Determinação de motivos de ligação à quitina em vicilinas de Canavalia ensiformis e Vigna unguiculata através de métodos in silico e relação com suas toxicidades para o bruquídeo Callosobruchus maculatus (Coleoptera:Bruchidae)

Chitin is an important structural component of the cellular wall of fungi and exoskeleton of many invertebrate plagues, such as insects and nematodes. In digestory systems of insects it forms a named matrix of peritrophic membrane. One of the most studied interaction models protein-carbohydrate is the model that involves chitin-binding proteins. Among the involved characterized domains already in this interaction if they detach the hevein domain (HD), from of Hevea brasiliensis (Rubber tree), the R&R consensus domain (R&R), found in cuticular proteins of insects, and the motif called in this study as conglicinin motif (CD), found in the cristallography structure of the β-conglicinin bounded with GlcNac. These three chitin-binding domains had been used to determine which of them could be involved in silico in the interaction of Canavalia ensiformis and Vigna unguiculata vicilins with chitin, as well as associate these results with the WD50 of these vicilins for Callosobruchus maculatus larvae. The technique of comparative modeling was used for construction of the model 3D of the vicilin of V. unguiculata, that was not found in the data bases. Using the ClustalW program it was gotten localization of these domains in the vicilins primary structure. The domains R&R and CD had been found with bigger homology in the vicilins primary sequences and had been target of interaction studies. Through program GRAMM models of interaction ( dockings ) of the vicilins with GlcNac had been gotten. The results had shown that, through analysis in silico, HD is not part of the vicilins structures, proving the result gotten with the alignment of the primary sequences; the R&R domain, although not to have structural similarity in the vicilins, probably it has a participation in the activity of interaction of these with GlcNac; whereas the CD domain participates directly in the interaction of the vicilins with GlcNac. These results in silico show that the amino acid number, the types and the amount of binding made for the CD motif with GlcNac seem to be directly associates to the deleterious power that these vicilins show for C. maculatus larvae. This can give an initial step in the briefing of as the vicilins interact with alive chitin in and exert its toxic power for insects that possess peritrophic membrane

Bioecology and conservation of Duck Mussel Species (Anodonta anatina L., 1758) in Northeastern Portugal

Freshwater mussel (Mollusca, Bivalvia, Unionoida) populations are one of the most endangered faunistic groups. Mussels play an important role in the functioning of aquatic ecosystems, because they are responsible for the filtration and purification of water. They have a complex life cycle, with a parasitic larvae and usually limited host fish species. The real status of these populations is still poorly understood worldwide. The objectives of the present work were the study of bioecology of duck mussel (Anodonta anatina L.) populations of Tua Basin (NE Portugal). It was made the characterization of the ecological status of Rabaçal, Tuela and Tua Rivers, selecting 15 sampling sites, equally distributed by the three rivers. Samplings were made in the winter of 2016, and several physico-chemical water parameters measured and two habitat quality indexes calculated (GQC and QBR indexes). Benthic macroinvertebrate communities were sampled based on the protocols established by the Water Framework Directive. Host fish populations for duck mussel were determined in laboratorial conditions, testing several native and exotic fish species. The results showed that several water quality variables (e.g. dissolved oxygen, conductivity, pH, total dissolved solids, and nutrients) can be used for the classification of river typology. Other responsive metrics were also determined to identify environmental degradation. For instances, hydromorphological conditions (GQC and QBR indexes) and biota related metrics (e.g. composition, distribution, abundance, diversity of invertebrate communities) contributed to the evaluation of the ecological integrity. The upper zones of Rabaçal and Tuela rivers were classified with excellent and good ecological integrity, while less quality was found in downstream zones. The host fish tests showed that only native species are effective hosts, essential for the conservation purposes of this mussel species. Threats, like pollution, sedimentation and river regularization (3 big dams are in construction or in filling phase), are the main cause of habitat loss for native mussel and fish populations in the future. Rehabilitation and mitigation measures are essential for these lotic ecosystems in order to preserve the prioritary habitats and the native species heavily threatened.

