The pre- and postembryonic development of Artemia franciscana (Anostraca: Artemiidae).

Artemia franciscana is a universal live feed in aquaculture, and it has been reported as an invasive species in many Asian hypersaline ecosystems. The present observations illustrated the pre- and postembryonic development stages of the A. franciscana population confined to the Indian saltern of Kelambakkam. We observed their growth patterns during various hydration periods with specific time intervals. Results showed differences in the development stages with respect to unique identity. Interestingly, a period of hydration showed notable cellular movement toward clockwise positions in the hydrating cysts. After 10 h of hydration, blastocoel appeared, accelerating the dynamic route of nuclei movement. At the end of the invagination, the embryo burst out of the cyst, and a sequence of emerging stages was noted. With reference to light microscopic observations, a series of developmental stages were observed, and each instar was documented by developing limb buds of nauplii. Excitingly, the 10th and 11th instar stages reveal sexual differentiation between male and female individuals. Thus, the laboratory culture study clearly documented the different developmental stages with their specific characteristic features. However, further molecular study would provide a cellular basis for understanding the early development of A. franciscana.

Validagao da espécie Dendrocephalus brasiliensis como organismo teste de água doce em estudos ecotoxicológicos para avaliagao de fármacos utilizados no tratamento da COVID-19 / Validation of the species Dendrocephalus brasiliensis as a freshwater test organism in ecotoxicological studies for evaluation of pharmaceuticals used in the treatment of COVID-19

Resumo Os contaminantes emergentes (CE), sao substáncias químicas (fármacos, produtos de higiene pessoal, drogas ilícitas entre outros) que estao presentes no ambiente como consequéncia da atividade antrópica e a falta de adequagao dos processos convencionais de tratamento de água e esgoto que nao logram remové-los eficientemente. Na atualidade o uso disseminado e desmedido de fármacos no tratamento da pandemia de COVID 19 tem aumentado a preocupagao dos impactos decorrentes da contaminagao por fármacos em ambientes aquáticos, consequéncia da liberagao no ambiente de grandes quantidades destes compostos. Assim, estudos de ecotoxicidade aquática sao fundamentais para avaliar o efeito de substáncias químicas tóxicas nas análises de impactos ambientais, sobretudo quando utilizado organismos representativos da biota aquática local, garantindo assim, maior confiabilidade e representatividade dos resultados obtidos. Diante disto, o objetivo deste trabalho foi validar a utili-dade do Dendrocephalus brasiliensis (Branchoneta) espécie autóctone do nordeste brasileiro como organismo teste para estudos de ecotoxicidade de fármacos utilizados no tratamento da COVID 19. Testes ecotoxicológicos utilizando D. brasiliensis foram realizados utilizando solugóes dos fármacos paracetamol, hidroxicloroquina, ivermectina e ibuprofeno, em concentragóes de 0,0025 até 600,0 mg/L seguindo os protocolos descritos pela Associagao Brasileira para Normas Técnicas (ABNT) para toxicidade aguda, protocolo padronizado para a realizagao do ensaio ecotoxicológicos utilizando como organismo teste a Daphnia magna, o qual foi empregada como referencia para comparar o padrao de resposta. Com os resultados obtidos foi realizado o cálculo da CL50-48h considerando como desfecho a morte dos organismos, ivermectina (< 0,0025 - < 0,0025), hidroxicloroquina (3,70 - 14,09), ibuprofeno (12,25 - 107,52), paracetamol (8,53 - 9,61), resultados CL50-48h mg/l D. magna e D. brasiliensis respectivamente. Os resultados obtidos mostraram um padrao diferenciado dependente da espécie e do fármaco analisado observando-se uma menor sensibilidade frente a exposigao da D. brasiliensis em comparagao a D. magna demonstrando a valia da D. brasiliensis como organismo teste. Pesquisas futuras dirigidas a analisar as potenciais interagóes destes fármacos em concentragóes ambientais reais sao necessárias para completar a validagao e ter uma aproximagao dos eventos acometidos em ambientes impactados por estes fármacos.

