DNA barcoding is now a common tool in parasitology and epidemiology, which require good methods for identification not only of parasites and pathogens but vectors and reservoirs. This special issue presents some advances and challenges in barcoding of microbes, parasites, and their vectors and reservoirs. DNA barcoding found new applications in disease ecology, conservation parasitology, environmental parasitology and in paleoparasitology. New technologies such as next-generation sequencing and matrix-assisted laser desorption–ionization time-of-flight have made it now possible to investigate large samples of specimens. By allowing the investigation of parasites at the interface between environment, biodiversity, animal and human health, barcoding and biobanking have important policy outcomes as well as ethics and legal implications. The special issue ‘Advances and challenges in the barcoding of parasites, vectors and reservoirs’ illustrates some recent advances and proposes new avenues for research in barcoding in parasitology.

Growing evidence demonstrates that bacterial species diversity is substantial, and many of these species are pathogenic in some contexts or hosts. At the same time, laboratories and museums have collected valuable animal tissue and ectoparasite samples that may contain substantial novel information on bacterial prevalence and diversity. However, the identification of bacterial species is challenging, partly due to the difficulty in culturing many microbes and the reliance on molecular data. Although the genomics revolution will surely add to our knowledge of bacterial systematics, these approaches are not accessible to all researchers and rely predominantly on cultured isolates. Thus, there is a need for comprehensive molecular analyses capable of accurately genotyping bacteria from animal tissues or ectoparasites using common methods that will facilitate large-scale comparisons of species diversity and prevalence. To illustrate the challenges of genotyping bacteria, we focus on the genus Bartonella, vector-borne bacteria common in mammals. We highlight the value and limitations of commonly used techniques for genotyping bartonellae and make recommendations for researchers interested in studying the diversity of these bacteria in various samples. Our recommendations could be applicable to many bacterial taxa (with some modifications) and could lead to a more complete understanding of bacterial species diversity.

Trypanosomes (genus Trypanosoma) are parasites of humans, and wild and domestic mammals, in which they cause several economically and socially important diseases, including sleeping sickness in Africa and Chagas disease in the Americas. Despite the development of numerous molecular diagnostics and increasing awareness of the importance of these neglected parasites, there is currently no universal genetic barcoding marker available for trypanosomes. In this review we provide an overview of the methods used for trypanosome detection and identification, discuss the potential application of different barcoding techniques and examine the requirements of the ‘ideal’ trypanosome genetic barcode. In addition, we explore potential alternative genetic markers for barcoding Trypanosoma species, including an analysis of phylogenetically informative nucleotide changes along the length of the 18S rRNA gene.

Cryptosporidium spp. (Apicomplexa) causing cryptosporidiosis are of medical and veterinary significance. The genus Cryptosporidium has benefited from the application of what is considered a DNA-barcoding approach, even before the term ‘DNA barcoding’ was formally coined. Here, the objective to define the DNA barcode diversity of Cryptosporidium infecting mammals is reviewed and considered to be accomplished. Within the Cryptosporidium literature, the distinction between DNA barcoding and DNA taxonomy is indistinct. DNA barcoding and DNA taxonomy are examined using the latest additions to the growing spectrum of named Cryptosporidium species and within-species and between-species identity is revisited. Ease and availability of whole-genome DNA sequencing of the relatively small Cryptosporidium genome offer an initial perspective on the intra-host diversity. The opportunity emerges to apply a metagenomic approach to purified field/clinical Cryptosporidum isolates. The outstanding question remains a reliable definition of Cryptosporidium phenotype. The complementary experimental infections and metagenome approach will need to be applied simultaneously to address Cryptosporidium phenotype with carefully chosen clinical evaluations enabling identification of virulence factors.

Leishmania kinetoplast DNA contains thousands of small circular molecules referred to as kinetoplast DNA (kDNA) minicercles. kDNA minicircles are the preferred targets for sensitive Leishmania detection, because they are present in high copy number and contain conserved sequence blocks in which polymerase chain reaction (PCR) primers can be designed. On the other hand, the heterogenic nature of minicircle networks has hampered the use of this peculiar genomic region for strain typing. The characterization of Leishmania minicirculomes used to require isolation and cloning steps prior to sequencing. Here, we show that high-throughput sequencing of single minicircle PCR products allows bypassing these laborious laboratory tasks. The 120 bp long minicircle conserved region was amplified by PCR from 18 Leishmania strains representative of the major species complexes found in the Neotropics. High-throughput sequencing of PCR products enabled recovering significant numbers of distinct minicircle sequences from each strain, reflecting minicircle class diversity. Minicircle sequence analysis revealed patterns that are congruent with current hypothesis of Leishmania relationships. Then, we show that a barcoding-like approach based on minicircle sequence comparisons may allow reliable identifications of Leishmania spp. This work opens up promising perspectives for the study of kDNA minicercles and a variety of applications in Leishmania research.

Leptospirosis is a zoonotic bacterial disease of global importance. A large spectrum of asymptomatic animal hosts can carry the infection and contribute to the burden of human disease. Environmental sources of human contamination also point to the importance of a hydrotelluric reservoir. Leptospirosis can be caused by as many as 15 different pathogenic or intermediate Leptospira species. However, classification of these bacteria remains complicated through the use of both serological and genetic classification systems that show poor correlation. With the advent of molecular techniques, DNA-based barcoding offers a conceptual framework that can be used for leptospirosis surveillance as well as source tracking. In this review, we summarize some of the current techniques, highlight significant successes and weaknesses and point to the future opportunities and challenges to successfully establish a widely applicable barcoding scheme for Leptospira.

