The genome of the extremophile Artemia provides insight into strategies to cope with extreme environments.

BACKGROUND: Brine shrimp Artemia have an unequalled ability to endure extreme salinity and complete anoxia. This study aims to elucidate its strategies to cope with these stressors. RESULTS AND DISCUSSION: Here, we present the genome of an inbred A. franciscana Kellogg, 1906. We identified 21,828 genes of which, under high salinity, 674 genes and under anoxia, 900 genes were differentially expressed (42%, respectively 30% were annotated). Under high salinity, relevant stress genes and pathways included several Heat Shock Protein and Leaf Embryogenesis Abundant genes, as well as the trehalose metabolism. In addition, based on differential gene expression analysis, it can be hypothesized that a high oxidative stress response and endocytosis/exocytosis are potential salt management strategies, in addition to the expression of major facilitator superfamily genes responsible for transmembrane ion transport. Under anoxia, genes involved in mitochondrial function, mTOR signalling and autophagy were differentially expressed. Both high salt and anoxia enhanced degradation of erroneous proteins and protein chaperoning. Compared with other branchiopod genomes, Artemia had 0.03% contracted and 6% expanded orthogroups, in which 14% of the genes were differentially expressed under high salinity or anoxia. One phospholipase D gene family, shown to be important in plant stress response, was uniquely present in both extremophiles Artemia and the tardigrade Hypsibius dujardini, yet not differentially expressed under the described experimental conditions. CONCLUSIONS: A relatively complete genome of Artemia was assembled, annotated and analysed, facilitating research on its extremophile features, and providing a reference sequence for crustacean research.

Unique chimeric composition of the trehalase gene from brine shrimp, Artemia franciscana.

To investigate the exon/intron structure of the Artemia trehalase gene, four overlapping clones were isolated from a genome library derived from an inbred strain of crustacean Artemia franciscana, and a 49 kb genetic area was re-constructed. The re-constructed area contained eight exons corresponding to the trehalase cDNA sequence that we had previously reported [1]. Comparative analysis of the Artemia trehalase gene with other animal trehalase genes revealed the existence of conserved exon/intron boundaries among different phyla. Comparison of the 5' UTR region of trehalase mRNA obtained by the 5' RACE method with the trehalase genes indicated the existence of a novel exon/intron boundary in the region designated "Exon I". Surprisingly, a part of a mitochondrial ribosomal protein gene (MRP-S33) was found to be inserted in the 5' UTR region of the trehalase gene. This sequence had the same polyadenylation signal that the Artemia MRP-S33 cDNAs did. Using the 3' RACE method, it was demonstrated that the poly (A) additional signal is still functional and that the chimeric mRNAs composed of the 5' UTR of the trehalase mRNA and of the 3' end derived from the MRP-S33 gene are transcribed.

Inbred strains of brine shrimp derived from Artemia franciscana: lineage, RAPD analysis, life span, reproductive traits and mode, adaptation, and tolerance to salinity changes.

Inbred strains of the brine shrimp were developed from dry dormant cysts of wild-type Artemia franciscana produced in the Great Salt Lake, U.S.A. The established strains were named GSL2, 4, and 7. They were raised in 2% natural sea salt solution at 28 degrees C under a long-day condition, and fed on food sold for Artemia. Ovoviviparous offspring (free-swimming nauplii) in each brood derived from full sib (sister x brother) matings were used for succeeding generations. The ordinal number of the filial generation increased at a rate of ten generations per year. The number was over 60, and the lineage was recorded. Random amplified polymorphic DNA (RAPD) analyses of the inbred strains revealed the uniqueness, homogeneity, and genetic similarity among them. Their life span, the time required to become sexually mature, brood size, mode of reproduction, and adaptation and tolerance to salinity changes were investigated. The inbred strains usually released free-swimming nauplii rather than spawning encysted gastrulae (dormant cysts). On the other hand, the opposite results were obtained from wild-type Artemia under the same conditions. Both adults and nauplii of the inbred strains appeared to be less adaptive and less tolerant to salinity changes compared to those of the wild type. The established inbred strains should provide a wider and deeper scope for Artemia biology in particular, and the life sciences in general.

