Morphological discrimination of species is problematic in many digenean taxa. Parasites of marine fish from the genus Lecithaster Lühe, 1901 are a good example of this. Our goal was to understand which species of Lecithaster infect fish in the White Sea, and reveal their life cycles. We collected specimens of maritae from nine fish species, analysed their morphology and sequenced 28S ribosomal DNA and internal transcribed spacer 2 (ITS2). Contrary to previous accounts, all of them belong to a single species, Lecithaster salmonis Yamaguti, 1934, which was previously only recorded from the Pacific. Morphologically, our maritae specimens were highly variable, sharing characters of L. salmonis, Lecithaster confusus Odhner, 1905 and Lecithaster gibbosus (Rudolphi, 1802) Lühe, 1901. This variability did not correlate with the moderate differences in ITS2 among the specimens, and neither did the fish host species. Members of the subfamily Salmoninae appear to be the best suited definitive hosts, judging from the intensity rates. The intermediate hosts were also discovered: the first is Cryptonatica affinis (Gmelin, 1791) and the second are planktonic copepods. These lifecycle data from the White Sea are consistent with L. salmonis species identification and with the distribution of this species in the North Pacific. The geographical range of L. salmonis seems to be interrupted, and we discuss possible ways of L. salmonis expansion.

Sarcoptic mange is a disease caused by an infectious parasite in the vicuñas (Vicugna vicugna) from South America. Although molecular studies have provided much information about the epidemiology of this disease, this information is still unknown in vicuñas. This study determined the prevalence and molecular characterization of Sarcoptes scabiei from vicuñas from Southern Peruvian Andes. During the 2018 shearing season, 181 vicuñas were clinically evaluated for lesions compatible with mange. Sarcoptes scabiei was detected in 35 (19.3%) vicuñas, and 50 mites from 25 vicuñas were selected for molecular analyses of the mitochondrial (cox1) and nuclear (ITS2) genetic markers. Molecular analyses of the cox1 and ITS2 sequences showed an identity of 94–99% and 99.8–100% with previous S. scabiei sequences registered in the GenBank, respectively. Sequence polymorphisms were more evident in the ITS2 than in the cox1, but only the cox1 had an association with the host. Phylogenetic analysis of S. scabiei cox1 sequences from vicuñas showed a cluster with S. scabiei cox1 sequences from canids, suggesting that the origin of S. scabiei from vicuña is associated with canid mites. This research is the first molecular analysis of S. scabiei from vicuñas. Future molecular studies will be necessary to determine the species variety, geographic segregation and host–parasite adaptation for this vicuña's mite.

The genus Isoparorchis (Family: Isoparorchiidae) contains trematodes infecting the air bladder of freshwater catfishes in Asia and Australia. Isoparorchis spp. rely on freshwater shrimps and fishes as intermediate hosts. There is limited information about parasitic infections in freshwater shrimp in Thailand, and Isoparorchis infection in an intermediate host in this country has never been reported. Thus, this study reports infections in freshwater shrimp (Macrobrachium lanchesteri and Caridina sp.), including overall prevalence, mean intensity, morphological characters and molecular analyses. The parasite specimens were analysed by studying their morphological characters, together with a molecular approach based on internal transcribed spacer 2 (ITS2) sequence data. The overall prevalence and mean intensity of Isoparorchis infections were 92% and 1.89, respectively. The metacercariae were identified as Isoparorchis sp. based on their morphological characters and supported by the comparison with published ITS2 sequences of Isoparorchis species. The phylogenetic studies based on the ITS2 region demonstrated that all of the Isoparorchis sp. specimens in this study are distinct from Isoparorchis species in previous reports. Moreover, we show for the first time that the freshwater shrimp M. lanchesteri serves as a second intermediate host of Isoparorchis sp. and we provide a morphological description and molecular characterization of Isoparorchis sp. metacercariae based on ITS2 sequence data to clarify the status of Isoparorchis sp. in Thailand.

The few Antarctic studies to date to have applied metabarcoding in Antarctica have primarily focused on microorganisms. In this study, for the first time, we apply high-throughput sequencing of environmental DNA to investigate the diversity of Embryophyta (Viridiplantae) DNA present in soil samples from two contrasting locations on Deception Island. The first was a relatively undisturbed site within an Antarctic Specially Protected Area at Crater Lake, and the second was a heavily human-impacted site in Whalers Bay. In samples obtained at Crater Lake, 84% of DNA reads represented fungi, 14% represented Chlorophyta and 2% represented Streptophyta, while at Whalers Bay, 79% of reads represented fungi, 20% represented Chlorophyta and < 1% represented Streptophyta, with ~1% of reads being unassigned. Among the Embryophyta we found 16 plant operational taxonomic units from three Divisions, including one Marchantiophyta, eight Bryophyta and seven Magnoliophyta. Sequences of six taxa were detected at both sampling sites, eight only at Whalers Bay and two only at Crater Lake. All of the Magnoliophyta sequences (flowering plants) represent species that are exotic to Antarctica, with most being plausibly linked to human food sources originating from local national research operator and tourism facilities.

