Publications by authors named "Aurelien Vigneron"

25 Publications

  • Page 1 of 1

Paratransgenic manipulation of a tsetse microRNA alters the physiological homeostasis of the fly's midgut environment.

PLoS Pathog 2021 Jun 9;17(6):e1009475. Epub 2021 Jun 9.

Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, Connecticut, United States of America.

Tsetse flies are vectors of parasitic African trypanosomes, the etiological agents of human and animal African trypanosomoses. Current disease control methods include fly-repelling pesticides, fly trapping, and chemotherapeutic treatment of infected people and animals. Inhibiting tsetse's ability to transmit trypanosomes by strengthening the fly's natural barriers can serve as an alternative approach to reduce disease. The peritrophic matrix (PM) is a chitinous and proteinaceous barrier that lines the insect midgut and serves as a protective barrier that inhibits infection with pathogens. African trypanosomes must cross tsetse's PM in order to establish an infection in the fly, and PM structural integrity negatively correlates with trypanosome infection outcomes. Bloodstream form trypanosomes shed variant surface glycoproteins (VSG) into tsetse's gut lumen early during the infection establishment, and free VSG molecules are internalized by the fly's PM-producing cardia. This process results in a reduction in the expression of a tsetse microRNA (miR275) and a sequential molecular cascade that compromises PM integrity. miRNAs are small non-coding RNAs that are critical in regulating many physiological processes. In the present study, we investigated the role(s) of tsetse miR275 by developing a paratransgenic expression system that employs tsetse's facultative bacterial endosymbiont, Sodalis glossinidius, to express tandem antagomir-275 repeats (or miR275 sponges). This system induces a constitutive, 40% reduction in miR275 transcript abundance in the fly's midgut and results in obstructed blood digestion (gut weights increased by 52%), a significant increase (p-value < 0.0001) in fly survival following infection with an entomopathogenic bacteria, and a 78% increase in trypanosome infection prevalence. RNA sequencing of cardia and midgut tissues from paratransgenic tsetse confirmed that miR275 regulates processes related to the expression of PM-associated proteins and digestive enzymes as well as genes that encode abundant secretory proteins. Our study demonstrates that paratransgenesis can be employed to study microRNA regulated pathways in arthropods that house symbiotic bacteria.
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http://dx.doi.org/10.1371/journal.ppat.1009475DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8216540PMC
June 2021

Single-cell RNA sequencing of from tsetse salivary glands unveils metacyclogenesis and identifies potential transmission blocking antigens.

Proc Natl Acad Sci U S A 2020 02 21;117(5):2613-2621. Epub 2020 Jan 21.

Department of Epidemiology of Microbial Diseases, Yale School of Public Health, Yale University, New Haven, CT 06520;

Tsetse-transmitted African trypanosomes must develop into mammalian-infectious metacyclic cells in the fly's salivary glands (SGs) before transmission to a new host. The molecular mechanisms that underlie this developmental process, known as metacyclogenesis, are poorly understood. Blocking the few metacyclic parasites deposited in saliva from further development in the mammal could prevent disease. To obtain an in-depth perspective of metacyclogenesis, we performed single-cell RNA sequencing (scRNA-seq) from a pool of 2,045 parasites collected from infected tsetse SGs. Our data revealed three major cell clusters that represent the epimastigote, and pre- and mature metacyclic trypanosome developmental stages. Individual cell level data also confirm that the metacyclic pool is diverse, and that each parasite expresses only one of the unique metacyclic variant surface glycoprotein (mVSG) coat protein transcripts identified. Further clustering of cells revealed a dynamic transcriptomic and metabolic landscape reflective of a developmental program leading to infectious metacyclic forms preadapted to survive in the mammalian host environment. We describe the expression profile of proteins that regulate gene expression and that potentially play a role in metacyclogenesis. We also report on a family of nonvariant surface proteins (Fam10) and demonstrate surface localization of one member (named SGM1.7) on mature metacyclic parasites. Vaccination of mice with recombinant SGM1.7 reduced parasitemia early in the infection. Future studies are warranted to investigate Fam10 family proteins as potential trypanosome transmission blocking vaccine antigens. Our experimental approach is translationally relevant for developing strategies to prevent other insect saliva-transmitted parasites from infecting and causing disease in mammalian hosts.
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http://dx.doi.org/10.1073/pnas.1914423117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7007551PMC
February 2020

Blind killing of both male and female Drosophila embryos by a natural variant of the endosymbiotic bacterium Spiroplasma poulsonii.

Cell Microbiol 2020 05 23;22(5):e13156. Epub 2020 Jan 23.

Global Health Institute, School of Life Sciences, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland.

Spiroplasma poulsonii is a vertically transmitted endosymbiont of Drosophila melanogaster that causes male-killing, that is the death of infected male embryos during embryogenesis. Here, we report a natural variant of S. poulsonii that is efficiently vertically transmitted yet does not selectively kill males, but kills rather a subset of all embryos regardless of their sex, a phenotype we call 'blind-killing'. We show that the natural plasmid of S. poulsonii has an altered structure: Spaid, the gene coding for the male-killing toxin, is deleted in the blind-killing strain, confirming its function as a male-killing factor. Then we further investigate several hypotheses that could explain the sex-independent toxicity of this new strain on host embryos. As the second non-male-killing variant isolated from a male-killing original population, this new strain raises questions on how male-killing is maintained or lost in fly populations. As a natural knock-out of Spaid, which is unachievable yet by genetic engineering approaches, this variant also represents a valuable tool for further investigations on the male-killing mechanism.
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http://dx.doi.org/10.1111/cmi.13156DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7187355PMC
May 2020

Increased temperatures reduce the vectorial capacity of mosquitoes for Zika virus.

