The genus Tanqua Blanchard, 1904, infests reptiles, particularly those inhabiting aquatic environments. This study examined a population of rainbow water snakes, Enhydris enhydris (Schneider, 1799), collected from southern Thailand. Adult nematodes consistent with Tanqua were found in the stomach. Various morphometric, meristic and qualitative morphological variables, including size, ratios, distances, cephalic appearance, the number of caudal papillae and other features, serve to distinguish the specimens from other species within the genus. In particular, Tanqua anomala and Tanqua diadema, which closely resemble our Tanqua specimens, can be differentiated by key diagnostic characteristics such as a retractable head, the distance from the anterior end to the cervical sac, the relative positions of caudal papillae and excretory pore, and the length of the uterus. Molecular analysis (COI and 18s rRNA genes) confirmed its status as a species of Tanqua, genetically distinct from Tanqua tiara, and matching the genetic sequence found in larvae of Tanqua sp. from a snakehead fish species from Bangladesh. Tanqua siamensis sp. nov. is described, supported by morphological traits, microscopic illustrations and genetic information. This study reports the first evidence of a caudal papillary pair in females. This species causes significant lesions on the stomach wall of the snake host, raising possible issues for snakes held in captivity regarding food hygiene and parasite protection.

During a 1-year study, Trichuris adults were obtained after necropsy of Arabian camels (Camelus dromedarius) from a slaughterhouse in Kuwait. Morphological and molecular identification was performed to confirm the identity of the Trichuris specimens obtained from C. dromedarius. Fifteen male Trichuris specimens were selected, and molecular identification was performed using mitochondrial cytochrome c oxidase subunit I, 12S ribosomal RNA, 16S ribosomal RNA genes and the nuclear internal transcribed spacer 2 (ITS2) region. Through phylogenetic analysis, 2 distinct groups were obtained using the mitochondrial genes, where group 1 showed a close relationship to Trichuris globulosa while group 2 showed a close relationship to Trichuris ovis, providing molecular evidence of a possible T. globulosa species complex. Additionally, the nuclear ITS2 region did not provide enough resolution to distinguish between the 2 groups of Trichuris specimens. Observation of morphological characters revealed variations in the shape of the male spicule sheath, where specimens present either a globular posteriorly truncated swelling or the absence of posteriorly truncated swelling. Moreover, the variations in male spicule sheath does not corroborate with the results of molecular data, suggesting the limited use of this character for identification of T. globulosa. In conclusion, molecular analysis suggests a possible species complex in T. globulosa, with the mitochondrial genetic markers successfully differentiating between the 2 groups. The limited use of the male spicule sheath as a diagnostic character for identification of T. globulosa is suggested.

The genus Encyclometra is one of the two genera in family Encyclometridae, known for parasitising the oesophagus, stomach and intestine of snakes. Among Encyclometra, the species present are: Encyclometra colubrimurorum, Encyclometra japonica, Encyclometra asymmetrica and Encyclometra bungara. Species discrimination within Encyclometra has predominantly relied on morphological differences, such as the length of the caeca and the position of the testes. Morphological overlaps exist among these species making species discrimination challenging. Additionally, the use of molecular information has been limited for Encyclometra. To determine the Encyclometra species infecting Enhydris enhydris from Thailand and Cambodia, morphological and molecular identification was conducted. Morphological characters and measurements were obtained from 30 Encyclometra adults, and they were compared with previous studies of other Encyclometra species. Novel sequences of E. bungara were generated using the nuclear 18S and 28S ribosomal RNA genes, and the mitochondrial cytochrome c oxidase subunit 1 gene. Our results revealed that the specimens could be morphologically identified as E. bungara, with support from molecular information obtained from the phylogenies of the 3 genetic markers employed. Molecular analysis showed that the Encyclometra specimens were distinct from E. colubrimurorum and E. japonica. Through morphological and molecular identification of the Encyclometra specimens found in E. enhydris from Thailand and Cambodia, we describe and provide a record of E. bungara in a new host and new locality. Additionally, novel molecular sequences were generated, revealing the phylogenetic position of E. bungara within the superfamily Gorgoderoidea.

