16S-23S rDNA Research Articles

First record of a mermithid nematode (Nematoda:Mermithidae) parasitizing winged females of gall‐forming aphids (Hemiptera:Aphididae:Eriosomatinae)

AbstractJuvenile mermithid nematodes were found to parasitize winged females (sexuparae) ofEriosoma auratumandTetraneura radicicola. The morphological characteristics of mermithid nematodes are briefly described. The 18S rDNA and 28S rDNA obtained from one nematode were sequenced and used to construct a Bayesian phylogenetic tree, on which the host ranges of mermithid nematodes were represented. Our study indicated that mermithid parasitism of sexuparae led to fewer and smaller sexual female embryos. This is the first record of a mermithid in relation to eriosomatine aphids and the fourth record with respect to Aphididae.

A study on Kudoa septempunctata infection from sashimi and sushi of olive flounder Paralichthys olivaceus in Busan, South Korea

Kudoa septempunctata has been reported as a new parasite in aquacultured olive flounder Paralichthys olivaceus, and also as a causative agent of food poisoning in humans. This paper investigated the infection of K. septempunctata in 216 sashimi and 20 sushi made of olive flounders in Busan, Korea. Among 236 samples, K. septempunctata was detected in eleven sashimi with 6-7 polar capsules by the microscopy. Among eleven sashimi, five sashimi were positive in Polymerase Chain Reaction (PCR) assay with the targets of 18S rDNA and 28S rDNA. The genotype of all the five PCR results is identified as the genotype ST3 which is common in Korea. K. septempunctata was found in olive flounders sashimi from Samcheonpo and Wando outside of Jeju Island. These findings would contribute to establish the standard of K. septempunctata for preventing food-borne outbreaks in advance and providing further preventive management for the seafood safety.

Insect Intestine Host Plant Pathogenic Bacteria that Cause Soft Rot of Neuboxbaumia tetetzo in Zapotitlan Salinas Valley, Puebla, Mexico

Tetecho (Neobuxbaumia tetetzo, Coulter, Backeberg) is an endemic columnar cactus in Zapotitlán Salinas Valley, Puebla, Mexico. Tetecho has an important role in the semi-arid ecosystem. For at least 8 years, tetecho cactus has shown symptoms of soft rot and necrosis. Insects were associated with the diseased plants. The main goal of the research was to identify insects associated with diseased tetecho and verify if they carry the pathogen. Insects were collected from 80 tetecho plants that showed typical symptoms. The insects were identified using taxonomic keys; bacteria in the intestine of the insects were identified by direct sequencing of IGS 16S-23S rDNA, gyrB, and rpoB genes. The insects and their frequency were larvae of Lagocheirus lugubris Dillon (Coleoptera: Cerambycidae) in 100% of the samples; larvae and adults of Chalcolepidius sp. Eschscholtz (Coleoptera: Elateridae) in 20%; and adults of Glyphidops flavifrons (Bigot) (Diptera: Neriidae) in 70% of the samples. Pseudomonas Migula was isolated from the intestine of larvae and adults of L. lugubris and G. flavifrons. Carnobacterium Collins et al. and Curtobacterium Yamada & Komagata were isolated from larvae of L. lugubris; Pectobacterium carotovorum subsp. brasiliense Nabhan et al. was inside larvae and adults of L. lugrubris and adults of G. flavifrons.

Genetic diversity and phylogenetic relationships among citrus germplasm in the Western Caucasus assessed with SSR and organelle DNA markers