Emerg Microbes Infect 2020 2;9(1):67-77. Epub 2020 Jan 2.

Griffin Laboratory, New York State Department of Health, Slingerlands, NY, USA.

Rapid and significant range expansion of both Zika virus (ZIKV) and its vector species has resulted in ZIKV being declared a global health threat. Mean temperatures are projected to increase globally, likely resulting in alterations of the transmission potential of mosquito-borne pathogens. To understand the effect of diurnal temperature range on the vectorial capacity of and for ZIKV, longevity, blood-feeding and vector competence were assessed at two temperature regimes following feeding on infectious blood meals. Higher temperatures resulted in decreased longevity of [Log-rank test, χ2, df 35.66, 5, < 0.001] and a decrease in blood-feeding rates of [Fisher's exact test, < 0.001]. Temperature had a population and species-specific impact on ZIKV infection rates. Overall, reared at the lowest temperature regime demonstrated the highest vectorial capacity (0.53) and the highest transmission efficiency (57%). Increased temperature decreased vectorial capacity across groups yet more significant effects were measured with relative to . The results of this study suggest that future increases in temperature in the Americas could significantly impact vector competence, blood-feeding and longevity, and potentially decrease the overall vectorial capacity of mosquitoes in the Americas.
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http://dx.doi.org/10.1080/22221751.2019.1707125DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6968261PMC
March 2020

Comparative genomic analysis of six Glossina genomes, vectors of African trypanosomes.

Genome Biol 2019 09 2;20(1):187. Epub 2019 Sep 2.

McDonnell Genome Institute, Washington University School of Medicine, St. Louis, MO, USA.

Background: Tsetse flies (Glossina sp.) are the vectors of human and animal trypanosomiasis throughout sub-Saharan Africa. Tsetse flies are distinguished from other Diptera by unique adaptations, including lactation and the birthing of live young (obligate viviparity), a vertebrate blood-specific diet by both sexes, and obligate bacterial symbiosis. This work describes the comparative analysis of six Glossina genomes representing three sub-genera: Morsitans (G. morsitans morsitans, G. pallidipes, G. austeni), Palpalis (G. palpalis, G. fuscipes), and Fusca (G. brevipalpis) which represent different habitats, host preferences, and vectorial capacity.

Results: Genomic analyses validate established evolutionary relationships and sub-genera. Syntenic analysis of Glossina relative to Drosophila melanogaster shows reduced structural conservation across the sex-linked X chromosome. Sex-linked scaffolds show increased rates of female-specific gene expression and lower evolutionary rates relative to autosome associated genes. Tsetse-specific genes are enriched in protease, odorant-binding, and helicase activities. Lactation-associated genes are conserved across all Glossina species while male seminal proteins are rapidly evolving. Olfactory and gustatory genes are reduced across the genus relative to other insects. Vision-associated Rhodopsin genes show conservation of motion detection/tracking functions and variance in the Rhodopsin detecting colors in the blue wavelength ranges.

Conclusions: Expanded genomic discoveries reveal the genetics underlying Glossina biology and provide a rich body of knowledge for basic science and disease control. They also provide insight into the evolutionary biology underlying novel adaptations and are relevant to applied aspects of vector control such as trap design and discovery of novel pest and disease control strategies.
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http://dx.doi.org/10.1186/s13059-019-1768-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6721284PMC
September 2019

Spatio-temporal distribution of Spiroplasma infections in the tsetse fly (Glossina fuscipes fuscipes) in northern Uganda.

PLoS Negl Trop Dis 2019 08 1;13(8):e0007340. Epub 2019 Aug 1.

Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, CT, United States of America.

Tsetse flies (Glossina spp.) are vectors of parasitic trypanosomes, which cause human (HAT) and animal African trypanosomiasis (AAT) in sub-Saharan Africa. In Uganda, Glossina fuscipes fuscipes (Gff) is the main vector of HAT, where it transmits Gambiense disease in the northwest and Rhodesiense disease in central, southeast and western regions. Endosymbionts can influence transmission efficiency of parasites through their insect vectors via conferring a protective effect against the parasite. It is known that the bacterium Spiroplasma is capable of protecting its Drosophila host from infection with a parasitic nematode. This endosymbiont can also impact its host's population structure via altering host reproductive traits. Here, we used field collections across 26 different Gff sampling sites in northern and western Uganda to investigate the association of Spiroplasma with geographic origin, seasonal conditions, Gff genetic background and sex, and trypanosome infection status. We also investigated the influence of Spiroplasma on Gff vector competence to trypanosome infections under laboratory conditions. Generalized linear models (GLM) showed that Spiroplasma probability was correlated with the geographic origin of Gff host and with the season of collection, with higher prevalence found in flies within the Albert Nile (0.42 vs 0.16) and Achwa River (0.36 vs 0.08) watersheds and with higher prevalence detected in flies collected in the intermediate than wet season. In contrast, there was no significant correlation of Spiroplasma prevalence with Gff host genetic background or sex once geographic origin was accounted for in generalized linear models. Additionally, we found a potential negative correlation of Spiroplasma with trypanosome infection, with only 2% of Spiroplasma infected flies harboring trypanosome co-infections. We also found that in a laboratory line of Gff, parasitic trypanosomes are less likely to colonize the midgut in individuals that harbor Spiroplasma infection. These results indicate that Spiroplasma infections in tsetse may be maintained by not only maternal but also via horizontal transmission routes, and Spiroplasma infections may also have important effects on trypanosome transmission efficiency of the host tsetse. Potential functional effects of Spiroplasma infection in Gff could have impacts on vector control approaches to reduce trypanosome infections.
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http://dx.doi.org/10.1371/journal.pntd.0007340DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6692048PMC
August 2019

Mutualist-Provisioned Resources Impact Vector Competency.

mBio 2019 06 4;10(3). Epub 2019 Jun 4.

