The plerocercoid larvae of Spirometra mansoni are etiological agents of human and animal sparganosis. Annexins are proteins with important roles in parasites. However, our knowledge of annexins in S. mansoni is still inadequate. In this study, 18 new members of the Annexin (ANX) family were characterized in S. mansoni. The clustering analysis demonstrated that all the SmANXs were divided into two main classes, consistent with the patterns of conserved motif organization. The 18 SmANXs were detected at all developmental stages (plerocercoid, adult, and egg) and displayed ubiquitous but highly variable expression patterns in all tissues/organs studied. The representative member rSmANX18 was successfully cloned and expressed. The protein was immunolocalized in the tegument and parenchyma of the plerocercoid and in the tegument, parenchyma, uterus and egg shell of adult worms. The recombinant protein can bind phospholipids with high affinity in a Ca2+-dependent manner, shows high anticoagulant activity and combines with FITC to recognize apoptotic cells. Annexin gene polymorphism and conservative core motif permutation were found in both cestodes and trematodes. SmANXs also revealed high genetic diversity among Platyhelminthes of medical interest. Our findings lay a foundation for further studies on the biological functions of ANXs in S. mansoni as well as other taxa in which ANXs occur.
UNASSIGNED: La famille des gènes des annexines chez Spirometra mansoni (Cestoda : Diphyllobothriidae) et son schéma phylogénétique parmi les Plathelminthes d’intérêt médical.
UNASSIGNED: Les larves plérocercoïdes de Spirometra mansoni sont des agents étiologiques de la sparganose humaine et animale. Les annexines sont des protéines jouant un rôle important chez les parasites. Cependant, nos connaissances sur les annexines chez S. mansoni sont encore insuffisantes. Dans cette étude, 18 nouveaux membres de la famille des annexines (ANX) ont été caractérisés chez S. mansoni. L’analyse de regroupement a démontré que tous les SmANX étaient divisées en deux classes principales, ce qui correspond aux modèles d’organisation des motifs conservés. Les 18 SmANX ont été détectées à tous les stades de développement (plérocercoïde, adulte et œuf) et présentaient des modèles d’expression omniprésents mais très variables dans tous les tissus/organes étudiés. Le membre représentatif rSmANX18 a été cloné et exprimé avec succès. La protéine a été immunolocalisée dans le tégument et le parenchyme du plérocercoïde ainsi que dans le tégument, le parenchyme, l’utérus et la coquille d’œuf des vers adultes. La protéine recombinante peut se lier aux phospholipides avec une affinité élevée de manière dépendante du Ca2+, présente une activité anticoagulante élevée et se combine avec le FITC pour reconnaître les cellules apoptotiques. Un polymorphisme du gène de l’annexine et une permutation conservatrice du motif central ont été trouvés chez les cestodes et les trématodes. Les SmANX ont également révélé une grande diversité génétique parmi les Plathelminthes d’intérêt médical. Nos résultats jettent les bases pour des études plus approfondies sur les fonctions biologiques des ANX chez S. mansoni ainsi que dans d’autres taxons dans lesquels les ANX sont présents.

Two adult male Puerto Rican crested anoles (Anolis cristatellus cristatellus) housed in a research facility were presented with debilitation and were euthanized. On autopsy, anole 1 had a large cystic white structure in the left pelvic limb, which protruded through the ruptured epidermis, and a large, poorly demarcated swelling in the right caudal abdomen. Anole 2 had masses in the mid-dorsum, caudal dorsum, left pelvic limb, and tail. These masses contained variably sized cestode larvae, which ruptured into the coelomic cavity. Evaluation of the larvae revealed a thickened and wrinkled anterior end, with a cleft-like invagination, consistent with either a plerocercoid sparganum or a tetrathyridium. Histologically, several cestode larvae were contained in the body wall of both anoles. These were up to 650 μm in diameter, with a thin tegument and a spongy parenchyma. The spongy parenchyma contained numerous, up to 30 μm diameter, sharply demarcated, basophilic-to-black structures (calcareous corpuscles). There was pneumonia and hepatitis in anole 2, suggestive of potential secondary infection subsequent to immunosuppression. Molecular amplification of the cytochrome C oxidase subunit 1 revealed 100% homology for the COX1 gene of the diphyllobothriid tapeworm Spirometra erinaceieuropaei, also known as Spirometra mansoni.

