Mosquito Attractants.

Vector control and personal protection against anthropophilic mosquitoes mainly rely on the use of insecticides and repellents. The search for mosquito-attractive semiochemicals has been the subject of intense studies for decades, and new compounds or odor blends are regularly proposed as lures for odor-baited traps. We present a comprehensive and up-to-date review of all the studies that have evaluated the attractiveness of volatiles to mosquitoes, including individual chemical compounds, synthetic blends of compounds, or natural host or plant odors. A total of 388 studies were analysed, and our survey highlights the existence of 105 attractants (77 volatile compounds, 17 organism odors, and 11 synthetic blends) that have been proved effective in attracting one or several mosquito species. The exhaustive list of these attractants is presented in various tables, while the most common mosquito attractants - for which effective attractiveness has been demonstrated in numerous studies - are discussed throughout the text. The increasing knowledge on compounds attractive to mosquitoes may now serve as the basis for complementary vector control strategies, such as those involving lure-and-kill traps, or the development of mass trapping. This review also points out the necessity of further improving the search for new volatile attractants, such as new compound blends in specific ratios, considering that mosquito attraction to odors may vary over the life of the mosquito or among species. Finally, the use of mosquito attractants will undoubtedly have an increasingly important role to play in future integrated vector management programs.

New ribosome-inactivating proteins and other proteins with protein synthesis-inhibiting activities.

Ribosome-inactivating proteins (RIPs) consist of three varieties. Type 1 RIPs are single-chained and approximately 30-kDa in molecular weight. Type 2 RIPs are double-chained and composed of a type 1 RIP chain and a lectin chain. Type III RIPs, such as maize b-32 barley and JIP60 which are produced as single-domain proenzymes, possess an N-terminal domain corresponding to the A domain of RIPs and fused to a C-terminal domain. In addition to the aforementioned three types of RIPs originating from flowering plants, there are recently discovered proteins and peptides with ribosome-inactivating and protein synthesis inhibitory activities but which are endowed with characteristics such as molecular weights distinctive from those of the regular RIPs. These new/unusual RIPs discussed in the present review encompass metazoan RIPs from Anopheles and Culex mosquitos, antimicrobial peptides derived from RIP of the pokeweed Phytolacca dioica, maize RIP (a type III RIP derived from a precursor form), RIPs from the garden pea and the kelp. In addition, RIPs with a molecular weight smaller than those of regular type 1 RIPs are produced by plants in the Cucurbitaceae family including the bitter gourd, bottle gourd, sponge gourd, ridge gourd, wax gourd, hairy gourd, pumpkin, and Chinese cucumber. A small type II RIP from camphor tree (Cinnamomum camphora) seeds and a snake gourd type II RIP with its catalytic chain cleaved into two have been reported. RIPs produced from mushrooms including the golden needle mushroom, king tuber mushroom, straw mushroom, and puffball mushroom are also discussed in addition to a type II RIP from the mushroom Polyporus umbellatus. Bacterial (Spiroplasma) RIPs associated with the fruitfly, Shiga toxin, and Streptomyces coelicolor RIP are also dealt with. The aforementioned proteins display a diversity of molecular weights, amino acid sequences, and mechanisms of action. Some of them are endowed with exploitable antipathogenic activities.

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.

Using MALDI-TOF MS to identify mosquitoes collected in Mali and their blood meals.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been recently described as an innovative and effective tool for identifying arthropods and mosquito blood meal sources. To test this approach in the context of an entomological survey in the field, mosquitoes were collected from five ecologically distinct areas of Mali. We successfully analysed the blood meals from 651 mosquito abdomens crushed on Whatman filter paper (WFPs) in the field using MALDI-TOF MS. The legs of 826 mosquitoes were then submitted for MALDI-TOF MS analysis in order to identify the different mosquito species. Eight mosquito species were identified, including Anopheles gambiae Giles, Anopheles coluzzii, Anopheles arabiensis, Culex quinquefasciatus, Culex neavei, Culex perexiguus, Aedes aegypti and Aedes fowleri in Mali. The field mosquitoes for which MALDI-TOF MS did not provide successful identification were not previously available in our database. These specimens were subsequently molecularly identified. The WFP blood meal sources found in this study were matched against human blood (n = 619), chicken blood (n = 9), cow blood (n = 9), donkey blood (n = 6), dog blood (n = 5) and sheep blood (n = 3). This study reinforces the fact that MALDI-TOF MS is a promising tool for entomological surveys.