Department of Epidemiology of Microbial Diseases, Yale School of Public Health, Yale University, New Haven, Connecticut, USA

Many symbionts supplement their host's diet with essential nutrients. However, whether these nutrients also enhance parasitism is unknown. In this study, we investigated whether folate (vitamin B) production by the tsetse fly ( spp.) essential mutualist, , aids auxotrophic African trypanosomes in completing their life cycle within this obligate vector. We show that the expression of folate biosynthesis genes changes with the progression of trypanosome infection within tsetse. The disruption of folate production caused a reduction in the percentage of flies that housed midgut (MG) trypanosome infections. However, decreased folate did not prevent MG trypanosomes from migrating to and establishing an infection in the fly's salivary glands, thus suggesting that nutrient requirements vary throughout the trypanosome life cycle. We further substantiated that trypanosomes rely on symbiont-generated folate by feeding this vitamin to , which exhibits low trypanosome vector competency and houses incapable of producing folate. Folate-supplemented flies were significantly more susceptible to trypanosome infection, further demonstrating that this vitamin facilitates parasite infection establishment. Our cumulative results provide evidence that provides a key metabolite (folate) that is "hijacked" by trypanosomes to enhance their infectivity, thus indirectly impacting tsetse species vector competency. Parasite dependence on symbiont-derived micronutrients, which likely also occurs in other arthropod vectors, represents a relationship that may be exploited to reduce disease transmission. Parasites elicit several physiological changes in their host to enhance transmission. Little is known about the functional association between parasitism and microbiota-provisioned resources typically dedicated to animal hosts and how these goods may be rerouted to optimize parasite development. This study is the first to identify a specific symbiont-generated metabolite that impacts insect vector competence by facilitating parasite establishment and, thus, eventual transmission. Specifically, we demonstrate that the tsetse fly obligate mutualist provisions folate (vitamin B) that pathogenic African trypanosomes exploit in an effort to successfully establish an infection in the vector's MG. This process is essential for the parasite to complete its life cycle and be transmitted to a new vertebrate host. Disrupting metabolic contributions provided by the microbiota of arthropod disease vectors may fuel future innovative control strategies while also offering minimal nontarget effects.
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http://dx.doi.org/10.1128/mBio.00018-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6550517PMC
June 2019

Immune Defenses of a Beneficial Pest: The Mealworm Beetle, .

Front Physiol 2019 12;10:138. Epub 2019 Mar 12.

UMR CNRS 6282 BioGéoSciences, Équipe Écologie Évolutive, Université Bourgogne-Franche Comté, Dijon, France.

The mealworm beetle, , is currently considered as a pest when infesting stored grains or grain products. However, mealworms are now being promoted as a beneficial insect because their high nutrient content makes them a viable food source and because they are capable of degrading polystyrene and plastic waste. These attributes make attractive for mass rearing, which may promote disease transmission within the insect colonies. Disease resistance is of paramount importance for both the control and the culture of mealworms, and several biotic and abiotic environmental factors affect the success of their anti-parasitic defenses, both positively and negatively. After providing a detailed description of 's anti-parasitic defenses, we review the main biotic and abiotic environmental factors that alter their presentation, and we discuss their implications for the purpose of controlling the development and health of this insect.
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http://dx.doi.org/10.3389/fphys.2019.00138DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6422893PMC
March 2019

Weevil prevents endosymbiont TCT dissemination and chronic host systemic immune activation.

Proc Natl Acad Sci U S A 2019 03 28;116(12):5623-5632. Epub 2019 Feb 28.

UMR0203, Biologie Fonctionnelle, Insectes et Interactions (BF2i), Institut National des Sciences Appliquées de Lyon (INSA-Lyon), Institut National de la Recherche Agronomique (INRA), University of Lyon (Univ Lyon), F-69621 Villeurbanne, France;

Long-term intracellular symbiosis (or endosymbiosis) is widely distributed across invertebrates and is recognized as a major driving force in evolution. However, the maintenance of immune homeostasis in organisms chronically infected with mutualistic bacteria is a challenging task, and little is known about the molecular processes that limit endosymbiont immunogenicity and host inflammation. Here, we investigated peptidoglycan recognition protein (PGRP)-encoding genes in the cereal weevil 's association with endosymbiont. We discovered that weevil generates three transcripts via alternative splicing and differential regulation. A secreted isoform is expressed in insect tissues under pathogenic conditions through activation of the PGRP-LC receptor of the immune deficiency pathway. In addition, cytosolic and transmembrane isoforms are permanently produced within endosymbiont-bearing organ, the bacteriome, in a PGRP-LC-independent manner. Bacteriome isoforms specifically cleave the tracheal cytotoxin (TCT), a peptidoglycan monomer released by endosymbionts. silencing by RNAi results in TCT escape from the bacteriome to other insect tissues, where it chronically activates the host systemic immunity through PGRP-LC. While such immune deregulations did not impact endosymbiont load, they did negatively affect host physiology, as attested by a diminished sexual maturation of adult weevils. Whereas was first described in pathogenic interactions, this work shows that, in an endosymbiosis context, specific bacteriome isoforms have evolved, allowing endosymbiont TCT scavenging and preventing chronic endosymbiont-induced immune responses, thus promoting host homeostasis.
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http://dx.doi.org/10.1073/pnas.1821806116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6431197PMC
March 2019

Colonization of the tsetse fly midgut with commensal Kosakonia cowanii Zambiae inhibits trypanosome infection establishment.

PLoS Pathog 2019 02 28;15(2):e1007470. Epub 2019 Feb 28.

Yale School of Public Health, Department of Epidemiology of Microbial Diseases, New Haven, Connecticut, United States of America.

