Imperial College London

Professor George K. Christophides

Faculty of Natural SciencesDepartment of Life Sciences

Professor of Infectious Diseases & Immunity
 
 
 
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Contact

 

+44 (0)20 7594 5342g.christophides

 
 
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Location

 

6167Sir Alexander Fleming BuildingSouth Kensington Campus

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Summary

 

Publications

Publication Type
Year
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119 results found

Witmer K, Fraschka SAK, Vlachou D, Bártfai R, Christophides GKet al., 2019, Epigenetic regulation underlying Plasmodium berghei gene expression during its developmental transition from host to vector

<jats:title>ABSTRACT</jats:title><jats:p>Epigenetic regulation of gene expression is an important attribute in the survival and adaptation of the malaria parasite <jats:italic>Plasmodium</jats:italic> in its human host. Our understanding of epigenetic regulation of gene expression in <jats:italic>Plasmodium</jats:italic> developmental stages beyond asexual replication in the mammalian host is sparse. We used chromatin immune-precipitation (ChIP) and RNA sequencing to create an epigenetic and transcriptomic map of the murine parasite <jats:italic>Plasmodium berghei</jats:italic> development from asexual blood stages to male and female gametocytes, and finally, to ookinetes. We show that heterochromatin 1 (HP1) almost exclusively associates with variantly expressed gene families at subtelomeric regions and remains stable across stages and various parasite lines. Variant expression based on heterochromatic silencing is observed only in very few genes. In contrast, the active histone mark histone 3 Lysine 9 acetylation (H3K9ac) is found between heterochromatin boundaries and occurs as a sharp peak around the start codon for ribosomal protein genes. H3K9ac occupancy positively correlates with gene transcripts in asexual blood stages, male gametocytes and ookinetes. Interestingly, H3K9ac occupancy does not correlate with transcript abundance in female gametocytes. Finally, we identify novel DNA motifs upstream of ookinete-specific genes thought to be involved in transcriptional activation upon fertilization.</jats:p>

Journal article

Debalke S, Habtewold T, Duchateau L, Christophides Get al., 2019, The effect of silencing immunity related genes on longevity in a naturally occurring Anopheles arabiensis mosquito population from southwest Ethiopia, Parasites & Vectors, Vol: 12, ISSN: 1756-3305

BackgroundVector control remains the most important tool to prevent malaria transmission. However, it is now severely constrained by the appearance of physiological and behavioral insecticide resistance. Therefore, the development of new vector control tools is warranted. Such tools could include immunization of blood hosts of vector mosquitoes with mosquito proteins involved in midgut homeostasis (anti-mosquito vaccines) or genetic engineering of mosquitoes that can drive population-wide knockout of genes producing such proteins to reduce mosquito lifespan and malaria transmission probability.MethodsTo achieve this, candidate genes related to midgut homeostasis regulation need to be assessed for their effect on mosquito survival. Here, different such candidate genes were silenced through dsRNA injection in the naturally occurring Anopheles arabiensis mosquitoes and the effect on mosquito survival was evaluated.ResultsSignificantly higher mortality rates were observed in the mosquitoes silenced for FN3D1 (AARA003032), FN3D3 (AARA007751) and GPRGr9 (AARA003963) genes as compared to the control group injected with dsRNA against a non-related bacterial gene (LacZ). This observed difference in mortality rate between the candidate genes and the control disappeared when gene-silenced mosquitoes were treated with antibiotic mixtures, suggesting that gut microbiota play a key role in the observed reduction of mosquito survival.ConclusionsWe demonstrated that interference with the expression of the FN3D1, FN3D3 or GPRGr9 genes causes a significant reduction of the longevity of An. arabiensis mosquito in the wild.

Journal article

Ruiz VMR, Sousa GL, Sneed SD, Farrant KV, Christophides GK, Povelones Met al., 2019, Stimulation of a protease targeting the LRIM1/APL1C complex reveals specificity in complement-like pathway activation in Anopheles gambiae, PLOS ONE, Vol: 14, ISSN: 1932-6203

Journal article

Christophides G, Rona L, Cechetto Carlos B, Souza-Neto Jet al., 2019, A comprehensive analysis of malaria transmission in Brazil, Pathogens and Global Health, ISSN: 2047-7724