Characterization of genetic diversity in germplasm collections is fundamental for an efficient management and utilization. The citrus germplasm in the Western Caucasus represent one of the most northerly citrus collections. The collection contains numerous cultivars specifically selected for frost-tolerance, which are valuable as genetic resources for citrus breeding programs worldwide. Currently, the field citrus collection consists of more than 200 accessions, but their taxonomic assignment, genetic structure and diversity has not been clarified.In our study, 130 different citrus accessions including 24 species or interspecific hybrids maintained and bred at the Federal Research Centre the Subtropical Scientific Centre of the Russian Academy of Sciences (FRC SSC RAS) were genetically investigated using 13 biparental (ncSSR) and 13 uniparental (orgDNA) markers. After STRUCTURE analysis, the single accessions were allocated to eight genetic clusters and their relationships were elucidated. This phylogenetic relationship was also confirmed by principal coordinates analysis (PCoA) and by dissimilarity analysis using DARwin. We identified twenty accessions as misclassified and reassigned them to the correct species. For the remaining 110 accessions the taxonomical assignment could be confirmed. A high genetic diversity was observed on basis of the nuclear ncSSR markers, while the detection of 19 different haplotypes allowed the distinct differentiation of the accessions within our collection. Our study showed that the combination of ncSSR and orgDNA was an efficient tool to estimate the genetic diversity, genetic structure and phylogeny in the FRC SSC RAS citrus germplasm collection. The results obtained are valuable for future management of the collection and the usage of individual accessions as genetic resource for citrus cultivar and rootstock breeding, especially in colder regions.

Morphological and Molecular Characterization of Heterobothrium indicus n. sp. (Monogenea: Diclidophoridae) and Its Phylogenetic Status

Background: A new species Heterobothrium indicus n. sp. was isolated from the gills of Upeneus moluccensis (Bleeker, 1855) from the Western Coast of India in Arabian Sea region. The monogenean parasite differs from other congeners by morphological features like the presence of asymmetrical haptoral region, 4th pair of clamp smallest than other three pairs of clamps, genital atrium with 8 hooks, number of testes 29-37 and absence of isthmus.Methods: During the survey of marine fishes at Arabian Sea region, new species of monogenean parasite was isolated from the gills of marine fish Upeneus moluccensis. The parasites were morphologically characterized with the help of light and phase contrast microscopy. 18S rDNA, 28S rDNA, mt COI, ITS1+5.8S and ITS2+5.8S gene regions of parasites were amplified, sequenced and compared with other diclidophorid taxa using different bioinformatics tools.Result: Phylogenetic tree analyses (NJ, ME and MP methods) of 18S rDNA, 28S rDNA and mt COI gene regions are complementing the morphological studies and clearly suggested the placement of this new species under subfamily Choricotylinae, family Diclidophoridae.

Development of a high-resolution molecular marker for tracking Pseudo-nitzschia pungens genetic diversity through comparative analysis of mitochondrial genomes

The harmful algal bloom (HAB) species Pseudo-nitzschia pungens is widely distributed in almost all continents. Accumulating evidence suggests that P. pungens has high genetic diversity and many strains can produce the toxin domoic acid (DA) that harms animals and humans. Nevertheless, different P. pungens strains cannot be distinguished using morphological features or using common molecular markers including 18S rDNA, 28S rDNA, ITS, cox1, and rbcL. As such, high-resolution molecular markers need to be developed to resolve P. pungens genetic diversity, facilitating accurate tracking of toxic P. pungens strains. We hypothesized that molecular markers with high resolution and high specificity can be designed through identifying regions with high genomic variations in the mitochondrial genome. Here, we describe the development of a new molecular marker Pseudo-nitzschia pungens mitochondrial 1 (ppmt1) with high resolution and high specificity through comparative analysis of mitochondrial genomes of nine P. pungens strains isolated from coastal regions of China. In conclusion, we have developed ppmt1 as a high-resolution and high-specificity molecular marker for tracking strains and genetic diversity of the HAB species P. pungens.

Redescription and New Host Record of Heteraxinoides atlanticus (Monogenea: Heteraxinidae) from the Gills of Nemipterus japonicus (Bloch) and Its Systematics

Background: Heteraxinoides atlanticus Gayevskaya et Kovaleva (1979) is described from the locality of Arabian Sea, India. This monogenean species is first time reported from India, infesting the gills of Nemipterus japonicus (Bloch). The present study redescribes H. atlanticus and its phylogenetic status using morphometric and molecular tools. Methods: A total of 35 parasites were collected from the 234 specimens of Nemipterus japonicus at Mumbai, India. The temporary and permanent mounts were prepared for the morphometric analyses of H. atlanticus. For PCR, the genomic DNA was isolated from the parasites using primers for 18S rDNA, 28S rDNA and mtCOI gene. The obtained DNA sequences were subjected to different bioinformatics softwares (BLASTn, Clustal Omega and MEGA) for phlyogenetic analyses.Result: Phylogenetic analyses with the help of partial 18S and 28S rDNA sequences of H. atlanticus and other available sequences of heteraxinids provided the better understanding of relationship in the family Heteraxinidae. Another species, H. karachiensis Hadi and Bilqees (2014) showed similar morphological features such as number of clamps and structure of genital atrium to H. atlanticus except few minor differences, so it must be considered as synonym of H. atlanticus and placed into Heteraxinidae instead of Axinidae.