Tsetse flies (Glossina spp.) vector pathogenic trypanosomes (Trypanosoma spp.) in sub-Saharan Africa. These parasites cause human and animal African trypanosomiases, which are debilitating diseases that inflict an enormous socio-economic burden on inhabitants of endemic regions. Current disease control strategies rely primarily on treating infected animals and reducing tsetse population densities. However, relevant programs are costly, labor intensive and difficult to sustain. As such, novel strategies aimed at reducing tsetse vector competence require development. Herein we investigated whether Kosakonia cowanii Zambiae (Kco_Z), which confers Anopheles gambiae with resistance to Plasmodium, is able to colonize tsetse and induce a trypanosome refractory phenotype in the fly. Kco_Z established stable infections in tsetse's gut and exhibited no adverse effect on the fly's survival. Flies with established Kco_Z infections in their gut were significantly more refractory to infection with two distinct trypanosome species (T. congolense, 6% infection; T. brucei, 32% infection) than were age-matched flies that did not house the exogenous bacterium (T. congolense, 36% infected; T. brucei, 70% infected). Additionally, 52% of Kco_Z colonized tsetse survived infection with entomopathogenic Serratia marcescens, compared with only 9% of their wild-type counterparts. These parasite and pathogen refractory phenotypes result from the fact that Kco_Z acidifies tsetse's midgut environment, which inhibits trypanosome and Serratia growth and thus infection establishment. Finally, we determined that Kco_Z infection does not impact the fecundity of male or female tsetse, nor the ability of male flies to compete with their wild-type counterparts for mates. We propose that Kco_Z could be used as one component of an integrated strategy aimed at reducing the ability of tsetse to transmit pathogenic trypanosomes.
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http://dx.doi.org/10.1371/journal.ppat.1007470DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6394900PMC
February 2019

What can a weevil teach a fly, and reciprocally? Interaction of host immune systems with endosymbionts in Glossina and Sitophilus.

BMC Microbiol 2018 11 23;18(Suppl 1):150. Epub 2018 Nov 23.

Univ Lyon, INSA-Lyon, INRA, BF2I, UMR0203, F-69621, Villeurbanne, France.

The tsetse fly (Glossina genus) is the main vector of African trypanosomes, which are protozoan parasites that cause human and animal African trypanosomiases in Sub-Saharan Africa. In the frame of the IAEA/FAO program 'Enhancing Vector Refractoriness to Trypanosome Infection', in addition to the tsetse, the cereal weevil Sitophilus has been introduced as a comparative system with regards to immune interactions with endosymbionts. The cereal weevil is an agricultural pest that destroys a significant proportion of cereal stocks worldwide. Tsetse flies are associated with three symbiotic bacteria, the multifunctional obligate Wigglesworthia glossinidia, the facultative commensal Sodalis glossinidius and the parasitic Wolbachia. Cereal weevils house an obligatory nutritional symbiosis with the bacterium Sodalis pierantonius, and occasionally Wolbachia. Studying insect host-symbiont interactions is highly relevant both for understanding the evolution of symbiosis and for envisioning novel pest control strategies. In both insects, the long co-evolution between host and endosymbiont has led to a stringent integration of the host-bacteria partnership. These associations were facilitated by the development of specialized host traits, including symbiont-housing cells called bacteriocytes and specific immune features that enable both tolerance and control of the bacteria. In this review, we compare the tsetse and weevil model systems and compile the latest research findings regarding their biological and ecological similarities, how the immune system controls endosymbiont load and location, and how host-symbiont interactions impact developmental features including cuticle synthesis and immune system maturation. We focus mainly on the interactions between the obligate symbionts and their host's immune systems, a central theme in both model systems. Finally, we highlight how parallel studies on cereal weevils and tsetse flies led to mutual discoveries and stimulated research on each model, creating a pivotal example of scientific improvement through comparison between relatively distant models.
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http://dx.doi.org/10.1186/s12866-018-1278-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6251153PMC
November 2018

Expression profiling of Trypanosoma congolense genes during development in the tsetse fly vector Glossina morsitans morsitans.

Parasit Vectors 2018 Jul 3;11(1):380. Epub 2018 Jul 3.

Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, CT, USA.

Background: The tsetse transmitted parasitic flagellate Trypanosoma congolense causes animal African trypanosomosis (AAT) across sub-Saharan Africa. AAT negatively impacts agricultural, economic, nutritional and subsequently, health status of the affected populace. The molecular mechanisms that underlie T. congolense's developmental program within tsetse are largely unknown due to considerable challenges with obtaining sufficient parasite cells to perform molecular studies.

Methods: In this study, we used RNA-seq to profile T. congolense gene expression during development in two distinct tsetse tissues, the cardia and proboscis. Indirect immunofluorescent antibody test (IFA) and confocal laser scanning microscope was used to localize the expression of a putative protein encoded by the hypothetical protein (TcIL3000_0_02370).

