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A new species of the nereidid annelid, genus Nicon Kinberg, 1866, from KIOST Seamount, Northwest Pacific deep water is described. Nicon is a genus characterized by lacking paragnaths or papillae on the pharynx and composed of nine species worldwide, distributed from shallow water to deep sea. Nicon ablepsia sp. nov. here described is characterized by the lack of eyes on the prostomium, prolonged tentacular cirri reaching to chaetiger 6, notochaetae homogomph spinigers, neurochaetae homogomph spinigers and heterogomph falcigers. Phylogenetic relationships of Nicon remain undetermined based on molecular data. In this study, we constructed molecular Maximum-Likelihood phylogenetic tree from 29 nereidid species based on four marker genes: mitochondrial 16S rRNA gene and cytochrome c oxidase subunit I (COI) gene; nuclear 18S rRNA gene and 28S rRNA gene. Our analysis suggest the Nicon is clustered within Nereidinae, and nereidinae is not recovered as monophyletic. A key to species of Nicon is provided.

The English grain aphid, Sitobion avenae (Fabricius), exhibits classic and dramatic phenotypic plasticity in wing development. Both genetic and environmental inputs contribute to the wing polyphenism in aphids, an extreme form of phenotypic plasticity in which a single genotype produces discrete winged and wingless morphs. Validated reference genes are needed to accurately normalize temporal and spatial variation in gene expression estimates by RT-qPCR. In this research, the stability of 11 candidate reference genes selected from S. avenae transcriptomes was evaluated under an array of abiotic and biotic conditions relevant to wing development. RefFinder, a comprehensive software integrating rankings from delta Ct, BestKeeper, NormFinder, and geNorm, offered a series of reference genes for every experimental condition. Overall, helicase (HEL) and ubiquitin ribosomal protein S27A fusion protein (RpS27) are suited for most of the conditions examined in this study, although exceptions do exist. Specifically, NADH dehydrogenase (Ap-NADH) and 28S ribosomal RNA (28S) are recommended for insecticide and antibiotic treatments, while ribosomal RNA L14 (RPL14) and 18S ribosomal RNA (18S) are selected for density treatment, respectively. This study provides a suite of reference genes to investigate the wing polyphenism in S. avenae, and is important for application of RT-qPCR in future experiments of novel tactics to control aphids.

Metagonimiasis, commonly seen in East Asian countries, is a parasitic disorder caused by definitive hosts’ ingestion of undercooked freshwater fishes. Recently, genetic analysis has proved 28S rRNA and cytochrome c oxidase subunit I (COI) mtDNA gene to be a successful marker differentiating species of the genus Metagonimus. In the present study, using specimens from the newly discovered Joseon Dynasty human remains of Goryeong, we obtained updated genetic data on genus Metagonimus, which was also prevalent during the Joseon period. The ancient DNA (aDNA) was retrieved from the coprolite sample of the seventeenth century, half-mummified individual discovered at Goryeong Country, South Korea. Cloning and sequencing were performed on PCR-amplified amplicons for M. yokogawai 28S rRNA and COI mtDNA gene. The consensus sequences were used for species identification and phylogenetic analysis using NCBI/BLAST and MEGA X software. Based on the COI mtDNA gene region, the Goryeong sequence was confirmed as belonging to M. yokogawai, as it was shown to form a separate cluster with other M. yokogawai taxa that are distinct also from M. takahashii and M. miyatai. In a series of our genetic analyses on genus Metagonimus using samples retrieved from Joseon-period cases, aDNA sequences of genus Metagonimus revealed in South Korea thus far are those of M. yokogawai, but not of M. miyatai or M. takahashii yet.

Trehala manna is the edible and trehalose-rich cocoons of a few Larinus species (Coleoptera: Curculionidae: Lixinae: Lixini), and manufactured by the feeding activity of larvae on the Echinops plants. Knowledge on the morphological and molecular properties of immature stages of Larinus species, especially trehala-constructing ones, is still very limited. Herein, the mature larva and pupa of Larinus hedenborgi Boheman, 1845, are morphologically described for the first time, illustrated and compared with known immature stages of other Larinus species. Morphological identification keys prepared for all known immature stages of Larinus species. The DNA sequences of three mitochondrial (COI, Cytb, and 16S RNA) and four nuclear (enolase, histon 4, 18S rRNA, and 28S rRNA) markers were generated for L. hedenborgi and compared with the available sequences in the GenBank. Comparative morphology revealed the affinity of L. hedenborgi with the L. vulpes, L. inaequalicollis, and L. capsulatus based on five larval and two pupal characters. Molecular analysis based on 1859 bp of mtDNA and 1618 bp of nDNA sequences indicated a close association between the L. hedenborgi and other Lixinae namely Larinus species. Although both morphological and molecular descriptions supported the taxonomic status of L. hedenborgi within weevils, sufficient molecular data must be available to allow further comparisons. This pioneering study can be expanded to a large-scale zoogeographic study using morpho-molecular characters, simultaneously with population-level sampling of all trehala-constructing Larinus species.

