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This research examines the rapid spread of the oak lace bug, Corythucha arcuata (Say, 1832), in Slovakia from 2020 to 2022. A native of the Nearctic region, this pest was first detected in Europe back in 2000 and has since extended across considerable part of the continent. Monitoring efforts revealed its presence in Slovakia in 2018, with subsequent surveys showing its distribution through the southern region and a northward expansion. The pest mainly infests oak trees, and in 2022, it was seen there on as many as 87% of the plots. Out of a total of 233 locations visited in the period 2020–2022, the bug was found on 169 sites, which represents 72.5%. The study identified the pedunculate and Turkey oak as primary host-plants. The results provided a clear trend from the bugs moving further north with each passing year, covering an estimated total distance of 84.5 km over the three-year timeframe. Factors influencing its spread included passive transport via vehicles and possible wind-assisted dispersal on a local scale. The study also highlights the need for continuous monitoring and management strategies to mitigate its impact on oak ecosystems.

Resistance of horse chestnut Aesculus hippocastanum ‘Mertelík’ to Cameraria ohridella was proven in the trial under semi-field conditions. Seedlings of vulnerable and resistant trees were grown together in rearing cages and were exposed to the strong attack from C. ohridella larvae. Significant differences in leaflet injuries were observed between susceptible and insect-resistant trees. Variety ‘Mertelík’ showed almost none or very small damages compared to high defoliation level in case of standard trees. The results confirmed that this resistant clone could be suitable for use under field conditions.

Coniferous forests in Europe have a considerable number of pests that attack trees of all ages from youngest up to oldest ones. One of them is the large pine weevil Hylobius abietis. This species is widespread in Europe and occurs from warm southern areas (Spain) up to northern countries (Sweden, Finland). Larvae of this species do not cause damage, they help in the decomposition process of stumps and roots. Adults are harmful as they consume bark of young coniferous seedlings, above all of spruce and pine. One adult can consume on average 75 mm2 of bark per day. Individuals can live up to 4 years, and during their lives they can damage and kill several tens of coniferous seedlings. Traditional chemical protection of seedlings against this pest is and will gradually be more and more restricted or forbidden. In this review, we describe this method as well as all other alternative methods including biological protection. We estimate that H. abietis causes annual damages in Europe of almost 120 million € and damages several tens of thousands of hectares of young forest stands.

The gypsy moth is one of the most serious pests in forests and fruit tree plantations over prevailing parts of the Northern Hemisphere. This work is based on a literature review, and presents history of gypsy moth Lymantria dispar L., observed in Slovak forests within the period 1945–2020. The life cycle, hosts, natural enemies, population dynamics of pests, impact of outbreaks on forests and different management methods used in the past are discussed. Since 1945, there were nine gypsy moth outbreaks in Slovakia. Between 1945 and 2020, a total of 155,034 ha of deciduous forests were touched with varying intensity, representing an average annual damage of 2,040 ha. The strongest outbreak culminated in 2004. Totally 51,479 ha were attacked in the period of 2000–2008. We have found outbreak periods that repeat with frequency of 7.8 ±2.2 years and the average outbreak phase lasts 3.1 ±1.1 years. The period between two subsequent outbreaks seems to be more or less constant and duration of the outbreak phase seems to be gradually shortened during the study period. Several factors influencing the gypsy moth population dynamics in Slovakia are discussed. The role of biological control by using entomopathogenic fungus Entomophaga maimaiga is described.

