Thus far, only one gene from the heat shock protein 70 (HSP70) family has been identified in Artemia franciscana. Here, we used the draft Artemia transcriptome database to search for other genes in the HSP70 family. Four novel HSP70 genes were identified and designated heat shock cognate 70 (HSC70), heat shock 70 kDa cognate 5 (HSC70-5), Immunoglobulin heavy-chain binding protein (BIP), and hypoxia up-regulated protein 1 (HYOU1). For each of these genes, we obtained nucleotide and deduced amino acid sequences, and reconstructed a phylogenetic tree. Expression analysis revealed that in the juvenile state, the transcription of HSP70 and HSC70 was significantly (P < 0.05) higher in a population of A. franciscana selectively bred for increased induced thermotolerance (TF12) relative to a control population (CF12). Following non-lethal heat shock treatment at the nauplius stage, transcription of HSP70, HSC70, and HSC70-5 were significantly (P < 0.05) up-regulated in TF12. In contrast, transcription of the other HSP70 family members in A. franciscana (BIP, HYOU1, and HSPA4) showed no significant (P > 0.05) induction. Gene expression analysis demonstrated that not all members of the HSP70 family are involved in the response to heat stress and selection and that especially altered expression of HSC70 plays a role in a population selected for increased thermotolerance.

Prior research on the microorganisms associated with the brine shrimp, Artemia franciscana, has mainly been limited to culture-based identification techniques or feeding studies for aquaculture. Our objective was to identify bacteria and archaea associated with Artemia adults and encysted embryos to understand the role of microbes in the Artemia life cycle and, therefore, their importance in a hypersaline food chain.

In the study, the complete mitochondrial genome of Artemia salina was reported for the first time. The mitochondrial genome of A. salina is 15,762 bp in length, with the typical structure of 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), and two ribosomal RNA genes, and a major non-coding region (CR). Phylogenetic analysis showed that A. salina has a much closer relationship with A. persimilis compared to other Artemia species. The complete cp genome sequence of A. salina reported here provided an essential resource for further population genetics research and germplasm conservation on Artemia.

Artemia is an industrially important genus used in aquaculture as a nutritious diet for fish and as an aquatic model organism for toxicity tests. However, despite the significance of Artemia, genomic research remains incomplete and knowledge on its genomic characteristics is insufficient. In particular, Artemia franciscana of North America has been widely used in fisheries of other continents, resulting in invasion of native species. Therefore, studies on population genetics and molecular marker development as well as morphological analyses are required to investigate its population structure and to discriminate closely related species. Here, we used the Illumina Hi-Seq platform to estimate the genomic characteristics of A. franciscana through genome survey sequencing (GSS). Further, simple sequence repeat (SSR) loci were identified for microsatellite marker development. The predicted genome size was ∼867 Mb using K-mer (a sequence of k characters in a string) analysis (K = 17), and heterozygosity and duplication rates were 0.655 and 0.809%, respectively. A total of 421467 SSRs were identified from the genome survey assembly, most of which were dinucleotide motifs with a frequency of 77.22%. The present study will be a useful basis in genomic and genetic research for A. franciscana.

Diapause-destined embryos of the crustacean Artemia franciscana cease development as gastrulae, encyst, and enter a resting stage characterized by greatly reduced metabolic activity and extreme stress resistance. To better understand diapause induction and maintenance in Artemia embryos gene expression was analyzed by subtractive hybridization at two days post-fertilization, a time early in this developmental process. Eighty-five of 264 cDNA clones sequenced matched GenBank entries and they fell into categories designated as environmental information processing, cellular processes, genetic information processing and metabolism. Semi-quantitative RT-PCR of cDNAs populating the subtractive library identified seventeen up-regulated and four down-regulated transcripts, the former including those encoding a human transcription cofactor homologue, three small heat shock proteins, putative cell growth suppressor proteins and several enzymes. As examples, p8 may modulate gene expression during diapause in Artemia embryos. BRCA1 associated protein-1 (BAP1) and other functionally related proteins may influence cell growth and division during transition into diapause, a time when these processes are inhibited, whereas small heat shock proteins protect embryos from stress. This study represents the first systematic molecular characterization of diapause in crustaceans. Several differentially expressed genes were identified, expanding the repertoire of proteins potentially modified during diapause and suggesting mechanistic pathways indigenous to the initiation and maintenance of this physiological state.

