An invasive bivalve with the potential to reconstruct chronologies of geomarkers in a large South American basin.

The Sr/Ca and 87Sr/86Sr ratios are used as natural indicators (geomarkers) in fish to reveal migratory patterns, due to significant relationships between these ratios measured in the water and in the fish calcified structures (otoliths, bones). The aim of this study was to assess the potential use of the Limnoperna fortunei shell as a proxy for monitoring the spatial and temporal variability of Sr/Ca and 87Sr/86Sr in water. These ratios were compared in water samples and bivalve shells proceeding from eight sites of four hydrogeological regions of the La Plata Basin (Argentina and Uruguay), collected in two hydrological periods (winter and summer) in order to depict the pattern of geographical and temporal variability and to evaluate the relationship between both matrices. Significant positive linear relationships were found between shell edge and water for 87Sr/86Sr (R2 = 0.96-0.97, p < 0.005) and Sr/Ca (R2 = 0.83-0.86, p < 0.05) in summer and winter. Slopes did not differ significantly from 1 (p > 0.05) for 87Sr/86Sr, while they differed from 1 for Sr/Ca in both seasons (p < 0.05), indicating that the elemental ratio would be influenced by endogenous/exogenous factors. No significant seasonal differences were found for both natural tags (p > 0.05), except for the lower Uruguay River (87Sr/86Sr) and outer estuary (Sr/Ca). These results indicate that the L. fortunei shell could be a valuable tool for monitoring spatio-temporal variations in water 87Sr/86Sr. Shell Sr/Ca shows high potential utility as a geomarker in freshwater, but additional assessments are needed to understand the impact of temperature and growth on this ratio.

eDNA-Based Early Detection Illustrates Rapid Spread of the Non-Native Golden Mussel Introduced into Beijing via Water Diversion.

The world's largest water diversion, the South-to-North Water Transfer Project (SNWTP) in China, has created an "invasion highway" to introduce invasive golden mussels (Limnoperna fortunei) from the Yangtze River basin to Beijing. To examine the spread and colonization patterns of this newly introduced invasive species, we conducted comprehensive environmental DNA (eDNA)-based early detection and conventional field surveys across all water bodies in five river basins in Beijing from 2020 to 2023. Our results indicated a rapid spread over the past four years. Among the 130 tested sites, the number of sites with positive signals from eDNA analysis exhibited an annual increase: Commencing with four infested sites identified through field surveys in 2019, eDNA analysis detected an additional 13, 11, and 10 positive sites in 2020, 2021, and 2022, respectively, and a substantial rise comprising an additional 28 sites in 2023. Conventional field surveys detected mussels 1-3 years later than eDNA-based analysis at 16 sites. Across all 16 sites, we detected a low population density ranging from 1 to 30 individuals/m2. These findings collectively indicate that the invasions by golden mussels in Beijing are still in their early stages. To date, golden mussels have successfully colonized four out of the five investigated river basins, including the Jiyun River (22.2% positive sites), North Canal River (59.6% positive sites), Chaobai River (40% positive sites), and Yongding River (63.6% positive sites), with the North Canal River and Yongding River being the most heavily infested. Currently, only the Daqing River basin remains uninfested. Given the significant number of infested sites and the ongoing transport of large new propagules via SNWTP, further rapid spread and colonization are anticipated across aquatic ecosystems in Beijing and beyond. Consequently, we call for the proper implementation of effective management strategies, encompassing early detection, risk assessment, and the use of appropriate control measures to mitigate the potential ecological and economic damages in invaded ecosystems.

eDNA-based detection reveals invasion risks of a biofouling bivalve in the world's largest water diversion project.