Functional and Nonfunctional Forms of CquiOR91, an Odorant Selectivity Subunit of Culex quinquefasciatus.

In Culex quinquefasciatus, CquiOR91 is the ortholog of 2 larvae-specific odorant receptors (ORs) from Anopheles gambiae (Agam\Or40, previously shown to respond to several odorant ligands including the broad-spectrum repellent N,N-diethyl-3-methylbenzamide, DEET) and Aedes aegypti (Aaeg\Or40). When we cloned full-length CquiOR91 from a Culex quinquefasciatus larval head RNA sample, we found 2 alleles of this OR, differing at 9 residues. Functional analysis using the Xenopus oocyte expression system and 2-electrode voltage clamp electrophysiology revealed one allele (CquiOR91.1) to be nonfunctional, whereas the other allele (CquiOR91.2) was functional. Receptors formed by CquiOR91.2 and Cqui\Orco responded to (-)-fenchone, (+)-fenchone, and DEET, similar to what has been reported for Agam\Or40. We also identified 5 novel odorant ligands for the CquiOR91.2 + Cqui\Orco receptor: 2-isobutylthiazole, veratrole, eucalyptol, d-camphor, and safranal, with safranal being the most potent. To explore possible reasons for the lack of function for CquiOR91.1, we generated a series of mutant CquiOR91.2 subunits, in which the residue at each of the 9 polymorphic residue positions was changed from what occurs in CquiOR91.2 to what occurs in CquiOR91.1. Eight of the 9 mutant versions of CquiOR91.2 formed functional receptors, responding to (-)-fenchone. Only the CquiOR91.2 Y183C mutant was nonfunctional. The reverse mutation (C183Y) conferred function on CquiOR91.1 , which became responsive to (-)-fenchone and safranal. These results indicate that the "defect" in CquiOR91.1 that prevents function is the cysteine at position 183.

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.

Hemoglobin-derived porphyrins preserved in a Middle Eocene blood-engorged mosquito.

Although hematophagy is found in ~14,000 species of extant insects, the fossil record of blood-feeding insects is extremely poor and largely confined to specimens identified as hematophagic based on their taxonomic affinities with extant hematophagic insects; direct evidence of hematophagy is limited to four insect fossils in which trypanosomes and the malarial protozoan Plasmodium have been found. Here, we describe a blood-engorged mosquito from the Middle Eocene Kishenehn Formation in Montana. This unique specimen provided the opportunity to ask whether or not hemoglobin, or biomolecules derived from hemoglobin, were preserved in the fossilized blood meal. The abdomen of the fossil mosquito was shown to contain very high levels of iron, and mass spectrometry data provided a convincing identification of porphyrin molecules derived from the oxygen-carrying heme moiety of hemoglobin. These data confirm the existence of taphonomic conditions conducive to the preservation of biomolecules through deep time and support previous reports of the existence of heme-derived porphyrins in terrestrial fossils.

Analysis of Pesticides and Toxic Heavy Metals Contained in Mosquito Coils.

In this study, 10 mosquito coils manufactured in China were obtained in Suriname, South America, where they are used extensively. The coils were analyzed for organics (allethrin, permethrin, and butylated hydroxytoluene) and heavy metals (Cr, Co, As, Cd, and Pb) by GC-MS and ICP-MS, respectively. Allethrin was the only target organic compound detected in all mosquito coils with concentrations ranging from ~1900 to ~4500 µg/g. The concentrations of heavy metals varied as follows (in µg/g): Cr: 2.9-9.4, Co: 0.1-1.2, Cu: 0.7-16.1, Se: 0.10-0.4, Ni: 2.1-5.8, As: 0.10-2.2, Cd: 0.10-0.2, and Pb: 1.1-3.6.

Identification of European mosquito species by MALDI-TOF MS.

MALDI-TOF MS profiling has proved to be efficient for arthropod identification at the species level. However, prior to entomological monitoring, the reference spectra database should cover relevant species. Here, 74 specimens were field-collected from 11 mosquito species captured in two distinct European areas and used either to increment our database or for blind tests. Misidentification was not noted, underlining the power of this approach. Nevertheless, three out of the 26 specimens used for the blind test did not reach the significant identification threshold value set, attributed to lower spectral quality. In the future, the quality control spectra parameters need to be defined to avoid not achieving significant threshold identification.

Culiseta subochrea as a bioindicator of metal contamination in Shadegan International Wetland, Iran (Diptera: Culicidae).