Since the commercialization of brine shrimp (genus Artemia) in the 1950s, this lineage, and in particular the model species Artemia franciscana, has been the subject of extensive research. However, our understanding of the genetic mechanisms underlying various aspects of their reproductive biology, including sex determination, is still lacking. This is partly due to the scarcity of genomic resources for Artemia species and crustaceans in general. Here, we present a chromosome-level genome assembly of A. franciscana (Kellogg 1906), from the Great Salt Lake, United States. The genome is 1 GB, and the majority of the genome (81%) is scaffolded into 21 linkage groups using a previously published high-density linkage map. We performed coverage and FST analyses using male and female genomic and transcriptomic reads to quantify the extent of differentiation between the Z and W chromosomes. Additionally, we quantified the expression levels in male and female heads and gonads and found further evidence for dosage compensation in this species.

Artemia (Anostraca) is among the most primitive and ancient groups of crustaceans. Artemia spp. play a dominant role in the ecosystems of hypersaline waters, and often they are the only animals in these extreme biotopes. Most ethological studies on Artemia have been conducted on nauplii and metanauplii. We made ethological observations on Artemia under laboratory conditions and in the natural waters of Crimea, where we studied growth and ontogenetic changes of swimming behavior. Growth occurred during the first 50 days up to a size of 9.5-10.5mm, after which time the size did not increase (some females lived up to 6.5 months). A strong positive relation was found between maximal speed and individual length, which varied between 0.4 and 10.5mm; it may be approximated by the power equation: Vmax=1.205·K(0.820), where Vmax is the maximal speed of Artemia (in mms(-1)) of the length K (in mm). There is no similar relation between average speed and length of Artemia. The average speed of adults was 40-60% lower in environments with microalgae compared to media without food. The duration of the "riding position" for mating pairs of Artemia urmiana in our experiments varied from 10 to 27 days. In lakes we observed different Artemia aggregations varying in size and form. We conclude that the swimming behavior of Artemia is quite complex and diverse, and develops during ontogeny.

While sexual reproduction is widespread among many taxa, asexual lineages have repeatedly evolved from sexual ancestors. Despite extensive research on the evolution of sex, it is still unclear whether this switch represents a major transition requiring major molecular reorganization, and how convergent the changes involved are. In this study, we investigated the phylogenetic relationship and patterns of gene expression of sexual and asexual lineages of Eurasian Artemia brine shrimp, to assess how gene expression patterns are affected by the transition to asexuality. We find only a few genes that are consistently associated with the evolution of asexuality, suggesting that this shift may not require an extensive overhauling of the meiotic machinery. While genes with sex-biased expression have high rates of expression divergence within Eurasian Artemia, neither female- nor male-biased genes appear to show unusual evolutionary patterns after sexuality is lost, contrary to theoretical expectations.

Different developmental stages of Artemia spp. (metanauplii, juveniles and adults) were bath-challenged with two isolates of the Lymphocystis disease virus (LCDV), namely, LCDV SA25 (belonging to the species Lymphocystis disease virus 3) and ATCC VR-342 (an unclassified member of the genus Lymphocystivirus). Viral quantification and gene expression were analyzed by qPCR at different times post-inoculation (pi). In addition, infectious titres were determined at 8 dpi by integrated cell culture (ICC)-RT-PCR, an assay that detects viral mRNA in inoculated cell cultures. In LCDV-challenged Artemia, the viral load increased by 2-3 orders of magnitude (depending on developmental stage and viral isolate) during the first 8-12 dpi, with viral titres up to 2.3 × 102 Most Probable Number of Infectious Units (MPNIU)/mg. Viral transcripts were detected in the infected Artemia, relative expression values showed a similar temporal evolution in the different experimental groups. Moreover, gilthead seabream (Sparus aurata) fingerlings were challenged by feeding on LCDV-infected metanauplii. Although no Lymphocystis symptoms were observed in the fish, the number of viral DNA copies was significantly higher at the end of the experimental trial and major capsid protein (mcp) gene expression was consistently detected. The results obtained support that LCDV infects Artemia spp., establishing an asymptomatic productive infection at least under the experimental conditions tested, and that the infected metanauplii are a vector for LCDV transmission to gilthead seabream.

