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Arthropod vectors and parasites are identified morphologically or, more recently, by molecular methods. Both methods are time consuming and require expertise and, in the case of molecular methods, specific devices. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) identification of bacteria has meant a major change in clinical microbiology laboratories because of its simplicity, speed and specificity, and its capacity to identify microorganisms, in some cases, directly from the sample (urine cultures, blood cultures). Recently, MALDI-TOF MS has been shown as useful for the identification of some parasites. On the other hand, the identification of vector arthropods and the control of their populations is essential for the control of diseases transmitted by arthropods, and in this aspect, it is crucial to have fast, simple and reliable methods for their identification. Ticks are blood-sucking arthropods with a worldwide distribution, that behave as efficient vectors of a wide group of human and animal pathogens, including bacteria, protozoa, viruses, and even helminths. They are capable of parasitizing numerous species of mammals, birds and reptiles. They constitute the second group of vectors of human diseases, after mosquitoes. MALDI-TOF MS has been shown as useful for the identification of different tick species, such as Ixodes, Rhipicephalus and Amblyomma. Some studies even suggest the possibility of being able to determine, through MALDI-TOF MS, if the arthropod is a carrier of certain microorganisms. Regarding mosquitoes, the main group of vector arthropods, the possibility of using MALDI-TOF MS for the identification of different species of Aedes and Anopheles has also been demonstrated. In this review, we address the possibilities of this technology for the identification of parasites and arthropod vectors, its characteristics, advantages and possible limitations. [ABSTRACT FROM AUTHOR]

Background: In the last decade, an innovative approach has emerged for arthropod identification based on matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Increasing interest in applying the original technique for arthropod identification has led to the development of a variety of procedures for sample preparation and selection of body parts, among others. However, the absence of a consensual strategy hampers direct inter-study comparisons. Moreover, these different procedures are confusing to new users. Establishing optimized procedures and standardized protocols for mosquito identification by MALDI-TOF MS is therefore a necessity, and would notably enable the sharing of reference MS databases. Here, we assess the optimal conditions for mosquito identification using MALDI-TOF MS profiling. Methods: Three homogenization methods, two of which were manual and one automatic, were used on three distinct body parts (legs, thorax, head) of two mosquito laboratory strains, Anopheles coluzzii and Aedes aegypti, and the results evaluated. The reproducibility of MS profiles, identification rate with relevant scores and the suitability of procedures for high-throughput analyses were the main criteria for establishing optimized guidelines. Additionally, the consequences of blood-feeding and geographical origin were evaluated using both laboratory strains and field-collected mosquitoes. Results: Relevant score values for mosquito identification were obtained for all the three body parts assayed using MALDI-TOF MS profiling; however, the thorax and legs were the most suitable specimens, independently of homogenization method or species. Although the manual homogenization methods were associated with a high rate of identification on the three body parts, this homogenization mode is not adaptable to the processing of a large number of samples. Therefore, the automatic homogenization procedure was selected as the reference homogenization method. Blood-feeding status did not hamper the identification of mosquito species, despite the presence of MS peaks from original blood in the MS profiles of the three body parts tested from both species. Finally, a significant improvement in identification scores was obtained for field-collected specimens when MS spectra of species from the same geographical area were added to the database. Conclusion: The results of the current study establish guidelines for the selection of mosquito anatomic parts and modality of sample preparation (e.g. homogenization) for future specimen identification by MALDI-TOF MS profiling. These standardized operational protocols could be used as references for creating an international MS database. [ABSTRACT FROM AUTHOR]

