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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.

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.

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.

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.

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.

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.

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.

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.

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.

Seven trichodorid species including a new one (Trichodorus hangzhouensissp. nov., T. nanjingensis, T. pakistanensis, T. cedarus, Paratrichodorus porosus, Nanidorus renifer and N. minor) were recovered from the rhizosphere of different hosts in 13 provinces of China. Each of the recovered species was characterized based on morphology and molecular data using rRNA gene sequences. Trichodorus hangzhouensissp. nov. is characterized by its males having medium-sized onchiostyle (46–49 µm) and three ventromedian cervical papillae (CP) anterior to the secretory-excretory (S-E) pore, CP1 located opposite the anterior part of isthmus, S-E pore opposite the isthmus or anterior end of pharyngeal bulb, spicules slightly ventrally curved, relatively small, 33.2 (32.0–34.5) µm long, wider slightly marked capitulum, lamina partially striated without bristles at striation; and females having rounded triangular sclerotized vaginal pieces with tips directed towards vulva, 1.5–2.0 µm sized, at about 1 µm apart, vulva pore-like in ventral view. Phylogenetic analysis based on D2-D3 28S rRNA gene sequences differentiated the new species among Trichodorus species from Europe, Asia and USA which formed a large clade. A review of the distribution of Trichodorus, Nanidorus and Paratrichodorus species revealed that T. cedarus, T. nanjingensis, T. pakistanensis and P. porosus are the most widespread species recorded from different provinces of China. This is the first extensive study of trichodorid species occurring in China.

Drechslera leaf spot (DLS) caused by Pyrenophora (Drechslera) species is one of the most serious diseases affecting Italian ryegrass (Lolium multiflorum) in China. Between 2015 and 2018, this disease was observed in three Italian ryegrass fields in the province of Sichuan, China. Average leaf disease incidence was approximately 1 to 12% but could range up to 100%. Symptoms appeared as brown or tan spots surrounded by a yellow halo, or brown to dark brown net blotch; subsequently, spots increased in number and size, and they later covered a large area of leaf, eventually causing leaf death. In this study, 86 strains of Pyrenophora fungi were isolated from leaf lesions of Italian ryegrass. Coupled with phylogenetic analysis of the internal transcribed spacer region, partial 28S ribosomal RNA gene, and glyceraldehyde-3-phosphate dehydrogenase gene, morphological characteristics showed that Pyrenophora dictyoides and P. nobleae are associated with Italian ryegrass in southwest China. Pathogenicity tests confirmed that both species can infect Italian ryegrass, causing leaf spot, whereas the virulence of the two species differed; P. nobleae showed lower pathogenicity to Italian ryegrass. This is the first time that these two Pyrenophora species were formally reported on Italian ryegrass based on both morphological and molecular characters. Overall, this study improves knowledge of the Pyrenophora species associated with Italian ryegrass and provides a foundation for control of this disease in the future.

In this short communication describing experiments carried out on the larvae of two insects, Unaspis euonymi Comstock (feeding on Euonymus japonicus Thunb.) and Dynaspidiotus britannicus Newstead (feeding on Laurus nobilis L.), we evaluate for the first time the efficiency of using DNA insecticides in the control of sap-sucking insects, including armored scale insects. Over a period of 10 days, high insect mortality was detected in both U. euonymi and D. britannicus, accompanied by a significant decrease in the concentration of target RNAs. At the same time, no visible changes were observed when the leaves of the host plants were subjected to treatment with DNA insecticides for one month. The results show the high efficiency of DNA insecticides used against hemipteran insect pests. It is noteworthy that the high efficiency of DNA insecticides and their low cost in comparison with RNA preparations provides a safe and extremely promising potential vehicle for the control of sap-sucking insects.

The genus Minibiotus is morphologically diverse, which may suggest its polyphyletic character. However, scarce genetic data and often also the lack of detailed morphological data currently do not allow for the verification of the relationships within this genus. Here, for the very first time, we provide an integrative description of a new Minibiotus species. Minibiotus ioculator sp. nov. from the Republic of South Africa differs from other congeners mainly by egg ornamentation with processes on the egg shell that resemble the hat of a royal jester. We also provide new taxonomic data on Minibiotus pentannulatus based on a population newly found in Tanzania, which constitutes the first African record of this species originally described from South America. Our study involved both classical taxonomic methods, which include morphological and morphometric analyses conducted with the use of light and scanning electron microscopy, and genetic data in the form of DNA sequences of four markers (three nuclear: 18S rRNA, 28S rRNA, ITS-2, and one mitochondrial: COI). The results of this study allow a discussion of species composition within Minibiotus and question the validity of the current diagnosis of the genus.

