Comparative transcriptomics suggest unique molecular adaptations within tardigrade lineages.

BACKGROUND: Tardigrades are renowned for their ability to enter cryptobiosis (latent life) and endure extreme stress, including desiccation and freezing. Increased focus is on revealing molecular mechanisms underlying this tolerance. Here, we provide the first transcriptomes from the heterotardigrade Echiniscoides cf. sigismundi and the eutardigrade Richtersius cf. coronifer, and compare these with data from other tardigrades and six eukaryote models. Investigating 107 genes/gene families, our study provides a thorough analysis of tardigrade gene content with focus on stress tolerance. RESULTS: E. cf. sigismundi, a strong cryptobiont, apparently lacks expression of a number of stress related genes. Most conspicuous is the lack of transcripts from genes involved in classical Non-Homologous End Joining. Our analyses suggest that post-cryptobiotic survival in tardigrades could rely on high fidelity transcription-coupled DNA repair. Tardigrades seem to lack many peroxins, but they all have a comprehensive number of genes encoding proteins involved in antioxidant defense. The "tardigrade unique proteins" (CAHS, SAHS, MAHS, RvLEAM), seem to be missing in the heterotardigrade lineage, revealing that cryptobiosis in general cannot be attributed solely to these proteins. Our investigation further reveals a unique and highly expressed cold shock domain. We hypothesize that the cold shock protein acts as a RNA-chaperone involved in regulation of translation following freezing. CONCLUSIONS: Our results show common gene family contractions and expansions within stress related gene pathways in tardigrades, but also indicate that evolutionary lineages have a high degree of divergence. Different taxa and lineages may exhibit unique physiological adaptations towards stress conditions involving possible unknown functional homologues and/or novel physiological and biochemical mechanisms. To further substantiate the current results genome assemblies coupled with transcriptome data and experimental investigations are needed from tardigrades belonging to different evolutionary lineages.

The germline mutational process in rhesus macaque and its implications for phylogenetic dating.

BACKGROUND: Understanding the rate and pattern of germline mutations is of fundamental importance for understanding evolutionary processes. RESULTS: Here we analyzed 19 parent-offspring trios of rhesus macaques (Macaca mulatta) at high sequencing coverage of ∼76× per individual and estimated a mean rate of 0.77 × 10-8de novo mutations per site per generation (95% CI: 0.69 × 10-8 to 0.85 × 10-8). By phasing 50% of the mutations to parental origins, we found that the mutation rate is positively correlated with the paternal age. The paternal lineage contributed a mean of 81% of the de novo mutations, with a trend of an increasing male contribution for older fathers. Approximately 3.5% of de novo mutations were shared between siblings, with no parental bias, suggesting that they arose from early development (postzygotic) stages. Finally, the divergence times between closely related primates calculated on the basis of the yearly mutation rate of rhesus macaque generally reconcile with divergence estimated with molecular clock methods, except for the Cercopithecoidea/Hominoidea molecular divergence dated at 58 Mya using our new estimate of the yearly mutation rate. CONCLUSIONS: When compared to the traditional molecular clock methods, new estimated rates from pedigree samples can provide insights into the evolution of well-studied groups such as primates.

Comparative Investigation of Copper Tolerance and Identification of Putative Tolerance Related Genes in Tardigrades.