Flea identification is a significant issue because some species are considered as important vectors of several human pathogens that have emerged or re‐emerged recently, such as Bartonella henselae (Rhizobiales: Bartonellaceae) and Rickettsia felis (Rickettsiales: Rickettsiaceae). Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) has been evaluated in recent years for the identification of multicellular organisms, including arthropods. A preliminary study corroborated the usefulness of this technique for the rapid identification of fleas, creating a preliminary database containing the spectra of five species of flea. However, longterm flea preservation in ethanol did not appear to be an adequate method of storage in the context of specimen identification by MALDI‐TOF MS profiling. The goal of the present work was to assess the performance of MALDI‐TOF MS in the identification of seven flea species [Ctenocephalides felis (Siphonaptera: Pulicidae), Ctenocephalides canis, Pulex irritans (Siphonaptera: Pulicidae), Archaeopsylla erinacei (Siphonaptera: Pulicidae), Leptopsylla taschenbergi (Siphonaptera: Ceratophyllidae), Stenoponia tripectinata (Siphonaptera: Stenoponiidae) and Nosopsyllus fasciatus (Siphonaptera: Ceratophyllidae)] collected in the field and stored in ethanol for different periods of time. The results confirmed that MALDI‐TOF MS can be used for the identification of wild fleas stored in ethanol. Furthermore, this technique was able to discriminate not only different flea genera, but also the two congeneric species C. felis and C. canis. [ABSTRACT FROM AUTHOR]

Abstract: Ecological studies require accurate identification of specimens. This is very time consuming when processing plankton, meiobenthos or soil biota samples due to the presence of a high number of minute specimens. A solution to this problem may be MALDI‐TOF MS, an emerging technique for identification of metazoan species. As an alternative to factory delivered software or clustering approaches, Random Forest (RF) models can be trained to identify species, using MALDI‐TOF data. However, in a real‐world scenario, RF models will fail in detecting species which were not included in the training dataset as well, thus producing false positives (misidentifications). We produced MALDI‐TOF MS spectra for meiofauna species and trained RF models, using MALDI‐TOF bins as predictors and species as multi‐level target class. We used the empirical beta distribution of the probability of class assignment in the model to design a post hoc test for false positive discovery. Two strategies increase the final accuracy of species identification: (1) “class smoothing” consisting of in silico observations of classes, created by bootstrapping the value of each predictor within each class and: (2) adding a “null class” to the training dataset by bootstrapping predictor values and shuffling predictor labels creating a class without multivariate signal. We prove that RF is an excellent method for species identification, using MALDI‐TOF MS data. The models are flexible enough to correctly classify observations created in silico by smoothing the classes. Our post hoc test unmasks false positive classifications successfully. Smoothing the classes and adding a null class to the training model attracts assignment of false positives to this class. In our example, a 100% false positive discovery could be achieved, while maintaining very high overall prediction accuracy. Combining MALDI‐TOF MS and RF models is a step towards a fully automatic species identification workflow that is particularly necessary for species‐rich communities of small organism for ecological studies but also for routine monitoring. The post hoc test for false positive discovery can be applied to any RF multilevel classification model, not only in a biological context. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Arthropod-borne diseases are important causes of morbidity and mortality. The identification of vector species relies mainly on morphological features and/or molecular biology tools. The first method requires specific technical skills and may result in misidentifications, and the second method is time-consuming and expensive. The aim of the present study is to assess the usefulness and accuracy of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry ( MALDI-TOF MS) as a supplementary tool with which to identify mosquito vector species and to invest in the creation of an international database. A total of 89 specimens belonging to 10 mosquito species were selected for the extraction of proteins from legs and for the establishment of a reference database. A blind test with 123 mosquitoes was performed to validate the MS method. Results showed that: (a) the spectra obtained in the study with a given species differed from the spectra of the same species collected in another country, which highlights the need for an international database; (b) MALDI-TOF MS is an accurate method for the rapid identification of mosquito species that are referenced in a database; (c) MALDI-TOF MS allows the separation of groups or complex species, and (d) laboratory specimens undergo a loss of proteins compared with those isolated in the field. In conclusion, MALDI-TOF MS is a useful supplementary tool for mosquito identification and can help inform vector control. [ABSTRACT FROM AUTHOR]