The Rosy Goatfish (Parupeneus rubescens) is considered to be one of the most common goatfish species in the Arabian Gulf used as seafood on Saudi Arabia’s fish markets. The purpose of this study was therefore to investigate one of the digenean species that infects this species of fish. It has been established that a new plagiorchiid species depends primarily on its morphological and morphometric characteristics within the Opecoelidae family and is classified as Paropecoelus saudiae referring to its host’s location. The present opecoelid species is characterized by having all the generic features within the genus Paropecoelus at morphological and morphometric levels. It could be differentiated from other species within this genus by the proportions of the different body parts, ratios of forebody/hindbody and oral/ventral suckers, location of oral sucker, number of marginal papillae on the ventral sucker, location and number of ovarian lobes, distribution and arrangement of uterine coils, extent of vitelline follicles, shape of seminal vesicle, and the terminal position of the genital pore. Due to the presence of some difficulties in the morphology of the closely related Paropecoelus species, the 18S and 28S rRNA gene-based molecular phylogenetic analysis was selected and analyzed to investigate the phylogenetic affinities and the taxonomic status of the recovered parasite species. The existing Paropecoelus species’ phylogenetic tree revealed a well-resolved distinct clade with other species belonging to The Opecoelidae family and deeply embedded within the Paropecoelus genus. The current study of the Paropecoelus species therefore reflects the third account of this genus as endoparasites of various species of the rosy goatfish. Keywords: Paropecoelus spp., Opecoelidae, Plagiorchiida, Arabian Gulf

It is still a challenge to reconstruct the deep phylogenetic relationships within spinicaudatans, and there are several different competing hypotheses regarding the interrelationships among Eocyzicidae, Cyzicidae s. s., Leptestheriidae, and Limnadiidae of the Suborder Spinicaudata. In order to explore the source of the inconsistencies, we focus on the sequence variation and the structure model of two rRNA genes based on extensive taxa sampling. The comparative sequence analysis revealed heterogeneity across species and the existence of conserved motifs in all spinicaudatan species. The level of intraspecific heterogeneity differed among species, which suggested that some species might have undergone a relaxed concerted evolution with respect to the 28S rRNA gene. The Bayesian analyses were performed on nuclear (28S rRNA, EF1α) and mitochondrial (16S rRNA, COI) genes. Further, we investigated compositional heterogeneity between lineages and assessed the potential for phylogenetic noise compared to signal in the combined data set. Reducing the non-phylogenetic signals and application of optimal rRNA model recovered a topology congruent with inference from the transcriptome data, whereby Limnadiidae was placed as a sister group to Leptestheriidae + Eocyzicidae with high support (topology I). Tests of alternative hypotheses provided implicit support for four competing topologies, and topology I was the best.

Phylogeographic studies contribute to addressing questions regarding the geographic patterns and evolutionary scenarios within and among species and also shed light on the taxonomic status of widely distributed species complexes. The pen shell Atrina pectinata species complex is a widely distributed and economically important bivalve in the northwestern Pacific. Previous phylogeographic studies have identified four genetically distinct cryptic species within the A. pectinata species complex along the coast of China, of which three cryptic species were distributed in the South China Sea. However, less attention has been given to their identification and delimitation. Herein, we report the phylogeography and taxonomic revision of the A. pectinata species complex in the South China Sea using DNA sequence data from mitochondrial cytochrome oxidase I (COI) and 16S ribosomal RNA (16S rRNA), and nuclear 28S ribosomal RNA (28S rRNA) gene markers. Using a combination of phylogenetic and DNA-based species delimitation analysis methods, we found strong support for four genetically valid species in the A. pectinata species complex and defined them as A. japonica, A. lischkeana, Atrina sp., and A. pectinata based on our results as well as on previous morphological and genetic studies. A demographic historical analysis showed that all three species in the South China Sea had populations that were relatively stable over time and then subjected to sudden expansion during the late Pleistocene (60,000–90,000 years ago). These results provide new insights into the systematics and evolution of the A. pectinata species complex and have important conservation and management implications.

