Distribution and molecular phylogeny of biliary trematodes (Opisthorchiidae) infecting native Lutra lutra and alien Neovison vison across Europe

The recent identification of Pseudamphistomum truncatum, (Rudolphi, 1819) (Trematoda: Opisthorchiidae) and Metorchis bilis (Braun, 1790) Odening, 1962 (synonymous with Metorchis albidus (Braun, 1893) Loos, 1899 and Metorchis crassiusculus (Rudolphi, 1809) Looss, 1899 (Trematoda: Opisthorchiidae)) in otters from Britain caused concern because of associated biliary damage, coupled with speculation over their alien status. Here, we investigate the presence, intensity and phylogeny of these trematodes in mustelids (principally otters) across Europe (Czech Republic, Denmark, France, Germany, Norway, Poland and Sweden and Britain). The trematodes were identified to species using the internal transcribed spacer II (ITS2) locus. Both parasites were found across Europe but at unequal frequency. In the German state of Saxony, eight out of eleven (73%) otters examined were infected with P. truncatum whilst this parasite was not found in either mink from Scotland (n = 40) or otters from Norway (n = 21). Differences in the phylogenies between the two species suggest divergent demographic histories possibly reflecting contrasting host diet or competitive exclusion, with M. bilis exhibiting greater mitochondrial diversity than P. truncatum. Shared haplotypes within the ranges of both parasite species probably reflect relatively unrestricted movements (both natural and anthropogenic) of intermediate and definitive hosts across Europe.     

Comparative cytogenetics of opisthorchid species (Trematoda, Opisthorchiidae)

In the present study karyotypes and chromosomes of five species of the family Opisthorchiidae (Opisthorchis felineus (Rivolta, 1884), O. viverrini (Poirier, 1886), Metorchis xanthosomus (Creplin, 1846), M. bilis (Braun, 1893), and Clonorchis sinensis (Cobbold, 1875)) were compared. Karyotypes of O. felineus, M. xanthosomus, M. bilis and C. sinensis consist of two pairs of large meta- and submetacentrics and five pairs of small chromosomes (2n = 14). The karyotype of O. viverrini is 2n = 12, which indicates a fusion of two chromosomes of opisthorchid ancestral karyotype. Analysis of mitotic and meiotic chromosomes was performed by heterologous in situ hybridization of microdissected DNA probes obtained from chromosomes 1 and 2 of O. felineus and chromosomes 1 and 2 of M. xanthosomus. Results of chromosome staining (C- and AgNOR-banding) and FISH of telomeric probes and ribosomal DNA probe on opisthorchid chromosomes were used for chromosome comparison. Data on chromosome number in opisthorchid species were also discussed.     

Life cycle,morphology of developmental stages of Metorchis ussuriensis sp. nov. (Trematoda: Opisthorchiidae), and phylogenetic relationships with other opisthorchiids

The complete life cycle and developmental stages of the fluke, Metorchis ussuriensis sp. nov. (Trematoda, Platyhelminthes), are herein described. The results of the present experiments showed that, for flukes from the Primorsky Region in the Russian southern Far East, the first intermediate hosts are the snails Parafossarulus spiridonovi and Boreoelona ussuriensis, and the second intermediate hosts are freshwater fish, tadpoles, and snails. The definitive host in this experiment was Anas platyrhynchos dom. Morphometric parameters of M. ussuriensis sp. nov. demonstrate similarities with Metorchis taiwanensis, but the two species differ in the sizes of their bodies, sizes of suckers of adult worms, and sizes of cercariae, as well as respective positions of the finfold in cercariae. Phylogenetic reconstructions and genetic distances using the cox1 gene sequences support the conclusion that M. ussuriensis sp. nov. is well distinguished from all other species of the genus Metorchis, while sequences of internal transcribed spacers (ITS1 and ITS2) failed to separate M. ussuriensis sp. nov., Metorchis bilis, and Metorchis xanthosomus. In addition, we sequenced 1,402 bp of the 28SrRNA gene of M. ussuriensis sp. nov. being the first 28S sequences in the genus Metorchis. Comparison to other trematodes suggests that 28SrRNA could proof suitable for the differentiation of trematode species.     