Results: Consistent with current knowledge, genes coding several variant surface glycoproteins (including metacyclic specific VSGs), and the surface coat protein, congolense epimastigote specific protein, were upregulated in parasites in the proboscis (PB-parasites). Additionally, our results indicate that parasites in tsetse's cardia (C-parasites) and PB employ oxidative phosphorylation and amino acid metabolism for energy. Several genes upregulated in C-parasites encoded receptor-type adenylate cyclases, surface carboxylate transporter family proteins (or PADs), transport proteins, RNA-binding proteins and procyclin isoforms. Gene ontology analysis of products of genes upregulated in C-parasites showed enrichment of terms broadly associated with nucleotides, microtubules, cell membrane and its components, cell signaling, quorum sensing and several transport activities, suggesting that the parasites colonizing the cardia may monitor their environment and regulate their density and movement in this tissue. Additionally, cell surface protein (CSP) encoding genes associated with the Fam50 'GARP', 'iii' and 'i' subfamilies were also significantly upregulated in C-parasites, suggesting that they are important for the long non-dividing trypomastigotes to colonize tsetse's cardia. The putative products of genes that were upregulated in PB-parasites were linked to nucleosomes, cytoplasm and membrane-bound organelles, which suggest that parasites in this niche undergo cell division in line with prior findings. Most of the CSPs upregulated in PB-parasites were hypothetical, thus requiring further functional characterization. Expression of one such hypothetical protein (TcIL3000_0_02370) was analyzed using immunofluorescence and confocal laser scanning microscopy, which together revealed preferential expression of this protein on the entire surface coat of T. congolense parasite stages that colonize G. m. morsitans' proboscis.

Conclusion: Collectively, our results provide insight into T. congolense gene expression profiles in distinct niches within the tsetse vector. Our results show that the hypothetical protein TcIL3000_0_02370, is expressed on the entire surface of the trypanosomes inhabiting tsetse's proboscis. We discuss our results in terms of their relevance to disease transmission processes.
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http://dx.doi.org/10.1186/s13071-018-2964-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6029126PMC
July 2018

A fine-tuned vector-parasite dialogue in tsetse's cardia determines peritrophic matrix integrity and trypanosome transmission success.

PLoS Pathog 2018 04 3;14(4):e1006972. Epub 2018 Apr 3.

Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, Connecticut, United States of America.

Arthropod vectors have multiple physical and immunological barriers that impede the development and transmission of parasites to new vertebrate hosts. These barriers include the peritrophic matrix (PM), a chitinous barrier that separates the blood bolus from the midgut epithelia and modulates vector-pathogens interactions. In tsetse flies, a sleeve-like PM is continuously produced by the cardia organ located at the fore- and midgut junction. African trypanosomes, Trypanosoma brucei, must bypass the PM twice; first to colonize the midgut and secondly to reach the salivary glands (SG), to complete their transmission cycle in tsetse. However, not all flies with midgut infections develop mammalian transmissible SG infections-the reasons for which are unclear. Here, we used transcriptomics, microscopy and functional genomics analyses to understand the factors that regulate parasite migration from midgut to SG. In flies with midgut infections only, parasites fail to cross the PM as they are eliminated from the cardia by reactive oxygen intermediates (ROIs)-albeit at the expense of collateral cytotoxic damage to the cardia. In flies with midgut and SG infections, expression of genes encoding components of the PM is reduced in the cardia, and structural integrity of the PM barrier is compromised. Under these circumstances trypanosomes traverse through the newly secreted and compromised PM. The process of PM attrition that enables the parasites to re-enter into the midgut lumen is apparently mediated by components of the parasites residing in the cardia. Thus, a fine-tuned dialogue between tsetse and trypanosomes at the cardia determines the outcome of PM integrity and trypanosome transmission success.
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http://dx.doi.org/10.1371/journal.ppat.1006972DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5898766PMC
April 2018

Uncovering Genomic Regions Associated with Infections in Wild Populations of the Tsetse Fly .

G3 (Bethesda) 2018 03 2;8(3):887-897. Epub 2018 Mar 2.

Department of Ecology and Evolutionary Biology, Yale University, New Haven, Connecticut 06511.

Vector-borne diseases are responsible for > 1 million deaths every year but genomic resources for most species responsible for their transmission are limited. This is true for neglected diseases such as sleeping sickness (Human African Trypanosomiasis), a disease caused by parasites vectored by several species of tseste flies within the genus We describe an integrative approach that identifies statistical associations between trypanosome infection status of () flies from Uganda, for which functional studies are complicated because the species cannot be easily maintained in laboratory colonies, and ∼73,000 polymorphic sites distributed across the genome. Then, we identify candidate genes involved in trypanosome susceptibility by taking advantage of genomic resources from a closely related species, (). We compiled a comprehensive transcript library from 72 published and unpublished RNAseq experiments of trypanosome-infected and uninfected flies, and improved the current transcriptome assembly. This new assembly was then used to enhance the functional annotations on the genome. As a consequence, we identified 56 candidate genes in the vicinity of the 18 regions associated with infection status in Twenty-nine of these genes were differentially expressed (DE) among parasite-infected and uninfected , suggesting that their orthologs in may correlate with disease transmission. These genes were involved in DNA regulation, neurophysiological functions, and immune responses. We highlight the power of integrating population and functional genomics from related species to enhance our understanding of the genetic basis of physiological traits, particularly in nonmodel organisms.
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http://dx.doi.org/10.1534/g3.117.300493DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5844309PMC
March 2018

Molecular characterization of tsetse's proboscis and its response to Trypanosoma congolense infection.

PLoS Negl Trop Dis 2017 Nov 20;11(11):e0006057. Epub 2017 Nov 20.

Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, CT, United States of America.

