Freshwater Environments Research Articles

Occurrence and characterization of microplastics in bottled drinking water

Microplastics (MPs) have been identified in diverse settings, including drinking water, freshwater, food, air, and marine environments. This study employed stereomicroscopic and µ-Raman techniques to analyze water samples from ten widely consumed bottled water brands, aiming to detect and characterize MPs. Additionally, the oral consumption of MPs per capita was estimated. The average abundance of MPs ranged from 9 ± 1.00 MPs/L to 3 ± 1.73 MPs/L across all brands. Various MPs shapes were observed, including fibers, fragments, films, and pellets, with fibers being predominant (58.928%). MPs were categorized by color into five groups (red, blue, black, yellow, and transparent), with red comprising approximately 35.714% of the total count. All identified MPs were less than 500 µm in size. µ-Raman analysis validated the presence of eight different polymer types in the samples, namely Polypropylene (PP) (37.5%), Polymethyl methacrylate (PMMA) (3.571%), polystyrene (PS) (33.928%), polycarbonate (PC) (3.571%), polybutylene1 (PB1) (14.2%), Isotactic polypropylene (iPP) (7.142), Nylone 6-α polymorph (αNY6) (5.357%), and polyvinyl alcohol (PVOH) (1.785%). The estimated daily intake of MPs per person was determined to be 0.42 MPs, translating to an annual intake of 153.3 MPs per person. The origin of MPs in bottled water was likely attributed to both raw water sources and packaging materials, underscoring the need for further investigation. Given the potential health implications of human exposure to drinking water MPs, careful consideration should be given to the use of plastic packaging for potable water.

Seasonal Variations in Physico- chemical Parameters of the Sediment of Thengapatnam Estuary, South-coastal Region of India

The tremendous biodiversity and ecological processes that depend on freshwater and marine environments are essential to human survival. The physicochemical parameters in both surface waters and sediments are important to understand estuarine dynamics. The present study was carried out to determine how the seasonal variation will affect the physicochemical parameters of sediments of five stations in Thengapatnam estuary. Sediment samples were collected for a period of one year from June 2014-May 2015 from five different sampling stations. The parameters such as pH, temperature, Nitrogen, Phosphorous, Potassium, Copper, Zinc, Iron and Manganese were analysed using standard procedures. The result expose that, soil sediment have high amount of potassium in all five stations. Sediment temperature varied from 25.7- 30.1°C. Hydrogen ion concentration ranged between 6.9 to 8.3, concentrations of nutrients viz. nitrate (2.4 – 6.4 mg/100g), phosphate (1.5 - 15.25 mg/100g) potassium (138 – 560 ppm) and concentration of metals like iron, zinc and manganese were found to be higher during monsoon season and lower during premonsoon seasons The overall findings highlight the need for a scientific solution to reduce the use of harmful chemicals and heavy metal contamination from the river system in order to preserve the biodiversity of freshwater and marine ecosystems.

Seasonal variation in airborne microbial communities of the Akiyoshido Cave: Lampenflora dispersed by phototrophic bioaerosols

Lampenflora, such as phototrophic microorganisms, multiply on speleothem surfaces under the show-cave lighting, causing deterioration of the natural and cultural heritages of the caves. Speleothems change color from white to green through microbial photosynthesis and are destroyed by microbial metabolisms. Aerosol transmission are suspected to disperses photosynthetic microorganisms. However, the mechanism underlying the process remains unclear, because the complexity of air ventilation affects the cave atmosphere. In the present study, we collected aerosol samples from the Akiyoshido Cave, which is a special natural monument in Japan and possesses various types of speleothems, to analyze microbial concentrations and communities in the cave atmosphere. Under a fluorescence microscope, phototrophic and heterotrophic microorganisms were observed in the aerosols with in the cave. The aerosol concentrations showed seasonal changes depending on air-flow variations in cave ventilation. High-throughput DNA sequencing targeting 16S rRNA genes revealed that the airborne bacterial communities inside the cave were dominated by members of the phyla Actinomycetota, Bacillota, and Pseudomonadota, originating from the external terrestrial, phyllosphere or freshwater environments, as well as its interior (guano). Cyanobacteria showed small relative abundances (from 1.0 to 10%) randomly in the aerosol samples at several locations in the cave, suggesting that the lampenflora-derived cyanobacteria were dispersed throughout the cave by the ventilation. Additionally, phototrophic microorganisms closely related to the relatives of the genera Leptolyngbya, Calothrix, and Chroococcidiopsis from the phylum Cyanobacteria were isolated from the aerosol samples. These results confirm that Cyanobacteria are one of the candidate microorganisms responsible for lampenflora dispersion and are “alive and airborne” in caves.

