Pearl Mussel Research Articles

Mass Mortality Events in Freshwater Pearl Mussel (Margaritifera margaritifera) Populations in Sweden: An Overview and Indication of Possible Causes

The freshwater pearl mussel Margaritifera margaritifera is an endangered species in Sweden with more than 600 known populations distributed in 16 out of 21 counties. Only approximately one-third of these populations are considered viable and healthy with signs of recent juvenile recruitment. From 2011 to 2017, research documented an increased mortality in this species, of up to 100% in some populations, but no etiological cause of these mortalities has been identified. With this paper, we provide current knowledge of locations where mass mortality of freshwater pearl mussel has been found in Sweden and discuss possible causes. Postmortem sampling and histopathological findings from two counties in 2016–17 detected lesions in digestive glands indicating a reduced capacity for nutrient uptake. Results from these macroscopic and microscopic investigations also indicate a reduction in, or a lack of, reproductive output compared with reference populations.

Annual gametogenic cycle of the freshwater pearl mussel, Lamellidens marginalis (Lamarck, 1819) collected from a perennial lentic habitat of Bangladesh

ABSTRACTWe investigated the annual gametogenic phenology of the freshwater pearl mussel, Lamellidens marginalis (Lamarck, 1819), collected from a lentic habitat at Mymensingh, Bangladesh, using biometry and histology through monthly sampling from August 2015 to July 2016. After biometric measurements, thin slices of dorso-ventral sections were cut from the middle of the mussels for histology to determine sex and level of gonadal maturation. The condition index (CI) ranged from 0.64 (March) to 0.99 (January) over the study period. The CI peaked three times (January, April and July) indicating that the mussels were ripe during these months and subsequent decreases in CI indicated spawning, which was consistent with histology. Both males and females exhibited similar patterns in terms of gonadal development, maturation and spawning activity. It was confirmed that natural populations of L. marginalis spawn throughout the year with remarkable temporal variations, except during December when the surface water temperature reaches annual minima (16.5°C). Highest spawning activity of L. marginalis was noted during February–March, May and July–November. The data obtained in the present study could be useful for the conservation managers of this commercial species by allowing harvesting of better quality mussels to be timed without interrupting major spawning activity.

Studies on morphological regime conditions of bi-modal grain size rivers: Challenges and new insights for freshwater pearl mussel habitats

Freshwater pearl mussels have high ecological demands on their habitat. For natural reproduction and distribution, they need a colmation-free porespace in the substrate and sufficient host fish for their parasitic stage of life. In Austria, river alterations and anthropogenic activities in the catchments are causing habitat degradation for both, the freshwater pearl mussel Margaritifera margaritifera and its host fish Salmo trutta. Due to the lack of reference stretches, sediment transport and sediment deposition in the main channel cannot be measured directly. In (semi-) natural river sections as well as in sections with anthropogenically caused over-width of the river bed, however, it can be observed that the river itself forms a two-stage ditch within a few years. This study examines the deposits of those rivers and describes hydraulic and sedimentological processes influencing the geometry within the main channel below bankfull stage. A possible correlation between the mean annual discharge and the instream geometry could be determined. A simple formula is given as a recommendation for action, which can be used for pre-dimensioning of channel geometry below the bankfull stage in case of river restoration of freshwater pearl mussel habitats. This equation also allows authorities to review planning services for ecologically negative interventions and to recommend a method of implementing measures below bankfull stage.

Effects of an extreme drought on the endangered pearl mussel Margaritifera margaritifera: a before/after assessment

Droughts can change the distribution and alter the behaviour, phenology and physiology of many species, leading in extreme cases to high mortalities or even extirpation. In this study, a before/after comparison was performed in two Portuguese river basins (Rabacal and Tuela) to assess the possible effects of an extreme drought on the abundance and size structure of two pearl mussel Margaritifera margaritifera populations, a Critically Endangered species in Europe. The hydrological year of 2017 was extremely dry in Portugal and this situation was responsible for a major reduction of the river flow and rapid decrease in the water levels during summer. One year after the drought, a significant reduction in abundance was observed (i.e. 27.6% for the Rabacal and 38.7% for the Tuela basin populations). However, no differences were detected in the size structure of the two pearl mussel populations. Droughts are predicted to increase in number and intensity and it is crucial to monitor M. margaritifera populations, given the low resistance (and possibly low resilience) of this species to these extreme conditions. Management measures that could help the conservation of pearl mussels are discussed.

