Philippa Kaur

Sampling and incubationFour rock samples were collected from the 3.7 km-deep Auka vent field in the Southern Pescadero Basin (23.956094N, 108.86192W)20,23. Sample NA091.008 was collected in 2017 on cruise NA091 with the Eexploration vessle Nautilus and incubated as described previously34. Samples 12,019 (S0200-R1), 11,719 (S0193-R2) and 11,868 (S0197-PC1), the latter representing a lithified nodule recovered from a sediment push core, were collected with Remotely operated vehicle SuBastian and Research vessel Falkor on cruise FK181031 in November 2018. These samples were processed shipboard and stored under anoxic conditions at 4 °C for subsequent incubation in the laboratory. In the laboratory, rock samples 12,019 and 11,719 were broken into smaller pieces under sterile conditions, immersed in N2-sparged sterilized artificial sea water and incubated under anoxic conditions with methane, as described previously for NA091.008 (ref. 34). Additional sampling information can be found in Supplementary Table 1. Mineralogical analysis by X-ray Powder Diffraction (XRD) identified barite in several of these samples, collected from two locations in the Auka vent field, including on the western side of the Matterhorn vent (11,719, NA091.008), and one oil-saturated sample (12,019) recovered from the sedimented flanks from the southern side of Z vent. Our analysis also includes metagenomic data from two sediment cores from the Auka vent field (DR750-PC67 and DR750-PC80) collected in April 2015 with the ROV Doc Ricketts and R/V Western Flyer (MBARI2015), previously published (ref. 23).Fluorescence in situ hybridizationSamples were fixed shipboard using freshly prepared paraformaldehyde (2 vol% in 3× Phosphate Buffer Solution (PBS), EMS15713) at 4 °C overnight, rinsed twice using 3× PBS, and stored in ethanol (50% in 1× PBS) at −20 °C until processing. Small pieces ( More

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Identification of Elovl genes from E. tetradactylum and E. rhadinumTotally, we successfully identified 9 Elovl genes, including elovl1a, elovl1b, elovl4a, elovl4b, elovl5, elovl6, elovl6l, elovl7a, and elovl8b, both from E. tetradactylum and E. rhadinum genome (Table 2). In E. rhadinum, the shortest and the longest putative CDS length among all Elovl genes was 810 bp and 2019 bp, respectively. Their encoded protein size ranged from 269 amino acids to 672 amino acids. The theoretical molecular weight of Elovl proteins varied from 31061.48 to 75051.42 Da, with the theoretical isoelectric points (pI) ranging from 7.86 to 9.59. Most of the Elovl proteins were characterized as stable and hydrophilic proteins. Signal peptide prediction analysis showed that the elovl1b, elovl5, and elovl6 contained signal peptide sequences. In addition to elovl8b, all Elovl proteins contained transmembrane domains ranging from 5 to 7. Almost all Elovl proteins were predicted to be endoplasmic reticulum-located except elovl8b, predominantly localized in the nucleus.Table 2 Basic information for the Elovl gene family members.Full size tableIn E. tetradactylum, the putative CDS length of Elovl genes ranged from 810 to 1824 bp, and their encoded protein size ranged from 269 amino acids to 409 amino acids. The molecular weight of Elovl proteins varied from 31049.42 to 68750.14 Da, with the pI ranging from 8.72 to 9.64. Like Elovl proteins in E. rhadinum, most elovl proteins were predicted to be stable and hydrophilic. Signal peptide prediction analysis revealed that elovl1a, elovl5, and elovl6 had signal peptide sequence, which was different from E. rhadinum that elovl1b contained signal peptide sequence, but elovl1a did not. In addition, seven members showed the same number of transmembrane structures with E. rhadinum, while the elovl8b contained three and elovl4b contained seven transmembrane structures in contrast to E. rhadinum. The elovl8b was predicted to be localized in nuclear, while other members were localized in the endoplasmic reticulum, similar to E. rhadinum.Evolution of divergence and conservation of Elovl genesDivergence and conservation accompany the process of species evolution. To elucidate the phylogenetic relationship of Elovl genes among different species, a maximum like-hood tree was constructed on the basis of 18 Elovl genes in E. tetradactylum and E. rhadinum and 106 publicly available Elovl protein sequences. As shown in Fig. 1, these Elovl genes can be divided into eight subfamilies, including elovl1a/1b, elovl2, elovl3, elovl4a, elovl5, elovl6/6 l, elovl7a/7b, elovl8a/8b. However, 6 subfamilies were presented in the Eleutheronema genus, and there was only one subtype for elovl7 (elovl7a) and elovl8 (elovl8b) in E. tetradactylum and E. rhadinum. The elovl3 was mainly identified in mammalians such as Homo sapiens and Mus musculus, while a recent study reported a full repertoire of