Abstract Emerging contaminants (EC) are chemical substances (pharmaceuticals, personal hygiene products, illicit drugs, among others) that are present in the environment because of human activity and the lack of adequacy of conventional water and sewage treatment processes that do not manage to remove them efficiently. Currently, the widespread and excessive use of drugs in the treatment of the COVID 19 pandemic has increased concern about the impacts resulting from contamination by drugs in aquatic environments, because of the release into the environment of large amounts of these compounds. Thus, aquatic ecotoxicity studies are essential to evaluate the effect of toxic chemical substances in the analysis of environmental impacts, especially when using representative organisms of the local aquatic biota, thus ensuring greater reliability and representativeness of the results obtained. In view of this, the objective of this work was to validate the usefulness of Dendrocephalus brasiliensis (Branchoneta), an autoch-thonous species from northeastern Brazil as a test organism for ecotoxicity studies of drugs used in the treatment of COVID 19. Ecotoxicological tests using D. brasiliensis were performed using drug solutions paracetamol, hydroxychloroquine, ivermectin and ibuprofen, in concentrations from 0.0025 to 600.0 mg/L following the protocols described by the Brazilian Association for Technical Norms (ABNT) for acute toxicity, standardized protocol for carrying out the ecotoxicological assay using as a test organism Daphnia magna, which was used as a reference to compare the response pattern. Based on the results obtained, the LC50-48h was calculated considering the death of organisms, ivermectin (< 0.0025 - < 0.0025), hydroxychloroquine (3.70 - 14.09), ibuprofen (12.25 - 107.52), paracetamol (8.53 - 9.61), results LC50-48h mg/l D. magna and D. brasiliensis respectively. The results obtained showed a differenti-ated pattern depending on the species and the analyzed drug, observing a lower sensitivity to exposure of D. brasiliensis compared to D. magna, demonstrating the value of D. brasiliensis as a test organism. Future research aimed at analyzing the potential interac-tions of these drugs at real environmental concentrations is necessary to complete the validation and to have an approximation of the events affected in environments impacted by these drugs.

Recognizing and counting Dendrocephalus brasiliensis (Crustacea: Anostraca) cysts using deep learning.

The Dendrocephalus brasiliensis, a native species from South America, is a freshwater crustacean well explored in conservational and productive activities. Its main characteristics are its rusticity and resistance cysts production, in which the hatching requires a period of dehydration. Independent of the species utilization nature, it is essential to manipulate its cysts, such as the counting using microscopes. Manually counting is a difficult task, prone to errors, and that also very time-consuming. In this paper, we propose an automatized approach for the detection and counting of Dendrocephalus brasiliensis cysts from images captured by a digital microscope. For this purpose, we built the DBrasiliensis dataset, a repository with 246 images containing 5141 cysts of Dendrocephalus brasiliensis. Then, we trained two state-of-the-art object detection methods, YOLOv3 (You Only Look Once) and Faster R-CNN (Region-based Convolutional Neural Networks), on DBrasiliensis dataset in order to compare them under both cyst detection and counting tasks. Experiments showed evidence that YOLOv3 is superior to Faster R-CNN, achieving an accuracy rate of 83,74%, R2 of 0.88, RMSE (Root Mean Square Error) of 3.49, and MAE (Mean Absolute Error) of 2.24 on cyst detection and counting. Moreover, we showed that is possible to infer the number of cysts of a substrate, with known weight, by performing the automated counting of some of its samples. In conclusion, the proposed approach using YOLOv3 is adequate to detect and count Dendrocephalus brasiliensis cysts. The DBrasiliensis dataset can be accessed at: https://doi.org/10.6084/m9.figshare.13073240.

Occurrence of Dendrocephalus brasiliensis Pesta, 1921 (Crustacea, Anostraca) in the Caras river, southern Ceara, Brazil

ABSTRACT Occurrence of Dendrocephalus brasiliensis Pesta, 1921 (Crustacea, Anostraca) in the Caras river, southern Ceara, Brazil. The specimens were collected in March and April 2014. The new occurrence extends the distribution and update area of occupancy of the species, which is characterized by a specific habitat: temporary lakes.

Toxic potential of the emerging contaminant nicotine to the aquatic ecosystem.

