Mitochondrial 16S rRNA Gene Research Articles

Molecular identification of ornamental loaches (Cypriniformes, Cobitoidei) of North East India using mitochondrial genes

North East (NE) India, referred as Biodiversity Hot Spot, is the natural habitat of a large variety of ornamental fishes. Among all the ornamental fishes available in NE India, Loaches are of great importance in the ornamental fish trade. In this study, 38 specimens from ten loaches species of North-eastern India are characterized using three mitochondrial genes i.e. cytochrome c oxidase subunit I (COI), 16S rRNA and cytochrome b (Cyt b). The average genetic distance values for all the three mitochondrial genes have increased gradually when moving up from species to genus to family levels. Among the genes, Cyt b gene showed relatively higher genetic distance values than mitochondrial 16S rRNA and COI genes. Some of the specimens of Botia and Lepidocephalichthys showed higher intraspecific genetic distance values compared to other specimens under analysis.

DNA metabarcoding for identification of species used in fish burgers.

The absence of morphological identification characters, together with the complexity of the fish supply chain make processed seafood vulnerable to cases of species substitution. Therefore, the authentication and the traceability of such products play a strategic role in ensuring quality and safety. The aim of the present study was to detect species used in the production of multi-species fish burgers and to evaluate mislabelling rates, using a DNA metabarcoding approach by sequencing a fragment of the 16S rRNA mitochondrial gene. The study highlighted the presence of 16 marine and 2 mammalian taxa with an overall mislabelling rate of 80%, including cases of species substitution, the undeclared presence of molluscs and of taxa whose use is not permitted by current Italian legislation. The presence of swine DNA as well as the inclusion of undeclared taxa potentially causing allergies raise concerns regarding consumer safety and protection regarding ethical or religious issues. Overall, the study shows that the application of DNA metabarcoding is a promising approach for successfully enforcing traceability systems targeting multi-species processed food and for supporting control activities, as a guarantee of an innovative food safety management system.

The hole is deeper: description of two new species within the Parastacus brasiliensis (von Martens, 1869) species complex with an integrative taxonomy approach.

In this contribution, we describe two new species of burrowing crayfish species complex Parastacus brasiliensis from forestry areas in the central region of the state of Rio Grande do Sul, southern Brazil. We used an integrative taxonomy approach with morphology and the mitochondrial DNA 16S rRNA gene and also evaluated their conservation status according to the IUCN Red List Criteria. Parastacus guapo sp. nov. was collected near the municipality of Pantano Grande and it differs from all other congeneric species in having an epistome anterolateral section with a large conical projection ending in a big sharp spine on both sides. It also differs from P. brasiliensis sensu stricto in the shape of the rostral surface, sub-orbital angle, postorbital carina straight, sub-orbital angle > 90, postorbital carinae prominent in anterior and middle portions, carpal spine present, the internal surface of chelipeds palm with sparse tubercules, areola narrow and pleon short. Parastacus gomesae sp. nov. was collected in the municipality of So Jernimo, it differs from all analysed species for rostral spine absent, epistome anteromedian lobe heptagonal and mandible incisive process with seven teeth (the second tooth from the anterior margin is the largest). It is also distinguished of P. brasiliensis sensu stricto in the sub-orbital angle > 90, postorbital carinae prominent in anterior and middle portions, 34 rows of verrucose tubercles irregularly distributed on the palm dorsal surface of chelipeds, areola narrow and pleon short. Phylogenetic relationships confirmed the distinct position of these new species to the already described species. The extent of occurrence (EOO) / area of occurrence (AOO) of P. guapo sp. nov and P. gomesae sp. nov. were estimated at 937 km / 1,87 km, and 2.107 km / 23,9 km respectively. The main threats identified were continued decline in the quality of habitats, resulting from deforestation and forestry areas. However, as we know only one point of occurrence for each new species, we suggest that both be categorized as Data Deficient.

Phylogeography of Nasutitermes ephratae (Termitidae: Nasutitermitinae) in neotropical region

The neotropical region ranks third in the number of termites and includes five different families. Of these, Termitidae is the most diverse and includes the species Nasutitermes ephratae, which is widespread in the neotropics. To date, only one study has been published about phylogeography in neotropical termites (N. corniger). Here, we explored the population genetic patterns of N. ephratae and also evaluated the phylogeographical processes involved in the evolutionary history of the species. We used the mitochondrial genes 16S rRNA and COII as molecular markers: these were sequenced for 128 samples of N. ephratae. We estimated the genetic diversity and divergence time as well as the demography and genetic structure. We also performed an ancestral area reconstruction and a haplotype network. The results showed high genetic variability, recent demographic expansion, and strong genetic structure. A dispersal route for the species, that occurred in both directions between South and Central America, was inferred. The results emphasize a temporary separation between the South and Central America populations that affected the origin of the current Central America populations. These populations were formed from different phylogeographic histories.