Elovl genes in the Colossoma macropomum genome, including elovl330. The loss of elovl2 occurred in the vast majority of marine fish lineages, which was only presented in a few fish species, such as C. carpio, D. rerio, S. salar, and S. grahami.Figure 1Phylogenetic tree for 18 Elovl proteins from E. tetradactylum and E. rhadinum, and 106 publicly available Elovl proteins from other species. All these proteins were aligned using ClustalW and then subjected to MEGAX for phylogenetic tree construction using the maximum like-hood method with 1000 replicates.Full size imageWe further performed the gene structure analysis to visualize the exon–intron structure of each gene, and the results revealed that the elovl8b had the largest intron number, while the elovl6/6 l subfamily genes contained three introns (Fig. 2a). Except for elovl8, Elovl genes belonging to the same subfamily shared a similar gene structure. Additionally, we identified ten motifs in Elovl genes, and the conversed motif types, numbers, and distributions in Elovl proteins were much more similar except for the elovl8b (Fig. 2b, TableS1). Two conserved motifs were found in the Elovl gene family except for elovl8b in E. rhadinum, which were related to the ELO domain via SMART evaluation analysis (Fig. 2c and d). Gene structural variation is important for gene evolution. In E. tetradactylum and E. rhadinum, Elovl genes showed similar gene structure, and the proteins shared similar motif compositions, indicating that the Elovl genes were highly conserved in the Eleutheronema genus.Figure 2Gene structure and conserved motifs diagram of Elovl genes. (a) Gene structure of Elovl genes. Exons were represented by pink boxes and introns by black lines; (b) Conserved motifs of Elovl proteins; (c and d) Logo representations of the ELO domains, motifs 1 and 2, respectively.Full size imageIn the process of evolution via natural selection, adaptation to certain environmental conditions likely drove the changes in endogenous capacity for LC-PUFA biosynthesis between marine and freshwater fishes31. The Elovl gene family has been functionally studied and characterized in a variety of fish species, and the member of the Elovl gene family of each species varied greatly. In the present study, for a comprehensive analysis of Elovl genes in the Eleutheronema genus, the Elovl gene ortholog clusters of mammals and various teleosts with different ecological niches and habitats were collected. The results showed that only seven Elovl genes (one gene for each subtype) were observed in mammals; however, more members were variably presented in teleosts, which might be related to the teleost-specific duplication. A previous study revealed that Sinocyclocheilus graham and C. carpio possessed the highest number of Elovl genes, containing 21 members of subtypes, resulting from an extra independent 4th whole-genome duplication event32, 33. Interestingly, only 9 Elovl genes were observed in Eleutheronema genus, the same as T. rubripes, possibly due to gene loss and the asymmetric acceleration of the evolutionary rate in one of the paralogs following the whole-genome duplication in some teleost fishes34. Additionally, the elovl2 and elovl3 were absent, but a novel subtype, elovl8, was present in most marine fishes. The elovl8, the most recently identified and novel active member of the Elovl protein family member, has been proposed to be a fish-specific elongase with two gene paralogs (elovl8a and elovl8b) described in teleost35. In Eleutheronema, we also found that the elovl8b was presented in E. tetradactylum and E. rhadinum, indicating the important roles in the LC-PUFAs biosynthesis of Eleutheronema fish. Similar results were also observed in rabbitfish and zebrafish20. The Elovl gene family member number in Eleutheronema genus is the same as T. rubripes, but less than I. punctatus (10), Gadus morhua (10), D. rerio (14), S. salar (18), and C. carpio (21), which might be due to the differential expansion events during the evolutions of fish species.Predicting the protein structure is a fundamental prerequisite for understanding the function and possible interactions of a protein. In the present study, the secondary structures as well as three-dimensional structures of Elovl proteins in both E. tetradactylum and E. rhadinum were predicted using the SOPMA and Phyre2 programs, respectively. The protein structures of all the candidate Elovl proteins were modeled at  > 90% confidence. The secondary structures of these proteins in E. tetradactylum revealed 40.86–50.30% alpha helixes, 28.10–28.10% random coil, 13.75–20.67% extended strand and 2.38–4.47% beta turn, while these ratios were predicted to be 47.55–53.27, 30.00–36.01, 6.99–18.12 and 2.38–4.75%, respectively, in E. rhadinum (Table 3). High ratio of alpha helixes and random coil in the Elovl protein structure might play