Cosmocerca spp. are common nematode parasites of amphibians. We provide herein molecular data for two species of Cosmocerca and describe a new species, Cosmocerca albopunctata n. sp., using light microscopy and molecular data (cytochrome c oxidase subunit 1 – COI mtDNA). Cosmocerca albopunctata n. sp. can be easily distinguished from other congeneric species by the combination of characteristics such as body size, length of spicules and gubernaculum, and the arrangements and number of caudal papillae (7 + 1:1 + 1:6). The phylogenetic results based on the partial COI mtDNA sequences clustered the new species in a monophyletic clade along with the other sequences of Cosmocerca spp. Therefore, our results contribute to the knowledge about the species diversity and genetic data for Cosmocerca spp. in the Neotropical region.

Digenetic trematodes are important parasites of humans and animals. They have complex life cycles and typically infect a gastropod as the first intermediate host. Bithynia siamensis goniomphalos, the first intermediate host of the liver fluke, Opisthorchis viverrini, harbours a wide variety of other trematode species. Morphological details of cercariae of 20 trematode taxa from B. s. goniomphalos, collected mainly in Thailand from 2009 to 2014, were provided in an earlier paper. Correct identification to the species or genus level based on morphology of these cercariae is generally not possible. Therefore, we used molecular data to improve identification and to investigate the diversity of the species of trematodes infecting B. s. goniomphalos. We were successful in extracting, amplifying and sequencing portions of the 28S ribosomal RNA (rRNA) gene for 19 of these 20 types of cercaria, and the internal transcribed spacer 2 region for 18 types. BLAST searches in GenBank and phylogenetic trees inferred from the 28S rRNA sequences identified members of at least nine superfamilies and 12 families. Only a few cercariae could be assigned confidently to genus or species on the basis of the sequence data. Matching sequence data from named adult trematodes will be required for definitive identification. There is clearly a great diversity of trematode species utilizing B. s. goniomphalos in Thailand.

Human strongyloidiasis is a deleterious gastrointestinal disease mainly caused by Strongyloides stercoralis infection. We aimed to study the possible transmission of S. stercoralis between humans and pet animals. We isolated Strongyloides from humans and domestic dogs in the same rural community in north-east Thailand and compared the nucleotide sequences of derived worms using portions of the mitochondrial cytochrome c oxidase subunit 1 (cox1) and 18S ribosomal RNA (18S rRNA) genes. Twenty-eight sequences from the 18S rRNA gene were obtained from worms derived from humans (n = 23) and dogs (n = 5), and were identical with S. stercoralis sequences (from Thailand, Cambodia, Lao PDR and Myanmar) published in the GenBank database. The 28 cox1 sequences from humans and dogs showed high similarity to each other. The available published cox1 sequences (n = 150), in combination with our 28 sequences, represented 68 haplotypes distributed among four clusters. The 28 samples from the present study represented eight haplotypes including four new haplotypes. Dogs and humans shared the same haplotypes, suggesting the possibility of zoonotic transmission from pet dogs to humans. This is of concern since dogs and humans live in close association with each other.