Faecal source apportionment using molecular methods: A proof of concept using the FEAST algorithm

Faecal contamination of freshwater and marine environments represents a significant risk for public health, recreational activity and food safety, and tools for evaluating complex multi-source contamination remain largely in the development phase. We evaluated the efficacy of the Fast Expectation Maximization (FEAST) microbial source tracking (MST) algorithm to apportion sources of faecal contamination among four mammalian species of interest in coastal waters in New Zealand. Using 16S ribosomal DNA metabarcoding of faecal samples from cows, fur seals, and sheep, as well as human wastewater, we aimed to differentiate and quantify the contribution of these sources in mixed faecal samples. Multivariate analysis confirmed significant differences in the microbial communities associated with each mammalian source, with specific bacterial classes indicative of different sources. The FEAST algorithm was tested using mixed DNA and mixed faecal samples, and we found that the algorithm correctly assigned the dominant source from all samples, but underestimated the dominant source's proportional contribution. This underestimation suggests the need for further refinement and validation to ensure accurate source apportionment in environmental samples where the faecal signal is likely to be a minor component. Despite these limitations, the findings of our study, in combination with the evidence from others who have tested the FEAST algorithm in environmental settings, indicates that it represents an advance on existing tools for microbial source tracking and may become a useful addition to the toolbox for environmental management.

Effects of Harmful Algal Blooms on Amphibians and Reptiles are Under-Reported and Under-Represented.

Harmful algal blooms (HABs) are a persistent and increasing problem globally, yet we still have limited knowledge about how they affect wildlife. Although semi-aquatic and aquatic amphibians and reptiles have experienced large declines and occupy environments where HABs are increasingly problematic, their vulnerability to HABs remains unclear. To inform monitoring, management, and future research, we conducted a literature review, synthesized the studies, and report on the mortality events describing effects of cyanotoxins from HABs on freshwater herpetofauna. Our review identified 37 unique studies and 71 endpoints (no-observed-effect and lowest-observed-effect concentrations) involving 11 amphibian and 3 reptile species worldwide. Responses varied widely among studies, species, and exposure concentrations used in experiments. Concentrations causing lethal and sublethal effects in laboratory experiments were generally 1 to 100 µg/L, which contains the mean value of reported HAB events but is 70 times less than the maximum cyanotoxin concentrations reported in the environment. However, one species of amphibian was tolerant to concentrations of 10,000 µg/L, demonstrating potentially immense differences in sensitivities. Most studies focused on microcystin-LR (MC-LR), which can increase systemic inflammation and harm the digestive system, reproductive organs, liver, kidneys, and development. The few studies on other cyanotoxins illustrated that effects resembled those of MC-LR at similar concentrations, but more research is needed to describe effects of other cyanotoxins and mixtures of cyanotoxins that commonly occur in the environment. All experimental studies were on larval and adult amphibians; there were no such studies on reptiles. Experimental work with reptiles and adult amphibians is needed to clarify thresholds of tolerance. Only nine mortality events were reported, mostly for reptiles. Given that amphibians likely decay faster than reptiles, which have tissues that resist decomposition, mass amphibian mortality events from HABs have likely been under-reported. We propose that future efforts should be focused on seven major areas, to enhance our understanding of effects and monitoring of HABs on herpetofauna that fill important roles in freshwater and terrestrial environments. Environ Toxicol Chem 2024;43:1936-1949. Published 2024. This article is a U.S. Government work and is in the public domain in the USA. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Multi‐Environment Quantification of Parasite and Intermediate Host DNA on Pasture for Fine‐Scale Disease Risk Assessment