Environmental DNA (eDNA) has increasingly been used to detect rare species (e.g., newly introduced nonindigenous species) in both terrestrial and aquatic ecosystems, often with distinct advantages over traditional methods. However, whether water eDNA signals can be used to inform invasion risks remains debatable owing to inherent uncertainties associated with the methods used and the varying conditions among study systems. Here, we sampled eDNA from canals of the central route of the South-to-North Water Diversion Project (hereafter SNWDP) in China to investigate eDNA distribution and efficacy to inform invasion risks in a unique lotic system. We first conducted a total of 16 monthly surveys in this system (two sites in the source reservoir and four sites in the main canal) to test if eDNA could be applied to detect an invasive, biofouling bivalve, the golden mussel Limnoperna fortunei. Second, we initiated a one-time survey in a sub-canal of the SNWDP using refined sampling (12 sites in ~22 km canal) and considered a few environmental predictors. We found that detection of target eDNA in the main canal was achieved up to 1100 km from the putative source population but was restricted to the warmer months (May-November). Detection probability exhibited a significant positive relationship with average daily minimum air temperature and with water temperature, consistent with the expected spawning season. eDNA concentration in the main canal generally fluctuated across months and sites and was generally higher in warmer months. Golden mussel eDNA concentration in the sub-canal decreased significantly with distance from the source and with increasing water temperature and became almost undetectable at ~22 km distance. Given the enormity of the SNWDP, golden mussels may eventually expand their distribution in the main canal, with established "bridgehead" populations facilitating further spread. Our findings suggest an elevated invasion risk of golden mussels in the SNWDP in warm months, highlighting the critical period for spread and, possibly, management.

Acute Exposure to Two Biocides Causes Morphological and Molecular Changes in the Gill Ciliary Epithelium of the Invasive Golden Mussel Limnoperna fortunei (Dunker, 1857).

Limnoperna fortunei, the golden mussel, is a bivalve mollusk considered an invader in South America. This species is responsible for ecological and economic damages due to its voluminous fouling capability. Chemical biocides such as MXD-100™ and sodium dichloroisocyanurate (NaDCC) are often used to control L. fortunei infestations in hydraulic systems. Thus, we proposed to investigate the effects of different periods (24, 48 and 72 h) of exposure to MXD-100™ (0.56 mg L-1) and NaDCC (1.5 mg L-1) on the gills of L. fortunei through morphological and molecular analyses. NaDCC promoted progressive morphological changes during the analyzed periods and only an upregulation of SOD and HSP70 expression during the first 24 h of exposure. MXD-100™ led to severe morphological changes from the first period of exposure, in addition to an upregulation of SOD, CAT, HSP70 and CYP expression during the first 24 h. In contrast, MXD-100™ led to a downregulation of CAT transcription between 24 and 48 h. In static conditions, NaDCC causes lethal damage after 72 h of exposure, and that exposure needs to be continuous to achieve the control of the species. Meanwhile, the MXD-100™ treatment presented several effects during the first 24 h, showing acute toxicity in a shorter period of time.

A chromosome-level assembly supports genome-wide investigation of the DMRT gene family in the golden mussel (Limnoperna fortunei).

BACKGROUND: The golden mussel (Limnoperna fortunei) is a highly invasive species that causes environmental and socioeconomic losses in invaded areas. Reference genomes have proven to be a valuable resource for studying the biology of invasive species. While the current golden mussel genome has been useful for identifying new genes, its high fragmentation hinders some applications. FINDINGS: In this study, we provide the first chromosome-level reference genome for the golden mussel. The genome was built using PacBio HiFi, 10X, and Hi-C sequencing data. The final assembly contains 99.4% of its total length assembled to the 15 chromosomes of the species and a scaffold N50 of 97.05 Mb. A total of 34,862 protein-coding genes were predicted, of which 84.7% were functionally annotated. A significant (6.48%) proportion of the genome was found to be in a hemizygous state. Using the new genome, we have performed a genome-wide characterization of the Doublesex and Mab-3 related transcription factor gene family, which has been proposed as a target for population control strategies in other species. CONCLUSIONS: From the applied research perspective, a higher-quality genome will support genome editing with the aim of developing biotechnology-based solutions to control invasion. From the basic research perspective, the new genome is a high-quality reference for molecular evolutionary studies of Mytilida and other Lophotrochozoa, and it may be used as a reference for future resequencing studies to assess genomic variation among different golden mussel populations, unveiling potential routes of dispersion and helping to establish better control policies.

ZnO nanoparticles alter redox metabolism of Limnoperna fortunei.

