Anopheline salivary protein genes and gene families: an evolutionary overview after the whole genome sequence of sixteen Anopheles species

Background: Mosquito saliva is a complex cocktail whose pharmacological properties play an essential role in blood feeding by counteracting host physiological response to tissue injury. Moreover, vector borne pathogens are transmitted to vertebrates and exposed to their immune system in the context of mosquito saliva which, in virtue of its immunomodulatory properties, can modify the local environment at the feeding site and eventually affect pathogen transmission. In addition, the host antibody response to salivary proteins may be used to assess human exposure to mosquito vectors. Even though the role of quite a few mosquito salivary proteins has been clarified in the last decade, we still completely ignore the physiological role of many of them as well as the extent of their involvement in the complex interactions taking place between the mosquito vectors, the pathogens they transmit and the vertebrate host. The recent release of the genomes of 16 Anopheles species offered the opportunity to get insights into function and evolution of salivary protein families in anopheline mosquitoes. Results: Orthologues of fifty three Anopheles gambiae salivary proteins were retrieved and annotated from 18 additional anopheline species belonging to the three subgenera Cellia, Anopheles, and Nyssorhynchus. Our analysis included 824 full-length salivary proteins from 24 different families and allowed the identification of 79 novel salivary genes and re-annotation of 379 wrong predictions. The comparative, structural and phylogenetic analyses yielded an unprecedented view of the anopheline salivary repertoires and of their evolution over 100 million years of anopheline radiation shedding light on mechanisms and evolutionary forces that contributed shaping the anopheline sialomes. Conclusions: We provide here a comprehensive description, classification and evolutionary overview of the main anopheline salivary protein families and identify two novel candidate markers of human exposure to malaria vectors worldwide. This anopheline sialome catalogue, which is easily accessible as hyperlinked spreadsheet, is expected to be useful to the vector biology community and to improve the capacity to gain a deeper understanding of mosquito salivary proteins facilitating their possible exploitation for epidemiological and/or pathogen-vector-host interaction studies

Generation, annotation, and analysis of ESTs from midgut tissue of adult female Anopheles stephensi mosquitoes

<p>Abstract</p> <p>Background</p> <p>Malaria is a tropical disease caused by protozoan parasite, <it>Plasmodium</it>, which is transmitted to humans by various species of female anopheline mosquitoes. <it>Anopheles stephensi </it>is one such major malaria vector in urban parts of the Indian subcontinent. Unlike <it>Anopheles gambiae</it>, an African malaria vector, transcriptome of <it>A. stephensi </it>midgut tissue is less explored. We have therefore carried out generation, annotation, and analysis of expressed sequence tags from sugar-fed and <it>Plasmodium yoelii </it>infected blood-fed (post 24 h) adult female <it>A. stephensi </it>midgut tissue.</p> <p>Results</p> <p>We obtained 7061 and 8306 ESTs from the sugar-fed and <it>P. yoelii </it>infected mosquito midgut tissue libraries, respectively. ESTs from the combined dataset formed 1319 contigs and 2627 singlets, totaling to 3946 unique transcripts. Putative functions were assigned to 1615 (40.9%) transcripts using BLASTX against UniProtKB database. Amongst unannotated transcripts, we identified 1513 putative novel transcripts and 818 potential untranslated regions (UTRs). Statistical comparison of annotated and unannotated ESTs from the two libraries identified 119 differentially regulated genes. Out of 3946 unique transcripts, only 1387 transcripts were mapped on the <it>A. gambiae </it>genome. These also included 189 novel transcripts, which were mapped to the unannotated regions of the genome. The EST data is available as ESTDB at <url>http://mycompdb.bioinfo-portal.cdac.in/cgi-bin/est/index.cgi</url>.</p> <p>Conclusion</p> <p>3946 unique transcripts were successfully identified from the adult female <it>A. stephensi </it>midgut tissue. These data can be used for microarray development for better understanding of vector-parasite relationship and to study differences or similarities with other malaria vectors. Mapping of putative novel transcripts from <it>A. stephensi </it>on the <it>A. gambiae </it>genome proved fruitful in identification and annotation of several genes. Failure of some novel transcripts to map on the <it>A. gambiae </it>genome indicates existence of substantial genomic dissimilarities between these two potent malaria vectors.</p