ABSTRACTParasite transmission occurs in complex environments comprising multiple matrices. Trematode parasites of ruminant livestock such as the liver fluke, Fasciola hepatica and the rumen fluke, Calicophoron daubneyi, show affinity with freshwater environments shared with their amphibious snail intermediate host, Galba truncatula. Isolation of environmental DNA (eDNA) from these parasites and their snail hosts in water draining from grazing land provides opportunities for improved molecular diagnostic detection and can help identify infection risks at farm level. The detection and quantification of eDNA from other environmental matrices has received less attention but would improve the understanding of parasite dynamics on pasture. Our study has considerably extended eDNA sampling methods for the detection of parasitic trematodes of ruminant livestock and their snail intermediate host by including for the first time the analysis of soil and herbage environmental samples alongside water collections. A droplet digital PCR (ddPCR) workflow was developed to detect parasite and snail eDNA from soil, herbage, and water collected from livestock farms. For the first time, C. daubneyi eDNA was isolated from agricultural soil alongside water samples and G. truncatula eDNA was detected in water, soil, and herbage samples. No environmental samples were positive for F. hepatica eDNA. Assessing multiple environmental matrices increased the number of positive sites. Future implementation of eDNA detection methods alongside traditional parasite diagnostics can underpin more holistic evaluations of the environmental components of parasite epidemiology and facilitate adaptation to changing disease patterns.

V9 Hypervariable Region Metabarcoding Primers for Euglenozoa and Metamonada

ABSTRACTShort amplicon sequencing is a commonly used method to study the diversity of organisms in various habitats. The hypervariable regions of the small subunit rRNA gene (18S rDNA) are the most general barcodes for eukaryotes, which can provide detailed taxonomic information across a wide range of eukaryotic diversity. However, some organisms are often missed by universal primers, which have difficulty amplifying their barcodes. In this study, specific primers were designed for the amplification of the highly diverse 18S‐V9 region of the Euglenozoa and Metamonada groups. The performance of the newly designed primers—V9Eug and V9Meta—was compared with the universal V9 primer on cultured communities derived from a range of freshwater environments of the Soos Natural Reserve and the Slavkov Forest in the Czech Republic. The V9Eug primer was more specific with Euglenozoa representing 91.8% of reads and 57.0% of OTUs, while the V9Meta primer showed lower specificity with only 48.4% of reads and 19.7% of OTUs assigned to Metamonada. Both the Euglenozoa and Metamonada primer pairs significantly improved recovery of their target groups compared to the universal V9 primer pair, detecting 2.7 and 1.8 times more OTUs, respectively. These results provide a more sensitive protocol for studying the diversity of these eukaryotic taxa.

From Origins to Impacts: A Comprehensive Review of Microplastics in Freshwater Environments

Microplastics are plastic particles of dimensions less than 5mm, increasingly recognized as omnipresent contaminants in freshwater systems. These small particles are sourced from microbeads provided by personal care products, industrial abrasives, and other secondary sources formed by the degradation of larger plastic items. The microplastics are contaminated in freshwater environments through runoff, atmospheric deposition, and the discharge of water treatment processes. The presence of microplastics in freshwater has greater potential to cause harm to aquatic environments and human health. The primary objective of this paper is to identify the microplastic pollution in freshwater resources and classify the sources of this pollution in freshwater resources, including both primary and secondary microplastics, and to evaluate the ecological impact of microplastics and their potential health risks to humans through food chain transfer. This review synthesizes the most recent findings on sources, impacts, and potential mitigation measures of microplastic pollution in freshwater ecosystems to emphasize the attentive work needed urgently in our efforts to conquer the environmental challenge. The mitigation strategies in practice thus focus on policy implementation and correct technology in wastewater treatment, raising public awareness to minimize the use of plastics and encourage good waste management. However, studies with much detail are still required to develop systematic methodologies for microplastic analysis and go far enough to find long-term solutions

Four novel species of Pleurotheciaceae collected from freshwater habitats in Jiangxi Province, China.

During an investigation of fungal diversity from freshwater environments in different regions in Jiangxi Province, China, four interesting species were collected. Morphology coupled with combined gene analysis of an ITS, LSU, SSU, and rpb2 DNA sequence data showed that they belong to the family Pleurotheciaceae. Four new species, Pleurotheciella ganzhouensis, Pla. irregularis, Pla. verrucosa, and Pleurothecium jiangxiense are herein described. Pleurotheciella ganzhouensis is characterized by its capsule-shaped conidia and short conidiophores, while Pla. irregularis has amorphous conidiophores and 3-septate conidia. Pleurotheciella verrucosa has cylindrical or verrucolose conidiogenous cells, 1-septate, narrowly fusiform, meniscus or subclavate conidia. Pleurothecium jiangxiense characterized in having conidiogenous cells with dense cylindrical denticles and short conidiophores. Pleurothecium obovoideum was transferred to Neomonodictys based on phylogenetic evidence. All species are compared with other similar species and comprehensive descriptions, micrographs, and phylogenetic data are provided.