Mineral and organic pollution in river Sabor (Northeastern Portugal): ecotoxicological effects on freshwater fauna

Aquatic ecosystems are final collectors of all kinds of pollution as an outcome of anthropogenic inputs, such us untreated industrial and municipal sewage and agricultural pollutants. There are several aquatic ecosystems that are threatened by mineral and organic pollution. In Northeastern Portugal, near Bragança, different watercourses are suffering negative impacts of human activities. It has been developed several studies in the monitoring of environmental impacts in these river basins, namely in Rio Fervença, affected by organic pollution, and in Portelo stream, affected, since 2009, by the collapse and continuous input of mining deposits. In this sense, the present study aimed to continue the monitoring study of ecological status of freshwater ecosystems of Northeastern Portugal, namely the following objectives: a) mineral pollution effects of mining deposits sudden incorporated into Portelo stream; b) organic pollution due to domestic and industrial inputs in River Fervença. Also, since fish are useful experimental models to evaluate toxicological mechanisms of contaminants, c) acute toxicity tests with Cu were conducted in laboratory conditions. During 2015/2016, it was made abiotic and biotic characterization of 16 sampling sites distributed by both Portelo and Fervença rivers, tributaries of main River Sabor (Douro Basin). Several physicochemical parameters were determined and Riparian Quality (QBR Index) and Channel Quality (GQC) Indexes were determined for habitat evaluation. Fish and invertebrate communities were sampled, according to protocols of Water Framework Directive (WFD). Several metrics were determined, with particular emphasis on the Biotic Index IBMWP and the Northern Portuguese Invertebrate Index (IPtIN). Acute toxicity tests were conducted with an Iberian fish species, common barbel (Luciobarbus bocagei) and some plasmatic electrolytes levels were evaluated, to assess their contribution to mitigate osmoregulatory adverse effects of Cu. Also, same electrolytes were measured after changing to clean water, in attempt to assess fish capacity to reverse this situation. Results obtained for both rivers showed a significant level of disturbance that affected decisively water, habitat and biological quality of aquatic ecosystems. Mineral and Organic Pollution in River Sabor (NE Portugal): Ecotoxicological Effects on Freshwater Fauna Due to this change of environmental conditions in Portelo stream (extreme pH values, high conductivity and presence of heavy metals), several biological metrics (e.g. taxonomic richness, abundance, diversity, evenness) confirmed, comparatively with reference sites, a substantial decrease on ecological integrity status. The same pattern was found for Fervença River; however other water parameters, namely the content of most limiting nutrients (e.g. N and P) seemed to have more influence in the composition and structure of macroinvertebrate and fish communities. In fact, despite the operation of the Sewage Treatment Plant of Bragança, Fervença River presented significant levels of disturbance that affected decisively the quality and ecological integrity of the aquatic ecosystem. The synergic effect of domestic and industrial pollution, intensive agriculture, regulation and degradation of aquatic and riparian habitats contributed to the decrease of ecological condition, namely in the downstream zones (after Bragança). The results for acute toxicity, showed that fish can change Na+ and K+ levels face to Cu exposition and, depending of Cu concentration tested, can also return to normal levels, providing some insights to that are believed to occurred in fish population, near the Portelo mines. The low ecological integrity status detected in the lotic ecosystems in NE Portugal as a result of mineral and organic pollution deserves the development of several measures for rehabilitation and improving of water quality. On the other hand, environmental education actions are needed to contribute to improvement of ecological integrity of the river and its conservation.

Molecular identification and localization of filamentous symbiotic bacteria associated with the hydrothermal vent annelid Alvinella pompejana

Alvinella pompejana is a polychaetous annelid that inhabits high temperature environments associated with active deep-sea hydrothermal vents along the East Pacific Rise. A unique and diverse epibiotic microflora with a prominent filamentous morphotype is found associated with the worm's dorsal integument. A previous study established the taxonomic positions of two epsilon proteobacterial phylotypes, 13B and 5A, which dominated a clone library of 16S rRNA genes amplified by PCR from the epibiotic microbial community of an A. pompejana specimen. In the present study deoxyoligonucleotide PCR primers specific for phylotypes 13B and 5A were used to demonstrate that these phylotypes are regular features of the bacterial community associated with A. pompejana. Assaying of other surfaces around colonies of A. pompejana revealed that phylotypes 13B and 5A are not restricted to A. pompejana. Phylotype 13B occurs on the exterior surfaces of other invertebrate genera and rock surfaces, and phylotype 5A occurs on a congener, Alvinella caudata. The 13B and 5A phylotypes were identified and localized on A. pompejana by in situ hybridization, demonstrating that these two phylotypes are, in fact, the prominent filamentous bacteria on the dorsal integument of A. pompejana. These findings indicate that the filamentous bacterial symbionts of A. pompejana are epsilon Proteobacteria which do not have an obligate requirement for A. pompejana.