Molecular phylogeny and morphology of Cephaleuros (Trentepohliales, Chlorophyta) from southern China

ABSTRACT Cephaleuros is a subaerial green alga widely distributed in tropical and subtropical regions. In this study, we investigated the taxonomy of Cephaleuros in southern China. Eight morphospecies (C. virescens, C. karstenii, C. expansus, C. drouetii, C. diffusus, C. tumidae-setae, C. lagerheimii and C. parasiticus) were identified according to previous descriptions, among which C. karstenii was the most frequently recorded. Subsequently, molecular phylogeny of Cephaleuros was inferred from 18S rDNA, ITS rDNA, 28S rDNA and rbcL sequences. Cephaleuros was subdivided into nine clades based on ITS analysis, which were recovered in phylogenies of two concatenated datasets (18S+ITS+28S and 18S+ITS+28S+rbcL). Molecular analyses revealed that Cephaleuros was composed of two main lineages, with one lineage comprising mostly samples of C. karstenii and C. virescens and the other comprising other species. The molecular phylogenies were not congruent with conventional morphological classifications, although some clades were distinguished by one or more diagnostic features. While the morphological features used to distinguish Cephaleuros spp were not reliable, the rbcL gene proved to be a good marker for identification of Cephaleuros. The ITS2 secondary structure prediction model showed that Cephaleuros spp have one ring structure with four helices, with Helix III being the longest without a branch in clades 3, 5 and 8. The present study represents the most detailed taxonomic assessment of Cephaleuros available to date. Further taxonomic research incorporating samples from the type locality is necessary for the reassessment of C. virescens.

Biogeography of the cosmopolitan terrestrial diatom Hantzschia amphioxys sensu lato based on molecular and morphological data

Until now, the reported diversity of representatives from the genus Hantzschia inhabiting soils from different parts of Eurasia was limited to the few species H. amphioxys, H. elongata and H. vivax and some of their infraspecific taxa. We have studied the morphology, ultrastructure and phylogeny of 25 soil diatom strains, which according to published description would be assigned to “H. amphioxys sensu lato” using 18S rDNA, 28S rDNA and rbcL. We show that strains are made up of seven different species of Hantzschia, including five new for science. Five strains were identified as H. abundans. This species has a slight curvature of the raphe near its external proximal ends. Four of the examined strains were represented by different populations of H. amphioxys and their morphological characteristics fully correspond with its isolectotype and epitype. The main specific features of this species include 21–25 striae in 10 μm, 6–11 fibulae in 10 μm, 40–50 areolae in 10 μm and internal proximal raphe endings bent in opposite directions. H. attractiva sp. nov., H. belgica sp. nov., H. parva sp. nov., H. pseudomongolica sp. nov. and H. stepposa sp. nov. were described based on differences in the shape of the valves, significant differences in dimensions, a lower number of striae and areolae in 10 μm and the degree and direction of deflection of the internal central raphe endings. Based on the study of the morphological variability and phylogeny of soil Hantzschia-species from different geographical locations we conclude that while some species such as H. amphioxys are truly cosmopolitan in their distributions, some sympatric populations of pseudocryptic taxa exist in the Holarctic.