BACKGROUND: Spirometra mansoni can parasitize animals and humans through food and water, causing parasitic zoonosis. Knowledge of the developmental process of S. mansoni is crucial for effective treatment; thus, it is important to characterize differential and specific proteins and pathways associated with parasite development.
METHODS: In this study, we performed a comparative proteomic analysis of the plerocercoid and adult stages using a tandem mass tag-based quantitative proteomic approach. Additionally, integrated transcriptomic and proteomic analyses were conducted to obtain the full protein expression profiles of different life cycle stages of the tapeworm.
RESULTS: Approximately 1166 differentially expressed proteins (DEPs) were identified in adults versus plerocercoids, of which 641 DEPs were upregulated and 525 were downregulated. Gene Ontology (GO), Clusters of Orthologous groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses indicated that most DEPs related to genetic information processing and metabolism of energy in adults seem to be more activated. In the plerocercoid stage, compared to metabolism, genetic information processing appears more dynamic. Protein-protein interaction (PPI) revealed six key proteins (phosphomannomutase, glutathione transferase, malate dehydrogenase, cytoplasmic, 40S ribosomal protein S15, ribosomal protein L15 and 60S acidic ribosomal protein P2) that may play active roles in the growth and development of S. mansoni. Finally, the combination of transcriptomic and proteomic data suggested that three pathways (ubiquitin-mediated proteolysis, phagosome and spliceosome) and five proteins closely related to these pathways might have a significant influence in S. mansoni.
CONCLUSIONS: These findings contribute to increasing the knowledge on the protein expression profiles of S. mansoni and provide new insights into functional studies on the molecular mechanisms of the neglected medical tapeworm.

Morphological characteristics and DNA sequencing were used to identify plerocercoids of a Schistocephalus sp. infecting slimy sculpin (Cottus cognatus) from northern New Brunswick and plerocercoids of Ligula intestinalis infecting blacknose dace (Rhinichthys atratulus) in Fundy National Park (FNP, New Brunswick). To our knowledge, no previous publications documented either cestode from New Brunswick, Canada. Blacknose dace represent a new host record for L. intestinalis. Identifications were made based on the presence or absence of segmentation and sequencing partial nicotinamide adenine dinucleotide dehydrogenase subunit 1 (ND1; mitochondrial DNA) and/or partial cytochrome c oxidase subunit 1 (COI; mitochondrial DNA). Plerocercoids from blacknose dace in FNP were identified as Ligula intestinalis based on >99% nucleotide identity with COI for this species in the NCBI GenBank database. Plerocercoids in slimy sculpin from northern New Brunswick were identified as a Schistocephalus sp. based on high nucleotide identity with congenerics in the NCBI GenBank database. The absence of GenBank entries with sufficient high percent identity to our specimens, and potential species hybrids in this genus, prevents species-level identification of Schistocephalus sp. plerocercoids currently. The absence of previous documentation of these cestodes might reflect recent environmental change promoting the transmission of these parasites that can modulate host fish behavior, induce sterility of host fishes, and contribute to epizootics.

Parasitic helminths modify host immune reactions to promote long-term parasitism. We previously purified a glycoprotein, plerocercoid-immunosuppressive factor (P-ISF), from the excretory/secretory products of Spirometra erinaceieuropaei plerocercoids and reported its cDNA and genomic DNA sequences. In this study, we isolated extracellular vesicles (EVs) from the excretory/secretory products of S. erinaceieuropaei plerocercoids and found that they suppressed the production of nitric oxide and the gene expression of tumor necrosis factor-α, interleukin-1β, and interleukin-6 in lipopolysaccharide-stimulated macrophages. EVs are membrane-bound vesicles 50-250 nm in diameter and are localized in the whole bodies of plerocercoids. EVs from plerocercoids encapsulate a variety of unidentified proteins and microRNAs (miRNAs), which are non-coding RNAs that play essential roles in post-transcriptional gene regulation. The miRNAs of the EVs were analyzed, and 334,137 sequencing reads were mapped to the genomes of other organisms. A total of 26 different miRNA families were identified, including miR-71, miR-10-5p, miR-223, and let-7-5p, which have been reported to have immunosuppressive effects. We confirmed that P-ISF was present in the supernatant but not in the EVs by western blotting with an anti-P-ISF antibody. These results suggest that S. erinaceieuropaei plerocercoids suppress host immunity by releasing P-ISF and EVs.

We report a case of Spirometra infection in a Samar cobra (Naja samarensis) imported from the Philippines, belonging to a zoological collection in the southern United States. Under a poor post-surgical prognosis, the snake was euthanized, and at necropsy plerocercoids of a Diphyllobotriidea were found in its subcutaneous tissues and musculature. Molecular and phylogenetic analyses of the complete cytochrome oxidase c subunit I (cox1) gene of the mitochondrial DNA confirmed that the isolate belonged to the genus Spirometra and was closely related to Spirometra mansoni isolates from Asian countries (bootstrap support = 99.4%). Considering the origin and clinical history and handling of the animal, the snake probably arrived infected in America. We suggest the inclusion of diagnostic imaging in the investigation of sparganosis in research and disease surveillance protocols applied in the pre- and post-quarantine period to asymptomatic animals imported from endemic areas.

