Comparing cestode infections and their consequences for host fitness in two sexual branchiopods: alien Artemia franciscana and native A. salina from syntopic-populations.

The American brine shrimp Artemia franciscana is invasive in the Mediterranean region where it has displaced native species (the sexual A. salina, and the clonal A. parthenogenetica) from many salt pond complexes. Artemia populations are parasitized by numerous avian cestodes whose effects have been studied in native species. We present a study from the Ebro Delta salterns (NE Spain), in a salt pond where both A. franciscana and native A. salina populations coexist, providing a unique opportunity to compare the parasite loads of the two sexual species in syntopy. The native species had consistently higher infection parameters, largely because the dominant cestode in A. salina adults and juveniles (Flamingolepis liguloides) was much rarer in A. franciscana. The most abundant cestodes in the alien species were Eurycestus avoceti (in adults) and Flamingolepis flamingo (in juveniles). The abundance of E. avoceti and F. liguloides was higher in the A. franciscana population syntopic with A. salina than in a population sampled at the same time in another pond where the native brine shrimp was absent, possibly because the native shrimp provides a better reservoir for parasite circulation. Infection by cestodes caused red colouration in adult and juvenile A. salina, and also led to castration in a high proportion of adult females. Both these effects were significantly stronger in the native host than in A. franciscana with the same parasite loads. However, for the first time, significant castration effects (for E. avoceti and F. liguloides) and colour change (for six cestode species) were observed in infected A. franciscana. Avian cestodes are likely to help A. franciscana outcompete native species. At the same time, they are likely to reduce the production of A. franciscana cysts in areas where they are harvested commercially.

Helminth parasites of Artemia franciscana (Crustacea: Branchiopoda) in the Great Salt Lake, Utah: first data from the native range of this invader of European wetlands.

The present study is the first survey on the role of Artemia franciscana Kellogg as intermediate host of helminth parasites in its native geographical range in North America (previous studies have recorded nine cestode and one nematode species from this host in its invasive habitats in the Western Mediterranean). Samples of Artemia franciscana were collected from four sites in the Great Salt Lake (GSL), Utah, across several months (June-September 2009). A. franciscana serves as intermediate host of five helminth species in this lake. Four of them are cestodes: three hymenolepidids, i.e. Confluaria podicipina (Szymanski, 1905) (adults parasitic in grebes), Hymenolepis (sensu lato) californicus Young, 1950 (adults parasitic in gulls), Wardium sp. (definitive host unknown, probably charadriiform birds), and one dilepidid, Fuhrmannolepis averini Spassky et Yurpalova, 1967 (adults parasitic in phalaropes). In addition, an unidentified nematode of the family Acuariidae was recorded. Confluaria podicipina is the most prevalent and abundant parasite at all sampling sites, followed by H. (s. l.) californicus. The species composition of the parasites and the spatial variations in their prevalence and abundance reflect the abundance and distribution of aquatic birds serving as their definitive hosts. The temporal dynamics of the overall helminth infections exhibits the highest prevalence in the last month of study at each site (August or September). This native population of A. franciscana from GSL is characterised with higher prevalence, intensity and abundance of the overall cestode infection compared to the introduced populations of this species in the Palaearctic Region. The values of the infection descriptors in the native population of A. franciscana are slightly lower or in some cases similar to those of the Palaearctic species Artemia parthenogenetica Barigozzi (diploid populations) and Artemia salina (Linnaeus) in their native habitats.

Laboratory generation of new parthenogenetic lineages supports contagious parthenogenesis in Artemia.

Contagious parthenogenesis-a process involving rare functional males produced by a parthenogenetic lineage which mate with coexisting sexual females resulting in fertile parthenogenetic offspring-is one of the most striking mechanisms responsible for the generation of new parthenogenetic lineages. Populations of the parthenogenetic diploid brine shrimp Artemia produce fully functional males in low proportions. The evolutionary role of these so-called Artemia rare males is, however, unknown. Here we investigate whether new parthenogenetic clones could be obtained in the laboratory by mating these rare males with sexual females. We assessed the survival and sex ratio of the hybrid ovoviviparous offspring from previous crosses between rare males and females from all Asiatic sexual species, carried out cross-mating experiments between F1 hybrid individuals to assess their fertility, and estimated the viability and the reproductive mode of the resulting F2 offspring. Molecular analysis confirmed the parentage of hybrid parthenogenetic F2. Our study documents the first laboratory synthesis of new parthenogenetic lineages in Artemia and supports a model for the contagious spread of parthenogenesis. Our results suggest recessive inheritance but further experiments are required to confirm the likelihood of the contagious parthenogenesis model.

