Lethal and behavioral effects of semi-purified microcystins, Micropeptin and apolar compounds from cyanobacteria on freshwater microcrustacean Thamnocephalus platyurus.

The mass proliferation of cyanobacteria, episodes known as blooms, is a concern worldwide. One of the most critical aspects during these blooms is the production of toxic secondary metabolites that are not limited to the four cyanotoxins recognized by the World Health Organization. These metabolites comprise a wide range of structurally diverse compounds that possess bioactive functions. Potential human and ecosystem health risks posed by these metabolites and co-produced mixtures remain largely unknown. We studied acute lethal and sublethal effects measured as impaired mobility on the freshwater microcrustaceans Thamnocephalus platyurus for metabolite mixtures from two cyanobacterial strains, a microcystin (MC) producer and a non-MC producer. Both cyanobacterial extracts, from the MC-producer and non-MC-producer, caused acute toxicity with LC50 (24 h) values of 0.50 and 2.55 mgdw_biomass/mL, respectively, and decreased locomotor activity. Evaluating the contribution of different cyanopeptides revealed that the Micropeptin-K139-dominated fraction from the MC-producer extract contributed significantly to mortality and locomotor impairment of the microcrustaceans, with potential mixture effect with other cyanopeptolins present in this fraction. In the non-MC-producer extract, compounds present in the apolar fraction contributed mainly to mortality, locomotor impairment, and morphological changes in the antennae of the microcrustacean. No lethal or sublethal effects were observed in the fractions dominated by other cyanopetides (Cyanopeptolin 959, Nostoginin BN741). Our findings contribute to the growing body of research indicating that cyanobacterial metabolites beyond traditional cyanotoxins cause detrimental effects. This underscores the importance of toxicological assessments of such compounds, also at sublethal levels.

A chromosome-level reference genome for the Versatile Fairy Shrimp, Branchinecta lindahli.

We present the novel reference genome of the Versatile Fairy Shrimp, Branchinecta lindahli. The Versatile Fairy Shrimp is a freshwater anostracan crustacean found across the western United States from Iowa to Oregon and from Alberta to Baja California. It is an ephemeral pool specialist, living in prairie potholes, irrigation ditches, tire treads, vernal pools, and other temporary freshwater wetlands. Anostracan fairy shrimp are facing global declines with 3 species in California on the Endangered Species list. This species was included in the California Conservation Genomics Project to provide an easily accessible reference genome, and to provide whole-genome resources for a generalist species, which may lead to new insights into Anostracan resiliency in the face of climate change. The final gapped genome comprises 15 chromosome-length scaffolds covering 98.63% of the 384.8 Mb sequence length, and an additional 55 unscaffolded contigs.

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.

A new fairy shrimp, Branchinecta piurae sp. nov. (Branchiopoda: Anostraca), from Peru.

South American species of Branchinecta that inhabit high altitude biotopes are so far represented by B. brushi and B. papillata. Here we describe a new species inhabiting these extreme habitats, B. piurae sp. nov., whose closest species is B. papillata. Differential characters of B. piurae are the lack, in males, of proximal and distal anteromedial bulges with spines at the second antenna basal antennomere which are conversely present in males of B. papillata; instead, the males of B. piurae have scattered, triangular spines over the anterior and medial surfaces at the distal half of the basal antennomere. In females of B. piurae, the dorsal surface of thoracic segments are provided with well-marked rough protuberances mainly on segments 3 to 10, features that are not observed in B. papillata. As far as is known, Branchinecta piurae sp. nov. is endemic to the Peruvian and Ecuadorian Andes, appearing at altitudes around 3,7003,800 m a.s.l, and latitudes from 247 to 504 S. The results of the genetic analyses based both on mitochondrial and nuclear markers provide a clear support for the morphological description of B. piurae as a new species. We also provide genetic sequences of B. papillata, B. ferox from Spain and B. orientalis from Spain and Mongolia, and show that Palaearctic Branchinecta species are not directly related to B. piurae and B. papillata. In the phylogeny derived from COI, the last two species form a sister clade to the American Branchinecta clade, including several North American species, or in the 16S phylogeny, only B. papillata is at the base. Our results suggest a different history about the genus in America than previous hypotheses. New molecular studies are needed that include additional South American species in order to better explore the diversity and biogeographical patterns of the genus Branchinecta.

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.