The quantity of some trace metals of mosquito larvae in Shadegan International Wetland from Iran was evaluated. Water, waterbed sediment, and mosquito larvae samplings were carried out from an urban site in the east of the wetland, using standard methods in December 2011. The identified Culiseta subochrea (Edwards) and Aedes caspius s.l. (Pallas) larvae, water, and waterbed sediment samples were analyzed for As, Cd, Co, Cr, Cu, Fe, Hg, Mn, Pb, and Zn trace metals using standard preparation and isolation procedure. Result showed that the waterbed sediment and Cu. subochrea larvae are polluted with all trace metals investigated except As and Hg. The trace metals bioaccumulated in the Cu. subochrea larvae range from 31.78 at the lowest level for Cr to 3822.7 at the highest level for Cd. In a conclusion, this is the first report confirmed that Cu. subochrea likely used as a bioindicator to trace metal pollution in marine ecosystems in the world, especially wetlands.

Evaluation of matrix-assisted laser desorption/ionization time of flight mass spectrometry for the identification of ceratopogonid and culicid larvae.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) was evaluated for the rapid identification of ceratopogonid larvae. Optimal sample preparation as evaluated with laboratory-reared biting midges Culicoides nubeculosus was the homogenization of gut-less larvae in 10% formic acid, and analysis of 0.2 mg/ml crude protein homogenate mixed with SA matrix at a ratio of 1:1.5. Using 5 larvae each of 4 ceratopogonid species (C. nubeculosus, C. obsoletus, C. decor, and Dasyhelea sp.) and of 2 culicid species (Aedes aegypti, Ae. japonicus), biomarker mass sets between 27 and 33 masses were determined. In a validation study, 67 larvae belonging to the target species were correctly identified by automated database-based identification (91%) or manual full comparison (9%). Four specimens of non-target species did not yield identification. As anticipated for holometabolous insects, the biomarker mass sets of adults cannot be used for the identification of larvae, and vice versa, because they share only very few similar masses as shown for C. nubeculosus, C. obsoletus, and Ae. japonicus. Thus, protein profiling by MALDI-TOF as a quick, inexpensive and accurate alternative tool is applicable to identify insect larvae of vector species collected in the field.

Resolving postglacial phylogeography using high-throughput sequencing.

The distinction between model and nonmodel organisms is becoming increasingly blurred. High-throughput, second-generation sequencing approaches are being applied to organisms based on their interesting ecological, physiological, developmental, or evolutionary properties and not on the depth of genetic information available for them. Here, we illustrate this point using a low-cost, efficient technique to determine the fine-scale phylogenetic relationships among recently diverged populations in a species. This application of restriction site-associated DNA tags (RAD tags) reveals previously unresolved genetic structure and direction of evolution in the pitcher plant mosquito, Wyeomyia smithii, from a southern Appalachian Mountain refugium following recession of the Laurentide Ice Sheet at 22,000-19,000 B.P. The RAD tag method can be used to identify detailed patterns of phylogeography in any organism regardless of existing genomic data, and, more broadly, to identify incipient speciation and genome-wide variation in natural populations in general.

MALDI-TOF mass spectrometry identification of mosquitoes collected in Vietnam.

BACKGROUND: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a tool that has revolutionised clinical microbiology and has recently been described as an innovative and effective approach to arthropod identification. METHODS: In this study, mosquitoes were captured in Vietnam using four different methods (human landing catch, CDC light traps, BG-Sentinel traps, animal-baited net traps). A total of 4215 mosquitoes were captured and morphologically identified as belonging to three genera: Aedes, Anopheles and Culex. We randomly selected 1253 mosquitoes, including 662 specimens of 14 Anopheles species, 200 specimens of two Aedes species and 391 morphologically unidentified Culex specimens, for molecular and MALDI-TOF MS analysis. The DNA from 98 mosquitoes (69 Anopheles specimens, 23 Culex specimens and six Aedes sp. specimens) was subjected to molecular analysis, either to confirm our morphological identification or the MALDI-TOF MS results, as well as to identify the Culex species that were morphologically identified at the genus level and to resolve the discrepancies between the morphological identification and the MALDI-TOF MS identification. RESULTS: High-quality MS spectra were obtained for 1058 of the 1253 specimens (84%), including 192/200 for Aedes, 589/662 for Anopheles and 277/391 for Culex. The blind test showed that 986/997 (99%) of the specimens were correctly identified by MALDI-TOF MS, with log score values ranging from 1.708 to 2.843. Eleven specimens of Culex could not be identified based on morphological features, MALDI-TOF MS or molecular analysis. CONCLUSIONS: This study enabled us to identify several species of mosquitoes from Vietnam using MALDI-TOF MS, and to enrich our database of MALDI-TOF MS reference spectra.