Encysted embryos (cysts) of the crustacean Artemia franciscana exhibit enormous tolerance to adverse conditions encompassing high doses of radiation, years of anoxia, desiccation and extreme salinity. So far, several mechanisms have been proposed to contribute to this extremophilia, however, none were sought in the lipid profile of the cysts. Here in, we used high resolution shotgun lipidomics suited for detailed quantitation and analysis of lipids in uncharacterized biological membranes and samples and assembled the total, mitochondrial and mitoplastic lipidome of Artemia franciscana cysts. Overall, we identified and quantitated 1098 lipid species dispersed among 22 different classes and subclasses. Regarding the mitochondrial lipidome, most lipid classes exhibited little differences from those reported in other animals, however, Artemia mitochondria harboured much less phosphatidylethanolamine, plasmenylethanolamines and ceramides than mitochondria of other species, some of which by two orders of magnitude. Alternatively, Artemia mitochondria exhibited much higher levels of phosphatidylglycerols and phosphatidylserines. The identification and quantitation of the total and mitochondrial lipidome of the cysts may help in the elucidation of actionable extremophilia-affording proteins, such as the 'late embryogenesis abundant' proteins, which are known to interact with lipid membranes.

There is wide interest in understanding how genetic diversity is generated and maintained in parthenogenetic lineages, as it will help clarify the debate of the evolution and maintenance of sexual reproduction. There are three mechanisms that can be responsible for the generation of genetic diversity of parthenogenetic lineages: contagious parthenogenesis, repeated hybridization and microorganism infections (e.g. Wolbachia). Brine shrimps of the genus Artemia (Crustacea, Branchiopoda, Anostraca) are a good model system to investigate evolutionary transitions between reproductive systems as they include sexual species and lineages of obligate parthenogenetic populations of different ploidy level, which often co-occur. Diploid parthenogenetic lineages produce occasional fully functional rare males, interspecific hybridization is known to occur, but the mechanisms of origin of asexual lineages are not completely understood. Here we sequenced and analysed fragments of one mitochondrial and two nuclear genes from an extensive set of populations of diploid parthenogenetic Artemia and sexual species from Central and East Asia to investigate the evolutionary origin of diploid parthenogenetic Artemia, and geographic origin of the parental taxa. Our results indicate that there are at least two, possibly three independent and recent maternal origins of parthenogenetic lineages, related to A. urmiana and Artemia sp. from Kazakhstan, but that the nuclear genes are very closely related in all the sexual species and parthenogegetic lineages except for A. sinica, who presumable took no part on the origin of diploid parthenogenetic strains. Our data cannot rule out either hybridization between any of the very closely related Asiatic sexual species or rare events of contagious parthenogenesis via rare males as the contributing mechanisms to the generation of genetic diversity in diploid parthenogenetic Artemia lineages.

Understanding the evolutionary origin and the phylogeographic patterns of asexual taxa can shed light on the origin and maintenance of sexual reproduction. We assessed the geographic origin, genetic diversity, and phylogeographic history of obligate parthenogen diploid Artemia parthenogenetica populations, a widespread halophilic crustacean.

Analysis of sensorimotor behavioral responses to stimuli such as light can provide an enhanced relevance during rapid prioritisation of chemical risk. Due to technical limitations, there have been, however, only minimal studies on using invertebrate phototactic behaviors in aquatic ecotoxicity testing. In this work, we demonstrate an innovative, purpose-built analytical system for a high-throughput phototactic biotest with nauplii of euryhaline brine shrimp Artemia franciscana. We also, for the first time, present a novel and dedicated bioinformatic approach that facilitates high-throughput analysis of phototactic behaviors at scale with great fidelity. The nauplii exhibited consistent light-seeking behaviors upon extinguishing a brief programmable light stimulus (5500K, 400 lux) without habituation. A proof-of-concept validation involving the short-term exposure of eggs (24 h) and instar I larval stages (6 h) to sub-lethal concentrations of insecticides organophosphate chlorpyrifos (10 µg/L) and neonicotinoid imidacloprid (50 µg/L) showed perturbation in light seeking behaviors in the absence of or minimal alteration in general mobility. Our preliminary data further support the notion that phototactic bioassays can represent an attractive new avenue in behavioral ecotoxicology because of their potential sensitivity, responsiveness, and low cost.