We investigated the prevalence, morphological characters and molecular classifications of trematode cercariae in freshwater snails randomly collected from 59 sampling localities in Bangkok from May 2018 to March 2019. We used a crushing technique to observe the cercarial stage inside each snail body and amplified the internal transcribed spacer 2 regions of cercarial DNA using polymerase chain reaction methodology. The associated phylogenetic tree was reconstructed using Bayesian inference analyses. A total of 517 of 15 621 examined snails were infected with trematode cercariae, and the infected snails were classified into 11 species of seven families with a 3.31% overall prevalence of the infection. The Bithynia siamensis siamensis snail displayed the highest prevalence of infection (16.16%), whereas the Physella acuta snail exhibited the lowest prevalence (0.08%) of infection. Eight morphological types of cercariae were observed. The highest prevalence of infection was observed in mutabile cercaria (1.86%). Based on molecular investigations, the phylogram revealed eight cercaria types assigned to at least nine digenean trematode families, of which five belong to groups of human intestinal flukes. Although, with the exception of schistosome cercaria, trematode cercariae are not known to directly damage humans, understanding the general biology of trematode cercariae (including diversity, distribution, infection rates and host range) is important and necessary for the prevention and control of parasitic transmission that impacts aquatic cultivations, livestock farming and human health.

Sequence data were combined with morphological analyses to identify two lepocreadiid trematode species from jellyfishes and fishes. Three species of jellyfish were captured within Port Phillip Bay, Australia, and three species of fish that feed on jellyfish were obtained from Moreton Bay (Queensland) and Port Phillip Bay and Portland (Victoria). The digeneans were distributed throughout most parts of the jellyfish. Opechona cf. kahawai Bray & Cribb, 2003 parasitized the scyphozoan jellyfish Aequorea eurodina and the scombrid fish Scomber australasicus. Cephalolepidapedon warehou Bray & Cribb, 2003 parasitized the scyphozoans Pseudorhiza haeckeli and Cyanea annaskala, and the centrolophid fishes Seriolella brama and Seriolella punctata. Intensities ranged from four to 96 in the jellyfish, and one to 30 in the fish. For both trematode species, internal transcribed spacer 2 of ribosomal DNA sequences from mature adults in the fishes matched those from metacercariae from the jellyfish. This is the first record of larval stages of C. warehou and O. cf. kahawai, and the first use of DNA sequencing to identify digenean trematode metacercariae from jellyfish. Three new host records are reported for C. warehou and two for O. cf. kahawai.

Thermal stress events threaten coral populations by disrupting symbiosis between the coral animal and microalgal symbionts in its tissues. These symbionts are key players in the response of the coral holobiont to elevated temperature. However, little is known about the microalgal symbiont type in select corals in the north-western Philippines and how they contribute to the differential responses of coral species. Based on sequencing of major ITS2 bands from DGGE, the dominant algal symbiont in Acropora digitifera, A. millepora, A. tenuis and Favites colemani was identified to be closely related to ITS2 type C3u, Montipora digitata contained ITS2 type C15, and Seriatopora caliendrum hosted ITS2 types similar to C3-Gulf and D1. Thin branching corals, such as A. tenuis and S. caliendrum, exhibited the greatest reduction in photochemical efficiency (Fv/Fm) and symbiont density at elevated temperature, followed by M. digitata and A. millepora, to a lesser extent. A. digitifera and F. colemani were least affected by the temperature treatment. Reduction in Fv/Fm and symbiont density was more apparent in A. tenuis and A. millepora than in M. digitata and F. colemani, although these species all host ITS2 type C3u symbionts. These results suggest that the impact of elevated temperature is influenced by factors apart from symbiont type. This highlights the importance of further studies on the diversity of corals and their microalgal symbionts in the region to gain insights into their potential resilience to recurring thermal stress events.