Subcutaneous nodular onchocercosis was detected and investigated in 17 Japanese Sika deer (Cervus nippon), captured in Gifu and Shiga Prefectures/Japan, in the period between 2016 and 2017. The worms were found in all the seventeen deer within characteristics subcutaneous nodules dispersed mainly in the back (especially in the lumbar region and flanks), with few scattered nodules were located at the forelimbs and neck. The all collected nodules were examined stereo-microscopically. The parasites were extracted from the nodules and identified through morphological and histopathological examinations. Molecular identification through sequencing of the following genes; internal transcribed spacer subunit 2 (ITS2)–28S ribosomal RNA (28S rRNA), cytochrome c oxidase subunit 1 (cox1) and mitochondrially encoded NADH dehydrogenase subunit 2 (NAD2) were performed. The histopathological, molecular and phylogenetic analysis demonstrated that, the filarial nematode isolated from Gifu and Shiga Prefectures in Japan is O. flexuosa. This is the first report about presence of O. flexuosa in Japanese Sika deer (Cervus nippon) in Gifu and Shiga Prefectures. SUPPLEMENTARY INFORMATION: The online version supplementary material available at 10.1007/s12639-021-01453-3.

Phyllodistomum is one of the most species-rich genera of parasitic platyhelminths, with 120 species described worldwide; they infect the urinary bladder of marine and freshwater fishes. As the number of new species within the genus has increased, morphological conservatism, and the lack of reliable diagnostic traits make the separation of species a challenging task. The increase of genetic data for Phyllodistomum species has permitted the use of an integrative taxonomy approach as a framework for species discovery and delimitation. DNA sequences (28S rRNA and COI mtDNA) were obtained from individuals of Phyllodistomum sampled in 29 locations across Middle America, and used in combination with morphology, host association and geographic distribution to uncover five new congeneric species. Morphologically, the new species are relatively similar; there are no unique morphological traits to readily distinguish them. We first investigated species boundaries through phylogenetic analyses of the independent and concatenated datasets; analyses recognised five candidate species showing reciprocal monophyly and strong clade support, particularly for COI data. The interspecific 28S rRNA and COI sequence divergence among the new species from 0.4 to 18.4% and from 5.1 to 27% respectively. These results were further validated by a Bayesian species delimitation approach. The five new species are well supported by molecular data used in combination with other sources of information such as host association and geographical distribution and are described herein as Phyllodistomum romualdae sp. nov., P. virmantasi sp. nov., P. isabelae sp. nov., P. scotti sp. nov., and P. simonae sp. nov.

A newly identified tardigrade species from China, Pilatobius nuominensis sp. nov., belongs to the group of species with cuticle of the dorsal and lateral caudal region with evident irregular polygonal sculpture. Nucleotide sequences of two nuclear (18S rRNA, 28S rRNA) and one mitochondrial (COI) DNA fragments of the new species are provided, which allows an independent verification of the taxonomic status of the new species. This is the first record of the genus Pilatobius in the Great Hinggan Mountains.

Anoplotaenia dasyuriBeddard, 1911 (Cestoda), from the Tasmanian devil,Sarcophilus harrisii(Boitard, 1842), is a taxonomic enigma, where a combination of morphological features, host type and geographical location have prevented it from being placed within a family and it is consideredincertae sedis, despite its accepted validity. We performed a phylogenetic analysis of threeA. dasyurispecimens collected from three Tasmanian devils using 18S and 28S rRNA sequences.Anoplotaenia dasyuriwas found to have closest affinity with the family Paruterinidae, especially the genusCladotaeniaCohn, 1901. The postulated theory of transfer of an ancestor ofAnoplotaeniaBeddard, 1911 transferring to the Tasmanian devil from an unrelated carnivorous host, such as an accipitriform or other carnivorous bird, is discussed and supported.

Epitranscriptomics is an emerging field of investigation dedicated to the study of post-transcriptional RNA modifications. RNA methylations regulate RNA metabolism and processing, including changes in response to environmental cues. Although RNA modifications are conserved from bacteria to eukaryotes, there is little evidence of a epitranscriptomic pathway in insects. Here we identified genes related to RNA m6 A (N6-methyladenine) and m5 C (5-methylcytosine) methylation machinery in seven bee genomes (Apis mellifera, Melipona quadrifasciata, Frieseomelitta varia, Eufriesea mexicana, Bombus terrestris, Megachile rotundata and Dufourea novaeangliae). In A. mellifera, we validated the expression of methyltransferase genes and found that the global levels of m6 A and m5 C measured in the fat body and brain of adult workers differ significantly. Also, m6 A levels were differed significantly mainly between the fourth larval instar of queens and workers. Moreover, we found a conserved m5 C site in the honeybee 28S rRNA. Taken together, we confirm the existence of epitranscriptomic machinery acting in bees and open avenues for future investigations on RNA epigenetics in a wide spectrum of hymenopteran species. This article is protected by copyright. All rights reserved.