Nicotine is a "life-style compound" widely consumed by human populations and, consequently, often found in surface waters. This fact presents a concern for possible effects in the aquatic ecosystems. The objective of this study was to assess the potential lethal and sublethal toxicity of nicotine in aquatic organisms from different trophic levels (Vibrio fischeri, Pseudokirchneriella subcapitata, Thamnocephalus platyurus, and Daphnia magna). The bioassays were performed by exposing the organisms to concentrations of nicotine in a range of 0.5-1000 µg/L. Results showed that nicotine, at tested concentration, was not acutely toxic to V. fischeri and T. platyurus. On the contrary, this substance exhibited toxicity to P. subcapitata and Daphnia magna. Thus, concentrations of nicotine of 100 and 200 µg/L promoted an inhibition in the growth of P. subcapitata. In addition, a concentration of 100 µg/L nicotine acted on the reproduction of the crustacean D. magna, by decreasing the number of juveniles produced by female. On the other hand, the results showed that concentrations equal to or greater than 10 µg/L induced the production of daphnids male offspring, which may indicate that nicotine is a weak juvenoid compound of the D. magna endocrine system. Furthermore, the result showed that concentrations tested of this chemical have the capacity to revert the effect of fenoxycarb, a strong juvenoid chemical insecticide. The results of the study revealed that nicotine can induce several changes in some of the most important key groups of the aquatic compartment, which can compromise, in a short time, the balance of aquatic ecosystem. Finally, a preliminary environmental risk assessment of this stimulant was performed from the highest measured concentration in surface water and the no observable effect concentration value in the most sensitive species, i.e., D. magna. This process revealed that nicotine can produce an important risk to aquatic organisms.

Shrimp oil extracted from the shrimp processing waste reduces the development of insulin resistance and metabolic phenotypes in diet-induced obese rats.

Diet-induced obesity, insulin resistance, impaired glucose tolerance, chronic inflammation, and oxidative stress represent the main features of type 2 diabetes mellitus. The present study was conducted to examine the efficacy and mechanisms of shrimp oil on glucose homeostasis in obese rats. Male CD rats fed a high-fat diet (52 kcal% fat) and 20% fructose drinking water were divided into 4 groups and treated with the dietary replacement of 0%, 10%, 15%, or 20% of lard with shrimp oil for 10 weeks. Age-matched rats fed a low-fat diet (10 kcal% fat) were used as the normal control. Rats on the high-fat diet showed impaired (p < 0.05) glucose tolerance and insulin resistance compared with rats fed the low-fat diet. Shrimp oil improved (p < 0.05) oral glucose tolerance, insulin response, and homeostatic model assessment-estimated insulin resistance index; decreased serum insulin, leptin, hemoglobin A1c, and free fatty acids; and increased adiponectin. Shrimp oil also increased (p < 0.05) antioxidant capacity and reduced oxidative stress and chronic inflammation. The results demonstrated that shrimp oil dose-dependently improved glycemic control in obese rats through multiple mechanisms.

Linderiella jebalae sp. nov. (Crustacea: Branchiopoda: Anostraca), a new species from the Rif mountains (northern Morocco).

A new species of fairy shrimp, Linderiella jebalae sp. nov., was found in temporary ponds located in the Rif region of northern Morocco. Morphological characters of females, such as the basal long warty outgrowth of the antennae, the highly developed cuticular papillae in the second genital segment, and the presence of a keel-shaped process directed posteriorly in dorsal side of the brood pouch, clearly distinguish this species from the others of the genus. In the case of males, the basiomedial antennal outgrowth and the distal segment of the antenna are diagnostic features for the species. Cyst ornamentation, characterized by thick, blunt and sometimes truncated spines, also separate this species. This is the fourth known Palaearctic Linderiella species, all of them described recently (from 1980s onwards) and located in a restricted area, from south-eastern France to Iberian Peninsula and Morocco.

RNA editing of microRNA prevents RNA-induced silencing complex recognition of target mRNA.

MicroRNAs (miRNAs) integrate with Argonaut (Ago) to create the RNA-induced silencing complex, and regulate gene expression by silencing target mRNAs. RNA editing of miRNA may affect miRNA processing, assembly of the Ago complex and target mRNA binding. However, the function of edited miRNA, assembled within the Ago complex, has not been extensively investigated. In this study, sequence analysis of the Ago complex of Marsupenaeus japonicus shrimp infected with white spot syndrome virus (WSSV) revealed that host ADAR (adenosine deaminase acting on RNA) catalysed A-to-I RNA editing of a viral miRNA (WSSV-miR-N12) at the +16 site. This editing of the non-seed sequence did not affect association of the edited miRNA with the Ago protein, but inhibited interaction between the miRNA and its target gene (wsv399). The WSSV early gene wsv399 inhibited WSSV infection. As a result, the RNA editing of miRNA caused virus latency. Our results highlight a novel example of miRNA editing in the miRNA-induced silencing complex.