Flood-induced buttertub spill reveals riverine macroplastic transport dynamics

During the July 2021 European floods approximately eight million empty dairy packaging (buttertubs) were flushed from a dairy processing facility in Belgium into the Vesdre river. Some were transported further downstream, into the Ourthe river and eventually the Meuse river. There are many unknowns when it comes to plastic transport in rivers, especially in response to floods. We therefore used this incident as an unique opportunity to study these buttertubs as a tracer for plastic transport dynamics in a riverine environment in response to an extreme flood event. Normally, it is unknown when and where individual plastic items found on riverbanks entered the environment. In this case, however, the ID stamps on the buttertups allowed for them to be traced back to the flooding of the factory. We studied the transport and deposition of these buttertubs in the Dutch Meuse over 2 years following the flood. We also collected buttertubs at different points in time to investigate their fragmentation and mass loss. Within 3 weeks of the flood, the buttertubs were transported up to 328 km from the spilling location. Overall, the majority (78%) of buttertubs we found within the first 3 weeks were deposited within less than 100 km of the point of emission. Over the following 2 years, the mean transport distance of the found buttertubs moved downstream from 100 km in July/August 2021, to 153 km in July 2023. The buttertubs average transport velocity decreased from 11.7 km/d within the first 3 weeks, to 0.2 km/d by July 2023. Based on the 89 buttertubs we collected and analyzed in detail over the 2 years, we did not find a significant mass loss. Of all 89 buttertubs found, 47 showed cracks and only 12 appeared to have pieces missing. This study shows that even during extreme flood events, the majority of spilled plastic litter is retained within a limited distance after being emitted into the river. The findings of this study can be utilized to improve plastic transport modelling, and overall better understand plastic transport in the freshwater environment.

The first record of gars (Ginglymodi: Lepisosteidae) from the Upper Cretaceous of Bulgaria reveals a wider paleogeographic distribution of lepisosteids within the European Archipelago

The bony-fishes of the clade Lepisosteidae, commonly called ‘gars’ or ‘gar-fish’, are a lineage of proficient piscivores with evolutionary history spanning about 150 million years, which are today represented by two genera inhabiting the freshwater and brackish areas of southeastern North America, Central America, and Cuba. This was not the case during the Late Cretaceous when lepisosteids were more diverse and had much wider geographical distribution. Gar fossils, albeit very fragmentary, are a common component of the Upper Cretaceous freshwater and brackish vertebrate assemblages in Europe, yet all of the pre-upper Campanian records come from the western and central parts of the Late Cretaceous European Archipelago. Here we describe new lepisosteid material from Bulgaria, comprising nine teeth and three scales found at the uppermost Santonian–lowermost Campanian Vrabchov Dol vertebrate locality. These fossils represent the easternmost record of gars within the European Archipelago to date. Despite being found in a lagoonal to foreshore deposits, the paleontological content of the locality, the incompleteness and preservational state of the material, as well as the predominantly non-marine ecology of modern and fossils gars suggest that these fossils belong to fishes which inhabited more inland, freshwater environments. The Vrabchov Dol lepisosteids remains are the first record of gars in Bulgaria and one of the rare documented occurrences of Mesozoic osteichthyans in the country. This material expands the paleobiogeographic distribution of the Lepisosteidae within the European Archipelago.

Assessing the direct and indirect effects of bigheaded carp (Hypophthalmichthys spp.) on freshwater food webs: A meta‐analysis