Acanthoparyphium shinanense n. sp. (Digenea: Echinostomatidae) is described from chicks experimentally infected with the metacercariae encysted in 2 brackish water clam species, Ruditapes philippinarum and Coecella chinensis, in the Republic of Korea. The metacercariae were round to oval, armed with 23 collar spines, and 0.216 (0.203-0.226) mm in diameter. From 5 chicks experimentally infected each with 200 metacercariae, 34 juvenile (5-day-old worms) and 104 adult flukes (7-day-old worms) were harvested from their small intestines, with the average worm recovery rate of 13.8%. The adult flukes were 3.18 (2.89-3.55) mm long and 0.68 (0.61-0.85) mm wide, with an elongated, posteriorly tapering body, and a prominent head collar armed with 23 collar spines arranged in a single uninterrupted row. The posterior testis of A. shinanense was longitudinally elongated, which is similar to Acanthoparyphium spinulosum Johnston, 1917 but unique from the other closely related species, including Acanthoparyphium tyosenense Yamaguti, 1939, Acanthoparyphium kurogamo Yamaguti, 1939, and Acanthoparyphium marilae Yamaguti, 1934. The eggs of A. shinanense were larger than those of A. spinulosum, and the anterior extent of 2 lateral groups of vitellaria was slightly more limited in A. shinanense than in A. spinulosum. Molecular analysis of nuclear and mitochondrial genes revealed low homology with A. spinulosum from USA (96.1% in 5.8S rRNA) and Ukraine (97.9% in 28S rRNA), Acanthoparyphium n. sp. from USA (98.0% in 28S rRNA), and Acanthoparyphium sp. from Australia, Kuwait, and New Zealand. Biological characteristics, including its first intermediate host and natural definitive hosts, as well as its zoonotic capability, should be elucidated.

White grub species, Melolontha cuprescens, Melolontha furcicauda, Melolontha indica and Polyphylla fullo are serious pests of different crops in north-western Himalaya. None of these species are, however, characterized at molecular level including DNA barcodes. Here we provide DNA barcodes for these species. The adults of these species were collected from different locations of Himachal Pradesh and described by molecular characterization. The mitochondrial Cytochrome c oxidase 1 (COX1) and 28S ribosomal RNA (28S rRNA) genes of these species were amplified and sequenced for molecular identification and phylogenetic analysis. The sequences of these genes from other species of these genera and other related genera were also retrieved from DNA databases and included in the analysis. The COX1 sequences of M. cuprescens were 99.88% similar to P. fullo, whereas those of M. furcicauda were 84.91% similar to M. indica. Among retrieved COX1 sequences, Melolontha hippocastani (HM120757 & KX087316) were closer to M. furcicauda (86.14–86.50%) and M. indica (85.74–86.04%) as compared to M. cuprescens (81.44–81.47%) and P. fullo (81.32–81.47%). Based on 28S rRNA gene sequences, M. indica showed 99.42% similarity to Melolontha melolontha (EU084231) as compared to M. cuprescens and M. furcicauda which were distantly related. The COX1 was a better DNA barcode for the identification of Melolontha and Polyphylla species. The 28S rRNA has a limited utility in DNA barcoding of Melolontha species.

The fungus genus Neoscytalidium is mainly distributed in (sub) tropical regions of the world and has been essentially considered as a phytopathogen. There are however several reports of human infection caused by Neoscytalidium spp. through direct or indirect contact with contaminated plants or soil. Reliable and accurate identification to species level is critical for implementing proper therapeutic strategies. In the present study we investigated the genotypes and in vitro antifungal susceptibility patterns of Neoscytalidium species identified from respiratory tracts of patients with various underlying diseases. The identity and diversity of the isolates were done using PCR and sequencing of five different loci (the ITS region, D1/D2 domains of 28S rRNA gene, and part of the beta tubulin, elongation factor 1α and chitin synthase genes). The in-vitro antifungal susceptibility was also performed using the Clinical and Laboratory Standards Institute (CLSI) M38-Ed3-2017 guidelines. Overall, 13 isolates were identified as Neoscytalidium species (eight N. dimidiatum and five N. novaehollandiae). Two sequence types (STs) were identified by the alignment of 1846 combined base pairs among 13 clinical isolates. All isolates classified as N. dimidiatum were clustered in ST6 (61.5%) and those of N. novaehollandiae were in ST7 (38.5%). Luliconazole was the most active antifungal in vitro against species. This is the first report of N. novaehollandiae isolation from respiratory tracts samples. Further study from other regions of the world with a larger set of clinical specimens is required to provide additional insight into diversity of Neoscytalidium species.

Using molecular markers and species delimitation analyses, a high diversity of bi-tentaculate Cirratulidae was discovered from the North-East Atlantic. Five new species are described: Chaetozone pseudosetosasp. nov., Chaetozone quintasp. nov., Chaetozone barentsensissp. nov., Chaetozone monteverdiisp. nov., and Chaetozone chambersaesp. nov. Several morphogroups are also described, even though the presence of cryptic diversity prevented naming of individual species. For each species presented, a molecular diagnostic is given from the universal barcode COI and, when available, the D1–D2 domains of the 28S rRNA. This increases the number of species in Chaetozone in northern European waters from ten to at least 17 species, the exact number of species remaining uncertain as taxonomic issues with older names remain unresolved.