Preparation of Fluorescent Microcystin Derivatives by Direct Arginine Labelling and Their Biological Evaluation.

Microcystin is the most prevalent toxin produced by cyanobacteria and poses a severe threat to livestock, humans and entire ecosystems. We report the preparation of a series of fluorescent microcystin derivatives by direct arginine labelling of the unprotected peptides at the guanidinium side chain. This new method allows a simple late-stage diversification strategy for native peptides devoid of protecting groups under mild conditions. A series of fluorophores were conjugated to microcystin-LR in good to very good yield. The fluorescent probes displayed biological activity comparable to that of unlabelled microcystin, in both phosphatase inhibition assays and toxicity tests on the crustacean Thamnocephalus platyurus. In addition, we demonstrate that the fluorescent probes penetrated Huh7 cells. Whole-animal imaging was performed on T. platyurus: labelled compound was mainly observed in the digestive tract.

LETHAL EFFECTS OF PESTICIDES ON THE NAUPLII FAIRY SHRIMP, BRANCHINELLA THAILANDENSIS.

During agricultural activities, large amounts of insecticides and herbicides are used for controlling pests. The overspraying of pesticides may contaminate freshwater bodies, as well as having a significant effect on aquatic animals. To determine the potential risk of pesticides, the acute toxicity of insecticides and herbicides on the nauplii fairy shrimp, Branchinella thailandensis were evaluated. The organisms were exposed to different concentrations of insecticides (chlorpyrifos and carbaryl), and herbicides (paraquat and 2,4-D) for 24 h, after which the LC50 values were compared. The acute toxicity tests determined that the LC50 values of chlorpyrifos and carbaryl after 24 h were 0.00186 mg/L and 0.21 mg/L, respectively. For herbicides, paraquat and 2,4-D, the LC50 values were 0.11 mg/L and 27.91 mg/L. Therefore, chlorpyrifos insecticides were found to be more toxic than herbicides (2,4-D and paraquat) with regard to the nauplii fairy shrimp. The high toxicity of pesticides has a long-term, and potentially damaging effect on aquatic organisms in the application areas.

Toxicity of two types of silver nanoparticles to aquatic crustaceans Daphnia magna and Thamnocephalus platyurus.

Although silver nanoparticles (NPs) are increasingly used in various consumer products and produced in industrial scale, information on harmful effects of nanosilver to environmentally relevant organisms is still scarce. This paper studies the adverse effects of silver NPs to two aquatic crustaceans, Daphnia magna and Thamnocephalus platyurus. For that, silver NPs were synthesized where Ag is covalently attached to poly(vinylpyrrolidone) (PVP). In parallel, the toxicity of collargol (protein-coated nanosilver) and AgNO3 was analyzed. Both types of silver NPs were highly toxic to both crustaceans: the EC50 values in artificial freshwater were 15-17 ppb for D. magna and 20-27 ppb for T. platyurus. The natural water (five different waters with dissolved organic carbon from 5 to 35 mg C/L were studied) mitigated the toxic effect of studied silver compounds up to 8-fold compared with artificial freshwater. The toxicity of silver NPs in all test media was up to 10-fold lower than that of soluble silver salt, AgNO3. The pattern of the toxic response of both crustacean species to the silver compounds was almost similar in artificial freshwater and in natural waters. The chronic 21-day toxicity of silver NPs to D. magna in natural water was at the part-per-billion level, and adult mortality was more sensitive toxicity test endpoint than the reproduction (the number of offspring per adult).

Comparative assessment of olive oil mill effluents from three-phase and two-phase systems, treated for hydrogen production.