Arm-specific dynamics of chromosome evolution in malaria mosquitoes

<p>Abstract</p> <p>Background</p> <p>The malaria mosquito species of subgenus <it>Cellia </it>have rich inversion polymorphisms that correlate with environmental variables. Polymorphic inversions tend to cluster on the chromosomal arms 2R and 2L but not on X, 3R and 3L in <it>Anopheles gambiae </it>and homologous arms in other species. However, it is unknown whether polymorphic inversions on homologous chromosomal arms of distantly related species from subgenus <it>Cellia </it>nonrandomly share similar sets of genes. It is also unclear if the evolutionary breakage of inversion-poor chromosomal arms is under constraints.</p> <p>Results</p> <p>To gain a better understanding of the arm-specific differences in the rates of genome rearrangements, we compared gene orders and established syntenic relationships among <it>Anopheles gambiae, Anopheles funestus</it>, and <it>Anopheles stephensi</it>. We provided evidence that polymorphic inversions on the 2R arms in these three species nonrandomly captured similar sets of genes. This nonrandom distribution of genes was not only a result of preservation of ancestral gene order but also an outcome of extensive reshuffling of gene orders that created new combinations of homologous genes within independently originated polymorphic inversions. The statistical analysis of distribution of conserved gene orders demonstrated that the autosomal arms differ in their tolerance to generating evolutionary breakpoints. The fastest evolving 2R autosomal arm was enriched with gene blocks conserved between only a pair of species. In contrast, all identified syntenic blocks were preserved on the slowly evolving 3R arm of <it>An. gambiae </it>and on the homologous arms of <it>An. funestus </it>and <it>An. stephensi</it>.</p> <p>Conclusions</p> <p>Our results suggest that natural selection favors specific gene combinations within polymorphic inversions when distant species are exposed to similar environmental pressures. This knowledge could be useful for the discovery of genes responsible for an association of inversion polymorphisms with phenotypic variations in multiple species. Our data support the chromosomal arm specificity in rates of gene order disruption during mosquito evolution. We conclude that the distribution of breakpoint regions is evolutionary conserved on slowly evolving arms and tends to be lineage-specific on rapidly evolving arms.</p

New Salivary Biomarkers of Human Exposure to Malaria Vector Bites

International audienc

Advances in Basic and Translational Research as Part of the Center for the Study of Complex Malaria in India.

The Center for the Study of Complex Malaria in India (CSCMi) is one of 10 International Centers of Excellence in Malaria Research funded by the National Institutes of Health since 2010. The Center combines innovative research with capacity building and technology transfer to undertake studies with clinical and translational impact that will move malaria control in India toward the ultimate goal of malaria elimination/eradication. A key element of each research site in the four states of India (Tamil Nadu, Gujarat, Odisha, and Meghalaya) has been undertaking community- and clinic-based epidemiology projects to characterize the burden of malaria in the region. Demographic and clinical data and samples collected during these studies have been used in downstream projects on, for example, the widespread use of mosquito repellants, the population genomics of Plasmodium vivax, and the serological responses to P. vivax and Plasmodium falciparum antigens that reflect past or present exposure. A focus has been studying the pathogenesis of severe malaria caused by P. falciparum through magnetic resonance imaging of cerebral malaria patients. Here we provide a snapshot of some of the basic and applied research the CSCMi has undertaken over the past 12 years and indicate the further research and/or clinical and translational impact these studies have had

The choreography of the chemical defensome response to insecticide stress: insights into the Anopheles stephensi transcriptome using RNA-Seq

Animals respond to chemical stress with an array of gene families and pathways termed "chemical defensome". In arthropods, despite many defensome genes have been detected, how their activation is arranged during toxic exposure remains poorly understood. Here, we sequenced the transcriptome of Anopheles stephensi larvae exposed for six, 24 and 48 hours to the LD50 dose of the insecticide permethrin to monitor transcriptional changes of defensome genes across time. A total of 177 genes involved in insecticide defense were differentially expressed (DE) in at least one time-point, including genes encoding for Phase 0, I, II, III and antioxidant enzymes and for Heat Shock and Cuticular Proteins. Three major patterns emerged throughout time. First, most of DE genes were down-regulated at all time-points, suggesting a reallocation of energetic resources during insecticide stress. Second, single genes and clusters of genes turn off and on from six to 48 hours of treatment, showing a modulated response across time. Third, the number of up-regulated genes peaked at six hours and then decreased during exposure. Our results give a first picture of how defensome gene families respond against toxicants and provide a valuable resource for understanding how defensome genes work together during insecticide stress

La risposta di difesa degli organismi agli insetticidi: dall’ecologia dello stress al controllo di artropodi vettori

