Microsporidian diversity in the aquatic isopod Asellus aquaticus.

We conducted a molecular survey on microsporidian diversity in different lineages (operational taxonomic units = OTUs) of Asellus aquaticus from 30 sites throughout Europe. Host body length was determined, and DNA was extracted from host tissue excluding the intestine and amplified by microsporidian-specific primers. In total, 247 A. aquaticus specimens were analysed from which 26.7% were PCR-positive for microsporidians, with significantly more infections in larger individuals. Prevalence ranged between 10 and 90%. At 9 sites, no microsporidians were detected. A significant relationship was found between the frequency of infected individuals and habitat type, as well as host OTU. The lowest proportion of infected individuals was detected in spring-habitats (8.7%, n = 46) and the highest in ponds (37.7%, n = 53). Proportion of infected individuals among host OTUs A, D and J was 31.7, 21.7 and 32.1%, respectively. No infections were detected in OTU F. Our results are, however, accompanied by a partially low sample size, as only a minimum of 5 individuals was available at a few locations. Overall, 17 different microsporidian molecular taxonomic units (MICMOTUs) were distinguished with 5 abundant isolates (found in 4­17 host individuals) while the remaining 12 MICMOTUs were "rare" and found only in 1­3 host individuals. No obvious spatio-genetic pattern could be observed. The MICMOTUs predominantly belonged to Nosematida and Enterocytozoonida. The present study shows that microsporidians in A. aquaticus are abundant and diverse but do not show obvious patterns related to host genetic lineages or geography.

Identifying Potential Hosts of Short-Branch Microsporidia.

Microsporidia are obligate parasites that are closely related to Fungi. While the widely known "long-branch" Microsporidia infect mostly metazoans, the hosts of "short-branch" Microsporidia are only partially characterized or not known at all. Here, we used network analyses from Neotropical rainforest soil metabarcoding data, to infer co-occurrences between environmental lineages of short-branch microsporidians and their potential hosts. We found significant co-occurrences with several taxa, especially with Apicomplexa, Cercozoa, and Fungi, as well as some Metazoa. Our results are the first step to identify potential hosts of the environmental lineages of short-branch microsporidians, which can be targeted in future molecular and microscopic studies.

Morphological comparison of genetically differentiated Polymorphus cf. minutus types.

In the present study, we analyzed the morphology of three genetic types of the bird-infecting acanthocephalan Polymorphus cf. minutus (PspT1, PspT2, PspT3), mainly based on the cystacanth-stage obtained from amphipods (Gammarus fossarum, Gammarus pulex, Gammarus roeselii, Echinogammarus spp.). Males and females were pooled as there was no considerable difference between the sexes concerning the hook measurements. Additionally, we conducted a laboratory infection of one domestic duck for each Polymorphus type, to compare their performance and localization in this host species, and to obtain adult specimens for morphological comparison. The recovery rate from the ducks 4 weeks after infection was 16% for PspT1, 23.8% for PspT2, and 25% for PspT3. The adult worms were gravid, and the females contained mature eggs. Hook size did not differ considerably between cystacanths and adults of the respective type. The three Polymorphus types could be distinguished based on the cystacanth stage by a linear discriminant analysis that included hook measurements, proboscis length, proboscis width, and number of longitudinal hook rows and hooks per row. Furthermore, PspT3 was more different from PspT1 and PspT2 than the latter types from each other. Mainly the number of longitudinal hook rows differed in PspT3 from the existing descriptions of P. minutus (mainly 14 vs. mainly 16 rows). Potentially, PspT3 could be a non-indigenous parasite that was introduced with G. roeselii and that adapted to use the indigenous G. pulex as a host, while PspT2 might have been introduced to central Europe together with Echinogammarus spp.

Assessing the response of an urban stream ecosystem to salinization under different flow regimes.