Pratylenchus bolivianus (Nematoda, Pratylenchidae) an important parasitic lesion nematode of ornamental and crop plants was found in association with rooibos (Aspalathus linearis) tea in the Cederberg region of South Africa. The population distribution and frequency of occurrence of P. bolivianus on the rooibos orchards were determined, and nematode characterization was done using a combination of traditional morphological characteristics, scanning electron microscopy (SEM), morphometrics and molecular marker by amplifying the D2-D3 expansion segment of the 28S ribosomal RNA gene. P. bolivianus occurred at 84.6% frequency in the sampled fields, with a mean population density that ranged between 10 and 770 lesion nematodes per 250 ml. The morphological features are similar to previous reports, with a slight variation in stylet length and ratio of ‘a’ due to intraspecific geographical variations. The en face view of the SEM shows pattern of the oral disc and first labial annule that is characteristic of P. bolivianus a pattern that falls under Group 2 classification. The phylogenetic relationships as inferred from Maximum Likelihood and Maximum Parsimony revealed a close relationship between the South African isolate of P. bolivianus and those published from other geographical locations. The study confirmed a morphological and genetic similarity between the amphimictic population of P. bolivianus from South Africa and those reported from Costa Rica.

The first data on a whole mitochondrial genome of Haploporidae, Parasaccocoelium mugili (Digenea: Haploporata: Haploporidae) was generated using the next-generation sequencing (NGS) approach. We sequenced the complete mitochondrial DNA (mtDNA) and ribosomal operon of Parasaccocoelium mugili, intestine parasite of mullet fish. The mtDNA of P. mugili contained 14,021 bp, including 12 protein-coding genes, two ribosomal genes, 22 tRNA genes, and non-coding region. The ribosomal operon of P. mugili was 8308 bp in length, including 18S rRNA gene (1981 bp), ITS1 rDNA (955 bp), 5.8S rRNA gene (157 bp), ITS2 rDNA (268 bp), 28S rRNA gene (4180 bp), and ETS (767 bp). We used the mtDNA protein-coding regions to make phylogenetic reconstructions of Haploporidae. Additionally, we performed the sequence cluster analysis based on codon usage bias of most of currently available mitochondrial genome data for trematodes. The observed gene arrangement in mtDNA sequence of P. mugili is identical to those of Plagiorchis maculosus (Rudolphi, 1802). Results of maximum likelihood (ML) phylogenetic analysis showed that P. mugili was closely related to Paragonimus species from the suborder Xiphidiata. The results of sequence cluster analysis based on codon usage bias showed that P. mugili has the highest similarity with Plagiorchis maculosus (Xiphidiata). Our results do not contradict to proposing a new suborder for Haploporoidea-Haploporata. On the basis of obtained results, the relationship between mitochondrial protein-coding gene rearrangements and synonymous nucleotide substitutions in mitochondrial genomes has been suggested.

Metacercariae of the zoogonid trematode Steganoderma cf. eamiqtrema ex crab Chionoecetes bairdi caught in the Sea of Okhotsk were described using morphological and molecular-genetic (ITS2 region, 28S rRNA and nd1 genes) data. These are the first molecular-genetic data for the genus Steganoderma. The studied trematodes differed from S. eamiqtrema in having a much larger body size. The phylogenetic analysis based on the 28S rRNA gene supported neither the current taxonomic hypothesis that Steganoderma belongs to the subfamily Lepidophyllinae nor the earlier views that the Steganodermatinae and the Lecithostaphylinae are synonymous. The topology of the phylogenetic tree shows that the Steganodermatinae and the Lecithostaphylinae are independent subfamilies. However, morphological differences between them are obscure. Until morphological evidence for the Steganodermatinae is found, we propose to distinguish the subfamily Lepidophyllinae sensu stricto with the genera Lepidophyllus and Urinatrema, and the subfamily Lecithostaphylinae sensu lato uniting all the other former lepidophyllines. Thus, for now, we propose to consider the Steganodermatinae as a conditional synonym for Lecithostaphylinae sensu lato.

The family Calliphoridae is a group of heterogenous calyptrate flies with a worldwide distribution including species of ecological, veterinary, medical, and forensic importance. Notorious for their parasitic habits, the larvae of many blowflies are characterised – like some other dipteran larvae – by their ability to develop in animal flesh. When parasitism affects a living host, it is termed “myiasis”. This has led the Calliphoridae to be considered as a pivotal family in its relationship with a man. Nevertheless, even after more than 50 years of research, the phylogenetic relationships among calliphorid subfamilies together with the evolutionary origin of myiasis remain unclear. In order to elucidate these problems, we constructed three phylogenetic trees by using nucleotide sequence data from cytochrome oxidase subunit one (COI), representing a mitochondrial conservative gene, and nuclear 28S subunit of ribosomal RNA gene (28S rRNA) in order to interpret the evolutionary profile of myiasis in the family Calliphoridae. The sequenced data represented species associated with ectoparasitic life-styles, either saprophagy or facultative and obligate parasitism. A total number of 50 accessions were collected for 28S rRNA, 56 for COI, and 38 for combined sequences phylogeny. Molecular Evolutionary Genetics Analysis (MEGA) software was used to align 2197 nucleotide positions of 28S rRNA and 1500 nucleotide positions of COI with a gap opening penalties and gap extension penalties equalling 20 and 0.1 respectively. The results reveal the non-monophyly of the family Calliphoridae despite the stable monophyletic status of the Chrysomyinae, Luciliinae, and Auchmeromyiinae. Also, our findings recommend ranking the Toxotarsinae as a separate family. Furthermore, comparative analysis of the phylogenetic trees shows that the habit of obligatory myiasis originated independently more than five times. This strengthens our hypothesis that the origin of eating fresh meat is a case of convergent evolution that has taken place after speciation events millions of years ago. Finally, estimating the divergence dates between lineages from molecular sequences provides a better chance of understanding their evolutionary biology.