Isolamento de uma quitinase extraída do cefalotórax do camarão marinho Litopenaeus schmitti (BURKENROAD, 1936) : avaliação de suas atividades antimicrobiana e larvicida

Chitinases are enzymes involved in degradation of chitin and are present in a range of organisms, including those that do not contain chitin, such as bacteria, viruses, plants and animals, and play important physiological and ecological roles. Chitin is hydrolyzed by a chitinolytic system classified as: endo-chitinases, exo-chitinases and N-acetyl-b-D-glucosaminidases. In this study a Litochitinase1 extracted from the cephalotorax of the shrimp Litopenaeus Schmitt was purified 987.32 times using ionexchange chromatography DEAE-Biogel and molecular exclusion Sephacryl S-200. These enzyme presented a molecular mass of about 28.5 kDa. The results, after kinetic assay with the Litochitinase1 using as substrate p-nitrophenyl-N-acetyl-b-Dglucosaminideo, showed apparent Km of 0.51 mM, optimal activity at pH ranging from 5.0 to 6.0, optimum temperature at 55°C and stability when pre-incubated at temperatures of 25, 37, 45, 50 and 55°C. The enzyme showed a range of stability at pH 4.0 to 5.5. HgCl2 inhibited Litochitinase1 while MgCl2 enhances its activity. Antimicrobial tests showed that Litochitinase1 present activity against gram-negative bacterium Escherichia coli in the 800 μg/mL concentration. The larvicidal activity against Aedes aegypti was investigated using crude extracts, F-III (50-80%) and Litochitinase1 at 24 and 48 hours. The results showed larvicidal activity in all these samples with EC50 values of 6.59 mg/mL for crude extract, 5.36 mg/mL for F-III and 0.71 mg/mL for Litochitinase1 at 24 hours and 3.22 and 0.49 mg/mL for the F-III and Litochitinase1 at 48 hours, respectively. Other experiments confirmed the presence of chitin in the midgut of Aedes aegypti larvae, which may be suffering the action of Litochitinase1 killing the larvae, but also the absence of contaminating proteins as serine proteinase inhibitors and lectins in the crude extract, F-III and Litochitinase1, indicating that the death of the larvae is by action of the Litochitinase1. We also observed that the enzymes extracted from intestinal homogenate of the larvae no have activity on Litochitinase1. These results indicate that the enzyme can be used as an alternative to control of infections caused by Escherichia coli and reducing the infestation of the mosquito vector of dengue.

Impact of Hypoxia on the Community Structure of Benthic Macroinvertebrates of Lagos Lagoon, Nigeria

Study of the Lagos lagoon was conducted for two years to investigate the impact of hypoxia on the benthic macroinvertebrates. Water and benthic samples were collected monthly along the study stretch and analysed in a standard laboratory. Temporal variation in water physico-chemistry was largely controlled by rainfall pattern while the spatial variation was influenced by proximity to the Harbour as well as the pollution sources and types. A total of 3,159 individuals comprising three phyla, five classes, nineteen families and twenty three species were recorded. Iddo I, Iddo II, Ogudu and Agboyi study stations recorded very low individuals, but relatively high number of polychaetes. Benthic macro- invertebrate community was dominated by the molluscs. Margalef’s index of species richness ranged from 0.79 to 2.57 while Shannon-Wiener index ranged from 0.40 to 2.19. Species evenness index ranged from 0.29 to 0.80. There was generally low biodiversity indicating the stressed nature of the study area.

Diagnosis of vibriosis in the era of genomics: lessons from invertebrates

Global changes linked to increases in temperature and ocean acidification, but also to more direct anthropogenic influences such as aquaculture, have caused a worldwide increase in the reports of Vibrio-associated illnesses affecting humans and also animals such as shrimp and molluscs. Investigation of the emergence of Vibrio pathogenesis events requires the analysis of microbial evolution at the gene, genome and population levels, in order to identify genomic modifications linked to increased virulence, resistance and/or prevalence, or to recent host shift. From a more applied point of view, the elucidation of virulence mechanisms is a prerequisite to devising prophylactic methods to fight infectious agents. In comparison with human pathogens, fairly little is known about the requirements for virulence in vibrios pathogenic to animals. However, the advent of genome sequencing, especially next-generation technologies,the possibility of genetically manipulating most of the Vibrio strains, and the recent availability of standardised animals for experimental infections have now compensated for the considerable delay in advancement of the knowledge of non-model pathogens such as Vibrio and have led to new scientific questions.

