Generalist parasites persist in degraded environments: a lesson learned from microsporidian diversity in amphipods.

The present study provides new insight into suitable microsporidian­host associations. It relates regional and continental-wide host specialization in microsporidians infecting amphipods to degraded and recovering habitats across 2 German river catchments. It provides a unique opportunity to infer the persistence of parasites following anthropogenic disturbance and their establishment in restored rivers. Amphipods were collected in 31 sampling sites with differing degradation and restoration gradients. Specimens were morphologically (hosts) and molecularly identified (host and parasites). Amphipod diversity and abundance, microsporidian diversity, host phylogenetic specificity and continental-wide ß-specificity were investigated and related to each other and/or environmental variables. Fourteen microsporidian molecular operational taxonomic units (MOTUs), mainly generalist parasites, infecting 6 amphipod MOTUs were detected, expanding the current knowledge on the host range by 17 interactions. There was no difference in microsporidian diversity and host specificity among restored and near-natural streams (Boye) or between those located in urban and rural areas (Kinzig). Similarly, microsporidian diversity was generally not influenced by water parameters. In the Boye catchment, host densities did not influence microsporidian MOTU richness across restored and near-natural sites. High host turnover across the geographical range suggests that neither environmental conditions nor host diversity plays a significant role in the establishment into restored areas. Host diversity and environmental parameters do not indicate the persistence and dispersal of phylogenetic host generalist microsporidians in environments that experienced anthropogenic disturbance. Instead, these might depend on more complex mechanisms such as the production of resistant spores, host switching and host dispersal acting individually or conjointly.

Possible seasonal and diurnal modulation of Gammarus pulex (Crustacea, Amphipoda) drift by microsporidian parasites.

In lotic freshwater ecosystems, the drift or downstream movement of animals (e.g., macroinvertebrates) constitutes a key dispersal pathway, thus shaping ecological and evolutionary patterns. There is evidence that macroinvertebrate drift may be modulated by parasites. However, most studies on parasite modulation of host drifting behavior have focused on acanthocephalans, whereas other parasites, such as microsporidians, have been largely neglected. This study provides new insight into possible seasonal and diurnal modulation of amphipod (Crustacea: Gammaridae) drift by microsporidian parasites. Three 72 h drift experiments were deployed in a German lowland stream in October 2021, April, and July 2022. The prevalence and composition of ten microsporidian parasites in Gammarus pulex clade E varied seasonally, diurnally, and between drifting and stationary specimens of G. pulex. Prevalence was generally higher in drifting amphipods than in stationary ones, mainly due to differences in host size. However, for two parasites, the prevalence in drift samples was highest during daytime suggesting changes in host phototaxis likely related to the parasite's mode of transmission and site of infection. Alterations in drifting behavior may have important implications for G. pulex population dynamics and microsporidians' dispersal. The underlying mechanisms are more complex than previously thought.

Parasitism enhances gastropod feeding on invasive and native algae while altering essential energy reserves for organismal homeostasis upon warming.

Marine bioinvasions are of increasing attention due to their potential of causing ecological and economic loss. The seaweed Gracilaria vermiculophylla has recently invaded the Baltic Sea, where, under certain conditions, it was found to outcompete the native alga Fucus vesiculosus. Parasites of grazers and temperature are among the potential factors which might indirectly modulate the interactions between these co-occurring algae through their single and combined effects on grazing rates. We tested the temperature and parasitism effects on the feeding of the gastropod Littorina littorea on F. vesiculosus vs. G. vermiculophylla. Uninfected and trematode-infected gastropods were exposed to 10, 16, 22, and 28 °C for 4 days while fed with either algae. Faeces production was determined as a proxy for grazing rate, and HSP70 expression, glycogen and lipid concentrations were used to assess the gastropod's biochemical condition. Gracilaria vermiculophylla was grazed more than F. vesiculosus. Trematode infection significantly enhanced faeces production, decreased glycogen concentrations, and increased lipid concentrations in the gastropod. Warming significantly affected glycogen and lipid concentrations, with glycogen peaking at 16 °C and lipids at 22 °C. Although not significant, warming and trematode infection increased HSP70 levels. Increased faeces production in infected snails and higher faeces production by L. littorea fed with G. vermiculophylla compared to those which fed on F. vesiculosus, suggest parasitism as an important indirect modulator of the interaction between these algae. The changes in the gastropod's biochemical condition indicate that thermal stress induced the mobilization of energy reserves, suggesting a possible onset of compensatory metabolism. Finally, glycogen decrease in infected snails compared to uninfected ones might make them more susceptible to thermal stress.