HC-SPH: A conserved serine protease homolog of S1 superfamily in the triangle-shell pearl mussel (Hyriopsis cumingii)

Serine proteases play central roles in immune defense in invertebrates through innate immunity, and are particularly important complement system in molluscs because their susceptibility to infection due to lack of an adaptive immune ability. A gene encoding the serine protease homolog from the triangle - shell pearl mussel (Hyriopsis cumingii) was identified and designated as HC-SPH in this study. Protein sequence analysis revealed that HC-SPH consists of a typical Tryp_SPc functional domain of serine protease of S1 family lead by a signal peptide, and the molecule shares a highly conserved sequence and structural organization with other members, including a cleavage site, 3 enzymatic active sites and 3 substrate binding sites, so that it was clustered into a trypsin-like serine protease subfamily of the S1 superfamily. Semi - quantitative analysis of the amplicons separated on agarose gel by comparing to the β-actin products revealed that the digestive gland had a strong expression while the gonads were seen as weak expression sites. Infected by Aeromonas hydrophila, the gene expression was significantly up - regulated in the kidney at the 6 hours post challenge (hpc), stomach at 12 hpc and gills at 24 hpc while the expression maintained steadily unchanged in the digestive gland. However, up to 48 hpc, the expression levels in all four tissues reached significantly high, and also joined by a high level of expression in intestine that was down - regulated before 24 hpc, to build up an enhanced immune defense. The complementary up - regulation of the gene expression in these tissues suggested a temporal and spatial reinforce model for HC-SPH in immune response.

Molecular cloning of complement component C3 gene from pearl mussel, Hyriopsis cumingii and analysis of the gene expression in response to tissue transplantation

Complement component C3 is well recognized as the central mediator of complement system, whose activation is responsible for the immune surveillance and elimination of non-self-antigens. In this study, C3 gene (HcC3) from a pearl making mussel, Hyriopsis cumingii, was successfully identified. The putative HcC3 possessed the canonical domains and highly conserved functional residues of C3 family members. In phylogenetic analysis, HcC3 was also clustered into C3 subfamily and separated from α2 macroglobulin clade. HcC3 gene was constitutively expressed in a wide range of tissues of pearl mussels, among which the immune-related tissues like hemocytes got highest expression. After allograft surgery of mantle tissues for aquaculture pearl production, the gene expression of HcC3 exhibited a rapid upregulation on day 1, dropped back on day 3, peaked the value on day 7, and restored to the level similar to control samples on day 14 after mantle allograft. The biphasic expression within the two weeks post the surgery suggests the important roles for HcC3 in alloimmune responses and an intricate complement activation mechanism in mollusks during tissue allograft.

Simple nonlethal identification criteria for two endangered freshwater pearl mussels, Margaritifera laevis and Margaritifera togakushiensis, in Hokkaido, northern Japan

AbstractSpecies identification is a fundamental process for ecological studies and conservation practices, and simple nonlethal identification criteria are important for endangered species. This study developed species identification criteria for two endangered freshwater pearl mussels (Margaritifera laevis and Margaritifera togakushiensis) based on linear discriminant functions (LDFs) that were established considering intraspecific regional morphological differences from sites at Hokkaido, northern Japan. We collected a total of 1,110 mussels from 52 rivers across 32 watersheds in two geographical regions (east and west). Shell morphologies (length, height, and width) of the collected mussels were measured, followed by species identification with gel electrophoresis banding patterns of 16S rRNA polymerase chain reaction products. LDFs were constructed for two size classes (≥40 mm and < 40 mm) using forward stepwise procedures to determine key morphological differences between the two species and consideration of whether regional morphological differences improved identification accuracies. The LDFs revealed that the morphologies of the two species were clearly distinguished by the relationship between height and length for both size classes. Region‐specific LDFs produced better identification accuracies. Regardless of region, the maximum length of M. laevis exceeded 100 mm, whereas all M. togakushiensis were < 100 mm in length. Identification accuracies of the established LDFs were high for each of the five length classes (0–20 mm, 20–40 mm, 40–60 mm, 60–80 mm, and 80–100 mm) with 85–96% (mean: 92%) accuracy in the east and 67–96% (mean: 80%) in the west. These criteria for species identification will progress future ecological studies and conservation practices for freshwater mussels.