Description of Alvaniawangi Xu, Qi & Kong, sp. nov. (Mollusca, Gastropoda, Littorinimorpha, Rissoidae) from the East China Sea.

Alvaniawangi Xu, Qi & Kong, sp. nov. (Mollusca, Gastropoda, Littorinimorpha, Rissoidae) was discovered within the intertidal zone in the Nanji Islands and Zhoushan Islands, Zhejiang Province, China. It has a radula characteristic of Alvania Risso, 1826, a protoconch sculptured with micro pits and lamellae between spiral lirae, and a teleoconch with growth lines and subobsolete cords. Specimens were examined using an integrative taxonomic approach incorporating morphological observations and phylogenetic analyses of concatenated mitochondrial 16S rRNA and nuclear 28S rRNA gene sequences. The findings suggest that the new species is sister to Alvaniacircinata A. Adams, 1861 and is probably endemic to the shallow waters of the East China Sea.

Sensitive and accurate DNA metabarcoding of parasitic helminth mock communities using the mitochondrial rRNA genes

Next-generation sequencing technologies have accelerated the pace of helminth DNA metabarcoding research, enabling species detection in bulk community samples. However, finding suitable genetic markers with robust species-level resolution and primers targeting a broad species range among parasitic helminths are some of the challenges faced. This study aimed to demonstrate the potential use of the mitochondrial 12S and 16S rRNA genes for parasitic helminth (nematodes, trematodes, cestodes) DNA metabarcoding. To demonstrate the robustness of the 12S and 16S rRNA genes for DNA metabarcoding, we determined the proportion of species successfully recovered using mock helminth communities without environment matrix and mock helminth communities artificially spiked with environmental matrices. The environmental matrices are human fecal material, garden soil, tissue, and pond water. Our results revealed the robustness of the mitochondrial rRNA genes, through the high sensitivity of the 12S rRNA gene, and the effectiveness of the 12S and 16S primers targeting platyhelminths. With the mitochondrial rRNA genes, a broad range of parasitc helminths were successfully detected to the species level. The potential of the mitochondrial rRNA genes for helminth DNA metabarcoding was demonstrated, providing a valuable gateway for future helminth DNA metabarcoding applications like helminth detection and biodiversity studies.

An unexpected new red-bellied Stumpffia (Microhylidae) from forest fragments in central Madagascar highlights remaining cryptic diversity.

The Madagascan endemic subfamily Cophylinae in the family Microhylidae, is an example of a taxonomic group for which much is still to be discovered. Indeed, the cophyline frogs present a large portion of Madagascar's cryptic and microendemic amphibian diversity, yet they remain understudied. A new red-bellied species of the microhylid frog genus Stumpffia is described from the central plateau of Madagascar. Visual encounter surveys in Ambohitantely and Anjozorobe in 2019 and 2020 identified this previously unknown Stumpffia species, which closely resembles Stumpffiakibomena known from Andasibe in the east. Stumpffialynnae sp. nov. adds another species to the red-bellied species complex, differing from S.kibomena by genetic differentiation in the mitochondrial 16S rRNA gene (3.6-3.9%) and distinct nuclear RAG1 haplotypes, as well as strongly by its advertisement call. The new species is known from across Ambohitantely Special Reserve and Anjozorobe Angavo protected area, but is known only from one complete specimen and eight individual tissue samples. Based on the rarity of the species, the small number of locations in which it has been found, and its disappearing forest habitat, its IUCN Red List classification is suggested as "Endangered". This species is the first Stumpffia described from Madagascar's central plateau, highlighting the importance of conserving the remnant forest fragments in this area and the ongoing need to survey and protect this threatened habitat type.