Telestula humilis (Thomson, 1927) is a rare deep-sea stoloniferan octocoral distributed in the eastern Atlantic. Here we compared seven putative colonies of this species collected off Spain with the lectotype from the Oceanographic Museum of Monaco and found them to be identical morphologically. Phylogenetic analyses on both full mitogenomes and a concatenated alignment containing two mtDNA genes (mtMutS and Cox1) and nuclear 28S rRNA gene recovered Telestula humilis sister to Incrustatus and Inconstantia rather than to other species of Telestula. This therefore supports its taxonomic reassignment to Pseudotelestula gen. nov. as Pseudotelestula humilis comb. nov. The taxonomic reassignment is also supported by subtle differences observed between the morphology of the colony and the sclerome of Pseudotelestula humilis comb. nov. and the two sister genera. The occurrence of an intrusion tissue with sclerites in the basal part of the gastric cavity of the adult polyps is shared among Telestula and Pseudotelestula gen. nov. However, Pseudotelestula gen. nov. has sclerites arranged in a collaret and points below the tentacles, the sclerites of the calyx wall and the stolon are plump warty spindles, and the intrusion tissue has long sticks and spindles with cone-like spines.

Trematodes of the genus Himasthla are usual parasites of coastal birds in nearshore ecosystems of northern European seas and the Atlantic coast of North America. Their first intermediate hosts are marine and brackish-water gastropods, while second intermediate hosts are various invertebrates. We analysed sequences of partial 28S rRNA and nad1 genes and the morphology of intramolluscan stages, particularly cercariae of Himasthla spp. parasitizing intertidal molluscs Littorina spp. in the White Sea, the Barents Sea and coasts of North Norway and Iceland. We showed that only three Himasthla spp. are associated with periwinkles in these regions. Intramolluscan stages of H. elongata were found in Littorina littorea, of H. littorinae, in both L. saxatilis and L. obtusata, and of Cercaria littorinae obtusatae, predominantly, in L. obtusata. Other Himasthla spp. previously reported from Littorina spp. in North Atlantic are either synonymous with one of these species or described erroneously. Based on a comparison of newly generated 28S rDNA sequences with GenBank data, rediae and cercariae of C. littorinae obtusatae were identified as belonging to H. leptosoma. Some previously unknown morphological features of young and mature rediae and cercariae of the three Himasthla spp. are described. We provide a key to the rediae and highlight characters important for identification of cercariae. Genetic diversity within the studied species was only partially determined by their specificity to the molluscan host. The nad1 network constructed for H. leptosoma lacked geographical structure, which is explained by a high gene flow owing to highly vagile definitive hosts, shorebirds.

In order to differentiate among Valeriana fauriei Briq. and other Eurasian medicinal valerian (V. dioica L., V. hardwickii Wall., V. jatamansi Jones, and V. officinalis L.), we attempted to establish DNA markers. DNA sequences for the psbA-trnH intergenic spacer region of chloroplast DNA (psbA-trnH) and 18S ribosomal RNA, internal transcribed spacer 1 (ITS1), 5.8S ribosomal RNA, internal transcribed spacer 2 (ITS2), and 28S ribosomal RNA of nuclear DNA in V. fauriei and other Eurasian medicinal valerian were compared. Using partial sequences of psbA-trnH (nucleotide positions 1-75 from the 5' end of the intergenic spacer region), V. fauriei and other Eurasian medicinal valerian could be correctly identified to the species level. In addition, the partial sequences of psbA-trnH in V. fauriei contained five different haplotypes, and it was possible to distinguish the origins of valerian from Japan and Eurasia (China and Korea). On the other hand, individuals had heterogeneous sequences of ITS1 and ITS2, making it impossible to use direct sequencing and DNA markers of ITS1 and ITS2 to distinguish species and origins of V. fauriei and other Eurasian medicinal valerian.

Till date 34 species of Pallisentis have been validated with almost negligible studies on its molecular phylogeny. The discovered species of the genus Pallisentis are confined only to the Asian continent. Many different species of Acanthocephala infecting commercially important freshwater as well as the marine fishes have been recorded from India mainly on the basis of morphology. The original record of morphological characteristics of Pallisentis nagpurensis (Bhalerao 1931) Baylis, 1933 can be traced back to the Bhalerao, 1931 from Channa striata (Ophiocephalus striatus) from the state of Uttar Pradesh, India without the separate documentation of male and female characteristics. Same species has also been reported to infect Channa striata from different parts of India as well as from the Indonesia and Ceylon (Baylis, 1933 and Fernando and Furtado, 1963). The present study compiles the morphological characteristics of male and female P. nagpurensis along with the comparison with similar species from same geographical region. This study also reports the first record of 18S, 28S rRNA and ITS1-5.8S-ITS2 gene sequences of P. nagpurensis generated for analyzing the phylogeny within this taxonomic group. P. nagpurensis together with the other species of Pallisentis reported from India distinctly formed a sub-clade separating it from the other members of same genus.

