Cox1 Sequences Research Articles

Canine piroplasmids: Molecular detection and laboratory characterization in dogs from Brasilia, Brazil, with the first molecular evidence of dog exposure to a novel opossum-associated Babesia sp.

Canine piroplasmid infections can be caused by Babesia spp., Theileria spp. and Rangelia vitalii. In Brazil, canine babesiosis caused by Babesia vogeli is endemic and reported throughout the country. On the other hand, Rangeliosis caused by R. vitalii has only been described so far in the South and Southeast regions. Despite that, studies analyzing the laboratory and molecular characterization of these hemoprotozoa are still scarce. To investigate the occurrence, the laboratory features, the molecular characterization, and the diversity of piroplasmids from Midwestern Brazil, a survey was performed using blood samples obtained from 276 domestic dogs from Brasília, Federal District, Midwestern Brazil. A broad-range quantitative PCR (qPCR) targeting the mitochondrial large subunit ribosomal DNA (LSU4) was used to detect piroplasmid DNA. The overall molecular occurrence of piroplasmids was 11.2% (31/276), with 9.7% (27/276) of the sequences identified as Babesia vogeli (98–100% identity to B. vogeli isolate from the USA). Based on a partial 18S rRNA sequence pairwise alignment (-250 bp), 1.4% (4/276) of the sequences showed only 76.8% identity with B. vogeli but 100% identity with opossum-associated Babesia sp. (MW290046–53). These findings suggest the exposure of dogs from Brazil to a recently described Babesia sp. isolated from white-eared opossum. None of the analyzed dogs was positive for Theileria spp. or R. vitalii. Subsequently, all positive sequences were submitted to three additional PCR assays based on the 18S rRNA, cox-1, and cytb genes, aiming at performing a haplotype network analysis. Haplotype network using cox-1 sequences showed the presence of six different haplotypes of B. vogeli; one of them was shared with isolates from Brazil, the USA, and India. When including animals co-infected with other vector-borne diseases, piroplasmid-positive dogs had 2.3 times higher chance of having thrombocytopenia than the negative ones. The molecular results demonstrated that the compared Babesia vogeli sequences showed a low variability as well as evidence of exposure to a putative novel opossum-associated Babesia sp. in dogs from Midwestern Brazil.

Limnotrachelobdella hypophthalmichthysa n. sp. (Hirudinida: Piscicolidae) on Gills of Bighead Carp Hypophthalmichthys nobilis in China.

We describe the characterization of a novel fish leech species found on the gills of bighead carp (Hypophthalmichthys nobilis) from lakes and reservoirs in China. This leech is morphologically similar to Limnotrachelobdella sinensis recorded on goldfish and common carp. However, there are 0-2 pairs of symmetrical or asymmetrical eyes and 10 pairs of pulsatile vesicles in the newly discovered leech, in remarkable contrast to L. sinensis. Except for bighead carp, where it demonstrated a higher than 90% prevalence, and silver carp (H. molitrix), where there was low infection, this leech was not detected on any other fish from the Qiandao reservoir in China that were examined during this investigation. Molecular analyses indicated 87.8% ITS sequence identity with L. sinensis and 85.0 and 86.1% COX1 sequence identity with L. sinensis and L. okae, respectively. The uncorrected p-distance based on the COX1 sequence was found to be 15.1 and 14.0% for L. sinensis and L. okae, respectively, suggesting interspecific variation. Phylogenetic analyses based on the combination of 18S and COX1 sequences showed that the newly discovered leech groups with Limnotrachelobdella species. Histopathological observation indicated that attachment of the leech on the gill rakers and gill arches causes a loss of connective tissue, hemorrhage, and ulceration. Based on the morphology, molecular analyses, and host specificity, we conclude that this leech is a new species of Limnotrachelobdella and named it Limnotrachelobdella hypophthalmichthysa n. sp.

Molecular characterization of Peronospora variabilis isolates infecting Chenopodium quinoa and Chenopodium album in Spain.

Quinoa is an expanding crop in Southern Spain. Downy mildew, caused by Peronospora variabilis, is the most important quinoa disease in Spain and worldwide. In Spain, this disease has also been observed oin the weed Chenopodium album. The objectives of this study were to unravel the origin of the P. variabilis isolates currently infecting quinoa in Southern Spain and to study their genetic diversity. We hypothesized that P. variabilis isolates infecting quinoa in Spain could have been introduced through the seeds of the quinoa varieties currently grown in the country, or, alternatively, that these isolates are endemic isolates, originally infecting C. album, that jumped to quinoa. In order to test these hypotheses, we sequenced the ITS, cox1 and cox2 regions of 33 P. variabilis isolates infecting C. quinoa and C. album in Southern Spain and analysed their phylogenetic relationship with isolates present in other countries infecting Chenopodium spp. cox1 gene sequences from all the Spanish P. variabilis isolates were identical and exhibited 9 SNPs compared to a single P. variabilis cox1 sequence found at GenBank. Phylogenetic analyses based on ITS rDNA region were not suitable to differentiate isolates according to their geographical origin or host. Cox2 sequences from P. variabilis Spanish isolates collected from C. quinoa and C. album were all identical and had a distinctive SNP in the last of four polymorphic sites that distinguished Spanish isolates from isolates from other countries. These results suggest that P. variabilis infecting quinoa in Southern Spain could be native isolates infecting originally C. album.