Tra i principali argomenti di studio della biologia vi sono i meccanismi con cui le cellule e gli organismi sono in grado di mantenere l'omeostasi in un ambiente avverso, ricco di stress chimici e fisici. “L’obbligo” di sopravvivenza che guida ogni organismo quando viene esposto ad agenti tossici, come sostanze chimiche tossiche endogene o composti xenobiotici, ha portato all’evoluzione di una serie di famiglie di geni e di pathways che, nell’insieme, prendono il nome di "defensoma chimico". Questa rete di difesa integrata, che permette ad ogni organismo di avvertire, trasformare ed eliminare le sostanze chimiche tossiche, è stata recentemente sottoposta ad approfondite indagini, in particolare modo negli artropodi, in merito alla loro grande importanza economica, ambientale e medico-veterinaria. Considerato il ruolo ancora oggi svolto dai composti chimici, come strumento per il controllo di numerosi pests, l'insorgenza della resistenza metabolica contro diversi insetticidi rappresenta un problema dal punto di vista del controllo d’insetti dannosi, da cui deriva il rischio di un’intensificazione nell’utilizzo degli stessi insetticidi, quindi problematiche ecologiche. Tra i principali meccanismi molecolari, appartenenti al complesso del defensoma chimico, vi sono gli ABC trasportatori, proteine integrali di membrana, atte a disintossicare la cellula da diversi composti nocivi con cui questa entra in contatto. Essi agiscono come prima linea di difesa nelle cellule esposte a uno stress chimico, trasportando le molecole tossiche all’esterno della cellula e riducendone di conseguenza la concentrazione intracellulare necessaria per provocare il danno all’organismo. Gli ABC trasportatori, ben noti e ampiamente indagati in altri animali, hanno suscitato interesse per quanto riguarda gli artropodi solo negli ultimi anni. Al momento, le indagini svolte hanno chiarito solo in parte il ruolo svolto da questi trasportatori nel complesso del defensoma, rimangono quindi diversi interrogativi irrisolti. Questo progetto di dottorato si pone come primo obiettivo quello d’indagare approfonditamente il ruolo di queste proteine di detossificazione e la loro risposta, in termini di espressione genica, all’insetticida di sintesi piretroide, permetrina, in due specie di zanzara vettrici di malaria, Anopheles stephensi e Anopheles gambiae sensu stricto. Una volta determinati i geni maggiormente coinvolti nei processi di detossificazione, si è posto come secondo obiettivo quello di sviluppare sistemi d’inibizione dell’espressione genica al fine di garantire una concentrazione adeguata dell’insetticida, in sede intracellulare, da cui deriverebbero sia un’intensificazione dell’azione dell’insetticida, sia la mancata realizzazione di condizioni di sotto-dosaggio, potenzialmente favorevoli all’insorgenza di resistenze. Per quanto riguarda An. stephensi, le ricerche hanno potuto trovare una base solida ed un background di confronto in lavori precedenti, in cui il grado di attivazione di un selezionato “pacchetto” di geni era già stato evidenziato in larve, in risposta al piretroide, a diversi tempi. Nel presente studio dunque abbiamo voluto indagare il pattern d’espressione degli stessi geni (sei, appartenenti alle sottofamiglie ABCB, ABCC, ABCG), correlato al tasso di mortalità, nello stadio adulto di An. stephensi, maschi e femmine, sottoposti al trattamento con permetrina. Le diverse pressioni selettive alle quali le molteplici forme sono sottoposte potrebbero influire sui valori e la dinamicità di espressione dei trasportatori. In An. gambiae s.s. si è voluto analizzare, per la prima volta, il coinvolgimento degli stessi geni in larve, tramite duplice approccio: induzione ed inibizione aspecifica. E’ stato quindi evidenziato il pattern di espressione nel tempo di cinque geni ABC (sottofamiglie ABCB, ABCC, ABCG) dopo induzione sia con sola permetrina, sia in combinazione con un inibitore aspecifico dei trasportatori ABC (verapamil). Le indagini svolte nell’ambito del primo obiettivo hanno permesso di identificare il trasportatore ABCG4 come target promettente ai fini di un’inibizione dei meccanismi di difesa, sia in An. stephensi che in An. gambiae. Il secondo obiettivo è stato perseguito attraverso esperimenti su larve di An. stephensi. Una prima fase di studio è consistita nella realizzazione di saggi d’inibizione attraverso siRNA specie- e sequenza-specifico. I risultati ottenuti hanno consolidato l’ipotesi di un marcato coinvolgimento di ABCG4 come prima difesa contro la permetrina nelle larve di An. stephensi. La seconda fase dello studio d’inibizione ha previsto l’utilizzo di un diverso tipo di oligonucleotide antisenso (Vivo-Morpholino), caratterizzato da una maggiore stabilità ed efficacia rispetto al siRNA e dunque più adatto per un eventuale utilizzo in campo. Maggiormente rispetto al siRNA, la molecola di Vivo-Morpholino ha potenziato l’azione della permetrina, permettendo di ottenere la medesima mortalità larvale in presenza di dosi ridotte d’insetticida. In conclusione, il lavoro svolto, oltre a determinare il quadro di espressione di geni coinvolti nella detossificazione in due specie di zanzare malariche, ha percorso i primi passi verso la possibilità di sfruttare l'inibizione dell'espressione di un ABC trasportatore, per la progettazione di nuovi approcci di controllo del vettore, al fine di ridurre le dosi d’insetticidi chimici impiegati. Sarà dunque opportuno effettuare indagini più approfondite, per lo sviluppo di larvicidi e di sistemi di delivery altamente ecocompatibili e dunque fruibili per future applicazioni in campo