Enterobiasis, caused by the nematode Enterobius vermicularis, is a common health problem among schoolchildren in Thailand. We provide the first molecular identification of this nematode from Thai schoolchildren and document genetic variation among E. vermicularis eggs using sequence analyses of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene and the nuclear ribosomal DNA second internal transcribed spacer (ITS2). A cross-sectional parasitological survey was conducted in schoolchildren (n = 491) in five regions of Thailand between May 2015 and December 2016. The diagnosis of Enterobius infection was made using the adhesive tape perianal swab technique. Enterobius eggs were recovered from 43 participants (8.75%). DNA was extracted from these eggs and the cox1 gene and partial ITS2 region amplified using the polymerase chain reaction (PCR). Nineteen amplified PCR products of the cox1 gene (441 bp) and 18 of the ITS2 region (623 bp) were subsequently sequenced. All sequences were identified as belonging to E. vermicularis based on database searches. Phylogenetic analysis and a median-joining network of available E. vermicularis cox1 sequences showed 66 haplotypes. We found haploclusters (types A and B) represented among the Thai sequences. Six haplotypes from Thailand fell into type A (of Nakano et al., 2006) (along with sequences from Japan and Korea) and five haplotypes into type B (with sequences from Japan, Iran, Czech Republic, Greece, Denmark and Sudan). The overall haplotype diversity (Hd) was 0.9888. Transmission of worms with type B haplotypes from primates to humans in Asia or from humans in Europe possibly occurs in Thailand.

The Ceratitis FAR complex (Diptera, Tephritidae) includes four economically important frugivorous flies (Ceratitis anonae, Ceratitis fasciventris, Ceratitis quilicii, Ceratitis rosa) whose immature stages and adult females cannot be properly resolved through morphological identification. In order to develop a simplified molecular tool for the identification of two of these species (C. rosa, C. quilicii), we selected a subset of six microsatellite markers out of a panel of 16 loci that were previously developed for the molecular differentiation of the taxa within the complex. These six markers were first tested in silico and then used for the actual genotyping of C. quilicii and C. rosa, resulting in the correct identification of all male reference specimens. Here, we propose an integrated morphological and molecular setup for the identification of the four species of the FAR complex. The decision map relies on preliminary DNA barcoding or morphological identification (when possible) to exclude species not belonging to the complex followed by (a) morphological identification of all adult male specimens and female C. anonae, (b) molecular identification via a panel of 16 microsatellite markers for immature stages, damaged vouchers and samples potentially including adult female C. fasciventris/C. quilicii/C. rosa and (c) molecular identification via a reduced panel of six microsatellite markers for samples including only C. quilicii and C. rosa. This simplified diagnostic setup was profitably implemented in the framework of the ERAfrica fruit fly project and will help correctly identify species within the FAR complex for their early detection and monitoring.

Strongyloides fuelleborni is a soil-transmitted nematode parasite of non-human primates. The worm is prevalent also in human populations in Africa and South-East Asia. In this study, we amplified and sequenced a portion of the 18S ribosomal RNA gene (rRNA) and of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene of Strongyloides adult males recovered from faecal samples from long-tailed macaques (Macaca fascicularis) in Thailand and Lao PDR. The prevalence in Thailand was 31.1% (55/177) and in Lao PDR it was 62.1% (41/66), with an overall prevalence of 39.5% (96/243). All 18S rRNA sequences that we obtained (n = 96) showed 100% identity with published S. fuelleborni sequences. The 96 cox1 sequences that we obtained represented 32 new haplotypes. When included with the 17 previously known haplotypes from S. fuelleborni, the cox1 sequences fell into four clusters, which had clear geographical structure. This is the first molecular confirmation of S. fuelleborni in long-tailed macaques in Thailand and Lao PDR. Clearly, awareness needs to be raised of the zoonotic potential of S. fuelleborni. A monitoring programme should be organized, taking into account the role of reservoir hosts (i.e. monkeys) in the natural background of human strongyloidiasis caused by S. fuelleborni.

We present the first demonstration of a general method for the chemical characterization of small surface features at high magnification via simultaneous collection of mass spectrometry (MS) imaging and tandem MS imaging data. High lateral resolution tandem secondary ion MS imaging is employed to determine the composition of surface features on poly(ethylene terephthalate) (PET) that precipitate during heat treatment. The surface features, probed at a lateral resolving power of<200 nm using a surface-sensitive ion beam, are found to be comprised of ethylene terephthalate trimer at a greater abundance than is observed in the surrounding polymer matrix. This is the first chemical identification of PET surface precipitates made without either an extraction step or the use of a reference material. The new capability employed for this study achieves the highest practical lateral resolution ever reported for tandem MS imaging.