Telomeric DNA in chromosomes of five opisthorchid species

The analysis of telomere repeat distribution in chromosomes of five opisthorchid species (Opisthorchis felineus (Rivolta, 1884), Opisthorchis viverrini (Poirier, 1886), Metorchis xanthosomus (Creplin, 1846), Metorchis bilis (Braun, 1890), Clonorchis sinensis (Cobbold, 1875)) was performed with fluorescent in situ hybridization (FISH) of labeled (TTAGGG)n DNA-probe and PNA telomere probe on mitotic and meiotic chromosomes of these species. It was shown that chromosome telomeres of all studied species contain large clusters of (TTAGGG)n telomeric repeats. Interstitial clusters of the (TTAGGG)n repeats have not been revealed in the chromosomes of any studied species even when FISH of PNA telomere probe on pachytene chromosomes was performed. Furthermore interstitial clusters of the (TTAGGG)n repeats have not been detected in the chromosomes of O. viverrini, one of chromosomes of this species is the result of a fusion of two ancestral opisthorchid chromosomes.     

Distribution of repetitive DNA sequences in chromosomes of five opisthorchid species (Trematoda, Opisthorchiidae)

Genomes of opisthorchid species are characterized by small size, suggesting a reduced amount of repetitive DNA in their genomes. Distribution of repetitive DNA sequences in the chromosomes of five species of the family Opisthorchiidae (Opisthorchis felineus 2n = 14 (Rivolta, 1884), Opisthorchis viverrini 2n = 12 (Poirier, 1886), Metorchis xanthosomus 2n = 14 (Creplin, 1846), Metorchis bilis 2n = 14 (Braun, 1890), Clonorchis sinensis 2n = 14 (Cobbold, 1875)) was studied with C- and AgNOR-banding, generation of microdissected DNA probes from individual chromosomes and fluorescent in situ hybridization on mitotic and meiotic chromosomes. Small-sized C-bands were discovered in pericentric regions of chromosomes. Ag-NOR staining of opisthorchid chromosomes and FISH with ribosomal DNA probe showed that karyotypes of all studied species were characterized by the only nucleolus organizer region in one of small chromosomes. The generation of DNA probes from chromosomes 1 and 2 of O. felineus and M. xanthosomus was performed with chromosome microdissection followed by DOP-PCR. FISH of obtained microdissected DNA probes on chromosomes of these species revealed chromosome specific DNA repeats in pericentric C-bands. It was also shown that microdissected DNA probes generated from chromosomes could be used as the Whole Chromosome Painting Probes without suppression of repetitive DNA hybridization. Chromosome painting using microdissected chromosome specific DNA probes showed the overall repeat distribution in opisthorchid chromosomes.     

High prevalence of zoonotic trematodes in roach (<Emphasis Type="Italic">Rutilus rutilus</Emphasis>) in the Gulf of Finland

The intention to increase roach (Rutilus rutilus) consumption is in focus for ecological and economic reasons in Finland. However, its safety as food has not been considered comprehensively. We collected and artificially digested 85 roach halves originating from the south-eastern coast of Finland, and found trematode metacercariae in 98.8% of the samples. Based on polymerase chain reaction (PCR) and sequencing of amplicons generated from the ITS2 gene region, zoonotic parasites of the family Opistorchiidae were identified as Pseudamphistomum truncatum and Metorchis bilis, and also non-zoonotic Holostephanus dubinini (family Cyathocotylidae) and Posthodiplostomum spp. (family Diplostomidae) were identified. The species identity of other trematodes found is currently being investigated. Mixed infections of several trematode species were common. The prevalence of morphologically identified zoonotic P. truncatum was 46%, and zoonotic M. bilis was found in one sequence sample. The high prevalence of zoonotic trematode metacercariae in roach from the Gulf of Finland is alarming. Only thoroughly cooked roach products can be recommended for human or animal consumption from the area.     

Opisthorchis felineus and Metorchis bilis are the main agents of liver fluke infection of humans in Russia

Liver fluke infections are gradually transforming from a local problem of individual geographic regions to a widespread problem. The observed expansion is likely to be connected with the ever-increasing intensity of traffic flow and migration of the infected carriers between cities, regions, and countries. Opisthorchis felineus, the trematode belonging to the family Opisthorchiidae, is a well known causative agent of the infection called opisthorchiasis. Metorchis bilis, also a member of the family Opisthorchiidae, causes metorchiasis, a disease very close to opisthorchiasis in symptomatology. Genetic markers can be used to develop methods for differential diagnostics of these diseases. However, the questions connected with epidemiology of these trematode infections, their clinical characteristics, prognosis and therapy remain open. This review briefs the general biological characteristics of O. felineus and M. bilis persisting in various countries of Eurasia, their geographical range, epidemiology and molecular diagnostics of these liver flukes.     