ON THE OCCURRENCE OF Scorpiops longimanus (POCOCK, 1893) (ARACHNIDA: SCORPIOPIDAE) IN MIZORAM, NORTHEAST INDIA

In this study, we confirm the occurrence of the tropical forest scorpion Scorpiops longimanus (Pocock, 1893) in Mizoram, India based on specimens collected from 13 different localities within the state. The total length of the animal was found to range from 42.22 mm up to 65.16 mm. The number of trichobothria on the ventral surface of the pedipalp patella and that of the external face alternates between 10–11 and 18–19, respectively. The pectines showed dissimilarities in the structure of the lamellae and in the number of teeth. We generated the first molecular data for the species and performed a maximum likelihood phylogenetic analysis using the partial mitochondrial 16S rRNA gene which showed the formation of a distinct clade of S. longimanus. The uncorrected k2p distance showed an intraspecific genetic variation of 2.9% among the species studies.

A new mandibulate termite Amitermes kavarattiensis sp. nov. (Blattodea: Isoptera: Termitidae) from Lakshadweep Island, India

ABSTRACT The subterranean, mandibulate termite genus Amitermes Silvestri (Blattodea: Isoptera: Termitidae) is hitherto known from India by only two species, Amitermes belli Desneux and Amitermes baluchistanicus Akhtar. This paper describes a new species Amitermes kavarattiensis Rituparna, Rajmohana & Amina sp. nov. collected from the Lakshadweep Islands, India. Morphological comparisons with both the known species are provided. A dichotomous key to identify all the five Amitermes species of Oriental region is also appended herewith. Additionally, three sequences of the partial fragment of mitochondrial 16S rRNA gene of the new species and five sequences of Amitermes belli have been generated for the first time. A phylogenetic tree with available sequences of Amitermes species is provided. http://www.zoobank.org/urn:lsid:zoobank.org:pub:E9743114-7B9E-4AE4-8750-90F86FC49EEE

Formal re-establishment of Macrocheiridae Dana, 1851 (Decapoda: Brachyura: Majoidea) for the giant spider crabMacrocheira kaempferi(Temminck, 1836) based on a reappraisal of morphological and genetic characters

AbstractThe Japanese spider crab, Macrocheira kaempferi (Temminck, 1836), the largest living arthropod in the world, was originally placed in the family Majidae Samouelle, 1819 as a monotypic subfamily, Macrocheirinae Dana, 1851. While various authors have treated the taxon as belonging to its own superfamily, family, subfamily, or even tribe, the consensus of modern taxonomic system has for the most part considered it to be simply a genus within Inachidae MacLeay, 1838 (superfamily Majoidea Samouelle, 1819). Strangely, however, considering its size and fame, its adult morphology has remained poorly studied. Recent larval and genetic studies have raised questions regarding its suprageneric status, suggesting an independent evolutionary position within Majoidea and the recognition of a separate family. We examined and compared a large suite of adult characters in Macrocheira De Haan, 1839 against other potentially related majoid groups, and found unique characters that support the larval evidence for monophyly of the genus. It is thus necessary to re-establish a monotypic Macrocheiridae for Macrocheira. A molecular phylogeny reconstructed from mitochondrial 16S rRNA gene is presented in further support of this conclusion, showing Macrocheira as sister to Oregoniidae Garth, 1958 and distant from Inachidae. A new diagnosis for the family is provided. Macrocheiridae differs from the morphologically most similar families, Inachidae and Oregoniidae, by characters associated with the carapace, thoracic sternum, male pleon, gonopod, and vulvae.

Characterization of mitochondrial 12S rRNA gene of yellow-striped chevrotain (Moschiola kathygre) and white-spotted chevrotain (Moschiola meminna) and development of a PCR-RFLP marker for the unambiguous identification of the species

Tragulids hold a significant place in the evolutionary history of mammals since they represent the basal branch of ruminants. Only three genera of tragulids are being extant to date such as Tragulus, Hyemoschus and Moschiola. In the genus Moschiola, Sri Lankan chevrotains (Moschiola meminna and Moschiola kathygre) are endemic to Sri Lanka while Moschiola indica is present in India. Sri Lankan chevrotains lack information on their population structure, distribution and molecular evidence on species identification. This leads to possible threats including habitat destruction, poaching and illegal hunting and trading under different names. In this study, genomic DNA from hair follicles was isolated from M. meminna and M. kathygre and 12S rRNA mitochondrial gene was amplified using universal primers. The PCR products were sequenced and phylogenetic analysis was done on Sri Lankan chevrotains for the first time. The sequences of Sri Lankan chevrotains share 99.7% similarity as they differ only in a single InDel present in M. meminna. In silico analysis of 12S rRNA region revealed that PCR-RFLP approach can be used to differentiate the Sri Lankan chevrotains from Indian chevrotain using the restriction enzymes; Rsa I, Sca I, Hinf I and Hind III. M. kathygre and M. meminna can be differentiated from each other by using Dra I.

