellular bacterium Piscirickettsia salmonis. Results Effects of the DFO treatment and P. salmonis on SHK-1 cells were gaged by assessing cytopathic effects, bacterial load and activity, and gene expression profiles of eight immune biomarkers at 4- and 7-days post infection (dpi) in the control group, groups receiving single treatments (DFO or P. salmonis) and their combination. The chelator appears to be well-tolerated by host cells, while it had a negative impact on the number of bacterial cells and associated cytotoxicity. DFO alone had minor effects on gene expression of SHK-1 cells, including an early activation of IL-1β at 4 dpi. In contrast to few moderate changes induced by single treatments (either infection or chelator), most genes had highest upregulation in the infected groups receiving DFO. The mildest induction of hepcidin-1 (antimicrobial peptide precursor and regulator of iron homeostasis) was observed in cells exposed to DFO alone, followed by P. salmonis infected cells while the addition of DFO to infected cells further increased the mRNA abundance of this gene. Transcripts encoding TNF-α (immune signaling) and iNOS (immune effector) showed sustained increase at both time points in this group while cathelicidin-1 (immune effector) and IL-8 (immune signaling) were upregulated at 7 dpi. The stimulation of protective gene responses seen in infected cultures supplemented with DFO coincided with the reduction of bacterial load and activity (judged by the expression of P. salmonis 16S rRNA), and damage to cultured host cells. Conclusion The absence of immune gene activation under normal iron conditions suggests modulation of host responses by P. salmonis. The negative effect of iron deficiency on bacteria likely allowed host cells to respond in a more protective manner to the infection, further decreasing its progression. Presented findings encourage in vivo exploration of iron chelators as a promising strategy against piscirickettsiosis.

Plasmodium yoelii nigeriensis: the effect of high and low intensity of infection upon the egg production and bloodmeal size of Anopheles stephensi during three gonotrophic cycles

Parasitology ◽  
1995 ◽  
Vol 111 (5) ◽  
pp. 555-562 ◽  
Author(s):  
J. C. Hogg ◽  
H. Hurd
Keyword(s):  
Egg Production ◽  
Anopheles Stephensi ◽  
Plasmodium Yoelii ◽  
Infected Mosquito ◽  
Intensity Of Infection ◽  
Related Factors ◽  
Low Intensity ◽  
Mosquito Mortality ◽  
Fecundity Reduction

SUMMARYAnopheles stephensi mosquitoes showed a reduction in fecundity over 3 successive gonotrophic cycles, after becoming infected with Plasmodium yoelii nigeriensis. This effect could be observed at high oocyst burdens (> 75) or at low oocyst burdens (mean of 4·36). Mean bloodmeal size of the infected mosquitoes was significantly reduced only when feeding upon a mouse with a high gametocytaemia and the conversion of the bloodmeal into eggs by the infected mosquitoes was disrupted. Patterns of infected mosquito mortality, over the 3 gonotrophic cycles, varied with severity of infection. Although in 1 case increased mortality and decreased bloodmeal size may have affected fecundity, this could not have accounted for all of the observed fecundity reduction. We propose that other, unknown parasite related factors, are involved.

scholarly journals The hemolymph of Biomphalaria snail vectors of schistosomiasis supports a diverse microbiome

2020 ◽  
Author(s):  
Frédéric D. Chevalier ◽  
Robbie Diaz ◽  
Marina McDew-White ◽  
Timothy JC. Anderson ◽  
Winka Le Clec’h
Keyword(s):  
Close Contact ◽  
Bacterial Load ◽  
Illumina Miseq ◽  
Disease Vectors ◽  
Tsetse Flies ◽  
Miseq Sequencing ◽  
Microbiome Composition ◽  
Laboratory Populations ◽  
Health And Disease ◽  
Microorganism Community

SUMMARYThe microbiome – the microorganism community that is found on or within an organism’s body – is increasingly recognized to shape many aspects of its host biology and is a key determinant of health and disease. Microbiomes modulate the capacity of insect disease vectors (mosquitos, tsetse flies, sandflies) to transmit parasites and disease. We investigate the diversity and abundance of microorganisms within the hemolymph (i.e. blood) of Biomphalaria snails, the intermediate host for Schistosoma mansoni, using Illumina MiSeq sequencing of the bacterial 16S V4 rDNA. We sampled hemolymph from 5 snails from 6 different laboratory populations of B. glabrata and one population of B. alexandrina. We observed 279.84 ± 0.79 amplicon sequence variants (ASVs) per snail. There were significant differences in microbiome composition at the level of individual snails, snail populations and species. Snail microbiomes were dominated by Proteobacteria and Bacteroidetes while water microbiomes from snail tank were dominated by Actinobacteria. We investigated the absolute bacterial load using qPCR: hemolymph samples contained 2,784 ± 339 bacteria per μL. We speculate that the microbiome may represent a critical, but unexplored intermediary in the snail-schistosome interaction as hemolymph is in very close contact to the parasite at each step of its development.

scholarly journals Glucose transporter GLUT1 influences Plasmodium berghei infection in Anopheles stephensi