The trematode Podocotyle atomon modulates biochemical responses of Gammarus locusta to thermal stress but not its feeding rate or survival.

Although parasitism is one of the most common species interactions in nature, the role of parasites in their hosts' thermal tolerance is often neglected. This study examined the ability of the trematode Podocotyle atomon to modulate the feeding and stress response of Gammarus locusta towards temperature. To accomplish this, infected and uninfected females and males of Gammarus locusta were exposed to temperatures (2, 6, 10, 14, 18, 22, 26, 30 °C) for six days. Shredding (change in food biomass) and defecation rates (as complementary measure to shredding rate) were measured as proxies for feeding activity. Lipid and glycogen concentrations (energy reserves), catalase (oxidative stress indicator), and phenoloxidase (an immunological response in invertebrates) were additionally measured. Gammarid survival was optimal at 10 °C as estimated by the linear model and was unaffected by trematode infection. Both temperature and sex influenced the direction of infection effect on phenoloxidase. Infected females presented lower phenoloxidase activity than uninfected females at 14 and 18 °C, while males remained unaffected by infection. Catalase activity increased at warmer temperatures for infected males and uninfected females. Higher activity of this enzyme at colder temperatures occurred only for infected females. Infection decreased lipid content in gammarids by 14 %. Infected males had significantly less glycogen than uninfected, while infected females showed the opposite trend. The largest infection effects were observed for catalase and phenoloxidase activity. An exacerbation of catalase activity in infected males at warmer temperatures might indicate (in the long-term) unsustainable, overwhelming, and perhaps lethal conditions in a warming sea. A decrease in phenoloxidase activity in infected females at warmer temperatures might indicate a reduction in the potential for fighting opportunistic infections. Results highlight the relevance of parasites and host sex in organismal homeostasis and provide useful insights into the organismal stability of a widespread amphipod in a warming sea.

Heat sensitivity of first host and cercariae may restrict parasite transmission in a warming sea.

To predict global warming impacts on parasitism, we should describe the thermal tolerance of all players in host-parasite systems. Complex life-cycle parasites such as trematodes are of particular interest since they can drive complex ecological changes. This study evaluates the net response to temperature of the infective larval stage of Himasthla elongata, a parasite inhabiting the southwestern Baltic Sea. The thermal sensitivity of (i) the infected and uninfected first intermediate host (Littorina littorea) and (ii) the cercarial emergence, survival, self-propelling, encystment, and infection capacity to the second intermediate host (Mytilus edulis sensu lato) were examined. We found that infection by the trematode rendered the gastropod more susceptible to elevated temperatures representing warm summer events in the region. At 22 °C, cercarial emergence and infectivity were at their optimum while cercarial survival was shortened, narrowing the time window for successful mussel infection. Faster out-of-host encystment occurred at increasing temperatures. After correcting the cercarial emergence and infectivity for the temperature-specific gastropod survival, we found that warming induces net adverse effects on the trematode transmission to the bivalve host. The findings suggest that gastropod and cercariae mortality, as a tradeoff for the emergence and infectivity, will hamper the possibility for trematodes to flourish in a warming ocean.

Transmission of Tetracapsuloides bryosalmonae (Myxozoa: Malacosporea) to Fredericella sultana (Bryozoa: Phylactolaemata) by various fish species.

Tetracapsuloides bryosalmonae is a myxozoan parasite of salmonids and freshwater bryozoans, which causes proliferative kidney disease (PKD) in the fish host. To test which fish species are able to transmit T. bryosalmonae to bryozoans, an infection experiment was conducted with 5 PKD-sensitive fish species from different genera. Rainbow trout Oncorhynchus mykiss, brown trout Salmo trutta, brook trout Salvelinus fontinalis, grayling Thymallus thymallus and northern pike Esox lucius were cohabitated with T. bryosalmonae-infected Fredericella sultana colonies and then subsequently cohabitated with statoblast-reared parasite free Bryozoa. Statoblasts from infected colonies were tested by PCR to detect cryptic stages of T. bryosalmonae, which may indicate vertical transmission of the parasite. In this study, brown trout and brook trout were able to infect Bryozoa, while there was no evidence that rainbow trout and grayling were able to do so. Few interstitial kidney stages of the parasite were detected by immunohistochemistry in brown trout and brook trout, while rainbow trout and grayling showed marked proliferation of renal interstitial tissue and macrophages with numerous parasite cells. Intraluminal stages in the kidney tubules were only detected in brown trout and rainbow trout. In contrast to previous observations, pike was not susceptible to PKD in these trials according to the results of T. bryosalmonae-specific PCR. No DNA of T. bryosalmonae was detected in any statoblast.