Intérêt biogéographique de la côte basque rocheuse

During recent decades, works on rocky shore biodiversity have been multiplied in the southern part of the Bay of Biscay and more precisely on intertidal and subtidal area were communities present a great interest. Necessity of conservation of coastal habitats and their communities and a growing pressure on coastal environments explain awareness of services provided by these ecosystems. Those communities are very sensitive to water quality change. Moreover, since the beginning of the XXI century various European directives require a good ecological status of coastal waters and conservation of their communities : Water Framework Directive (WFD), Marine Strategy Framework Directive (MSFD) and conservation of habitats with Directive Habitat Fauna and Flora (DHFF).... Integrated environmental status assessment approach is needed for this requirement in front of specific component at the regional scale of the Bay of Biscay. This analyze, at this regional scale, bring a particular interest to follow some biological groups in front of their ecological sensitivity. Among them, some example are listed like algae, invertebrate as species of mollusc opistobranch and fishes of the family blennidae are targets of interest for future monitoring. The biogeographic specificity of species of these groups is to present strong ecological requirements, in a trophic point of view for example, as well as boundary in local distribution in the southern part of the Bay of Biscay. Thus, monitoring of their distribution and abundance should be a relevant indicator of environmental change. If the presence of individuals is relatively easy to implement, monitoring in terms of abundance are more complex to develop to obtain representative data in coastal areas. The mobile character of the individual and their high location variability based on fluctuating environmental conditions is a challenge that needs to be considered. Interest concerns both the development of their number and their migration to the north for species in northern limit, and/or disappearance for species in southern distribution limits. Moreover, acquisition of knowledge on the taxonomy of local species is a way to improve biodiversity knowledge and assessment of global change as climatic change.

Physiological Effects of Alcohol on Crayfish Escape Circuitry

Alcohol is one of the oldest and most widely used drugs on the planet, but the cellular mechanisms by which it affects neural function are still poorly understood. Unlike other drugs of abuse, alcohol has no specific receptor in the nervous system, but is believed to operate through GABAergic and serotonergic neurotransmitter systems. Invertebrate models offer circuits of reduced numerical complexity and involve the same cell types and neurotransmitter systems as vertebrate circuits. The well-understood neural circuits controlling crayfish escape behavior offer neurons that are modulated by GABAergic inhibition, thus making tail-flip circuitry an effective circuit model to study the cellular mechanisms of acute alcohol exposure. Crayfish are capable of two stereotyped, reflexive escape behaviors known as tail-flips that are controlled by two different pairs of giant interneurons, the lateral giants (LG) and the medial giants (MG). The LG circuit has been an established model in the neuroscience field for more than 60 years and is almost completely mapped out. In contrast, the MG is still poorly understood, but has important behavioral implications in social behavior and value-based decision making. In this dissertation, I show that both crayfish tail-flip circuitry are physiologically sensitive to relevant alcohol concentrations and that this sensitivity is observable on the single cell level. I also show that this ethyl alcohol (EtOH) sensitivity in the LG can be changed by altering the crayfish’s recent social experience and by removing descending inputs to the LG. While the MG exhibits similar physiological sensitivity, its inhibitory properties have never been studied before this research. Through the use of electrophysiological and pharmacological techniques, I show that the MG exhibits many similar inhibitory properties as the LG that appear to be the result of GABA-mediated chloride currents. Finally, I present evidence that the EtOH-induced changes in the MG are blocked through pre-treatment of the potent GABAA receptor agonist, muscimol, which underlines the role of GABA in EtOH’s effects on crayfish tail-flip circuitry. The work presented here opens the way for crayfish tail-flip circuitry to be used as an effective model for EtOH’s acute effects on aggression and value-based decision making.