A population of Xiphinemella esseri, recently collected under the canopy of associated live oak trees in north Florida, was studied and described with an integrative approach, including the first molecular study of the genus. This Florida population is characterized by its 2.30 to 3.32 mm long body, labial disc well developed, lip region offset by constriction, and 16.5 to 17.5 μm broad, odontostyle 46 to 49 μm long with minute aperture, neck 288 to 296 μm long, pharyngeal expansion occupying 28 to 30% of total neck length, uterus a tripartite tube-like structure, pars refringens vaginae absent, vulva transverse (V = 45.4-49.7%), tail short and rounded (18-28 μm, c = 94-158, c′ = 0.6-0.9), spicules 41 to 45 μm long, and 8 to 10 irregularly spaced ventromedian supplements bearing hiatus. The phylogenetic analysis inferred from the D2-D3 expansion segments of 28S rRNA gene and 18S rRNA gene sequences showed that X. esseri clustered with other dorylaims from the family Leptonchidae. A brief discussion about the distribution and biological considerations of X. esseri is also provided.

Sampling for needle nematodes was carried out in a grapevine area in Thessaloniki, North Greece and two nematode species of Longidorus (L. pauli and L. pisi) were collected. Nematodes were extracted from 500 cm3 of soil by modified sieving and decanting method, processed to glycerol and mounted on permanent slides, and subsequently identified morphologically and molecularly. Nematode DNA was extracted from single individuals and PCR assays were conducted to amplify D2-D3 expansion segments of 28S rRNA, ITS1 rRNA, and partial mitochondrial coxI regions. Morphology and morphometry data obtained from these populations were consistent with L. pauli and L. pisi identifications. To our knowledge, this is the first report of L. pauli for Greece, and the second world report after the original description from Idleb, Syria, extending the geographical distribution of this species in the Mediterranean Basin., Spanish authors acknowledge grant 201740E042, ‘Análisis de diversidad molecular, barcoding, y relaciones filogenéticas de nematodos fitoparásitos en cultivos mediterráneos’ from Spanish National Research Council (CSIC).

Cactodera torreyanae Cid del Prado Vera & Subbotin, 2014 cysts were discovered during a Pale Potato Cyst Nematode (PCN) survey conducted by Minnesota Department of Agriculture as part of the Animal and Plant Health Inspection Service (APHIS) efforts to survey states for the presence of PCN. The soil samples were collected from a potato field, located in Karlstad, Kittson County, Minnesota, USA. Two out of 175 vials submitted for identification to the Mycology and Nematology Genetic Diversity and Biology Laboratory (MNGDBL) contained few cysts and juveniles of C. torreyanae. Cysts were dark brown in color, lemon-shaped to elongated with distinct vulval cone. Vulva with denticles present around fenestra, cyst length to width ratio between 1.6 and 2.3 and anus distinct. The juveniles had rounded stylet knobs, some sloping slightly posteriorly. The molecular analysis included sequence and phylogenetic analysis of ITS rRNA, D2-D3 expansion segments of 28S rRNA and COI of mtDNA genes. The nematode species was identified by both morphological and molecular means as Cactodera torreyanae. To the best of our knowledge this represents the first report of Cactodera torreyanae from the United States and first report of this cyst nematode species from potato fields. Definite host plant for this nematode remains unknown.

During the winter and spring of 2021, the root-knot nematodes were extracted from samples of galled roots of potted American pitcher plants (Sarracenia sp.). Samples were collected from a botanical garden nursery in Los Angeles County, California. The root-knot nematode was identified by molecular methods as Meloidogyne haplanaria. In the USA, M. haplanaria was initially found in Texas, and subsequently reported from Arkansas and Florida. Molecular characterization of the Californian M. haplanaria isolate was done using the analysis of the D2-D3 of 28S rRNA, ITS rRNA, mitochondrial l-rRNA, COI, and nad5 gene sequences. Some rRNA gene clusters of M. haplanaria were similar with those of M. arenaria. Possible hybridization events within mitotic parthenogenetic root-knot nematodes are discussed. This study confirmed that reliable diagnostics of M. haplanaria should be based on mtDNA sequence analysis. This is a first report of M. haplanaria from Sarracenia sp. and California. Consequently, this nematode was considered to be eradicated from this botanical garden nursery and the State of California.