Meloidogyne partityla on Pecan Isozyme Phenotypes and Other Host

Meloidogyne sp. from five pecan (Carya illinoensis) orchards in Texas were distinctive in host range and iszoyme profiles from common species of Meloidogyne but were morphologically congruent with Meloidogyne partityla Kleynhans, a species previously known only in South Africa. In addition to pecan, species of walnut (Juglans hindsii and J. regia) and hickory (C. ovata) also were hosts. No reproduction was observed on 15 other plant species from nine families, including several common hosts of other Meloidogyne spp. Three esterase phenotypes and two malate dehydrogenase phenotypes of M. partityla were identified by polyacrylamide gel electrophoresis. Each of these isozyme phenotypes was distinct from those of the more common species M. arenaria, M. hapla, M. incognita, and M. javanica.     

Population structure of Hymenoscyphus pseudoalbidus and its genetic relationship to Hymenoscyphus albidus

The ascomycete fungus Hymenoscyphus pseudoalbidus (anamorph Chalara fraxinea) is responsible for ash dieback currently expanding over large parts of Europe. Our objective was to investigate the genetic structure of H. pseudoalbidus and to examine its relationship to the species H. albidus, known as a saprotroph. The study comprised 181 isolates of H. pseudoalbidus collected within the diseased area, 17 H. albidus isolates from six apothecia, collected outside the diseased area in Norway, and nine apothecia of H. pseudoalbidus collected in Sweden. By analysis of microsatellite markers developed for this study, combined with AP-PCR using the M13 primer, we demonstrated sexual heterothally in H. pseudoalbidus, detected high gene flow and low geographic structure of the H. pseudoalbidus population and found indications of a founder effect. Also, substantial genetic differences were detected between the two species of fungi; only four of seven microsatellite markers developed for H. pseudoalbidus were amplified for H. albidus, and no alleles were shared among the species. Furthermore, AP-PCR banding patterns were distinctly different for the two species. We conclude that even though the two fungi have a similar habitat and are morphologically virtually identical, they do not share a recent common ancestor.     

An invasive Mimosa in India does not adopt the symbionts of its native relatives

Background and Aims

The large monophyletic genus Mimosa comprises approx. 500 species, most of which are native to the New World, with Central Brazil being the main centre of radiation. All Brazilian Mimosa spp. so far examined are nodulated by rhizobia in the betaproteobacterial genus Burkholderia. Approximately 10 Mya, transoceanic dispersal resulted in the Indian subcontinent hosting up to six endemic Mimosa spp. The nodulation ability and rhizobial symbionts of two of these, M. hamata and M. himalayana, both from north-west India, are here examined, and compared with those of M. pudica, an invasive species.

Methods

Nodules were collected from several locations, and examined by light and electron microscopy. Rhizobia isolated from them were characterized in terms of their abilities to nodulate the three Mimosa hosts. The molecular phylogenetic relationships of the rhizobia were determined by analysis of 16S rRNA, nifH and nodA gene sequences.

Key Results

Both native Indian Mimosa spp. nodulated effectively in their respective rhizosphere soils. Based on 16S rRNA, nifH and nodA sequences, their symbionts were identified as belonging to the alphaproteobacterial genus Ensifer, and were closest to the ‘Old World’ Ensifer saheli, E. kostiensis and E. arboris. In contrast, the invasive M. pudica was predominantly nodulated by Betaproteobacteria in the genera Cupriavidus and Burkholderia. All rhizobial strains tested effectively nodulated their original hosts, but the symbionts of the native species could not nodulate M. pudica.

Conclusions

The native Mimosa spp. in India are not nodulated by the Burkholderia symbionts of their South American relatives, but by a unique group of alpha-rhizobial microsymbionts that are closely related to the ‘local’ Old World Ensifer symbionts of other mimosoid legumes in north-west India. They appear not to share symbionts with the invasive M. pudica, symbionts of which are mostly beta-rhizobial.     