2020 ◽  
Author(s):  
Mengfei Wang ◽  
Jingwen Wang
Keyword(s):  
Transcriptome Analysis ◽  
Plasmodium Berghei ◽  
Anopheles Stephensi ◽  
Glucose Transporter ◽  
Vector Competence ◽  
Expression Patterns ◽  
Blood Feeding ◽  
Temporal Expression ◽  
Expression Of Genes ◽  
Global Influence

Abstract Background: Sugar feeding provides energy for mosquitoes. Facilitated glucose transporters (GLUTs) are responsible for the uptake of glucose in animals. However, the knowledge of GLUTs function in Anopheles mosquito is limited. Methods: Phylogenetic analysis of GLUTs in Anopheles stephensi (Asteglut) was performed by the maximum likelihood and Bayesian method. The spatial and temporal expression patterns of the four Astegluts were analyzed by qPCR. The function of Asteglut1 was examined using a dsRNA-mediated RNA interference method. Transcriptome analysis was used to investigate the global influence of Asteglut1 on mosquito physiology. Results: We identified 4 glut genes, Asteglut1 , Asteglutx , Asteglut3 and Asteglut4 in An. stephensi . Asteglut1, Asteglut3 and Asteglut4 were mainly expressed in the midgut. Plasmodium berghei infection differentially regulated the expression of Astegluts with significant downregulation of Asteglut1 and Asteglut4 , while upregulation of Asteglutx . Only knocking down Asteglut1 facilitated Plasmodium berghei infection in An. stephensi . This might be due to the accumulation of glucose prior to blood feeding in dsAsteglut1-treated mosquitoes. Our transcriptome analysis revealed that knockdown of Asteglut1 differentially regulated expression of genes associated with multiple functional clusters, especially those related to detoxification and immunity. The dysregulation of multiple pathways might contribute to the increased P. berghei infection. Conclusions: Our study shows that Asteglut1 participates in defense against P. berghei in An. stephensi . The regulation of Asteglut1 on vector competence might through modulating multiple biological processes, such as detoxification and immunity.

scholarly journals Glucose transporter GLUT1 influences Plasmodium berghei infection in Anopheles stephensi

2020 ◽  
Author(s):  
Mengfei Wang ◽  
Jingwen Wang
Keyword(s):  
Transcriptome Analysis ◽  
Plasmodium Berghei ◽  
Anopheles Stephensi ◽  
Glucose Transporter ◽  
Vector Competence ◽  
Expression Patterns ◽  
Blood Feeding ◽  
Temporal Expression ◽  
Expression Of Genes ◽  
Global Influence

Abstract Background: Sugar feeding provides energy for mosquitoes. Facilitated glucose transporters (GLUTs) are responsible for the uptake of glucose in animals. However, the knowledge of GLUTs function in Anopheles mosquito is limited. Methods: Phylogenetic analysis of GLUTs in Anopheles stephensi (Asteglut) was performed by the maximum likelihood and Bayesian method. The spatial and temporal expression patterns of the four Astegluts were analyzed by qPCR. The function of Asteglut1 was examined using a dsRNA-mediated RNA interference method. Transcriptome analysis was used to investigate the global influence of Asteglut1 on mosquito physiology. Results: We identified 4 glut genes, Asteglut1 , Asteglutx , Asteglut3 and Asteglut4 in An. stephensi . Asteglut1, Asteglut3 and Asteglut4 were mainly expressed in the midgut. Plasmodium berghei infection differentially regulated the expression of Astegluts with significant downregulation of Asteglut1 and Asteglut4 , while upregulation of Asteglutx . Only knocking down Asteglut1 significantly increased the susceptibility of An. stephensi to Plasmodium berghei infection. This might be due to the accumulation of glucose prior to blood feeding in dsAsteglut1-treated mosquitoes. Our transcriptome analysis revealed that knockdown of Asteglut1 differentially regulated expression of genes associated with multiple functional clusters including detoxification and immunity. The dysregulation of multiple pathways might contribute to the increased P. berghei infection. Conclusions: Our study shows that Asteglut1 is essential in defense against P. berghei in An. stephensi . The regulation of Asteglut1 on vector competence might through modulating multiple biological processes, including detoxification and immunity.

scholarly journals Glucose transporter Glut1 influences Plasmodium berghei infection in Anopheles stephensi

2020 ◽  
Author(s):  
Mengfei Wang ◽  
Jingwen Wang
Keyword(s):  
Transcriptome Analysis ◽  
Plasmodium Berghei ◽  
Serine Proteases ◽  
Anopheles Stephensi ◽  
Glucose Transporter ◽  
Vector Competence ◽  
Expression Patterns ◽  
Mosquito Vector ◽  
Temporal Expression ◽  
Expression Of Genes