Malaria remains a serious public health problem in Brazil despite a significant drop in the number of cases in the past decade. We conduct a comprehensive analysis of malaria transmission in Brazil to highlight the epidemiologically most relevant components that could help tackle the disease. We consider factors impacting on the malaria burden and transmission dynamics including the geographical occurrence of both autochthonous and imported infections, the distribution and abundance of malaria vectors and records of natural mosquito infections with Plasmodium. Our analysis identifies three discrete malaria transmission systems related to the Amazon rainforest, Atlantic rainforest and Brazilian coast, respectively. The Amazonian system accounts for 99% of all malaria cases in the country. It is largely due to autochthonous P. vivax and P. falciparum transmission by mosquitoes of the Nyssorhynchus subgenus, primarily Anopheles darlingi. Whilst P. vivax transmission is widespread, P. falciparum transmission is restricted to hotspot areas mostly in the States of Amazonas and Acre. This system is the major source of P. vivax exportation to the extra-Amazonian regions that are also affected by importation of P. falciparum from Africa. The Atlantic system comprises autochthonous P. vivax transmission typically by the bromeliad-associated mosquitoes An. cruzii and An. bellator of the Kerteszia subgenus. An. cruzii also transmits simian malaria parasites to humans. The third, widespread but geographically fragmented, system is found along the Brazilian coast and comprises P. vivax transmission mainly by An. aquasalis. We conclude that these geographically and biologically distinct malaria transmission systems require specific strategies for effective disease control.

Journal article

Habtewold T, Tapanelli S, Ellen KG M, Astrid H, Nikolai W, George K Cet al., 2019, Streamlined SMFA and mosquito dark-feeding regime significantly improve malaria transmission-blocking assay robustness and sensitivity, Malaria Journal, Vol: 18, ISSN: 1475-2875

BackgroundThe development of malaria transmission-blocking strategies including the generation of malaria refractory mosquitoes to replace the wild populations through means of gene drives hold great promise. The standard membrane feeding assay (SMFA) that involves mosquito feeding on parasitized blood through an artificial membrane system is a vital tool for evaluating the efficacy of transmission-blocking interventions. However, despite the availability of several published protocols, the SMFA remains highly variable and broadly insensitive.MethodsThe SMFA protocol was optimized through coordinated culturing of Anopheles coluzzii mosquitoes and Plasmodium falciparum parasite coupled with placing mosquitoes under a strict dark regime before, during, and after the gametocyte feed.ResultsA detailed description of essential steps is provided toward synchronized generation of highly fit An. coluzzii mosquitoes and P. falciparum gametocytes in preparation for an SMFA. A dark-infection regime that emulates the natural vector-parasite interaction system is described, which results in a significant increase in the infection intensity and prevalence. Using this optimal SMFA pipeline, a series of putative transmission-blocking antimicrobial peptides (AMPs) were screened, confirming that melittin and magainin can interfere with P. falciparum development in the vector.ConclusionA robust SMFA protocol that enhances the evaluation of interventions targeting human malaria transmission in laboratory setting is reported. Melittin and magainin are identified as highly potent antiparasitic AMPs that can be used for the generation of refractory Anopheles gambiae mosquitoes.

Journal article

Nash A, Urdaneta Mignini G, Beaghton A, Hoermann A, Papathanos P, Christophides G, Windbichler Net al., 2019, Integral Gene Drives for population replacement, Biology Open, Vol: 8, ISSN: 2046-6390

A first generation of CRISPR-based gene drives has now been tested in the laboratory in a number of organisms, including malaria vector mosquitoes. Challenges for their use in the area-wide genetic control of vector-borne disease have been identified, including the development of target site resistance, their long-term efficacy in the field, their molecular complexity, and practical and legal limitations for field testing of both gene drive and coupled anti-pathogen traits. We have evaluated theoretically the concept of integral gene drive (IGD) as an alternative paradigm for population replacement. IGDs incorporate a minimal set of molecular components, including drive and anti-pathogen effector elements directly embedded within endogenous genes – an arrangement that in theory allows targeting functionally conserved coding sequences without disrupting their function. Autonomous and non-autonomous IGD strains could be generated, optimized, regulated and imported independently. We performed quantitative modeling comparing IGDs with classical replacement drives and show that selection for the function of the hijacked host gene can significantly reduce the establishment of resistant alleles in the population, while drive occurring at multiple genomic loci prolongs the duration of transmission blockage in the face of pre-existing target site variation. IGD thus has potential as a more durable and flexible population replacement strategy.