Origin and genetic diversity of diploid parthenogenetic Artemia in Eurasia.

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.

Bird migratory flyways influence the phylogeography of the invasive brine shrimp Artemia franciscana in its native American range.

Since Darwin's time, waterbirds have been considered an important vector for the dispersal of continental aquatic invertebrates. Bird movements have facilitated the worldwide invasion of the American brine shrimp Artemia franciscana, transporting cysts (diapausing eggs), and favouring rapid range expansions from introduction sites. Here we address the impact of bird migratory flyways on the population genetic structure and phylogeography of A. franciscana in its native range in the Americas. We examined sequence variation for two mitochondrial gene fragments (COI and 16S for a subset of the data) in a large set of population samples representing the entire native range of A. franciscana. Furthermore, we performed Mantel tests and redundancy analyses (RDA) to test the role of flyways, geography and human introductions on the phylogeography and population genetic structure at a continental scale. A. franciscana mitochondrial DNA was very diverse, with two main clades, largely corresponding to Pacific and Atlantic populations, mirroring American bird flyways. There was a high degree of regional endemism, with populations subdivided into at least 12 divergent, geographically restricted and largely allopatric mitochondrial lineages, and high levels of population structure (Φ ST of 0.92), indicating low ongoing gene flow. We found evidence of human-mediated introductions in nine out of 39 populations analysed. Once these populations were removed, Mantel tests revealed a strong association between genetic variation and geographic distance (i.e., isolation-by-distance pattern). RDA showed that shared bird flyways explained around 20% of the variance in genetic distance between populations and this was highly significant, once geographic distance was controlled for. The variance explained increased to 30% when the factor human introduction was included in the model. Our findings suggest that bird-mediated transport of brine shrimp propagules does not result in substantial ongoing gene flow; instead, it had a significant historical role on the current species phylogeography, facilitating the colonisation of new aquatic environments as they become available along their main migratory flyways.

A revision of Artemia biodiversity in Macaronesia.

In a biogeographical context, the term Macaronesia broadly embraces the North Atlantic archipelagos of the Azores, Madeira, Selvagens, the Canary Islands, and Cape Verde. The peculiar arid climatic conditions in some of these places have led to the development of marine salt exploitations, which can be counted among the hypersaline habitats of the brine shrimp Artemia (Branchiopoda, Anostraca). Parthenogenetic populations of this anostracan were described in the Canary Islands during the last decades of the 20th century, while the American Artemia franciscana species was recently found in the Cape Verde archipelago. Following an invasive pattern, this exotic species has recently reached the Canary Islands, too. This paper reports information dealing with biotope loss (solar saltworks) in this biogeographical region, together with possible consequences concerning the arrival of invasive species, two factors that frequently promote dramatic biodiversity losses. The discussion of this threat focuses mainly on the Canary Islands archipelago where native species of Artemia still exist.

Characterization of the organic contamination pattern of a hyper-saline ecosystem by rapid screening using gas chromatography coupled to high-resolution time-of-flight mass spectrometry.

In this paper, gas chromatography coupled to high-resolution time-of-flight mass spectrometry (GC-TOF MS) has been applied to evaluate organic pollution in a hyper-saline aquatic environment. Firstly, a target screening was made for a list of 150 GC-amenable organic micro-contaminants, including PAHs, octyl/nonyl phenols, PCBs, PBDEs, and a notable number of pesticides, such us insecticides (organochlorines, organophosphorus, carbamates and pyrethroids), herbicides (triazines and chloroacetanilides), fungicides and several transformation products. This methodology was applied to brine samples, with a salt content from 112 g/L to saturation, and to samples from Artemia populations (crustacean Anostraca) collected during 1 year from three sampling stations in saltworks bodies sited in the Ebro river delta. Around 50 target contaminants, belong to chemical families included in the list of priority substances within the framework on European water policy. Additionally, a non-target analysis was performed in both types of samples with the objective of investigating the presence of other non-selected organic compounds taking advantage of the potential of GC-TOF MS (high sensitivity in full-spectrum acquisition mode, accurate mass measurements) for searching unknowns. Organophosphorus pesticides were the contaminants more frequently detected in brine samples. Other compounds usually present in urban and industrial wastewaters, like caffeine, methylparaben, butylated-hydroxytoluene and N-butylbenzenesulfonamide were also detected in brines. The herbicide simazine and the insecticide chlorpyrifos were among the contaminants detected in Artemia samples. Results of this work reveal a potential threat to vulnerable populations inhabiting the hyper-saline ecosystem. The valuable contribution of GC-TOF MS in environmental analysis, allowing the rapid screening of a large number of organic contaminants, is also demonstrated in this paper.

