MALDI-TOF MS protein profiling for the rapid identification of Chagas disease triatomine vectors and application to the triatomine fauna of French Guiana.

Triatomines are haematophagous insects involved in the transmission of Trypanosoma cruzi, the aetiological agent of Chagas disease. The vector competence of these arthropods can be highly variable, depending on the species. A precise identification is therefore crucial for the epidemiological surveillance of T. cruzi and the determination of at-risk human populations. To circumvent the difficulties of morphological identification and the lack of comprehensiveness of the GenBank database, we hereby propose an alternative method for triatomine identification. The femurs of the median legs of triatomines from eight different species from French Guiana were subjected to matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis. Method evaluation was performed on fresh specimens and was applied to dry specimens collected between 1991 and 2003. Femur-derived protein extracts provided reproducible spectra within the same species along with significant interspecies heterogeneity. Validation of the study by blind test analysis provided 100% correct identification of the specimens in terms of the species, sex and developmental stage. MALDI-TOF mass spectrometry appears to be a powerful tool for triatomine identification, which is a major step forward in the fight against Chagas disease.

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.

Transmission potential of Rickettsia felis infection by Anopheles gambiae mosquitoes.

A growing number of recent reports have implicated Rickettsia felis as a human pathogen, paralleling the increasing detection of R. felis in arthropod hosts across the globe, primarily in fleas. Here Anopheles gambiae mosquitoes, the primary malarial vectors in sub-Saharan Africa, were fed with either blood meal infected with R. felis or infected cellular media administered in membrane feeding systems. In addition, a group of mosquitoes was fed on R. felis-infected BALB/c mice. The acquisition and persistence of R. felis in mosquitoes was demonstrated by quantitative PCR detection of the bacteria up to day 15 postinfection. R. felis was detected in mosquito feces up to day 14. Furthermore, R. felis was visualized by immunofluorescence in salivary glands, in and around the gut, and in the ovaries, although no vertical transmission was observed. R. felis was also found in the cotton used for sucrose feeding after the mosquitoes were fed infected blood. Natural bites from R. felis-infected An. gambiae were able to cause transient rickettsemias in mice, indicating that this mosquito species has the potential to be a vector of R. felis infection. This is particularly important given the recent report of high prevalence of R. felis infection in patients with "fever of unknown origin" in malaria-endemic areas.

Medical Entomology: A Reemerging Field of Research to Better Understand Vector-Borne Infectious Diseases.

In the last decade, the Chikungunya and Zika virus outbreaks have turned public attention to the possibility of the expansion of vector-borne infectious diseases worldwide. Medical entomology is focused on the study of arthropods involved in human health. We review here some of the research approaches taken by the medical entomology team of the University Hospital Institute (UHI) Méditerranée Infection of Marseille, France, with the support of recent or representative studies. We propose our approaches to technical innovations in arthropod identification and the detection of microorganisms in arthropods, the use of arthropods as epidemiological or diagnostic tools, entomological investigations around clinical cases or within specific populations, and how we have developed experimental models to decipher the interactions between arthropods, microorganisms, and humans.

Candidatus Coxiella massiliensis Infection.

Bacteria genetically related to Coxiella burnetii have been found in ticks. Using molecular techniques, we detected Coxiella-like bacteria, here named Candidatus Coxiella massiliensis, in skin biopsy samples and ticks removed from patients with an eschar. This organism may be a common agent of scalp eschar and neck lymphadenopathy after tick bite.

Identification of blood meal sources in the main African malaria mosquito vector by MALDI-TOF MS.