Journal article

Christophides GK, Nakhleh J, Osta MA, 2017, The serine protease homolog CLIPA14 modulates the intensity of the immune response in the mosquito Anopheles gambiae, Journal of Biological Chemistry, Vol: 292, Pages: 18217-18226, ISSN: 0021-9258

CLIP domain serine protease homologues (cSPHs) are positive and negative regulators of Anopheles gambiae immune responses mediated by the complement-like protein TEP1 against Plasmodium malaria parasites and other microbial infections. We have previously reported that the SPH CLIPA2 is a negative regulator of the TEP1-mediated response by showing that CLIPA2 knockdown (kd) enhances mosquito resistance to infections with fungi, bacteria and Plasmodium parasites. Here, we identify another SPH, CLIPA14, as a novel regulator of mosquito immunity. We found that CLIPA14 is a hemolymph protein that is rapidly cleaved following a systemic infection. CLIPA14 kd mosquitoes elicited a potent melanization response against Plasmodium berghei ookinetes and exhibited significantly increased resistance to Plasmodium infections as well as to systemic and oral bacterial infections. The activity of the enzyme phenoloxidase, which initiates melanin biosynthesis, dramatically increased in the hemolymph of CLIPA14 kd mosquitoes in response to systemic bacterial infections. Ookinete melanization and hemolymph phenoloxidase activity were further increased after co-silencing CLIPA14 and CLIPA2, suggesting that these two SPHs act in concert to control the melanization response. Interestingly, CLIPA14 RNAi phenotypes and its infection-induced cleavage were abolished in a TEP1 loss-of-function background. Our results suggest that a complex network of SPHs functions downstream of TEP1 to regulate the melanization reaction.

Journal article

Ukegbu CV, Giogalli M, Yassine H, Luis Ramirez J, Taxiarchi C, Barillas-Mury C, Christophides GKet al., 2017, Plasmodium berghei P47 is essential for ookinete protection from the Anopheles gambiae complement-like response, Scientific Reports, Vol: 7, ISSN: 2045-2322

Malaria is a mosquito-borne disease affecting millions of people every year. The rodent parasite Plasmodium berghei has served as a model for human malaria transmission studies and played a pivotal role in dissecting the mosquito immune response against infection. The 6-cysteine protein P47, known to be important for P. berghei female gamete fertility, is shown to serve a different function in Plasmodium falciparum, protecting ookinetes from the mosquito immune response. Here, we investigate the function of P. berghei P47 in Anopheles gambiae mosquito infections. We show that P47 is expressed on the surface of both female gametocytes and ookinetes where it serves distinct functions in promoting gametocyte-to-ookinete development and protecting ookinetes from the mosquito complement-like response, respectively. The latter function is essential, as ookinetes lacking P47 are targeted for killing while traversing the mosquito midgut cells and eliminated upon exposure to hemolymph proteins of the complement-like system. Silencing key factors of the complement-like system restores oocyst development and disease transmission to rodent hosts. Our data establish a dual role of P. berghei P47 in vivo and reinforce the use of this parasite to study the impact of the mosquito immune response on human malaria transmission.

Journal article

Ukegbu CV, Akinosoglou KA, Christophides GKC, Vlachou Det al., 2017, Plasmodium berghei PIMMS2 promotes ookinete invasion of the Anopheles gambiae mosquito midgut, Infection and Immunity, Vol: 85, ISSN: 1098-5522

Mosquito midgut stages of the malaria parasite present an attractive biological system to study host-parasite interactions and develop interventions to block disease transmission. Mosquito infection ensues upon oocyst development that follows ookinete invasion and traversal of the mosquito midgut epithelium. Here, we report the characterization of PIMMS2 (Plasmodium Invasion of Mosquito Midgut Screen candidate 2), a Plasmodium berghei protein with structural similarities to subtilisin-like proteins. PIMMS2 orthologs are present in the genomes of all plasmodia and are mapped between the subtilisin-encoding genes SUB1 and SUB3. P. berghei PIMMS2 is specifically expressed in zygotes and ookinetes and is localized on the ookinete surface. Loss of PIMMS2 function through gene disruption by homologous recombination leads to normal development of motile ookinetes that exhibit severely impaired capacity to traverse the mosquito midgut and transform to oocysts. Genetic complementation of the disrupted locus with a mutated PIMMS2 allele reveals that amino acid residues corresponding to the putative subtilisin-like catalytic triad are important but not essential for the protein function. Our data demonstrate that PIMMS2 is a novel ookinete-specific protein that promotes parasite traversal of the mosquito midgut epithelium and establishment of mosquito infection.