Development of a multiplex PCR assay to discriminate native Hymenoscyphus albidus and introduced Hymenoscyphus fraxineus in Britain and assess their distribution

Two Hymenoscyphus species are associated with ash (Fraxinus) in Europe; the recently arrived ash dieback pathogen Hymenoscyphus fraxineus and apparently native Hymenoscyphus albidus. However, in parts of Europe invasive H. fraxineus appears to be replacing H. albidus which may, therefore, face extinction. We designed primers based on calmodulin sequences for use in singleplex and multiplex PCR assays to detect and discriminate between these two morphologically very similar species. The multiplex assay was tested against 50 isolates and 26 apothecial field samples of H. albidus/H. fraxineus from Europe and Asia, plus isolates of Hymenoscyphus fructigenus, Hymenoscyphus koreanus and Hymenoscyphus occultus. Use of the assay confirmed both H. albidus and H. fraxineus were present across southern Britain between 2014 and 2015, with H. albidus detected at eight geographically distinct sites. The sensitivity, specificity and potential applications of the multiplex assay are discussed.     

Diet shift induced rapid evolution of size and function in a predatory bird

A predator’s body size correlates with its prey size. Change in the diet may call for changes in the hunting mode and traits determining hunting success. We explored long-term trends in sternum size and shape in the northern goshawk by applying geometric morphometrics. Tetraonids, the primary prey of the goshawk, have decreased and been replaced by smaller birds in the diet. We expected that the size of the goshawk has decreased accordingly more in males than females based on earlier observations of outer morphology. We also expected changes in sternum shape as a function of changes in hunting mode. Size of both sexes has decreased during the preceding decades (1962?2008), seemingly reflecting a shift in prey size and hunting mode. Female goshawks hunting also mammalian prey tend to have a pronouncedly “Buteo-type” sternum compared to males preying upon birds. Interestingly, the shrinkage of body size resulted in an increasingly “Buteo-type” sternum in both sexes. In addition, the sternum shape in birds that died accidentally (i.e., fit individuals) was more Buteo-type than in starved ones, hinting that selection was towards a Buteo-type sternum shape. We conclude that these observed patterns are likely due to directional selection driven by changes in the diet towards smaller and more agile prey. On the other hand, global warming is predicted to also cause a decrease in size, thus these two scenarios are inseparable. Because of difficulties in studying fitness-related phenotypic changes of large raptors in the field, time series of museum exemplars collected over a wide geographical area may give answers to this conundrum.     

Metazoan parasites of Neogobius fishes in the Slovak section of the River Danube

Metazoan parasites were investigated in three non‐native fishes (monkey goby Neogobius fluviatilis, bighead goby Neogobius kessleri and round goby Neogobius melanostomus) collected from the former and current main channel of the River Danube and from the River Hron, Slovakia, in November 2003. Thirteen parasite species were identified: Triaenophorus crassus, Diplostomum sp., Tylodelphys clavata, Metorchis xanthosomus, Nicolla skrjabini, Gyrodactylus proterorhini, Pomphorhynchus laevis, Contracoecum sp., Raphidascaris acus, Anguillicola crassus, Unio tumidus, Anodonta anatina and Pseudoanodonta complanata. The maximum parasite diversity was found in N. fluviatilis. Total parasite abundance was significantly higher in N. kessleri, but no significant differences among sampling sites were observed. Pomphorhynchus laevis and glochidia of Anodonta anatina reached 100% prevalence in N. kessleri in the new channel of the Danube and, in general, these species were also the most prevalent parasites in all three goby species. For endoparasites, gobies served mostly as intermediate (digenean, cestodes and nematodes) or paratenic (acantocephalan and nematodes) hosts. All parasite species found are common parasites in the Middle Danube basin. No parasites specific to Neogobius, known from their native populations, were observed.     

Morphological and Molecular Characterization of Lymnaeid Snails and Their Potential Role in Transmission of Fasciola spp. in Vietnam

Freshwater snails of the family Lymnaeidae play an important role in the transmission of fascioliasis worldwide. In Vietnam, 2 common lymnaeid species, Lymnaea swinhoei and Lymnaea viridis, can be recognized on the basis of morphology, and a third species, Lymnaea sp., is known to exist. Recent studies have raised controversy about their role in transmission of Fasciola spp. because of confusion in identification of the snail hosts. The aim of this study is, therefore, to clarify the identities of lymnaeid snails in Vietnam by a combination of morphological and molecular approaches. The molecular analyses using the second internal transcribed spacer (ITS2) of the nuclear ribosomal DNA clearly showed that lymnaeids in Vietnam include 3 species, Austropeplea viridis (morphologically identified as L. viridis), Radix auricularia (morphologically identified as L. swinhoei) and Radix rubiginosa (morphologically identified as Lymnaea sp.). R. rubiginosa is a new record for Vietnam. Among them, only A. viridis was found to be infected with Fasciola spp. These results provide a new insight into lymnaeid snails in Vietnam. Identification of lymnaeid snails in Vietnam and their role in the liver fluke transmission should be further investigated.     

Host Suitability of the Olive Cultivars Arbequina and Picual for Plant-Parasitic Nematodes

Host suitability of olive cultivars Arbequina and Picual to several plant-parasitic nematodes was studied under controlled conditions. Arbequina and Picual were not suitable hosts for the root-lesion nematodes Pratylenchus fallax, P. thornei, and Zygotylenchus guevarai. However, the ring nematode Mesocriconema xenoplax and the spiral nematodes Helicotylenchus digonicus and H. pseudorobustus reproduced on both olive cultivars. The potential of Meloidogyne arenaria race 2, M. incognita race 1, and M. javanica, as well as P. vulnus and P. penetrans to damage olive cultivars, was also assessed. Picual planting stocks infected by root-knot nematodes showed a distinct yellowing affecting the uppermost leaves, followed by a partial defoliation. Symptoms were more severe on M. arenaria and M. javanica-infected plants than on M. incognita-infected plants. Inoculation of plants with 15,000 eggs + second-stage juveniles/pot of these Meloidogyne spp. suppressed the main height of shoot and number of nodes of Arbequina, but not Picual. Infection by each of the two lesion nematodes (5,000 nematodes/pot) or by each of the three Meloidogyne spp. suppressed (P < 0.05) the main stem diameter of both cultivars. On Arbequina, the reproduction rate of Meloidogyne spp. was higher (P < 0.05) than that of Pratylenchus spp.; on Picual, Pratylenchus spp. reproduction was higher (P < 0.05) than that of Meloidogyne spp.     

Evaluation of 31 Potential Biofumigant Brassicaceous Plants as Hosts for Three Meloiodogyne Species

Brassicaceous cover crops can be used for biofumigation after soil incorporation of the mowed crop. This strategy can be used to manage root-knot nematodes (Meloidogyne spp.), but the fact that many of these crops are host to root-knot nematodes can result in an undesired nematode population increase during the cultivation of the cover crop. To avoid this, cover crop cultivars that are poor or nonhosts should be selected. In this study, the host status of 31 plants in the family Brassicaceae for the three root-knot nematode species M. incognita, M. javanica, and M. hapla were evaluated, and compared with a susceptible tomato host in repeated greenhouse pot trials. The results showed that M. incognita and M. javanica responded in a similar fashion to the different cover cultivars. Indian mustard (Brassica juncea) and turnip (B. rapa) were generally good hosts, whereas most oil radish cultivars (Raphanus. sativus ssp. oleiferus) were poor hosts. However, some oil radish cultivars were among the best hosts for M. hapla. The arugula (Eruca sativa) cultivar Nemat was a poor host for all three nematode species tested. This study provides important information for chosing a cover crop with the purpose of managing root-knot nematodes.     

Use of cyclopoid copepods for mosquito control

The New Orleans Mosquito Control Board mass produces Mesocyclops longisetus and Macrocyclops albidus for introduction to mosquito breeding sites as a routine part of control operations. Mesocyclops longisetus is used in tires that collect rainwater; M. albidus is used in temporary pools. Field trials in a Spartina marsh, rice fields, and residential roadside ditches in Louisiana suggest that M. longisetus and M. albidus could be of use to control larvae of Anopheles spp. and Culex quinquefasciatus. Mesocyclops longisetus has proved to be effective for Aedes aegypti control in cisterns, 55-gallon drums, and other domestic containers in Honduras.     

Reproduction and potential range expansion of walnut twig beetle across the Juglandaceae

Biological invasions by insects that vector plant pathogens have altered the composition of natural and urban forests. Thousand cankers disease is a new, recent example and is caused by the complex of walnut twig beetle, Pityophthorus juglandis, and the fungus, Geosmithia morbida, on susceptible hosts, notably some Juglans spp. and Pterocarya spp. Host colonization by P. juglandis may be particularly important for disease development, but the beetle’s host range is not known. In the United States and Italy, this insect has expanded its geographic range by colonizing naïve hosts. The objective of this study was to characterize limits to, and variation within, the host range of P. juglandis and infer the extent to which hosts might constrain the geographic distribution of the insect. We examined colonization and reproduction by P. juglandis in no-choice laboratory experiments with 11 Juglans spp., one Pterocarya sp., and two Carya spp. over 2 years and found that all but the Carya spp. were hosts. Reproduction was generally greater on Juglans californica, J. hindsii, and J. nigra, than on J. ailantifolia, J. cathayensis, J. cinerea, J. major, J. mandshurica, J. microcarpa, or J. regia. Escape of an insect vector into populations of evolutionary-naïve hosts can facilitate rapid range expansion by the pest and massive mortality to hosts. Multi-continental plantings of suitable species may facilitate geographic range expansion of P. juglandis and place other, native Juglans spp. at risk.     

Intra-specific variation of Kudoa spp. (Myxosporea: Multivalvulida) from apogonid fishes (Perciformes), including the description of two new species, K. cheilodipteri n. sp. and K. cookii n. sp., from Australian waters

Kudoa spp. from the musculature and intestinal mucosa of species of the teleost family Apogonidae were examined for their taxonomic identity. Two novel species are characterised: Kudoa cheilodipteri n. sp. from the musculature of Cheilodipterus quinquelineatus Cuvier, Ostorhinchus cyanosoma (Bleeker) and O. aureus (Lacépède); and Kudoa cookii n. sp. from the submucosa of the intestines of O. cookii (Macleay) only. Both species are characterised using morphology, small subunit ribosomal DNA (SSU rDNA), large subunit ribosomal DNA (LSU rDNA), and biological characters. Three new host records, O. cyanosoma, O. aureus and Apogon doederleini, and associated geographical, morphological and genetic data are also provided for Kudoa whippsi Burger & Adlard, 2010. Morphological and molecular intra-specific variation of all isolates assigned to K. whippsi is also examined. Phylogenetic analyses further support the idea that tissue tropism is a distinguishing character between morphologically similar species; species reported here display close relatedness to morphologically similar species infecting the same tissue within their hosts.     

Systematic examination of the cardiopulmonary,urogenital, muscular and gastrointestinal parasites of the Eurasian otters (Lutra lutra) in Denmark,a protected species recovering from a dramatic decline

The Eurasian otter (Lutra lutra) is a protected species in Denmark and at present, the population is recovering due to conservation efforts. The Danish otters are mainly found in the continental part of Denmark (Jutland), but establishment in the main islands (Fyn and Zealand) has been observed. While there is a lack of systematic studies on the parasite fauna of otters in Denmark, this study aims to screen otters for their parasite fauna, especially those of zoonotic and/or veterinary importance. Thirty-three otter carcasses, road-killed (n = 30), found dead (n = 2) and shot (n = 1), were collected between June 2013 and May 2014 and examined for cardiopulmonary, urogenital, gastrointestinal, and muscle helminths by post mortem examination. Faecal samples were analysed by modified concentration McMaster technique and direct immunofluorescence test for Giardia and Cryptosporidium. At least one parasite was found in 75.8% of animals. The parasite fauna included 13 species, consisting of five nematodes: Molineus patens (30.3%), Aonchotheca putorii (27.3%), Strongyloides sp. (24.2%), Physaloptera sp. (12.1%), Eucoleus aerophilus (10.0%); one cestode: Schistocephalus solidius (6.1%); four trematodes: Metorchis bilis (33.3%), Isthimiophora melis (15.2%), Cryptocotyle sp. (3.0%), Plagiorchis sp. (3.0%); one acanthocephalan: Acanthocephalus ranae (18.2%); and two protozoans: Giardia spp. (3.1%), and Eimeria spp. (3.1%). The study showed that otters carry parasites of zoonotic and veterinary importance. Many of these parasites can also infect native carnivores and birds, and the distribution of these parasites may be affected if the otter population continue to increase in Denmark.