The effect of fairy shrimp “branchoneta” (Dendrocephalus brasiliensis) as the initial diet of tambaqui postlarvae

The effect of fairy shrimp “branchoneta” as the first exogenous feed for tambaqui postlarvae was evaluated. A hundred and eighty larvae were distributed in 12 aquariums, containing 1.5L of water and the stocking density was 10 larvae per liter. The trials lasted for 20 days and postlarvae were submitted to three diets (T1: Commercial feed, T2: branchoneta, and T3: Commercial feed with branchoneta), with a completely randomized design with four replicates for each treatment. During the trials, the water quality parameters were kept within ideal conditions for the species. The tambaqui postlarvae submitted to T2 and T3 showed better survival rates respectively. Regarding the postlarval development, larvae fed with only branchoneta had a better final weight (3.48 mg), daily weight gain (0.14 mg day-1), and the best specific growth rate (8.62%). The results also showed that the tambaqui postlarvae fed with only commercial feed developed less efficiently due to lower rates of survival and growth. The present study indicates that the branchoneta promoted greater development and survival rates of the tambaqui postlarvae and therefore, it presents good potential as a live food for the species.

The effect of fairy shrimp “branchoneta” (Dendrocephalus brasiliensis) as the initial diet of tambaqui postlarvae

The effect of fairy shrimp “branchoneta” as the first exogenous feed for tambaqui postlarvae was evaluated. A hundred and eighty larvae were distributed in 12 aquariums, containing 1.5L of water and the stocking density was 10 larvae per liter. The trials lasted for 20 days and postlarvae were submitted to three diets (T1: Commercial feed, T2: branchoneta, and T3: Commercial feed with branchoneta), with a completely randomized design with four replicates for each treatment. During the trials, the water quality parameters were kept within ideal conditions for the species. The tambaqui postlarvae submitted to T2 and T3 showed better survival rates respectively. Regarding the postlarval development, larvae fed with only branchoneta had a better final weight (3.48 mg), daily weight gain (0.14 mg day-1), and the best specific growth rate (8.62%). The results also showed that the tambaqui postlarvae fed with only commercial feed developed less efficiently due to lower rates of survival and growth. The present study indicates that the branchoneta promoted greater development and survival rates of the tambaqui postlarvae and therefore, it presents good potential as a live food for the species.(AU)

A new species of Archaebranchinecta (Anostraca: Branchinectidae) from the South American Altiplano.

A detailed morphological comparison was carried out among specimens of several samples of Archaebranchinecta Rogers Coronel, 2011 from the Altiplano of Peru, Bolivia, and Argentina. Surprisingly, striking differences were found between Peruvian samples collected near the western shore of Lake Titicaca, and those from Bolivia taken southwardly, near the east coasts of River Desaguadero and Lake Poopó. Accordingly, the new species Archaebranchinecta aimara sp. nov. is described, representing the second specific entity of a genus that so far included only A. pollicifera (Harding, 1940). The main differential features between both species include: (a) size and shape of the three processes of basal segment of the male second antenna; (b) shape and protrusion degree of the pair of medioventral bulges in the male genital segments; (c) presence or absence of strong ventrolateral spine on the second genital segment of female; and (d) presence or absence of a pair of ventrolateral outgrowths in the brood pouch. This research contributes to our understanding of the biodiversity and endemism of the unique Altiplano region of South America.

Genetic and phenotypic diversity of Branchinecta sandiegonensis (Crustacea: Anostraca) in the vernal pools of Baja California, México.

Branchinecta sandiegonensis is a passively dispersed species that occurs in the vernal pool complexes of southern California, the USA, and northwestern Baja California, México. The fragmented distribution of these vernal pool complexes could limit the gene flow, generating high genetic structure and morphometric variation across the landscape. Here we estimate the genetic and phenotypic variation of B. sandiegonensis in the southern part of its range. We sampled 15 vernal pools from four geographic regions of the Baja California Peninsula. We genotyped 150 individuals using nuclear microsatellites and 31 individuals using the mitochondrial COI region. We also conducted a morphometric analysis on a sample of 232 individuals. We found moderate levels of genetic diversity and different patterns of structure depending upon the spatial scale of analysis. Demographic models suggest contrasting trends among populations. Phenotypically, we found high levels of heterogeneity in body size of fairy shrimps within and across the regions. Our findings highlight that vernal pools in Baja California are important reservoirs of genetic and phenotypic diversity for B. sandiegonensis. The interplay between gene flow and genetic drift may have influenced the patterns we detected in the southern part of the range of this species.

Acute ecotoxicity bioassay using Dendrocephalus brasiliensis: alternative test species for monitoring of contaminants in tropical and subtropical freshwaters.