Abstract The rapid growth of aquaculture over the last century has led to the widespread introduction of aquatic species to freshwater environments. Bighead carp (Cyprinidae: Hypophthalmichthys nobilis) and silver carp (Cyprinidae: Hypophthalmichthys molitrix), collectively referred to as bigheaded carp, are among the top finfishes produced in aquaculture. These filter feeders possess feeding structures that allow them to efficiently remove plankton and other particles from the water. Unfortunately, when introduced into the wild and outside their historical range, their feeding traits generally result in the exploitation and suppression of phytoplankton and zooplankton, reducing food resources for native invertebrates and vertebrates. We conducted a meta‐analysis of published experiments to examine how bigheaded carp directly and indirectly affect organisms in aquatic food webs to better understand their ecological role within ecosystems. Specifically, we addressed the following questions: (1) What is the overall magnitude and direction of bigheaded carp effects on different aquatic organisms? (2) Do bigheaded carp affect organisms in planktonic and benthic habitats? (3) Are effects similar between bighead carp and silver carp? (4) Are effects consistent among experimental arenas (i.e., mesocosm, ponds)? We detected direct and indirect effects of bigheaded carp across multiple trophic levels. Large zooplankton often exhibited the strongest negative effects, with taxa‐specific differences between carp species. The suppression of zooplankton cascaded down to primary producers, resulting in an increase in phytoplankton (i.e., indirect effect). However, we observed no compensatory increases in rotifers when large zooplankton were reduced. Growth rates of planktivorous native fishes were the slowest when stocked with bigheaded carp. Our results further indicated that egested waste from bigheaded carp appeared to subsidise (i.e., direct effect) some benthic invertebrates such as Chironomidae midges. Effects of carp on zooplankton were generally similar between mesocosms and ponds. Invasive bigheaded carp greatly restructure their surrounding food web, both positively and negatively, through a complex suite of direct and indirect effects. These changes manifested across multiple trophic levels and differing habitats. Our analysis provides a more complete understanding of how these invasive carps change their surroundings by exploiting food resources and transferring materials from planktonic to benthic habitats. Taken together, the suite of direct and indirect effects shows how an invasive species can create imbalances within aquatic ecosystems, often to the detriment of native taxa. Bigheaded carp are invasive in many countries across the globe because of their popularity in aquaculture. Our findings reaffirm the threat these planktivores pose to native freshwater organisms.

Aliphatic and polycyclic aromatic compounds in coal and coal-based solid wastes: Relationship with coal-forming paleoenvironment and implications for environmental pollution

In this study, coal and coal-based solid wastes (coal gangue, fly ash, bottom ash, desulfurized gypsum and tar residue) were collected from major coal mines, power plants and coking plants in Lianghuai mining area (LH), China, and were analyzed for 76 polycyclic aromatic compounds (PACs), 27 n-alkanes and 2 isoprenoids (phytane and pristane). The total n-alkanes concentrations and ∑76 PACs in raw coals (640 ± 600 and 180 ± 87 μg/g) were higher than those in coal-based solid wastes (47 ± 40 and 24 ± 25 μg/g), but were lower than those in tar residue (3700 and 63,000 μg/g). It was discovered that the depositional paleoenvironment in LH was mostly a lacustrine and freshwater environment with oxidizing conditions and mixed organic matter input, but the Huainan coalfield had stronger oxidizing conditions and more input of terrestrial organic matter than that of the Huaibei coalfield. Alkylated PACs made up 56 ± 12 % of the ∑76PACs in raw coals, whereas solid wastes mainly consisted of 16 EPA PAHs (66 ± 16 %). Coal combustion and gangue weathering altered the structural properties of n-alkanes and PACs, resulting in a significant loss of n-alkanes and PACs, a higher proportion of parent PACs, and an increased abundance of short n-alkanes in the products (No apparent change of n-alkanes composition was observed through gangue weathering). The toxicity of PACs in raw coal and its solid wastes in LH from high to low was tar residue, raw coal, coal gangue, and coal-fired products. This investigation further confirmed that traditional diagnostic ratios may distort source information, and that they should not be used to assess PACs sources from raw coal particles or coal gangues, but rather to identify combustion sources near the point source. In addition, Retene/(Retene + Chrysene) < 0.03 may indicate direct contamination of raw coal particles.