Three new species of the thorid shrimp genus Lebbeus White, 1847 (Decapoda: Caridea) from Nemuro Strait, Hokkaido, Japan, with supplemental note on L. fujimotoi Matsuzaki, Hibino amp; Komai, 2015.

Based on a collection of deep-water caridean shrimps captured by commercial baited traps and gill nets operated in Nemuro Strait, southwestern part of the Sea of Okhotsk, three new species of the thorid shrimp genus Lebbeus White, 1847, L. rufomaculosus n. sp., L. subtilis n. sp. and L. tenuipes n. sp., are described and illustrated. Integrative approach using morphological comparison and genetic analyses with the mitochondrial COI and 16S rRNA genes is taken to clarify specific identities of those newly recognized taxa. In addition, an aberrant male specimen of L. fujimotoi Matsuzaki, Hibino Komai, 2015 is reported, of which the identity is confirmed by genetic analyses.

Molecular analysis of Lepidopleuruscajetanus (Poli, 1791) (Polyplacophora, Leptochitonidae) from the Mediterranean and near Atlantic.

In the present paper we used a molecular data set (including mitochondrial partial 16S rRNA and COI gene sequences) to examine the genetic structure of Lepidopleuruscajetanus (Poli, 1791) (Polyplacophora, Leptochitonidae) - a distinctive shallow water chiton and member of the basal branching Lepidopleurida, which is widespread in and adjacent to the Mediterranean. The analyses of the two mt-standard marker fragments resolved two main discrete clusters reported as L.cajetanus s.s. and L.aff.cajetanus, respectively. Lepidopleuruscajetanus s.s. is widespread throughout the area under study, while the second distinct lineage apparently co-occurs on the eastern Spanish mainland coast of the Balearic Sea. This result is discussed comparing our data with those reported, in 2014, by Fernández and colleagues who described L.cajetanus as exhibiting "a 'chaotic patchiness' pattern defined by a high genetic variability with locality-exclusive haplotypes, high genetic divergence, and a lack of geographic structure". Although genetic data alone are not sufficient to draw any definitive conclusions, nevertheless we believe that present results shed new light on L.cajetanus which apparently shows more geographically patterned genetic structure than supposed so far.

THE GENUS MACROBRACHIUM (CRUSTACEA, CARIDEA, PALAEMONIDAE): A TAXONOMIC SYNOPSIS FROM THE CRADLE OF EVOLUTION IN AFRICA TO ASIA AND THE AMERICAS.

The genus Macrobrachium like all other decapod groups is very speciose. This make its systematic fairly cumbersome especially because the major morphological organ used in the classication is dependent on the ambient environment and also very plastic. The congruence therefore between classication based on morphology and molecular markers is an essential new process for deducing valid systematics of members of the group. The 16S rRNA mitochondrial gene is perhaps the most frequently used molecular marker for the systematics of the group. For this research work, we sequenced the three major Macrobrachium species found in Nigeria, namely the Macrobrachium vollenhovenii, Macrobrachium dux and Macrobrachium macrobrahion and used the obtained sequences to mine other sequences that were 95% similar to our sequences to deduce the phylogenetic afnities in fourty four species of Macrobrachium species found in four continents namely Africa, Asia, North and South America. There was conict between morphological and molecular systematics in the Nigerian species as consistently the Macrobrachium dux and Macrobrachion macrobrachion were grouped together making them at best ecotypes of the same species. Very high afnities also existed between the Asian species as they grouped together consistently using three different methods of phylogenetic inference (maximum likelihood, minimum evolution and maximum parsimony). The African species also mapped closely with the north American species consistently. One very clear conclusion is that the genetic divergence between the grouping is small, evidence that the genus is just evolving. Further research need to include more African species as there is a dearth of sequences from African species in the Genebank.

New record of a rarely collected caridean shrimp Bathypalaemonella pandaloides (Rathbun, 1906) (Decapoda: Bathypalaemonellidae) from the West Mariana Ridge, northwestern Pacific.