Schistosome invasive stages, cercariae, leave intermediate snail hosts, penetrate the skin of definitive hosts, and transform to schistosomula which migrate to the final location. During invasion, cercariae employ histolytic and other bioactive products of specialized holocrine secretory cells - postacetabular (PA) and circumacetabular (CA) penetration glands. Although several studies attempted to characterize protein composition of the in vitro-induced gland secretions in Schistosoma mansoni and Schistosoma japonicum, the results were somewhat inconsistent and dependent on the method of sample collection and processing. Products of both gland types mixed during their secretion did not allow localization of identified proteins to a particular gland. Here we compared proteomes of separately isolated cercarial gland cells of the avian schistosome Trichobilharzia szidati, employing laser-assisted microdissection and shotgun LC-MS/MS, thus obtaining the largest dataset so far of the representation and localization of cercarial penetration gland proteins. We optimized the methods of sample processing with cercarial bodies (heads) first. Alizarin-pre-stained, chemically non-fixed samples provided optimal results of MS analyses, and enabled us to distinguish PA and CA glands for microdissection. Using 7.5 × 10 6  μm 3 sample volume per gland replicate, we identified 3347 peptides assigned to 792 proteins, from which 461 occurred in at least two of three replicates in either gland type (PA = 455, 40 exclusive; CA = 421, six exclusive; 60 proteins differed significantly in their abundance between the glands). Peptidases of five catalytic types accounted for ca. 8% and 6% of reliably identified proteins in PA and CA glands, respectively. Invadolysin, nardilysin, cathepsins B2 and L3, and elastase 2b orthologs were the major gland endopeptidases. Two cystatins and a serpin were highly abundant peptidase inhibitors in the glands. While PA glands generally had rich enzymatic equipment, CA glands were conspicuously abundant in venom allergen-like proteins., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Helminth neuroinfections represent serious medical conditions, but the diversity of the host-parasite interplay within the nervous tissue often remains poorly understood, partially due to the lack of laboratory models. Here, we investigated the neuroinvasion of the mouse spinal cord by Trichobilharzia regenti (Schistosomatidae). Active migration of T. regenti schistosomula through the mouse spinal cord induced motor deficits in hindlimbs but did not affect the general locomotion or working memory. Histological examination of the infected spinal cord revealed eosinophilic meningomyelitis with eosinophil-rich infiltrates entrapping the schistosomula. Flow cytometry and transcriptomic analysis of the spinal cord confirmed massive activation of the host immune response. Of note, we recorded striking upregulation of the major histocompatibility complex II pathway and M2-associated markers, such as arginase or chitinase-like 3. Arginase also dominated the proteins found in the microdissected tissue from the close vicinity of the migrating schistosomula, which unselectively fed on the host nervous tissue. Next, we evaluated the pathological sequelae of T. regenti neuroinvasion. While no demyelination or blood-brain barrier alterations were noticed, our transcriptomic data revealed a remarkable disruption of neurophysiological functions not yet recorded in helminth neuroinfections. We also detected DNA fragmentation at the host-schistosomulum interface, but schistosomula antigens did not affect the viability of neurons and glial cells in vitro. Collectively, altered locomotion, significant disruption of neurophysiological functions, and strong M2 polarization were the most prominent features of T. regenti neuroinvasion, making it a promising candidate for further neuroinfection research. Indeed, understanding the diversity of pathogen-related neuroinflammatory processes is a prerequisite for developing better protective measures, treatment strategies, and diagnostic tools., Competing Interests: The authors have declared that no competing interests exist.

Schistosomula (the post-infective stages) of the neurotropic schistosome Trichobilharzia regenti possess multiple isoforms of cathepsin B1 peptidase (TrCB1.1-TrCB1.6) with involvement in nutrient digestion. The comparison of substrate preferences of TrCB1.1 and TrCB1.4 showed that TrCB1.4 had a very narrow substrate specificity and after processing it was less effective toward protein substrates when compared to TrCB1.1. Self-processing of both isoforms could be facilitated by sulfated polysaccharides due to a specific binding motif in the pro-sequence. Trans-activation by heterologous enzymes was also successfully employed. Expression profiling revealed a high level of transcription of genes encoding the enzymatically inactive paralogs TrCB1.5 and TrCB1.6. The transcription level of TrCB1.6 was comparable with that of TrCB1.1 and TrCB1.2, the most abundant active isoforms. Recombinant TrCB1.6wt, a wild type paralog with a Cys 29 -to-Gly substitution in the active site that renders the enzyme inactive, was processed by the active TrCB1 forms and by an asparaginyl endopeptidase. Although TrCB1.6wt lacked hydrolytic activity, endopeptidase, but not dipeptidase, activity could be restored by mutating Gly 29 to Cys 29 . The lack of exopeptidase activity may be due to other mutations, such as His 110 -to-Asn in the occluding loop and Asp 224 -to-Gly in the main body of the mature TrCB1.6, which do not occur in the active isoforms TrCB1.1 and TrCB1.4 with exopeptidase activity. The catalytically active enzymes and the inactive TrCB1.6 paralog formed complexes with chicken cystatin, thus supporting experimentally the hypothesis that inactive paralogs could potentially regulate the activity of the active forms or protect them from being inhibited by host inhibitors. The effect on cell viability and nitric oxide production by selected immune cells observed for TrCB1.1 was not confirmed for TrCB1.6. We show here that the active isoforms of TrCB1 have different affinities for peptide substrates thereby facilitating diversity in protein-derived nutrition for the parasite. The inactive paralogs are unexpectedly highly expressed and one of them retains the ability to bind cystatins, likely due to specific mutations in the occluding loop and the enzyme body. This suggests a role in sequestration of inhibitors and protection of active cysteine peptidases., (Copyright © 2020 Dvořáková, Leontovyč, Macháček, O'Donoghue, Šedo, Zdráhal, Craik, Caffrey, Horák and Mikeš.)

