First record of Anopheles claviger (Meigen, 1804) (Diptera, Culicidae) in Karelia, Northwestern Russia.

Anopheles claviger (Meigen, 1804) (Diptera, Culicidae) is widespread in the western Palaearctic Region, but it was recorded in Karelia (Russia) for the first time. This record is one of the northernmost ones in the Palaearctic Region and Russia, updates the northern border of the An. claviger range. Mosquitoes were collected from July to September 2023 in the southern Karelia (the village of Gomselga, Kondopoga District, and Petrozavodsk) using Krishtal trap (from human) and Mosquito Magnet® trap (Pioneer design, Octenol as attractant). Seven females of An. claviger were collected in Gomselga; one specimen was sampled from Petrozavodsk City parks. Morphological identification of eight females was verified by COI and ITS2 sequences. Phylogenetic analysis of ITS2 and COI sequences confirmed the collected specimens to An. claviger s. s., clustering in both cases in a strongly supported clade clearly differentiated from the closely related species An. petragnani. The high diversity of An. claviger haplotypes from Karelia is in agreement with data from other geographical regions and shows that the records of this species in Gomselga and Petrozavodsk are not accidental.

Effect of cryopreservation on Odc1 and RhoA genes expression in diapausing mouse blastocysts.

The possibility of embryo cryopreservation is important for applying the genome resource banking (GRB) concept to those mammalian species that exhibit embryonal diapause in their early development. Odc1 encodes ODC1, which is a key enzyme in polyamine synthesis. RhoA is an essential part of Rho/ROCK system. Both Odc1 and RhoA play an important role in preimplantation embryo development. Studying these systems in mammalian species with obligate or experimentally designed embryonic diapause may provide insight into the molecular machinery underlying embryo dormancy and re-activation. The effect of cryopreservation procedures on the expression of the Odc1 and RhoA in diapausing embryos has not been properly studied yet. The purpose of this work is to address the possibility of cryopreservation diapausing embryos and to estimate the expression of the Odc1 and RhoA genes in diapausing and non-diapausing embryos before and after freeze-thaw procedures using ovariectomized progesterone treated mice as a model. Both diapausing and non-diapausing in vivo-derived embryos continued their development in vitro after freezing-thawing as evidenced by blastocoel re-expansion. Although cryopreservation dramatically decreased the expression of the Odc1 and RhoA genes in non-diapausing embryos, no such effects have been observed in diapausing embryos where these genes were already at the low level before freeze-thaw procedures. Future studies may attempt to facilitate the re-activation of diapausing embryos, for example frozen-thawed ones, specifically targeting Odc1 or Rho/ROCK system.

Single-Standard Quantification Strategy for Lignin Dimers by Supercritical Fluid Chromatography with Charged Aerosol Detection.

The increased interest in utilizing lignin as a feedstock to produce various aromatic compounds requires advanced chemical analysis methods to provide qualitative and quantitative characterization of lignin samples along different technology streamlines. However, due to the lack of commercially available chemical standards, routine quantification of industrially relevant lignin oligomers in complex lignin samples remains a challenge. This study presents a novel method for universal quantification of lignin dimers based on supercritical fluid chromatography with charged aerosol detection (CAD). A series of lignin-derived dimeric compounds that have been reported from reductive catalytic fractionation (RCF) were synthesized and used as standards. The applicability of using linear regression instead of quadratic calibration curves was evaluated over a concentration range of 15-125 mg/L, demonstrating that the former calibration method is as appropriate as the latter. The response factors of lignin dimeric compounds were compared to assess the uniformity of the CAD signal, revealing that the CAD response for the tested lignin dimers did not differ substantially. It was also found that the response factors were not dependent on the number of methoxy groups or linkage motifs, ultimately enabling the use of only one calibrant for these compounds. The importance of chromatographic peak resolution in CAD was stressed, and the use of a digital peak sharpening technique was adopted and applied to address this challenge. The developed method was verified and used for the quantification of lignin dimers in an oil obtained by a RCF of birch sawdust.

Phylogenetic assessment of some Palearctic Allocreadium spp. (Trematoda, Gorgoderoidea: Allocreadiidae).