In International guidelines for standard ecotoxicological bioassays, Daphnia magna is the most applied microcrustacea for assessing toxicity of different pollutants. However, in research realized in tropical and subtropical areas, autochthonous species must be prioritized because they are adapted to the specificities of ecosystems. In this sense, the present study aimed to assess and compare (with D. magna) the sensitivity of the tropical species Dendrocephalus brasiliensis as alternative test species for monitoring of contaminants in tropical and subtropical freshwaters, by carrying out acute toxicity tests with different pollutants. According results, D. brasiliensis presented EC50-48 h values lower than D. magna for all substances tested, indicating higher sensitivity of the tropical organism in relation to the temperate organism. Furthermore, comparing the results obtained with data from other studies, D. brasiliensis is more sensitive to the chemicals tested than D. magna and has similar sensitivity to Pseudosida ramosa and Ceriodaphnia dubia, common species in tropical areas. In view of this, we suggest that D. brasiliensis can be used as alternative test species for monitoring of contaminants in tropical and subtropical freshwaters.

Branchinecta uruguayensis Rogers Racz Lorenz, 2015 is a junior synonym of B. achalensis César, 1985 (Branchiopoda: Anostraca: Branchinectidae).

As part of preparation for identification keys to the branchiopod crustaceans of the Neotropical Region, we re-examined many species of Anostraca that appeared to be closely related. Our examination caused us to re-evaluate the status and relationship of two branchinectid species.

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

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.

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.

Thamnocephalus chacosaltensis, a new species of Anostraca (Crustacea: Branchiopoda) from Salta province (Argentina).

Thamnocephalus chacosaltensis, a new species of fairy shrimp, was found in a temporary turbid pond in the Rivadavia Department, Salta province, Argentina (24º13'19.3''S; 62º 52'14.3''W), belonging to the Chaco salteño. The males present a well developed frontal appendage. Their First (FB) and Third (TB) branches show features diagnostic for the species. FB shows three sub-branches; the most proximal one is coiled, lacks spines all along, gradually thins in distal direction and flattens in the distal half; distally, the flattened portion is folded and ends blunt, bearing a small spine at the tip. TB is a broad-based triangular, thin, much flattened and folded outgrowth, with a small spine at the apex. Male genital and abdominal segments lack medio-ventral spines near their posterior margins. Comparisons with the other species of the genus are established.

A new species of Branchinecta (Crustacea: Anostraca) from Uruguay.

Branchinecta uruguayensis, a new species of fairy shrimp, is described from a single seasonally astatic aquatic habitat in coastal Uruguay. This is the first record of an anostracan from the nation of Uruguay. Male B. uruguayensis n. sp. are separated from other Branchinecta species by the form of the second antennae. Branchinecta uruguayensis n. sp. share the distal antennomere lobiform projection with B. papillata Rogers, De los Rios, & Zúñiga, 2008 and B. achalensis César, 1985. Branchinecta uruguayensis n. sp. females are readily separated from all other Branchinecta by the presence of posteriolateral thoracic spines and lateral crenulations on the first abdomen segment margins. A key is provided to the Branchinectidae of South America.

Morphological variation in males of Dendrocephalus orientalis (Anostraca: Thamnocephalidae): Implications for species identification.

The genus Dendrocephalus Daday, 1908 consists of 17 described species. The morphology of the frontal appendage and the first pairs of thoracopods of males are the main characteristics used for species identification. The objectives of this study were to increase knowledge of the morphology of Dendrocephalus orientalis and to propose a new identification key for the species occurring in Brazil. Specimens were collected in temporary ponds within the conservation unit Monument Natural Grota do Angico (MNGA), which straddles the borders of the municipalities of Poço Redondo and Canindé de São Francisco, State of Sergipe, Brazil. During the study period, 560 male individuals of D. orientalis were sampled. The specimens showed variations in the number of spines on the eyes and the proximal surface of the male frontal appendage. On the eye, the number of spines ranged from 0 to 2; morphotypes with 1 and 2 spines predominated in the samples, with 50.5% and 46.2%, respectively. The number of spines on each eye was the same within each individual. On the proximal surface of the males frontal appendage the following numbers of spines on each arm were recorded: 1 and 1 (57.86%); 2 and 1 (30.89%); 2 and 2 (9.46%); 1 and 0 (1.25%); 3 and 2 (0.36%); 2 and 0 (0.18%). Based on these variations, we conclude that the number of spines on the eyes and the frontal appendage arms cannot be used as a diagnostic character for species identification. On the other hand, the first pairs of thoracopods and sub-branches 1V and 2A of the frontal appendage should be included in keys, since these structures did not show significant intraspecific morphological variation. 

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.