Parvatrema spp. (Digenea, Gymnophallidae) with parthenogenetic metacercariae: diversity, distribution and host specificity in the palaearctic.

There are several species of gymnophallid digeneans in the genus Parvatrema that are unique in developing metacercariae that reproduce by parthenogenesis in the second intermediate host. Transmission of these digeneans takes place in coastal ecosystems of the North Pacific and North Atlantic seas. The first intermediate hosts are bivalves, the second ones are gastropods, and the definitive hosts are migratory birds. We integrated data accumulated over 25 years of research and differentiated a complex of five closely related species. They differ in the molluscan second intermediate hosts, distribution ranges, and life cycles patterns. The type I life cycle includes two generations of parthenogenetic metacercariae, followed by development of metacercariae which are invasive for the definitive host. In the type II life cycle, the number of generations of parthenogenetic metacercariae is unlimited, and they can also produce cercariae. These cercariae emerge into the environment and can infect new individuals of the second intermediate host. We conclude that the type I life cycle is a derived option that has evolved as a better fit to transmission in the unstable conditions in the intertidal zone. Another evolutionary trend in Parvatrema is transition from inhabiting the extrapallial space of the gastropod second intermediate host to endoparasitism in its mantle and internal organs. rDNA sequence analysis highlighted that Parvatrema spp. with parthenogenetic metacercariae form a monophyletic clade and suggested the Pacific origin of the group, with two transfers to the North Atlantic and colonisation of new second intermediate host species. Apparently the group formed in the late Pliocene-Pleistocene and diversified as a result of recurrent isolation in inshore refugia during glacial periods. We argue that parthenogenetic metacercariae in Parvatrema may serve as a model for early digenean evolution, demonstrating the first steps of adopting the molluscan first intermediate host and becoming tissue parasites.

Virtual screening and identification of promising therapeutic compounds against drug-resistant Mycobacterium tuberculosis ß-ketoacyl-acyl carrier protein synthase I (KasA).

The emergence of new Mycobacterium tuberculosis (Mtb) strains resistant to the key drugs currently used in the clinic for tuberculosis treatment can substantially reduce the probability of therapy success, causing the relevance and importance of studies on the development of novel potent antibacterial agents targeting different vulnerable spots of Mtb. In this study, 28,860 compounds from the library of bioactive molecules were screened to identify novel potential inhibitors of ß-ketoacyl-acyl carrier protein synthase I (KasA), one of the key enzymes involved in the biosynthesis of mycolic acids of the Mtb cell wall. In doing so, we used a structure-based virtual screening approach to drug repurposing that included high-throughput docking of the C171Q KasA enzyme with compounds from the library of bioactive molecules including the FDA-approved drugs and investigational drug candidates, assessment of the binding affinity for the docked ligand/C171Q KasA complexes, and molecular dynamics simulations followed by binding free energy calculations. As a result, post-modeling analysis revealed 6 top-ranking compounds exhibiting a strong attachment to the malonyl binding site of the enzyme, as evidenced by the values of binding free energy which are significantly lower than those predicted for the KasA inhibitor TLM5 used in the calculations as a positive control. In light of the data obtained, the identified compounds are suggested to form a good basis for the development of new antitubercular molecules of clinical significance with activity against the KasA enzyme of Mtb.Communicated by Ramaswamy H. Sarma.

Repurposing Navitoclax to block SARS-CoV-2 fusion and entry by targeting heptapeptide repeat sequence 1 in S2 protein.

Along with the long pandemic of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has come the dilemma of emerging viral variants of concern (VOC), particularly Omicron and its subvariants, able to deftly escape immune surveillance and the otherwise protective effect of current vaccines and antibody drugs. We previously identified a peptide-based pan-CoV fusion inhibitor, termed as EK1, able to bind the HR1 region in viral spike (S) protein S2 subunit. This effectively blocked formation of the six-helix bundle (6-HB) fusion core and, thus, showed efficacy against all human coronaviruses (HCoVs). EK1 is now in phase 3 clinical trials. However, the peptide drug generally lacks oral availability. Therefore, we herein performed a structure-based virtual screening of the libraries of biologically active molecules and identified nine candidate compounds. One is Navitoclax, an orally active anticancer drug by inhibition of Bcl-2. Like EK1 peptide, it could bind HR1 and block 6-HB formation, efficiently inhibiting fusion and infection of all SARS-CoV-2 variants tested, as well as SARS-CoV and MERS-CoV, with IC50 values ranging from 0.5 to 3.7 µM. These findings suggest that Navitoclax is a promising repurposed drug candidate for development as a safe and orally available broad-spectrum antiviral drug to combat the current SARS-CoV-2 and its variants, as well as other HCoVs.

Species complexes and life cycles of digenetic trematodes from the family Derogenidae.