Nanoparticles such as zinc oxide nanoparticles (ZnO-NP) that are incorporated in consumer and industrial products have caused concern about their potential ecotoxicological impact when released into the environment. Bivalve mollusks are susceptible targets for nanoparticle toxicity since nanomaterials can enter the cells by endocytosis mechanisms. The aim of this study was to evaluate the influence of ZnO-NP on the redox metabolism in Limnoperna fortunei and the DNA damage after exposure to ZnO-NP. Adult bivalves were incubated with 1-, 10-, and 50-µg mL-1 ZnO-NP for 2, 4, and 24 h. Ionic Zn release, enzymatic and non-enzymatic antioxidant activity, oxidative damage, and DNA damage were evaluated. Oxidative damage to proteins and lipids were observed after 4-h exposure and returned to baseline levels after 24 h. Superoxide dismutase levels decreased after 4-h exposure and increased after 24 h. No significant alteration was observed in the catalase activity or even DNA double-strand cleavage. The dissociation of ZnO may occur after 24 h, releasing ionic zinc (Zn2+) by hydrolysis, which was confirmed by the increase in the ionic Zn concentration following 24-h exposure. In conclusion, ZnO-NP were able to induce oxidative stress in exposed golden mussels. The golden mussel can modulate its own antioxidant defenses in response to oxidative stress and seems to be able to hydrolyze the nanoparticles and consequently, release Zn2+ into the cellular compartment.

Effects of ultrasound on invasive golden mussel Limnoperna fortunei mortality and tissue lesions.

Biofouling by the invasive golden mussel Limnoperna fortunei deleteriously affects artificial water systems, but few effective, environmentally friendly antifouling strategies exist. We propose ultrasound for control of this invasive mussel and report minimum exposure times to kill juveniles and adults at ultrasonic powers ranging 300-600 W from a fixed distance of 8.5 cm. Analysis using a PMA + RT-qPCR assay revealed the formation of tissue lesions in response to ultrasound, with gill tissue more prone to injury than adductor muscle tissue. Shell microstructure determined using scanning electron microscopy (SEM) + energy dispersive X-ray spectroscopy (EDS) is plywood-like, with a thicker shell and increased numbers of prism and nacre layers in adult mussels that provide greater resistance to ultrasound, reducing mortality and tissue lesions. Our results suggest L. fortunei biomass could be effectively reduced by ultrasound, especially for early life-history stages without, or with only immature shells.

Fatty acid response of the invasive bivalve Limnoperna fortunei fed with Microcystis aeruginosa exposed to high temperature.

The success of Limnoperna fortunei as an invasive freshwater bivalve species is related to its physiological plasticity to endure changes in environmental conditions. The aim of this study was to investigate the physiological responses of L. fortunei after feeding on Microcystis aeruginosa grown at 26 °C (control) and 29 °C during 10 days. At the beginning, we measured biomass, fatty acids (FAs) composition on Cyanobacteria grown at both temperatures at different time intervals. Afterwards, mussels were fed with the thawed M. aeruginosa cells and their FA profile was measured after 15 days of feeding. M. aeruginosa exposed to 29 °C had the highest content of the FAs 18:2ω6 and cis-18:1ω9. The FA profile of the consumer L. fortunei fed with M. aeruginosa cultures grown at 29 °C was also significantly different to those fed with cultures grown at 26 °C, with a significant increased Eicosapentaenoic acid (EPA, 20:5ω3) and Arachidonic acid (ARA, 20:4ω6) concentrations. L. fortunei was already known to be physiologically adapted to live at 29 °C, but our results also shown a high biosynthesis of EPA and ARA (increase of 70 and 40% respectively, compared with 26 °C) and avoided the lipid peroxidation of both FAs. This increased EPA and ARA biosynthesis may be an important source of ω3 and ω6 polyunsaturated FAs (PUFAs) for higher trophic levels, such as the pelagic fishes or birds that mainly prey on these mussels. The transfer of the cyanobacterial response at higher temperature to higher trophic levels will influence the overall functioning of freshwater bodies.

Invasive species versus pollutants: Potential of Limnoperna fortunei to degrade glyphosate-based commercial formulations.