First Report of Phytophthora occultans Causing Dieback of Buxus sempervirens in the Czech Republic.

In Nov 2011, and then recurrently since Sep 2020, an extensive decline has been recorded in boxwood (Buxus sempervirens), sometimes with several dozens of damaged individuals planted in private gardens and public areas and purchased in amateur markets in the Czech Republic. The leaves of the plants first showed orange-bronze discoloration, then dried and fell off, and the affected plants died. The roots, collars and stems of these plants had dark brown to black necrotic lesions. Phytophthora occultans Man in 't Veld & K. Rosend. was consistently isolated on selective medium PARPNH (Jung et al. 1996) directly from segments of symptomatic collar tissues and from rhododendron leaf pieces used to bait excised roots. On 20% V8 agar (V8A) and on carrot agar (CA), colonies had a stellate pattern. Radial growth at 25°C was 9.4 mm/day on V8A and 5.3 mm/day on CA. The cardinal growth temperatures were min. 7°C, optimum 25 to 27°C, and max. 32°C. The isolates were homothallic and produced on CA colorless globose oogonia ranging from 25.4 to 36.4 µm (n = 40) in diam. Oospores were slightly aplerotic and measured (n = 40) 22.5 to 31.9 µm in diam., with a 0.9 to 1.5 µm thick wall. The antheridia were predominantly paragynous and averaged 11.5 × 9.9 µm (height × width, n = 40). Noncaducous sporangia were obpyriform, ovoid, elongated to irregular and semipapillate, sometimes bipapillate and measured (n = 40) 31.4 to 73.4 × 17.8 to 32.1 µm, and the L:B ratio was 1.9 to 2.0. Chlamydospores and hyphal swellings were not observed. The morphological characteristics resembled those described for P. occultans (Man in't Veld et al. 2015). The isolates were deposited in the Czech Collection of Phytopathogenic Oomycetes (CCPO) under accession nos. 551.11, 1158.20, 1176.21, 1201.21, 1218.21, 1236.21 and 1261.22. For molecular identification, the internal transcribed spacer (ITS) region, cytochrome oxidase subunit 1 gene (COX1), and translation elongation factor-1α (EF) gene from all isolates were amplified and sequenced using the primer pairs ITS4/ITS5 (White et al. 1990), COXF-CIT/COXR-CIT (Man in't Veld et al. 2015), and ELONGF1/ELONGR1 (Kroon et al. 2004), respectively. The resulting sequences of representative isolates P1158.20 and P1176.21 were deposited in GenBank (accession nos. MW750576 and OP326036 for ITS, ON862131 and OP313505 for COX1 and MW762616 and ON862132 for EF). BLASTn searches of GenBank, using the partial ITS, COX1, and EF sequences, revealed 100, 100, and 99% sequence identity, respectively, to P. occultans ex-holotype culture CBS101557 accessions JX978155, JX978156 and KF650770 (Man in't Veld et al. 2015). Concatenated sequences of the three genes were used to conduct a phylogenetic analysis using the maximum likelihood method in MEGA 11 (Tamura et al. 2021). The isolates were identified as P. occultans based on morphology and a multigene phylogenetic analysis. Koch´s postulates were confirmed by a soil infestation test. Healthy 2-year-old B. sempervirens plants were inoculated (9 plants per isolate and control, isolates no. 1158.20, 1176.21, 1261.22) with three 5-mm-diam. V8A mycelial plugs by inserting into the substrate near the collar. Control plants were treated with sterile agar plugs. All plants were kept in a greenhouse at 25°C and exposed to 24 h of flooding up to collar once a week. All inoculated plants showed wilting, collar lesions and root rot occurred after 21 days, while control plants remained healthy. The pathogen was reisolated from infected plants and confirmed by molecular identification. P. occultans was found for the first time in 1998 on Buxus sempervirens in the Netherlands and later in Belgium, the United Kingdom, Germany and Romania (Man in´t Veld et al. 2015, Nechwatal et al. 2014), as well as in the USA (Reeser et al. 2015, Gitto et al. 2018). This is the first report of P. occultans in the Czech Republic. This pathogen likely poses another significant threat to boxwood cultivation in addition to the previously invaded Cydalima perspectalis and Calonectria pseudonaviculata.