The best way to study digenean diversity combines molecular genetic methods, life-cycle studies and elaborate morphological descriptions. This approach has been barely used for one of the most widespread digenean taxa parasitizing fish ­ the superfamily Hemiuroidea. Here, we applied the integrative approach to the hemiuroideans from the family Derogenidae parasitizing fish at the White and Barents Seas. Analysis of 28S, 18S, 5.8S rDNA, ITS2 and cox1 gene sequences from sexually adult worms (maritae) showed genetic heterogeneity for 2 derogenid species known from this area: Derogenes varicus and Progonus muelleri. Thus, 2 pairs of genetic lineages were found: DV1 and DV2, PM1 and PM2, respectively. Data from other regions indicate that 2 more lineages of D. varicus probably exist. Based on previous records from the White and Barents Seas, we hypothesized that the cercariae found in the moonsnails (family Naticidae) belong to the Derogenidae and may help to differentiate these lineages as species. According to our results, Cercaria appendiculata from Cryptonatica affinis matched DV1, similar nameless cercariae from Euspira pallida and Amauropsis islandica matched DV2, and Cercaria octocauda from C. affinis matched PM1. We provide new data on the structure of these cercariae and discuss the life-cycle pattern of the studied digeneans.

The Pacific Notocotylus atlanticus (Digenea: Notocotylidae).

Recently two unidentified Notocotylus species have been recovered from a mallard in Hokkaido, Japan. Preliminary data indicated that one of them may belong to N. atlanticus-a species found in Europe and on the Atlantic coast of North America. Now we have sequenced partial cox1 for several European N. atlanticus isolates and performed a barcode gap analysis. It has shown the conspecificity of European and Japanese worms, bringing up discussion on distribution, transmission and host specificity in this species.

Life cycle truncation in Digenea, a case study of Neophasis spp. (Acanthocolpidae).

Truncated life cycles may emerge in digeneans if the second intermediate host is eliminated, and the first intermediate host, the mollusc, takes up its role. To understand the causes of this type of life cycle truncation, we analyzed closely related species of the genus Neophasis (Acanthocolpidae) with three-host and two-host life cycles. The life cycle of Neophasis anarrhichae involves two hosts: wolffishes of the genus Anarhichas as the definitive host and the common whelk Buccinum undatum as the intermediate host. Neophasis oculata, a closely related species with a three-host life cycle, would be a suitable candidate for the comparison, but some previous data on its life cycle seem to be erroneous. In this study, we aimed to redescribe the life cycle of N. oculata and to verify the life cycle of N. anarrhichae using molecular and morphological methods. Putative life cycle stages of these two species from intermediate hosts were linked with adult worms from definitive hosts using ribosomal molecular data: 18S, ITS1, 5.8S-ITS2, 28S. These markers did not differ within the species and were only slightly different between them. Intra- and interspecific variability was also estimated using mitochondrial COI gene. In the constructed phylogeny Neophasis spp. formed a common clade with two other genera of the Acanthocolpidae, Tormopsolus and Pleorchis. We demonstrated that the first intermediate hosts of N. oculata were gastropods Neptunea despecta and B. undatum (Buccinoidea). Shorthorn sculpins Myoxocephalus scorpius were shown to act as the second intermediate and definitive hosts of N. oculata. The previous reconstruction of the two-host life cycle of N. anarrhichae was reaffirmed. We suggest that life cycle truncation in N. anarrhichae was initiated by an acquisition of continuous morphogenesis in the hermaphroditic generation and supported by a strong prey-predator relationship between A. lupus and B. undatum.

It is marine: distinguishing a new species of Catatropis (Digenea: Notocotylidae) from its freshwater twin.

The morphology of sexual adults is the cornerstone of digenean systematics. In addition, life cycle data have always been significant. The integration of these approaches, supplemented with molecular data, has allowed us to detect a new species that many researchers may have previously seen, but not recognized. Sexual adults from common eiders that we found in northern European seas were extremely similar to other notocotylids, but the discovery of their intermediate host, a marine snail, revealed the true nature of this material. Here we describe sexual adults, rediae and cercariae of Catatropis onobae sp. nov. We discuss how 'Catatropis verrucosa' should be regarded, justify designation of the new species C. onobae for our material and explain why it can be considered a cryptic species. The phylogenetic position of C. onobae within Notocotylidae, along with other evidence, highlights the challenges for the taxonomy of the family, for which two major genera appear to be polyphyletic and life cycle data likely undervalued.

New type of xiphidiocercariae (Digenea: Microphalloidea) from South Vietnam.