By-products of a two-phase and a three-phase olive oil mill process treated in an anaerobic fermentation system for hydrogen production, were evaluated by three bioassays: the zebrafish Danio rerio embryo test and two microbiotests, Thamnotoxkit F and Daphtoxkit F™ pulex. Samples from both processes were classified as "very toxic" with LC(50) values ranging from 1.52% (T. platyurus 24 h test) to 4.48% (D. pulex 48 h-LC50). Toxicity values were differently correlated to physicochemical parameters showing different degree of influence. The treated effluents of both process systems remained very toxic showing the necessity for further treatment, aiming to environmentally safe discharges.

Comparisons of stress proteins and soluble carbohydrate in encysted embryos of Artemia franciscana and two species of Parartemia.

We compared stress proteins (p26, artemin, hsp70) and alcohol-soluble carbohydrates (ASC) in cysts of Artemia franciscana and two as yet un-named species populations of Parartemia, the brine shrimp endemic to Australia. The small stress proteins and molecular chaperones, p26 and artemin, previously thought to be restricted to Artemia, and present in very large amounts in its encysted embryos (cysts), were also detected by western blotting in Parartemia cysts, even though roughly 85-100 million years have passed since these genera diverged. We interpret this finding as further evidence for the adaptive importance of these proteins in coping with the severe stresses these encysted embryos endure. As expected, hsp70 was present in all three groups of cysts, but apparently at somewhat lower concentrations in those of Parartemia. Based on measurements of ASC we propose that the disaccharide trehalose, critical for desiccation tolerance in many animal cells, has probably also been maintained in the metabolic repertoire of Parartemia whose cysts have well developed tolerance to severe desiccation.

Identification and characterization of a prawn white spot syndrome virus gene that encodes an envelope protein VP31.

Based on a combination of SDS-PAGE and mass spectrometry, a protein with an apparent molecular mass of 31 kDa (termed as VP31) was identified from purified shrimp white spot syndrome virus (WSSV) envelope fraction. The resulting amino acid (aa) sequence matched an open reading frame (WSV340) of the WSSV genome. This ORF contained 783 nucleotides (nt), encoding 261 aa. A fragment of WSV340 was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein with a 6His-tag, and then specific antibody was raised. Western blot analysis and the immunoelectron microscope method (IEM) confirmed that VP31 was present exclusively in the viral envelope fraction. The neutralization experiment suggested that VP31 might play an important role in WSSV infectivity.

Eclosión de quistes en dos especies de Dendrocephalus (Anostraca: Thamnocephalidae) de uso potencial como alimento en acuacultura / Eclossion of cysts in two species of Dendrocephalus (Anostraca: Thamnocephalidae) in a potential use as food in aquaculture

The use of Artemia salina nauplii as live food has significantly aided culture of commercial fish and shrimps in recent years. However, reported deficiencies in the nutritional value of some strains originated the study of freshwater fairy shrimps as an alternative to Artemia. This study presents cyst biometry for Dendrocephalus geayi and D. spartaenovae (Anostraca: Thamnocephalidae), and the effects of some physicochemical variables on cyst hatching. The biometric characteristics of D. geayi and D. spartaenovae are within the size range of commercial Artemia strains. Favorable conditions to hatch D. geayi are 30 degrees C and conductivity near that of distilled water (<5 micromhos). For D. spartaenovae, these conditions are 28 degrees C and 280 micromhos. A very low salinity such as 1 per thousand inhibits hatching in both species.

[Eclossion of cysts of two species of Dendrocephalus (Anostraca: Thamnocephalidae) of potential use as food in aquaculture]. / Eclosión de quistes en dos especies de Dendrocephalus (Anostraca: Thamnocephalidae) de uso potencial como alimento en acuacultura.

The use of Artemia salina nauplii as live food has significantly aided culture of commercial fish and shrimps in recent years. However, reported deficiencies in the nutritional value of some strains originated the study of freshwater fairy shrimps as an alternative to Artemia. This study presents cyst biometry for Dendrocephalus geayi and D. spartaenovae (Anostraca: Thamnocephalidae), and the effects of some physicochemical variables on cyst hatching. The biometric characteristics of D. geayi and D. spartaenovae are within the size range of commercial Artemia strains. Favorable conditions to hatch D. geayi are 30 degrees C and conductivity near that of distilled water (<5 micromhos). For D. spartaenovae, these conditions are 28 degrees C and 280 micromhos. A very low salinity such as 1 per thousand inhibits hatching in both species.