Molecular characterization of oriental eyeworm (Thelazia callipaeda) detected from raccoon (Procyon lotor) and Japanese raccoon dog (Nyctereutes viverrinus) in Kanto region, Japan

BackgroundThe oriental eyeworm Thelazia callipaeda (Spirurida: Thelaziidae) is an emerging parasitic ocular nematode of carnivores and humans. In domestic animals and humans, the infection causes varying degrees of inflammation and lacrimation, and wild carnivores represent an important reservoir. In this study we examined the infection status and molecular characterization of T. callipaeda in two urban carnivores, raccoons Procyon lotor and wild Japanese raccoon dogs Nyctereutes viverrinus, in the Kanto region of Japan.MethodsFrom January 2020 to December 2021, 193 carcasses including 178 raccoons and 15 raccoon dogs were examined for the presence of worms in the eye. The worms from infected animals (one worm per host) were morphologically identified as T. callipaeda. Worms (1–5 worms per host) were subjected to genetic analysis using mitochondrial cytochrome c oxidase subunit I gene sequences.ResultsThe prevalence of T. callipaeda in raccoons and Japanese raccoon dogs was 20.2% (36/178) and 13.3% (2/15), respectively. The cox1 sequences from 56 worms from 38 animals revealed three haplotypes (h9, h10, and h12). Analysis of multiple worms for five raccoons showed co-infection of two different haplotypes (h9 and h10) in a single host. Comparing our data with published sequences, three sequences obtained from raccoons and raccoon dogs shared the same haplotypes as those reported in humans, dogs, and cats in Japan.ConclusionsOur findings show a high prevalence of T. callipaeda in raccoons, suggesting that this invasive carnivore species serves as an important natural reservoir of T. callipaeda in the Kanto region of Japan, an area with the highest human population of the country.Graphical

DNA Barcoding Reveals Occurrence of Cardiocephaloides sp. (Digenea: Strigeidae) Infecting the Great Cormorant Phalacrocorax carbo (L. 1758) in Mwanza Gulf, Lake Victoria

Cormorants (Pelecaniformes) are widely distributed worldwide, occurring as coastal birds in inland waters and marine environments, and have been implicated in the transmission of some aquatic parasites. As such, the objective of the present work was to investigate the occurrence and morphological variations in Cardiocephaloides (Digenea: Strigeidae), parasites of the great cormorant Phalacrocorax carbo. The Cardiocephaloides specimens used for molecular analysis (DNA barcoding (cox1) region) were obtained from the intestines of the great cormorants collected from Mwanza Gulf in Lake Victoria. Morphological examination of Cardiocephaloides specimens showed the possible co-existence of four morphospecies belonging to the genus Cardiocephaloides. However, detailed analyses of the cox1 sequences, phylogeny and haplotypes revealed that all four morphospecies belonged to a single unknown species of Cardiocephaloides. This paper provides the first report on the great cormorant Phalacrocorax carbo, serving as the definitive host for Cardiocephaloides in freshwater systems. The findings also reveal that the diversity of Cardiocephaloides in Africa is higher than earlier reported. Moreover, it highlights the need for more research in Tanzania to divulge snail and fish species involved in the life cycles of digenean species occurring in birds and other vertebrates in aquatic environments.
 Keywords: Digenean trematodes; Strigeidae; Phalacrocorax carbo; Cardiocephaloides; cox1

Taxonomic revision of Tinocladia (Ectocarpales s.l., Phaeophyceae): merger of Tinocladia with Eudesme and description of E. pseudocrassa sp. nov

ABSTRACT A molecular phylogeny of Tinocladia and Eudesme based on specimens covering a large proportion of the known species was done using mitochondrial cox1 and cox3, chloroplast atpB, psaA, psbA and rbcL genes and 5.8S rDNA and its ITS2 region sequences. The phylogeny revealed a close phylogenetic relationship between the two genera and the occurrence of a cryptic species within the generitype T. crassa. Eudesme species (E. borealis, E. shandongensis and the generitype E. virescens) showed a sister relationship with the northern hemisphere Tinocladia (T. crassa, including a cryptic sister species T. pseudocrassa sp. nov., and T. sanrikuensis), whereas southern hemisphere Tinocladia (T. australis, T. falklandica, T. novae-zelandiae, and a cryptic species from New Zealand) nested in the Eudesme clade. Morphologically, Tinocladia species have been distinguished from Eudesme by their denser medullary filaments and more extensive subcortical layer, but our molecular phylogenetic analyses suggest that these features do not correlate with genetic differences. The cox3 sequence divergence between northern hemisphere Tinocladia and the lineage that includes Eudesme and southern hemisphere Tinocladia was comparable to or smaller than those within selected ectocarpalean genera. We therefore propose to merge Tinocladia with Eudesme and resurrect Eudesme crassa (Suringar) Okamura for T. crassa. Although the two independent lineages of T. crassa do not show marked morphological differences, they are genetically isolated even in sympatric populations. We therefore propose to treat them as independent species. In this paper we neotypify Tinocladia crassa (=Eudesme crassa), describe E. pseudocrassa sp. nov., and determine that the Californian population of E. crassa is an introduction from Northeast Asia. HIGHLIGHTS •A taxonomic revision of Eudesme and Tinocladia, based on genetic and morpho-anatomy, provides evidence to merge Tinocladia with Eudesme. •The finding of cryptic species in New Zealand highlights the need for further taxonomic studies in this region. •The genetic data have revealed the occurrence of a cryptic species within T. crassa in Japan.