In this work we analyzed the relationship among native arbuscular mycorrhizal fungi (AMF) and vine roots affected by esca, a serious grapevine trunk disease. The AMF symbiosis was analyzed on the roots of neighboring plants (symptomatic and asymptomatic to esca) in 14 sites of three vineyards in Marche region (central–eastern Italy). The AMF colonization intensity, identified by non-vital staining, showed higher value in all esca symptomatic plants (ranging from 24.6% to 61.3%) than neighboring asymptomatic plants (from 17.4% to 57.6%). The same trend of Glomeromycota phylum abundance was detected by analyzing fungal operational taxonomic units (OTUs) linked to the AMF community, obtained by amplicon high throughput analysis of ITS 1 region. Overall, the highest amount of OTUs was detected on roots from symptomatic plants (0.42%), compared to asymptomatic roots (0.29%). Specific primer pairs for native Rhizophagus irregularis and Funneliformis mosseae AMF species, were designed in 28S rRNA and large subunit (LSU) ribosomal RNA, respectively, and droplet digital PCR protocol for absolute quantification was set up. A higher number of DNA copies of both fungal species were detected more frequently in symptomatic than asymptomatic vines. Our study suggests a relationship between esca and native AMF in grapevine. These results underline the importance of native rhizosphere microbial communities for a better knowledge of grapevine esca disease.

Summary Two species of digenetic trematodes of the genus Pleurogenoides viz., P. cyanophlyctiShinad & Prasadan (2018a) and P. euphlyctiShinad & Prasadan (2018b) have been described from India. Information regarding the molecular data of various species of the genus Pleurogenoides Travassos, 1921 is virtually lacking. This study addresses the application of molecular markers to validate the phylogenetic position of P. cyanophlycti and P. euphlycti. In the present study, two species P. cyanophlycti and P. euphlycti were collected between January 2016 to October 2017, infecting the freshwater frogs inhabiting the Western Ghats, India. In the present study, the two species were identifi ed morphologically and by PCR amplification of the 28S ribosomal RNA gene. Phylogenetic tree results clearly demonstrate that both P. cyanophlycti and P. euphlycti belongs to the family Pleurogenidae Looss, 1899. Based on these results, we presented and discussed the phylogenetic relationships of P. cyanophlycti and P. euphlycti within family Pleurogenidae from India. Phylogenetic analyses showed that P. cyanophlycti and P. euphlycti cluster according to their vertebrate host and revealed an important congruence between the phylogenetic trees of Pleurogenoides and of their vertebrate hosts. P. cyanophlycti and P. euphlycti clearly constitute a separate, sister branch with other species of the genera, Pleurogenoides, Pleurogenes (=Candidotrema), Prosotocus and Brandesia. The present study firstly provides important information about the molecular study and phylogenetic analysis of P. cyanophlycti and P. euphlycti. This study will also serve as a baseline for Pleurogenoides species identifi cation for further studies.

A new genus Fasciodontia is described to classify Xylodon bugellensis, known mostly from Europe, and one new species, F. brasiliensis, collected in Atlantic rainforests of Brazil. A new combination F. bugellensis is proposed and the lectotype for this taxon was selected. The genus is characterized by fascicles of skeletal-like hyphae in aculeal trama and thick-walled basidiospores. The generic concept was supported by Bayesian and Maximum Likelihood molecular phylogenetic reconstructions based on the ITS region and D1/D2 domains of 28S rRNA gene. The morphological distinctions between Lyomyces pruni and F. bugellensis (earlier considered as conspecific) and their phylogenetic independence are described and illustrated. Additionally, Deviodontia pilaecystidiata is put into the genus Kneiffiella; Hyphodontia mongolica is referred to synonyms of K. pilaecystidiata. The phylogenetic position of Hastodontia halonata, Kneiffiella alienata, and Lyomyces fimbriatus are discussed for the first time, and the new combination Lyomyces fimbriatus is introduced.