Influence of darkness on embryonic diapause termination in dormant Artemia cysts with no experience of desiccation.

One of the environmental cues for Artemia embryonic diapause termination (EDT), the nature of which is barely understood, is the absence of light, although light-dark cycles (photoperiod) have been examined in inducing embryonic diapause. The influence of constant darkness (DD) on EDT was investigated in laboratory-produced cysts of Artemia franciscana. In a previous report (Nambu et al., 2008. J Exp Zool 309A:17-24 ), we described a facilitated effect of pretreatment of DD (5-14 days) on EDT in Artemia cysts exposed to a standard rearing condition. In this article, the influence of pretreatment of DD (1-150 days) was investigated in detail by bioassay of hatching. It was found that all the DD conditions significantly promoted EDT. Either the plateau value of the percent hatch increased in response to each DD condition, or the rearing period to attain the half value of the plateau value became shorter. It was also found that DD (1-100 days) led to cyclic changes of EDT: the logistic functions of hatching were cyclically regulated and 30 days-DD cycles of relative values of their parameters were observed synchronously. The effect of DD-duration was not cumulative, and a couple of optimum conditions for higher EDT levels were obtained. The probability of Artemia's encountering DD and the possible meaning of cyclic regulation of EDT by DD in Artemia are discussed.

Influence of temperature and darkness on embryonic diapause termination in dormant Artemia cysts that have never been desiccated.

Environmental cues for embryonic diapause termination (EDT) were investigated in the laboratory-produced encysted dormant embryos of the brine shrimp, Artemia franciscana. The cysts were spawned and kept throughout in a 2% sea salt solution. They were activated by various temperatures of the temperate zones or by continuous dark condition (DD), resulting in a state of EDT, and were thereafter able to resume their subsequent development and hatch under appropriate conditions. The level of EDT was conveniently assayed by a hatch test observed within 2 days at 28 degrees C under continuous light condition (LL). A cold treatment of the newly spawned dormant cysts, at 4 degrees C under DD for more than 100 days, resulted in more than 95% hatch of the dormant cysts. Similar treatments of the dormant cysts but at room temperature or 28 degrees C led to significantly different results (30-40% hatch). Almost all the residual non-hatched cysts derived from the above could hatch after an additional cold treatment (at 4 degrees C under DD for about 100 days). This might prove to be latent partial bivoltine in Artemia. Meanwhile, a rearing condition (28 degrees C under LL) induced the newly spawned cysts to hatch scatteredly at and after 1 month, resulting in 22% cumulative hatch on the 92nd day after spawning. When the newly spawned dormant cysts were pretreated at 28 degrees C under DD for 5 or 14 days and then reared at 28 degrees C under LL, the cumulative hatch significantly increased (60%). These results are discussed with respect to probable diapause regulator(s) involved in EDT.

Influence of photoperiod and temperature on reproductive mode in the Brine shrimp, Artemia franciscana.

Brine shrimp, Artemia, exhibit two modes of reproduction: oviparity (diapause cyst production) and ovoviviparity (live larvae release). Environmental conditions determining these developmental routes are poorly understood, so we investigated the effects of photoperiod and temperature on reproductive mode. Nauplii of A. franciscana were hatched from cysts produced in the Great Salt Lake, Utah, and raised in 2% natural sea salt water under photoperiods of 24, 14, 12, or 10 h at 28 degrees or 20 degrees C. Mating pairs of mature shrimp were isolated and reared continuously under those conditions. The mode of reproduction shown by each pair was determined daily throughout their life span, and found to be greatly affected by photoperiod, and less influenced by temperature. The relative degree of oviparity increased as the photoperiod became shorter at both temperatures. In contrast, the degree of ovoviviparity was higher as the photoperiod became longer at both temperatures. The critical photoperiod appears to be between 12 and 14 h. For all photoperiods examined, the degree of oviparity was higher at 28 degrees C than at 20 degrees C, whereas the degree of ovoviviparity was greater at 20 degrees C than at 28 degrees C.