An important step towards developing a successful integrated pest management (IPM) programme for the control of banana pseudostem weevil, Odoiporus longicollis (Olivier), a serious pest of banana in India, is the study of the population structure of the pest. In the present study, the genetic variation among 30 individual weevils of O.longicollis collected from six different locations was assessed by analysing the primary nucleotide sequences of the rDNA ITS1 and ITS2 regions. AMOVA, Mantel test, and the maximum likelihood trees of the haplotypes failed to reveal any phylogeographic structuring, which was confirmed by haplotype analysis and genetic differentiation estimates. The results indicate that the locations are not differentiated, i.e. there is gene flow between the locations. The star-shaped networks revealed a signature of demographic expansion that was confirmed by the different demographic tests. These results provide important information, which is essential for the development of suitable strategies for the control of this banana pest, as well as management of its resistance to insecticides.

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.

Prodiplosis longifila is reported as a pest of a wide range of species cultivated in America, including citrus, solanaceous species and asparagus. This species has different behavioural traits that are primarily centred on the oviposition habit and the feeding of larvae, which can change depending on the host. However, scarce information is available on population studies and the natural history of this insect, and uncertainty exists about the taxonomic identity and the geographic distribution of this species. The main objective was to perform a phylogenetic and genetic study of P. longifila populations and to define whether the North American and South American populations belong to the same species or whether a differentiation process had occurred due to geographic distance. A second objective was to determine whether this species showed genetic differentiation by host specialization in South America. The phylogenetic and population analyses based on DNA barcodes (cytochrome oxidase I gene) and a region of the ribosomal DNA (ITS2) revealed divergent clades attributable to geographic distance and host specificity. The North American and South American P. longifila insects were confirmed to be genetically distinct, and the genetic distances exceeded the values expected for intraspecific variation. In South America, the population analysis of P. longifila from tomato, sweet pepper (Solanaceae), Tahiti lime and key lime (Rutaceae) hosts evidenced high genetic differentiation between populations associated with different hosts and an absence of gene flow between these groups, suggesting the corresponding formation of cryptic species.

Lecithodendrium linstowi is one of the most prevalent and abundant trematodes of bats, but the larval stages and intermediate hosts have not been identified. We present the first molecular and morphological characterization of the cercariae of L. linstowi based on a phylogenetic analysis of partial fragments of LSU and ITS2 rDNA. The first intermediate host was incriminated as Radix balthica by DNA barcoding using cox1 and ITS2 sequences, although the snail morphologically resembled Radix peregra, emphasizing the requirement for molecular identification of lymnaeids as important intermediate hosts of medical and veterinary impact. The application of molecular data in this study has enabled linkage of life cycle stages and accurate incrimination of the first intermediate host.

Mentha (Nepetoideae, Lamiaceae) is a taxonomically complex genus consisting of 29 species and 11 different hybrids including a number of economically important species among them medical plants used for more than 250 years. Because of its high incidence of polyploidy, diverse morphology, high degree of chromosome number variations and interspecies hybridization, Mentha is considered to be a taxonomical challenge. In this paper, we describe cloning and sequencing of internal transcribed spacers (ITS) 1 and 2 of three mint species: Mentha aquatic L., Mentha longifolia L. (Huds) and Mentha piperita L., collected from natural habitats of Bulgaria. The applicability of ITS1 and ITS2 for molecular fingerprinting and further comparative studies, including the newly obtained ITS1 and ITS2 sequences, was tested in phylogenetic analysis of all eight Mentha species, found in Bulgarian flora.

Frankliniella schultzei Trybom is a polyphagous pest and vector of tospoviruses worldwide. It occurs in dark and pale colour forms that are morphologically similar but differ in vector competency and geographic spread. In Kenya and other tropical regions, mixed populations of both colour forms are observed in similar habitats, so are considered as one species. To ascertain the taxonomic status of the two colour forms, they were characterized using morphological, molecular, biological and ecological approaches. Morphological characterization revealed differences between the colour forms on eight features and they separated into distinct clusters through principal component analysis. Restriction fragment length polymorphism of the internal transcribed spacer region (ITS-RFLP) analysis revealed differences between the two colour forms and was confirmed by differences in ITS2 sequences. Virgin pale females had female offspring (thelytoky), while virgin dark females had male offspring (arrhenotoky). Interbreeding of dark males with pale females resulted in pale females, indicating absence of interbreeding between the two colour forms. Laboratory colonies of pale forms lacked males and further analysis of F. schultzei males from Ipomoea setosa flowers in the field indicated the presence of dark males and the absence of pale males. Field surveys in Kenya indicated differences in distribution and host plant preferences among the colour forms. Lack of interbreeding, distinct host preferences and distribution, and morphological and molecular differences indicate that the two colour forms of F. schultzei could be different species. The results highlight the need for combining morphological, biological, molecular and ecological characteristics for resolving taxonomic status of closely related insects.