The intensive use of glyphosate in industrial agriculture may lead to freshwater contamination, encouraging studies of its toxic effect on non-target aquatic organisms. Glyphosate-based commercial formulations contain adjuvants, making them even more toxic than the active ingredient (a.i.) itself. The golden mussel Limnoperna fortunei is a freshwater invasive species which has been found to increase glyphosate dissipation in water and to accelerate eutrophication. The aim of this study is to evaluate the capability of L. fortunei to reduce the concentration of glyphosate in two commercial formulations, Roundup Max® and Glifosato Atanor®. Results were compared with the decay of the a.i. alone and in presence of mussels. Evasive response and toxicity tests were performed in a first set of trials to analyze the response of L. fortunei exposed to Roundup Max® and Glifosato Atanor®. Subsequently, we conducted a 21-day degradation experiment in 2.6-L microcosms applying the following treatments: 6 mg L-1 of technical-grade glyphosate (G), Glifosato Atanor® (A), Roundup Max® (R), 20 mussels in dechlorinated tap water (M), and the combination of mussels and herbicide either in the technical-grade (MG) or formulated form (MA and MR) (all by triplicate). Samples were collected at days 0, 1, 7, 14 and 21. No significant differences in glyphosate decay were found between treatments with mussels (MG: 2.03 ± 0.40 mg L-1; MA: 1.60 ± 0.32 mg L-1; MR: 1.81 ± 0.21 mg L-1), between glyphosate as a.i. and the commercial formulations, and between the commercial formulations, suggesting that the adjuvants did not affect the degrading potential of L. fortunei. In addition to the acceleration of glyphosate dissipation in water, there was an increase in the concentration of dissolved nutrients in water (N-NH4+ and P-PO43-) even higher than that caused by the filtering activity of the mussels, probably resulting from stress or from the degradation of glyphosate and adjuvants. We believe that a larger bioavailability of these nutrients due to glyphosate metabolization mediated by mussels would accelerate eutrophication processes in natural water bodies. The approach used here, where L. fortunei was exposed to two commercial formulations actually used in agricultural practices, sheds light on the potential impact of glyphosate decay on water bodies invaded by this species.

Acute Toxicity of [Formula: see text], [Formula: see text], and [Formula: see text] to the Golden Mussel Limnoperna fortunei (Dunker 1857).

In this study we test the sensitivity of three sizes of golden mussel (Limnoperna fortunei), an introduced species in Argentina, to a 96-h exposure to [Formula: see text], [Formula: see text], and [Formula: see text]. We also analysed the relative sensitivity of L. fortunei compared to other freshwater bivalve equivalent sensitivity data. The ANOVA results showed that both factors, heavy metal and size, had significant effects (p = 0.0013 and p = 0.0091, respectively) on the mortality of the golden mussel. Tukey's test showed significant differences for [Formula: see text] treatment and the smallest size class (7 mm [Formula: see text]). The relative sensitivity analysis showed that [Formula: see text] values for the smallest size class of L. fortunei exposed to [Formula: see text] and [Formula: see text] were in the low range, with values of 11.40 mg/L and 12.65 mg/L, respectively. In the case of [Formula: see text] (1.66 mg/L), its [Formula: see text] was in the medium-low range of the freshwater bivalve sensitivity distribution.

The golden mussel proteome and its response to niclosamide: Uncovering rational targets for control or elimination.

The Asian invasive species Limnoperna fortunei (Dunker, 1857), known as the golden mussel, causes great economic and environmental damage due to its fixative capacity and accelerated proliferation. Molecular studies for the control of larval and adult forms are of great economic, scientific and technological interest. Here, we first report on the compositional analysis of the L. fortunei proteome obtained through shotgun analysis using LC-MS/MS. Among those 2790 proteins identified, many of them related to secretory processes and membrane receptors. Our second approach consisted in exposing the mollusc to the molluscicide niclosamide to evaluate the induced proteomic alterations. Exposure to niclosamide at 0.25 mg/L for 24 h resulted in a pronounced differential abundance of proteins when compared to those obtained when exposure was reduced to 4 h at 2.3 mg/L. In total, 342 proteins were found differentially expressed in the responsive individuals as revealed by label-free quantitative proteomics. Regarding the affected cell processes were: cell division and differentiation, cytoskeletal organization and compartment acidification (upregulated), and energy metabolism (downregulated). Our findings constitute the first inventory of the expressed proteome of the golden mussel and have the potential to contribute with a more rational proposition of molecular targets for control and monitoring of this species. SIGNIFICANCE: With the recent availability of transcriptomic and genomic data applied to L. fortunei the timing is right to interrogate its putative gene repertoire using proteomic techniques. These have the potential to validate the existence of the predicted genes, infer their relative abundance and quantify their levels as a response to environmental stressors or various agents. Here we provided an inventory of the golden mussel proteome and evaluated its response to the molluscicide niclosamide. The obtained results open new avenues for intervention aimed at its control or elimination, particularly by targeting the various cellular processes that were uncovered.