BACKGROUND: In China, the plerocercoid of the cestode Spirometra mansoni is the main causative agent of human and animal sparganosis. However, the population genetic structure of this parasite remains unclear. In this study, we genotyped S. mansoni isolates with the aim to improve current knowledge on the evolution and population diversity of this cestode.
METHODS: We first screened 34 perfect simple sequence repeats (SSRs) using all available omic data and then constructed target sequencing technology (Target SSR-seq) based on the Illumina NovaSeq platform. Next, a series of STRUCTURE. clustering, principal component, analysis of molecular variance and TreeMix analyses were performed on 362 worm samples isolated from 12 different hosts in 16 geographical populations of China to identify the genetic structure.
RESULTS: A total of 170 alleles were detected. The whole population could be organized and was found to be derived from the admixture of two ancestral clusters. TreeMix analysis hinted that possible gene flow occurred from Guizhou (GZ) to Sichuan (SC), SC to Jaingxi (JX), SC to Hubei (HB), GZ to Yunnan (YN) and GZ to Jiangsu (JS). Both neighbor-joining clustering and principal coordinate analysis showed that isolates from intermediate hosts tend to cluster together, while parasites from definitive hosts revealed greater genetic differences. Generally, a S. mansoni population was observed to harbor high genetic diversity, moderate genetic differentiation and a little genetic exchange among geographical populations.
CONCLUSIONS: A Target SSR-seq genotyping method was successfully developed, and an in-depth view of genetic diversity and genetic relationship will have important implications for the prevention and control of sparganosis.

The mitochondria of adult and plerocercoid Spirometra mansoni were characterized in isolated mitochondria and in situ by electron microscopic histochemistry with special attention to the respiratory chain. Although the specific activities of the constituent enzyme complexes of succinate oxidase are fairly similar in adult and plerocercoid mitochondria, those of succinate oxidase and NADH-FRD are approximately 4- and 25-fold higher in adult mitochondria than in plerocercoid mitochondria, respectively. Quinone analysis by high performance liquid chromatography and mass spectrometry showed that adult and plerocercoid mitochondria contained both rhodoquinone-10 and ubiquinone-10 at concentrations of 4.98 and 0.106 nmol mg-1 for adult, and 0.677 and 0.137 nmol mg-1 for plerocercoid, respectively. Inhibition studies on the succinate-oxidase system of adult mitochondria showed that they possessed both cyanide-sensitive and -insensitive succinate oxidases, the latter of which produces hydrogen peroxide. Adult mitochondria, when NADH was used as a substrate, were shown to produce hydrogen peroxide, and the production of hydrogen peroxide decreased to undetectable levels in the presence of fumarate. The specific activities of NADH-fumarate reductase and cytochrome c oxidase were significantly higher in mature proglottids than in immature and gravid proglottids. Isopycnic density-gradient centrifugation analyses and in situ electron microscopic histochemistry revealed that both adult and plerocercoid mitochondria were heterogeneous in terms of respiratory function and physicochemical properties. The physiological significance of adult and plerocercoid mitochondria is discussed in relation to the oxygen tension of their parasitic habitats.

The plerocercoid larvae of the tapeworm Spirometra erinaceieuropaei can parasitize humans and animals and cause serious parasitic zoonosis. However, our knowledge of the developmental process of S. erinaceieuropaei is still inadequate. To better characterize differential and specific genes and pathways associated with parasite development, a comparative transcriptomic analysis of the plerocercoid stage and the adult stage was performed using RNA-seq and de novo analysis. Approximately 13,659 differentially expressed genes (DEGs) were identified in plerocercoids versus adults, of which 6455 DEGs were upregulated and 7204 were downregulated. DEGs involved in parasite immunoevasion were more active in plerocercoid larvae than in adults, while DEGs associated with metabolic activity were upregulated in adults. Gene Ontology (GO) and Kyoto Encyclopedia of Genes (KEGG) analyses revealed that most DEGs involved in protein phosphorylation/dephosphorylation and the Wnt signalling pathway were much more active in plerocercoid larvae. The molecular functions of upregulated unigenes in adults were mainly enriched for metabolic activities. qPCR validated that the expression levels of 10 selected DEGs were consistent with those in RNA-seq, confirming the accuracy of the RNA-seq results. Our results contributed to increasing the knowledge on the S. erinaceieuropaei gene repertoire and expression profile and also provide valuable resources for functional studies on the molecular mechanisms of S. erinaceieuropaei.

Human sparganosis is a food-borne parasitic disease caused by the plerocercoids of Spirometra species. Clinical diagnosis of sparganosis is crucial for effective treatment, thus it is important to identify sensitive and specific antigens of plerocercoids. The aim of the current study was to identify and characterize the immunogenic proteins of Spirometra erinaceieuropaei plerocercoids that were recognized by patient sera. Crude soluble extract of the plerocercoids were separated using 2-dimensional gel electrophoresis coupled with immunoblot and mass spectrometry analysis. Based on immunoblotting patterns and mass spectrometry results, 8 antigenic proteins were identified from the plerocercoid. Among the proteins, cysteine protease protein might be developed as an antigen for diagnosis of sparganosis.