Males and females of Artemia franciscana, a crustacean commonly used in the aquarium trade, are highly dimorphic. Sex is determined by a pair of ZW chromosomes, but the nature and extent of differentiation of these chromosomes is unknown. Here, we characterize the Z chromosome by detecting genomic regions that show lower genomic coverage in female than in male samples, and regions that harbor an excess of female-specific SNPs. We detect many Z-specific genes, which no longer have homologs on the W, but also Z-linked genes that appear to have diverged very recently from their existing W-linked homolog. We assess patterns of male and female expression in two tissues with extensive morphological dimorphism, gonads, and heads. In agreement with their morphology, sex-biased expression is common in both tissues. Interestingly, the Z chromosome is not enriched for sex-biased genes, and seems to in fact have a mechanism of dosage compensation that leads to equal expression in males and in females. Both of these patterns are contrary to most ZW systems studied so far, making A. franciscana an excellent model for investigating the interplay between the evolution of sexual dimorphism and dosage compensation, as well as Z chromosome evolution in general.

The understanding of sex determination and differentiation in animals has recently made remarkable strides through the use of advanced research tools. At the gene level, the Mab-3-related transcription factor (Dmrt) gene family, which encodes for the typical DNA-binding doublesex/Mab-3 (DM) domain in their protein, is known for its contribution to sex determination and differentiation in insects. In this study, DNA-binding DM domain screening has identified eight transcripts from Artemia franciscana transcriptomic that encode proteins containing one conserved DNA-binding DM domain. The genome mapping confirmed that these eight transcripts are transcribed from six different loci on the A. franciscana genome assembly. One of those loci, the Af.dsx-4 locus, is closely related to Doublesex, a gene belonging to the Dmrt gene family. This locus could be transcribed into three alternative transcripts, namely Af.dsx4, Af.dsxF and Af.dsxM. While Af.dsx4 and Af.dsxF could putatively be translated to form an identical Af.dsxF protein of 186 aa long, Af.dsxM translates for an Af.dsxM protein of 289 aa long but shares a DNA-binding DM domain. Interestingly, Af.dsxF and Af.dsxM are confirmed as sex-specific transcripts, Af.dsxF is only present in females, and Af.dsxM is only present in male individuals. The results suggest that the sex-specific splicing mechanism of the doublesex described in insects is also present in A. franciscana. Af.dxs-4 locus can be used in further studies to clarify the sex determination pathways in A. fracnciscana.

This study aims to measure the effect of toxic aqueous solutions of metals on the mobility of Artemia salina nauplii by using digital image processing. The instrument consists of a camera with a macro lens, a dark chamber, a light source and a laptop computer. Four nauplii were inserted into a macro cuvette, which contained copper, cadmium, iron and zinc ions at various concentrations. The nauplii were then filmed inside the dark chamber for two minutes and the video sequence was processed by a motion tracking algorithm that estimated their mobility. The results obtained by this system were compared to the mortality assay of the Artemia salina nauplii. Despite the small number of tested organisms, this system demonstrates great sensitivity in quantifying the mobility of the nauplii, which leads to significantly lower EC50 values than those of the mortality assay. Furthermore, concentrations of parts per trillion of toxic compounds could be detected for some of the metals. The main novelty of this instrument relies in the sub-pixel accuracy of the tracking algorithm that enables robust measurement of the deterioration of the mobility of Artemia salina even at very low concentrations of toxic metals.