Journal article

Gendrin MEM, Turlure F, Rodgers FH, Cohuet A, Morlais I, Christophides GKet al., 2017, The peptidoglycan recognition proteins PGRPLA and PGRPLB regulate Anopheles immunity to bacteria and affect infection by Plasmodium, Journal of Innate Immunity, Vol: 9, Pages: 333-342, ISSN: 1662-8128

Peptidoglycan recognition proteins (PGRPs) form a family of immune regulators that is conserved from insects to mammals. In the malaria vector mosquito Anophelescoluzzii, the peptidoglycan receptor PGRPLC activates the Imd pathway limiting both the microbiota load and Plasmodiuminfection. Here, we carried out an RNAi screen to examine the role of all seven Anopheles PGRPs in infections with Plasmodium berghei and Plasmodium falciparum. We show that, in addition to PGRPLC, PGRPLA and PGRPS2/S3 also participate in antiparasitic defenses, and that PGRPLB promotes mosquito permissiveness to P. falciparum. We also demonstrate that following a mosquito blood feeding, which promotes growth of the gut microbiota, PGRPLA and PGRPLB positively and negatively regulate the activation of the Imd pathway, respectively. Our data demonstrate that PGRPs are important regulators of the mosquito epithelial immunity and vector competence.

Journal article

Rodgers FH, Gendrin M, Wyer CAS, Christophides GKet al., 2017, Microbiota-induced peritrophic matrix regulates midgut homeostasis and prevents systemic infection of malaria vector mosquitoes, PLOS Pathogens, Vol: 13, ISSN: 1553-7366

Manipulation of the mosquito gut microbiota can lay the foundations for novel methods for disease transmission control. Mosquito blood feeding triggers a significant, transient increase of the gut microbiota, but little is known about the mechanisms by which the mosquito controls this bacterial growth whilst limiting inflammation of the gut epithelium. Here, we investigate the gut epithelial response to the changing microbiota load upon blood feeding in the malaria vector Anopheles coluzzii. We show that the synthesis and integrity of the peritrophic matrix, which physically separates the gut epithelium from its luminal contents, is microbiota dependent. We reveal that the peritrophic matrix limits the growth and persistence of Enterobacteriaceae within the gut, whilst preventing seeding of a systemic infection. Our results demonstrate that the peritrophic matrix is a key regulator of mosquito gut homeostasis and establish functional analogies between this and the mucus layers of the mammalian gastrointestinal tract.

Journal article

Habtewold T, Groom Z, Christophides G, 2017, Immune resistance and tolerance strategies in malaria vector and non-vector mosquitoes, Parasites & Vectors, Vol: 10, ISSN: 1756-3305

BackgroundThe Anopheles gambiae complex consists of species that vary greatly in their capacity to transmit malaria. The mosquito immune system has been identified as a key factor that can influence whether Plasmodium infection establishes within the mosquito vector. This study was designed to investigate the immune responses of An. coluzzii, An. arabiensis and An. quadriannulatus mosquitoes. The first two mosquito species are major vectors of malaria in sub-Saharan Africa, while the third is thought to be a non-vector.MethodsAll three mosquito species were reared in mixed cultures. Their capacity to eliminate P. berghei and regulate midgut bacteria was examined.ResultsOur results revealed large differences in mosquito resistance to P. berghei. In all three mosquito species, immune reactions involving the complement system were triggered when the number of parasites that mosquitoes were challenged with exceeded a certain level, i.e. immune tolerance threshold. This threshold was markedly lower in An. quadriannulatus compared to An. coluzzii and An. arabiensis. We also demonstrated that the level of immune tolerance to P. berghei infection in the haemolymph is inversely correlated with the level of immune tolerance to microbiota observed in the midgut lumen after a blood meal. The malaria non-vector mosquito species, An. quadriannulatus was shown to have a much higher level of tolerance to microbiota in the midgut than An. coluzzii.ConclusionsWe propose a model whereby an increased tolerance to microbiota in the mosquito midgut results in lower tolerance to Plasmodium infection. In this model, malaria non-vector mosquito species are expected to have increased immune resistance in the haemocoel, possibly due to complement priming by microbiota elicitors. We propose that this strategy is employed by the malaria non-vector mosquito, An. quadriannulatus, while An. coluzzii has reduced tolerance to bacterial infection in the midgut and consequently reduced immune resistan

Journal article

Erguler K, Chandra NL, Proestos Y, Lelieveld J, Christophides GK, Parham PEet al., 2017, A large-scale stochastic spatiotemporal model for Aedes albopictus-borne chikungunya epidemiology, PLOS ONE, Vol: 12, ISSN: 1932-6203

Journal article

Habtewold T, Groom Z, Christophides G, Immune resistance and tolerance strategies in malaria vector and non-vector mosquitoes, Parasite & Vectors

