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Jong Soo Park
Kyungpook National University
Corresponding Author
ORCiD: https://orcid.org/0000-0001-6253-5199
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Background: The brine shrimp Artemia salina can thrive in a variety of salinities and is commonly distributed in natural hypersaline lakes and solar salterns. The zooplankter A. salina proves to be a filter feeder, consuming the alga Dunaliella and prokaryotes and plays a critical role in the hypersaline food web. However, the high salinity adaptation mechanisms of A. salina remain poorly understood through transcriptome analysis. Here, we examined the gene expression patterns of A. salina adults that were salt-adapted for 2–4 weeks at five salinities (35, 50, 100, 150, and 230 psu), and generated long-read isoform sequencing (IsoSeq) data to construct a high-quality transcriptome assembly of A. salina. The patterns of A. salina along the salinity gradient provide evidence for halotolerant and euryhaline adaptations at the genetic level.
Results: We confirmed that the activity of sodium/potassium ATPase was up-regulated at the genetic level in high salinity waters. Interestingly, genes related to beta-mannosidase and mannose activities were also up-regulated, suggesting that mannose and mannose derivatives may be accumulated as organic osmolytes. Alternatively, considering that glucose and galactose-related activities were suppressed at high salinities, mannose may be the primary sugar involved in the glycolytic pathway under such conditions. This result further supports the theory that mannose is the main energy source used by A. salina in highly saline environments. The gene expression patterns of A. salina may also be affected by increased thickness of the cuticle, increased numbers of mitochondria, and low dissolved oxygen in high salinity waters. Furthermore, the cellular response of A. salina to acclimation to intermediate salinities depends on the number and type of genes expressed; differential expression patterns are likely to fluctuate at the population level.
Conclusions: Our results provide a high-quality transcriptome assembly of the cosmopolitan brine shrimp Artemia salina at five different salinities (35, 50, 100, 150, and 230 psu) for the first time. The gene expression patterns of salt-adapted A. salina display greater osmoregulation process complexity than we thought. Furthermore, A. salina represents a potential model organism to study locally adapted populations at various salinities.
Keywords
Artemia salina, crustacean, differential expressed genes, euryhaline, halotolerant, transcriptome, osmoregulation
Figure 1
Figure 2
Figure 3
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Figure 5
This is a list of supplementary files associated with this preprint. Click to download.
- Additionalfile1.mp4
Additional File 1. Locomotion of Artemia salina specimens in artificial saline water at 75 psu.
- Additionalfile2.xlsx
Additional File 2: Table S1. Information on RNA sequencing data (Illumina Novaseq6000). Table S2. Analysis of BUSCO (Benchmarking Universal Single-Copy Orthologs) gene sets. Table S3. Functional annotations of up- and down-regulated genes with increasing salinity from 35 psu to 230 psu. Table S4. Enriched gene ontology (GO) terms (p-value < 0.05) of up- and down-regulated genes with increasing salinity from 35 psu to 230 psu. Table S5. Frequency of acidic, basic, hydrophilic, and hydrophobic amino acids in proteomes of pancrustacean species. Table S6. Enriched gene ontology (GO) terms (p-value < 0.05) of genes with U-shaped and inverted U-shaped expression patterns. Only terminal GO annotations are shown. Table S7. Potential salinity response genes in Artemia salina transcriptome. Gene candidates were selected by comparisons with Artemia salinity response gene candidates in previous studies.
- Additionalfile3.pdf
Additional File 3: Figure S1. The de novo transcriptome assembly pipeline employed in this study. Hybrid transcriptome assembly was created using Trinity assembler (v2.8.4). Figure S2. Enrichment of gene ontology (GO) terms (topGO R package; Fisher test, p-value < 0.05) of up- and down-regulated genes with increasing salinity from 35 psu to 230 psu. a Up-regulated genes related to molecular function. b Up-regulated genes related to biological processes, c Up-regulated genes related to cellular components. d Down-regulated genes related to molecular function, e Down-regulated genes related to biological processes. f Down-regulated genes related to cellular components. Figure S3. Phylogenetic tree of carotenoid isomerooxygenases in metazoans. Homologous proteins of A. salina were collected from BLASTp results (NR database, top 500 hits; e-value cutoff = 1.e-05). Figure S4. Analysis of KEGG metabolic pathways in up- and down-regulated genes with increasing salinity from 35 psu to 230 psu, showing genes with U-shaped and inverted U-shaped expression patterns along salinity gradient (up-inc: up-regulated genes with increasing salinity; dn-inc: down-regulated genes with increasing salinity; U-50, U-100, and U-150: genes with U-shaped expression patterns having minima at 50 psu, 100 psu, and 150 psu, respectively; iU-50, iU-100, and iU-150: genes with inverted U-shaped expression patterns having maxima at 50 psu, 100 psu, and 150 psu, respectively).
- Additionalfile4.xlsx
Additional File 4. The predicted proteins and their coding sequences of Artemia salina in this study.