Tsetse flies (Glossina spp.) transmit parasitic African trypanosomes (Trypanosoma spp.), including Trypanosoma congolense, which causes animal African trypanosomiasis (AAT). AAT detrimentally affects agricultural activities in sub-Saharan Africa and has negative impacts on the livelihood and nutrient availability for the affected communities. After tsetse ingests an infectious blood meal, T. congolense sequentially colonizes the fly's gut and proboscis (PB) organs before being transmitted to new mammalian hosts during subsequent feedings. Despite the importance of PB in blood feeding and disease transmission, little is known about its molecular composition, function and response to trypanosome infection. To bridge this gap, we used RNA-seq analysis to determine its molecular characteristics and responses to trypanosome infection. By comparing the PB transcriptome to whole head and midgut transcriptomes, we identified 668 PB-enriched transcripts that encoded proteins associated with muscle tissue, organ development, chemosensation and chitin-cuticle structure development. Moreover, transcripts encoding putative mechanoreceptors that monitor blood flow during tsetse feeding and interact with trypanosomes were also expressed in the PB. Microscopic analysis of the PB revealed cellular structures associated with muscles and cells. Infection with T. congolense resulted in increased and decreased expression of 38 and 88 transcripts, respectively. Twelve of these differentially expressed transcripts were PB-enriched. Among the transcripts induced upon infection were those encoding putative proteins associated with cell division function(s), suggesting enhanced tissue renewal, while those suppressed were associated with metabolic processes, extracellular matrix and ATP-binding as well as immunity. These results suggest that PB is a muscular organ with chemosensory and mechanosensory capabilities. The mechanoreceptors may be point of PB-trypanosomes interactions. T. congolense infection resulted in reduced metabolic and immune capacity of the PB. The molecular knowledge on the composition and putative functions of PB forms the foundation to identify new targets to disrupt tsetse's ability to feed and parasite transmission.
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http://dx.doi.org/10.1371/journal.pntd.0006057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5695773PMC
November 2017

Unravelling the relationship between the tsetse fly and its obligate symbiont : transcriptomic and metabolomic landscapes reveal highly integrated physiological networks.

Proc Biol Sci 2017 Jun;284(1857)

Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, CT 06520, USA

Insects with restricted diets rely on obligate microbes to fulfil nutritional requirements essential for biological function. Tsetse flies, vectors of African trypanosome parasites, feed exclusively on vertebrate blood and harbour the obligate endosymbiont Without , tsetse are unable to reproduce. These symbionts are sheltered within specialized cells (bacteriocytes) that form the midgut-associated bacteriome organ. To decipher the core functions of this symbiosis essential for tsetse's survival, we performed dual-RNA-seq analysis of the bacteriome, coupled with metabolomic analysis of bacteriome and haemolymph collected from normal and symbiont-cured (sterile) females. Bacteriocytes produce immune regulatory peptidoglycan recognition protein () that protects , and a multivitamin transporter () that can aid in nutrient dissemination. overexpress a molecular chaperone (GroEL) to augment their translational/transport machinery and biosynthesize an abundance of B vitamins (specifically B-, B-, B- and B-associated metabolites) to supplement the host's nutritionally deficient diet. The absence of contributions disrupts multiple metabolic pathways impacting carbohydrate and amino acid metabolism. These disruptions affect the dependent downstream processes of nucleotide biosynthesis and metabolism and biosynthesis of -adenosyl methionine (SAM), an essential cofactor. This holistic fundamental knowledge of the symbiotic dialogue highlights new biological targets for the development of innovative vector control methods.
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http://dx.doi.org/10.1098/rspb.2017.0360DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5489720PMC
June 2017

Symbiont-induced odorant binding proteins mediate insect host hematopoiesis.

Elife 2017 01 12;6. Epub 2017 Jan 12.

Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, United States.

Symbiotic bacteria assist in maintaining homeostasis of the animal immune system. However, the molecular mechanisms that underlie symbiont-mediated host immunity are largely unknown. Tsetse flies ( spp.) house maternally transmitted symbionts that regulate the development and function of their host's immune system. Herein we demonstrate that the obligate mutualist, , up-regulates expression of in the gut of intrauterine tsetse larvae. This process is necessary and sufficient to induce systemic expression of the hematopoietic RUNX transcription factor and the subsequent production of crystal cells, which actuate the melanotic immune response in adult tsetse. Larval indigenous microbiota, which is acquired from the environment, regulates an orthologous hematopoietic pathway in their host. These findings provide insight into the molecular mechanisms that underlie enteric symbiont-stimulated systemic immune system development, and indicate that these processes are evolutionarily conserved despite the divergent nature of host-symbiont interactions in these model systems.
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http://dx.doi.org/10.7554/eLife.19535DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5231409PMC
January 2017

Transcriptome Profiling of Trypanosoma brucei Development in the Tsetse Fly Vector Glossina morsitans.

PLoS One 2016 21;11(12):e0168877. Epub 2016 Dec 21.

Department of Epidemiology of Microbial Diseases, School of Public Health, Yale University, New Haven, Connecticut, United States of America.

African trypanosomes, the causative agents of sleeping sickness in humans and nagana in animals, have a complex digenetic life cycle between a mammalian host and an insect vector, the blood-feeding tsetse fly. Although the importance of the insect vector to transmit the disease was first realized over a century ago, many aspects of trypanosome development in tsetse have not progressed beyond a morphological analysis, mainly due to considerable challenges to obtain sufficient material for molecular studies. Here, we used high-throughput RNA-Sequencing (RNA-Seq) to profile Trypanosoma brucei transcript levels in three distinct tissues of the tsetse fly, namely the midgut, proventriculus and salivary glands. Consistent with current knowledge and providing a proof of principle, transcripts coding for procyclin isoforms and several components of the cytochrome oxidase complex were highly up-regulated in the midgut transcriptome, whereas transcripts encoding metacyclic VSGs (mVSGs) and the surface coat protein brucei alanine rich protein or BARP were extremely up-regulated in the salivary gland transcriptome. Gene ontology analysis also supported the up-regulation of biological processes such as DNA metabolism and DNA replication in the proventriculus transcriptome and major changes in signal transduction and cyclic nucleotide metabolism in the salivary gland transcriptome. Our data highlight a small repertoire of expressed mVSGs and potential signaling pathways involving receptor-type adenylate cyclases and members of a surface carboxylate transporter family, called PADs (Proteins Associated with Differentiation), to cope with the changing environment, as well as RNA-binding proteins as a possible global regulators of gene expression.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0168877PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5176191PMC
July 2017

Mammalian African trypanosome VSG coat enhances tsetse's vector competence.