Ability of near-infrared spectroscopy and chemometrics to predict the age of mosquitoes reared under different conditions.

BACKGROUND: Practical, field-ready age-grading tools for mosquito vectors of disease are urgently needed because of the impact that daily survival has on vectorial capacity. Previous studies have shown that near-infrared spectroscopy (NIRS), in combination with chemometrics and predictive modeling, can forecast the age of laboratory-reared mosquitoes with moderate to high accuracy. It remains unclear whether the technique has utility for identifying shifts in the age structure of wild-caught mosquitoes. Here we investigate whether models derived from the laboratory strain of mosquitoes can be used to predict the age of mosquitoes grown from pupae collected in the field. METHODS: NIRS data from adult female Aedes albopictus mosquitoes reared in the laboratory (2, 5, 8, 12 and 15 days-old) were analysed against spectra from mosquitoes emerging from wild-caught pupae (1, 7 and 14 days-old). Different partial least squares (PLS) regression methods trained on spectra from laboratory mosquitoes were evaluated on their ability to predict the age of mosquitoes from more natural environments. RESULTS: Models trained on spectra from laboratory-reared material were able to predict the age of other laboratory-reared mosquitoes with moderate accuracy and successfully differentiated all day 2 and 15 mosquitoes. Models derived with laboratory mosquitoes could not differentiate between field-derived age groups, with age predictions relatively indistinguishable for day 1-14. Pre-processing of spectral data and improving the PLS regression framework to avoid overfitting can increase accuracy, but predictions of mosquitoes reared in different environments remained poor. Principal components analysis confirms substantial spectral variations between laboratory and field-derived mosquitoes despite both originating from the same island population. CONCLUSIONS: Models trained on laboratory mosquitoes were able to predict ages of laboratory mosquitoes with good sensitivity and specificity though they were unable to predict age of field-derived mosquitoes. This study suggests that laboratory-reared mosquitoes do not capture enough environmental variation to accurately predict the age of the same species reared under different conditions. Further research is needed to explore alternative pre-processing methods and machine learning techniques, and to understand factors that affect absorbance in mosquitoes before field application using NIRS.

Application of the Droplet Digital Polymerase Chain Reaction (ddPCR) Platform for Detection and Quantification of Vertebrate Host DNA in Engorged Mosquitoes.

Hematophagous arthropod bloodmeal identification has remained a challenge in the field of vector biology, but these studies are important to understand blood feeding patterns of arthropods, spatial, and temporal patterns in arbovirus transmission cycles, and risk of human and veterinary disease. We investigated the use of an existing vertebrate primer set for use on the droplet digital polymerase chain reaction (ddPCR) platform, to explore the use of this technology in the identification and quantification of vertebrate DNA in mosquito blood meals. Host DNA was detectable 48-h post-engorgement in some mosquitoes by ddPCR, compared with 24-h post-engorgement using traditional PCR. The capability of ddPCR for absolute quantification of template DNA offers unique potential applications of this new technology to field studies on the ecology of vector-borne diseases, but currently with limited scope.

Insect anionic septapeptides suppress DENV replication by activating antiviral cytokines and miRNAs in primary human monocytes.

Dengue viruses (DENVs) have threatened 2/3 of the world population for decades. Thus, combating DENV infection with either antiviral therapy or protective vaccination is an urgent goal. In the present study, we investigated the anti-DENV activity of insect cell-derived anionic septapeptides from C6/36 mosquito cell cultures persistently infected with DENV. These molecules were previously shown to protect C6/36 and Vero cells against DENV infection. We found that treatment with these septapeptides strongly and rapidly downregulated the multiplication of DENV-1 16007, DENV-3 16562, and DENV-4 1036 but not that of DENV-2 16681 in primary human monocytes. This inhibitory effect was likely mediated through various routes including the increased production of antiviral cytokines (IFN-I), activation of mononuclear cell migration, and upregulation of the expression of antiviral miRNAs (has-miR-30e*, has-miR-133a, and has-miR-223) and inflammation-related miRNAs (has-miR-146a and has-miR-147). In conclusion, anionic septapeptides exerted anti-DENV activity in human monocytes through the upregulation of innate immune responses and the activation of several previously reported antiviral and inflammation-related miRNAs.

