Ciliates in the subclass Hypotrichia have long been difficult to classify as they are one of the most polymorphic and highly differentiated groups, leading to their systematics remaining unresolved. Phylogenetic relationships within the hypotrich family Strongylidiidae have been ambiguous due to discordance between the morphological and genetic data. In this study, a new strongylidiid genus Heterouroleptus is established, mainly based on the novel mode of origin of the ventral cirral rows: left ventral cirral row (LVR) originates from frontal-ventral-transverse cirral anlagen (FVTA) III (anterior portion), IV (middle portion), and V (rear portion); right ventral cirral row comes from the entire FVTA VI. A new species, Heterouroleptus weishanensis gen. nov., sp. nov., is investigated along with the morphometric and molecular data from a population of Strongylidium wuhanense. Eight new sequences and nuclear gene markers (single-gene and multi-gene) are provided to analyze the phylogenetic relationships of strongylidiids, with the COI gene utilized to uncover further genetic information at species level and below. The results reveal that: (1) Strongylidiidae is monophyletic and has a close relationship with Dorsomarginalia; (2) Heterouroleptus gen. nov. forms a clade that is sister to all the other strongylidiids; (3) Hemiamphisiella Foissner, 1988 and Pseudouroleptus Hemberger, 1985 should not be synonyms, and both genera should be subdivided due to their variable morphological characteristics; (4) LVR originating from three anlagen is a plesiomorphy of Strongylidiidae. The discovery of the origin of the LVR not only contributes to the establishment of the genus Heterouroleptus, but also helps to improve the diagnosis of the family Strongylidiidae.
UNASSIGNED: The online version contains supplementary material available at 10.1007/s42995-024-00243-z.

According to the latest taxonomy of Spirometra species, six species (lineages) have been tentatively classified as valid. These species are Spirometra erinaceieuropaei, S. folium, S. mansoni, undescribed Spirometra sp. 1, and S. decipiens complex 1 and 2. Among these species, the undescribed species was first discovered as plerocercoid larvae in wild boars in Japan and further studies have confirmed that this species is a new taxon belonging to the genus Spirometra. Here, we describe Spirometra asiana sp. nov., which is difficult to distinguish morphologically from known Spirometra species. However, it is genetically easily distinct from other Spirometra species, thus facilitating identification. We also emphasize that S. mansoni and S. asiana, but not S. erinaceieuropaei, are etiological agents that cause human sparganosis and/or spirometrosis in Asia.

Dibothriocephalus latus and Dibothriocephalus dendriticus are found throughout the temperate and sub-arctic zones of the northern hemisphere, but they are also found in the southern core countries of South America, Chile and Argentina. Genetic characteristics of D. latus and D. dendriticus from South America have yet to be fully defined. The present study aimed to understand the genetic characteristics of D. latus and D. dendriticus from Chile by haplotype network analysis of mitochondrial cytochrome c oxidase subunit I gene (cox1) and cytochrome b gene (cob), as well as their origins. Dibothriocephalus latus and D. dendriticus plerocercoid larvae were obtained from feral and/or wild salmonids captured in Lake Llanquihue in Región de Los Lagos, and Lake Panguipulli in Región de Los Ríos, located south of central Chile. Haplotype analysis of D. latus revealed that H1 in cox1 and H2 in cob are the key haplotypes common to D. latus across the world, including Chile, and both genes exhibited limited genetic diversity in D. latus. It was assumed that D. latus was brought into South America by European and Russian immigrants in the 19th century as previously reported. In contrast, both the cox1 and cob of D. dendriticus display considerable genetic diversity, with no common haplotypes between D. dendriticus populations from Chile and the northern hemisphere. More intriguingly, two cob haplotypes (H24, H25) detected in Chilean D. dendriticus were closely linked to haplotypes (H30, H31) detected in North American D. dendriticus, strongly implying that D. dendriticus in Chile was brought by piscivorous migrating birds from North America. It has also been estimated that the D. dendriticus from Chile genetically diverged from the D. dendriticus from the northern hemisphere approximately 1.11 million years ago, long before humans migrated to the southern parts of South America.