Genetic and morphological analyses uncover a new record and a cryptic species in Allonais (Clitellata: Naididae)

Allonais inaequalis (Stephenson, 1911) is a well-known “morpho-species” of the subfamily Naidinae with a wide distribution in the world. In this study we describe it, as a new record, from China, and we compare molecular data of A. inaequalis from two geographic populations in China and one in Peru, and we re-evaluate the phylogenetic status of Allonais. Sequence data of both mitochondrial (COI, 12S rDNA and 16S rDNA) and nuclear (18S rDNA and 28S rDNA) from 26 species of naidids (representing 14 of the 22 genera currently recognized) and 7 outgroup taxa were used. The combined data set were analyzed with Bayesian inference and Maximum Likelihood. The different phylogenetic analyses gave largely congruent trees, and they corroborate monophyly of Allonais. The results show that the genetic distances among the three geographic populations of A. inaequalis for COI are 0.0%-7.8% and for 28S vary between 0.0% and 0.8%. Allonais inaequalis falls into two groups, one from China, and one from Peru. We suggest that the Chinese morphotype more resembles A. inaequalis from India, where the species was first described, whereas the species in Peru is likely to be another species.

DNA barcoding approach to characterize microalgae isolated from freshwater systems in Ecuador

ABSTRACT The use of suitable DNA barcodes and the generation of databases with reference sequences have been considered a promissory approach for the identification of Chlorophyta and Cyanophyta microalgae. In this study, we carried out a molecular characterization and identification of strains isolated from freshwater systems in Ecuador using a dual barcode method. The target sequences for Chlorophyta were 18S rDNA and rbcL genes, and 16S rDNA and 16S–23S rDNA intergenic spacer (ITS) for Cyanophyta. We reported these DNA barcodes for 20 different Molecular Operational Taxonomic Units (MOTUs) for Chlorophyta and 10 for Cyanophyta. Our results show that the 18S V4 hypervariable region (300 bp) is sufficient for differentiating between isolates, but rbcL is a determinant for genus identification in Scenedesmaceae and Chlorellaceae strains. In Cyanophyta, both barcodes enabled the genus-level assignment of 9 out of 10 MOTUs. These results highlight the necessity of a second barcode additional to small ribosomal subunit sequences to improve molecular identification. Furthermore, the present study significantly contributes to the body of Ecuadorian barcode sequences of microalgae that are currently documented, making them available for future comparative diversity studies.

Detection and molecular characterization of phytoplasmas associated with stone fruit trees in northwest of Iran

During surveys conducted from 2015 to 2018 in stone fruit orchards of East Azerbaijan province in the northwest of Iran, several stone fruit trees showing phytoplasma typical symptoms such as witches'-broom, little leaf, leaf rolling, leaf scorching, dieback, decline, yellowing, reddening and rosette were sampled. PCR and nested PCR analyses using phytoplasma universal primers confirmed the phytoplasma infection in some of symptomatic almond, peach, plum and sweet cherry trees. Sequence analyses of some 485 bp and 1239 bp 16S–23S rDNA fragments amplified in nested PCR, confirmed that ʻCandidatus Phytoplasma asterisʼ, ʻCa. Phytoplasma aurantifoliaʼ and ʻCa. Phytoplasma phoeniciumʼ were associated with almond, ʻCa. Phytoplasma phoeniciumʼ was associated with peach, ʻCa. Phytoplasma asterisʼ and ʻCa. Phytoplasma trifoliiʼ were associated with plum and ʻCa. Phytoplasma asterisʼ was associated with sweet cherry trees. Virtual RFLP analyses indicated that stone fruit phytoplasmas in East Azerbaijan are belonging to 16SrI–B, 16SrII-A, 16SrIX-C, 16SrXII-A and 16SrVI-A subgroups. This research is the first report of infection by ʻCa. Phytoplasma asterisʼ in almond and infection by ʻCa. Phytoplasma trifolliʼ in plum around the world and the first report of ʻCa. Phytoplasma aurantifoliaʼ and ʻCa. Phytoplasma trifoliiʼ presence in the northwest of Iran.