We found unusual digenean intramolluscan stages, sporocysts and cercariae, in gastropods Sulcospira dautzenbergiana (Morelet) (Caenogastropoda: Pachychilidae) from Southern Vietnam and named them Cercaria cattieni 1. These cercariae have a stylet and thus belong to the Xiphidiata. However, such combination of characters as extremely large body size and I-shaped excretory bladder has not been found before in any other xiphidiocercariae. We obtained COI, ITS1, 5.8S + ITS2, and 28S rDNA sequences for C. cattieni 1. The latter allowed us to specify the phylogenetic position of the discovered cercariae: C. cattieni 1 falls within the superfamily Microphalloidea and is most closely grouped to Pachypsolus irroratus (Rudolphi, 1819) (Pachypsolidae), the sea turtle parasite. Information on the family Pachypsolidae is limited. Judging from the molecular phylogeny, C. cattieni 1 might be the larva of the Pachypsolidae, documented for the first time.

First elucidation of the life cycle in the family Brachycladiidae (Digenea), parasites of marine mammals.

Digeneans of the family Brachycladiidae are cosmopolitan parasites restricted to marine mammals. Their life cycles are unknown. Phylogenetically, Brachycladiidae are closely related to Acanthocolpidae, parasites of marine teleost fishes. Acanthocolpida typically possess three-host life cycles with gastropods of the superfamily Buccinoidea acting as the first intermediate hosts for most species, and either fishes or bivalves acting as the second intermediate hosts. A few species previously identified as Neophasis differ from other Acanthocolpidae in having naticid gastropods as first intermediate hosts, and both fishes and bivalves as second ones. We assumed that this may indicate an incorrect life cycle description and revised previous data on rediae and cercariae of Neophasis spp. from Cryptonatica affinis (Naticidae) and metacercariae from cardiid bivalves at the White Sea using molecular and morphological approaches. Sequence comparison showed that rediae and cercariae from C. affinis resembling some representatives of the genus Neophasis and metacercariae from bivalves resembling Neophasis oculata belong to the brachycladiid species Orthosplanchnus arcticus. Thus, the life cycle of O. arcticus proceeds as follows: seals serve as the definitive host, C. affinis as the first intermediate host and cardiid bivalves as the second. We found one more type of redia and cercaria in C. affinis which, by molecular evidence, also belongs to Brachycladiidae and is closely related to O. arcticus. Here we refer to them as Brachycladiidae gen. sp. 1 WS. We suggest that Brachycladiidae gen. sp. 1 WS may belong to either Orthosplanchnus or Odhneriella, with beluga whales possibly being the definitive host. Morphological features of O. arcticus and Brachycladiidae gen. sp. 1 WS cercariae are summarised and matched with published data on putatively brachycladiid cercariae. We compare and discuss the diversity of life cycle patterns among Brachycladiidae and Acanthocolpidae, and show that they differ not only in the type of definitive host, but also in both intermediate hosts.

Short communication: New data support phylogeographic patterns in a marine parasite Tristriata anatis (Digenea: Notocotylidae).

Intraspecific diversity in parasites with heteroxenous life cycles is guided by reproduction mode, host vagility and dispersal, transmission features and many other factors. Studies of these factors in Digenea have highlighted several important patterns. However, little is known about intraspecific variation for digeneans in the marine Arctic ecosystems. Here we analyse an extended dataset of partial cox1 and nadh1 sequences for Tristriata anatis (Notocotylidae) and confirm the preliminary findings on its distribution across Eurasia. Haplotypes are not shared between Europe and the North Pacific, suggesting a lack of current connection between these populations. Periwinkle distribution and anatid migration routes are consistent with such a structure of haplotype network. The North Pacific population appears ancestral, with later expansion of T. anatis to the North Atlantic. Here the parasite circulates widely, but the direction of haplotype transfer from the north-east to the south-west is more likely than the opposite. In the eastern Barents Sea, the local transmission hotspot is favoured.

Transatlantic discovery of Notocotylus atlanticus (Digenea: Notocotylidae) based on life cycle data.

Digenean parasites feature a series of stages with a distinct appearance, reproduction mode, and lifestyle that together constitute their well-known, complex life cycle. Species descriptions of Digenea have always been based on one of these stages-the marita, or sexually reproducing adult in the final host. However, in some cases, data on the life cycle are essential for the differential diagnosis of closely related species. Here, we present the case of Notocotylus atlanticus, where different stages of its life cycle were discovered for the first time since the species description, and across the Atlantic. We used a material from a naturally infected intertidal marine snail, Ecrobia ventrosa, and several waterfowl species and also carried out infection experiments. For morphological studies, we employed light microscopy, SEM, and CLSM; molecular data obtained include sequences of ITS1 and 28S rRNA gene. We demonstrate that N. atlanticus adult worm morphology is barely sufficient to distinguish it from several other species. Cercariae morphology and identity of the first intermediate hosts provide crucial additional information. According to our preliminary phylogenetic reconstructions, two notocotylid lineages are associated with two major gastropod lineages-the Caenogastropoda and the Heterobranchia. The traditional character to identify notocotylid genera (structure of ventral organs) fails to explain the phylogeny and thus requires reassessment. Further reliable morphological, life cycle and molecular data on other species are likely to reveal more patterns in notocotylid systematics, host specificity, and evolution.