Refuge in the sāqya: Irrigation canals as habitat for one of the world's 100 most threatened species

Anthropogenic habitats may function as a refuge for some species, including freshwater mussels (Bivalvia, Unionida). In this study we assessed possible differences in density, size and condition index of the Moroccan freshwater pearl mussel Pseudunio marocanus (Pallary, 1918), formerly Margaritifera marocana, colonizing anthropogenic (sāqya = irrigation canal) and natural (Bouhlou River) habitats in Morocco. Results showed that the individuals colonizing the irrigation canal located on the left bank have a significantly higher density and condition index when compared to natural conditions, but no differences were found regarding size. Since this species is listed as critically endangered by the IUCN, being also considered among the 100 most endangered organisms in the planet, the presence of P. marocanus in anthropogenic habitats has high conservation importance. In fact, and despite no Moroccan freshwater pearl mussels were found on the irrigation canal located in the right bank, the one located on the left bank present stable conditions for the settlement of juveniles and, if managed carefully, for the future survival of the species. However, local authorities reported frequent dredging and cleaning activities by local farmers on this sāqya without any special attention devoted to Moroccan freshwater pearl mussels (or any other species) conservation. Therefore, the results reported here can be used by scientists, managers, politicians and local people to promote future management actions that enhance the Moroccan freshwater pearl mussel protection and guarantee their future survival, including on anthropogenic habitats.

Effect of glochidia infection on growth of fish: freshwater pearl mussel Margaritifera margaritifera and brown trout Salmo trutta

Effect of freshwater mussels’ (Unionoida) glochidia on the growth of fish host has remained poorly studied. We compared the specific growth rate of the juvenile, PIT-marked brown trout (Salmo trutta) between uninfected controls to those experimentally infected (average initial intensity of infection 8000 fish−1) with Margaritifera margaritifera glochidia, kept in high and low feeding. Growth and mortality of fish were monitored for 10 months. Our hypothesis was that glochidiosis would impair the growth of fish. According to our hypothesis, infected fish gained statistically significantly less weight than the control fish throughout the experiment. A proportional increase in weight of control individuals was 11% higher than that of the infected fish. However, neither the feeding regime (high, low) nor the period (September–November, November–March, March–May), had a significant effect on the growth difference between control and infected fish. As the effect of infection on the growth of fish was subtle and no effect on host mortality was detected either, this may turn public opinion favorable for M. margaritifera conservation even if the salmonid host population is important for commercial or recreational fishing.

Molecular Characterization of Two Toll Receptors in Hyriopsis cumingii and Their Potential Roles in Antibacterial Response.

Tolls/Toll-like receptors (TLRs) play a key role in innate immunity by detecting the invading microbes and subsequently activating downstream signaling cascades. In this study, two new molluscan Toll members (designed as HcToll6 and HcToll7) were identified from triangle-shell pearl mussel (Hyriopsis cumingii). The obtained HcToll6 full-length cDNA was 3207 bp consisting of a 2223 bp open reading frame (ORF) that encoded a peptide of 740 amino acids. HcToll7 cDNA is a 3216 bp molecule that contains an ORF of 2139 bp encoding a protein of 712 amino acids. The deduced HcToll6 and HcToll7 proteins share two common structures: extracellular leucine-rich repeat (LRR) domains and intracellular Toll/interleukin-1 receptor (TIR) domain. Quantitative real-time PCR results showed that HcToll6 and HcToll7 were mainly expressed in the hepatopancreas and the gills, and they responded rapidly to bacterial stimulation. RNA interference by dsRNA results revealed that HcToll6 and HcToll7 RNAi strongly decreased the expression of lysozyme (HcLyso) and defensin (HcDef) in the gills of RNAi-treated mussels with Vibrio parahaemolyticus challenge. As a pattern recognition receptor, the prokaryotic expressed the recombinant LRR domains of HcToll6 and HcToll7 (rHcToll6-LRR and rHcToll7-LRR) could bind to Gram-positive and Gram-negative bacteria and had a strong tendency to recognize lipopolysaccharide (LPS) and peptidoglycan (PNG). rHcToll6-LRR and rHcToll7-LRR exhibited a significant in vitro bactericidal activity against V. parahaemolyticus and Staphylococcus aureus. These findings provide useful information to characterize Tolls in mussels.