A new sacoglossan species of the genus Elysia Risso, 1818 from southern Italy is described based on its external morphology, color pattern, radular teeth, and reproductive system. Specimens of this new species were previously attributed to Elysia viridis (Montagu, 1804) because of morphological similarity, but maximum-likelihood and Bayesian analyses based on four genes (mitochondrial cytochrome c oxidase subunit I, 16S rRNA, nuclear 28S rRNA, Histone 3), including 28 additional species of Elysia from the Atlantic, showed that they are distinct. In addition, to determine the identity of these specimens, different species delimitation analyses [Automatic Barcode Gap Discovery (ABGD), General Mixed Yule Coalescent (GMYC), Bayesian Poisson Tree Processes, (bPTP)] were carried out. The molecular results support the validity of this new species of Elysia from the Mediterranean Sea, named Elysia rubeni sp. nov. This new species is characterized by a bright light greenish surface color due to the abundance of kleptoplasts. It can be distinguished by its bright blue specks and minute sparkling yellow dots scattered along the body, as well as its smooth and greyish rhinophores.

Hemicycliophora paraconida n. sp. from the Tilekenar forest in northern Iran, is described and illustrated. The new species is characterized by having female body length of 907–1197 µm, 286–341 body annuli and a 96–115 µm long stylet. Lateral fields with three longitudinal lines forming two rows of blocks with breaks and anastomosis of transverse striae; each annulus in lateral field with four subcuticular ovate markings. Tail elongated uniformly conoid, with distinct annuli and narrowly rounded to acute terminus. Male possess robust and semi-circular spicules and a well-developed penial tube. Based on molecular and morphological compari-sons, the new species comes close to seven species of the genus namely: H. conida, H. iwia, H. ovata, H. hellenica, H. poranga, H. ornamenta and H. halophila, but it differs from them by the body size, R related indices, stylet length, lateral field structure, vulva position and tail shape. Molecular analyses based on sequences of D2-D3 expansion segments of 28S rRNA gene supported its morphological status as a new species and revealed that the new species is closest to the H. conida clade in the dendrogram inferred using 28S D2-D3 gene.

Phoretic nematodes associated with two mass-occurring populations of the millipede Parafontaria laminata were examined, focusing on Pristionchus spp. The nematodes that propagated on dissected millipedes were genotyped using the D2-D3 expansion segments of the 28S ribosomal RNA gene. Four Pristionchus spp. were detected: P. degawai, P. laevicollis, P. fukushimae, and P. entomophagus. Of the four, P. degawai dominated and it was isolated from more than 90% of the millipedes examined. The haplotypes of partial sequences of mitochondrial cytochrome oxidase subunit I examined for Pristionchus spp. and P. degawai showed high haplotype diversity.

Background Triatomines are natural vectors of Chagas disease and are mainly prevalent in the Americas. In China, previous data from decades ago showed that there were two species of triatomine bugs, Triatoma rubrofasciata and T. sinica. However, the distribution, genetic characteristics and public health implications of triatomines in China are still relatively unknown. In order to gain knowledge on the distribution, genetic characteristics and public health implications of the triatomines in Guangxi, China, an entomological-epidemiological study and genetic research was conducted. Methods Different methods were used to elucidate the distribution of triatomines in Guangxi including consultations with county-level Center for Disease Prevention and Control staff and village doctors, the distribution of educational material on triatomines though the internet and social media apps such as Wechat and QQ, and conducting manual inspections and light trapping to collect triatomines. The morphological characteristics of the collected triatomines were identified under light microscopy. The mitochondrial 16S rRNA, cytochrome b (cytb) genes and nuclear 28S rRNA gene were amplified, sequenced and used in phylogenetic analyses. Results A total of 305 triatomines were captured from 54 different sites in 13 cities in Guangxi. All collected bugs were identified as T. rubrofasciata based on morphology. Most triatomine collection sites were around or inside houses. Four triatomines bite cases were observed during the investigation indicating that triatomine bites are common, the bites can cause serious anaphylaxis and skin papules and urticaria, suggesting a systemic skin response. The 16S rRNA, 28S rRNA and cytb sequence analyses of T. rubrofasciata from Guangxi and other countries showed that T. rubrofasciata sequences from different regions exhibit a high similarity, with no geographical differences. The phylogenetic tree based on the 16S rRNA and cytb genes showed that T. rubrofasciata sequences from different regions and continents were in the same cluster, indicating no differentiation among different geographical populations. Conclusions Our study showed that T. rubrofasciata is widely distributed in Guangxi and that people are commonly bitten by this insect in some regions. This highlights the need to enhance surveillance for and control of T. rubrofasciata and to strengthen the monitoring of imported Trypanosoma cruzi in China. The 16S rRNA, 28S rRNA and cytb sequence analyses of T. rubrofasciata from different regions and continents suggested that T. rubrofasciata populations exhibit high similarity, and the clustering in the phylogenetic analyses indicates that T. rubrofasciata has a close ancestor originating in the Americas.