An insight into the sialotranscriptome of the non-blood feeding Toxorhynchites amboinensis mosquito.

All adult mosquitoes take sugar meals, and most adult females also take blood meals to develop eggs. Salivary glands (SG) of males are thus much smaller and do not contain many of the antihemostatic and antiinflammatory compounds found in females. In the past 5 years, transcriptome analyses have identified nearly 70 different genes expressed in adult female SG. For most of these, no function can be assigned in either blood or sugar feeding. Exceptionally, Toxorhynchites mosquitoes are unusual in that they never feed on blood, and the SG of adults are identical in both sexes. Transcriptome analysis of the adult SG of this mosquito was performed to increase knowledge of the evolution of blood feeding--and to identify polypeptide families associated with sugar feeding--in mosquitoes.

Improvement of mosquito identification by MALDI-TOF MS biotyping using protein signatures from two body parts.

BACKGROUND: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry technology (MALDI-TOF MS) is an innovative tool that has been shown to be effective for the identification of numerous arthropod groups including mosquitoes. A critical step in the implementation of MALDI-TOF MS identification is the creation of spectra databases (DB) for the species of interest. Mosquito legs were the body part most frequently used to create identification DB. However, legs are one of the most fragile mosquito compartments, which can put identification at risk. Here, we assessed whether mosquito thoraxes could also be used as a relevant body part for mosquito species identification using a MALDI-TOF MS biotyping strategy; we propose a double DB query strategy to reinforce identification success. METHODS: Thoraxes and legs from 91 mosquito specimens belonging to seven mosquito species collected in six localities from Guadeloupe, and two laboratory strains, Aedes aegypti BORA and Aedes albopictus Marseille, were dissected and analyzed by MALDI-TOF MS. Molecular identification using cox1 gene sequencing was also conducted on representative specimens to confirm their identification. RESULTS: MS profiles obtained with both thoraxes and legs were highly compartment-specific, species-specific and species-reproducible, allowing high identification scores (log-score values, LSVs) when queried against the in-house MS reference spectra DB (thorax LSVs range: 2.260-2.783, leg LSVs range: 2.132-2.753). CONCLUSIONS: Both thoraxes and legs could be used for a double DB query in order to reinforce the success and accuracy of MALDI-TOF MS identification.

Rapid identification of medically important mosquitoes by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

BACKGROUND: Accurate and rapid identification of dipteran vectors is integral for entomological surveys and is a vital component of control programs for mosquito-borne diseases. Conventionally, morphological features are used for mosquito identification, which suffer from biological and geographical variations and lack of standardization. We used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for protein profiling of mosquito species from North India with the aim of creating a MALDI-TOF MS database and evaluating it. METHODS: Mosquito larvae were collected from different rural and urban areas and reared to adult stages. The adult mosquitoes of four medically important genera, Anopheles, Aedes, Culex and Armigerus, were morphologically identified to the species level and confirmed by ITS2-specific PCR sequencing. The cephalothoraces of the adult specimens were subjected to MALDI-TOF analysis and the signature peak spectra were selected for creation of database, which was then evaluated to identify 60 blinded mosquito specimens. RESULTS: Reproducible MALDI-TOF MS spectra spanning over 2-14 kDa m/z range were produced for nine mosquito species: Anopheles (An. stephensi, An. culicifacies and An. annularis); Aedes (Ae. aegypti and Ae. albopictus); Culex (Cx. quinquefasciatus, Cx. vishnui and Cx. tritaenorhynchus); and Armigerus (Ar. subalbatus). Genus- and species-specific peaks were identified to create the database and a score of > 1.8 was used to denote reliable identification. The average numbers of peaks obtained were 55-60 for Anopheles, 80-100 for Aedes, 30-60 for Culex and 45-50 peaks for Armigeres species. Of the 60 coded samples, 58 (96.67%) were correctly identified by MALDI-TOF MS with a score > 1.8, while there were two unreliable identifications (both Cx. quinquefasciatus with scores < 1.8). CONCLUSIONS: MALDI-TOF MS appears to be a pragmatic technique for accurate and rapid identification of mosquito species. The database needs to be expanded to include species from different geographical regions and also different life-cycle stages to fully harness the technique for entomological surveillance programs.