Genetic diversity of Kappaphycus malesianus (Solieriaceae, Rhodophyta) from the Philippines

Kappaphycus farming for carrageenan production is characterized by a strong selective pressure at the genetic level. Traits of agronomic importance are compromised due to domestication bottlenecks and the subsequent events of possible selective breeding of founding cultivars. Kappaphycus malesianus is farmed in Malaysia and the Philippines, and is distributed within the Malesian region. While the majority of genetically characterized specimens of this species are from Malaysia, those from the Philippines are poorly explored. Here, we assessed the genetic diversity of K. malesianus from the Philippines based on cox1 sequences. Of the 15 identified haplotypes, 14 specimens represent three novel haplotypes (wild specimens) that form a group distinct from the main clade comprising most K. malesianus haplotypes known to date. An additional haplotype from a cultivated specimen was identical to that of the most widely distributed haplotype. Our findings demonstrate that the K. malesianus is genetically more diverse than previously recognized. It is expected that higher genetic diversity may be revealed through additional sampling from a wider geographic range and careful application of integrative approaches. Future selective breeding programs in Kappaphycus would benefit from the incorporation of the genetic resources, as provided in this study.

Update on the genetic diversity and population structure of Echinococcus granulosus in Gansu Province, Tibet Autonomous Region, and Xinjiang Uygur Autonomous Region, Western China, inferred from mitochondrial cox1, nad1, and nad5 sequences.

The identification of additional Echinococcus granulosus sensu lato (s.l.) complex species/genotypes in recent years raises the possibility that there might be more variation among this species in China than is currently understood. The aim of this study was to explore intra- and inter-species variation and population structure of Echinococcus species isolated from sheep in three areas of Western China. Of the isolates, 317, 322, and 326 were successfully amplified and sequenced for cox1, nad1, and nad5 genes, respectively. BLAST analysis revealed that the majority of the isolates were E. granulosus s.s., and using the cox1, nad1, and nad5 genes, respectively, 17, 14, and 11 isolates corresponded to Elodea canadensis (genotype G6/G7). In the three study areas, G1 genotypes were the most prevalent. There were 233 mutation sites along with 129 parsimony informative sites. A transition/transversion ratio of 7.5, 8, and 3.25, respectively, for cox1, nad1, and nad5 genes was obtained. Every mitochondrial gene had intraspecific variations, which were represented in a star-like network with a major haplotype with observable mutations from other distant and minor haplotypes. The Tajima's D value was significantly negative in all populations, indicating a substantial divergence from neutrality and supporting the demographic expansion of E. granulosus s.s. in the study areas. The phylogeny inferred by the maximum likelihood (ML) method using nucleotide sequences of cox1-nad1-nad5 further confirmed their identity. The nodes assigned to the G1, G3, and G6 clades as well as the reference sequences utilized had maximal posterior probability values (1.00). In conclusion, our study confirms the existence of a significant major haplotype of E. granulosus s.s. where G1 is the predominant genotype causing of CE in both livestock and humans in China.

Globisporangium and Pythium Species Associated with Yield Decline of Pyrethrum (Tanacetum cinerariifolium) in Australia

Pyrethrum (Tanacetum cinerariifolium) cultivation in Australia, which accounts for the majority of global production of natural insecticidal pyrethrins, is affected by a persistent yield decline which in part is caused by a complex of pathogens. Globisporangium and Pythium species were isolated from crown and roots of pyrethrum plants showing stunting and brown discoloration of crown tissue, and from soil adjacent to diseased plants from yield-decline-affected sites in Tasmania and Victoria, Australia. Ten known Globisporangium species (Globisporangium attrantheridium, G. erinaceum, G. intermedium, G. irregulare, G. macrosporum, G. recalcitrans, G. rostratifingens, G. sylvaticum, G. terrestris and G. ultimum var. ultimum), two new Globisporangium species (Globisporangium capense sp. nov. and Globisporangium commune sp. nov.) and three Pythium species (Pythium diclinum/lutarium, P. tracheiphilum and P. vanterpoolii) were identified through morphological studies and multigene phylogenetic analyses using ITS and Cox1 sequences. Globisporangium ultimum var. ultimum, G. sylvaticum, G. commune sp. nov. and G. irregulare were most abundant. Globisporangium attrantheridium, G. macrosporum and G. terrestris were reported for the first time in Australia. Seven Globisporangium species were pathogenic on both pyrethrum seeds (in vitro assays) and seedlings (glasshouse bioassays), while two Globisporangium species and three Pythium species only caused significant symptoms on pyrethrum seeds. Globisporangium irregulare and G. ultimum var. ultimum were the most aggressive species, causing pyrethrum seed rot, seedling damping-off and significant plant biomass reduction. This is the first report of Globisporangium and Pythium species causing disease in pyrethrum globally and suggests that oomycete species in the family Pythiaceae may have an important role in the yield decline of pyrethrum in Australia.