Stabilization of nitrite in the presence of the nitrification inhibitor allylthiourea (ATU) in freshwater nitrification rate measurements

AbstractNitrification rate measurements provide critical information on the performance of an environmental process central to the N cycle and are best studied using isotope labeling techniques. However, combining the high sensitivity of isotope labeling techniques with selected inhibition of nitrifiers as a whole or of specific nitrifier guilds has not been established in limnology. This can be achieved with different concentrations of the commonly used nitrification inhibitor allylthiourea (ATU). In the 15N‐ammonium oxidation technique, the converted isotope label is typically captured in an excess pool of 14N‐nitrite. Here, we assessed how different storage conditions affect the stability of the nitrite pool in freshwater samples treated with ATU. When stored frozen, the nitrite pool was rapidly destabilized to 25–31% after 7 d of storage and even to less than 5% after storage exceeding 90 d for samples treated with ATU, thus making them unusable for rate determinations in these cost and labor‐intensive experiments. In comparison, this was not the case in marine samples or freshwater samples not treated with ATU, where the nitrite pool remained stable. Building on these results, we tested two options to stabilize nitrite during the storage of freshwater samples. The nitrite pool was stable if samples were stored at 4°C instead of freezing. We recommend this option for short‐term storage. For long‐term storage, samples should be supplemented with 0.5 mmol L−1 NaCl to increase salinity before freezing. As in marine samples, this stabilized the nitrite pool. Our results provide important guidance for the storage of non‐saline samples used for nitrification rate measurements in freshwater environments.

Polystyrene nanoparticles with different particle sizes cause autophagy by ROS/ERS/FOXO1 axis in the Cyprinus carpio kidney affecting immunological function

Microplastic pollution poses challenges for ecosystems worldwide, and nanoplastics (NPs, 1–1000 nm) have been identified as persistent pollutants. However, although some studies have described the hazards of NPs to aquatic organisms, the toxicological processes of NPs in the common carp kidney and the biotoxicity of differently sized NPs remain unclear. In this study, we used juvenile common carp as an in vivo model that were constantly exposed to freshwater at 1000 μg/L polystyrene nanoparticle (PSNP) concentrations (50, 100, and 400 nm) for 28 days. Simultaneously, we constructed an in vitro model utilizing grass fish kidney cells (CIK) to study the toxicological effects of PSNPs of various sizes. We performed RT-PCR and Western blot assays on the genes involved in FOXO1, HMGB1, HIF-1α, endoplasmic reticulum stress, autophagy, and immunoreaction. According to these results, exposure to PSNPs increased reactive oxygen species (ROS) levels, and the carp kidneys experienced endoplasmic reticulum stress. Additionally, PSNPs promoted renal autophagy by activating the ROS/ERS/FOXO1 (ERS: endoplasmic reticulum stress) pathway, and it affected immunological function by stimulating the ROS/HMGB1/HIF-1α signaling pathway. This study provides new insights into the contamination hazards of NPs in freshwater environments, as well as the harm they pose to the human living environments. The relationship between particle size and the degree of damage caused by PSNPs to organisms is a potential future research direction.

Network analysis to reveal the most commonly detected compounds in predator-prey pairs in freshwater and marine mammals and fish in Europe

Marine and freshwater mammalian predators and fish samples, retrieved from environmental specimen banks (ESBs), natural history museum (NHMs) and other scientific collections, were analysed by LIFE APEX partners for a wide range of legacy and emerging contaminants (2545 in total). Network analysis was used to visualize the chemical occurrence data and reveal the predominant chemical mixtures for the freshwater and marine environments. For this purpose, a web tool was created to explore these chemical mixtures in predator-prey pairs. Predominant chemicals, defined as the most prevalent substances detected in prey-predator pairs were identified through this innovative approach. The analysis established the most frequently co-occurring substances in chemical mixtures from AP&P in the marine and freshwater environments. Freshwater and marine environments shared 23 chemicals among their top 25 predominant chemicals. Legacy chemical, including perfluorooctanesulfonic acid (PFOS), brominated diphenyl ethers (BDEs), polychlorinated biphenyls (PCBs), hexachlorobenzene and mercury were dominant chemicals in both environments. Furthermore, N-acetylaminoantipyrine was a predominant pharmaceutical in both environments. The LIFE APEX chemical mixture application (https://norman-data.eu/LIFE_APEX_Mixtures) was proven to be useful to establish most prevalent compounds in terms of number of detected counts in prey-predator pairs. Nonetheless, further research is needed to establish food chain associations of the predominant chemicals.

Conditions for establishing fin primary cell cultures in a wide range of ray-finned fishes.

