Mosquito Vectors (Diptera: Culicidae) and Mosquito-Borne Diseases in North Africa.

Mosquitoes (Diptera: Culicidae) are of significant public health importance because of their ability to transmit major diseases to humans and animals, and are considered as the world's most deadly arthropods. In recent decades, climate change and globalization have promoted mosquito-borne diseases' (MBDs) geographic expansion to new areas, such as North African countries, where some of these MBDs were unusual or even unknown. In this review, we summarize the latest data on mosquito vector species distribution and MBDs affecting both human and animals in North Africa, in order to better understand the risks associated with the introduction of new invasive mosquito species such as Aedes albopictus. Currently, 26 mosquito species confirmed as pathogen vectors occur in North Africa, including Aedes (five species), Culex (eight species), Culiseta (one species) and Anopheles (12 species). These 26 species are involved in the circulation of seven MBDs in North Africa, including two parasitic infections (malaria and filariasis) and five viral infections (WNV, RVF, DENV, SINV and USUV). No bacterial diseases have been reported so far in this area. This review may guide research studies to fill the data gaps, as well as helping with developing effective vector surveillance and controlling strategies by concerned institutions in different involved countries, leading to cooperative and coordinate vector control measures.

Identification of Algerian field-caught mosquito vectors by MALDI-TOF MS.

Vector-borne diseases represent a real threats worldwide, in reason of the lack of vaccine and cure for some diseases. Among arthropod vectors, mosquitoes are described to be the most dangerous animal on earth, resulting in an estimated 725,000 deaths per year due to their borne diseases. Geographical position of Algeria makes this country a high risk area for emerging and re-emerging diseases, such as dengue coming from north (Europe) and malaria from south (Africa). To prevent these threats, rapid and continuous surveillance of mosquito vectors is essential. For this purpose we aimed in this study to create a mosquito vectors locale database using MALDI-TOF mass spectrometry technology for rapid identification of these arthropods. This methodology was validated by testing 211 mosquitoes, including four species (Aedes albopictus, Culex pipiens, Culex quinquefasciatus, and Culiseta longiareolata), in two northern wilayahs of Algeria (Algiers and Bejaia). Species determination by MALDI TOF MS was highly concordant with reference phenotypic and genetic methods. Using this MALDI-TOF MS tool will allow better surveillance of mosquito species able to transmit mosquito borne diseases in Algeria.

Virome Diversity among Mosquito Populations in a Sub-Urban Region of Marseille, France.

Some mosquito species have significant public health importance given their ability to transmit major diseases to humans and animals, making them the deadliest animals in the world. Among these, the Aedes (Ae.) genus is a vector of several viruses such as Dengue, Chikungunya, and Zika viruses that can cause serious pathologies in humans. Since 2004, Ae. albopictus has been encountered in the South of France, and autochthonous cases of Dengue, Chikungunya, and Zika diseases have recently been reported, further highlighting the need for a comprehensive survey of the mosquitoes and their associated viruses in this area. Using high throughput sequencing (HTS) techniques, we report an analysis of the DNA and RNA viral communities of three mosquito species Ae. albopictus, Culex (Cx.) pipiens, and Culiseta (Cs.) longiareolata vectors of human infectious diseases in a small sub-urban city in the South of France. Results revealed the presence of a significant diversity of viruses known to infect bacteria, plants, insects, and mammals. Several novel viruses were detected, including novel members of the Rhabdoviridae, Totiviridae, Iflaviviridae, Circoviridae, and Sobemoviridae families. No sequence related to major zoonotic viruses transmitted by mosquitoes was detected. The use of HTS on arthropod vector populations is a promising strategy for monitoring the emergence and circulation of zoonoses and epizooties. This study is a contribution to the knowledge of the mosquito microbiome.

Identification of Aedes mosquitoes by MALDI-TOF MS biotyping using protein signatures from larval and pupal exuviae.