Despite metabarcoding being widely used to analyse bacterial community composition, its application in parasitological research remains limited. What interest there has been has focused on previously intractable research settings where traditional methods are inappropriate, for example, in longitudinal studies and studies involving endangered species. In settings such as these, non-invasive sampling combined with metabarcoding can provide a fast and accurate assessment of component communities. In this paper we review the use of metabarcoding in the study of helminth communities in wild mammals, outlining the necessary procedures from sample collection to statistical analysis. We highlight the limitations of the metabarcoding approach and speculate on what type of parasitological study would benefit from such methods in the future.

Mosquitoes’ importance as vectors of pathogens that drive disease underscores the importance of precise and comparable methods of taxa identification among their species. While several molecular targets have been used to study mosquitoes since the initiation of PCR in the 1980s, its application to mosquito identification took off in the early 1990s. This review follows the research's recent journey into the use of mitochondrial DNA (mtDNA) cytochrome oxidase 1 (COI or COX1) as a DNA barcode target for mosquito species identification – a target whose utility for discriminating mosquitoes is now escalating. The pros and cons of using a mitochondrial genome target are discussed with a broad sweep of the mosquito literature suggesting that nuclear introgressions of mtDNA sequences appear to be uncommon and that the COI works well for distantly related taxa and shows encouraging utility in discriminating more closely related species such as cryptic/sibling species groups. However, the utility of COI in discriminating some closely related groups can be problematic and investigators are advised to proceed with caution as problems with incomplete lineage sorting and introgression events can result in indistinguishable COI sequences appearing in reproductively independent populations. In these – if not all – cases, it is advisable to run a nuclear marker alongside the mtDNA and thus the utility of the ribosomal DNA – and in particular the internal transcribed spacer 2 – is also briefly discussed as a useful counterpoint to the COI.

Hybridization events between Schistosoma species (Digenea, Platyhelminthes) are reported with increasing frequency, largely due to improved access to molecular tools. Nevertheless, little is known about the distribution and frequency of hybrid schistosomes in nature. Screening for hybrids on a large scale is complicated by the need for nuclear and mitochondrial sequence information, precluding a ‘simple’ barcoding approach. Here we aimed to determine and understand the spatiotemporal distribution of Schistosoma haematobium × Schistosoma bovis hybrids in the Senegal River Basin. From ten villages, distributed over the four main water basins, we genotyped a total of 1236 schistosome larvae collected from human urine samples using a partial mitochondrial cox1 fragment; a subset of 268 parasites was also genotyped using ITS rDNA. Hybrid schistosomes were unevenly distributed, with substantially higher numbers in villages bordering Lac de Guiers than in villages from the Lampsar River and the Middle Valley of the Senegal River. The frequency of hybrids per village was not linked with the prevalence of urinary schistosomiasis in that village. However, we did find a significant positive association between the frequency of hybrids per village and the prevalence of Schistosoma mansoni. We discuss the potential consequences of adopting a barcoding approach when studying hybrids in nature.

Ancient samples present a number of technical challenges for DNA barcoding, including damaged DNA with low endogenous copy number and short fragment lengths. Nevertheless, techniques are available to overcome these issues, and DNA barcoding has now been used to successfully recover parasite DNA from a wide variety of ancient substrates, including coprolites, cesspit sediment, mummified tissues, burial sediments and permafrost soils. The study of parasite DNA from ancient samples can provide a number of unique scientific insights, for example: (1) into the parasite communities and health of prehistoric human populations; (2) the ability to reconstruct the natural parasite faunas of rare or extinct host species, which has implications for conservation management and de-extinction; and (3) the ability to view in ‘real-time’ processes that may operate over century- or millenial-timescales, such as how parasites responded to past climate change events or how they co-evolved alongside their hosts. The application of DNA metabarcoding and high-throughput sequencing to ancient specimens has so far been limited, but in future promises great potential for gaining empirical data on poorly understood processes such as parasite co-extinction.

While some species of parasites can be identified to species level from archaeological remains using microscopy (i.e. Enterobius vermicularis, Clonorchis sinensis), others can only be identified to family or genus level as different species produce eggs with similar morphology (i.e. Tænia sp. and Echinococcus sp.). Molecular and immunological approaches offer the possibility to provide more precise determination at the species level. They can also identify taxa when classic parasite markers such as eggs or cysts have been destroyed over time. However, biomolecules can be poorly preserved and modern reference DNA is available only for a limited number of species of parasites, leading to the conclusion that classic microscopic observation should be combined with molecular analyses. Here we present a review of the molecular approaches used over the past two decades to identify human pathogenic helminths (Ascaris sp., Trichuris sp., E. vermicularis, Fasciola sp. etc.) or protists (Giardia sp., Trypanosoma sp., Leishmania sp. etc.). We also discuss the prospects for studying the evolution of parasites with genetics and genomics.

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.

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.

Research on emerging infectious diseases calls for a work on collections of pathogens (including hosts or vectors from which the pathogens were isolated), related to human and animal health, to wildlife or on the environmental material. In this respect, the adoption of a One Health perspective is determined by the need for a common approach to consider the collection, storage and use of pathogens coming from human or non-human sources, and particularly when the same pathogen is taken from different environments. In response to this development, our purpose is to delineate a flexible regulation framework concerning collections of pathogens from various origins or hosts and their associated data in order to facilitate scientific work and research partnerships. The legal and ethical cutting-edge research on Biomedical Big Data is particularly stimulating when it comes to address challenges related to collections or biobanks of pathogens such as prior informed consent and accessibility, Material Transfer Agreement or benefit sharing.