Strigeid trematodes of the genus Apatemon Szidat, 1928 are intestinal parasites of fish-eating birds, utilizing various fish species as second intermediate hosts. In this study, we report morphometrical and molecular characterization of Apatemon sp. metacercariae parasitizing killifish Nothobranchius furzeri (Cyprinodontiformes: Nothobranchiidae) in south-east Mozambique. Metacercariae obtained from the cerebral cavity of killifish and two adult individuals isolated from experimentally infected ducklings were used for detailed morphological and molecular description, both resulting in generic affiliation to Apatemon. This is the first molecularly confirmed record of this trematode genus in Africa. Considering the morphological variability and wide host range of individual Apatemon species, the combination of both morphological and molecular analyses is indispensable for valid identification of this parasite. The results of our molecular analysis together with phylogenetic reconstruction indicated the presence of a new African lineage, reflecting potentially high diversity within the genus Apatemon comparable with other digenean genera., (Copyright © 2019. Published by Elsevier B.V.)

Trichobilharzia species are parasitic flatworms (called schistosomes or flukes) that cause important diseases in birds and humans, but very little is known about their molecular biology. Here, using a transcriptomics-bioinformatics-based approach, we explored molecular aspects pertaining to the nutritional requirements of Trichobilharzia szidati ('visceral fluke') and T. regenti ('neurotropic fluke') in their avian host. We studied the larvae of each species before they enter (cercariae) and as they migrate (schistosomules) through distinct tissues in their avian (duck) host. Cercariae of both species were enriched for pathways or molecules associated predominantly with carbohydrate metabolism, oxidative phosphorylation and translation of proteins linked to ribosome biogenesis, exosome production and/or lipid biogenesis. Schistosomules of both species were enriched for pathways or molecules associated with processes including signal transduction, cell turnover and motility, DNA replication and repair, molecular transport and/or catabolism. Comparative informatic analyses identified molecular repertoires (within, e.g., peptidases and secretory proteins) in schistosomules that can broadly degrade macromolecules in both T. szidati and T. regenti, and others that are tailored to each species to selectively acquire nutrients from particular tissues through which it migrates. Thus, this study provides molecular evidence for distinct modes of nutrient acquisition between the visceral and neurotropic flukes of birds.

Background: Representatives of the trematode family Fasciolidae are responsible for major socio-economic losses worldwide. Fascioloides magna is an important pathogenic liver fluke of wild and domestic ungulates. To date, only a limited number of studies concerning the molecular biology of F. magna exist. Therefore, the objective of the present study was to determine the complete mitochondrial (mt) genome sequence of F. magna, and assess the phylogenetic relationships of this fluke with other trematodes based on the mtDNA dataset., Findings: The complete F. magna mt genome sequence is 14,047 bp. The gene content and arrangement of the F. magna mt genome is similar to those of Fasciola spp., except that trnE is located between trnG and the only non-coding region in F. magna mt genome. Phylogenetic relationships of F. magna with selected trematodes using Bayesian inference (BI) was reconstructed based on the concatenated amino acid sequences for 12 protein-coding genes, which confirmed that the genus Fascioloides is closely related to the genus Fasciola; the intergeneric differences of amino acid composition between the genera Fascioloides and Fasciola ranged 17.97-18.24 %., Conclusions: The determination of F. magna mt genome sequence provides a valuable resource for further investigations of the phylogeny of the family Fasciolidae and other trematodes, and represents a useful platform for designing appropriate molecular markers.

Infection with Fascioloides magna (Digenea) causes serious damage to liver tissue in definitive hosts represented by ruminants, especially cervids. The distribution of F. magna includes the indigenous areas in North America, and the areas to which F. magna was introduced-Central Europe, Southeast Europe, and Italy. The North American intermediate host of F. magna, the freshwater snail Pseudosuccinea columella (Lymnaeidae), is an invasive species recorded in South America, the Caribbean, Africa, Australia, and west and Southeast Europe. In Europe, Galba truncatula is the snail serving for transmission, but P. columella has potential to become here a new intermediate host of F. magna. Little is known about interactions between F. magna and P. columella. In this study, the susceptibility of P. columella (Oregon, USA) to the infection by a single miracidium of the Czech strain of F. magna and the influence of F. magna on snail fecundity, shell height, and survival were evaluated. The data show that the Oregon strain of P. columella is a highly suitable host for the Czech strain of F. magna, with the infection rate of 74 %. In addition, a negative effect on survival rate of infected snails was recorded only in the late phase of infection. The infection was accompanied by a major reduction in egg mass production and by a decrease in the number of eggs per egg mass. The shell height of infected snails did not significantly differ from that in unexposed controls.