Effects of the acanthocephalan Polymorphus minutus and the microsporidian Dictyocoela duebenum on energy reserves and stress response of cadmium exposed Gammarus fossarum.

Amphipods are commonly parasitized by acanthocephalans and microsporidians and co-infections are found frequently. Both groups of parasites are known to have severe effects on their host. For example, microsporidians can modify host sex ratio and acanthocephalans can manipulate the behavior of the amphipod to promote transmission to the final host. These effects influence host metabolism in general and will also affect the ability of amphipods to cope with additional stressors such as environmental pollution, e.g., by toxic metals. Here we tested the effects of sub-lethal concentrations of cadmium on glycogen and lipid levels, as well as on the 70kDa heat shock protein (hsp70) response of field collected Gammarus fossarum, which were naturally infected with microsporidians and the acanthocephalan Polymorphus minutus. Infected and uninfected G. fossarum were exposed to a nominal Cd concentration of 4 µg/L, which resembled measured aqueous Cd concentration of 2.9 µg/L in reconstituted water for 7 d at 15 °C in parallel to an unexposed control. After exposure gammarids were snap frozen, weighed, sexed and tested for microsporidian infection by PCR. Only individuals containing the microsporidian Dictyocoela duebenum were used for the further biochemical and metal analyses. P. minutus infected amphipods were significantly smaller than their uninfected conspecifics. Mortality was insignificantly increased due to cadmium exposure, but not due to parasite infection. Microsporidian infection in combination with cadmium exposure led to increased glycogen levels in female gammarids. An increase of glycogen was also found due to interaction of acanthocephalan and microsporidian infection. Elevated lipid levels were observed in all groups infected with microsporidians, while acanthocephalans had the opposite effect. A positive correlation of lipid and glycogen levels was observed. The general stress response measured in form of hsp70 was significantly increased in microsporidian infected gammarids exposed to cadmium. P. minutus did not affect the stress response of its host. Lipid levels were correlated negatively with hsp70 response, and indicated a possible increased stress susceptibility of individuals with depleted energy reserves. The results of our study clearly demonstrate the importance of parasitic infections, especially of microsporidians, for ecotoxicological research.

Invaders, natives and their enemies: distribution patterns of amphipods and their microsporidian parasites in the Ruhr Metropolis, Germany.

BACKGROUND: The amphipod and microsporidian diversity in freshwaters of a heterogeneous urban region in Germany was assessed. Indigenous and non-indigenous host species provide an ideal framework to test general hypotheses on potentially new host-parasite interactions, parasite spillback and spillover in recently invaded urban freshwater communities. METHODS: Amphipods were sampled in 17 smaller and larger streams belonging to catchments of the four major rivers in the Ruhr Metropolis (Emscher, Lippe, Ruhr, Rhine), including sites invaded and not invaded by non-indigenous amphipods. Species were identified morphologically (hosts only) and via DNA barcoding (hosts and parasites). Prevalence was obtained by newly designed parasite-specific PCR assays. RESULTS: Three indigenous and five non-indigenous amphipod species were detected. Gammarus pulex was further distinguished into three clades (C, D and E) and G. fossarum more precisely identified as type B. Ten microsporidian lineages were detected, including two new isolates (designated as Microsporidium sp. nov. RR1 and RR2). All microsporidians occurred in at least two different host clades or species. Seven genetically distinct microsporidians were present in non-invaded populations, six of those were also found in invaded assemblages. Only Cucumispora dikerogammari and Dictyocoela berillonum can be unambiguously considered as non-indigenous co-introduced parasites. Both were rare and were not observed in indigenous hosts. Overall, microsporidian prevalence ranged from 50% (in G. roeselii and G. pulex C) to 73% (G. fossarum) in indigenous and from 10% (Dikerogammarus villosus) to 100 % (Echinogammarus trichiatus) in non-indigenous amphipods. The most common microsporidians belonged to the Dictyocoela duebenum- /D. muelleri- complex, found in both indigenous and non-indigenous hosts. Some haplotype clades were inclusive for a certain host lineage. CONCLUSIONS: The Ruhr Metropolis harbours a high diversity of indigenous and non-indigenous amphipod and microsporidian species, and we found indications for an exchange of parasites between indigenous and non-indigenous hosts. No introduced microsporidians were found in indigenous hosts and prevalence of indigenous parasites in non-indigenous hosts was generally low. Therefore, no indication for parasite spillover or spillback was found. We conclude that non-indigenous microsporidians constitute only a minimal threat to the native amphipod fauna. However, this might change e.g. if C. dikerogammari adapts to indigenous amphipod species or if other hosts and parasites invade.