Background Species of Sarcocystis are parasitic protozoa in poikilothermic and homeothermic animals. Out of the 26 valid species in birds as intermediate hosts, none has been reported in those of the order Musophagiformes, such as the great blue turaco Corythaeola cristata (Vieillot, 1816), which is a bird endemic to Central and Western Africa. The examination of great blue turacos imported from the Central Africa Republic to Czech Republic allowed the morphological and molecular characterization of a new species of Sarcocystis. Methods Four turacos imported from the Central Africa Republic to a private breeder (Czech Republic) underwent parasitological examination for the presence of sarcocysts through wet mounts of breast, heart and leg muscles. Found parasites were molecularly and histologically studied by four loci (18S rRNA, 28S rRNA, ITS1 and cox1) and haematoxylin and eosin staining, respectively. Results Three out of four examined birds harboured numerous sarcocysts in the breast and leg muscles. No macroscopic lesions where observed. Sarcocysts were microscopic, elongate and ribbon-shaped with a wall characterised by the presence of finger-shaped villar protrusions and filled with numerous elongate, banana-shaped bradyzoites, 11.87–14.84 × 2.05–2.92 µm in size. The new species was most closely related to Sarcocystisalbifronsi, Sarcocystisanasi, Sarcocystisatraii, Sarcocystischloropusae, Sarcocystisrileyi, Sarcocystiswenzeli and Sarcocystis sp. isolate from chicken in the four loci. Conclusions To our knowledge, this is the first species of Sarcocystis found in a musophagiform bird worldwide. Genetically, S. cristata sp. nov. represents a distinct species. Phylogenetic analyses are useful for predicting potential definitive hosts of the new Sarcocystis species. graphical Abstract

Mealybugs (Hemiptera: Pseudococcidae) are small piercing–sucking insects that infesting a wide range of crops and vegetables and their species are similar, which makes it difficult for non-specialists to distinguish between them. Samples were collected of infested okra plants from five districts at Sharkia Governorate, Egypt. This study aimed to study the distribution of the cotton mealybug, Phenacoccus solenopsis Tinsley and its associated parasitoid as well as the identification of this species by molecular characterization due to the high degree of morphological similarity between different closely related species of mealybugs. It was found that the Parasitism has a big influence on the occurrence of this insect infestation and this species of mealybug was confirmed by amplified and sequenced DNA loci known to be informative for species identification by provides information of 28S ribosomal RNA gene sequence. Consequently, the success of IPM programs against mealybugs depends on the correct definition of this pest through genetic characterization to choose the best control strategies. On the other hand, the difficulty of correctly identifying these species leads to its difficulty in choosing appropriate methods of control, especially biological control where each pest has a specific species of its natural enemy, which controls it biologically.

The genus Boehmiella was initially described as a member of the family Trichostrongylidae. Subsequently, it was assigned to the subfamily Haemonchinae in the family Haemonchidae. We analyzed parasites of spiny tree-rats, Mesomys hispidus, collected in the Amazon rainforest, which were identified as B. wilsoni based on integrative taxonomy. Using morphology, morphometry, and scanning electron microscopy (SEM), we added new data to the original description of the species. We also inferred phylogenetic hypotheses for its relationships within the Trichostrongylina, based on partial nuclear 18S and 28S rRNA genes, through Maximum Likelihood and Bayesian analyses. In conclusion, B. wilsoni does not belong to the family Haemonchidae, nor is it closely related to any other trichostrongylin family, and therefore, we propose the establishment of a new family, Boehmiellidae fam. nov., to which the genus Boehmiella is allocated., Graphical abstract Image 1, Highlights • Helminth found in arboreal rodent of the Amazon region, with description of new host, new geographical distribution, new taxonomic data, and proposal of a new family to allocate the genus according to molecular and morphological analyses.

Of the four species in the genus Cadlina present in the northwestern Pacific region, C. japonica has been the only species recorded from South Korea. For the purpose of investigating Cadlina in Korean waters, specimens were collected from the Korean East Sea (Sea of Japan) by scuba diving. The radula and morphology of these specimens were examined by stereoscopic and scanning electron microscopy. Based on morphology, three species were identified in Korean waters, including the new species, Cadlina koreanasp. nov., C. umiushi (first record in South Korea), and C. japonica. Cadlina koreanasp. nov. somewhat resembles C. umiushi but differs in both its morphology as well as the structure of its radula. The background color of Cadlina koreanasp. nov. is translucent white, tubercles on the dorsum are opaque white and the yellow marginal band is absent. The radular formula of Cadlina koreanasp. nov. is 57 × 23.1.23 with a rectangular rachidian tooth. In addition, mitochondrial cytochrome c subunit 1 (COI), 16S ribosomal RNA (16S rRNA), and nuclear 28S ribosomal RNA (28S rRNA) gene sequences were generated and used for analysis of Automatic Barcode Gap Discovery (ABGD) and reconstruction of the phylogenetic tree. Morphological distinction and genetic analyses confirm that three Cadlina species are present in Korean waters of which Cadlina koreana is a new species.

The present study was to investigate the copepodid species infecting rosy goatfish Parupeneus rubescens, one of the most economically important fishes in the Arabian Gulf. A copepodid species identified from the examined fish specimens belongs to the Taeniacanthidae family and is labeled as Taeniacanthus yamagutii Shiino, 1957, mainly depending on its morphological, morphometric, and ultrastructural characteristics, in particular the presence of maxilliped claw with a conspicuous digitiform process at the base, the terminal process of the second maxilla stout, and a setiform element at the tip of each exopod spine of legs 2–4. In order to ensure the accurate identification and exact taxonomic characterization of this species, the 28S rRNA gene sequence was analyzed. The result revealed that the present copepodid species belong to the Taenicanthidae family and has a close relationship with Taeniacanthus yamagutii (gb∣ KR048852.1) in the same taxon. The present study demonstrated that the rosy goatfish is a host for Taeniacanthus species, which will be helpful to prevent this parasitic infection.