Eurasian brine shrimp (genus Artemia) have closely related sexual and asexual lineages of parthenogenetic females, which produce rare males at low frequencies. Although they are known to have ZW chromosomes, these are not well characterized, and it is unclear whether they are shared across the clade. Furthermore, the underlying genetic architecture of the transmission of asexuality, which can occur when rare males mate with closely related sexual females, is not well understood. We produced a chromosome-level assembly for the sexual Eurasian species Artemia sinica and characterized in detail the pair of sex chromosomes of this species. We combined this new assembly with short-read genomic data for the sexual species Artemia sp. Kazakhstan and several asexual lineages of Artemia parthenogenetica, allowing us to perform an in-depth characterization of sex-chromosome evolution across the genus. We identified a small differentiated region of the ZW pair that is shared by all sexual and asexual lineages, supporting the shared ancestry of the sex chromosomes. We also inferred that recombination suppression has spread to larger sections of the chromosome independently in the American and Eurasian lineages. Finally, we took advantage of a rare male, which we backcrossed to sexual females, to explore the genetic basis of asexuality. Our results suggest that parthenogenesis is likely partly controlled by a locus on the Z chromosome, highlighting the interplay between sex determination and asexuality.

The maintenance of sex is paradoxical as sexual species pay the "twofold cost of males" and should thus quickly be replaced by asexual mutants reproducing clonally. However, asexuals may not be strictly clonal and engage in "cryptic sex," challenging this simple scenario. We study the cryptic sex life of the brine shrimp Artemia parthenogenetica, which has once been termed an "ancient asexual" and where no genetic differences have ever been observed between parents and offspring. This asexual species rarely produces males, which can hybridize with sexual females of closely related species and transmit asexuality to their offspring. Using such hybrids, we show that recombination occurs in asexual lineages, causing loss-of-heterozygosity and parent-offspring differences. These differences cannot generally be observed in field-sampled asexuals because once heterozygosity is lost, subsequent recombination leaves no footprint. Furthermore, using extensive paternity tests, we show that hybrid females can reproduce both sexually and asexually, and transmit asexuality to both sexually and asexually produced offspring in a dominant fashion. Finally, we show that, contrary to previous reports, field-sampled asexual females also rarely reproduce sexually (rate ∼2‰). Overall, most previously known facts about Artemia asexuality turned out to be erroneous. More generally, our findings suggest that the evidence for strictly clonal reproduction of asexual species needs to be reconsidered, and that rare sex and consequences of nonclonal asexuality, such as gene flow within asexuals, need to be more widely taken into account in more realistic models for the maintenance of sex and the persistence of asexual lineages.

The aim of this work was to determine the toxic effect of the most used herbicides on marine organisms, the bacterium Aliivibrio fischeri, and the crustacean Artemia salina. The effect of these substances was evaluated using a luminescent bacterial test and an ecotoxicity test. The results showed that half maximal inhibitory concentration for A. fischeri is as follows: 15minIC50 (Roundup® Classic Pro) = 236 μg·L-1, 15minIC50 (Kaput® Premium) = 2475 μg·L-1, 15minIC50 (Banvel® 480 S) = 2637 μg·L-1, 15minIC50 (Lontrel 300) = 7596 μg·L-1, 15minIC50 (Finalsan®) = 64 μg·L-1, 15minIC50 (glyphosate) = 7934 μg·L-1, 15minIC50 (dicamba) = 15,937 μg·L-1, 15minIC50 (clopyralid) = 10,417 μg·L-1, 15minIC50 (nonanoic acid) = 16,040 μg·L-1. Median lethal concentrations for A. salina were determined as follows: LC50 (Roundup® Classic Pro) = 18 μg·L-1, LC50 (Kaput® Premium) = 19 μg·L-1, LC50 (Banvel® 480 S) = 2519 μg·L-1, LC50 (Lontrel 300) = 1796 μg·L-1, LC50 (Finalsan®) = 100 μg·L-1, LC50 (glyphosate) = 811 μg·L-1, LC50 (dicamba) = 3705 μg·L-1, LC50 (clopyralid) = 2800 μg·L-1, LC50 (nonanoic acid) = 7493 μg·L-1. These findings indicate the need to monitor the herbicides used for all environmental compartments.

Recently, there has been a worrying increase in the pollution of the aquatic ecosystem caused by emerging contaminants (ECs) detected in wastewater effluent discharges. Although traces of ECs in waters have been found in low concentrations, it leads to negative effects for nontarget organisms. Antihistamines are a class of drugs largely used, whose metabolites are widespread in the aquatic ecosystem. The aim of the study was to evaluate the short-term effects of promethazine hydrochloride on nauplii of Artemia sp. A high percentage of mortality and morphological alterations were found. The results suggest a possible correlation between exposure to antihistamine and an acceleration of larval development.