The genus Allocreadium is a group of digenetic trematodes whose adult representatives mainly parasitize the intestines of freshwater fishes. The aim of this research is to reconstruct the phylogeny of four Palearctic species of this genus, Allocreadium dogieli, Allocreadium isoporum, Allocreadium papilligerum, and Allocreadium sp. ex Oreoleuciscus potanini from Mongolia. The DNA sequences of the 28S rRNA gene and the rDNA ITS2 region were obtained and then analyzed for phylogenetic inference. The analysis is complemented with the morphological descriptions for all four species. Phylogenetic analyses show that the newly obtained isolate of A. isoporum appeared to be genetically similar to previously obtained isolates of A. isoporum. Allocreadium dogieli probably belongs to the same evolutionary lineage of Allocreadium as A. crassum, while A. papilligerum to the same evolutionary lineage as Alocreadium transversale ex Cobitis taenia from Lithuania, but the detailed species composition of these lineages requires further elucidation. Allocreadium sp. was genetically close to Allocreadium sp. ex P. phoxinus from Primorski Krai, Russia, and a group of these Allocreadium had a sister relationship with Allocreadium khankaiensis. Our findings contradict some recent hypotheses about the phylogeography of Allocreadium spp.

Another plea for 'best practice' in molecular approaches to trematode systematics: Diplostomum sp. clade Q identified as Diplostomum baeri Dubois, 1937 in Europe.

DNA sequence data became an integral part of species characterization and identification. Still, specimens associated with a particular DNA sequence must be identified by the use of traditional morphology-based analysis and correct linking of sequence and identification must be ensured. Only a small part of DNA sequences of the genus Diplostomum (Diplostomidae) is based on adult isolates which are essential for accurate identification. In this study, we provide species identification with an aid of morphological and molecular (cox1, ITS-5.8S-ITS2 and 28S) characterization of adults of Diplostomum baeri Dubois, 1937 from naturally infected Larus canus Linnaeus in Karelia, Russia. Furthermore, we reveal that the DNA sequences of our isolates of D. baeri are identical with those of the lineage Diplostomum sp. clade Q , while other sequences labelled as the 'D. baeri' complex do not represent lineages of D. baeri. Our new material of cercariae from Radix balthica (Linnaeus) in Ireland is also linked to Diplostomum sp. clade Q. We reveal that D. baeri is widely distributed in Europe; as first intermediate hosts lymnaeid snails (Radix auricularia (Linnaeus), R. balthica) are used; metacercariae occur in eye lens of cyprinid fishes. In light of the convoluted taxonomy of D. baeri and other Diplostomum spp., we extend the recommendations of Blasco-Costa et al. (2016, Systematic Parasitology 93, 295­306) for the 'best practice' in molecular approaches to trematode systematics. The current study is another step in elucidating the species spectrum of Diplostomum based on integrative taxonomy with well-described morphology of adults linked to sequences.

New record of Tylodelphys metacercariae (Diplostomidae) from Perccottus glenii (Odontobutidae) and their phylogenetic assessment.

Metacercariae of Tylodelphys sp. were found in the abdominal cavity of the Chinese sleeper (Perccottus glenii) collected in Liaoning Province and Inner Mongolia Autonomous Region of China. The sequences of the mitochondrial cox1 gene and ribosomal ITS1-5.8S rDNA-ITS2 region were obtained and used for molecular identification and phylogenetic assessment of this parasite species. Results of phylogenetic analyses based on ITS and cox1 markers showed that the metacercariae of Tylodelphys sp. ex P. glenii from China were conspecific with specimens of Tylodelphys sp. collected by Sokolov et al. (2013) from the same fish-host species captured earlier in West Siberia, Russia. The examined Tylodelphys sp. ex. P. glenii is the only member of the genus whose metacercariae parasitise the abdominal cavity of fish in northern Eurasia. Tylodelphys sp. ex P. glenii clustered with T. darbyi, T. immer, T. podicipina, and Tylodelphys sp. of Soldánová et al., 2017 based on mitochondrial DNA markers, and with T. darbyi, T. immer, T. kuerepus, and T. schreuringi using nuclear DNA markers.

Myxidium shedkoae Sokolov, 2013 (Myxozoa: Myxidiidae), a parasite of the gallbladder of Perccottus glenii Dybowski, 1877 (Actinoptrygii: Odontobutidae): Supplementary data on morphology and phylogenetic position based on 18s rDNA sequence analysis.