Effect of multiple microsporidian infections and temperature stress on the heat shock protein 70 (hsp70) response of the amphipod Gammarus pulex.

BACKGROUND: Increasing temperatures can be a significant stressor for aquatic organisms. Amphipods are one of the most abundant and functionally important groups of freshwater macroinvertebrates. Therefore, we conducted a laboratory experiment with Gammarus pulex, naturally infected with microsporidians. METHODS: In each group, 42 gammarids were exposed to 15°C and 25°C for 24 h. Sex of gammarids was determined and microsporidian infections were detected by specific PCR. To quantify stress levels of the amphipods, the 70 kDa heat shock proteins (hsp70) were analyzed by western blot. RESULTS: More males than females were detected in the randomized population sample (ratio of females/males: 0.87). No mortality occurred at 15°C, while 42.9% of gammarids died at 25°C. Sequences of three microsporidians (M1, M2, M3) were detected in this G. pulex population (99.7%-100% sequence identity to Microsporidium spp. from GenBank). Previous studies showed that M3 is vertically transmitted, while M1 and M2 are presumably horizontally transmitted. Prevalences, according to PCR, were 27.0%, 37.8% and 64.9% for Microsporidium sp. M1, M2 and M3, respectively. Cumulative prevalence was 82.4%. Multiple infections with all three microsporidians in single gammarids were detected with a prevalence of 8.1%, and bi-infections ranged between 12.2% and 25.7%. In dead gammarids, comparatively low prevalences were noted for M1 (males and females: 11.1%) and M2 (females: 11.1%; males 0%), while prevalence of M3 was higher (females: 66.7%; males: 88.9%). No significant effect of host sex on microsporidian infection was found.Significant effects of temperature and bi-infection with Microsporidium spp. M2 + M3 on hsp70 response were detected by analysis of the whole sample (15°C and 25°C group) and of M2 + M3 bi-infection and gammarid weight when analyzing the 25°C group separately. None of the parameters had a significant effect on hsp70 levels in the 15°C group. CONCLUSION: This study shows that some microsporidian infections in amphipods can cause an increase in stress protein level, in addition to other stressors. Although more harmful effects of combined stressors can be expected, experimental evidence suggests that such an increase might possibly have a protective effect for the host against acute temperature stress.

Effect of different temperatures on the expression of the newly characterized heat shock protein 90 (Hsp90) in L3 of Anisakis spp. isolated from Scomber australasicus.

Anisakid nematodes are distributed worldwide in a wide variety of marine fishes and they are known to cause the zoonotic disease, anisakiasis. The temperature control is commonly applied for prevention and control of anisakiasis. To analyze the cellular response to temperature stress in Anisakis, the heat shock protein 90 (Hsp90) was chosen in the present study, as it plays a key role in many cellular processes and responds to stress conditions such as heat or cold shock. Anisakids were sampled from spotted mackerel Scomber australasicus caught from the coastal waters of Yilan, in northeastern Taiwan (25 °N, 121 °E). Anisakid nematodes were pre-identified morphologically and later molecularly by PCR-RFLP. In total, we obtained six species of the genus Anisakis, A. typica, A. pegreffii, A. paggiae, A. brevispiculata, A. physeteris, and a recombinant genotype between A. pegreffii and A. simplex sensu stricto. Thereby we provide new host and locality records for A. paggiae, A. brevispiculata and A. physeteris. The Hsp90 genes of five species (except the recombinant genotype) were cloned by rapid amplification of cDNA ends (RACE) and their deduced amino acid sequences were further characterized. Quantitative real-time PCR and Western blot analysis were used to examine the expression levels of the Hsp90 in A. pegreffii under different temperature conditions. Quantitative RT-PCR showed that Hsp90 transcript levels increased slightly under heat shock (50 °C) treatment, and increased gradually during the first 3h, and thereafter, returned to its baseline value at 37 °C. Under cold shock (4 °C) treatment, the mRNA expression of Hsp90 did not change significantly. In addition, we found a clear time-dependent Hsp90 protein expression pattern of A. pegreffii exposed to high temperature. Our results suggest that the mRNA and protein expression patterns of Hsp90 are related to the temperature, and are especially significantly increased under heat stress.

Invasion biology meets parasitology: a case study of parasite spill-back with Egyptian Fasciola gigantica in the invasive snail Pseudosuccinea columella.