Host specificity drives genetic structure in a freshwater mussel

Parasites often depend on their hosts for long distance transport, and genetic population structure can be strongly affected by host specificity and dispersal. Freshwater pearl mussel (Margaritifera margaritifera) populations have previously been found to naturally infest either primarily Atlantic salmon (‘salmon-mussel’) or exclusively brown trout (‘trout-mussel’) across a wide geographic range. Here, we experimentally test whether this intraspecific variation in natural infestation can be explained by host specificity in freshwater pearl mussel. Our experiments show that when both host species were exposed to larvae from salmon- and trout-mussel respectively, salmon-mussel larvae almost never infested brown trout and vice versa. This suggests that host specificity can explain variation in natural infestation among the studied freshwater pearl mussel populations. Host specificity provides a link to the species’ variable population genetic structure, as mussel populations limited to Atlantic salmon, the host with stronger dispersal, show higher genetic diversity and weaker differentiation than populations limited to brown trout as host.

Quantification of mantle attachment scars on the Margaritifera margaritifera (Margaritiferidae, Bivalvia) shell

A characteristic feature shared by all Margaritiferidae mussels consists in the presence of scars on the internal surface of their shells. These scars mark places of mantle attachment by specific epithelial cells. According to some authors, such parameters of mantle attachment scars as the density and degree of their manifestation can be used for the purposes of species identification. To this end, quality indicators (numerous or few, pronounced or poorly visible scars) are typically used; however, the use of quantitative criteria is preferable. This work was aimed at developing quantitative indicators of mantle attachment scars in the Margaritifera margaritifera freshwater pearl mussels for the populations of the Syuskyuyanyoki and Livoyoki rivers (Karelia). It is shown that the density and size of mantle attachment scars are approximately the same for all the investigated shell samples. These parameters display the absence of dependence on the age and size of molluscs. The regression coefficients for the linear dependence of the density or size of mantle attachment scars on the age or length of the shell show no significant difference from 0. For all the investigated molluscs, the density and size of mantle attachment scars are established to vary within the range of 0.37–1.16 scars/cm2 (0.71 ± 0.03 scars/cm2 on average) and 215–690 µm (417 ± 2 µm on average), respectively. According to a two-factor analysis of variance, the distribution of mantle attachment scars appears to be irregular across the shell surface. At the same time, a significant (p < 0.001) increase in their density is observed in the posterior and ventral directions, as well as diagonally in the posterior ventral direction. Issues associated with the application of the investigated parameters for the purposes of mollusc species identification are discussed.

An unexpected host for the endangered giant freshwater pearl mussel Margaritifera auricularia (Spengler, 1793) as a conservation tool

Abstract The giant freshwater pearl mussel Margaritifera auricularia (GFPM) is one of the most endangered bivalve species in the world. Originally occurring in many European rivers, the GFPM is a relict now restricted to a few ageing populations in France and Spain in which natural reproduction is almost absent. Like most unionoid mussels, the GFPM needs host fish for the development of their parasitic larvae (glochidia). The European sturgeon (Acipenser sturio), the only known native host fish of the GFPM in France, is essentially extinct. Therefore, the aim of this study was to identify other hosts that could be responsible for the few cases of recent recruitment. Natural infestation of wild fishes in three French rivers was assessed to identify potential hosts of M. auricularia, while artificial infestation experiments were conducted on the sea lamprey (Petromyzon marinus) and the wels catfish (Silurus glanis) to determine their compatibility as hosts. Among the 29 fish species assessed for natural infestation, only the three‐spined stickleback (Gasterosteus aculeatus) and the European eel (Anguilla anguilla) carried M. auricularia glochidia. In the artificial infestation experiments, living juvenile mussels were collected from both P. marinus and S. glanis. The number of juveniles collected from a single P. marinus specimen (13,827) suggests that this species is a highly efficient host. As with previously known hosts, newly identified ones also appear to have a relationship with marine environments. The present findings suggest that P. marinus has played a key role in preventing the total extinction of M. auricularia in France, and indicate the potential use of P. marinus in conservation strategies aimed at reintroducing or stabilizing populations of this rare mollusc.