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This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Research
Jong Soo Park
Kyungpook National University
Corresponding Author
ORCiD: https://orcid.org/0000-0001-6253-5199
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Posted 13 Oct, 2020
No community comments so far
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PDF Downloads: ...
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Subject Areas
Animal Science
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background: The brine shrimp Artemia salina can thrive in a variety of salinities and is commonly distributed in natural hypersaline lakes and solar salterns. The zooplankter A. salina proves to be a filter feeder, consuming the alga Dunaliella and prokaryotes and plays a critical role in the hypersaline food web. However, the high salinity adaptation mechanisms of A. salina remain poorly understood through transcriptome analysis. Here, we examined the gene expression patterns of A. salina adults that were salt-adapted for 2–4 weeks at five salinities (35, 50, 100, 150, and 230 psu), and generated long-read isoform sequencing (IsoSeq) data to construct a high-quality transcriptome assembly of A. salina. The patterns of A. salina along the salinity gradient provide evidence for halotolerant and euryhaline adaptations at the genetic level.
Results: We confirmed that the activity of sodium/potassium ATPase was up-regulated at the genetic level in high salinity waters. Interestingly, genes related to beta-mannosidase and mannose activities were also up-regulated, suggesting that mannose and mannose derivatives may be accumulated as organic osmolytes. Alternatively, considering that glucose and galactose-related activities were suppressed at high salinities, mannose may be the primary sugar involved in the glycolytic pathway under such conditions. This result further supports the theory that mannose is the main energy source used by A. salina in highly saline environments. The gene expression patterns of A. salina may also be affected by increased thickness of the cuticle, increased numbers of mitochondria, and low dissolved oxygen in high salinity waters. Furthermore, the cellular response of A. salina to acclimation to intermediate salinities depends on the number and type of genes expressed; differential expression patterns are likely to fluctuate at the population level.
Conclusions: Our results provide a high-quality transcriptome assembly of the cosmopolitan brine shrimp Artemia salina at five different salinities (35, 50, 100, 150, and 230 psu) for the first time. The gene expression patterns of salt-adapted A. salina display greater osmoregulation process complexity than we thought. Furthermore, A. salina represents a potential model organism to study locally adapted populations at various salinities.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
This is a list of supplementary files associated with this preprint. Click to download.
- Additionalfile1.mp4
Additional File 1. Locomotion of Artemia salina specimens in artificial saline water at 75 psu.
- Additionalfile2.xlsx
Additional File 2: Table S1. Information on RNA sequencing data (Illumina Novaseq6000). Table S2. Analysis of BUSCO (Benchmarking Universal Single-Copy Orthologs) gene sets. Table S3. Functional annotations of up- and down-regulated genes with increasing salinity from 35 psu to 230 psu. Table S4. Enriched gene ontology (GO) terms (p-value < 0.05) of up- and down-regulated genes with increasing salinity from 35 psu to 230 psu. Table S5. Frequency of acidic, basic, hydrophilic, and hydrophobic amino acids in proteomes of pancrustacean species. Table S6. Enriched gene ontology (GO) terms (p-value < 0.05) of genes with U-shaped and inverted U-shaped expression patterns. Only terminal GO annotations are shown. Table S7. Potential salinity response genes in Artemia salina transcriptome. Gene candidates were selected by comparisons with Artemia salinity response gene candidates in previous studies.
- Additionalfile3.pdf
Additional File 3: Figure S1. The de novo transcriptome assembly pipeline employed in this study. Hybrid transcriptome assembly was created using Trinity assembler (v2.8.4). Figure S2. Enrichment of gene ontology (GO) terms (topGO R package; Fisher test, p-value < 0.05) of up- and down-regulated genes with increasing salinity from 35 psu to 230 psu. a Up-regulated genes related to molecular function. b Up-regulated genes related to biological processes, c Up-regulated genes related to cellular components. d Down-regulated genes related to molecular function, e Down-regulated genes related to biological processes. f Down-regulated genes related to cellular components. Figure S3. Phylogenetic tree of carotenoid isomerooxygenases in metazoans. Homologous proteins of A. salina were collected from BLASTp results (NR database, top 500 hits; e-value cutoff = 1.e-05). Figure S4. Analysis of KEGG metabolic pathways in up- and down-regulated genes with increasing salinity from 35 psu to 230 psu, showing genes with U-shaped and inverted U-shaped expression patterns along salinity gradient (up-inc: up-regulated genes with increasing salinity; dn-inc: down-regulated genes with increasing salinity; U-50, U-100, and U-150: genes with U-shaped expression patterns having minima at 50 psu, 100 psu, and 150 psu, respectively; iU-50, iU-100, and iU-150: genes with inverted U-shaped expression patterns having maxima at 50 psu, 100 psu, and 150 psu, respectively).
- Additionalfile4.xlsx
Additional File 4. The predicted proteins and their coding sequences of Artemia salina in this study.
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