BACKGROUND: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) biotyping is an innovative strategy, applied successfully for the identification of numerous arthropod families including mosquitoes. The effective mosquito identification using this emerging tool was demonstrated possible at different steps of their life-cycle, including eggs, immature and adult stages. Unfortunately, for species identification by MS, the euthanasia of the mosquito specimen is required. METHODS: To avoid mosquito euthanasia, the present study assessed whether aedine mosquitoes could be identified by MALDI-TOF MS biotyping, using their respective exuviae. In this way, exuviae from the fourth-instar and pupal stages of Aedes albopictus and Aedes aegypti were submitted to MALDI-TOF MS analysis. RESULTS: Reproducible and specific MS spectra according to aedine species and stage of exuviae were observed which were objectified by cluster analyses, composite correlation index (CCI) tool and principal components analysis (PCA). The query of our reference MS spectra database (DB) upgraded with MS spectra of exuviae from fourth-instar larvae and pupae of both Aedes species revealed that 100% of the samples were correctly classified at the species and stage levels. Among them, 93.8% (135/144) of the MS profiles reached the threshold log score value (LSV > 1.8) for reliable identification. CONCLUSIONS: The extension of reference MS spectra DB to exuviae from fourth-instar and pupal stages made now possible the identification of mosquitoes throughout their life-cycle at aquatic and aerial stages. The exuviae presenting the advantage to avoid specimen euthanasia, allowing to perform complementary analysis on alive mosquitoes.

Molecular detection of avian spirochete Borrelia anserina in Argas persicus ticks in Algeria.

Argasid ticks are one of the most important poultry ectoparasites. They affect poultry directly through blood meal and indirectly through the transmission of pathogens essentially Borrelia anserina, agent of avian borreliosis, one of the most widespread poultry diseases in the world, and is of great economic importance. This study was conducted between April 2014 and March 2015 in the region of Ksar El Boukhari, Algeria, in order to investigate the presence of soft ticks in laying hen farms and to detect B. anserina bacteria using molecular tools. DNA was extracted and screened for the presence of Borrelia spp. DNA by real-time polymerase chain reaction (qPCR). Borrelia spp. screening was performed using primers and probe targeting the 16S rRNA gene. A total of 83 traditional laying hen farms were visited, of which 39 (46.98 %) were found infested with A. persicus tick. Molecular analysis revealed that 2/34 (5.88 %) of ticks were infected by B. anserina. None of the ticks tested were positive for Rickettsia spp., and Coxiella burnetii. These results constitute the first report in Algeria of A. persicus harboring B. anserina.

Co-infection of bacteria and protozoan parasites in Ixodes ricinus nymphs collected in the Alsace region, France.

Fifty nymphal Ixodes ricinus ticks collected in Alsace, France, identified by morphological criteria and using MALDI-TOF MS, were tested by PCR to detect tick-associated bacteria and protozoan parasites. Seventy percent (35/50) of ticks contained at least one microorganism; 26% (9/35) contained two or more species. Several human pathogens were identified including Borrelia burgdorferi s.s. (4%), Borrelia afzelii (2%), Borrelia garinii (2%), Borrelia valaisiana (4%), Borrelia miyamotoi (2%), Rickettsia helvetica (6%) and "Babesia venatorum" (2%). Bartonella spp. (10%) and a Wolbachia spp. (8%) were also detected. The most common co-infections involved Anaplasmataceae with Borrelia spp. (4%), Anaplasmataceae with Bartonella spp. (6%) and Anaplasmataceae with Rickettsia spp. (6%). Co-infection involving three different groups of bacteria was seen between bacteria of the family Anaplasmataceae, Borrelia spp. and Bartonella spp. (2%). Results highlight the panel of infectious agents carried by Ixodes ricinus. Co-infection suggests the possibility of transmission of more than one pathogen to human and animals during tick blood feeding.

Longitudinal monitoring of environmental factors at Culicidae larval habitats in urban areas and their association with various mosquito species using an innovative strategy.

BACKGROUND: To prevent the risk of mosquito-borne disease outbreaks, larval source management remains the most sustainable and effective mosquito control strategy. The present study aimed to determine the influence of environmental characteristics of mosquito larval habitats in an urban area of Marseille, France. Fourteen sites containing water were monitored every 2 weeks from May to October 2015 for mosquito species occurrence and larval density, and environmental parameters were measured at each visit. Rapid and accurate species identification of mosquito larvae was performed using an innovative MALDI-TOF MS method. RESULTS: A total of 6753 larvae (L1-L4) and pupae were collected, of which 35.8% (n = 2418) were speciated using MALDI-TOF MS. Correct identifications were obtained for 2259 specimens (93.4%). A total of five mosquito species were found, including Aedes (Ae.) albopictus, Culex (Cx.) p. pipiens, Cx. hortensis, Cx. impudicus, and Culiseta (Cs.) longiareolata. Larvae of the Culex genus were predominant in both density and distribution. Small, shaded pools of shallow water favored Ae. albopictus colonization, whereas the wide distribution of Cx. p. pipiens demonstrated that this species was weakly influenced by environmental changes. CONCLUSIONS: The present work confirms that MALDI-TOF MS is a useful tool for mosquito speciation and suggests that understanding the environmental factors associated with the occurrence and density of mosquito species at the larval stage in Marseille may aid in the future implementation of mosquito control programs. © 2018 Society of Chemical Industry.