important roles in fatty acids biosynthesis in fish, in accordance with the literature for the order Perciformes in Perca fluviatilis36. Additionally, the secondary structure pattern of Elovl proteins in the candidate E. tetradactylum and E. rhadinum species were highly similar (Fig. 3), indicating the probable similar biological functions as well as highly evolutionarily conserved Elovl genes in Eleutheronema species.Table 3 Properties of the secondary structures of Elovl proteins.Full size tableFigure 3The secondary structure pattern, including alpha helix (blue color), random coil (purple color), extended strand (red color), and beta turn (green color), of Elovl proteins in E. tetradactylum and E. rhadinum.Full size imageThe 3D model results showed that all predicted Elovl proteins had complex 3D structures, composing of multiple secondary structures including alpha-helices, random coils, and others (Fig. 4). The Elovl proteins of different subfamilies showed different 3D configurations. The 3D structures of Elovl proteins also revealed the presence of the conserved domain in each Elovl protein, which showed a typical three-dimensional frame comprising of various parallel alpha-helixes. To assay the quality and accuracy of the predicted 3D model for the candidate Elovl proteins, the Ramachandran plot analysis was employed (Figure S1). In model validation, the qualities of the Elovl proteins model varied from 90 to 98% based on the Ramachandran plot analysis, suggesting the reasonably good quality and reliability of the predicted 3D models. These results indicated that the predicted 3D model of Elovl proteins could provide valuable information for the further comprehensive studies of molecular function in the fatty acids biosynthesis in Eleutheronema species. Additionally, the comparisons between these structures in E. tetradactylum and E. rhadinum suggested that the Elovl proteins encompassed the conserved structures. In addition, gene duplication resulted in obvious 3D structural variation in the duplicated genes, such as Elovl4 (elovl4a and elovl4b), Elovl6 (elovl6 and elovl6l). The ascertained variations were revealed in duplicated Elovl proteins, and the diversities in these proteins structure may reflect their different obligations in the fatty acid biosynthesis and other biological processes.Figure 4Three-dimensional modeling of Elovl proteins in E. tetradactylum and E. rhadinum. All models have confidence levels above 90%.Full size imageTo explore the functional selection pressures acting on Elovl gene family, Ka, Ks, and Ka/Ks ratios were calculated for each gene. Generally, Ka/Ks  1 indicates positive selection. In this study, we found that all the Ka/Ks ratios for each gene were less than 0.5, suggesting that they were subjected to strong purifying selection during evolution, and their functions might be evolutionarily conserved (Fig. 5). Therefore, theoretically, the Elovl genes in the Eleutheronema genus had eliminated deleterious mutations in the population through purification selection. Similar results were also observed in Elovl gene family of Gymnocypris przewalskii that no positive selection trace was detected in most members except elovl211. Moreover, elovl6l and elovl8b showed a higher average Ka/Ks ratio than the other seven members, indicating that the evolution of elovl6l and elovl8b might be much less conservative and thereby could provide more variants for natural selection in Eleutheronema species.Figure 5The evolutionary rates of the Elovl genes in (a) E. tetradactylum and (b) E. rhadinum. The Ka, Ks, and Ka/Ks values were demonstrated in boxplots with error lines.Full size imageChromosomal location, collinearity, and protein–protein interaction network analysis of Elovl genesAs shown in Fig. 6a and b, Elovl genes were randomly and unevenly distributed on seven chromosomes in both E. tetradactylum and E. rhadinum, including Chr5, Chr6, Chr8, Chr10, Chr11, Chr13, and Chr25. The Chr5 and Chr6 harbored two Elovl genes (elovl1b and elovl8b in Chr5, elovl5 and elovl6l in Chr6), while other chromosomes each carried a single Elovl gene. Collinearity relationship analysis was performed to further investigate the gene duplication events within the Elovl gene family. The results revealed that a pair of segmental duplication genes (elovl4a/4b) showed collinear relationships. A chromosome-wide collinearity analysis also showed that the chromosomes were highly homologous between E. tetradactylum and E. rhadinum, including the Elovl gene family (Figure S2). To infer the protein interaction within Elovl gene family, we constructed the protein–protein interaction (PPI) network of the Elovl proteins based on the interaction relationship of the homologous Elovl proteins in zebrafish. The results showed that Elovl genes had close interaction with other members except for the elovl4a/4b