A perfect day for zebrafish : neuromodulation of sleep in a diurnal vertebrate

Every day, we shift among various states of sleep and arousal to meet the many demands of our bodies and environment. A central puzzle in neurobiology is how the brain controls these behavioral states, which are essential to an animal's well-being and survival. Mammalian models have predominated sleep and arousal research, although in the past decade, invertebrate models have made significant contributions to our understanding of the genetic underpinnings of behavioral states. More recently, the zebrafish (Danio rerio), a diurnal vertebrate, has emerged as a promising model system for sleep and arousal research.

In this thesis, I describe two studies on sleep/arousal pathways that I conducted using zebrafish, and I discuss how the findings can be combined in future projects to advance our understanding of vertebrate sleep/arousal pathways. In the first study, I discovered a neuropeptide that regulates zebrafish sleep and arousal as a result of a large-scale effort to identify molecules that regulate behavioral states. Taking advantage of facile zebrafish genetics, I constructed mutants for the three known receptors of this peptide and identified the one receptor that exclusively mediates the observed behavioral effects. I further show that the peptide exerts its behavioral effects independently of signaling at a key module of a neuroendocrine signaling pathway. This finding contradicts the hypothesis put forth in mammalian systems that the peptide acts through the classical neuroendocrine pathway; our data further generate new testable hypotheses for determining the central nervous system or alternative neuroendocrine pathways involved.

Second, I will present the development of a chemigenetic method to non-invasively manipulate neurons in the behaving zebrafish. I validated this technique by expressing and inducing the chemigenetic tool in a restricted population of sleep-regulating neurons in the zebrafish. As predicted by established models of this vertebrate sleep regulator, chemigenetic activation of these neurons induced hyperactivity, whereas chemigenetic ablation of these neurons induced increased sleep behavior. Given that light is a potent modulator of behavior in zebrafish, our proof-of-principle data provide a springboard for future studies of sleep/arousal and other light-dependent behaviors to interrogate genetically-defined populations of neurons independently of optogenetic tools.

Genetic considerations on the introduction of farmed fish in marine protected areas: The case of study of white seabream restocking in the Gulf of Castellammare (Southern Tyrrhenian Sea)

Human exploitation has drastically reduced the abundance and distribution of several marine fish and invertebrate populations through overfishing and habitat destruction. Restocking can potentially mitigate these impacts and help to reconstitute depleted stocks but genetic repercussions must be considered. In the present study, the degree of genetic similarity between white seabream (Diplodus sargus Linnaeus 1758) individuals reared for restocking purposes and the receiving population in the Gulf of Castellammare fishery reserve (Sicily, Italy) was assessed using microsatellites. We also inferred the spatial pattern of the genetic structure of D. sargus and connectivity along Sicilian coasts. The farmed population showed significant heterozygosity deficiency in 6 loci and an important reduction in the number of alleles, which could indicate an incipient inbreeding. Both the farmed population and the target one for restocking (Castellammare fishery reserve), showed high and significant values of genetic differentiation due to different allele frequencies, number of privative alleles and total number of alleles. These findings indicate a low degree of genetic similarity between both populations, therefore this restocking initiative is not advisable. The genetic connectivity pattern, highly consistent with oceanographic currents, identified two distinct metapopulations of white seabream around Sicily. Thus it is recommended to utilize broods from the same metapopulation for restocking purposes to provide a better genetic match to the wild populations.

Purificação e caracterização parcial de uma B-D glicosidase de Artemia franciscana com ação sobre celobiose e lactose