To date, most molecular investigations of schistosomatids have focused principally on blood flukes (schistosomes) of humans. Despite the clinical importance of cercarial dermatitis in humans caused by Trichobilharzia regenti and the serious neuropathologic disease that this parasite causes in its permissive avian hosts and accidental mammalian hosts, almost nothing is known about the molecular aspects of how this fluke invades its hosts, migrates in host tissues and how it interacts with its hosts' immune system. Here, we explored selected aspects using a transcriptomic-bioinformatic approach. To do this, we sequenced, assembled and annotated the transcriptome representing two consecutive life stages (cercariae and schistosomula) of T. regenti involved in the first phases of infection of the avian host. We identified key biological and metabolic pathways specific to each of these two developmental stages and also undertook comparative analyses using data available for taxonomically related blood flukes of the genus Schistosoma. Detailed comparative analyses revealed the unique involvement of carbohydrate metabolism, translation and amino acid metabolism, and calcium in T. regenti cercariae during their invasion and in growth and development, as well as the roles of cell adhesion molecules, microaerobic metabolism (citrate cycle and oxidative phosphorylation), peptidases (cathepsins) and other histolytic and lysozomal proteins in schistosomula during their particular migration in neural tissues of the avian host. In conclusion, the present transcriptomic exploration provides new and significant insights into the molecular biology of T. regenti, which should underpin future genomic and proteomic investigations of T. regenti and, importantly, provides a useful starting point for a range of comparative studies of schistosomatids and other trematodes.

Background: Fascioloides magna is a pathogenic fluke introduced to Europe ca 140 years ago. As it is spreading over the continent, new intermediate and definitive hosts might be involved in transmission of the parasite. In Europe, several studies reported potential new intermediate snail hosts (Radix spp.) for F. magna, and also several cases of fascioloidosis of wild and domestic animals were published. However, the data based on molecular and histological analyses confirming these findings remained unreported. This study aims to refer to unique findings of F. magna in European snails and domestic animals (the first observation in the Czech Republic in the last 30 years) and demonstrate the use of molecular techniques in determination of F. magna., Results: Two snails of R. labiata naturally infected with F. magna were found; mature cercariae and daughter rediae were observed. Maturity of cercariae was checked by histological methods, however, their ability to encyst was not confirmed. Co-infection of F. magna and Fasciola hepatica in the liver of two highland cattle bulls was proved. Adult fasciolid flukes producing eggs were found in the liver pseudocysts (F. magna) and the bile ducts (F. hepatica). Identification of intermediate hosts, intramolluscan stages, adult flukes and eggs was performed by sequencing the ITS2 region. Connection of F. magna pseudocysts with the gut (via the bile ducts) was not confirmed by means of histological and coprological examinations., Conclusions: For the first time, Radix labiata was confirmed as the snail host for F. magna under natural conditions and, together with the finding of F. magna infection in cattle, we can expect further transmission of F. magna from wildlife to livestock in localities shared by these hosts.

Lymnaeid snails of the genus Radix serve as intermediate hosts of some schistosomes and fasciolids. In Europe, delineation of species within the genus Radix is unresolved and, therefore, spectrum of snail hosts susceptible to trematode infections is under discussion. We used and compared three criteria for species delineation using snails collected at 43 localities. (a) Sequence analysis of ITS-2 rDNA disclosed that the collected snails belong to four species - R. auricularia (Linnaeus, 1758), R. peregra (Müller, 1774), R. lagotis (Schrank, 1803) and R. labiata (Rossmaessler, 1835) (criteria and names are based on the work of Bargues et al. 2001). Occurrence of R. peregra in the Czech Republic was confirmed by molecular data for the first time. (b) Characterization of reproductive system disclosed differences in location, size and shape of bursa copulatrix and its ductus. Unfortunately, some R. labiata specimens shared morphological features of reproductive organs with R. lagotis. (c) Statistical analysis of shell morphology proved that significant differences exist among particular species. One prediction model showed that correct classification of species may be achieved in 82-84% of cases. However, identification of individual snails in the field (without knowledge of respective snail population and use of statistical tools) still remains a complicated issue due to overlaps of shell characteristics. Concerning the role in trematode transmission, R. lagotis, R. labiata and R. peregra are susceptible to Trichobilharzia regenti. Also, successful experimental infections of R. lagotis and R. labiata by Fascioloides magna were accomplished.