This paper is the first report on the molecular characterisation of myxozoan parasites from the odontobutid fish Chinese (Amur) sleeper (Perccottus glenii Dybowski, 1877). The authors determined the partial 18S rDNA sequence of Myxidium shedkoae Sokolov, 2013 from the gallbladder of the fish. Phylogenies reconstructed using maximum likelihood and Bayesian inference analysis revealed that M. shedkoae belongs to the hepatic biliary group of myxozoans (after Kristmundsson and Freeman, 2013) as a member of the clade consisting of Zschokkella sp. KLT-2014, Myxidium truttae and Zschokkella nova. Some new morphological features of the parasite are also presented.

Nematodes in the mallard (Anas platyrhynchos Linnaeus, 1758) and the common goldeneye (Bucephala clangula Linnaeus, 1758) (Anatidae) from Northern Europe.

There are first data on nematodes of Anas platyrhynchos Linnaeus, 1758 (mallard) and Bucephala clangula Linnaeus, 1758 (common goldeneye) from Northern Europe (Ladoga Lake region). The ducks were found to be infected with nine nematode species. A. platyrhynchos hosted eight nematode species and B. clangula was host to four nematode species. All species except Capillaria anatis were found in the region for the first time. Nematodes Amidostomum acutum, Streptocara crassicauda, and Tetrameres fissispina parasitized on both hosts and were the most abundant. The biggest number of parasites revealed was biohelminths with a direct life cycle.

Program freezing of diapausing embryos in the mouse.

Embryonal diapause is a characteristic feature of about 130 mammalian species. However, very few studies have addressed cryopreservation of diapausing embryos. This work is aimed to apply program freezing to blastocysts obtained from CD1 mice, which were at diapause state after ovariectomy and the subsequent hormonal therapy. Blastocysts collected from non-operated mice of the same strain served as controls. Some diapausing as well as non-diapausing frozen-thawed blastocysts demonstrated blastocoel re-expansion after 24 h of in vitro culture (IVC) indicating their viability after cryopreservation. Raman spectroscopy assessment of phenylalanine accumulation revealed that the fraction of new synthesized proteins was lower for non-frozen as well as for frozen-thawed diapausing blastocysts compared to non-diapausing ones. Although protein metabolism was reduced in diapausing embryos, most of the protein synthesis remained active. Cell number increased after 24 h of IVC in non-frozen as well as in the frozen-thawed blastocysts of the control but not of the diapause group. However, cell numbers were increased in frozen-thawed diapausing blastocysts after 47 h of IVC in a medium supplemented with putrescine. This indicates viability of frozen-thawed diapausing embryos after cryopreservation. Besides, protein metabolism was not affected by cryopreservation in diapausing and non-diapausing murine embryos indicating their viability. Our results demonstrated the possibility of successful cryopreservation of diapausing murine embryos.

New Salinity Tolerant Species of Gyrodactylus (Platyhelminthes, Monogenea) on Intertidal and Supratidal Fish Species from the Chilean Coast.

PURPOSE: The intertidal and supratidal coastal zone challenges the osmoregulatory capacity of aquatic inhabitants. Four new species of Gyrodactylus ectoparasites on two intertidal fishes from Chile are described based on molecular and morphological analyses. METHODS: Monogeneans were found from two fish species, the clingfish Sicyases sanguineus Müller & Troschel, 1843 and the combtooth blenny Scartichthys viridis Valenciennes, 1836. The morphology was described by drawings, and minimal measurements. The parasites were barcoded via the sequencing of the ribosomal DNA over ITS1-5.8S-ITS2. RESULTS: The air-breathing clingfish S. sanguineus carried Gyrodactylus amphibius sp. nov., hiding in the ventral sucker formed by the modified pectoral fins of the fish. The intertidal combtooth blenny S. viridis carried three other new species: Gyrodactylus scartichthi sp. nov., Gyrodactylus viridae sp. nov., and Gyrodactylus zietarae sp. nov. CONCLUSION: The four new species were all phylogenetically related with the previously described G. chileani Zietara et al. 2012 on triplefin Helcogrammoides chilensis Cancino, 1960 in the same habitat. Thus, the five Chilean Pacific Gyrodactylus species formed a statistically well-supported (100%) monophyletic clade together with three geographically distant species recorded in Europe. The Chilean Pacific parasites are not related to G. salinae and G. magadiensis, parasites described in extreme osmotic stress environments earlier.