Oligonychus punicae and Oligonychus perseae (Acari: Tetranychidae) are the most important mite species affecting avocado orchards in Mexico. Here we used nucleotide sequence data from segments of the nuclear ribosomal internal transcribed spacers (ITS1 and ITS2) and mitochondrial cytochrome oxidase subunit I (COI) genes to assess the phylogenetic relationships between both sympatric mite species and, using only ITS sequence data, examine genetic variation and population structure in both species, to test the hypothesis that, although both species co-occur, their genetic population structures are different in both Michoacan state (main producer) and Mexico state. Phylogenetic analysis showed a clear separation between both species using ITS and COI sequence information. Haplotype network analysis done on 24 samples of O. punicae revealed low genetic diversity with only three haplotypes found but a significant geographical population structure confirmed by analysis of molecular variance (AMOVA) and Kimura-2-parameter (K2P) analyses. In addition, a Mantel test revealed that geographical isolation was a factor responsible for the genetic differentiation. In contrast, analyses of 22 samples of O. perseae revealed high genetic diversity with 15 haplotypes found but no geographical structure confirmed by the AMOVA, K2P and Mantel test analyses. We have suggested that geographical separation is one of the most important factors driving genetic variation, but that it affected each species differently. The role of the ecology of these species on our results, and the importance of our findings in the development of monitoring and control strategies are discussed.

Clonorchiasis, caused by Clonorchis sinensis, is a key foodborne zoonosis, which is mainly found in China, Korea and Vietnam. Detection of this parasite from the second intermediate host, the freshwater fish is the common method for epidemiological surveys of this parasite, but is time consuming, labour intensive and easily leads to misdiagnosis. In this study, we have developed a rapid, sensitive and reliable molecular method for the diagnosis of C. sinensis from its first intermediate hosts, freshwater snails, based on a loop-mediated isothermal amplification (LAMP) method. The specific amplified fragment from genomic DNA of C. sinensis did not cross-react with those from other relevant trematodes and a range of hosts (freshwater fish, shrimps and snails) of C. sinensis living in similar environments. The detection limit of the LAMP method was as low as 10 fg which was 1000 times more sensitive than conventional PCR, which was also demonstrated by successful application to field samples. These results show that the LAMP method is a more sensitive tool than conventional PCR for the detection of C. sinensis infection in the first intermediate hosts and, due to a simpler protocol, is an ideal molecular method for field-based epidemiological surveys of this parasite.

Pseudococcidae (mealybugs) is a large taxonomic group, including a number of agronomic pests. Taxonomic identification of mealybug species is a recurrent problem and represents a major barrier to the establishment of adequate pest management strategies. We combined molecular analysis of three DNA markers (28S-D2, cytochrome oxidase I and internal transcribed spacer 2) with morphological examination, for the identification of 176 specimens collected from 40 mealybug populations infesting various crops and ornamental plants in Egypt and France. This combination of DNA and morphological analyses led to the identification of 17 species: seven in Egypt (Planococcus citri (Risso), Planococcus ficus (Signoret), Maconellicoccus hirsutus (Green), Ferrisia virgata (Cockerell), Phenacoccus solenopsis Tinsley, Phenacoccus parvus Morrison and Saccharicoccus sacchari (Cockerell)) and 11 in France (Planococcus citri, Pseudococcus viburni Signoret, Pseudococcus longispinus (Targioni-Tozzetti), Pseudococcus comstocki (Kuwana), Rhizoecus amorphophalli Betrem, Trionymus bambusae (Green), Balanococcus diminutus (Leonardi), Phenacoccus madeirensis Green, Planococcus vovae (Nasonov), Dysmicoccus brevipes (Cockerell) and Phenacoccus aceris Signoret), Pl. citri being found in both countries. We also found genetic variation between populations considered to belong to the same species, justifying further investigation of the possible occurrence of complexes of cryptic taxa.