Multiclonal spread of Klebsiella pneumoniae across hospitals in Khartoum, Sudan

ObjectivesMultidrug-resistant (MDR) Klebsiella pneumoniae is increasing worldwide with poorly characterised epidemiology in many parts of the world, particularly in Africa. This study aimed to investigate the molecular epidemiology of K. pneumoniae, to identify the diversity of sequence types (ST), and to detect carbapenem resistance genes in major regional hospitals in Khartoum, Sudan. MethodsKlebsiella pneumoniae isolates (n = 117) were cultured from four hospitals in Khartoum, from April 2015 to October 2016. The isolates were characterised by sequencing of 16S-23S rDNA internal transcribed spacer (ITS) region. Molecular epidemiology was determined by multilocus sequence typing (MLST), and analysed by maximum likelihood phylogeny (PhyML). Antimicrobial susceptibility was determined by disk diffusion. Isolates phenotypically resistant to carbapenem were screened for carbapenemase genes: blaNDM, blaOXA48, blaIMP, blaVIM and blaGES by PCR. ResultsITS sequencing confirmed the 117 isolates as K. pneumoniae. MLST revealed 52 different STs grouped in four distinct clusters by PhyML. All isolates were MDR, and carbapenemase-producing K. pneumoniae (CP-KP) isolates accounted for 44/117 (37.6%) mostly harbouring blaNDM (28/44) and blaOXA-48 (7/44), with several isolates harbouring multiple genes. ConclusionMDR and CP-KP K. pneumoniae is widespread in Khartoum hospitals, with a diverse population of 52 STs clustering in four major lineages. There is an urgent need for systematic epidemiological studies of drug-resistant infections across all healthcare institutions in Sudan to inform local infection prevention and control strategies.

Development of a Genus-Specific Brucella Real-Time PCR Assay Targeting the 16S-23S rDNA Internal Transcribed Spacer from Different Specimen Types.

The aim of this study was to develop a 16S-23S ribosomal deoxyribonucleic acid internal transcribed spacer (ITS) quantitative polymerase chain reaction (qPCR) assay for the early diagnosis and rapid screening of brucellosis. Blood, milk, and tissue samples were spiked with B. abortus biovar 1 (B01988-18 strain) to determine the analytical sensitivity and specificity of the assay. The 95% limit of detection of the ITS qPCR assay was highest in tissue, followed by blood, then milk, i.e., 0.48, 4.43, and 15.18 bacteria/PCR reaction, respectively. The diagnostic performance of the assay was compared to the Brucella cell surface protein (BCSP) 31 qPCR assay and bacterial culture. Out of 56 aborted foetal tissue samples from bovine, ovine, and caprine, 33% (19/56) were positive for Brucella spp. The sensitivity and specificity of the ITS qPCR assay was 87% and 95% respectively, compared to 92% and 89% for the BCSP31 qPCR assay and 47% and 55% for bacterial culture, respectively. The assay was efficient, sensitive, and specific, making it a valuable tool in the early detection of the Brucella pathogen.

The Development of a Genetic Linkage Map of Redcurrant (Ribes rubrum L.) by Means of SSR and SNP DNA Markers

This work was aimed at compiling the world’s first linkage map of redcurrant with the use of microsatellite and SNP DNA markers. As a result of mapping, eight linkage groups with an LOD level of 10 were constructed, which included 299 DNA markers, including 12 microsatellite and 287 SNP markers; the total length of all linkage groups was 741 cM; the average was 93 cM. In seven of the eight linkage groups obtained, at least one microsatellite DNA marker was mapped. Most of the microsatellite loci localized on the redcurrant map correspond to their position on the blackcurrant linkage groups (loci e3-B02, g1-A01, and g1-L12 are located on the same linkage group no. 5; loci g2-J08, g2-B20, e1-O21, and e1-O01 on the resulting map are each in a separate linkage group 2, 3, 4, and 6, respectively), with the exception of the loci mapped at blackcurrant on linkage group 1, which in our work were unmapped. The g2-J11 locus in our studies amplifies fragments in two ranges, which suggests its duplication in the genome of the studied samples. This locus was mapped in two linkage groups of redcurrant. Two new, not previously mapped and unpublished microsatellite loci were also localized on linkage map of redcurrant. In the future, it is planned to localize economically useful traits in the redcurrant genome.

Molecular and morphological description of Sarcocystis kutkienae sp. nov. from the common raven (Corvus corax).