Global fish consumption is increasing in tandem with population growth, resulting in the dilemma of overfishing. Overfished high-value fish are often replaced with other fish in markets. Therefore, the accurate identification of fish products in the market is important. In this study, full-DNA and mini-DNA barcoding were used to detect fish product fraud in Guiyang, Guizhou Province, People\'s Republic of China. The molecular results revealed that 39 (20.42%) of the 191 samples were inconsistent with the labels. The percentages of mislabeling of fresh, frozen, cooked, and canned fish products were 11.70, 20.00, 34.09, and 50.00%, respectively. The average Kimura two-parameter distances of mini-DNA barcoding within species and within genera were 0.56 and 6.42%, respectively, and those of full-DNA barcoding were 0.53 and 7.25%, respectively. Commercial fraud was evident in this study; most high-priced fish were replaced with low-priced fish with similar features. Our findings indicate that DNA barcoding is an effective tool for identifying fish products and could be used to enhance transparency and fair trade in domestic fisheries.

OBJECTIVE: The aim of this study was to characterize Leucocytozoon caulleryi from backyard chickens in Khon Kaen Province, Thailand.
METHODS: Tissue samples were collected from backyard chickens suspected to have leucocytozoonosis and subjected to histopathology examination. The BLAST Tool at NCBI GenBank (Basic Local Alignment Research Tools) (http://www.ncbi.nlm.nih.gov/BLAST) was used to identify the nucleotide sequence of the partial cytochrome c oxidase subunit I (cox I) gene. A Phylogenetic tree for analysis of L. caulleryi was constructed by using MEGA 7.0 software (https://www.megasoftware.net/).
RESULTS: The necropsy results revealed the subcutaneous hemorrhages of pectoral muscles, multifocal hemorrhages of the thymus and pectoral muscles, hemorrhage of the proventriculus and peritoneal cavity, and megaloschizonts of the pancreas and intestine, including subcapsular hemorrhages of the liver. Microscopic examination revealed numerous megaloschizonts of Leucocytozoon spp. in the pectoral muscles, intestine, pancreas, and thymus. Molecular analysis of the partial cox I gene showed that the causal agent was closely related to L. caulleryi reported in Japan.
CONCLUSIONS: From these results, L. caulleryi was determined to be the causal agent of leucocytozoonosis and was closely associated with L. caulleryi reported in Japan.

The systematics of tapeworms in the genus Spirometra has been progressing with the accumulation of molecular genetics data, but the taxonomic status of many nominal species remains under debate. We report morphological and molecular-phylogenetic data for a Spirometra species collected from a domestic cat (Felis silvestris catus) in Chiloé Island, Chile. The Spirometra species was shown to be genetically conspecific with Spirometra decipiens complex 1 found in a Pampas fox (Lycalopex gymnocercus) from Argentina, and was closely related to a Hoary fox (Lycalopex vetulus) and rattlesnake (Crotalus durissus) from Brazil. Therefore, the presence of S. decipiens complex 1 was molecularly confirmed for the first time in Chile. The findings of the present study add useful information for the systematics of poorly known Spirometra species in South America.

A 92-year-old Japanese woman presented with a mass in the left breast, and sparganosis was suspected by biopsy of the mass. The mass disappeared once, but it reappeared at the same site one year later. For a definitive diagnosis, the mass was surgically removed, and a sparganum-like worm was detected. The causative agent was confirmed as Spirometra Type I (most probably Spirometra mansoni) by mitochondrial DNA analysis. The serological examination also proved the case as sparganosis. Considering the presence of two Spirometra species (Type I and II) in Asia, particularly Japan, molecular analysis of the causative agents is highly recommended to understand the epidemiology, infection sources, and pathogenicity in humans in both species, if the parasite specimens are available.