Abstract Background Sugar feeding provides energy for mosquito. Facilitated glucose transporters (GLUT) are responsible for cellular uptake of glucose. However, the knowledge of GLUT function in Anopheles mosquito is limited. Methods Phylogenetic analysis of GLUTs in Anopheles stephensi (AsteGlut) was performed by the maximum likelihood and Bayesian method. The spatial and temporal expression patterns of three Astegluts were analyzed by qPCR. The function of AsteGlut1 was examined using a dsRNA-mediated RNA interference method. Transcriptome analysis was used to understand the influence of AsteGlut1 on mosquito vector competence. Results We identified 3 glut genes, Asteglut1 , Asteglutx and Asteglut3 in An. stephensi . Asteglut1 and Asteglut3 were mainly localized in the midgut. The expression of all three Astegluts were strongly induced after blood meal. All three genes were knocked down successfully, but only abrogation of Asteglut1 significantly increased the susceptibility of An. stephensi to Plasmodium berghei infection. Our transcriptome analysis revealed that knockdown of Asteglut1 differentially regulated expression of genes associated with the functional clusters including detoxification, serine proteases, and immunity. The dysregulation of multiple pathways might contribute to the increased P. berghei infection. Conclusions Our study shows that Asteglut1 plays a role in defense against P. berghei in An. stephensi . The regulation of Asteglut1 on vector competence might through modulating multiple biological processes, including detoxification and immunity.

Variation in the vector competence of Aedes polynesiensis for Wuchereria bancrofti

Parasitology ◽  
1995 ◽  
Vol 111 (1) ◽  
pp. 19-29 ◽  
Author(s):  
A.-B. Failloux ◽  
M. Raymond ◽  
A. Ung ◽  
P. Glaziou ◽  
P. M. V. Martin ◽  
...  
Keyword(s):  
Mortality Rate ◽  
Experimental Infection ◽  
Vector Competence ◽  
Geographic Origin ◽  
Wuchereria Bancrofti ◽  
Infective Stage ◽  
Intermediate Hosts ◽  
Mosquito Mortality ◽  
Aedes Polynesiensis ◽  
Different Levels

SUMMARYThe vector competences of 6 geographic strains of Aedes polynesiensis for Wuchereria bancrofti were studied using two types of experimental infections. Experimental infection of laboratory-bred mosquitoes fed on the carriers' forearms with different levels of microfilaraemia showed that microfilariae (mf) uptake was directly proportional to the carrier's mf density and, as mf densities decreased, concentration capacity of Ae. polynesiensis increased. It was also shown that infection has an important effect on mosquito mortality, and that the mortality rate differed among mosquito strains. In infections using artificial feeders, the mf uptake was closely regulated, thus showing differences in the vectorial efficiency of Ae. polynesiensis related to the geographic origin of the mosquito strain. The mosquitoes from the Society archipelago were more efficient intermediate hosts than geographically distant strains when infected with W. bancrofti from an island within the archipelago (Tahiti). Mosquito strains from the Society archipelago developed the highest proportion of infective-stage larvae and exhibited the lowest mortality rate when infected with sympatric Tahitian W. bancrofti.

scholarly journals Poor vector competence of the human flea, Pulex irritans, to transmit Yersinia pestis

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Adélaïde Miarinjara ◽  
David M. Bland ◽  
James R. Belthoff ◽  
B. Joseph Hinnebusch
Keyword(s):  
Human Blood ◽  
Blood Meal ◽  
Vector Competence ◽  
Bacterial Load ◽  
Bacterial Biofilm ◽  
Rat Blood ◽  
Pulex Irritans ◽  
High Prevalence ◽  
Phase Transmission ◽  
Host Blood

Abstract Background The human flea, Pulex irritans, is widespread globally and has a long association with humans, one of its principal hosts. Its role in plague transmission is still under discussion, although its high prevalence in plague-endemic regions and the presence of infected fleas of this species during plague outbreaks has led to proposals that it has been a significant vector in human-to-human transmission in some historical and present-day epidemiologic situations. However, based on a limited number of studies, P. irritans is considered to be a poor vector and receives very little attention from public health policymakers. In this study we examined the vector competence of P. irritans collected from foxes and owls in the western United States, using a standard protocol and artificial infection system. Methods Wild-caught fleas were maintained in the laboratory and infected by allowing them to feed on human or rat blood containing 2 × 108 to 1 × 109Y. pestis/ml. The fleas were then monitored periodically for infection rate and bacterial load, mortality, feeding rate, bacterial biofilm formation in the foregut (proventricular blockage), and ability to transmit Y. pestis after their single infectious blood meal. Results P. irritans were susceptible to infection, with more than 30% maintaining high bacterial loads for up to 20 days. Transmission during this time was infrequent and inefficient, however. Consistent with previous studies, a low level of early-phase transmission (3 days after the infectious blood meal) was detected in some trials. Transmission at later time points was also sporadic, and the incidence of proventricular blockage, required for this mode of transmission, was low in fleas infected using rat blood and never occurred in fleas infected using human blood. The highest level of blockage and transmission was seen in fleas infected using rat blood and allowed to feed intermittently rather than daily, indicating that host blood and feeding frequency influence vector competence. Conclusions Our results affirm the reputation of P. irritans as a feeble vector compared to rodent flea species examined similarly, and its vector competence may be lower when infected by feeding on bacteremic human blood. Graphic abstract

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