Proc Natl Acad Sci U S A 2016 06 16;113(25):6961-6. Epub 2016 May 16.

Department of Epidemiology of Microbial Diseases, Yale School of Public Health, Yale University, New Haven, CT 06520;

Tsetse flies are biological vectors of African trypanosomes, the protozoan parasites responsible for causing human and animal trypanosomiases across sub-Saharan Africa. Currently, no vaccines are available for disease prevention due to antigenic variation of the Variant Surface Glycoproteins (VSG) that coat parasites while they reside within mammalian hosts. As a result, interference with parasite development in the tsetse vector is being explored to reduce disease transmission. A major bottleneck to infection occurs as parasites attempt to colonize tsetse's midgut. One critical factor influencing this bottleneck is the fly's peritrophic matrix (PM), a semipermeable, chitinous barrier that lines the midgut. The mechanisms that enable trypanosomes to cross this barrier are currently unknown. Here, we determined that as parasites enter the tsetse's gut, VSG molecules released from trypanosomes are internalized by cells of the cardia-the tissue responsible for producing the PM. VSG internalization results in decreased expression of a tsetse microRNA (mir-275) and interferes with the Wnt-signaling pathway and the Iroquois/IRX transcription factor family. This interference reduces the function of the PM barrier and promotes parasite colonization of the gut early in the infection process. Manipulation of the insect midgut homeostasis by the mammalian parasite coat proteins is a novel function and indicates that VSG serves a dual role in trypanosome biology-that of facilitating transmission through its mammalian host and insect vector. We detail critical steps in the course of trypanosome infection establishment that can serve as novel targets to reduce the tsetse's vector competence and disease transmission.
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http://dx.doi.org/10.1073/pnas.1600304113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4922192PMC
June 2016

Weevil endosymbiont dynamics is associated with a clamping of immunity.

BMC Genomics 2015 Oct 19;16:819. Epub 2015 Oct 19.

Université de Lyon, INSA-Lyon, INRA, UMR203 BF2I, Biologie Fonctionnelle Insectes et Interactions, F-69621, Villeurbanne, France.

Background: Insects subsisting on nutritionally unbalanced diets have evolved long-term mutualistic relationships with intracellular symbiotic bacteria (endosymbionts). The endosymbiont population load undergoes changes along with insect development. In the cereal weevil Sitophilus oryzae, the midgut endosymbionts Sodalis pierantonius drastically multiply following adult metamorphosis and rapidly decline until total elimination when the insect achieves its cuticle synthesis. Whilst symbiont load was shown to timely meet insect metabolic needs, little is known about the host molecular and immune processes underlying this dynamics.

Methods: We performed RNA sequencing analysis on weevil midguts at three representative phases of the endosymbiont dynamics (i.e. increase, climax and decrease). To screen genes which transcriptional changes are specifically related to symbiont dynamics and not to the intrinsic development of the midgut, we further have monitored by RT-qPCR sixteen gene transcript levels in symbiotic and artificially non-symbiotic (aposymbiotic) weevils. We also localized the endosymbionts during the elimination process by fluorescence microscopy.

Results: Functional analysis of the host differentially expressed genes by RNA sequencing showed that the main transcriptional changes occur during endosymbiont growth phase and affect cell proliferation, apoptosis, autophagy, phagocytosis, and metabolism of fatty acids and nucleic acids. We also showed that symbiont dynamics alters the expression of several genes involved in insect development. Our results strengthened the implication of apoptosis and autophagy processes in symbiont elimination and recycling. Remarkably, apart from the coleoptericin A that is known to target endosymbionts and controls their division and location, no gene coding antimicrobial peptide was upregulated during the symbiont growth and elimination phases.

Conclusion: We show that endosymbiont dynamics parallels numerous transcriptional changes in weevil developing adults and affects several biological processes, including metabolism and development. It also triggers cell apoptosis, autophagy and gut epithelial cell swelling and delamination. Strikingly, immunity is repressed during the whole process, presumably avoiding tissue inflammation and allowing insects to optimize nutrient recovery from recycled endosymbiont.
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http://dx.doi.org/10.1186/s12864-015-2048-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4617454PMC
October 2015

Systemic infection generates a local-like immune response of the bacteriome organ in insect symbiosis.

J Innate Immun 2015 23;7(3):290-301. Epub 2015 Jan 23.

Biologie Fonctionnelle Insectes et Interactions, UMR203 BF2I, INRA, INSA-Lyon, Université de Lyon, Villeurbanne, France.

Endosymbiosis is common in insects thriving in nutritionally unbalanced habitats. The cereal weevil, Sitophilus oryzae, houses Sodalis pierantonius, a Gram-negative intracellular symbiotic bacterium (endosymbiont), within a dedicated organ called a bacteriome. Recent data have shown that the bacteriome expresses certain immune genes that result in local symbiont tolerance and control. Here, we address the question of whether and how the bacteriome responds to insect infections involving exogenous bacteria. We have established an infection model by challenging weevil larvae with the Gram-negative bacterium Dickeya dadantii. We showed that D. dadantii infects host tissues and triggers a systemic immune response. Gene transcript analysis indicated that the bacteriome is also immune responsive, but it expresses immune effector genes to a lesser extent than the systemic and intestinal responses. Most genes putatively involved in immune pathways remain weakly expressed in the bacteriome following D. dadantii infection. Moreover, quantitative PCR experiments showed that the endosymbiont load is not affected by insect infection or the resulting bacteriome immune activation. Thus, the contained immune effector gene expression in the bacteriome may prevent potentially harmful effects of the immune response on endosymbionts, whilst efficiently protecting them from bacterial intruders.
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http://dx.doi.org/10.1159/000368928DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6738764PMC
January 2016

Insects recycle endosymbionts when the benefit is over.