Molecular techniques are useful tools for solving taxonomic confusion among species. Polymerase chain reaction (PCR) and polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) methods were applied for the identification of barnyardgrass, early watergrass, and late watergrass. Total DNA was extracted from 266 accessions, which were collected from different rice growing areas of Turkey. The two primer sets (trn-a and trn-b1, and trn-c and trn-d) specific to a target region of the intergenic spacer between trnT (UGU) and trnL (UAA) and the entire intron region of trnL (UAA), respectively, were used in PCR amplifications. Of the 266 accessions of Echinochloa spp., only eight accessions gave a similar fragment size, which was slightly shorter than 495 bp. The PCR product obtained with the primers trn-a and trn-b1 gave two fragments when EcoRI restriction enzyme was used in barnyardgrass and early watergrass. However, not all accessions of late watergrass were digested with this enzyme. In contrast to EcoRI, the PCR product obtained using the trn-c and trn-d primer set was digested into two fragments by using AluI restriction enzyme in all accessions of late watergrass; whereas, it was not digested in barnyardgrass and early watergrass. This molecular differentiation among barnyardgrass, early watergrass, and late watergrass supports the hypothesis that late watergrass is not a synonym of early watergrass in Turkish accessions.

Though Crassostrea oysters have been distributed and cultured worldwide, their taxonomy is still difficult and often inaccurately determined because of the high level of phenotypic plasticity of the shell morphology. With the help of mitochondrial DNA, two novel species of Crassostrea oysters (C. hongkongensis and C. zhanjiangensis) were recently recognized, which suggests that the species diversity of Indo-Pacific oysters could be underestimated. Utilizing a combination of shell characteristics, a molecular marker (mitochondrial cox1 gene) and phylogenetic analysis, we identified a mangrove-distributed novel Crassostrea oyster species, C. dianbaiensis. The shell morphology of the new species is phenoplastic, as seen in other congeneric oysters. The left valve of this oyster is usually deeply cupped, and the right valve is usually slightly concave. The body size is classified as medium and is approximately 5–10 cm in height (estimated from 20 individuals). One distinctive feature of C. dianbaiensis is that the adductor muscle scars vary in color from dark purple to white in the right valve, but always appear white in the left valve. Based on the cox1 phylogenetic tree, C. dianbaiensis is inferred to be a new member of the Southeast Asia tropical oysters and is believed to be the northernmost distributed species among the tropical oysters. This study provided basal information for future studies, which are necessary to better understand the faunal characteristics, population and roles of this oyster in nearshore ecosystems.

An ovigerous Maja spider crab was captured during a fishery research cruise in the North Alboran Sea, western Mediterranean, near the city of Malaga. From morphological characters this individual was identified as Maja brachydactyla. Prior to the specimen being returned alive back to sea, it was measured, photographed, and a portion of a pleopod excised for genetic analysis. The latter confirmed the identification as correct. An additional seven specimens collected from Caleta de Velez in the vicinity of Malaga were morphologically examined, confirming the presence of M. brachydactyla in the Alboran Sea. This finding would represent the first evidence of the species occurring in the western Mediterranean Sea, with apparently well-established populations.