The deep-sea caridean shrimp Bathypalaemonella pandaloides (Rathbun, 1906) (Bathypalaemonellidae) was heretofore known only from Hawaii (type locality) and French Polynesia. In this article, we report this rarely collected species on the basis of a single male specimen from the Ritto Seamount on the West Mariana Ridge, northwestern Pacific, at a depth of 657 m, collected by means of a remotely operated vehicle (ROV). The specimen is intact, enabling us to describe the characteristic major pereopod 2 of the species for the first time. The closest relative is B. zimmeri Balss, 1914, known only from the western Indian Ocean; fewer accessory spiniform setae on the pereopods 35 dactyli and the less produced distal lamella of the antennal scaphocerite distinguish B. pandaloides from B. zimmeri. K2P divergences of the mitochondrial COI and 16S rRNA genes in taxa attributed to Bathypalaemonella Balss, 1914 are presented. The present specimen was collected from an anthipatharian octocoral colony, Leiopathes sp. (Cnidaria), suggesting a commensal relationship.

Metabarcoding the Antarctic Peninsula biodiversity using a multi-gene approach

Marine sediment communities are major contributors to biogeochemical cycling and benthic ecosystem functioning, but they are poorly described, particularly in remote regions such as Antarctica. We analysed patterns and drivers of diversity in metazoan and prokaryotic benthic communities of the Antarctic Peninsula with metabarcoding approaches. Our results show that the combined use of mitochondrial Cox1, and 16S and 18S rRNA gene regions recovered more phyla, from metazoan to non-metazoan groups, and allowed correlation of possible interactions between kingdoms. This higher level of detection revealed dominance by the arthropods and not nematodes in the Antarctic benthos and further eukaryotic diversity was dominated by benthic protists: the world’s largest reservoir of marine diversity. The bacterial family Woeseiaceae was described for the first time in Antarctic sediments. Almost 50% of bacteria and 70% metazoan taxa were unique to each sampled site (high alpha diversity) and harboured unique features for local adaptation (niche-driven). The main abiotic drivers measured, shaping community structure were sediment organic matter, water content and mud. Biotic factors included the nematodes and the highly abundant bacterial fraction, placing protists as a possible bridge for between kingdom interactions. Meiofauna are proposed as sentinels for identifying anthropogenic-induced changes in Antarctic marine sediments.

Establishment and multiapproach characterization of Amblyomma sculptum (Acari: Ixodidae) cell line (ASE-14) from Brazil

The establishment and characterization of the ASE-14 cell line derived from embryos of Amblyomma sculptum is described here. Primary cultures were started, and after 60 days of culturing a confluent monolayer was formed and the first subculture was then carried out. After this, new subcultures were carried out every 4 weeks. Cryopreservation of cells was successful only after the 14th subculture. We compared the chromosomes of the ASE-14 cell line with those of parental ticks. Cytogenetic analysis revealed occurrences of variable and increased diploid numbers in the ASE-14 cell line in comparison with adult ticks, probably through polyploidization events, chromosome fusions and translocations, which allowed generation of cells with distinct diploid numbers. Confirmation of the origin of the A. sculptum cell line was obtained through conventional PCR and sequencing of a fragment of the mitochondrial 16S rRNA gene. In addition, no DNA from Anaplasma marginale, Anaplasma spp., Babesia/Theileria spp., Bartonella spp., Coxiella spp., Ehrlichia canis, Mycoplasma spp. or Rickettsia spp. was detected in the cells through PCR assays. Cytological analyses were performed using live phase contrast microscopy and cytocentrifuge smears stained with Giemsa, while periodic acid-Schiff and bromophenol blue staining techniques were used to detect polysaccharides and protein, respectively. In conclusion, a new cell line derived from embryos of A. sculptum was generated and characterized in this study. The ASE-14 cell line was deposited in the Tick Cell Biobank at the University of Liverpool, and in the Tick Cell Biobank South America Outpost at the Oswaldo Cruz Foundation (FIOCRUZ). The ASE-14 cell line is an important addition to the existing panel of tick cell lines and can be used as a tool for advancing research in various areas of the virology, bacteriology, biology and control of this tick.