First Description of the Metacercaria of Nematostrigea serpens serpens (Nitzsch, 1819) (Trematoda, Strigeidae) and Phylogenetic Affiliation of Nematostrigea vietnamiensis Zhokhov & Mishina, 2011.

BACKGROUND: The genus Nematostrigea comprises trematodes parasitising in fish-eating birds of Europe, Africa, and North America. Their life cycle is unknown. PURPOSE: To provide the first description of metacercariae of Nematostrigea serpens serpens, a nominative subspecies of the type species of Nematostrigea, and to record metacercaria of Nematostrigea vietnamiensis, with molecular data. METHODS: Encysted metacercariae of N. serpens serpens and N. vietnamiensis were collected from fish in Russia and Vietnam, respectively, and were processed, identified, and documented using standard morphological techniques. The 28S rRNA gene of metacercariae of both species and the cox1 gene of metacercariae an earlier studied adult of N. serpens serpens were sequenced and used for phylogenetic analysis. RESULTS: Metacercaria of N. serpens serpens have a spatulate body with a foliate forebody and a short hindbody, two long lateral rectilinear pseudosuckers, and the holdfast organ with bifurcated anterior and entire posterior external lobes. The analysis of the cox1and 28S rRNA gene sequences unequivocally showed the conspecificity of metacercariae and adult stage of N. serpens serpens. Based on the 28S rRNA gene sequence-based phylogenetic analysis, the genus Nematostrigea is a sister taxon to the group of strigeid genera Cotylurus + Ichthyocotylurus. Morphological and phylogenetic data demonstrated that N. vietnamiensis does not belong to the genus Nematostrigea and is possibly be affiliated with the crassiphialine trematodes. CONCLUSION: This is the first record of metacercariae of N. serpens serpens. N. vietnamiensis, renamed Prodiplostomulum vietnamiense comb. nov., must be moved to the crassiphialine 'Prodiplostomulum'-type metacercariae group.

Morphological and molecular differentiation of Diplostomum spp. metacercariae from brain of minnows (Phoxinus phoxinus L.) in four populations of northern Europe and East Asia.

Metacercariae of trematodes from the genus Diplostomum are major helminth pathogens of freshwater fish, infecting the eye or the brain. The taxonomy of the genus Diplostomum is complicated, and has recently been based mainly on the molecular markers. In this study, we report the results of the morphological and molecular genetic analysis of diplostomid metacercaria from the brain of the minnow Phoxinus phoxinus from three populations in Fennoscandia (Northern Europe) and one population in Mongolia (East Asia). We obtained the data on the polymorphism of the partial mitochondrial cox1 gene and ribosomal ITS1-5.8S-ITS2 region of these parasites. РСА-based morphological analysis revealed that the parasites in the Asian and the European groups of Diplostomum sp. were distinctly different. Metacercariae from the brain of Mongolian minnows were much larger than those from the brain of Fennoscandian minnows but had much fewer excretory granules. Considering that the two study regions were separated by a distance of about 4500 km, we also tested the genetic homogeneity of their host, the minnow, using the mitochondrial cytb gene. It was shown that Diplostomum-infected minnows from Mongolia and Fennoscandia represented two previously unknown separate phylogenetic lineages of the genus Phoxinus. Both molecular and morphological analysis demonstrated that the parasites from Fennoscandia belonged the species Diplostomum phoxini, while the parasites from Mongolia belonged to a separate species, Diplostomum sp. MТ.Each of the two studied Diplostomum spp. was associated with a specific, and previously unknown, genealogical lineage of its second intermediate host, P. phoxinus.

Redescription of Proctophantastes gillissi (Overstreet et Pritchard, 1977) (Trematoda: Zoogonidae) with discussion on the systematic position of the genus Proctophantastes Odhner, 1911.

The redescription of Proctophantastes gillissi (Overstreet et Pritchard, 1977) (Trematoda: Zoogonidae) was made on specimens collected from Muraenolepis marmorata Günther, 1880 (Gadiformes) caught in the Ross Sea and the Amundsen Sea (Antarctic). The fish is a new host of this parasite. Phylogenetic relations of P. gillissi were inferred by Maximum Likelihood and Bayesian inference analysis of partial sequences from 28S rDNA. The findings from analysis of the molecular data are not consistent with the traditional point of view about the position of the genus Proctophantastes Odhner, 1911 in the subfamily Lepidophyllinae. The taxonomical position of the genus needs further revision.