Tolerance of native and invasive bivalves under herbicide and metal contamination: an ex vivo approach.

The literature indicates that exotic species have a greater tolerance to environmental stressors compared with native species. In recent decades, the introduction of contaminants into the environment has increased as a result of industrialization. The objective of this study was to verify the resistance of bivalve mollusks from freshwater native (Anodontites trapesialis) and exotic (Limnoperna fortunei) species to chemical contamination using an ex vivo/in vitro approach. Gill and muscle tissues were exposed to two different types of environmental stressors, copper (metal), and Roundup Transorb® (herbicide). The tissues were submitted to a cytotoxicity test in which the lysosomal integrity was assessed, from the adaptation of a method to isolated cells, and multixenobiotic resistance (MXR) test which evaluated cellular defense. In the exotic species, only copper at 9000 µg/L and Roundup Transorb® at 5000 µg/L were cytotoxic. In the native species, copper cytotoxicity at 900 and 9000 µg/L and Roundup Transorb® at 50 and 5000 µg/L were observed. Results were the same in both tissues. The MXR, responsible for the extrusion of contaminants (cell defense), was inhibited in both species when exposed to the contaminants, this cell defense system seems to be more inhibited in the native species, when exposed to both pollutants, indicating greater sensitivity. Therefore, cytotoxicity may be related to the lack of capacity of cellular defense. In relation to lysosomal integrity, the native species was more sensitive to cytotoxic pollutants, where a greater number of experimental conditions of metals and herbicide showed cytotoxicity, as well as more experimental situations inhibited its ability to defend itself.

Biosseguridade para sistemas de produção de peixes em tanque-rede em função da colmatação agravada por Limnoperna fortunei / Biosecurity for fishfarming cage systems due to the highly clogged screen aggravated by Limnoperna fortunei

Este trabalho avaliou a colmatação por Limnoperna fortunei em diferentes materiais para confecção de telas de arame, a dinâmica da colmatação pelo molusco e a relação custo-benefício dos materiais usados em tanques-rede. Utilizaram-se amostras de telas de simples torção de malha 19mm, de cinco tipos diferentes de revestimentos, colocadas na barragem de Salto Caxias, no Rio Iguaçu, estado do Paraná, entre julho de 2012 e julho de 2014. A cada seis meses, foi verificado o ganho de peso das telas em razão da colmatação, além da contagem do número de mexilhões aderidos. Após as coletas dos dados, foi realizado o teste estatístico de Kruskal-Wallis para se avaliar o ganho de peso das telas devido à colmatação. Realizou-se também uma pesquisa com 21 empresas para se obter o preço comercializado de telas para confecção de tanques-rede e se avaliar a expectativa de durabilidade dos materiais. Verificou-se que os diferentes materiais avaliados para as telas influenciam na colmatação e aderência do mexilhão-dourado; além disso, constatou-se que a tela mais eficiente quanto ao custo/ano é a de arame galvanizado plastificado e, para baixa colmatação, a de arame Bezinal.(AU)

This research evaluated degree of clogging by Limnoperna fortunei in different materials for wire mesh fabrication, the dynamics of mollusk sealing and the cost-benefit ratio of these materials used in tank-nets. Samples of single-twist screens of 19 mm mesh were used, from five different types of coatings were placed in the Salto Caxias dam on the Iguaçu River in the Paraná state, between July 2012 and July 2014. Every six months, it was verified the weight gain of the screens due to clogging and counting of the number of mussels adhered. After the data collection was performed Kruskal-Wallis statistical test to evaluate the weight gain of the screens due to clogging. In addition, a survey was carried out with 21 companies to obtain the commercialized price of screens for the production of net tanks and to evaluate the expected durability of the materials. The different materials evaluated for the screens influenced the sealing and adhesion of the golden mussel. In addition, the most cost-per-year screen is that of plastic-coated galvanized wire and for low clogging it is the Bezinal wire.(AU)

Chemical oxidants affect byssus adhesion in the highly invasive fouling mussel Limnoperna fortunei.