The liver fluke Fasciola gigantica is a trematode parasite of ruminants and humans that occurs naturally in Africa and Asia. Cases of human fascioliasis, attributable at least in part to F. gigantica, are significantly increasing in the last decades. The introduced snail species Galba truncatula was already identified to be an important intermediate host for this parasite and the efficient invader Pseudosuccinea columella is another suspect in this case. Therefore, we investigated snails collected in irrigation canals in Fayoum governorate in Egypt for prevalence of trematodes with focus on P. columella and its role for the transmission of F. gigantica. Species were identified morphologically and by partial sequencing of the cytochrome oxidase subunit I gene (COI). Among all 689 snails found at the 21 sampling sites, P. columella was the most abundant snail with 296 individuals (42.96%) and it was also the most dominant species at 10 sites. It was not found at 8 sites. Molecular detection by PCR and sequencing of the ITS1-5.8S-ITS2 region of the ribosomal DNA (rDNA) revealed infections with F. gigantica (3.38%), Echinostoma caproni (2.36%) and another echinostome (7.09%) that could not be identified further according to its sequence. No dependency of snail size and trematode infection was found. Both high abundance of P. columella in the Fayoum irrigation system and common infection with F. gigantica might be a case of parasite spill-back (increased prevalence in local final hosts due to highly susceptible introduced intermediate host species) from the introduced P. columella to the human population, explaining at least partly the observed increase of reported fascioliasis-cases in Egypt. Eichhornia crassipes, the invasive water hyacinth, which covers huge areas of the irrigation canals, offers safe refuges for the amphibious P. columella during molluscicide application. As a consequence, this snail dominates snail communities and efficiently transmits F. gigantica.

Merging species? Evidence for hybridization between the eel parasites Anguillicola crassus and A. novaezelandiae (Nematoda, Anguillicolidea).

BACKGROUND: The eel parasitic nematodes Anguillicola crassus (originating from Asia) and Anguillicola novaezelandiae (originating from New Zealand) were both introduced to Europe, but occurred in sympatry only in Lake Bracciano in Italy, where they both infected the European eel (Anguilla anguilla). A. novaezelandiae was introduced to the lake in 1975 and disappeared soon after A. crassus was also found there in 1993. We tested the hypothesis if hybridization of the two species might be an explanation for the findings at Lake Bracciano. FINDINGS: After laboratory infection of one European eel with 10 third stage larvae of each parasite, two living female and 4 male adults of each species were found to co-occur in the swim bladder after 222 days post exposure. In 9 out of 17 eggs, isolated in total from uteri of the two A. novaezelandiae females, alleles were detected by microsatellite analysis that are characteristic for A. crassus, suggesting the hybrid origin of these eggs. In contrast, none of the eggs isolated from A. crassus females possessed alleles different from those found in A. crassus adults, but it was revealed that one female can be inseminated by several males. CONCLUSION: Our results show that A. crassus and A. novaezelandiae can co-infect a single eel and can mature together in the same swim bladder. We also provide evidence for the possibility of hybridization of A. crassus males with A. novaezelandiae females. Therefore, hybridization might be an explanation for the disappearance of A. novaezelandiae from Lake Bracciano.

Comparison of the susceptibility of brown trout (Salmo trutta) and four rainbow trout (Oncorhynchus mykiss) strains to the myxozoan Tetracapsuloides bryosalmonae, the causative agent of proliferative kidney disease (PKD).

Differences in susceptibility to the myxozoan parasite Tetracapsuloides bryosalmonae, the causative agent of proliferative kidney disease (PKD), between four strains of rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta) were evaluated. Fish were exposed to water enzootic for the parasite in the field for 5 days and were subsequently transferred to the laboratory. Relative parasite load was determined after 2, 3 and 4 weeks post-exposure (wpe) by quantitative real-time PCR (qPCR) of kidney samples and number of parasite stages was determined in immunohistochemical stained sections of kidney, liver and spleen tissues. According to qPCR results, the highest amount of parasite DNA per equal amount of host tissue at all time points was measured in brown trout. Two of the rainbow trout strains showed lower relative parasite load than all other groups at the beginning of the experiment, but the parasite multiplied faster in these strains resulting in an equal level of relative parasite load for all rainbow trout strains at 4 wpe. A weak negative correlation of fish size and parasite load was detected. Only in samples of a few fish, single stages of T. bryosalmonae were found in sections stained by immunohistochemistry impeding quantitative evaluation of parasite numbers by this method. The results indicate a differential resistance to T. bryosalmonae between the rainbow trout strains investigated and between rainbow trout and brown trout.