Gigantorhynchus echinodiscus (Diesing, 1851) is a parasite of anteaters in South America. Although described by Diesing in 1851, there is still a lack of taxonomic and phylogenetic information regarding this species. In the present study, we redescribe G. echinodiscus collected from a giant anteater, Myrmecophaga tridactyla Linnaeus, 1758, from the Brazilian Cerrado (Savannah) in the State of São Paulo by light and scanning electron microscopy. In addition, phylogenies were inferred from partial DNA gene sequence of the nuclear large subunit ribosomal RNA gene (28S rRNA). We provide for the first time details of the proboscis with a crown having 18 large hooks and numerous small hooks, a lateral papilla at the base of the proboscis, a ringed pseudo-segmented body, large testes, cemented glands in pairs, and a non-segmented region in the posterior end of the body, which contributed to the diagnosis of the species. Molecular phylogenetic analyses recovered G. echinodiscus forming a well-supported monophyletic group with Mediorhynchus sp., which was congruent with morphological studies that allocate both genera within the family Gigantorhynchidae. In conclusion, the present work adds new morphological and molecular information, emphasizing the importance of adopting integrative taxonomic approaches in studies of Acanthocephala., Graphical abstract Image 1, Highlights • Redescription of Gigantorhynchus echinodischus from Brazilian giant anteater. • First molecular data of the genus Gigantorhynchus with 28S rRNA partial gene. • Phylogenetic relationships of Gigantorhynchidae are assessed.

Foliar nematodes represent a minor feeding group within the genus Aphelenchoides Fischer, 1894. The facultative plant parasitic species A. blastophthorus can cause crinkling of leaves, reduced vigor, and stunting of agricultural and ornamental plants. Here we report the first finding of A. blastophthorus in leaves, crowns, and roots of strawberry plants collected in Switzerland in 2018. Species identification was confirmed by morphological and morphometric characterization supported by molecular barcoding of 18S ribosomal RNA (18S), 28S ribosomal RNA (28S), and cytochrome c oxidase I (COI) gene fragment analyses. Phylogenetic analysis of 18S indicated that A. blastophthorus was grouped within close distance to A. fragariae, a well-known foliar nematode affecting strawberry plants. Furthermore, the newly generated molecular barcodes of the partial 28S and COI of A. blastophthorus will support species identification in the future.

The knowledge of the diversity and distribution of tardigrades on Madagascar is rather poor. To date, only 13 tardigrade taxa have been reported from this region (including one Milnesium species). We examined 46 specimens belonging to two new-to-science species of the genus Milnesium described herein using an integrative approach, including classical morphology and molecular marker (COI, ITS-2 and 28S rRNA) analysis. The species were found in two moss and lichen samples collected in the Ivohibory forest in Fianarantsoa Province. Milnesium matheusisp. nov., with claw configuration [3-3]–[3-3] and rather wide buccal tube, morphologically is most similar to: Mil. beatae Roszkowska, Ostrowska & Kaczmarek, 2015, Mil. bohleberi Bartels, Nelson, Kaczmarek & Michalczyk, 2014, Mil. eurystomum Maucci, 1991, Mil. shilohae Meyer, 2015 and Mil. tumanovi Pilato, Sabella & Lisi, 2016; however, it differs from these by morphometric characteristics. Milnesium wrightaesp. nov., by the presence of four points on secondary branches of claws IV, is most similar to Mil. quadrifidum Nederström, 1919. However, Mil. wrightaesp. nov. differs from Mil. quadrifidum by claw configuration ([4-4]–[4-4] in Mil. quadrifidum vs. [3-3]–[4-4] in Mil. wrightaesp. nov.), but also by the position of the fourth points on secondary branches of claws IV, which are located near the base of the claw in the new species and near the top of the claw in Mil. quadrifidum. Genotypic analysis showed that Mil. matheusisp. nov. is most similar to Milnesium sp. (28S rRNA), Mil. variefidum (COI) and Mil. t. tardigradum (ITS-2) while Mil. wrightaesp. nov. is most similar to Milnesium sp. (28S rRNA), Mil. variefidum (COI) and Mil. matheusi (ITS-2). Five Milnesium taxa are recorded from the African region, including the two new species from Madagascar reported in this study.