Quantification of mantle attachment scars on the Margaritifera margaritifera (Margaritiferidae, Bivalvia) shell

A characteristic feature shared by all Margaritiferidae mussels consists in the presence of scars on the internal surface of their shells. These scars mark places of mantle attachment by specific epithelial cells. According to some authors, such parameters of mantle attachment scars as the density and degree of their manifestation can be used for the purposes of species identification. To this end, quality indicators (numerous or few, pronounced or poorly visible scars) are typically used; however, the use of quantitative criteria is preferable. This work was aimed at developing quantitative indicators of mantle attachment scars in the Margaritifera margaritifera freshwater pearl mussels for the populations of the Syuskyuyanyoki and Livoyoki rivers (Karelia). It is shown that the density and size of mantle attachment scars are approximately the same for all the investigated shell samples. These parameters display the absence of dependence on the age and size of molluscs. The regression coefficients for the linear dependence of the density or size of mantle attachment scars on the age or length of the shell show no significant difference from 0. For all the investigated molluscs, the density and size of mantle attachment scars are established to vary within the range of 0.37–1.16 scars/cm2 (0.71 ± 0.03 scars/cm2 on average) and 215–690 µm (417 ± 2 µm on average), respectively. According to a two-factor analysis of variance, the distribution of mantle attachment scars appears to be irregular across the shell surface. At the same time, a significant (p < 0.001) increase in their density is observed in the posterior and ventral directions, as well as diagonally in the posterior ventral direction. Issues associated with the application of the investigated parameters for the purposes of mollusc species identification are discussed.

Cellular and humoral immune responses of meagre (Argyrosomus regius) juveniles to bacterial infection with Photobacterium damselae piscicida

For pearl culture of bivalve Hyriopsis cumingii, implantation of the sabio may cause nucleus discharge and increased host death rates. We performed a transcriptome analysis of the pearl sac of H. cumingii for 30 days after mantle implantation; 293863 unigenes were obtained, and 27176 unigenes were identified using nr, nt, KO, Swiss-Prot, Pfam, GO, and KOG databases. We detected 4878 differentially expressed genes (DEGs) through pairwise comparisons. We speculated that the physical condition of the recipient mussels returned to normal in about one month; the period was divided into six vital phases (0, 2 h–6 h, 12 h–24 h, 48 h to 7 days, 14 days and 30 days) on the basis of the overall similarities in DEGs. We compared the DEGs between time points and identified key immune-related genes. Our findings provide information on the immunological reactions induced by implantation in pearl mussels.

Water mill canals as habitat for Margaritifera margaritifera: Stable refuge or an ecological trap?

Abstract Anthropogenic habitats may serve as a refuge for aquatic species, including freshwater mussels (Bivalvia, Unionida). Evaluating the role of anthropogenic habitats is a fundamental, but still ignored, conservation issue given the pace that humans have been converting natural ecosystems. In this study, possible differences in abundance, size and condition index of the freshwater pearl mussel Margaritifera margaritifera colonizing anthropogenic (water mill canals) and natural (Tuela River) habitats were assessed. No differences were found in the abundance of freshwater pearl mussels colonizing both habitats, but individuals present in the water mill canals have a significantly higher condition index and size. Water mill canals seem to provide stable conditions for the settlement, growth and survival of freshwater pearl mussels. However, the occurrence of an exceptional drought during the late summer of 2017 was responsible for an almost 100% mortality in one of the two water mill canals surveyed in this study. Therefore, and during extreme climatic events, these anthropogenic structures may function as an ecological trap for freshwater pearl mussels. This study can be used by managers to promote future actions that enhance freshwater pearl mussel protection and guarantee their survival, including on anthropogenic habitats.

Fish hosts, glochidia features and life cycle of the endemic freshwater pearl mussel Margaritifera dahurica from the Amur Basin

Margaritiferidae is a small freshwater bivalve family with 16 species. In spite of a small number of taxa and long-term history of research, several gaps in our knowledge on the freshwater pearl mussels still exist. Here we present the discovery of host fishes for Margaritifera dahurica, i.e. Lower Amur grayling, sharp-snouted lenok, and blunt-snouted lenok. The host fishes were studied in rivers of the Ussuri Basin. The identification of glochidia and fish hosts was confirmed by DNA analysis. The life cycle of M. dahurica and its glochidia are described for the first time. The SEM study of glochidia revealed that the rounded, unhooked Margaritifera dahurica larvae are similar to those of the other Margaritiferidae. Margaritifera dahurica is a tachytictic breeder, the larvae of which attach to fish gills during the Late August – September and finish the metamorphosis in June. Ancestral host reconstruction and a review of the salmonid - pearl mussel coevolution suggest that the ancestral host of the Margaritiferidae was a non-salmonid fish, while that of the genus Margaritifera most likely was an early salmonid species or their stem lineage. The overfishing of lenoks and graylings appears to be the most significant threat for this rare mussel species.