Until now, two Sarcocystis species, S. cornixi and S. corvusi, were known to employ members of the family Corvidae as intermediate hosts. Between 2013 and 2019, having examined leg muscles of 23 common ravens in Lithuania, sarcocysts were detected in 18 birds (78.3%). Using light microscopy, transmission electron microscopy (TEM), and molecular analysis (three genetic loci, 18S rDNA, 28S rDNA, and ITS1), sarcocysts found in the common raven were described as a new species S. kutkienae. Under a light microscope, the observed sarcocysts were ribbon-shaped (1500-8147 × 53-79 μm) and had a wavy striated cyst wall that reached up to 1.5 μm. Lancet-shaped bradyzoites were 7.7 × 2.2 μm (6.1-9.0 × 1.2-3.0 μm) in size. Ultrastructurally, the sarcocyst wall was 1.5-1.8 μm in thickness and had conical-like protrusions with minute invaginations of a parasitophorous vacuolar membrane. The cyst wall was type 1e-like. Limited genetic variability was observed between the 18S rDNA and 28S rDNA sequences of S. kutkienae and other Sarcocystis spp. using birds as intermediate hosts. In contrast, S. kutkienae could be clearly identified by comparing sequences. At this locus, sequences of S. kutkienae shared the highest similarity (89.5-89.7%) with those of S. cornixi. Phylogenetic analysis showed thatS. kutkienae was most closely related to Sarcocystis spp. that employs birds as intermediate and definitive hosts. The issue relating to which species might serve as definitive hosts of S. kutkienae in Lithuania is addressed.

Chromosomal mapping of 5S and 18S-5.8S-25S rRNA genes in Saccharina japonica (Phaeophyceae) as visualized by dual-color fluorescence in situ hybridization

It has been reported that there was a linkage of 5S rRNA gene to 18S-5.8S-25S rRNA gene in a few of species in Ochrophyta. In regard to the usual two positions of linked 5S rDNA to the 3' end of 25S rDNA, two pairs of primers were designed for amplification to verify this linkage of two genes in a kelp cultivar of Saccharina japonica, one of species in Ochrophyta. This result supplemented the previous report that 5S rDNA was unlinked to 25S rDNA in this kelp. In order to simultaneously visualize this unlinkage of two genes, dual-color fluorescence in situ hybridization (FISH) technique was applied to the cytogenetics of S. japonica. Dual-color FISH images showed that two and four hybridization signals were present in the kelp gametophyte and sporophyte, respectively, metaphase nuclei hybridized simultaneously with the labeled probes of 18S rDNA and 5S rDNA. Both haploid and diploid karyotypes in decreasing length of chromosomes showed that 18S-5.8S-25S rDNA was localized at the interstitial region of Chromosome 23, whereas 5S rDNA resided at the sub-telomeric region of Chromosome 27. These karyotypes suggested that the kelp nuclear genome had only one locus of each rRNA gene, and their loci on different chromosomes indicated the physical unlinkage of 5S rDNA to 18S-5.8S-25S rDNA in this kelp. Therefore, dual-color FISH seems to be a powerful technique for the discrimination and pairing of chromosomes featured in both small size and nearly identical shape in S. japonica.

Morphological and molecular characterization of Sarcocystis wenzeli in chickens (Gallus gallus) in China

BackgroundThere has been considerable confusion concerning the number and classification of Sarcocystis spp. in chickens. Scarce nucleotide data of Sarcocystis spp. from chickens are provided in GenBank. The study aimed to investigate the morphological and molecular characteristics of Sarcocystis spp. found in chickens in China.MethodsTissues from 33 chickens were collected in 2019. Sarcocysts were observed using light (LM) and transmission electron microscopy (TEM). Individual sarcocysts from different chickens were selected for DNA extraction, and five loci, 18S rDNA, 28S rDNA, ITS1 region, the mitochondrial cox1 gene and the apicoplastic rpoB gene, were amplified from each sarcocyst, sequenced and analyzed.ResultsOnly S. wenzeli was found in 14 of 33 (42.4%) chickens. Under LM, the sarcocysts were microscopic and exhibited palisade-like villar protrusions measuring 1.5–2.8 μm. Ultrastructurally, the sarcocyst wall contained numerous stubby hill-like villar protrusions. The protrusions included scattered microtubules, which extended from the tips of the protrusions into the ground substance. The five loci were successfully sequenced and the sequences deposited in GenBank. At 18S rDNA, ITS1 and cox1, the most similar sequences in GenBank were those of Sarcocystis sp. obtained from the brains of chickens, i.e. 99.9–100%, 98.1–98.5% and 99.3% identity, respectively. The five loci (18S rDNA, 28S rDNA, ITS1, cox1 and rpoB) showed different levels of interspecific sequence similarity with other closely related species of Sarcocystis (e.g. 99.8%, 99.0–99.2%, 89.3–89.7%, 98.5%, and 97.5%, respectively, with S. anasi). Phylogenetic analysis based on four of the loci (18S rDNA, cox1, rpoB and ITS1) revealed that S. wenzeli formed an independent clade with Sarcocystis spp. that utilize geese or ducks as intermediate hosts and canines as the known or presumed definitive host.ConclusionsTo our knowledge, the sequences of 28S rDNA and rpoB reported here constitute the first records of genetic markers of Sarcocystis spp. in chickens. Based on molecular analysis, S. wenzeli might be responsible for the neurological disease in chickens, and ITS1 and rpoB are more suitable for discriminating it from closely related Sarcocystis spp. Phylogenetic analysis revealed that S. wenzeli presents a close relationship with Sarcocystis spp. in geese or ducks.