Artisanal salt production in Aveiro/Portugal - an ecofriendly process.

Solar salinas are man-made systems exploited for the extraction of salt, by solar and wind evaporation of seawater. Salt production achieved by traditional methods is associated with landscapes and environmental and patrimonial values generated throughout history. Since the mid-twentieth century, this activity has been facing a marked decline in Portugal, with most salinas either abandoned or subjected to destruction, making it necessary to find a strategy to reverse this trend.It is, however, possible to generate revenue from salinas at several levels, not merely in terms of good quality salt production, but also by obtaining other products that can be commercialized, or by exploring their potential for tourism, and as research facilities, among others. Furthermore, with an adequate management, biodiversity can be restored to abandoned salinas, which constitute important feeding and breeding grounds for resident and migratory aquatic birds, many of which are protected by European Community Directives.The aims of this manuscript are to present a brief overview on the current state of sea salt exploitation in Portugal and to stress the importance of recovering these salinas for the conservation of this particular environment, for the regional economy, the scientific community and the general public. The Aveiro salina complex is presented in detail, to exemplify salina structure and functioning, as well as current problems and potential solutions for artisanal salinas.

Non-target screening of organic contaminants in marine salts by gas chromatography coupled to high-resolution time-of-flight mass spectrometry.

Gas chromatography coupled to time-of-flight mass spectrometry (GC-TOF MS) has been applied to characterize the organic pollution pattern of marine salt samples collected in saltworks from the Spanish Mediterranean coast. After dissolving the samples in water, a solid-phase extraction was applied reaching with a 250-preconcentration factor. The screening methodology allowed the detection of sample components without any kind of pre-selection of target pollutants. The identity of components detected was established by accurate mass measurements and comparison of experimental full-acquisition spectra with theoretical MS libraries. Several organic pollutants were identified in the samples, like plasticizers - potentially toxic to humans - and fragrances -included within the group of pharmaceuticals and personal care products-, among others. Our results indicate that these contaminants can be found in the marine salt after the crystallization process. GC-TOF MS is a powerful technique for wide-scope screening of (semi)volatile, low-polar organic contaminants, able to investigate the presence of a large number of compounds. Searching of contaminants is not restricted to a target list of compounds. Therefore, unexpected contaminants can be discovered in an efficient way, with better sensitivity and selectivity than other conventional analytical techniques, and making use of the powerful qualitative information provided by full-spectrum acquisition at accurate mass.

Participation of metanauplii and juvenile individuals of Artemia parthenogenetica (Branchiopoda) in the circulation of avian cestodes.

Adult crustaceans of the genus Artemia (brine shrimps) are intermediate hosts in the life cycle of cestode species parasitic in aquatic birds as their definitive hosts. However, there are no data on the role of larval and juvenile brine shrimps in the transmission of avian helminth parasites. In order to examine the possible role of early developmental stages (nauplii, metanauplii and juveniles) of Artemia for the circulation of avian cestodes, the natural cestode infection in the population of Artemia parthenogenetica from La Mata Lagoon, Mediterranean coast of Spain, was studied. Metacestodes (cysticercoids) of four cestode species were recorded in adult brine shrimps: Flamingolepis liguloides and Flamingolepis flamingo (hymenolepidids parasitic in flamingos), Confluaria podicipina (a hymenolepidid species parasitic in grebes) and Eurycestus avoceti (a dilepidid species parasitic in avocets, stilts, plovers and, to a lesser extent, in flamingos). No cysticercoids were found in nauplii. Two species, F. liguloides and F. flamingo, were found in metanauplii and juvenile brine shrimps. Only 36.3% of the cysticercoids of F. liguloides occurred in adult brine shrimps; the remaining 63.7% were parasitic in metanauplii (39.6%) and juveniles (24.1%). Similarly, the metacestodes of F. flamingo were also distributed among various age groups: in adults (44.4% of cysticercoids), juveniles (27.8%) and metanauplii (27.8%). These results indicate that the early developmental stages of Artemia have an important role for the circulation of certain parasite species. No cysticercoids of C. podicipina and E. avoceti were recorded in larval and juvenile brine shrimps. The selective infestation of larval brine shrimps with flamingo parasites is probably associated with the feeding behaviour of definitive hosts, which are filtering predators; in contrast, grebes and waders pick brine shrimps individually one by one. The possible underlying mechanism for selective infestation of metanauplii and adults by certain cestode species is associated with the size of parasite eggs, allowing only cestode species with small eggs to be ingested by larval brine shrimps.

Evolutionary origin and phylogeography of the diploid obligate parthenogen Artemia parthenogenetica (Branchiopoda: Anostraca).

BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: We analysed a partial sequence of the Cytochrome c Oxidase Subunit I mitochondrial gene from an extensive set of localities (including Eurasia, Africa, and Australia), and examined their phylogeographic patterns and the phylogenetic relationships of diploid A. parthenogenetica and its closest sexual relatives. Populations displayed an extremely low level of mitochondrial genetic diversity, with one widespread haplotype shared by over 79% of individuals analysed. Phylogenetic and phylogeographic analyses indicated a multiple and recent evolutionary origin of diploid A. parthenogenetica, and strongly suggested that the geographic origin of parthenogenesis in Artemia was in Central Asia. Our results indicate that the maternal sexual ancestors of diploid A. parthenogenetica were an undescribed species from Kazakhstan and A. urmiana. CONCLUSIONS/SIGNIFICANCE: We found evidence for multiple origin of parthenogenesis in Central Asia. Our results indicated that, shortly after its origin, diploid A. parthenogenetica populations underwent a rapid range expansion from Central Asia towards the Mediterranean region, and probably to the rest of its current geographic distribution. This contrasts with the restricted geographic distribution, strong genetic structure, and regional endemism of sexual Artemia lineages and other passively dispersed sexual continental aquatic invertebrates. We hypothesize that diploid parthenogens might have reached their current distribution in historical times, with a range expansion possibly facilitated by an increased availability of suitable habitat provided by anthropogenic activities, such as the spread of solar saltworks, aided by their natural dispersal vectors (i.e., waterbirds).

Phylogeography and local endemism of the native Mediterranean brine shrimp Artemia salina (Branchiopoda: Anostraca).

There has been a recent appreciation of the ecological impacts of zooplanktonic species invasions. The North American brine shrimp Artemia franciscana is one such alien invader in hyper-saline water ecosystems at a global scale. It has been shown to outcompete native Artemia species, leading to their local extinction. We used partial sequences of the mitochondrial Cytochrome c Oxidase Subunit 1 (COI or cox1) gene to investigate the genetic diversity and phylogeography of A. salina, an extreme halophilic sexual brine shrimp, over its known distribution range (Mediterranean Basin and South Africa) and to assess the extent of local endemism, the degree of population structure and the potential impact of traditional human saltpan management on this species. We also examined the phylogenetic relationships in the genus Artemia using COI sequences. Our results show extensive regional endemism and indicate an early Pleistocene expansion of A. salina in the Mediterranean Basin. Subsequent population isolation in a mosaic of Pleistocene refugia is suggested, with two or three refugia located in the Iberian Peninsula. Two instances of long-distance colonization were also observed. Surprisingly, given its strong phylogeographical structure, A. salina showed a signature of correlation between geographical and genetic distance. Owing to strong 'priority effects', extensive population differentiation is retained, despite dispersal via migrant birds and human management of saltpans. The foreseeable expansion of A. franciscana is likely to be followed by substantial loss of genetic diversity in Mediterranean A. salina. Large genetic divergences between Mediterranean and South African A. salina suggest that the latter deserves species status.

Regulation and function of lysine-substituted Na,K pumps in salt adaptation of Artemia franciscana.

The brine shrimp Artemia thrives at extreme conditions of up to 300 g/l salt in hypersaline lakes, but the molecular aspects of this salt adaptation are not clarified. To examine the influence of salt on the expression of two isoforms of Na,K-ATPase, adult Artemia franciscana were cultured for 39 days with the microalga Dunaliella salina as fodder at increasing salt from 30 to 280 g/l. Quantitative reverse-transcriptase polymerase chain reaction showed that the abundance of mRNA of the lysine-substituted alpha(2)(KK)-subunit was very low at 30 g/l salt but rose steeply in the range of 70-200 g/l to a level at 200-280 g/l salt, similar to the abundance of the mRNA of the alpha(1)(NN)-subunit, which was insignificantly affected by increasing salt. Site-directed mutagenesis showed that Asn324Lys and Asn776Lys in the alpha(1)-subunit of pig kidney Na,K-ATPase reduced the stoichiometry of (204)Tl binding from 2 to about 1 Tl(+)(K(+)) per alpha-subunit and Na(+)-dependent phosphorylation from ATP to 25-30%. In structure models, the epsilon-amino group of Lys776 is located at cation site 1 in the E(1)P form and near cation site 2 in the E(2) conformation, while the side chain of Lys324 points away from the cation sites. Salt-induced expression of the alpha(2)(KK)-subunit Na,K-ATPase in A. franciscana may reduce the Na(+)/ATP ratio and enable the Na,K pump to extrude Na(+) against steeper gradients and, thus, contribute to salt adaptation.