Field application of MALDI-TOF MS on mosquito larvae identification

Abstract

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.

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

“…Larvae and pupae were placed individually in microtubes (collection microtubes × 96, Qiagen) and homogenized according to the standardized protocols . After homogenization, debris was pelleted by a quick spin centrifugation for 1 min, and 1.5 µL of supernatant of each sample was deposited in duplicate on the MALDI‐TOF target plate (Bruker Daltonics, Wissembourg, France) as previously described . To control for differences of the target plate, matrix quality and MALDI‐TOF apparatus performance, matrix solution was loaded in duplicate onto each MALDI‐TOF plate with and without a bacterial test standard (Bruker Bacterial Test Standard, ref: #8255343).…”

“…Protein mass profiles were obtained using a Microflex LT MALDI‐TOF mass spectrometer (Bruker Daltonics, Bremen, Germany). Setting parameters were as previously described . Each spectrum was automatically acquired using the AutoXecute of the Flex Control v.2.4 software (Bruker Daltonics).…”

“…Three situations were possible: (i) LSVs greater than 1.8 were considered reliable for species identification as previously described, (ii) LSVs between 1.6 and 1.8 were subjected to additional manual controls. To be considered reliable, LSVs from duplicate tests must both be included in the range 1.6–1.8 with identical first top hits for species identification as previously described . Additionally, cluster analyses were performed to confirm species classification.…”

“…Each larva selected for molecular analysis was rinsed with sterile water and DNA was individually extracted using the QIAamp DNA tissue extraction kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Molecular identification of mosquito larvae at the species level was performed by sequencing the PCR product of a fragment of the cytochrome c oxidase I gene (COI) as described previously . The sequences were assembled and analyzed using the ChromasPro software version 1.7.7 (Technelysium Pty.…”

“…This tool has the advantage of being fast and reliable, and it requires low‐cost reagents . Moreover, the proof of principle for the application of this tool to identify mosquito larvae collected in stagnant water sites from Marseille has been demonstrated …”