The major aim of the present study was to determine by molecular methods whether the wide and narrow types of macroscopic sarcocysts in Spanish sheep belonged to different species, that is, Sarcocystis gigantea and Sarcocystis medusiformis, respectively. Additionally, we wanted to identify and characterize molecularly the species forming microscopic sarcocysts and determine the phylogenetic placement of all species found. Portions of the oesophagus, diaphragm and hind legs containing macroscopic sarcocysts were collected from slaughtered culled ewes at an abattoir in the Province of Madrid, Central Spain, but both macroscopic and microscopic sarcocysts were isolated for molecular examination. Genomic DNA from 63 sarcocysts (21 macroscopic, 42 microscopic) were examined at the cytochrome c oxidase subunit I gene (cox1), while selected isolates of each species found were further examined at the 18S and 28S ribosomal RNA (rRNA) genes. The 63 sarcocysts comprised five cox1 sequence types, each corresponding to a particular sarcocyst type, and thus represented five Sarcocystis spp. The slender fusiform and thick macrocysts belonged to S. medusiformis and S. gigantea, respectively. The microscopic sarcocysts belonged to Sarcocystis arieticanis, Sarcocystis tenella and a Sarcocystis mihoensis-like species with slanting thorn-like cyst wall protrusions, which was characterised molecularly for the first time. Based on its phylogenetic position, the S. mihoensis-like species probably uses corvids as definitive hosts.

Three new species and one subspecies of the genus Amynthas are described from Guangxi Zhuang Autonomous Region, China: The new species are: Amynthas maximus Qiu & Dong, sp. nov. and Amynthas tortuosus Qiu & Dong, sp. nov., and Amynthas shengtangmontis Dong & Jiang, sp. nov., the subspecies is Amynthas shengtangmontis minusculus subsp. nov. All have four pairs of spermathecal pores in 5/6-8/9, which indicates that they should belong to the corticis-group. Their morphological characteristics are compared to other similar species in the corticis-group from China and other Asian countries, such as Amynthas pulvinus Sun & Jiang, 2013, Amynthas homosetus (Chen, 1938), Amynthas corticis (Kinberg, 1867), Amynthas dorsualis Sun & Qiu, 2013, and Amynthas carnosus (Goto & Hatai, 1899). In addition, the results presented are confirmed by the pairwise comparison of COI barcode sequences. The pairwise distances between each new species and the other eighteen corticis-group species are greater than 14.7% on average. Furthermore, the pairwise distance between A. shengtangmontis shengtangmontis and A. shengtangmontis minusculus is 10.7-11.4%.

Samples of diaphragm were collected from 53 sika deer from Gifu Prefecture, Japan; 220 sarcocysts were isolated, examined in wet mounts and classified according to their cyst wall protrusions. The sarcocysts were then examined molecularly in order to assign them to different species. All but 11 of the 220 sarcocysts were initially identified by means of a multiplex PCR assay targeting cox1 of five species, whereas the remaining 11 sarcocysts were identified by standard PCR and sequencing. DNA from selected sarcocysts was used for PCR amplification and sequencing of cox1 (59 sequences) and 18S rDNA (23 sequences). The 220 sarcocysts comprised seven major cox1 sequence types or species. Types 4 and 7 were assigned to the known species Sarcocystis pilosa and Sarcocystis ovalis, whereas types 1, 3 and 5 were considered to represent three new species, for which the names Sarcocystis japonica, Sarcocystis matsuoae and Sarcocystis gjerdei have been proposed. Types 2 and 6 were most similar to Sarcocystis tarandi and Sarcocystis taeniata, respectively, but could not be unequivocally assigned to these species. Sarcocysts belonging to S. japonica were macroscopic with fairly thick finger-like protrusions, whereas most sarcocysts of the six other species were microscopic. Sarcocysts of S. cf. tarandi and S. matsuoae were spindle-shaped and possessed thin finger-like cyst-wall protrusions. Sarcocysts of S. pilosa and S. gjerdei had similar hair-like protrusions, whereas those of S. cf. taeniata had a smooth surface. Sarcocysts of S. japonica, S. pilosa, S. cf. tarandi, S. gjerdei, S. matsuoae, S. cf. taeniata and S. ovalis were found in 50 (94.3%), 29 (54.7%), 22 (41.5%), 10 (18.9%), 8 (15.1%), 6 (11.3%) and 1 (1.9%) of the 53 sika deer examined, respectively. An improved multiplex PCR assay targeting cox1 was developed, through which the seven Sarcocystis spp. found in the present study could be identified.