Journal article

Gendrin MEM, Christophides, Linenberg, Inbaret al., 2016, Larval diet affects mosquito development and permissiveness to Plasmodium infection, Scientific Reports, Vol: 6, ISSN: 2045-2322

The larval stages of malaria vector mosquitoes develop in water pools, feeding mostly on microorganisms and environmental detritus. Richness in the nutrient supply to larvae influences the development and metabolism of larvae and adults. Here, we investigated the effects of larval diet on the development, microbiota content and permissiveness to Plasmodium of Anopheles coluzzii. We tested three fish diets often used to rear mosquitoes in the laboratory, including two pelleted diets, Dr. Clarke’s Pool Pellets and Nishikoi Fish Pellets, and one flaked diet, Tetramin Fish-Flakes. Larvae grow and develop faster and produce bigger adults when feeding on both types of pellets compared with flakes. This correlates with a higher microbiota load in pellet-fed larvae, in agreement with the known positive effect of the microbiota on mosquito development. Larval diet also significantly influences the prevalence and intensity of Plasmodium berghei infection in adults, whereby Nishikoi Fish Pellets-fed larvae develop into adults that are highly permissive to parasites and survive longer after infection. This correlates with a lower amount of Enterobacteriaceae in the midgut microbiota. Together, our results shed light on the influence of larval feeding on mosquito development, microbiota and vector competence; they also provide useful data for mosquito rearing.

Journal article

Dahiya N, Chianese G, Abay SM, Taglialatela-Scafati O, Esposito F, Lupidi G, Bramucci M, Quassinti L, Christophides G, Habluetzel A, Lucantoni Let al., 2016, In vitro and ex vivo activity of an Azadirachta indica A.Juss. seed kernel extract on early sporogonic development of Plasmodium in comparison with azadirachtin A, its most abundant constituent, PHYTOMEDICINE, Vol: 23, Pages: 1743-1752, ISSN: 0944-7113

Journal article

Kohl A, Pondeville E, Schnettler E, Crisanti A, Supparo C, Christophides GK, Kersey PJ, Maslen GL, Takken W, Koenraadt CJ, Oliva CF, Busquets N, Abad FX, Failloux AB, Levashina EA, Wilson AJ, Veronesi E, Pichard M, Arnaud Marsh S, Simard F, Vernick KDet al., 2016, Advancing vector biology research: a community survey for future directions, research applications and infrastructure requirements., Pathogens and Global Health, Vol: 110, Pages: 164-172, ISSN: 2047-7732

Vector-borne pathogens impact public health, animal production, and animal welfare. Research on arthropod vectors such as mosquitoes, ticks, sandflies, and midges which transmit pathogens to humans and economically important animals is crucial for development of new control measures that target transmission by the vector. While insecticides are an important part of this arsenal, appearance of resistance mechanisms is increasingly common. Novel tools for genetic manipulation of vectors, use of Wolbachia endosymbiotic bacteria, and other biological control mechanisms to prevent pathogen transmission have led to promising new intervention strategies, adding to strong interest in vector biology and genetics as well as vector-pathogen interactions. Vector research is therefore at a crucial juncture, and strategic decisions on future research directions and research infrastructure investment should be informed by the research community. A survey initiated by the European Horizon 2020 INFRAVEC-2 consortium set out to canvass priorities in the vector biology research community and to determine key activities that are needed for researchers to efficiently study vectors, vector-pathogen interactions, as well as access the structures and services that allow such activities to be carried out. We summarize the most important findings of the survey which in particular reflect the priorities of researchers in European countries, and which will be of use to stakeholders that include researchers, government, and research organizations.

Journal article

Akorli J, Gendrin M, Pels NAP, Yeboah-Manu D, Christophides GK, Wilson MDet al., 2016, Seasonality and Locality Affect the Diversity of Anopheles gambiae and Anopheles coluzzii Midgut Microbiota from Ghana, PLOS One, Vol: 11, ISSN: 1932-6203

Symbiotic bacteria can have important implications in the development and competence of disease vectors. In Anopheles mosquitoes, the composition of the midgut microbiota is largely influenced by the larval breeding site, but the exact factors shaping this composition are currently unknown. Here, we examined whether the proximity to urban areas and seasons have an impact on the midgut microbial community of the two major malaria vectors in Africa, An. coluzzii and An. gambiae. Larvae and pupae were collected from selected habitats in two districts of Ghana during the dry and rainy season periods. The midgut microbiota of adults that emerged from these collections was determined by 454-pyrosequencing of the 16S ribosomal DNA. We show that in both mosquito species, Shewanellaceae constituted on average of 54% and 73% of the midgut microbiota from each site in the dry and rainy season, respectively. Enterobacteriaceae was found in comparatively low abundance below 1% in 22/30 samples in the dry season, and in 25/38 samples in the rainy season. Our data indicate that seasonality and locality significantly affect both the diversity of microbiota and the relative abundance of bacterial families with a positive impact of dry season and peri-urban settings.