Ventral concavity and musculature arrangement in notocotylid maritae (Digenea: Notocotylidae).

Maritae of family Notocotylidae have no ventral sucker, an organ that serves for attachment in many other digeneans. Instead, attachment is accomplished through the ventral concavity which is formed by the whole body. Musculature providing such an attachment was previously described only in part, and specific patterns of body-wall and internal muscle arrangement in notocotylids have never been studied before. In this paper we describe muscle system organization in maritae of three notocotylid species by means of fluorescent actin staining and confocal microscopy. A very special U-shaped pattern of body-wall longitudinal muscle fibres is found to support the ventral concavity. Together with very prominent dorsoventral musculature this pattern must play a key role in actions of ventral concavity. Other details concerning the muscle system are provided, including the musculature of ventral protrusions, oral sucker, walls of digestive tract and reproductive organs. We discuss functionality of the musculature and also make comparisons with cercariae of this family.

Musculature arrangement and locomotion in notocotylid cercariae (Digenea: Notocotylidae) from mud snail Ecrobia ventrosa (Montagu, 1803).

Cercariae of digenean family Notocotylidae are characterized by a set of morphological traits which make them easily distinguishable from any other. One of the key features is absence of ventral sucker. This affects basic ways of locomotion and attachment. To understand how these functions are fulfilled we studied musculature arrangement in cercariae of two species by means of fluorescent-phalloidin staining and confocal microscopy. We used Cercaria Notocotylidae sp. No. 11 and 12 Deblock, 1980 from mud snails Ecrobia (=Hydrobia) ventrosa. Information on gross morphology (especially body-tail junction) and basic behavioural patterns of these cercariae is also updated. Major special features of musculature are associated with the ventral concavity: extreme development of dorsoventral muscle fibres and formation of annular arrangement of longitudinal muscle fibres on the ventral side. Additional body-wall and internal muscle bundles in the anterior region are also specific for notocotylid cercariae and seem to play important role in twisting movements during substratum testing. Musculature of dorsal adhesive pockets, oral sucker and tail is also described. These results are discussed in relation to observed locomotory patterns.

Life cycle and biology of Tristriata anatis (Digenea: Notocotylidae): morphological and molecular approaches.

Notocotylids are common digeneans parasitising birds and mammals. They have a two-host life cycle with cercariae encysting in the open. Particular life cycles remain unknown for majority of notocotylid species, including a common parasite of sea ducks Tristriata anatis. Here we resolve the life cycle of T. anatis by means of D2 LSU, ITS1 and CO1 DNA sequence analysis, showing that the first intermediate hosts for this species are periwinkles Littorina spp. Morphological descriptions of rediae and cercariae are provided for the first time, and we also supplement the existing morphological data on adults. Apart from differential diagnosis, we discuss some features of cercariae and rediae biology, geographical distribution and host range. Our molecular data confirm that genus Tristriata is monotypic and that T. anatis has circumpolar distribution. CO1 sequence analysis has shown that isolation exists between the Atlantic and Pacific populations of T. anatis, suggesting that there are geographical races. We suppose that their formation may be linked to the Last Ice Age events, when trans-Arctic bird migrations ceased and periwinkle ranges shrunk. These made transfer of parasites across the Arctic impossible, and it still has not resumed. We discuss the possible influence of host vagility and adults' lifespan on digeneans' potential for geographical colonisation.

Genetic and environmental determinants of host use in the trematode Maritrema novaezealandensis (Microphallidae).

Factors constraining host specificity are poorly understood. Intraspecific variation in host preferences in generalist parasites may reveal which factors affect patterns of host use, and thus the evolution of specialization. Here, laboratory experiments examined genetic variation in host preferences and the effect of a refugium against infection on host use. Firstly, 6 cercarial clones of the trematode Maritrema novaezealandensis (ranging widely in heterozygosities) were exposed simultaneously to 2 alternative hosts, the amphipods Heterophoxus stephenseni and Paracalliope novizealandiae, to assess host preferences and fitness correlations with parasite heterozygosity. All clones showed a distinct preference for H. stephenseni, though the extent of this preference varied among clones. No clear association was found between heterozygosity and either parasite infection success or preference for a particular host. Secondly, cercariae were exposed to the same 2 amphipods in both the presence and absence of sand (refugium for H. stephenseni). Without sand, infection levels were significantly higher in H. stephenseni than in P. novizealandiae. With sand, H. stephenseni was able to hide, offsetting the parasite's intrinsic preferences for this host. These results demonstrate the existence of genetic variation in host preferences, as well as the effect of environmental variables on observed patterns of host use.