Biofouling by the invasive mussel species Limnoperna fortunei has caused severe negative consequences in various freshwater ecosystems, but there is still a lack of effective antifouling strategies for this species, particularly in drinking water treatment systems where many existing strategies cannot be used. Chemical oxidants have been proposed to potentially control fouling mussels by influencing 3,4­dihydroxyphenylalanine oxidation involved in byssus adhesion. To test this potential control strategy, L. fortunei adults were exposed to four oxidants: sodium hypochlorite (NaClO), potassium permanganate (KMnO4), hydrogen peroxide (H2O2) and chloramine T (CAT) at concentrations of 0.5, 1.0, 3.0 and 5.0 mg L-1 for one week. The results showed that low concentrations of KMnO4 stimulated foot protein secretion, which was beneficial to byssus production (number and length). NaClO and H2O2, but not KMnO4 and CAT, significantly inhibited byssus production and reduced breaking force, causing an increased byssus shedding rate. However, only NaClO dissolved byssus and altered the failure location from the byssal thread to adhesive plaque, even when exposed at low concentrations, showing its great impacts on byssus adhesion. Further analysis of polyphenoloxidase activities and gene expression profiles of LfBP-1, LfFP-2 and LfBP-3 revealed that chemical oxidants affected byssus adhesion by altering Dopa oxidation and foot protein gene expressions. This study, therefore, suggests that a low concentration of NaClO can be used as an alternative and environmentally friendly chemical for controlling L. fortunei biofouling by weakening byssus adhesion.

Impact of interaction between Limnoperna fortunei and Roundup Max® on freshwater phytoplankton: An in situ approach in Salto Grande reservoir (Argentina).

The joint impact of the glyphosate-based commercial formulation Roundup Max® and the invasive mussel Limnoperna fortunei on phytoplankton and water quality was assessed in Salto Grande reservoir, a scenario were both stressors coexist. We performed an in situ mesocosm approach, through a 7-day experiment using 400-L enclosures. The following treatments were applied by triplicate: addition of 250 mussels (M); addition of 5 mg L-1 of active ingredient (a.i.) in Roundup Max® (R); addition of 250 mussels and 5 mg L-1 of a.i. in Roundup Max® (MR), and controls, without any addition (C). R showed higher total phosphorus (TP) and ammonium nitrogen (NNH4+) concentrations due to the herbicide input, and a significant increase in algal abundance, biovolume and chlorophyll a levels (Chl-a). In M mussels grazed on phytoplankton, which resulted in subsequent phosphates (SRP) release. A decrease in species diversity was observed in R and M with respect to C. In MR, there were higher TP and NNH4+ concentrations, a decrease in biovolume, an antagonistic effect on Chl-a and a synergistic effect on phytoplankton abundance. Species diversity and evenness showed a significant decrease due to the explosive growth of a small and opportunistic Chlorophyta, Spermatozopsis exsultans. The dominance of this species may be due to negative selectivity for S. exsultans and/or release of potential competitors by L. fortunei, and to the input of nutrients by Roundup Max® and/or removal of competitors by its toxicity.

Biochemical responses of the golden mussel Limnoperna fortunei under dietary glyphosate exposure.

The aim of this study was to analyze the biochemical alterations in the golden mussel Limnoperna fortunei under dietary glyphosate exposure. Mussels were fed during 4 weeks with the green algae Scenedesmus vacuolatus previously exposed to a commercial formulation of glyphosate (6 mg L-1 active principle) with the addition of alkyl aryl polyglycol ether surfactant. After 1, 7, 14, 21 and 28 days of dietary exposure, glutathione-S-transferase (GST), catalase (CAT), superoxide dismutase (SOD), acetylcholinesterase (AChE), carboxylesterases (CES) and alkaline phosphatase (ALP) activities, glutathione (GSH) content and damage to lipids and proteins levels were analyzed. A significant increase (72%) in the GST activity and a significant decrease (26%) in the CES activity in the mussels fed on glyphosate exposed algae for 28 days were observed. The ALP activity was significantly increased at 21 and 28 days of dietary exposure (48% and 72%, respectively). GSH content and CAT, SOD and AchE activities did not show any differences between the exposed and non exposed bivalves. No oxidative damage to lipids and proteins, measured as TBARS and carbonyl content respectively, was observed in response to glyphosate dietary exposure. The decrease in the CES activity and the increases in GST and ALP activities observed in L. fortunei indicate that dietary exposure to glyphosate provokes metabolic alterations, related with detoxification mechanisms.

Byssus Structure and Protein Composition in the Highly Invasive Fouling Mussel Limnoperna fortunei.