Integrated transcriptome analysis of immunological responses in the pearl sac of the triangle sail mussel (Hyriopsis cumingii) after mantle implantation

For pearl culture of bivalve Hyriopsis cumingii, implantation of the sabio may cause nucleus discharge and increased host death rates. We performed a transcriptome analysis of the pearl sac of H. cumingii for 30 days after mantle implantation; 293863 unigenes were obtained, and 27176 unigenes were identified using nr, nt, KO, Swiss-Prot, Pfam, GO, and KOG databases. We detected 4878 differentially expressed genes (DEGs) through pairwise comparisons. We speculated that the physical condition of the recipient mussels returned to normal in about one month; the period was divided into six vital phases (0, 2 h–6 h, 12 h–24 h, 48 h to 7 days, 14 days and 30 days) on the basis of the overall similarities in DEGs. We compared the DEGs between time points and identified key immune-related genes. Our findings provide information on the immunological reactions induced by implantation in pearl mussels.

Search for hidden pearl mussel populations at the Russian section of the Baltic Sea

The freshwater pearl mussel Margaritifera margaritifera (L., 1758) is considered to be a critically endangered species. It is important to obtain information on the distribution and status of the surviving populations of the species, but in Russia this work is still problematic due to the size of the country’s territory, combined with the insufficient number of available specialists. “Searching rules” have been proposed to remotely identify potential pearl mussel habitats in rivers. The most important criteria are relating to the lakes found at the rivers’ sources, as well as to the extent of the deforestation along river basins. About 200 watercourses of the Russian section of the Baltic Sea basin were evaluated using these “rules”. As a result, two previously unknown, viable populations were discovered (comprising about 53,000 specimens), while the absence of pearl mussels was made evident among some rivers which were traditionally believed to be their habitats. The obtained data provides an update on the status of pearl mussels in Russian territories located south of the 61 st degree of latitude. There are 10 populations of this species; 4 of them are almost extinct, 6 are relatively numerous but declining. The total number of pearl mussels hardly exceeds 90,000 individuals.

Downstream transport and seasonal variation in freshwater pearl mussel (Margaritifera margaritifera) eDNA concentration

AbstractEnvironmental DNA (eDNA) can be used to detect the presence and abundance of aquatic organisms from water samples. Before implementing this methodology as a tool for monitoring, more knowledge is needed on variation in eDNA concentrations in relation to species abundance and potential confounding factors. Shedding and decay of eDNA may vary extensively over the season and are dependent on environmental factors such as water temperature and on biological processes such as activity level and reproduction. In lotic systems, eDNA concentrations are also affected by downstream transport of eDNA. Sessile freshwater mussels provide an ideal study system for investigating the relationship between species spatial distribution and eDNA concentrations in lotic systems. We quantified freshwater pearl mussel (Margaritifera margaritifera) eDNA concentrations at four localities in a natural river with detailed knowledge of mussel spatial distribution: (a) upstream of the known species distribution, just downstream (b) a small and (c) a large aggregation and (d) 1,700 m downstream of the large aggregation. To study seasonal variation, we quantified eDNA concentrations during three periods: (a) in late spring, with cold water and relatively inactive mussels; (b) in mid‐summer, with higher water temperature and active mussel filtration; and (c) in late summer, during the release of larvae. Species detection was highly reliable, with no detection of eDNA upstream of the species distribution and complete detection downstream of the large aggregation. Detection success of the small aggregation was low, with 13% of the samples testing positive. Downstream transport was efficient, with no significant decrease in eDNA concentrations over 1,700 m river distance. Seasonal variation was strong, with a 20‐fold increase in eDNA concentrations from late spring to late summer, during reproduction. Our results highlight both the potential and challenges of eDNA monitoring in lotic systems.