Needle nematodes have an economic importance by causing damage to a wide range of natural and cultivated plants not only by directly feeding on root cells, but also by transmitting plant nepoviruses. Two new Longidorus nematodes, Longidorus oakcrassus n. sp. and Longidorus oakgracilis n. sp., are described and illustrated from populations associated with the rhizosphere of Pyrenean oak (Quercus pyrenaica Wild.) in southern Spain. The taxonomic position of both new species within the genus was assigned using an integrative approach. Morphologically, L. oakcrassus n. sp. is characterized by a female with a large and robust body size (9.2–12.2 mm), lip region anteriorly flattened to slightly rounded and almost continuous or slightly offset by a depression with body contour, ca 25.5–32.0 μm wide, amphidial fovea with slightly asymmetrical lobes, stylet composed by an odontostyle moderately long (110.0–133.5 μm) and odontophore weakly developed, pharynx short ending in a terminal pharyngeal bulb with common arrangement of pharyngeal glands, tail short almost hemispherical shape. Longidorus oakgracilis n. sp. is characterized by having a moderately long and thin female body (5.4–7.9 mm in length), a bluntly-rounded lip region, set off from body contour by a slight depression, amphidial fovea funnel-shaped without lobe, odontostyle moderately long (94.0–106.0 μm), pharyngeal bulb with common arrangement of pharyngeal glands, short tail, bluntly hemispherical. The presence of males is common in both species. Integrative diagnosis was based on molecular data using D2-D3 expansion domains of the 28S rRNA, 18S rRNA, ITS1 rRNA and partial coxI gene sequences and morphology. Although different gene markers show variations in the phylogenetic relationships, phylogeny indicated that L. oakcrassus n. sp. is phylogenetically related with several species described from the Iberian Peninsula, including L. oakgracilis n. sp., which is clustered with L.cf. olegi, L. lusitanicus and L. silvestris., This research was financially supported by grant 201740E042, “Análisis de diversidad molecular, barcoding, y relaciones filogenéticas de nematodos fitoparásitos en cultivos mediterráneos” from Spanish National Research Council (CSIC). Authors thank G. León Ropero and J. Martín Barbarroja (IAS-CSIC) for the excellent technical assistance. The first author acknowledges the China Scholarship Council (CSC) for financial support. The second author is a recipient of Humboldt Research Fellowship for Postdoctoral Researchers at Helmholtz Centre for Environmental Research-UFZ, Leipzig, Germany. The fourth author acknowledges Spanish Ministry of Economy and Competitiveness for the “Ramon y Cajal” Fellowship RYC-2017-22228.

Camels were formerly considered resistant to most of the diseases commonly affecting livestock. Recently, camels were found to be susceptible to a large number of pathogenic agents. The aim of this work is to identify the causative agent of meat lesion encountered in one humped male camels carcasses at abattoir in Matrouh governorate, Egypt. Out of 50 carcass of slaughtered male camel, 12 meat lesions were macroscopically found. Blackening and necrotic area lesions were exposed to routine mycological examination. This revealed the presence of Cryptococcus in one lesion. Cryptococcus infection is an opportunistic infection that occurs primarily among patients who are immunosuppressed or getting access through accidental penetration of skin barriers. The isolated fungi was tested by PCR amplification of the ITS region and sequencing of 28S ribosomal RNA gene. The alignment of the fungal isolates 28S region sequences revealed 94% identity with Cryptococcus cerealis in the gene bank library. For the best of our knowledge, this is the first isolation of C. cerealis from camel meat lesion in Egypt.