Urban streams are exposed to a variety of anthropogenic stressors. Freshwater salinization is a key stressor in these ecosystems that is predicted to be further exacerbated by climate change, which causes simultaneous changes in flow parameters, potentially resulting in non-additive effects on aquatic ecosystems. However, the effects of salinization and flow velocity on urban streams are still poorly understood as multiple-stressor experiments are often conducted at pristine rather than urban sites. Therefore, we conducted a mesocosm experiment at the Boye River, a recently restored stream located in a highly urbanized area in Western Germany, and applied recurrent pulses of salinity along a gradient (NaCl, 9 h daily of +0 to +2.5 mS/cm) in combination with normal and reduced current velocities (20 cm/s vs. 10 cm/s). Using a comprehensive assessment across multiple organism groups (macroinvertebrates, eukaryotic algae, fungi, parasites) and ecosystem functions (primary production, organic-matter decomposition), we show that flow velocity reduction has a pervasive impact, causing community shifts for almost all assessed organism groups (except fungi) and inhibiting organic-matter decomposition. Salinization affected only dynamic components of community assembly by enhancing invertebrate emigration via drift and reducing fungal reproduction. We caution that the comparatively small impact of salt in our study can be due to legacy effects from past salt pollution by coal mining activities >30 years ago. Nevertheless, our results suggest that urban stream management should prioritize the continuity of a minimum discharge to maintain ecosystem integrity. Our study exemplifies a holistic approach for the assessment of multiple-stressor impacts on streams, which is needed to inform the establishment of a salinity threshold above which mitigation actions must be taken.

Comparing Microsporidia-targeting primers for environmental DNA sequencing.

Metabarcoding is a powerful tool to detect classical, and well-known "long-branch" Microsporidia in environmental samples. Several primer pairs were developed to target these unique microbial parasites, the majority of which remain undetected when using general metabarcoding primers. Most of these Microsporidia-targeting primer pairs amplify fragments of different length of the small subunit ribosomal RNA (SSU-rRNA) gene. However, we lack a broad comparison of the efficacy of those primers. Here, we conducted in silico PCRs with three short-read (which amplify a few-hundred base pairs) and two long-read (which amplify over a thousand base pairs) metabarcoding primer pairs on a variety of publicly available Microsporidia sensu lato SSU-rRNA gene sequences to test which primers capture most of the Microsporidia diversity. Our results indicate that the primer pairs do result in slight differences in inferred richness. Furthermore, some of the reverse primers are also able to bind to microsporidian subtaxa beyond the classical Microsporidia, which include the metchnikovellidan Amphiamblys spp., the chytridiopsid Chytridiopsis typographi and the "short-branch" microsporidian Mitosporidium daphniae.

Title: Comparaison des amorces ciblant les Microsporidies pour le séquençage de l'ADN environnemental. Abstract: Le métabarcoding est un outil puissant pour détecter les microsporidies classiques et bien connues à « longues branches ¼ dans les échantillons environnementaux. Plusieurs paires d'amorces ont été développées pour cibler ces parasites microscopiques exceptionnels, dont la majorité restent indétectables lors de l'utilisation d'amorces générales de métabarcoding. La plupart de ces paires d'amorces ciblant les microsporidies amplifient des fragments de différentes longueurs du gène de la petite sous-unité de l'ARN ribosomal (SSU-rRNA). Cependant, nous manquons d'une comparaison générale de l'efficacité de ces amorces. Ici, pour tester quelles amorces capturent la plus grande partie de la diversité des microsporidies, nous avons réalisé des PCR in silico avec trois paires d'amorces de métabarcoding à lecture courte (qui amplifient quelques centaines de paires de bases) et deux paires d'amorces de métabarcoding à lecture longue (qui amplifient plus d'un millier de bases), sur une variété de séquences du gène SSU-rRNA de Microsporidia sensu lato accessibles au public. Nos résultats indiquent que les paires d'amorces entraînent de légères différences dans la richesse déduite. En outre, certaines des amorces inverses sont également capables de se lier à des sous-taxons de microsporidies au-delà des Microsporidia classiques, notamment les Metchnikovellidae Amphiamblys spp., le Chytridiopsida Chytridiopsis typographi et la microsporidie à « branches courtes ¼ Mitosporidium daphniae.

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.

The Asymmetric Response Concept explains ecological consequences of multiple stressor exposure and release.

Our capacity to predict trajectories of ecosystem degradation and recovery is limited, especially when impairments are caused by multiple stressors. Recovery may be fast or slow and either complete or partial, sometimes result in novel ecosystem states or even fail completely. Here, we introduce the Asymmetric Response Concept (ARC) that provides a basis for exploring and predicting the pace and magnitude of ecological responses to, and release from, multiple stressors. The ARC holds that three key mechanisms govern population, community and ecosystem trajectories. Stress tolerance is the main mechanism determining responses to increasing stressor intensity, whereas dispersal and biotic interactions predominantly govern responses to the release from stressors. The shifting importance of these mechanisms creates asymmetries between the ecological trajectories that follow increasing and decreasing stressor intensities. This recognition helps to understand multiple stressor impacts and to predict which measures will restore communities that are resistant to restoration.