Choricotyle australiensis Roubal, Armitage & Rohde, 1983, a diclidophorid monogenean species, is redescribed and genetically characterised using the partial nuclear 28S ribosomal RNA gene (28S rRNA) and a fragment of mitochondrial cytochrome c oxidase subunit 1 (cox1) gene sequences for specimens collected from Chrysophrys auratus (Forster) off Australia and New Zealand. Previous studies have either provided morphological or genetic results, whereas this study combines morphological and advanced molecular methods. A total of 70 Ch. auratus were examined with 22 individuals of C. australiensis recovered from the gills (overall prevalence of 23%). This study has provided the first evidence for the exploration of mitochondrial cox1 region for C. australiensis. Comparison of the newly generated sequences with other available data supported the distinction of C. australiensis among diclidophorid Furhmann, 1928 species thus confirming its taxonomic status.

Background European species of the large genus Phyllodistomum Braun, 1899 had historically been erected based solely on morphological characters. Unfortunately, many of them are still poorly known and inadequately described. Molecular approaches are critical to delineate species which were impossible to differentiate based on morphology alone. Methods New samples of adult Phyllodistomum spp. were collected from the urinary bladder and/or ureters of European freshwater fishes and fixed to conduct a light and scanning electron microscopy study, and to obtain sequences of nuclear (ITS2 spacer and 28S rRNA gene), to be analysed in the context of a molecular phylogeny. Results Based on new findings, a new species of Phyllodistomum from the urinary bladder of the European perch, Perca fluviatilis, in Volga River basin, Russia, is described. Additionally, new data on the morphology and tegumental surface topography of P. macrocotyle (Lühe, 1909) Odhner, 1911 from ureters of the common rudd, Scardinius erythrophthalmus, is presented. The host range of P. folium, confirmed by DNA analysis, is extended to other cyprinid fish species. Conclusions The present study has again shown that species of the genus Phyllodistomum are in dire need of revision based on both molecular analysis and detailed morphological redescriptions of the forms attributed to the genus. Morphologically, P. kupermani n. sp. most closely resembles P. pseudofolium, a highly host-specific parasite of Gymnocephalus cernuus (L.), but molecular phylogenetic analyses based on ITS2 and 28S rDNA sequences showed that these species are distantly related. Phyllodistomum kupermani n. sp. was found to be phylogenetically most closely related to the type-species of Phyllodistomum, P. folium. Phylogenetic analyses revealed that Phyllodistomum kupermani n. sp. and P. folium formed a clade with other freshwater species for which cystocercous cercariae develop in bivalves of the family Sphaeriidae. The micromorphology and tegumental surface topography of P. macrocotyle revealed in the present study provide a valuable taxonomic criterion for congeneric species differentiation.

The chromosomes of the Indian muntjac (Muntiacus muntjak, 2n = 6 in females and 2n = 7 in males) are described using the methods of G-, C-, CDAG-, and AgNOR-staining, and in situ localization of 18S, 5.8S, 28S rRNA genes.

High‐throughput sequencing has become an accurate method for the identification of species present in soil, water, faeces, gut or stomach contents. However, information at the species level is limited due to the choice of short barcodes and based on the idea that DNA is too degraded to allow longer sequences to be amplified. We have therefore developed a long DNA metabarcoding method based on the sequencing of short reads followed by de novo assembly, which can precisely identify the taxonomic groups of organisms associated with complex diets, such as omnivorous individuals. The procedure includes 11 different primer pairs targeting the COI gene, the large subunit of the ribulose‐1,5‐bisphosphate carboxylase gene, the maturase K gene, the 28S rRNA and the trnL‐trnF chloroplastic region. We validated this approach using 32 faeces samples from an omnivorous reptile, the European pond turtle (Emys orbicularis, L. 1758). This metabarcoding approach was assessed using controlled experiments including mock communities and faecal samples from captive feeding trials. The method allowed us to accurately identify prey DNA present in the diet of the European pond turtles to the species level in most of the cases (82.4%), based on the amplicon lengths of multiple markers (168–1,379 bp, average 546 bp), and produced by de novo assembly. The proposed approach can be adapted to analyse various diets, in numerous conservation and ecological applications. It is consequently appropriate for detecting fine dietary variations among individuals, populations and species as well as for the identification of rare food items.

A new species of Cryptops, C. (Cryptops) legagussp. nov., occurs in caves in the Koanaka and Gcwihaba Hills in northwestern Botswana. Bayesian molecular phylogenetics using 18S rRNA, 28S rRNA, 16S rRNA and cytochrome c oxidase subunit I corroborates a morphological assignment to the subgenus Cryptops and closest affinities to southern temperate species in South Africa, Australia and New Zealand. The new species is not conspicuously modified as a troglomorph.