Tardigrades are microscopic aquatic animals renowned for their tolerance toward extreme environmental conditions. The current study is the first to investigate their tolerance toward heavy metals and we present a novel tardigrade toxicant tolerance assay based on activity assessments as a measure of survival. Specifically, we compare tolerance toward copper in four species representing different evolutionary lineages, habitats and adaptation strategies, i.e., a marine heterotardigrade, Echiniscoides sigismundi, a limno-terrestrial heterotardigrade, Echiniscus testudo, a limno-terrestrial eutardigrade, Ramazzottius oberhaeuseri, and a marine eutardigrade, Halobiotus crispae. The latter was sampled at a time of year, when the population is predominantly represented by aberrant P1 cysts, while the other species were in normal active states prior to exposure. Based on volume measurements and a general relation between body mass and copper tolerance, expected tardigrade EC50 values were estimated at 0.5-2 µg l-1. Following 24 h of exposure, tolerance was high with no apparent link to lineage or habitat. EC50s (95% CI), 24 h after exposure, were estimated at 178 (168-186) and 310 (295-328) µg l-1, respectively, for E. sigismundi and R. oberhaeuseri, whereas E. testudo and H. crispae were less affected. Highest tolerance was observed in H. crispae with a mean ± s.e.m. activity of 77 ± 2% (n = 3) 24 h after removal from ~3 mg l-1 copper, suggesting that tardigrade cysts have increased tolerance toward toxicants. In order to identify putative tolerance related genes, an E. sigismundi transcriptome was searched for key enzymes involved in osmoregulation, antioxidant defense and copper metabolism. We found high expression of Na/K ATPase and carbonic anhydrase, known targets for copper. Our transcriptome, furthermore, revealed high expression of antioxidant enzymes, copper transporters, ATOX1, and a Cu-ATPase. In summary, our results indicate that tardigrades express well-known key osmoregulatory enzymes, supporting the hypothesis that copper inhibits sodium turnover as demonstrated for other aquatic organisms. Tardigrades, nevertheless, have high tolerance toward the toxicant, which is likely linked to high expression of antioxidant enzymes and an ability to enter dormant states. Tardigrades, furthermore, seem to have a well-developed battery of cuproproteins involved in copper homeostasis, providing basis for active copper sequestering and excretion.

Shotgun assembly of the assassin bug Brontostoma colossus mitochondrial genome (Heteroptera, Reduviidae).

The complete mitochondrial genome of the assassin bug Brontostoma colossus (Distant, 1902) (Heteroptera: Reduviidae) has been sequenced using a genome-skimming approach on an Illumina Hiseq 2000 platform. Fifty-four additional heteropteran mitogenomes, including five assassin bug species, were retrieved to allow for comparisons and phylogenetic analyses. The mitochondrial genome of B. colossus was determined to be 16,625 bp long, and consists of 13 protein-coding genes (PCGs), 23 transfer-RNA genes (tRNAs), two ribosomal-RNA genes (rRNAs), and one control region. The nucleotide composition is biased toward adenine and thymine (A+T=73.4%). Overall, architecture, nucleotide composition and genome asymmetry are similar among all available assassin bug mitogenomes. All PCGs have usual start-codons (Met and Ile). Three T and two TA incomplete termination codons were identified adjacent to tRNAs, which was consistent with the punctuation model for primary transcripts processing followed by 3' polyadenylation of mature mRNA. All tRNAs exhibit the classic clover-leaf secondary structure except for tRNASer(AGN) in which the DHU arm forms a simple loop. Two notable features are present in the B. colossus mitogenome: (i) a 131 bp duplicated unit including the complete tRNAArg gene, resulting in 23 potentially functional tRNAs in total, and (ii) a 857 bp duplicated region comprising 277 bp of the srRNA gene and 580 bp of the control region. A phylogenetic analysis based on 55 true bug mitogenomes confirmed that B. colossus belongs to Reduviidae, but contradicted a widely accepted hypothesis. This highlights the limits of phylogenetic analyses based on mitochondrial data only.

Seasonal alterations of landfill leachate composition and toxic potency in semi-arid regions.

The present study investigates seasonal variations of leachate composition and its toxic potency on different species, such as the brine shrimp Artemia franciscana (formerly Artemia salina), the fairy shrimp Thamnocephalus platyurus, the estuarine rotifer Brachionus plicatilis and the microalgal flagellate Dunaliella tertiolecta. In specific, leachate regularly collected from the municipal landfill site of Aigeira (Peloponissos, Greece) during the year 2011, showed significant alterations of almost all its physicochemical parameters with time. Further analysis showed that seasonal alterations of leachate composition are related with the amount of rainfall obtained throughout the year. In fact, rainfall-related parameters, such as conductivity (Cond), nitrates (NO(3)(-)), total nitrogen (TN), ammonium (NH(4)-N), total dissolved solids (TDS) and the BOD(5)/NH(4)-N ratio could merely reflect the leachate strength and toxicity, as verified by the significant correlations occurred among each of them with the toxic endpoints, 24 h LC(50) and/or 72 h IC(50), obtained in all species tested. According to the result of the present study, it could be suggested that the aforementioned leachate parameters could be used independently, or in combination as a low-cost effective tools for estimating leachate strength and toxic potency, at least in the case of semi-arid areas such as the most of the Mediterranean countries.