Genetic integrity is still maintained in natural populations of the indigenous wild apple species Malus sylvestris (Mill.) in Saxony as demonstrated with nuclear SSR and chloroplast DNA markers.

Malus sylvestris (Mill.) is the only indigenous wild apple species in Central Europe. Agriculture, forestry, and urbanization increasingly endanger Malus sylvestris natural habitats. In addition, the risks of cross‐hybridization associated with increase in the cultivation of the domesticated apple Malus × domestica (Borkh.) threaten the genetic integrity of M. sylvestris. The present study investigated the number of hybrids, genetic diversity, and genetic structure of 292 putative M. sylvestris that originate from five different natural M. sylvestris populations in Saxony, Germany. All samples were genetically analyzed using nine nuclear microsatellite markers (ncSSR) and four maternally inherited chloroplast markers (cpDNA) along with 56 apple cultivars commonly cultivated in Saxony. Eighty‐seven percent of the wild apple accessions were identified as pure M. sylvestris. The cpDNA analysis showed six private haplotypes for M. sylvestris, whereas three haplotypes were present in M. sylvestris and M. × domestica. The analysis of molecular variance (AMOVA) resulted in a moderate (ncSSR) and great (cpDNA) variation among pure M. sylvestris and M. × domestica individuals indicating a low gene flow between both species. The genetic diversity within the pure M. sylvestris populations was high with a weak genetic structure between the M. sylvestris populations indicating an unrestricted genetic exchange between these M. sylvestris populations. The clear distinguishing of M. sylvestris and M. ×domestica confirms our expectation of the existence of pure M. sylvestris accessions in this area and supports the argument for the implementation of preservation measures to protect the M. sylvestris populations in Saxony.

Mikrocytos mytilicoli n.sp. (Cercozoa, Mikrocytida, Mikrocytiidae) infecting the copepod Mytilicola intestinalis (Arthropoda, Cyclopoida, Mytilicolidae), a symbiont of Mytilus galloprovincialis in Galicia (NW Spain)

During a histopathological survey of Mytilus galloprovincialis in Galicia (NW Spain), microcells were observed infecting several organs of the symbiont copepod Mytilicola intestinalis. Positive results of PCR assay with specific primers for genus Mikrocytos and a clear signal of in situ hybridization with MACKINI-1 digoxigenin- labelled DNA probe (DIG-ISH) indicated a protozoan parasite of Mikrocytos genus. The ultrastructural study revealed intra and extracellular locations, polymorphic nuclei, intracellular round vesicles in the cytoplasm and absence of mitochondria. The present paper reports the characterization of the Mikrocytos sp. infecting M. intestinalis and proposes a novel species in the genus: Mikrocytos mytilicoli n. sp. A sequence of 18S-28S rDNA was obtained with 95.6% maximum identity (query cover 100%) with Mikrocytos mackini. Phylogenetic analysis showed that M. mytilicoli n. sp. and M. mackini share a common ancestor. However, comparison of the ITS1 rDNA region showed low similarity (75.8%) with M. mackini, which, combined with differences in ultrastructural details, host and geographic location, support the designation of a new species. This is the first description of a microcytid parasite of the genus Mikrocytos from a non-bivalve host.