Taxonomic study of Scytosiphon (Phaeophyceae) from temperate coasts of Argentina.

Scytosiphon is a common intertidal genus widely distributed on temperate coasts worldwide. Recently, eight species have been delimited with molecular tools. Although S. lomentaria is the only species that predominates in the macroalgal literature of the Southwestern Atlantic Ocean (SwAO), unpublished molecular data obtained for a population study of S. lomentaria revealed hidden species diversity of Scytosiphon among the individuals collected from four localities at the SwAO. The aim of this study was to revise the identity and phylogenetic relationships of Scytosiphon from temperate coasts of the SwAO using DNA data. Thalli were collected from the Argentinean coast between 39° S and 43° S, from which cox1 and rbcL gene sequences were obtained. Phylogenies and haplotype networks were inferred and morphology of gametophytes was studied. Four species were recognized, S. lomentaria, S. promiscuus, S. shibazakiorum, and one species that belongs to a complex of species known as "Scytosiphon Atlantic complex." This complex was known to occur only in the North Atlantic, however, the results found in this study revealed that it has an extended distribution range that includes the southern hemisphere, where its populations have high genetic diversity and unique haplotypes. The morphological differences among the four species were subtle; denoting that previous Scytosiphon records from the SwAO attributed to the renowned S. lomentaria could represent different species. In addition, sex ratio and genome-wide single nucleotide polymorphisms (SNPs) analyses were done for populations of S. promiscuus presumably introduced to the SwAO, and the results indicated that they included female-dominant parthenogenetic populations, which were probably introduced from Japan.

Elucidating the life cycle of opossum parasites: DNA sequences reveal the involvement of planorbid snails as intermediate hosts of Rhopalias spp. (Trematoda: Echinostomatidae) in Brazil.

Echinostomatid digeneans belonging to the genus Rhopalias are intestinal trematodes found mainly in opossums in the New World. The genus comprises seven species, but their life cycles and intermediate hosts have been unknown until now. During our long-term study carried out in freshwater habitats within the state of Minas Gerais, Southeast Brazil, echinostomatid cercariae lacking collar spines were found in planorbid snails Biomphalaria glabrata, Biomphalaria straminea, Drepanotrema lucidum and Gundlachia ticaga in six different batches of snail samples collected between 2010 and 2019. Morphologically, the larvae reported herein are morphologically consistent with each other and characterized by the presence of 2-3 large ovoid or spherical corpuscles in each main duct of the excretory system, resembling to Cercaria macrogranulosa previously described from the same region of Brazil. Partial sequences of the ITS (ITS1-5.8S-ITS2) region and 28S gene of the nuclear ribosomal RNA operon, and partial sequences of mitochondrial nad1 and cox1 genes were obtained and compared with the data available for members of the family Echinostomatidae. Nuclear markers indicate that all samples of cercariae evaluated in the present study can be assigned to Rhopalias, but distinct from North American isolates of Rhopalias macracanthus, Rhopalias coronatus and Rhopalias oochi (divergence 0.2-1.2% in 28S and 0.8-4.7% in ITS). The lack of differences verified in both 28S and ITS in 5 out 6 studied samples suggested that they belong to the same species. However, nad1 sequences revealed that our cercariae correspond to three distinct species of Rhopalias (interspecific divergence: 7.7-9.9%), named here as Rhopalias sp. 1, found in B. straminea and G. ticaga, Rhopalias sp. 2 found in B. glabrata and D. lucidum, and Rhopalias sp. 3 also found in D. lucidum. They also differ by 10.8-17.2% from a North American isolate of R. macracanthus sequenced in this study. The cox1 sequences obtained for Rhopalias sp. 1 and Rhopalias sp. 2 (but not Rhopalias sp. 3) reveal that they are distinct from North American isolates of R. macracanthus (genetic divergence 16.3-16.5% and 15.6-15.7%, respectively), R. coronatus (9.2-9.3% and 9.3-9.5%) and Rhopalias oochi (9.0% and 9.5-10.1%). Encysted metacercariae with general morphology similar to that of the body of cercariae were found in tadpoles of Rhinella sp. from the same stream where snails harbored Rhopalias sp. 2, suggesting that the amphibians could act as second intermediate hosts of species of Rhopalias. Data obtained provide the first insights into the life cycle of this unusual echinostomatid genus.