Journal article

Gendrin MEM, Yerbanga RS, Ouedrogo JB, Lefèvre T, Cohuet A, Christophides GKet al., 2016, Differential effects of azithromycin, doxycycline and co-trimoxazole in ingested blood on the vectorial capacity of malaria mosquitoes, Open Forum Infectious Diseases, Vol: 3, ISSN: 2328-8957

Background The gut microbiota of malaria vector mosquitoes grows after a blood meal and limits Plasmodium infection. We previously showed that penicillin and streptomycin in the ingested blood affect bacterial growth and positively impact mosquito survival and permissiveness to Plasmodium. Here, we examine the effects of doxycycline, azithromycin and co-trimoxazole. All three antibiotics are used in mass-drug administration programs and have antimicrobial activities against bacteria and various stages of malaria parasites.MethodsThe effects of blood meal supplementation with antibiotics on the mosquito microbiota, lifespan and permissiveness to Plasmodium falciparum were assessed. ResultsIngestion of any of the three antibiotics significantly affected the mosquito microbiota. Azithromycin decreased P. falciparum infection load and mosquito lifespan, while at high concentrations, doxycycline increased P. falciparum infection load. Co-trimoxazole negatively impacted infection intensity but had no reproducible effect on mosquito lifespan.ConclusionsOur data suggest that the overall effect of antibiotic treatment on parameters critical for mosquito vectorial capacity is drug-specific. The negative effect of azithromycin on malaria transmission is consistent with current efforts for disease elimination, while additional, larger scale investigations are required before conclusions can be drawn about doxycycline.

Journal article

Habtewold T, Duchateau L, Christophides GK, 2016, Flow cytometry analysis of the microbiota associated with the midguts of vector mosquitoes, Parasites & Vectors, Vol: 9, ISSN: 1756-3305

BackgroundThe scientific interest to understand the function and structure of the microbiota associated with the midgut of mosquito disease vectors is increasing. The advancement of such a knowledge has encountered challenges and limitations associated with conventional culture-based and PCR techniques. MethodFlow cytometry (FCM) combined with various cell marking dyes have been successfully applied in the field of ecological microbiology to circumvent the above shortcomings. Here, we describe FCM technique coupled with live/dead differential staining dyes SYBR Green I (SGI) and Propidium Iodide (PI) to quantify and study other essential characteristics of the mosquito gut microbiota. Results A clear discrimination between cells and debris, as well as between live and dead cells was achieved when the midgut homogenate was stained with 5x103 dilution of the SGI and 30 µM concentration of the PI. Reproducibly, FCM event collections produced discrete populations including non-fluorescent cells, SYBR positive cells, PI fluorescing cells and cells that fluoresce both in SYBR and PI, all these cell populations representing, respectively, background noise, live bacterial, dead cells and inactive cells with partial permeability to PI. The FCM produced a strong linear relationship between cell counts and their corresponding dilution factors (R² = 0.987), and the technique has a better precision compared to qRT-PCR. The FCM count of the microbiota reached a peak load at 18 h post-feeding and started declining at 24 h. The present FCM technique also successfully applied to quantify bacterial cells in fixed midgut samples that were homogenized in 4% PFA. ConclusionThe FCM technique described here offers enormous potential and possibilities of integration with advanced molecular biochemical techniques for the study of the microbiota community in disease vector mosquitoes.

Journal article

Erguler K, Smith-Unna SE, Waldock J, Proestos Y, Christophides GK, Lelieveld J, Parham PEet al., 2016, Large-Scale Modelling of the Environmentally-Driven Population Dynamics of Temperate Aedes albopictus (Skuse), PLOS One, Vol: 11, ISSN: 1932-6203