Field application of MALDI-TOF MS on mosquito larvae identification.

In recent years, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has emerged as an efficient tool for arthropod identification. Its application for field monitoring of adult mosquitoes was demonstrated, but identification of larvae has been limited to laboratory-reared specimens. Study aim was to test the success of MALDI-TOF MS in correctly identifying mosquito larvae collected in the field. Collections were performed at 13 breeding sites in urban areas of Marseille, a city in the South of France. A total of 559 larvae were collected. Of these, 73 were accurately morphologically identified, with confirmation either by molecular identification (n = 31) or analysis with MALDI-TOF MS (n = 31) and 11 were tested using both methods. The larvae identified belonged to six species including Culiseta longiareolata, Culex pipiens pipiens, Culex hortensis, Aedes albopictus, Ochlerotatus caspius and Anopheles maculipennis. A high intra-species reproducibility and inter-species specificity of whole larva MS spectra was obtained and was independent of breeding site. More than 92% of the remaining 486 larvae were identified in blind tests against the MS spectra database. Identification rates were lower for early and pupal stages, which is attributed to lower protein abundance and metamorphosis, respectively. The suitability of MALDI-TOF MS for mosquito larvae identification from the field has been confirmed.

Assessment of MALDI-TOF MS biotyping for Borrelia burgdorferi sl detection in Ixodes ricinus.

Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) has been demonstrated to be useful for tick identification at the species level. More recently, this tool has been successfully applied for the detection of bacterial pathogens directly in tick vectors. The present work has assessed the detection of Borrelia burgdorferi sensu lato in Ixodes ricinus tick vector by MALDI-TOF MS. To this aim, experimental infection model of I. ricinus ticks by B. afzelii was carried out and specimens collected in the field were also included in the study. Borrelia infectious status of I. ricinus ticks was molecularly controlled using half-idiosome to classify specimens. Among the 39 ticks engorged on infected mice, 14 were confirmed to be infected by B. afzelii. For field collection, 14.8% (n = 12/81) I. ricinus ticks were validated molecularly as infected by B. burgdorferi sl. To determine the body part allowing the detection of MS protein profile changes between non-infected and B. afzelii infected specimens, ticks were dissected in three compartments (i.e. 4 legs, capitulum and half-idiosome) prior to MS analysis. Highly reproducible MS spectra were obtained for I. ricinus ticks according to the compartment tested and their infectious status. However, no MS profile change was found when paired body part comparison between non-infected and B. afzelii infected specimens was made. Statistical analyses did not succeed to discover, per body part, specific MS peaks distinguishing Borrelia-infected from non-infected ticks whatever their origins, laboratory reared or field collected. Despite the unsuccessful of MALDI-TOF MS to classify tick specimens according to their B. afzelii infectious status, this proteomic tool remains a promising method for rapid, economic and accurate identification of tick species. Moreover, the singularity of MS spectra between legs and half-idiosome of I. ricinus could be used to reinforce this proteomic identification by submission of both these compartments to MS.

Standardization of sample homogenization for mosquito identification using an innovative proteomic tool based on protein profiling.

The rapid spread of vector-borne diseases demands the development of an innovative strategy for arthropod monitoring. The emergence of MALDI-TOF MS as a rapid, low-cost, and accurate tool for arthropod identification is revolutionizing medical entomology. However, as MS spectra from an arthropod can vary according to the body part selected, the sample homogenization method used and the mode and duration of sample storage, standardization of protocols is indispensable prior to the creation and sharing of an MS reference spectra database. In the present study, manual grinding of Anopheles gambiae Giles and Aedes albopictus mosquitoes at the adult and larval (L3) developmental stages was compared to automated homogenization. Settings for each homogenizer were optimized, and glass powder was found to be the best sample disruptor based on its ability to create reproducible and intense MS spectra. In addition, the suitability of common arthropod storage conditions for further MALDI-TOF MS analysis was kinetically evaluated. The conditions that best preserved samples for accurate species identification by MALDI-TOF MS were freezing at -20°C or in liquid nitrogen for up to 6 months. The optimized conditions were objectified based on the reproducibility and stability of species-specific MS profiles. The automation and standardization of mosquito sample preparation methods for MALDI-TOF MS analyses will popularize the use of this innovative tool for the rapid identification of arthropods with medical interest.