Biofouling mediated by byssus adhesion in invasive bivalves has become a global environmental problem in aquatic ecosystems, resulting in negative ecological and economic consequences. Previous studies suggested that mechanisms responsible for byssus adhesion largely vary among bivalves, but it is poorly understood in freshwater species. Understanding of byssus structure and protein composition is the prerequisite for revealing these mechanisms. Here, we used multiple methods, including scanning electron microscope, liquid chromatography-tandem mass spectrometry, transcriptome sequencing, real-time quantitative PCR, inductively coupled plasma mass spectrometry, to investigate structure, and protein composition of byssus in the highly invasive freshwater mussel Limnoperna fortunei. The results indicated that the structure characteristics of adhesive plaque, proximal and distal threads were conducive to byssus adhesion, contributing to the high biofouling capacity of this species. The 3,4-dihydroxyphenyl-α-alanine (Dopa) is a major post-transnationally modification in L. fortunei byssus. We identified 16 representative foot proteins with typical repetitive motifs and conserved domains by integrating transcriptomic and proteomic approaches. In these proteins, Lfbp-1, Lffp-2, and Lfbp-3 were specially located in foot tissue and highly expressed in the rapid byssus formation period, suggesting the involvement of these foot proteins in byssus production and adhesion. Multiple metal irons, including Ca2+, Mg2+, Zn2+, Al3+, and Fe3+, were abundant in both foot tissue and byssal thread. The heavy metals in these irons may be directly accumulated by L. fortunei from surrounding environments. Nevertheless, some metal ions (e.g., Ca2+) corresponded well with amino acid preferences of L. fortunei foot proteins, suggesting functional roles of these metal ions by interacting with foot proteins in byssus adhesion. Overall, this study provides structural and molecular bases of adhesive mechanisms of byssus in L. fortunei, and findings here are expected to develop strategies against biofouling by freshwater organisms.

Differential impact of Limnoperna fortunei-herbicide interaction between Roundup Max® and glyphosate on freshwater microscopic communities.

Multiple anthropogenic stressors act simultaneously on the environment, with consequences different from those caused by single-stressor exposure. We investigated how the combination of the invasive mussel Limnoperna fortunei and a widely applied herbicide, Roundup Max®, affected freshwater microscopic communities and water quality. Further, we compared these results with those induced by the combination of the mussel and technical-grade glyphosate. We carried out a 34-day experiment in outdoor mesocosms, applying the following six treatments: 6 mg L(-1) of technical-grade glyphosate (G), the equivalent concentration of glyphosate in Roundup Max® (R), 100 mussels (M), the combination of mussels and herbicide either in the technical-grade or formulated form (MG and MR, respectively), and control (C). Herbicides significantly increased total phosphorus in water; R and MR showed greater initial total nitrogen and ammonium. R increased picoplankton abundance and caused an eightfold increase in phytoplankton, with high turbidity values; G had a lower effect on these variables. Herbicide-mussel combination induced an accelerated dissipation of glyphosate in water (MG 6.36 ± 0.83 mg G g DW(-1) day(-1) and MR 5.16 ± 1.26 mg G g DW(-1) day(-1)). A synergistic effect on ammonium was observed in MR but not in MG. MR and MG had an antagonistic effect on phytoplankton, which showed a drastic reduction due to grazing, as revealed by M. We provide evidence of differential effects of Roundup Max® and technical-grade glyphosate over water quality and microscopic communities, and in combination with mussels. However, in the combination of mussels and herbicides, mussels seem to play a leading role. In the presence of L. fortunei, the effects of higher nutrient availability provided by herbicides addition were counteracted by the filtration activity of mussels, which released nutrients, grazed on picoplankton and phytoplankton, and boosted the development of other primary producers, periphyton and metaphyton.