The Asian tiger mosquito, Aedes albopictus, is a highly invasive vector species. It is a proven vector of dengue and chikungunya viruses, with the potential to host a further 24 arboviruses. It has recently expanded its geographical range, threatening many countries in the Middle East, Mediterranean, Europe and North America. Here, we investigate the theoretical limitations of its range expansion by developing an environmentally-driven mathematical model of its population dynamics. We focus on the temperate strain of Ae. albopictus and compile a comprehensive literature-based database of physiological parameters. As a novel approach, we link its population dynamics to globally-available environmental datasets by performing inference on all parameters. We adopt a Bayesian approach using experimental data as prior knowledge and the surveillance dataset of Emilia-Romagna, Italy, as evidence. The model accounts for temperature, precipitation, human population density and photoperiod as the main environmental drivers, and, in addition, incorporates the mechanism of diapause and a simple breeding site model. The model demonstrates high predictive skill over the reference region and beyond, confirming most of the current reports of vector presence in Europe. One of the main hypotheses derived from the model is the survival of Ae. albopictus populations through harsh winter conditions. The model, constrained by the environmental datasets, requires that either diapausing eggs or adult vectors have increased cold resistance. The model also suggests that temperature and photoperiod control diapause initiation and termination differentially. We demonstrate that it is possible to account for unobserved properties and constraints, such as differences between laboratory and field conditions, to derive reliable inferences on the environmental dependence of Ae. albopictus populations.

Journal article

Lombardo F, Christophides GK, 2016, Novel factors of Anopheles gambiae haemocyte immune response to Plasmodium berghei infection, Parasites & Vectors, Vol: 9, ISSN: 1756-3305

BackgroundInsect haemocytes mediate cellular immune responses (e.g., phagocytosis) and contribute to the synthesis of humoral immune factors. In previous work, a genome-wide molecular characterization of Anopheles gambiae circulating haemocytes was followed by functional gene characterization using cell-based RNAi screens. Assays were carried out to investigate the role of selected haemocyte-specific or enriched genes in phagocytosis of bacterial bio-particles, expression of the antimicrobial peptide cecropin1, and basal and induced expression of the mosquito complement factor LRIM1 (leucine-rich repeat immune gene I).FindingsHere we studied the impact of a subset of genes (37 candidates) from the haemocyte-specific dsRNA collection on the development of Plasmodium in the mosquito by in vivo gene silencing. Our screening identifies 10 novel factors with a role in the mosquito response to Plasmodium. Analysis of in vivo screening phenotypes reveals a significant anti-correlation between the prevalence of oocysts and melanised ookinetes.ConclusionsAmong novel immune genes are putative pattern recognition proteins (leucine-rich repeat, fibrinogen-domain and R-type lectins), immune modulation and signalling proteins (LPS-induced tumor necrosis factor alpha factor, LITAF and CLIP proteases), and components of extracellular matrix such as laminin and collagen. Additional identified proteins such as the storage protein hexamerin and vesicular-type ATPase (V-ATPase) are associated for the first time with the mosquito response against Plasmodium.

Journal article

Povelones M, Osta MA, Christophides GK, 2016, The Complement System of Malaria Vector Mosquitoes, PROGRESS IN MOSQUITO RESEARCH, Editors: Raikhel, Publisher: ACADEMIC PRESS LTD-ELSEVIER SCIENCE LTD, Pages: 223-242

Book chapter

Fofana A, Gendrin M, Sangare I, Sanou S, Wangraoua S, Drabo A, Christophides G, Ouedraogo JB, Yerbanga RSet al., 2015, CHARACTERIZATION AND ANTIBIORESISTANCE OF CULTURABLE MICROFLORA BACTERIA IN THE MIDGUT OF ADULT ANOPHELES GAMBIAE AND AN. COLUZZII, Publisher: AMER SOC TROP MED & HYGIENE, Pages: 264-265, ISSN: 0002-9637

Conference paper

Povelones M, Farrant KV, Christophides GK, 2015, STIMULATION OF A SERINE PROTEASE TARGETING THE LRIM1/APL1C COMPLEX REVEALS SPECIFICITY IN COMPLEMENT-LIKE PATHWAY ACTIVATION IN ANOPHELES GAMBIAE, Publisher: AMER SOC TROP MED & HYGIENE, Pages: 417-417, ISSN: 0002-9637

Conference paper

Habtewold T, Groom Z, Duchateau L, Christophides GKet al., 2015, Detection of viable Plasmodium ookinetes in the midguts of Anopheles coluzzi using PMA-qrtPCR, Parasites & Vectors, Vol: 8, ISSN: 1756-3305