-D-glucosidase (EC 3.2.1.21) is one of the most interesting glycosidases, especially for hydrolysis cellobiose releasing glucose, is last step degradation of cellulose. This function makes the -D-glucosidase is of great interest as a versatile industrial biocatalyst, being critical to various bio-treatment / biorefinery processes, such as bioethanol production. Hen in the report, a -D-glucosidase was extracts from protein extracted of the invertebrate marine Artemia franciscana was purified and characterized with a combination of precipitation with ammonium sulfate (0 - 30%, 30 to 50%, 50 to 80%), the fraction saturated in the range of 30 to 50% (called F-II) was applied in a molecular exclusion chromatography, in Sephacryl S-200, the fractions corresponding to the first peak of activity of -D-glucosidase were gathered and applied in a chromatography of ion exchange in Mono Q; the third peak this protein obtained chromatography, which coincides with the peak of activity of -D-glucosidase was held and applied in a gel filtration chromatography Superose 12 where the first peak protein, which has activity of -D-glucosidase was rechromatography on Superose 12. This enzyme is probably multimerica, consisting of three subunit molecular mass of 52.7 kDa (determined by SDS-PAGE) with native molecular mass of 157 kDa (determined by gel filtration chromatography on Superose 12 under the system FPLC). The enzyme was purified 44.09 times with a recovery of 1.01%. Using up p-nitrophenyl-β-D-glucopiranoside as substrate obtained a Km apparent of 0.229 mM and a Vmax of 1.109 mM.60min-1.mL-1mM. The optimum pH and optimum temperature of catalysis of the synthetic substrate were 5.0 and 45 °C, respectively. The activity of the -D-glucosidase was strongly, inhibited by silver nitrate and N- etylmaleimide, this inhibition indicates the involvement of radical sulfidrila the hydrolysis of synthetic substrate. The -D-glucosidase of Artemia franciscana presented degradativa action on celobiose, lactose and on the synthetic substrate -nitrophenyl-β-D-glucopiranoside indicating potential use of this enzyme in the industry mainly for the production of bioethanol (production of alcohol from the participating cellulose), and production hydrolysate milk (devoid of milk lactose)

Everglades R-EMAP 2005

The Everglades R-EMAP project for year 2005 produced large quantities of data collected at 232 sampling sites. Data collection and analysis is an on-going long-term activity conducted by scientists of different disciplines at irregular intervals of several years. The data sets collected for 2005 include bio-geo-chemical (including mercury and hydro period), fish, invertebrate, periphyton, and plant data. Each sampling site is associated with a location, a description of the site to provide a general overview and photographs to provide a pictorial impression. The Geographic Information Systems and Remote Sensing Center(GISRSC) at Florida International University (FIU) has designed and implemented an enterprise database for long-term storage of the project�s data in a central repository, providing the framework of data storage for the continuity of future sampling campaigns and allowing integration of new sample data as it becomes available. In addition GISRSC provides this interactive web application for easy, quick and effective retrieval and visualization of that data.

Biodiversidad estacional del macrozoobentos mesolitoral de fondos blandos en la playa de Salaverry, La Libertad – Perú

La biodiversidad del macrozoobentos en el mesolitoral de la playa de Salaverry se determinó durante setiembre 2015 a marzo 2016. Se establecieron cuatro transectos (A, B, C y D) y se fijaron tres estaciones. Además se determinó la biodiversidad estacional a través de los índices de Shannon - Wiener (H'), Margalef (d'), Equidad de Pielou (J'), Simpson (λ) y de Similitud de Bray- Curtis. Las muestras fueron colectadas quincenalmente, utilizando un cilindro de Penchaszadeh de 0,028 m2. Asi también se tomaron datos de temperatura, salinidad y granulometría. Las especies se identificaron utilizando claves taxonómicas de invertebrados marinos. Se registraron especies de las clases Polychaeta, Nemertea, Gastropoda, Bivalvia y Malacostraca (Subphylum Crustacea). La biodiversidad del macrozoobentos para primavera fue de 2,51 bits/ind y para verano de 1,91 bits/ind con una riqueza de 22 especies.

DNA barcoding: an effective tool to overcome morphological identification constraints in the assessment of the ecological quality

DNA barcoding has the potential to overcome taxonomic challenges in biological community assessments. However, fulfilling that potential requires successful amplification of a large and unbiased portion of the community. In this study, we attempted to identify mitochondrial gene cytochrome c oxidase I (COI) barcodes from 1024 benthic invertebrate specimens belonging to 54 taxa from low salinity environments of the Mira estuary and Torgal riverside (SW Portugal). Up to 17 primer pairs and several reaction conditions were attempted among specimens from all taxa, with amplification success defined as a single band of approximately 658 bp visualized on a pre-cast agarose gel, starting near the 5' end of the COI gene and suitable for sequencing. Amplification success was achieved for 99.6% of the 54 taxa, though no single primer was successful for more than 88.9% of the taxa. However, only 68.5% of the specimens within these taxa successfully amplified. Inhibition factors resulting from a non-purified DNA extracted and inexistence of species-specific primers for COI were pointed as the main reasons for an unsuccessful amplification. These results suggest that DNA barcoding can be an effective tool for application in low salinity environments where taxa such as chironomids and oligochaetes are challenging for morphological identification. Nevertheless, its implementation is not simple, as methods are still being standardized and multiple species