Phylogenomic and Evolutionary Insights into Lipoprotein Lipase (LPL) Genes in Tambaqui: Gene Duplication, Tissue-Specific Expression and Physiological Implications

 Comment 1: Line 133: To be accurate the phylogenetic analysis was done with LPL aa sequences.

 Response1: We thank the reviewer for his/her kind comments. We agree with the reviewer and have revised the sentence for clarity and accuracy. The updated sentence (line 132-135) now reads:

 “This procedure produced multiple BLAST hits for LPL amino acid sequences in each of the protein databases, which were individually examined and retained in FASTA format for phylogenetic analysis.”

Comment 2: Line 202 (and results): The authors use “read depth” as a proxy to compare lpl expression across teleosts; read depth values were obtained from the Phylofish database. Although read depth relates with the number of reads that map to a specific gene, it also varies according to the sequencing depth (i.e. total number of reads per sample). Is the sequencing depth across species/tissues comparable?

 Response 2: We thank the reviewer for your comments. We agree that sequencing depth can influence read depth values, which may limit direct quantitative comparisons across species or tissues if library construction and sequencing were not standardized. However, the PhyloFish database was specifically designed to enable interspecies comparisons of gene expression by using a consistent RNA-seq protocol across all 23 fish species, including (i) the same set of ten tissues, (ii) identical library preparation and sequencing chemistry, and (iii) comparable sequencing depth (Pasquier et al., 2016). These methodological controls minimize technical variability and allow mean depth values to be used as a reasonable proxy for cross-tissue expression patterns. In our study, we used average depth data primarily as an exploratory and qualitative reference to visualize the tissue-specific expression of lpl paralogs across teleosts. To clarify this point, we edited the sentence in line 211-215 as follows:

Revised: “The average depth values for each lpl copy were obtained from the RNA-seq libraries available in the PhyloFish database (https://phylofish.sigenae.org/) using the TBLASTN algorithm with protein sequences as queries. These values were used qualitatively to visualize tissue-specific expression patterns, as sequencing depth across species and tissues was standardized in PhyloFish; nonetheless, differences in transcriptome completeness and expression dynamics may still limit direct quantitative comparisons (Supplementary Figure 1).”

Comment 3: Please standardize gene naming to improve readability: e.g. line 295 lpl and lpl-like and line 300 lpl1 and lpl2.

Response 3: Thank you for your observation. We have revised the manuscript to standardize gene nomenclature throughout the text, including line 296. The names lpl1, and lpl2 are now used consistently to improve clarity and readability.

Comment 4: Line 341: “reflecting functional divergence and post-duplication adaptation”, although syntenic rearrangement can affect gene regulation, I find the authors should be careful when directly affirming that rearrangement reflects functional divergence. The data is robust and adequate to infer orthologies, not function.

Response 4: We appreciate the reviewer’s valuable comment and fully agree that syntenic rearrangements alone are not sufficient to confirm functional divergence. Our intention was to suggest a possible link between the observed gene context variation and functional differentiation, not to assert it definitively. In light of this, we have revised the sentence for greater accuracy and to avoid overinterpretation. The revised sentence in lines Line 336-340 now reads:

Revised: “The genes adjacent to lpl1b, tpm4, rab8a, cib3 (upstream), and ap1m1, klf2b, eps15l1, crt3, rx2 (downstream) are conserved in all teleosts that retained this copy, except for Mexican tetra, which shares only upstream genes in synteny. However, despite these variations, the syntenic organization of tpm4-lpl1b-ap1m1 is highly conserved among the teleost species analyzed”

Comment 5: Line 410: correct Lp11a

Response 5: Thank you for pointing that out. The typographical error in "Lp11a" has been corrected (Line 407)

Comment 6: Line 458: remove extra “.”

Response 6: Thank you for the observation. The extra period has been removed (Lines 224 and 451).

 Comment 7: Line 462/654: The instability indexes are described at length, but what are the putative consequences of being stable/unstable? The author suggests “tailored to each species physiological and environmental needs.” Could it be tissue-dependent?

Response 7: We thank the reviewer for this insightful question. We agree that the implications of protein stability merit further clarification. To address this, we have expanded the discussion to briefly consider possible functional consequences (Lines 678-685). While the instability index is a predictive in silico parameter and does not necessarily reflect in vivo degradation rates, it can provide indirect clues about protein turnover, structural dynamics, or adaptation to specific cellular contexts.