Meroplankton community studies in the Antarctic have primarily focused on the coastal waters of both the Antarctic Peninsula and the Ross Sea. The New Zealand International Polar Year - Census of Antarctic Marine Life (IPY-CAML) voyage to the Ross Sea during the late summer (February–March) 2008 provided the first meroplankton samples from three regions in the deep, oceanic waters of the Ross Sea (shelf, slope and adjacent offshore Antarctic waters of Admiralty Seamount and Scott Island). We used a combined morphological and molecular approach to identify 36 larval operational taxonomic units based on sequences from three loci (16S, 18S, COI), and exclude early developmental stages of holoplankton. Overall, larval abundance was lower than previous Antarctic studies (5.19 specimens per 100 m3), with larvae most abundant in the first 200 m of the water column and most diverse in the shelf region. Multivariate analysis revealed significant differences in the meroplankton community between regions and depth ranges, but with low similarity within these groupings; differences between water masses were undetectable due to the confounding effect with both region and depth. The influence of nearby benthic populations (e.g. the acorn barnacle Bathylasma corolliforme) and/or locally abundant taxa (e.g. the nudibranch Tergipes antarcticus) was evident in the meroplankton community.

Cupped oysters (Ostreidae, genus Crassostrea) occupy nearshore marine and estuarine habitats worldwide, providing many ecosystem goods and services as well as being a commercially important group of bivalves. In this study, the species identification of an “adulterant” oyster with small body size, which is often misidentified as a “young individual” of other sympatric species, including C. sikamea and C. hongkongensis, was determined for the first time, based on molecular markers (partial mitochondrial cox1 and rrnL genes), phylogenetic analysis, and morphometric approaches. This novel species, C. zhanjiangensis, commonly known as the “cat ear oyster” in Guandu (a famous estuarine oyster farming region of Zhanjiang, Guangdong Province, China), appears to potentially influence the efficiency of C. hongkongensis spat collection due to niche competition on spat collection devices. Phylogenetic analyses confirm its status as the most basal taxon of the Indo-Pacific Crassostrea. A comparative study of the shell characteristics of C. zhanjiangensis, and other Crassostrea species revealed several distinctive morphological traits, including a generally smaller body size, a deeply cupped left valve, and a right valve that is convex in adults but flat in young individuals. Other distinctive features of the new species include life cycle traits that are unique compared with the sympatric C. hongkongensis and C. sikamea species, such as a higher growth rate in the fast growth phase after settlement, followed by a significantly slower growth rate and mass mortality during subsequent life stages. This study provides the basic information necessary for further ecological and population genetic studies on this new species.

Myxozoans are a group of diverse, spore-forming metazoan microparasites bound to aquatic environments. Sphaerospora dykovae (previously S. renicola) causes renal sphaerosporosis and acute swim bladder inflammation (SBI) in juvenile Cyprinus carpio carpio, in central Europe. A morphologically similar species with comparably low pathogenicity, S. angulata has been described from C. c. carpio, Carassius auratus auratus and Carassius gibelio. To clarify uncertainties and ambiguities in taxon identification in these hosts we decided to re-investigate differences in spore morphology using a statistical approach, in combination with SSU and LSU rDNA sequence analyses. We found that developing spores of S. angulata and S. dykovae cannot be distinguished morphologically and designed a duplex PCR assay for the cryptic species that demonstrated S. dykovae is specific to C. c. carpio, whereas S. angulata infects C. a. auratus and C. gibelio. The molecular identification of myxozoan blood stages in common carp and goldfish, which had previously been ascribed to Sphaerospora spp. showed that approximately 75% of blood stages were from non-sphaerosporid coelozoic species infecting these cyprinids and more than 10% were from an alien species, Myxobilatus gasterostei, developing in sticklebacks. We hereby report non-selective myxozoan host invasion and multi-species infections, whose role in SBI still requires clarification.