Cellular development of the germinal epithelium during the gametogenic cycle of the golden mussel Limnoperna fortunei (Bivalvia: Mytilidae)

AbstractThe golden mussel Limnoperna fortunei is an invasive species that has quickly dispersed and colonized several potential different habitats distributed all over the world, causing environmental and economic impacts. Thus, in order to contribute to a better understanding of the reproductive aspects of L. fortunei, we described the cellular dynamic of the male and female germinal epithelium during the annual reproductive life history of this species, with the use of high resolution histology. An approximate of 1 200 specimens of L. fortunei were sampled periodically on the upper Paraná River floodplain (Brazil), from March 2010 to December 2012. Based on the activity of the germinal epithelium and consequent germ cell development, this study has resulted in the recognition of the following reproductive phases: Developing, Spawning Capable, Regressing and Regenerating. In the characterization of these phases, the following germ cells were described for males: spermatogonia, primary and secondary spermatocytes, spermatids and spermatozoa. Cell nests, oogonia, early prophase oocytes, previtellogenic oocytes and vitellogenic oocytes (early vitellogenic oocytes, middle vitellogenic oocytes and full-grown oocytes) were described for females. The morphological data and reproductive parameters obtained, showed the value of the cellular dynamics of the germinal epithelium, for the understanding of the cyclic gonadal events during the adult reproductive life of the mollusk in general. These results on the gametogenesis of this invasive species may be a fundamental tool for the development of control strategies and programs implementation, to reduce their proliferation and impacts in natural local environments. Rev. Biol. Trop. 64 (2): 521-536. Epub 2016 June 01.

ResumenEl mejillón dorado Limnoperna fortunei (Dunker, 1857) es una especie invasora que se ha dispersado rápidamente y ha colonizado diferentes hábitats potenciales distribuidos por todo el mundo, causando impactos ambientales y económicos. El control de esta especie depende del conocimiento de los aspectos reproductivos. Entre marzo 2010 y diciembre 2012 fueron muestreados periodicamente alrededor de 1 200 especímenes de L. fortunei en la planicie de inundación del Alto Rio Paraná, Brasil. Con el fin de contribuir con una mejor comprensión de los aspectos reproductivos de L. fortunei, se describió la dinámica celular del epitelio germinativo masculino y femenino durante la vida reproductiva anual de esta especie, mediante histología de alta resolución basada en la actividad del epitelio germinal y el consecuente desarrollo de las células germinales. En este estudio se reconocieron las siguientes fases reproductivas: desarrollo, capacidad de desove, regresión y regeneración. En los machos se describieron las siguientes células germinales: espermatogonias, espermatocitos primarios y secundarios, espermátidas y espermatozoides. Mientras que en las hembras se estudiaron: nidos celulares, ovogonias, ovocitos profásicos tempranos, ovocitos pre-vitelogénicos y ovocitos vitelogénicos (ovocitos vitelogénicos tempranos, ovocitos vitelogénicos y ovocitos completamente desarrollados).Los datos morfológicos y parámetros reproductivos que se muestran aquí confirman cómo la dinámica celular del epitelio germinal permite la comprensión de los acontecimientos cíclicos gonadales durante la vida reproductiva de adultos del molusco, en general. El conocimiento de la gametogénesis de esta especie invasora puede convertirse en una herramienta fundamental para el desarrollo de estrategias de control y aplicación de programas para disminuir su proliferación en ambientes naturales.

Titanium dioxide nanoparticles induce genotoxicity but not mutagenicity in golden mussel Limnoperna fortunei.

The widespread use of titanium dioxide nanoparticles (TiO2-NP) in consumer products is the cause of its appearance in wastewater and effluents, reaching the aquatic environment. The evaluation of the biological impact of TiO2-NP and the need to understand its ecotoxicological impact to the aquatic ecosystem are of major concern. Bivalve mollusks may represent a target group for nanoparticle toxicity. Limnoperna fortunei (golden mussel), a freshwater bivalve organism that has been employed in biomonitoring environmental conditions. Comet assay, micronucleus test and oxidative damage to lipids and proteins were performed after the golden mussel was exposed to TiO2-NP (1, 5, 10 and 50µgmL(-1)). The results demonstrate that TiO2-NP can damage the DNA of haemocytes after 2h of exposure and the genotoxic activity significantly increased after 4h exposure to TiO2-NP, at all the TiO2-NP concentrations. TiO2-NP was ineffective in causing mutagenicity in the haemolymph cells of golden mussel. The increase in the lipid peroxidation levels and carbonyl proteins after the exposure to TiO2-NP indicates the induction of oxidative stress at 2h exposure with similar results to all TiO2-NP concentrations, but these effects did not occur at 4h exposure. These results demonstrated that, although TiO2-NP is not mutagenic to golden mussel, it does induce DNA damage and oxidative stress in these organisms.