Curr Biol 2014 Oct 18;24(19):2267-73. Epub 2014 Sep 18.

Biologie Fonctionnelle Insectes et Interactions, UMR203 BF2I, INRA, INSA-Lyon, Université de Lyon, 69621 Villeurbanne, France. Electronic address:

Symbiotic associations are widespread in nature and represent a driving force in evolution. They are known to impact fitness, and thereby shape the host phenotype. Insects subsisting on nutritionally poor substrates have evolved mutualistic relationships with intracellular symbiotic bacteria (endosymbionts) that supply them with metabolic components lacking in their diet. In many species, endosymbionts are hosted within specialized host cells, called the bacteriocytes, and transmitted vertically across host generations. How hosts balance the costs and benefits of having endosymbionts, and whether and how they adjust symbiont load to their physiological needs, remains largely unexplored. By investigating the cereal weevil Sitophilus association with the Sodalis pierantonius endosymbiont, we discover that endosymbiont populations intensively multiply in young adults, before being rapidly eliminated within few days. We show that young adults strongly depend on endosymbionts and that endosymbiont proliferation after metamorphosis matches a drastic host physiological need for the tyrosine (Tyr) and phenylalanine (Phe) amino acids to rapidly build their protective exoskeleton. Tyr and Phe are precursors of the dihydroxyphenylalanine (DOPA) molecule that is an essential component for the cuticle synthesis. Once the cuticle is achieved, DOPA reaches high amounts in insects, which triggers endosymbiont elimination. This elimination relies on apoptosis and autophagy activation, allowing digestion and recycling of the endosymbiont material. Thus, the weevil-endosymbiont association reveals an adaptive interplay between metabolic and cellular functions that minimizes the cost of symbiosis and speeds up the exoskeleton formation during a critical phase when emerging adults are especially vulnerable.
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http://dx.doi.org/10.1016/j.cub.2014.07.065DOI Listing
October 2014

Host gene response to endosymbiont and pathogen in the cereal weevil Sitophilus oryzae.

BMC Microbiol 2012 Jan 18;12 Suppl 1:S14. Epub 2012 Jan 18.

Background: Insects thriving on nutritionally poor habitats have integrated mutualistic intracellular symbiotic bacteria (endosymbionts) in a bacteria-bearing tissue (the bacteriome) that isolates the endosymbionts and protects them against a host systemic immune response. Whilst the metabolic and physiological features of long-term insect associations have been investigated in detail over the past decades, cellular and immune regulations that determine the host response to endosymbionts and pathogens have attracted interest more recently.

Results: To investigate bacteriome cellular specificities and weevil immune responses to bacteria, we have constructed and sequenced 7 cDNA libraries from Sitophilus oryzae whole larvae and bacteriomes. Bioinformatic analysis of 26,886 ESTs led to the generation of 8,941 weevil unigenes. Based on in silico analysis and on the examination of genes involved in the cellular pathways of potential interest to intracellular symbiosis (i.e. cell growth and apoptosis, autophagy, immunity), we have selected and analyzed 29 genes using qRT-PCR, taking into consideration bacteriome specificity and symbiosis impact on the host response to pathogens. We show that the bacteriome tissue accumulates transcripts from genes involved in cellular development and survival, such as the apoptotic inhibitors iap2 and iap3, and endosomal fusion and trafficking, such as Rab7, Hrs, and SNARE. As regards our investigation into immunity, we first strengthen the bacteriome immunomodulation previously reported in S. zeamais. We show that the sarcotoxin, the c-type lysozyme, and the wpgrp2 genes are downregulated in the S. oryzae bacteriome, when compared to aposymbiotic insects and insects challenged with E. coli. Secondly, transcript level comparison between symbiotic and aposymbiotic larvae provides evidence that the immune systemic response to pathogens is decreased in symbiotic insects, as shown by the relatively high expression of wpgrp2, wpgrp3, coleoptericin-B, diptericin, and sarcotoxin genes in aposymbiotic insects.

Conclusions: Library sequencing significantly increased the number of unigenes, allowing for improved functional and genetic investigations in the cereal weevil S. oryzae. Transcriptomic analyses support selective and local immune gene expression in the bacteriome tissue and uncover cellular pathways that are of potential interest to bacteriocyte survival and homeostasis. Bacterial challenge experiments have revealed that the systemic immune response would be less induced in a symbiotic insect, thus highlighting new perspectives on host immunity in long-term invertebrate co-evolutionary associations.
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http://dx.doi.org/10.1186/1471-2180-12-S1-S14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3287511PMC
January 2012

Antimicrobial peptides keep insect endosymbionts under control.

Science 2011 Oct;334(6054):362-5

INSA-Lyon, INRA, UMR203 BF2I, Biologie Fonctionnelle Insectes et Interactions, F-69621 Villeurbanne, France.

Vertically transmitted endosymbionts persist for millions of years in invertebrates and play an important role in animal evolution. However, the functional basis underlying the maintenance of these long-term resident bacteria is unknown. We report that the weevil coleoptericin-A (ColA) antimicrobial peptide selectively targets endosymbionts within the bacteriocytes and regulates their growth through the inhibition of cell division. Silencing the colA gene with RNA interference resulted in a decrease in size of the giant filamentous endosymbionts, which escaped from the bacteriocytes and spread into insect tissues. Although this family of peptides is commonly linked with microbe clearance, this work shows that endosymbiosis benefits from ColA, suggesting that long-term host-symbiont coevolution might have shaped immune effectors for symbiont maintenance.
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http://dx.doi.org/10.1126/science.1209728DOI Listing
October 2011
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