Background: The tick population of Africa includes several important genera belonging to the family Ixodidae. Many of these ticks are vectors of protozoan and rickettsial pathogens including Theileria parva that causes East Coast fever, a debilitating cattle disease endemic to eastern, central and southern Africa. Effective surveillance of tick-borne pathogens depends on accurate identification and mapping of their tick vectors. A simple and reproducible technique for rapid and reliable differentiation of large numbers of closely related field-collected ticks, which are often difficult and tedious to discriminate purely by morphology, will be an essential component of this strategy. Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) is increasingly becoming a useful tool in arthropod identification and has the potential to overcome the limitations of classical morphology-based species identification. In this study, we applied MALDI-TOF MS to a collection of laboratory and field ticks found in Eastern Africa. The objective was to determine the utility of this proteomic tool for reliable species identification of closely related afrotropical ticks. Methods: A total of 398 ixodid ticks from laboratory maintained colonies, extracted from the hides of animals or systematically collected from vegetation in Kenya, Sudan and Zimbabwe were analyzed in the present investigation. The cytochrome c oxidase I (COI) genes from 33 specimens were sequenced to confirm the tentatively assigned specimen taxa identity on the basis of morphological analyses. Subsequently, the legs of ticks were homogenized and analyzed by MALDI-TOF MS. A collection of reference mass spectra, based on the mass profiles of four individual ticks per species, was developed and deposited in the spectral database SARAMIS™. The ability of these superspectra (SSp.) to identify and reliably validate a set of ticks was demonstrated using the remaining individual 333 ticks. Results: Ultimately, ten different tick species within the genera Amblyomma, Hyalomma, Rhipicephalus and Rhipicephalus (Boophilus) based on molecular COI typing and morphology were included into the study analysis. The robustness of the 12 distinct SSp. developed here proved to be very high, with 319 out of 333 ticks used for validation identified correctly at species level. Moreover, these novel SSp. allowed for diagnostic specificity of 99.7 %. The failure of species identification for 14 ticks was directly linked to low quality mass spectra, most likely due to poor specimen quality that was received in the laboratory before sample preparation. Conclusions: Our results are consistent with earlier studies demonstrating the potential of MALDI-TOF MS as a reliable tool for differentiating ticks originating from the field, especially females that are difficult to identify after blood feeding. This work provides further evidence of the utility of MALDI-TOF MS to identify morphologically and genetically highly similar tick species and indicates the potential of this tool for large-scale monitoring of tick populations, species distributions and host preferences. [ABSTRACT FROM AUTHOR]

Calanoid copepods play an important role in the pelagic ecosystem making them subject to various taxonomic and ecological studies, as well as indicators for detecting changes in the marine habitat. For all these investigations, valid identification, mainly of sibling and cryptic species as well as early life history stages, represents a central issue. In this study, we compare species identification methods for pelagic calanoid copepod species from the North Sea and adjacent regions in a total of 333 specimens. Morphologically identified specimens were analysed on the basis of nucleotide sequences (i.e. partial mitochondrial cytochrome c oxidase subunit I ( COI) and complete 18S r DNA) and on proteome fingerprints using the technology of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry ( MALDI- TOF MS). On all three molecular approaches, all specimens were classified to species level indicated by low intraspecific and high interspecific variability. Sequence divergences in both markers revealed a second Pseudocalanus species for the southern North Sea identified as Pseudocalanus moultoni by COI sequence comparisons to Gen Bank. Proteome fingerprints were valid for species clusters irrespective of high intraspecific variability, including significant differences between early developmental stages and adults. There was no effect of sampling region or time; thus, trophic effect, when analysing the whole organisms, was observed in species-specific protein mass spectra, underlining the power of this tool in the application on metazoan species identification. Because of less sample preparation steps, we recommend proteomic fingerprinting using the MALDI- TOF MS as an alternative or supplementary approach for rapid, cost-effective species identification. [ABSTRACT FROM AUTHOR]