Molecular Characterization of Mitochondrial Cytochrome c oxidase subunit 1 (Cox1) gene from Trichostrongylus Species (Nematoda: Trichostrongylidae) in Northern Iran

The objective of the present study was to identify Trichostrongylus species by molecular analysis and also phylogenetic relationships of Trichostrongylus species by mitochondrial Cytochrome c oxidase subunit 1 (Cox1) gene in Guilan province, northern Iran. Abomasum and duodenum contents of 144 livestock were collected from sheep, goats, and cattle in Guilan province. Morphological survey was performed for initial screening. Total DNA was extracted, and the partial region of Cox1 gene was amplified and sequenced. Genetic diversity was calculated and phylogenetic analysis of the data on nucleotide sequence was conducted by MEGA7 software. Three species of Trichostrongylus including T. colubriformis, T. vitrinus, and T. axei were identified by morphological characteristics. The genetic divergence within the species in the present study was observed for T. axei (0-2.5%), T. colubriformis (0.77%), and T. vitrinus (0%). The mean inter-species difference between the three species of Trichostrongylus obtained in this study was 14.4-15.4%. The Cox1 sequences of the members of Trichostrongylus spp. were highly variable and this could be used as a valuable measure to achieve a proper assessment on biodiversity. Sequence data generation from other species of Trichostrongylus will be needed to reconstruct the phylogenetic relationships of this genus of nematodes.

Molecular identification of Sarcocystis species in sika deer (Cervus nippon) of free-ranging populations in Germany and Austria.

In this study, for the first time, Sarcocystis species were identified molecularly in sika deer (Cervus nippon) that form free-ranging populations in several countries of Europe. Diaphragm muscle samples from 151 deer from 10 populations in Germany and Austria were examined for sarcocysts. By one-gram methylene-blue staining, sarcocysts were recorded in samples of 114 animals (75.5%) which originated from all populations. Sarcocysts were more often (p < 0.0001) recorded in yearling and adult deer than in calves. Infection intensity was generally low with ~ 70% of the sarcocyst positive deer harbouring ≤ 10 sarcocysts per 1-gram diaphragm muscle. Based on cox1 sequence comparison, 10 species of Sarcocystis, all previously reported parasitizing cervids, were identified: S. elongata, S. entzerothi, S. hjorti, S. iberica, S. japonica, S. linearis, S. morae, S. pilosa, S. silva and S. truncata. The prevailing S. hjorti was detected in sika deer of all 10 populations. The identification in sika deer of S. hjorti, S. iberica, S. elongata, S. linearis, S. morae and S. silva constitutes new host records. With the additional species records of this study, the highest number of Sarcocystis species, at least 16, was identified in this host.

Description of Sarcocystis platyrhynchosi n. sp. (Apicomplexa: Sarcocystidae) from domestic ducks Anas platyrhynchos (Anseriformes: Anatidae) in China

BackgroundLimited data are currently available on protozoan parasites of the genus Sarcocystis that infect their avian hosts within the order Anseriformes (waterfowl). To date, no Sarcocystis species has been recorded in ducks in China.MethodsLeg muscles were sampled from 26 domestic ducks (Anas platyrhynchos) in China in 2021. Morphological characteristics of sarcocysts detected in the muscle tissue were described using light microscopy (LM) and transmission electron microscopy (TEM). Genomic DNA was extracted from single sarcocysts obtained from different ducks, and three genetic markers, 18S ribosomal DNA (18S rDNA), 28S ribosomal DNA (28S rDNA) and mitochondrial (mt) cytochrome oxidase subunit 1 (cox1), were amplified and cloned for sequence analyses.ResultsSarcocysts were observed by LM in only three of the 28 samples (10.7%). These sarcocysts had a thick cyst wall with numerous brush-like villar protrusions (vps) of 3.8–4.3 μm in length (n = 30) on the cyst surface. TEM observation showed that the sarcocysts had lanceolated vps. Each vps narrowed in the stalk and contained a bundle of microtubules that extended into the ground substance. Comparisons of the new sequences with those deposited in GenBank showed that the most similar sequences were those of Sarcocystishalieti in the great cormorant Phalacrocorax carbo and European starling Sturnus vulgaris, and Sarcocystis calchasi in the domestic pigeon (Columba livia) at the 18S rDNA (99.1% identity); Sarcocystiswenzeli from the domestic chicken Gallus gallus at the 28S rDNA (95.9–96.0% identity); and Sarcocystis speeri from the opossum at the mtcox1 (98.2% identity). The new 18S rDNA, 28S rDNA and mitochondrial cox1 sequences shared up to 99.0%, 95.6% and 97.7% identity, respectively, with those of Sarcocystis spp. obtained from Anseriformes avian hosts. Phylogenetic analysis inferred from the sequences of the three genetic markers placed the organism within a group of Sarcocystis spp. obtained from avian or carnivorous intermediate hosts and avian, marsupial or carnivorous definitive hosts. Based on the morphological observation and molecular analyses, the organism found in the Chinese domestic ducks was regarded as a new species and named Sarcocystis platyrhynchosi n. sp.ConclusionsBased on morphology and sequence analyses, the microcysts diagnosed in the domestic ducks examined in this study were named as a new species. This is the first record of Sarcocystis spp. from waterfowl in China. Sarcocysts of similar morphology occur frequently in different Anseriformes birds, and the relationships among these species need to be further clarified in future studies using more molecular markers.Graphical

First report of rust caused by Puccinia paullula on Monstera deliciosa and M. adansonii in Japan.