BACKGROUND: The identification of blood meal sources in malaria vectors is critical to better understanding host/vector interactions and malaria epidemiology and control. Currently, the identification of mosquito blood meal origins is based on time-consuming and costly techniques such as precipitin tests, ELISA and molecular tools. Although these tools have been validated to identify the blood meal and trophic preferences of female Anopheles mosquitoes, they present several limitations. Recently, matrix-assisted, laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was successfully used as a quick and accurate tool for arthropod identification, including mosquitoes. The aim of the present work was to test whether MALDI-TOF MS could also be applied to identification of blood meal sources from engorged mosquitoes. METHODS: Abdomen proteins extracted from Anopheles gambiae (stricto sensu, S molecular form) that were either unengorged or artificially engorged on seven distinct types of vertebrate blood (human, horse, sheep, rabbit, mouse, rat, dog) were submitted for MALDI-TOF MS. RESULTS: The comparison of mass spectrometry (MS) spectra from mosquito abdomens collected 1 h post-feeding, were able to discriminate blood meal origins. Moreover, using Aedes albopictus specimens, abdominal protein MS spectra from engorged mosquitoes were found specific to host blood source and independent of the mosquito species. A sequential analysis revealed stability of mosquito abdominal protein spectra up to 24 h post-feeding. CONCLUSIONS: These results indicate that MALDI-TOF MS could determine feeding patterns of freshly engorged mosquitoes up to 24 h post-blood meal. The MALDI-TOF MS technique appears to be an efficient tool for large epidemiological surveillance of vector-borne diseases and outbreak source identification.

A High-Performance Vacuum Cleaner for Bed Bug Sampling: A Useful Tool for Medical Entomology.

Arthropods can be captured by two modes: a passive mode using traps or an active mode mainly based on the use of mouth or powered aspirators. These apparatuses are useful tools for collecting large numbers of crawling, flying, resting, or jumping arthropod specimens, particularly small specimens, such as mosquitoes or sandflies, for laboratory experiments or breeding. Different aspirator models are used to collect various arthropod specimens. However, to our knowledge, no specific system is currently available for the reliable sampling of live bed bugs in the field. Thus, we described a new system based on a classic autonomous house aspirator that requires few modifications for the collecting bed bugs. The low weight and size of this apparatus is advantageous, and it provides for rapid and secure bed bug sampling for medical entomology purposes.

Acquisition and excretion of Bartonella quintana by the cat flea, Ctenocephalides felis felis.

Bartonella quintana is transmitted by the infected faeces of body lice. Recently, this bacterium was detected in cat fleas (Ctenocephalides felis) and in two humans with chronic adenopathy whose only risk factor was contact with cat fleas. In this study, a total of 960 C. felis were divided into 12 groups (2 control groups and 10 infected groups) each containing 80 fleas. The fleas were fed B. quintana-inoculated human blood at different dilutions (≈3.6 × 10(4) - 8.4 × 10(9) bacteria) for 4 days via an artificial membrane. Subsequently, all flea groups were fed uninfected blood until day 13 postinfection (dpi). On day 3 pi, B. quintana was detected with two specific genes by quantitative PCR in 60-100% of randomly chosen fleas per dilution: 52% (26/50) in the infected fleas in Trial 1 and 90% (45/50) of the fleas in Trial 2. B. quintana was also identified by molecular and culture assays in flea faeces. The average number of B. quintana as determined by qPCR decreased until the 11th dpi and was absent in both trials at the 13th dpi. Bacteria were localized only in the flea gastrointestinal gut by specific immunohistochemistry. Our results indicate that cat fleas can acquire B. quintana by feeding and release viable organisms into their faeces. Therefore, fleas may play a role as vectors of trench fever or other clinical manifestations that are caused by B. quintana. However, the biological role of C. felis in the transmission of B. quintana under natural conditions is yet to be defined.

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.

Viral metagenomics on animals as a tool for the detection of zoonoses prior to human infection?

Many human viral infections have a zoonotic, i.e., wild or domestic animal, origin. Several zoonotic viruses are transmitted to humans directly via contact with an animal or indirectly via exposure to the urine or feces of infected animals or the bite of a bloodsucking arthropod. If a virus is able to adapt and replicate in its new human host, human-to-human transmissions may occur, possibly resulting in an epidemic, such as the A/H1N1 flu pandemic in 2009. Thus, predicting emerging zoonotic infections is an important challenge for public health officials in the coming decades. The recent development of viral metagenomics, i.e., the characterization of the complete viral diversity isolated from an organism or an environment using high-throughput sequencing technologies, is promising for the surveillance of such diseases and can be accomplished by analyzing the viromes of selected animals and arthropods that are closely in contact with humans. In this review, we summarize our current knowledge of viral diversity within such animals (in particular blood-feeding arthropods, wildlife and domestic animals) using metagenomics and present its possible future application for the surveillance of zoonotic and arboviral diseases.