The cixiid species Reptalus quinquecostatus, R. cuspidatus, R. panzeri and R. melanochaetus are widely distributed in Europe and are receiving growing attention because of their potential role as phytoplasma vectors. Identifying the Reptalus species is restricted to a few specialist entomologists and relies on the morphology of the male genitalia, hampering the identification of juveniles and females. This study provides the tools for species discrimination by integrating the morphological description, which is primarily for the genus identification, with new molecular assays, based on both ribosomal and mitochondrial DNA. PCR-RFLP assays carried out on the mitochondrial cytochrome oxidase I gene (COI) with AluI provided species-specific profiles for the four Reptalus species. Amplification of a ribosomal internal transcribed spacer (ITS2) region produced species-specific fragments of different sizes for R. quinquecostatus, R. melanochaetus, R. cuspidatus and R. panzeri. The digestion of the ITS2 PCR product with TaqI allowed the discrimination of these latter two species. This molecular identification key ensures reliable results and can be successfully applied not only to adults, but also to the nymphs feeding on the roots. The identification of the nymphs (i) extends the collection period of these monovoltine species to the whole year (adults are present for a short summer period) and (ii) allows the unambiguous identification of their actual host plants because nymphs are steady on the root system while adults tend to disperse onto other plants. Fast and reliable identification of the Reptalus species provides useful help in monitoring activities and, therefore, in designing rational control strategies to protect crops from phytoplasma infection.

Pouzolzia rugulosa (Wedd.) Acharya & Kravtsova, comb. nov., is transferred from the genus Boehmeria on the grounds of the anatomy and morphology of the fruit, correlated with molecular data. A revised set of anatomical and morphological characters is provided to distinguish between Boehmeria and Pouzolzia. Pouzolzia rugulosa shares morphological similarities with a group of serrate-leaved taxa in that genus known from Asia and Oceania. It is a shrub or small tree and is found along the southern slopes of the Himalayas from Himachal Pradesh in northern India to the mountains of northern Burma. It occurs in forest at elevations of up to 1900 m, mainly at forest edges and in clearings. A full description of Pouzolzia rugulosa, including drawings and SEM images of the fruit, is provided, together with a key.

Accurate identification of aspermic Fasciola forms in Japan remains difficult because of their morphological variations. In order to characterize the forms genetically, nucleotide sequences of ribosomal internal transcribed spacer (ITS1 and ITS2) and mitochondrial cytochrome c oxidase I (COI) and NADH dehydrogenase I (NDI) genes in 34 liver flukes from 16 prefectures in Japan were analysed. Two major forms represented by Fsp 1 and Fsp 2 had sequences identical to or closely resembling those of F. hepatica and F. gigantica, respectively, in all the 4 DNA markers and were mainly distributed in northern and eastern-western parts of Japan, respectively. Fsp 1 and Fsp 2 would have been introduced into Japan with infected cattle of 2 distinct lineages via the Korean Peninsula and spread through limited parts of Japan (northern and eastern-western parts) together with the movement of each cattle lineage. The Japanese form (Fsp 1/2), which showed heterozygosity in ribosomal DNA and Fsp 2 haplotype in mitochondrial DNA, may have originated in interspecific cross hybridization between paternal F. hepatica and maternal F. gigantica.

In this study, we conducted phylogenetic analyses of nematode parasites within the genus Dictyocaulus (superfamily Trichostrongyloidea). Lungworms from cattle (Bos taurus), domestic sheep (Ovis aries), European fallow deer (Dama dama), moose (Alces alces), musk ox (Ovibos moschatus), red deer (Cervus elaphus), reindeer (Rangifer tarandus) and roe deer (Capreolus capreolus) were obtained and their small subunit ribosomal RNA (SSU) and internal transcribed spacer 2 (ITS2) sequences analysed. In the hosts examined we identified D. capreolus, D. eckerti, D. filaria and D. viviparus. However, in fallow deer we detected a taxon with unique SSU and ITS2 sequences. The phylogenetic position of this taxon based on the SSU sequences shows that it is a separate evolutionary lineage from the other recognized species of Dictyocaulus. Furthermore, the analysis of the ITS2 sequence data indicates that it is as genetically distinct as are the named species of Dictyocaulus. Therefore, either this taxon needs to be recognized as a new species, or D. capreolus, D. eckerti and D. viviparus need to be combined into a single species. Traditionally, the genus Dictyocaulus has been placed as a separate family within the superfamily Trichostrongyloidea. The present molecular phylogenetic analyses support the placement as a separate family, but the current data do not support the placement of the Dictyocaulidae within the Trichostrongyloidea without a reassessment of the placement of the superfamily Strongyloidea. While D. eckerti has been regarded as the one and only lungworm species of cervids, this study showed that 4 host species including 3 members of Cervidae (moose, reindeer, red deer) and 1 Bovidae (musk ox) were infected with this parasite. Host ranges of D. viviparus (cattle), D. filaria (sheep) and D. capreolus (moose and roe deer) were more restricted. No clear pattern of co-evolution between the dictyocaulid taxa and their bovid and cervid hosts could be determined.