Monstera deliciosa Liebm. and M. adansonii Schott. (Araceae) have been cultivated for the commercial production in Hachijo Island, Tokyo, Japan, since the 1950s. A rust disease producing uredinial sori was found on the leaves of M. deliciosa and M. adansonii in the fields on Hachijo Island in February 2021. Symptoms were first observed on the leaf surfaces as small chlorotic spots, which expanded and became brown to reddish-brown, and produced uredinia with abundant urediniospores. The disease occurred on the whole island including the farm land, symptom incidence ranged from 5 to 30%. To confirm the pathogenicity of this rust fungus, ten plant species of Araceae (M. deliciosa, M. adansonii, Alocasia macrorrhizos, Anthurium andreauum, Dieffenbachia maculata, Epipremnum mirabile, E. pinnatum, Philodendron scandens, Spathiphyllum sp., Syngonium podophyllum) were inoculated with urediniospores obtained from infected samples on M. deliciosa and M. adansonii. Urediniospores were suspended in distilled water (1 × 106 conidia/ml) and sprayed on healthy plants. The inoculated plants were kept in a dark chamber at about 25°C for 48 h, and then transferred to a greenhouse. After 40 days, uredinia were reproduced only on M. deliciosa and M. adansonii. Morphological characteristics of the urediniospores obtained from inoculated monstera plants matched those obtained from the field plants and used as inoculum, thus fulfilling Koch's postulates. No symptoms were observed on the other plant species inoculated. For identification of the rust fungus, dry specimens obtained from both naturally infected plants and inoculated plants were used for morphological observations. Urediniospores with pedicels emerging from the stomata were densely aggregated, globose, and 24.8-29.3 µm (n = 30). Their walls were brown, echinulate, and 1.4-2.2 µm thick. Telia were found in the specimens collected in July and August. The teliospores with pedicels emerging from the stomata were two-celled, ellipsoid, and 21.3-27.5 × 19.5-23.4 µm. Their walls were pale yellow, smooth, and 1.0-1.9 µm thick. Morphological differences among the specimens on M. deliciosa and M. adansonii were not observed. These morphological characteristics were consistent with the description of P. paullula (Sydow and Sydow 1913). For molecular identification, the large subunit (LSU) rDNA and cytochrome oxidase III (cox3) were amplified. LRust1R, LR3, and LR5 (Vilgalys and Hester 1990) primers were used for sequencing of LSU region. Cox3_F (5'-GTTCAGTATGTTATTTTAACG-3') and cox3_R (5'-ATAGGAATAGTCAAACAACATC-3') primers were constructed here based on the P. paullula sequences (KX999927) for the cox3 region. The sequences were deposited in the GenBank as accession numbers OK509070, OK509071, LC663719 and LC663720. BLAST analysis showed that LSU and cox3 sequences shared 98.8% (902/913 nts) and 100% (632/632 nts) identities with P. paullula, KX999886 and KX999927 (Marin-Felix et al. 2017). This is the first report of a rust disease of M. deliciosa and M. adansonii caused by P. paululla in Japan. The rust fungi has been reported as the pathogen on the species of Amorphophallus, Rhaphidophora and Monstera (Sydow and Sydow 1913, Shaw 1991, Chen et al. 2009). The occurrence of this disease should be monitored because it can reduced yield and commercial value of monstera plants. All the specimens used in the experiments were deposited in the National Museum of Nature and Science, Japan (TNS-F82068-82077).

Eastern Colombian Páramo Liodessus Guignot, 1939 diving beetles are genetically structured, but show signs of hybridization, with description of new species and subspecies (Coleoptera, Dytiscidae).

We studied Liodessus diving beetles from six eastern Colombian Páramo areas, as well as from the Altiplano. We discovered a highly characteristic new species, based on male genital morphology, Liodessussantarositasp. nov., in the Páramo de Guantiva-Rusia. Specimens from the Altiplano around Bogotá, and the Páramos of Almorzadero, Chingaza, Matarredonda, Rabanal y Rio Bogotá and Sumapaz form one clade of genetically similar populations based on mitochondrial Cox1 sequence data. The individuals of this clade are sub-structured according to their geographic distribution. The populations differ from each other mainly in terms of body size and coloration and, at most, subtly in their genital morphology. In two cases, we find putative hybrid populations between Altiplano and Páramo areas. We suggest that the different Páramo populations are in an early phase of speciation, and perhaps already genetically isolated in some cases. They are here assigned subspecies status to highlight these ongoing processes pending more comprehensive geographic sampling and use of genomic data. We refer to this clade as the Liodessusbogotensis complex, containing Liodessusb.bogotensis Guignot, 1953; Liodessusb.almorzaderossp. nov.; Liodessusb.chingazassp. nov.; Liodessusb.lacunaviridis Balke et al., 2021, stat. nov.; Liodessusb.matarredondassp. nov., and Liodessusb.sumapazssp. nov.