Morphological, molecular, and MALDI-TOF MS identification of bed bugs and associated Wolbachia species from Cameroon.

After vanishing from the public eye for more than 50 years, bed bugs have resurged to become one of the most widely discussed and heavily researched insect pests in the world. This study presents the basic information of infestations of tropical bed bugs, Cimex hemipterus (Hemiptera: Cimicidae), in Cameroon. A total of 248 immature stage and adult bed bug specimens were collected from households and a travel agency in Yaoundé and Douala, Cameroon. The ability of MALDI-TOF MS to identify bed bugs was tested using heads for adults and cephalothoraxes for immature stages. Microorganism screening was performed by qPCR and confirmed by regular PCR and sequencing. Based on morphometrical criteria, four stages of immature bed bugs are represented. Of the 248 bed bug specimens morphologically identified as Cimex hemipterus, 246 (77 males, 65 females and 104 immature specimens) were submitted to MALDI-TOF MS analysis. Of the 222 adults and immature specimens tested, 122 (59.9 %) produced good quality MALDI-TOF MS spectra (35 adults and 87 immature specimens). Blind testing allowed species level identification of 98.21 % of adult and immature C. hemipterus. Among the bacteria tested, only Wolbachia DNA was found in 12/246 (4.8 %) bed bugs. More surveys in the country are warranted to assess the true level of bed bug infestations, in order to take appropriate action for their control.

Identification of termites from Gabon using MALDI-TOF MS.

Termites are one of the most common pests that damage wood and other cellulosic materials. Although Africa has more varieties of termite species than any other continent, few entomological studies have been conducted in Gabon. Identifying termites poses significant difficulties for entomologists. The aim of this study was to evaluate the reliability and confirm the significance of MALDI-TOF MS in identifying fresh termites collected in equatorial Africa. A total of 108 termites were collected from 13 termite nests during a field mission in 2021 in Lekedi and Bongoville, Gabon. Termites were morphologically identified and subjected to MALDI-TOF MS, then molecular analyses using the COI and 12S rRNA genes. Four termite species were morphologically identified in this study: Pseudacanthotermes militaris, Macrotermes muelleri, Macrotermes nobilis, and Noditermes indoensis. However, when using molecular biology, only three species were identified, namely Macrotermes bellicosus, P. militaris, and N. indoensis, because the specimens initially identified as M. muelleri and M. nobilis were found to be M. bellicosus. The MALDI-TOF MS spectral profiles of the termites were all of good quality, with intra-species reproducibility and inter-species specificity. The spectra of 98 termites were blind tested against our upgraded database, which included the spectra of ten termite specimens. All tested spectra were correctly matched to their respective species, with log score values (LSVs) ranging from 1.649 to 2.592. The mean LSV was 2.215 ± 0.203, and the median was 2.241. However, 95.91% (94/98) of our spectra had LSVs above 1.8. This study demonstrates how a proteomic approach can overcome termites' molecular and morphological identification limitations and serve as a useful taxonomic tool.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry for rapid identification of tick vectors.