BackgroundMosquito infection with malaria parasites depends on complex interactions between the mosquito immune response, the parasite developmental program and the midgut microbiota. Simultaneous monitoring of the parasite and bacterial dynamics is important when studying these interactions. PCR based methods of genomic DNA (gDNA) have been widely used, but their inability to discriminate between live and dead cells compromises their application. The alternative method of quantification of mRNA mainly reports on cell activity rather than density.MethodQuantitative real-time (qrt) PCR in combination with Propidium Monoazide (PMA) treatment (PMA-qrtPCR) has been previously used for selectively enumerating viable microbial cells. PMA penetrates damaged cell membranes and intercalates in the DNA inhibiting its PCR amplification. Here, we tested the potential of PMA-qrtPCR to discriminate between and quantify live and dead Plasmodium berghei malarial parasites and commensal bacteria in the midgut of Anopheles coluzzii Coetzee & Wilkerson 2013 (formerly An. gambiae M-form).ResultsBy combining microscopic observations with reverse transcriptase PCR (RT-PCR) we reveal that, in addition to gDNA, mRNA from dead parasites also persists inside the mosquito midgut, therefore its quantification cannot accurately reflect live-only parasites at the time of monitoring. In contrast, pre-treating the samples with PMA selectively inhibited qrtPCR amplification of parasite gDNA, with about 15 cycles (Ct-value) difference between PMA-treated and control samples. The limit of detection corresponds to 10 Plasmodium ookinetes. Finally, we show that the PMA-qrtPCR method can be used to quantify bacteria that are present in the mosquito midgut.ConclusionThe PMA-qrtPCR is a suitable method for quantification of viable parasites and bacteria in the midgut of Anopheles mosquitoes. The method will be valuable when studying the molecular interactions between the mosquito, the malaria parasite

Journal article

Abay SM, Dahiya N, Dembo E, Dori G, Christophides G, Esposito F, Lucantoni L, Lupidi G, Ogboi S, Ouedraogo R, Ouedraogo J-B, Sinisi A, Taglialatela-Scafati O, Yerbanga S, Habluetzel Aet al., 2015, Malaria transmission blocking activity of sesquiterpene lactones from Vernonia amygdalina, TROPICAL MEDICINE & INTERNATIONAL HEALTH, Vol: 20, Pages: 150-151, ISSN: 1360-2276

Journal article

Dahiya N, Tapanelli S, Chianese G, Abay SM, Taglialatela-Scafati O, Esposito F, Lupidi G, Christophides G, Lucantoni L, Yerbanga SR, Habluetzel Aet al., 2015, Transmission blocking effects of Azadirachta indica limonoids on early sporogonic development of Plasmodium: activity and bioavailability of seed fractions and isolated compounds, TROPICAL MEDICINE & INTERNATIONAL HEALTH, Vol: 20, Pages: 178-179, ISSN: 1360-2276

Journal article

Ukegbu CV, Cho J-S, Christophides GK, Vlachou Det al., 2015, Transcriptional silencing and activation of paternal DNA during Plasmodium berghei zygotic development and transformation to oocyst, Cellular Microbiology, Vol: 17, Pages: 1230-1240, ISSN: 1462-5822

The malaria parasite develops sexually in the mosquito midgut upon entry with the ingested blood meal before it can invade the midgut epithelium and embark on sporogony. Recent data have identified a number of distinct transcriptional programmes operating during this critical phase of the parasite life cycle. We aimed at characterizing the parental contribution to these transcriptional programmes and establish the genetic framework that would guide further studies of Plasmodium zygotic development and ookinete-to-oocyst transition. To achieve this we used in vitro and in vivo cross-fertilization experiments of various parasite lines expressing fluorescent reporters under the control of constitutive and stage-specific promoters. The results revealed that the zygote/ookinete stage exhibits a maternal phenotype with respect to constitutively expressed reporters, which is derived from either maternal mRNA inheritance or transcription of the maternal allele. The respective paternal alleles are silenced in the zygote/ookinete but reactivated after midgut invasion and transformation to oocyst. Transcripts specifically produced in the zygote/ookinete are synthesized de novo by both parental alleles. These findings highlight a putative role of epigenetic regulation of Plasmodium zygotic development and add substantially to the emerging picture of the molecular mechanisms regulating this important stage of malaria transmission.

Journal article

Abay SM, Lucantoni L, Dahiya N, Dori G, Dembo EG, Esposito F, Lupidi G, Ogboi S, Ouedraogo RK, Sinisi A, Taglialatela-Scafati O, Yerbanga RS, Bramucci M, Quassinti L, Ouedraogo JB, Christophides G, Habluetzel Aet al., 2015, Plasmodium transmission blocking activities of Vernonia amygdalina extracts and isolated compounds, MALARIA JOURNAL, Vol: 14, ISSN: 1475-2875

Journal article

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