Identification and prevalence investigation of cyathocotylid trematode (Trematoda: Digenea: Cyathocotylidae) in Carassius auratus in Wuhu, China.

The family Cyathocotylidae trematode is a world-widely distributed parasite whose adults are mainly found in fish, reptiles, birds, and mammals in both freshwater and marine environments. However, little is known of the prevalence of these trematode in China. For the first time, we found Cyathocotylidae trematode in the Wuhu area. Therefore, we investigated the prevalence of metacercariae in Carassius auratus from Zhang Lake, Kui Lake, Qingyi River, and Yangtze River (Wuhu Section) in Wuhu area. A total of 392 one-year-old C. auratus were tested in January, April, July, and October 2019, respectively. After the fish were euthanized, the back, chest, and tail muscles were used for preliminary screening for the presence of metacercariae by direct compression method. Metacercariae were isolated by the artificial digestion method and then morphologically and molecularly identified by cox1 (642bp) and ITS2 (418bp) sequences amplification by PCR. Based on morphological features and sequence analysis, they were identified as cyathocotylid metacercariae. The four water sources have different degrees of prevalence throughout the year, the prevalence ranges from 28.45% (29/102) in Yangtze River to 50.55% (46/91) in Zhang Lake, with an average prevalence of 40.56% (159/392). There were also differences in the prevalence in different months: The highest occurred in July, which was 81.63% (80/98), while the lowest occurred in January, which was 8.89% (8/90). This study can provide basic data for the prevention and control of cyathocotylid trematode in this area.

Morphometry and Molecular Identification of Haemonchus Cobb, 1898 (Trichostrongylidae: Nematoda) Isolates from Small Ruminants in Tanzania Based on Mitochondrial cox 1 and rRNA-ITS genes.

The genus Haemonchus is the major abomasal parasite of ruminants responsible for substantial economic losses in tropical and temperate regions. This study was conducted to clarify the morphometry and molecular characterisation of Haemonchus species isolated from sheep in Babati district, Tanzania. A total of 486 trichostrongylid nematodes were recovered from five sheep. Of the total worms, 106 nematodes were distinguished by 37 males and 69 females. The asymmetrical length of dorsal ray and the distance of bulb at the apex of spicules were used for identification of males. In females, the linguiform vulvar flap was the most predominant with 33 out of 69 (48%) compared with knobbed morph type which was 25/69 (36%) and smooth morph type with 11/69 (16%). Partial cox1 sequence fragments of Haemonchus contortus isolates showed 98.8%, 99.3%, 99.7%, 99.5%, 99.3%, and 98.4% in male, smooth, knobbed, linguiform A, linguiform B, and linguiform C, respectively; with the average nucleotide divergence ranged from 1.03 to 2.35%. The amplified fragments of ITS-2 genes in knobbed, linguiform A, and smooth morphotypes revealed 99.4%, 98.5%, and 98.3%, respectively. Phylogenetic analysis was evaluated by employing Bayesian inference and maximum-likelihood, and the tree was distinctly separated into three clusters focusing on H. contortus in cluster I within the family Haemonchidae. Genetic drifting, mutation, and modification of the morphological features of the Haemonchus species described to have an impact on the development of drug resistance. Species identification is necessary to understand which species infect animal host. We recommend more studies on the parasites intensity and the strategies for controlling Haemonchus species in Tanzania.

Two-Step Identification of N-, S-, R- and T-Cytoplasm Types in Onion Breeding Lines Using High-Resolution Melting (HRM)-Based Markers.

High-resolution melting (HRM) analysis is a powerful detection method for fast, high-throughput post-PCR analysis. A two-step HRM marker system was developed for identification of the N-, S-, R- and T-cytoplasms of onion. In the first step for the identification of N-, S- and R-cytoplasms, one forward primer was designed to the identical sequences of both cox1 and orf725 genes, and two reverse primers specific to the polymorphic sequences of cox1 and orf725 genes were used. For the second step, breeding lines with N-cytoplasm were evaluated with primers developed from the orfA501 sequence to distinguish between N- and T-cytoplasms. An amplicon with primers to the mitocondrial atp9 gene was used as an internal control. The two-step HRM marker system was tested using 246 onion plants. HRM analysis showed that the most common source of CMS, often used by Russian breeders, was S-cytoplasm; the rarest type of CMS was R-cytoplasm; and the proportion of T-cytoplasm among the analyzed breeding lines was 20.5%. The identification of the cytoplasm of a single plant by phenotype takes from 4 to 8 years. The HRM-based system enables quick and easy distinguishing of the four types of onion cytoplasm.