We have modified the manuscript as follows:

Revised: “While the instability index is a theoretical prediction and does not directly determine in vivo protein degradation, lower stability may suggest a higher turnover rate, potentially affecting enzyme availability and activity under different physiological demands (Guruprasad et al., 1990). These variations may reflect specific adaptations in the optimal activity of lipases and temperature stability, tailored to each species physiological and environmental needs [60,61,62,63]. Additionally, such variation might also be tissue-dependent, as certain tissues could require more dynamic regulation of lipid metabolism than others (Weil et al., 2013).”

Comment 8: Figure 6: provide efficiency values for RT-PCR reations

Response 8: We thank the reviewer for this observation. We have now included the amplification efficiency values in the legend of Figure 6 and in the Materials and Methods section (lines 186–190).

 Revised: Amplification efficiency for each primer set was calculated from five-point, 1:4 serial dilution curves, using pooled liver cDNA for lpl1a and lpl2a, and pooled ovary and testis cDNA for lpl1b. The RT-qPCR assays showed high linearity and efficiency for all target genes, with amplification efficiencies of 101.39% (lpl1a), 97.75% (lpl1b), 95.99% (lpl2a), and 100.88% (β-actin).

Comment 9: Line 589: protein symbols not in italics

Response 9: Thank you for pointing this out. We have carefully reviewed the manuscript and corrected the formatting to ensure that gene symbols are italicized and protein symbols are presented in regular font. Specifically, in line 578 (589 in the original document) and throughout the manuscript, we verified and standardized the use of italics and capitalization according to accepted gene and protein nomenclature guidelines.

Comment 10: Line 612, Table S1, S2: Replace “basal teleost” by “Early branching teleost”

Response 10: Done. The term "basal teleost" was replaced with "early branching teleost" throughout the manuscript and in Supplementary Tables S1 and S2 to ensure consistency and reflect current phylogenetic terminology.

Comment 11: Line 631: The maintenance of syntenic blocks is more likely to be conserved between closely-related species, this does not necessarily reflect specific functional adaptations.

Response 11: We appreciate the reviewer’s comment and agree that the conservation of syntenic blocks, especially among closely related species, should not be interpreted as direct evidence of functional adaptation. We have revised the text to adopt a more cautious interpretation, focusing on the potential for structural constraints and evolutionary pressures without making definitive claims about functional divergence (Lines 649-654).
Revised: “The specific retention of adjacent genes in the lpl1a and lpl1b regions of tambaqui (Colossoma macropomum) and red-bellied piranha (Pygocentrus nattereri), both members of the Serrasalmidae family, compared to other teleosts, may reflect conserved genomic architecture, which is more likely to be maintained between closely-related species. These patterns may reflect structural constraints or lineage-specific regulatory evolution, rather than direct functional adaptation.”

 Comment 12: Line 699: Is there a relationship between glycosylation patterns in tambaqui and specific tissue expression/tissue environment (e.g. gonads vs liver)? 

Response 12: We thank the reviewer for this insightful question. We have incorporated a clarification in the revised manuscript to better link the glycosylation patterns of lpl1a and lpl1b with their tissue-specific expression profiles in tambaqui. Although the functional implications of glycosylation were initially discussed from a comparative and evolutionary perspective, we agree that tissue-specific expression data support a more targeted interpretation. In tambaqui, lpl1a is more highly expressed in the liver, while lpl1b is not detected in the liver but is predominantly expressed in the gonads. Given that Lpl1b contains more predicted N-glycosylation sites than Lpl1a, it is plausible that these differences reflect tissue-specific post-translational requirements, possibly related to the local microenvironment or physiological demands of lipid metabolism in the gonads. This connection has now been briefly mentioned in the glycosylation discussion and is further explored in the gene expression results section (Lines 779-787).

Revised: “Furthermore, these tissue-expression differences between lpl1a and lpl1b are consistent with the variations in the glycosylation patterns predicted for the tambaqui amino acid sequence, which may suggest tissue-specific adaptations. The higher number of predicted glycosylation sites for Lpl1b, compared to Lpl1a, could imply differentiated post-translational modifications, possibly influenced by the distinct physiological needs of the tissues. The preferential expression of lpl1b in the gonads, in line with the glycosylation patterns, suggests that glycosylations might contribute to modulating enzymatic activity, potentially adjusting the function of lpl1b in different physiological environments, such as gonadal development.”