Root-knot nematodes (RKN) are serious pests of tomato production in Kenya. Accurate identification of the plant parasitic nematodes is important for their effective management. A study conducted to assess their prevalence and identify RKN occurring on tomatoes in Central Kenya showed infestation of the crop in all the three districts. Of the total sampled plants (N = 900), the RKN infestation level ranged from 28 to 62%. RKN disease severity ranged from 2.5 to 5.3 in all the locations. Meloidogyne incognita,M. javanica and M. arenaria were the only species found infesting tomatoes in these areas. Female nematodes sampled from the symptomatic root system that had root galls characteristic of RKN were analysed by isozyme phenotypes of esterase (EST) and malate dehydrogenase (MDH). EST phenotype was polymorphic and enabled identification of the three different species, while MDH was monomorphic. Polymerase chain reaction-restriction fragment length polymorphism with region between mitochondrial cytochrome oxidase subunit II and large subunit ribosomal RNA (mtDNA COII-LSUrRNA) using HinfI showed that all isolates of M. incognita could be digested into three restriction fragments of about 1300, 400 and 100 bp, except for one species that showed an additional restriction site, giving four fragments of 900, 420, 380 and 100 bp. The 1800 bp PCR product of M. javanica was not digested by HinfI. Meloidogyne arenaria (EST phenotype A1) PCR product was digested into two restriction fragments of about 1700 and 100 bp, while M. arenaria (EST phenotype A2) had two restriction fragments at 1100 and 100 bp.

Urtica dioica and Convolvulus arvensis are the main host plants of Hyalesthes obsoletus and play an important role in the epidemiology of Bois noir of grapevines. The earliest survey, which was carried out to compare the phenology of nymphal instars on U. dioica and C. arvensis, had highlighted some problems in the identification of the instars. Therefore, the correct identification of nymphs to species and instar level became a preliminary aim of this research. Adults and nymphs attributable to H. obsoletus were collected during 2008–2010 in three flatland vineyard habitats of northern Italy on U. dioica, C. arvensis and Artemisia verlotorum. Nymphs and morphologically identified adults of H. obsoletus were submitted to molecular identification. Morphometric and morphological studies were carried out on nymphs collected in the field or obtained in laboratory rearings. Molecular methods not only confirmed the identity of adults, but also allowed the assignment of the nymphs to this species. Morphometric and morphological characteristics (e.g. body and head-thoracic lengths, number of thoracic pits) showed the existence of five nymphal instars. Morphometric differences between newly hatched and older first-instar nymphs were observed. A key to distinguish the five instars was proposed. Evident differences between H. obsoletus nymphs studied here and elsewhere were identified. According to differences in adult-flight period, an earlier phenology of nymphs on C. arvensis than on U. dioica was observed. In particular, the typical overwintering instar was the second on U. dioica and the third on C. arvensis.

Mealybugs are major pests of grapevines worldwide. They cause economic losses by lowering the cosmetic value of fruits, reducing yields, transmitting viruses and resulting in the quarantine or rejection of produce in international trade. Knowledge of the species present in a vineyard is important for the adjustment of management strategies. We surveyed and accurately characterized the mealybugs infesting vineyards in one of the main production areas of Chile; 164 mealybugs were sampled from 26 vineyards in four regions of Chile and identified by DNA sequencing for two markers (cytochrome oxidase I and internal transcribed spacer 2) and morphological examination. Pseudococcus viburni (Signoret) was the most common species, followed by Pseudococcus meridionalis Prado and Pseudococcus cribata González. Molecular variability at the COI and ITS2 loci was observed in both P. viburni and P. cribata. A comparison of haplotypes of P. viburni worldwide provides support for a recent hypothesis that this species is native to South America, a finding with direct consequences for management. Neither Pseudococcus longispinus (Targioni & Tozzetti) nor Planococcus ficus Signoret were found.

Podonomus Philippi was recently found in Brazilian inselbergs. In this study, we used sequences from the barcode region of cytochrome c oxidase I (COI) to assess Podonomus molecular diversity from mountains in Southeastern Brazil. Our results revealed the adult female and the larvae of Podonomus pepinellii Roque and Trivinho-Strixino, 2004 and extended the known geographical distribution of the species. Moreover, we found high molecular divergence in two populations located in the Serra da Mantiqueira and Serra do Espinhaço mountain range when compared with the only species recorded to Brazil, indicating the possibility of two more species to be described.