Ray-finned fishes (Actinopterygii) represent the most diverse vertebrate lineage that show extensive variations in physiology, ways of life, and adaptations to marine and freshwater environments, and several species have been established as biological research models. The in vitro culture of cells is fundamental for several fields of biological research, being an alternative for studies that use animals. Hundreds of fish cell lines have been established using specific methods for each cell type and species. Here is described a protocol which can be used commonly for obtaining cell cultures from the caudal fin of a wide range of ray-finned fishes including marine and freshwater species. Conditions for sample collection, microbial disinfection, tissue dissociation, plating and incubation, cryopreservation and thawing, and karyotyping are described in detail. Primary cell cultures were developed for 20 species grouped into 12 different orders. Eleven of these species have been cultivated in vitro for the first time. In the beginning, the fish cell cultures showed different capacities of proliferation among them; however throughout the passages, most cultures began to have a similar proliferation rate. Throughout the passages, it was noticed that cells similar to fibroblasts began to predominate. The great proliferative ability of these cultures reveals their potential to become cell lines. The culture of A. mexicanus, for example, has been proliferating for months and is already in its 65th passage. Moreover, these cell cultures showed conserved diploid chromosome numbers in comparison with in vivo descriptions which suggest these cultures have stable karyotypes. Therefore, these cultures have potential to be used in several fields, such as toxicology, cytogenetics, genomics, pathology, immunology, cellular agriculture, and conservation, and this method has the potential to be expanded to species not yet tested, as well as to other organs.

Diversity of Free-Living Amoebae in New Zealand Groundwater and Their Ability to Feed on Legionella pneumophila.

Free-living amoebae (FLA) are common in both natural and engineered freshwater ecosystems. They play important roles in biofilm control and contaminant removal through the predation of bacteria and other taxa. Bacterial predation by FLA is also thought to contribute to pathogen dispersal and infectious disease transmission in freshwater environments via the egestion of viable bacteria. Despite their importance in shaping freshwater microbial communities, the diversity and function of FLA in many freshwater ecosystems are poorly understood. In this study, we isolated and characterized FLA from two groundwater sites in Canterbury, New Zealand using microbiological, microscopic, and molecular techniques. Different methods for groundwater FLA isolation and enrichment were trialed and optimized. The ability of these isolated FLA to predate on human pathogen Legionella pneumophila was assessed. FLA were identified by 18S metagenomic amplicon sequencing. Our study showed that Acanthamoeba spp. (including A. polyphaga) and Vermamoeba veriformis were the main FLA species present in both groundwater sites examined. While most of the isolated FLA co-existed with L. pneumophila, the FLA populations in the L. pneumophila co-culture experiments predominantly consisted of A. polyphaga, Acanthamoeba spp., Naegleria spp., V. vermiformis, Paravahlkampfia spp., and Echinamoeba spp. These observations suggest that FLA may have the potential to act as reservoirs for L. pneumophila in Canterbury, New Zealand groundwater systems and could be introduced into the local drinking water infrastructure, where they may promote the survival, multiplication, and dissemination of Legionella. This research addresses an important gap in our understanding of FLA-mediated pathogen dispersal in freshwater ecosystems.

Impact of Calotropis procera on Behaviour of Leeches in Freshwater Environment

Impact of Calotropis procera on Behaviour of Leeches in Freshwater Environment

Phylogeny, morphology, and behavior of the new ciliate species Stentor stipatus.

The study of evolution at the cellular level traditionally has focused on the evolution of metabolic pathways, endomembrane systems, and genomes, but there has been increasing interest in evolution of more complex cellular structures and behaviors, particularly in the eukaryotes. Ciliates have major advantages for such studies due to their easily visible surface patterning and their dramatic and complex behaviors that can be easily analyzed. Among the ciliates, the genus Stentor epitomizes the features that are useful for studying evolution: they are widespread in freshwater environments, easy to visualize because of their large size, and capable of complex behaviors such as learning, decision-making, and phototaxis. Here, we introduce the discovery of a new species within this genus: Stentor stipatus, so named for their distinctive dark brown aggregates. We present morphological, phylogenetic, ecological, and behavioral characterizations of these cells. The S. stipatus clade has a bootstrap value of 93 and is phylogenetically distinct from S. amethystinus, the closest related species which shares a sequence similarity of 98.9%. S. stipatus is capable of phototaxis and can also habituate more quickly than S. coeruleus, the Stentor species in which most habituation studies have previously been conducted. These findings expand our understanding of Stentor species diversity, natural history, and demonstrate common principles of complex behavior that are present in single-celled organisms.