and elovl8b (Fig. 6c), which suggested that they might participate in diverse functions by interacting with other proteins. Thus far, elovl4a and elovl4b were widely identified in most fish, which could effectively elongate PUFA substrates37. In addition, the elovl4a/4b were identified to be homologous proteins of zebrafish, indicating that the elovl4 subtype was highly conserved during evolution and played important roles in the biosynthesis of LC-PUFA in Eleutheronema.Figure 6Chromosomal location and collinearity analysis of Elovl gene family in (a) E. tetradactylum and (b) E. rhadinum. Colored boxes represented chromosomes. Segmental duplication genes are connected with grey lines; (c) a protein–protein interaction network for Elovl genes based on their orthologs in zebrafish.Full size imageExpression patterns of ELOVL genes in different tissuesIn the present study, we aimed to determine the expression patterns and gained insights into the potential functions of Elovl genes in the brain, eye, gill, heart, kidney, liver, muscle, stomach, and intestine. The expression patterns of Elovl genes in different tissues and species were distinct, suggesting the diverse roles during fish development (Fig. 7a and b). In our present study, the elovl1a and elvovl1b were expressed in a relatively narrow range of tissues, including the liver, stomach, and intestine. Some Elovl genes had much higher relative expression rates, e.g., elovl1a and elovl7a. The elovl4a was primarily distributed in the brain and eye, slightly expressed in gills while hardly detectable in other tissues, consistent with previous studies37, 38, which might play an important role in endogenous biosynthesis of LC-PUFA in the neural system of fish. In contrast to elovl4a, elovl4b was ubiquitously, instead of tissue-specific, expressed in most tissues while hardly examined in the heart and kidney. The elovl4a and elovl4b were two commonly paralogues in evolutionarily diverged fish species, and the striking difference in expression patterns between elovl4a and elovl4b might be due to the potential functional divergence of these two paralogues. In addition, elovl8b, the novel active member of the Elovl protein family, was expressed in several tissues, suggesting the essential roles in LC-PUFAs biosynthesis of teleost as indicated by a previous study20. Moreover, the differences in expression patterns among different Elovl genes indicated that these genes might possibly undergo functional divergence during evolution in the Eleutheronema genus. Overall, our present study firstly provided the preliminary organ-specific expression data of the Elovl gene family in E. tetradactylum and E. rhadinum, which could provide the foundation for further clarifying the function of these genes in the evolutionary development of the Eleutheronema genus.Figure 7qPCR assessment of tissue distribution of elovl1a, elovl1b, elovl4a, elovl4b, elovl5, elovl6, elovl6l, elovl7a, and elovl8b gene expression in (a) E. tetradactylum and (b) E. rhadinum for various tissues including the brain, eye, gill, heart, kidney, liver, muscle, stomach, and intestine.Full size image More

Our main objective was to test the SJH (positing that adolescents living in “traditional”, non-industrial environments will more closely fulfil their “biological/natural” sleep requirements25,26) by comparing sleep deprivation among adolescents in rural and urban societies. The SJH argues that adolescent “biological/natural” sleep quotas and circadian cycles can be ascertained from free days, when sleep patterns are minimally shaped by social commitments5,37. Therefore, we predicted that sleep deprivation would be rare in the more rural agricultural settings of Puebla and Campeche but more frequent among participants in Mexico City. Likewise, we predicted that we would not see sleep deprivation on free days among any of the rural participants.Our predictions were not supported, instead, we found that short sleep quotas during school nights are common in both rural agricultural settings, with over 75% of adolescents in each group sleeping  More

Authors and AffiliationsStatistics Division, Food and Agriculture Organization of the United Nations, Rome, ItalyFrancesco N. Tubiello, Giulia Conchedda, Leon Casse & Giorgia De SantisDigitization and Informatics Division, Food and Agriculture Organization of the United Nations, Rome, ItalyHao Pengyu & Chen ZhongxinInternational Institute for Applied Systems Analysis, Laxenburg, AustriaSteffen FritzGeospatial Unit, Land and Water Division, Food and Agriculture Organization of the United Nations, Rome, ItalyDouglas MuchoneyAuthorsFrancesco N. TubielloGiulia ConcheddaLeon CasseHao PengyuChen ZhongxinGiorgia De SantisSteffen FritzDouglas MuchoneyCorresponding authorCorrespondence to
Francesco N. Tubiello. More

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