Comment 1: Gene Nomenclature

Gene naming—especially when proposing changes—should follow established nomenclature guidelines to maintain consistency across species. Renaming the lpl gene (an ortholog of human LPL) to lpl1, as proposed in this manuscript, may disrupt cross-species comparability and create unnecessary confusion. Furthermore, the gene referred to as lpl-like (renamed lpl2 in the manuscript) shares structural similarities with lpl, but its function remains uncharacterized. The absence of a clear ortholog in tetrapods further complicates any functional inference, making it premature to rename this gene lpl2 without additional functional evidence.

In this reviewer’s opinion, assigning the name lpl to a gene implies functional conservation with its mammalian counterpart, which may not be appropriate in this case. Additionally, if the authors wish to rename lpl to lpl1, they should have submitted a request for nomenclature approval to ZFIN (Zebrafish Nomenclature Conventions), as outlined in the guidelines:
? https://zfin.atlassian.net/wiki/spaces/general/pages/1818394635/ZFIN+Zebrafish+Nomenclature+Conventions

Such approval would provide greater clarity, ensure community-wide consistency, and offer more confidence in accepting the proposed nomenclature changes.


Response1: We thank the reviewer for the thoughtful and constructive comment regarding gene nomenclature. We fully agree that standardized and community-approved nomenclature is essential for ensuring clarity and consistency across species.

In our study, we refer to lpl1 and lpl2 to distinguish two paralogous genes found in teleosts and other non-tetrapod vertebrates. These genes do not result from the teleost-specific whole genome duplication, but rather originate from an ancient tandem duplication that predates the divergence between sarcopterygians and actinopterygians. This inference is supported by our phylogenetic analyses, which place lpl1 and lpl2 in well-supported and distinct clades, with conserved synteny observed in multiple vertebrate lineages.

The nomenclature lpl1 and lpl2 has precedent in previous studies in fish species such as medaka, red seabream, and Fugu (e.g., Wang et al., 2015; Oku et al., 2006; Kaneko et al., 2013), where both copies are retained and differentially expressed. In our manuscript, we adopted these names to facilitate evolutionary and comparative genomic analyses and to maintain consistency with established literature. We emphasize that lpl1 corresponds to the ortholog of the single LPL gene found in mammals, whereas lpl2 refers to its paralog that was retained in specific lineages following the ancestral duplication.

We acknowledge that ZFIN guidelines discourage the use of suffixes such as “a/b” or “.1/.2” when duplications predate the ray-finned/lobe-finned divergence. However, given the absence of a one-to-one orthologous relationship to mammalian LPL in species where both copies are present, and the importance of distinguishing between these paralogs, we opted for the lpl1/lpl2 designation. This usage is not intended to imply functional equivalence with mammalian LPL or to represent a formal nomenclature proposal.

To avoid confusion, we have revised the manuscript to explicitly state that these gene names are used only to distinguish ancient paralogs and that functional divergence of lpl2 remains to be experimentally validated. We have also removed any language suggesting formal gene symbol proposals and clarified our intent to submit a nomenclature request to ZFIN in the future should further functional and comparative evidence support such an action.

We believe that these revisions strengthen the manuscript by improving clarity while maintaining a biologically meaningful distinction between the lpl paralogs in teleosts and other vertebrates.

We have revised the manuscript and removed any formal nomenclature proposals in the results section (lines 234-236). Additionally, to clarify our naming rationale and improve overall readability, we enhanced the Discussion section (lines 587–592) with a detailed explanation of the evolutionary origin of the gene copies and the temporary use of the terms LPL1 and LPL2. We also included lines 747–753 in the Tissue-Specific Expression section to align our terminology with previous literature, where these paralogs were referred to as lpl1 and lpl2, and to note their similar expression patterns in other teleosts. These additions should help clarify the context and consistent use of gene/protein names throughout the manuscript.










Comment 2: qPCR Normalization

The authors report using only one reference gene for the qPCR expression analysis. However, it is widely accepted that using at least two validated reference genes is necessary to ensure accurate normalization and reliable results. Relying on a single reference gene—especially one whose stability has not been rigorously confirmed—can lead to misleading interpretations, as expression levels may vary depending on tissue type, developmental stage, or external conditions. Best practices typically involve selecting a panel of candidate reference genes and then evaluating their stability using established algorithms (such as geNorm or/and NormFinder) to identify the most appropriate housekeeping genes for the specific experimental context.

Thus, in this reviewer’s opinion, the expression analysis and normalization of LPL transcripts in the current manuscript require further validation. