Modification of the chemically induced inflammation assay reveals the Janus face of a phenol rich fulvic acid.

Inflammation is an essential process as a reaction towards infections or wounding. Exposure to hazardous environmental pollutants can lead to chronic inflammations, where the resolving phase is delayed or blocked. Very contradictory studies have been reported on the pro- and anti-inflammatory effects of humic substances (HSs) leading to significant disagreements between researchers. To a certain extent, this can be attributed to the chemical heterogeneity of this group of xenobiotics. Here we show for the first time that pro- and anti-inflammatory effects can occur by one HSs. We adapted an assay that uses green fluorescence-labeled zebrafish larvae and CuSO4 to indue an inflammation. In wild-type larvae, exposure to 50 µM CuSO4 for 2 h activated the production of reactive oxygen species, which can be monitored with a fluorescence dye (H2DCFDA) and a microplate reader. This allows not only the use of wild-type fish but also a temporal separation of copper exposure and inflammatory substance while retaining the high throughput. This modified assay was then used to evaluate the inflammatory properties of a fulvic acid (FA). We found, that the aromatic structure of the FA protects from inflammation at 5 and 50 mg C/L, while the persistent free radicals enhance the copper-induced inflammation at ≥ 300 mg C/L.

Fulvic acid accelerates hatching and stimulates antioxidative protection and the innate immune response in zebrafish larvae.

Aquaculture plays a pivotal role in covering dietary animal protein demands and restocking endangered fish populations. However, high mortality takes place at the earliest life stages: prior and immediately after hatching. Improving growth and health parameters by immunostimulants is widely used in older fish, but rarely studied in larvae. Fulvic acids (FAs) are natural substances found in soil and water. Using zebrafish as a model organism, we evaluated the effects of exposure to a FA at concentrations ranging from 1 to 500 mg C/L (mg dissolved organic carbon per liter) on embryonic development. Furthermore, the concentration of reactive oxygen species (ROS) inside the larvae as well as the molecular mechanisms involved in growth, immune response, and antioxidative protection were determined at 5, 50, and 500 mg C/L. 20 to 200 mg C/L accelerated the hatching, which was mediated by increased expression of ifg-1, gh, and he1-α. Furthermore, lyz and mpx were significantly increased at 5 and 50 mg C/L. A concentration of 500 mg C/L induced genes involved in the protection against ROS (nrf-2, keap-1, cat, sod-1), increased the concentration of ROS inside the larvae and caused tissue damage and mortality. Interestingly, 50 mg C/L activated ROS protection as well (nrf-2, sod-2), while no increase of ROS was found in the larvae. Our results show, that FA at low to medium concentrations can increase the health of larvae, but becomes detrimental at higher concentrations.

Eye fluke infection changes diet composition in juvenile European perch (Perca fluviatilis).

Intraspecific diet specialization, usually driven by resource availability, competition and predation, is common in natural populations. However, the role of parasites on diet specialization of their hosts has rarely been studied. Eye flukes can impair vision ability of their hosts and have been associated with alterations of fish feeding behavior. Here it was assessed whether European perch (Perca fluviatilis) alter their diet composition as a consequence of infection with eye flukes. Young-of-the-year (YOY) perch from temperate Lake Müggelsee (Berlin, Germany) were sampled in two years, eye flukes counted and fish diet was evaluated using both stomach content and stable isotope analyses. Perch diet was dominated by zooplankton and benthic macroinvertebrates. Both methods indicated that with increasing eye fluke infection intensity fish had a more selective diet, feeding mainly on the benthic macroinvertebrate Dikerogammarus villosus, while less intensively infected fish appeared to be generalist feeders showing no preference for any particular prey type. Our results show that infection with eye flukes can indirectly affect interaction of the host with lower trophic levels by altering the diet composition and highlight the underestimated role of parasites in food web studies.

Innate immunity, oxidative stress and body indices of Eurasian perch Perca fluviatilis after two weeks of exposure to artificial light at night.

Artificial light at night (ALAN) can disrupt biological rhythms of fish and other vertebrates by changing the light information of the nocturnal environment. Disrupted biorhythms can impair the immune system of vertebrates as it has been shown for conditions with continuous illumination or long-day photoperiod in many vertebrates, including fish. Nonetheless, this has not been shown so far for typical ALAN scenarios with high light intensities during day and low light intensities at night. Therefore, in this study, proxies for the innate immune system and oxidative stress as well as body indices of Eurasian perch Perca fluviatilis were measured under a wide range of intensities of nocturnal illumination. The authors found no changes in parameters of the innate immune system and no significant changes in proxies for oxidative stress after 2-week exposures to nocturnal illuminance ranging from 0.01 lx to 1 lx in one experiment or from 1 lx to 100 lx in a second experiment. A decrease in the hepato-somatic index at the highest tested light intensity of 100 lx compared to the dark control was the only significant difference in all parameters among treatments. After 2 weeks of exposure, ALAN does not seem to seriously challenge the innate immune system and seems to cause less oxidative stress than expected. The results of this study contradict the findings from other studies applying continuous illumination or long-day photoperiod and highlight the importance of further research in this field. Because ALAN represents a sustained modulation of the environment that may have cumulative effects over time, long-term studies are required for a better understanding of how ALAN modulates the health of fish.

Phenol-rich fulvic acid as a water additive enhances growth, reduces stress, and stimulates the immune system of fish in aquaculture.

Aquaculture has become imperative to cover the demands for dietary animal protein. Simultaneously, it has to overcome prejudices from excessive use of antibiotics and environmental impacts. Natural supplements are traditionally applied orally. In this study, we demonstrated another pathway: the gills. Humic substances are immunostimulants and a natural part of every aquatic ecosystem, making them ideal to be used as bath stimulants. Five and 50 mg C/L of a fulvic acid-rich humic substance was added for 28 days to the water of juvenile rainbow trout (Oncorhynchus mykiss). This fulvic acid is characterized by a high content of phenolic moieties with persistent free radicals and a high electron exchange capacity. The high concentration of the fulvic acid significantly increased growth and reduced the food conversion ratio and the response to a handling-stressor. Phagocytosis and potential killing activity of head kidney leukocytes were increased, as well as the total oxyradical scavenging capacity (TOSC) and lysozyme activity in the gills. In conclusion, immunostimulation via gills is possible with our fulvic acid, and the high phenolic content improved overall health and stress resistance of fish.

Encapsulation of Anguillicola crassus reduces the abundance of adult parasite stages in the European eel (Anguilla anguilla).

Encapsulation of the parasitic nematode Anguillicola crassus Kuwahara, Niimi & Hagaki is commonly observed in its native host, the Japanese eel (Anguilla japonica Temminck & Schlegel). Encapsulation has also been described in a novel host, the European eel (A. anguilla L.), and there is evidence that encapsulation frequency has increased since the introduction of A. crassus. We examined whether encapsulation of A. crassus provides an advantage to its novel host in Lake Müggelsee, NE Germany. We provide the first evidence that encapsulation was associated with reduced abundance of adult A. crassus. This pattern was consistent in samples taken 3 months apart. There was no influence of infection on the expression of the two metabolic genes studied, but the number of capsules was negatively correlated with the expression of two mhc II genes of the adaptive immune response, suggesting a reduced activation. Interestingly, eels that encapsulated A. crassus had higher abundances of two native parasites compared with non-encapsulating eels. We propose that the response of A. anguilla to infection by A. crassus may interfere with its reaction to other co-occurring parasites.

Gene expression response to a nematode parasite in novel and native eel hosts.

Invasive parasites are involved in population declines of new host species worldwide. The high susceptibilities observed in many novel hosts have been attributed to the lack of protective immunity to the parasites which native hosts acquired during their shared evolution. We experimentally infected Japanese eels (Anguilla japonica) and European eels (Anguilla anguilla) with Anguillicola crassus, a nematode parasite that is native to the Japanese eel and invasive in the European eel. We inferred gene expression changes in head kidney tissue from both species, using RNA-seq data to determine the responses at two time points during the early stages of infection (3 and 23 days postinfection). At both time points, the novel host modified the expression of a larger and functionally more diverse set of genes than the native host. Strikingly, the native host regulated immune gene expression only at the earlier time point and to a small extent while the novel host regulated these genes at both time points. A low number of differentially expressed immune genes, especially in the native host, suggest that a systemic immune response was of minor importance during the early stages of infection. Transcript abundance of genes involved in cell respiration was reduced in the novel host which may affect its ability to cope with harsh conditions and energetically demanding activities. The observed gene expression changes in response to a novel parasite that we observed in a fish follow a general pattern observed in amphibians and mammals, and suggest that the disruption of physiological processes, rather than the absence of an immediate immune response, is responsible for the higher susceptibility of the novel host.

Eye fluke (Tylodelphys clavata) infection impairs visual ability and hampers foraging success in European perch.

Visual performance and environmental conditions can influence both behavioral patterns and predator-prey interactions of fish. Eye parasites can impair their host's sensory performance with important consequences for the detection of prey, predators, and conspecifics. We used European perch (Perca fluviatilis) experimentally infected with the eye fluke Tylodelphys clavata and evaluated their feeding behavior and competitive ability under competition with non-infected conspecifics, in groups of four individuals, for two different prey species (Asellus aquaticus and Daphnia magna). To test whether the effect of T. clavata infection differs at different light conditions, we performed the experiments at two light intensities (600 and 6 lx). Foraging efficiency of perch was significantly affected by infection but not by light intensity. The distance at which infected fish attacked both prey species was significantly shorter in comparison to non-infected conspecifics. Additionally, infected fish more often unsuccessfully attacked A. aquaticus. Although the outcome of competition depended on prey species, there was a general tendency that non-infected fish consumed more of the available prey under both light intensities. Even though individual prey preferences for either A. aquaticus or D. magna were observed, we could not detect that infected fish change their prey preference to compensate for a reduced competitive foraging ability. As infection of T. clavata impairs foraging efficiency and competitive ability, infected fish would need to spend more time foraging to attain similar food intake as non-infected conspecifics; this presumably increases predation risk and potentially enhances transmission success to the final host.

Potash mining effluents and ion imbalances cause transient stress in adult common roach, Rutilus rutilus.

A present ecological issue causing secondary salinization in different countries is the discharge of effluents by the potash mining industry. In Germany, the River Werra is used as a sink for potash mining discharges containing high concentrations of ions, predominantly Cl-, K+, Na+, and Mg2+ resulting in a strong decline of the biodiversity and abundance of local species. However, hardly anything is known about the acute and chronic physiological effects of high concentrations and imbalances of ions being prevalent in potash mining effluents in fish. Therefore, the stress response and selected immune and growth parameters were investigated in standardized laboratory experiments. A native freshwater fish species, Rutilus rutilus, was exposed to concentrations of the high currently allowed (HT) and lowered future thresholds (LT) and three different ion solutions (containing high Mg2+ (Mg), high K+ (K) and high Mg2++K+ (Mg+K) concentrations) for four different exposure times (24 h, 7 d, 21 d, 8 wk). Tank water (additionally after 9 and 12 h) and plasma cortisol, glucose and protein, hematocrit and hemoglobin were determined after each exposure time. Furthermore, plasma lysozyme and head kidney leucocyte respiratory burst activity (only after 21 d) were evaluated as well as growth parameters. A transient stress response was induced in almost all groups. Tank water cortisol was elevated after 9 h in HT, LT and Mg+K and in HT after 12 h, whereas glucose concentrations increased after 24 h in all exposure groups except K. HT led to enhanced hematocrit and hemoglobin content after 24 h. Plasma protein, immune system and growth were not affected in any group. None of the ion solutions induced acute toxicity but most triggered typical acute stress reactions. Rather the sum of high ion concentrations than single ions challenged the fish. Even though the effects observed in adult roach were only transient and indicate acclimatization under laboratory conditions, adverse effects observed in the river are evident and further research on physiological endpoints including reproductive parameters and impacts on younger life stages seem to be needed to scientifically base protective thresholds.

Experimental infection with Anguillicola crassus alters immune gene expression in both spleen and head kidney of the European eel (Anguilla anguilla).

Invasive parasites have been implicated in the declines of several freshwater species. The swim bladder nematode Anguillicola crassus was introduced into Europe in the 1980s and is considered a threat to the European eel (Anguilla anguilla). Infection affects stress resistance and swimming behaviour. European eels produce an immune response against the parasite during the late stages of infection and after repeated infections. We used RNA-seq to examine the molecular response to infection during the poorly understood early stage and identify expression of genes and associated processes that are modified in two immune organs of European eels 3 days post infection with A. crassus. In the spleen, 67 genes were differentially expressed, 32 of which were annotated. Most of these were involved in immune processes and their regulation. Other differentially expressed genes in the spleen were important for heme metabolism and heme turn-over. In the head kidney, 257 genes (134 annotated) were differentially expressed. Several of these were associated with immune functions. Other differentially expressed genes in the head kidney were related to renal function, in particular osmoregulation and paracellular flow. We conclude that the early response of European eels to A. crassus is complex and involves various processes aside from the immune system. We identified molecular changes occurring early during the infection and identified candidate genes and processes which will facilitate future studies aimed at determining the factors affecting European eel viability in the face of this invasive parasite.

Impact of trematode infections on periphyton grazing rates of freshwater snails.

In freshwater ecosystems, snails can significantly influence the competition between primary producers through grazing of periphyton. This activity can potentially be modified by trematodes, a large group of parasites which mostly use molluscs as the first intermediate host. Available studies, however, show contradictory effects of trematodes on snail periphyton grazing. Here, we used four different freshwater snail-trematode systems to test whether a general pattern can be detected for the impact of trematode infections on snail periphyton grazing. In our experimental systems, mass-specific periphyton grazing rates of infected snails were higher, lower, or similar to rates of non-infected conspecifics, suggesting that no general pattern exists. The variation across studied snail-trematode systems may result from differences on how the parasite uses the resources of the snail and thus affects their energy budget. Trematode infections can significantly alter the grazing rate of snails, where, depending on the system, the mass-specific grazing rate can double or halve. This underlines both, the high ecological relevance of trematodes and the need for comprehensive studies at the species level to allow an integration of these parasite-host interactions into aquatic food web concepts.

The eye fluke Tylodelphys clavata affects prey detection and intraspecific competition of European perch (Perca fluviatilis).

Parasites that occupy the eyes of fish have the potential to affect visual perception and consequently alter the host's behaviour, as these organs provide information about their surroundings. In an experimental study, the feeding behaviour of European perch (Perca fluviatilis) infested with the eye fluke Tylodelphys clavata was examined. The results showed that an individual's ability to identify and approach food items was negatively affected by the infestation intensity of T. clavata. Additionally, the foraging success of an individual was reduced in competition with another, less heavily infested conspecific, when the same food resource was exploited. These alterations in the ability to locate food may have important consequences on the feeding strategy of the fish. Furthermore, the impaired feeding capability caused by T. clavata may also increase the predation risk as heavily infested fish need to spend more time foraging to attain a rate of food intake equivalent to less infested conspecifics.

The adjuvant effect of low frequency ultrasound when applied with an inactivated Aeromonas salmonicida vaccine to rainbow trout (Oncorhynchus mykiss).

Vaccine adjuvants are classified according to their properties of either inducing the persistence of antigens within the animal after immunisation and/or activation of the animal's immune response. The adjuvant effect of low intensity low frequency sonophoresis (LFS) was tested in rainbow trout using an Aeromonas salmonicida bacterin vaccine administered by immersion vaccination using LFS at 37 kHz. The adjuvant effect obtained with LFS was compared with that of normal immersion or intraperitoneal injection vaccination. Quantitative PCR was used to measure bacterial DNA in vaccinated fish up to 35 days post-vaccination, while RT-qPCR was used to assess gene expression during the early and late immune response post-vaccination. Results showed that antigen uptake in the gills was significantly higher in the group exposed to low intensity LFS compared to the other two vaccination groups 15 min post-vaccination, but this initially high uptake did not persist over the rest of the experiment. In the kidney, by comparison, the vast majority of the samples analysed did not show the presence or persistence of the bacterin. Showing that the route of vaccine uptake using the A. salmonicida bacterin, does not influence the persistence of the bacterin in the gills or the kidney. On the other hand, LFS induced a higher inflammatory response and T-helper cell activation, characterized by a significant up-regulation of interleukin-8 (IL-8), IL-1ß and CD-4, respectively. The expression of Ig-M, Ig-T and Ig-D was up-regulated in gills (being significant for Ig-M), but not in the spleen and kidney of the sonicated group. Conversely, Ig-M was up-regulated in the spleen of the non-sonicated groups, but not in the sonicated group. This highlights the ability of ultrasound to enhance mucosal immunity. It remains to be established whether the up-regulation of Ig-M in gills would be sufficient to offer protection in fish infected with A. salmonicida.

Nematode eel parasite found inside acanthocephalan cysts--a "Trojan horse" strategy?

BACKGROUND: The invasive eel parasite Anguillicoloides crassus (syn. Anguillicola crassus) is considered one of the major causes for the decline of the European eel (Anguilla anguilla) panmictic population. It impairs the swim bladder function and reduces swimming performance of its host. The life cycle of this parasite involves different intermediate and paratenic hosts. Despite an efficient immune system of the paratenic fish hosts acting against infections with A. crassus, levels of parasitized eels remain high in European river systems. Recently, the round goby Neogobius melanostomus (Gobiidae) has become dominant in many rivers in Europe and is still spreading at a rapid pace. This highly invasive species might potentially act as an important, so far neglected paratenic fish host for A. crassus. METHODS: Based on own observations and earlier single sightings of A. crassus in N. melanostomus, 60 fresh individuals of N. melanostomus were caught in the Rhine River and examined to assess the infection levels with metazoan parasites, especially A. crassus. Glycerin preparations were used for parasite identification. RESULTS: The parasite most frequently found in N. melanostomus was the acanthocephalan Pomphorhynchus sp. (subadult stage) which occurred mainly encysted in the mesenteries and liver. Every third gobiid (P = 31.7%) was infected by A. crassus larvae (L3) which exclusively occurred inside the acanthocephalan cysts. No intact or degenerated larvae of A. crassus were detected elsewhere in the goby, neither in the body cavity and mesenteries nor in other organs. Affected cysts contained the acanthocephalan larvae and 1-12 (mI =3) living A. crassus larvae. Additionally, encysted larvae of the nematode Raphidascaris acus were detected in the gobies, but only in the body cavity and not inside the acanthocephalan cysts. CONCLUSIONS: Based on our observations, we suggest that A. crassus might actively bypass the immune response of N. melanostomus by invading the cysts of acanthocephalan parasites of the genus Pomphorhynchus using them as "Trojan horses". Providing that eels prey on the highly abundant round goby and that the latter transfers viable infective larvae of A. crassus, the new paratenic host might have a strong impact on the epidemiology of A. crassus.

Enhanced Aeromonas salmonicida bacterin uptake and side effects caused by low frequency sonophoresis in rainbow trout (Oncorhynchus mykiss).

Low frequency sonophoresis (LFS) has been recognized as one of the most advanced technologies in transdermal delivery of substances, due to the modification of the stratum corneum lipid bilayer, in focal skin applications in mammals. Based on these findings, LFS has been suggested as a potential technology to be used for enhancement in immersion fish vaccination. In contrast to mammals where LFS is applied to discrete regions of the skin, in fish the whole individual needs to be exposed for practical purposes. The current study evaluated the impact of LFS at 37 kHz on the uptake of an Aeromonas salmonicida bacterin and side effects of the treatment in rainbow trout. Quantitative real time PCR (qPCR) and immunohistochemistry were used to examine the bacterin uptake into skin and gill tissue. Side effects were assessed by behavioural examination, histology and blood serum analysis. The sonication intensity of 171 mW/cm² was enough for increasing skin permeability, but caused heavy erratic swimming and gill haemorrhages. Sonication intensities as low as 105 mW/cm² did not modify skin permeability and enhanced the bacterin uptake into the gill tissue by factor 15 compared to conventional immersion. Following sonication, the gill permeability for the bacterin decreased after 20 min and 120 min by factor 3 and 2, respectively. However, during sonication, erratic swimming of the fish raised some concerns. Further reduction of the sonication intensity to 57 mW/cm² did not induce erratic swimming, and the bacterin uptake into the gill tissue was still increased by factor 3. In addition, a decreasing albumin-globulin ratio in the serum of the rainbow trout within 40 min revealed that LFS leads to an inflammatory response. Consequently, based on both increased bacterin uptake and the inflammatory response, low intensity LFS has the potential to enhance vaccine immunity without significant side effects.

Low frequency ultrasound and UV-C for elimination of pathogens in recirculating aquaculture systems.

Low frequency ultrasound (LFUS) was evaluated as a novel disinfection technique within recirculating aquaculture systems both individually and combined with UV-C. Dose-dependent inactivation rates were determined for the total viable counts and model organisms representing different taxa of common fish parasites: the ciliate Paramecium sp., second larval stage (L2) of the nematode Anguillicola crassus and metanauplii of Artemia sp. Application of LFUS up to 19 kJ/L did not reduce the number of colony forming units (CFU), whilst UV-C irradiation was highly effective. Pre-treatment with LFUS reduced the mean size of suspended solids in aquaculture water and thus increased the germicidal effect of UV-C by up to 0.6 log units. LFUS was effective against the eukaryotic organisms, and the dose-dependent inactivation could be well described by functions of an exponential decay. However, the efficiency of LFUS differed greatly between species. A LFUS dose of 1.9 kJ/L (consumed energy) was sufficient to inactivate Artemia by 99%, but a ten times higher dose was necessary to inactivate 95% and 81% of Paramecium and Anguillicola larvae, respectively. In clear water, the energetic efficiency of UV-C (emitted by a low pressure lamp) against Paramecium and Anguillicola larvae was higher compared to LFUS, but LFUS was more efficient against Artemia. However, the efficiency of LFUS against ciliates or nematode larvae would be similar or even higher than UV-C in highly turbid water or if less efficient medium pressure lamps are used. This study shows that LFUS can be applied safely at energy densities that are effective against a wide range of parasites like ciliates, nematodes and crustaceans. The combination of LFUS and UV-C could provide an appropriate water treatment with regards to all relevant pathogens in recirculating aquaculture systems.

Progestogens cause immunosuppression of stimulated carp (Cyprinus carpio L.) leukocytes in vitro.

The involvement of steroid hormones in direct and indirect regulation and modulation of immune responses is well recognized in mammals. Here, we demonstrate that progestogens are capable of influencing the innate immunity in fish as well. Therefore, we confirmed the known immunosuppressive effects of natural progesterone (P4), and compared them to influences of 17alpha,20beta-dihydroxy progesterone (DHP4) and the synthetic progestins, medroxyprogesterone acetate (MPA) and levonorgestrel (LEV), on NO release by in vitro-stimulated carp leukocytes derived from both, head and trunk kidney, respectively. DHP4 known as the main maturation-inducing steroid in many teleosts potently inhibited the NO release by carp leukocytes. The synthetic progestin MPA, which may also be environmentally relevant due to its world-wide use in hormonal contraception, significantly decreased NO formation by head and trunk kidney cells. In contrast, LEV showed no significant influence on NO release by head and trunk kidney leukocytes. The observed immunosuppressive actions of progestogens on NO production were compared to the known impairment by natural and synthetic glucocorticoids. Determining the potential impact of progestogens on mRNA expression of iNOS by means of semi-quantitative reverse transcription polymerase chain reactions (RT-PCR) revealed downregulation of proinflammatory type I immune response characteristics at high concentrations. These findings demonstrate for the first time that similar to the known effects of natural progesterone synthetic progestogens are also able to influence immune signaling cascades in fish, and provide evidence that these steroids are capable of influencing mRNA expression of iNOS. The induction of a regulatory type II immune response by progestogens is a striking example of interference of female steroid-mediated events with the piscine immune system. Furthermore, the identification of a partial sequence of a membrane-associated progestogen receptor (mPR) in carp leukocytes by RT-PCR indicates a specific mechanism underlying the observed effects of progestogens on these immune cells.

Parasite community and mortality of overwintering young-of-the-year roach (Rutilus rutilus).

The composition and development of the parasite community of overwintering young-of-the-year (YOY) roach (Rutilus rutilus) was studied in outdoor artificial ponds. Cumulative mortality of the roach from November until May was about 20%. The prevalences of the epizoic ciliates Trichodina sp. and Apiosoma sp., and of the monogeneans Dactylogyrus suecicus, Dactylogyrus nanus, and Gyrodactylus sp. in general tended to peak in the beginning of April at a water temperature of about 8 C. However, mean parasite intensity remained constant over this interval. Prevalence of metacercariae of the digeneans Diplostomum cf. spathaceum and Tylodelphys clavata remained constant with values around 60% and 10%, respectively. Larvae of the nematode Philometra obturans and the cestode Ligula intestinalis and females of the copepod Neoergasilus japonicus were found sporadically. A concept of 2 thresholds, a critical condition factor (attained by a temperature-induced energy deficiency), and a condition-dependent critical infection intensity, which determine winter mortality of YOY, is suggested.