Widely used, short 16S rRNA mitochondrial gene fragments yield poor and erratic results in phylogenetic estimation and species delimitation of amphibians

BackgroundThe 16S mitochondrial rRNA gene is the most widely sequenced molecular marker in amphibian systematic studies, making it comparable to the universal CO1 barcode that is more commonly used in other animal groups. However, studies employ different primer combinations that target different lengths/regions of the 16S gene ranging from complete gene sequences (~ 1500 bp) to short fragments (~ 500 bp), the latter of which is the most ubiquitously used. Sequences of different lengths are often concatenated, compared, and/or jointly analyzed to infer phylogenetic relationships, estimate genetic divergence (p-distances), and justify the recognition of new species (species delimitation), making the 16S gene region, by far, the most influential molecular marker in amphibian systematics. Despite their ubiquitous and multifarious use, no studies have ever been conducted to evaluate the congruence and performance among the different fragment lengths.ResultsUsing empirical data derived from both Sanger-based and genomic approaches, we show that full-length 16S sequences recover the most accurate phylogenetic relationships, highest branch support, lowest variation in genetic distances (pairwise p-distances), and best-scoring species delimitation partitions. In contrast, widely used short fragments produce inaccurate phylogenetic reconstructions, lower and more variable branch support, erratic genetic distances, and low-scoring species delimitation partitions, the numbers of which are vastly overestimated. The relatively poor performance of short 16S fragments is likely due to insufficient phylogenetic information content.ConclusionsTaken together, our results demonstrate that short 16S fragments are unable to match the efficacy achieved by full-length sequences in terms of topological accuracy, heuristic branch support, genetic divergences, and species delimitation partitions, and thus, phylogenetic and taxonomic inferences that are predicated on short 16S fragments should be interpreted with caution. However, short 16S fragments can still be useful for species identification, rapid assessments, or definitively coupling complex life stages in natural history studies and faunal inventories. While the full 16S sequence performs best, it requires the use of several primer pairs that increases cost, time, and effort. As a compromise, our results demonstrate that practitioners should utilize medium-length primers in favor of the short-fragment primers because they have the potential to markedly improve phylogenetic inference and species delimitation without additional cost.

Molecular taxonomic identification and species-level phylogeny of the narrow-mouthed frogs of the genus Rhombophryne (Anura: Microhylidae: Cophylinae) from Madagascar

The study of diamond frogs (genus Rhombophryne, endemic to Madagascar) has been historically hampered by the paucity of available specimens, because of their low detectability in the field. Over the last 10 years, 13 new taxa have been described, and 20 named species are currently recognized. Nevertheless, undescribed diversity within the genus is probably large, calling for a revision of the taxonomic identification of published records and an update of the known distribution of each lineage. Here we generate DNA sequences of the mitochondrial 16S rRNA gene of all specimens available to us, revise the genetic data from public databases, and report all deeply divergent mitochondrial lineages of Rhombophryne identifiable from these data. We also generate a multi-locus dataset (including five mitochondrial and eight nuclear markers; 9844 bp) to infer a species-level phylogenetic hypothesis for the diversification of this genus and revise the distribution of each lineage. We recognize a total of 10 candidate species, two of which are identified here for the first time. The genus Rhombophryne is here proposed to be divided into six main species groups, and phylogenetic relationships among some of them are not fully resolved. These frogs are primarily distributed in northern Madagascar, and most species are known from only few localities. A previous record of this genus from the Tsingy de Bemaraha (western Madagascar) is interpreted as probably due to a mislabelling and should not be considered further unless confirmed by new data. By generating this phylogenetic hypothesis and providing an updated distribution of each lineage, our findings will facilitate future species descriptions, pave the way for evolutionary studies, and provide valuable information for the urgent conservation of diamond frogs.

A new real-time PCR assay to specifically detect crustaceans in vegan raw materials and vegan shrimps

Contamination of vegan products and vegan shrimps with real shrimps and crustaceans must be avoided to comply with vegan claims, even more for preventing strong allergenic reactions. Therefore, the detection of crustaceans must be reliable and sensitive enough for authenticity, traceability, and food safety purposes. A new real-time PCR assay was developed targeting the mitochondrial 16S rRNA gene of crustaceans and was optimised to avoid critical mismatches with primers and probe. By testing several crustacean species and common food ingredients, the method was demonstrated to be specific to crustaceans only. To comply with the limit of non-vegan contamination established at 0.1% (w/w) by the European Vegetarian Union, dedicated cut-off CT values were determined on vegan raw materials and on vegan shrimps spiked with crustacean materials. The method reached a sensitivity ≤ 0.0005% (w/w), which was further confirmed on reference materials containing a similar amount of crustacean.