A method for rapid species identification of ticks may help clinicians predict the disease outcomes of patients with tick bites and may inform the decision as to whether to administer postexposure prophylactic antibiotic treatment. We aimed to establish a matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) spectrum database based on the analysis of the legs of six tick vectors: Amblyomma variegatum, Rhipicephalus sanguineus, Hyalomma marginatum rufipes, Ixodes ricinus, Dermacentor marginatus, and Dermacentor reticulatus. A blind test was performed on a trial set of ticks to identify specimens of each species. Subsequently, we used MALDI-TOF MS to identify ticks obtained from the wild or removed from patients. The latter tick samples were also identified by 12S ribosomal DNA (rDNA) sequencing and were tested for bacterial infections. Ticks obtained from the wild or removed from patients (R. sanguineus, I. ricinus, and D. marginatus) were accurately identified using MALDI-TOF MS, with the exception of those ticks for which no spectra were available in the database. Furthermore, one damaged specimen was correctly identified as I. ricinus, a vector of Lyme disease, using MALDI-TOF MS only. Six of the 14 ticks removed from patients were found to be infected by pathogens that included Rickettsia, Anaplasma, and Borrelia spp. MALDI-TOF MS appears to be an effective tool for the rapid identification of tick vectors that requires no previous expertise in tick identification. The benefits for clinicians include the more targeted surveillance of patients for symptoms of potentially transmitted diseases and the ability to make more informed decisions as to whether to administer postexposure prophylactic treatment.

Chemical characterization and acaricide potential of essential oil from aerial parts of Tagetes patula L. (Asteraceae) against engorged adult females of Rhipicephalus sanguineus (Latreille, 1806).

Rhipicephalus sanguineus, commonly known as the brown dog tick, is one of the most widely distributed species of tick. In dogs, it can cause anemia and provide the transmission of pathogenic microorganisms such as Babesia canis, Ehrlichia canis, Hepatozoon canis, Anaplasma platys, and Mycoplasma haemocanis. To man, it can transmit the intracellular parasites Rickettsia rickettsii and Rickettsia conorii, the causative agents of the Rocky Mountain spotted fever in the Americas and Mediterranean and spotted fever in Europe and North Africa. Its control is performed by applying synthetic formulations composed of pyrethroids; however, continued use of these products results in environmental damage and acquisition of resistance. Alternatively, studies with botanical insecticides have been increasingly recurrent. Therefore, this study aimed to test the efficacy of essential oil of Tagetes patula, a ruderal species widely described in the literature for its insecticidal properties, in engorged females of R. sanguineus by the adults immersion test (AIT) and impregnated paper disk test (IPDT). The essential oil used, through gas chromatography coupled to mass spectrometry, revealed the presence of 55 compounds, being the 4-vinyl guaiacol and gamma terpinene the majority ones. The AIT compared to the IPDT was more efficient in inhibiting oviposition of tick; however, the eggs laid by the females submitted to saturated atmosphere with essential oil, from IPDT, not hatched, interrupted their development cycle. Besides being a pioneer work, the results presented here contributes to new researches, aiming the incorporation of essential oil in an acaricide for use in the environment.

A new challenge for hospitals in southeast France: monitoring local populations of Aedes albopictus to prevent nosocomial transmission of dengue or chikungunya.

Aedes albopictus was first identified in southern France in 2004, inducing an emerging risk for autochthonous transmission around imported cases of dengue or chikungunya, and also for mosquito-borne nosocomial transmission in hospitals. Aedes albopictus has been present in Marseille since September 2009 and in Aix-en-Provence since August 2010. Because of the possible admission of viremic patients with dengue or chikungunya in the hospitals of these cities, a mosquito survey was conducted in 2011 in 2 of the hospitals, with the use of mosquito traps. Aedes albopictus was detected with Eisenhans II traps and egg traps in both hospitals during the warm season.

A new species of Kodormus Barber, with a redescription of the genus, taxonomic notes, and a key to the species of the genus (Hemiptera, Heteroptera, Reduviidae, Stenopodainae).

Kodormusdavidmartinsisp. nov. is described. Taxonomic notes on the other species of Kodormus Barber, 1930, including the description of their male genitalia, are provided. The record of Kodormusbruneosus Barber, 1930 from Brazil and information about the female of the species are presented for the first time. A redescription of Kodormus and a key for its species are provided. Photographs of the holotypes of K.barberi (Costa Lima, 1941), K.bruneosus, and of a paratype of K.oscurus Maldonado & Bérenger, 1996 are presented.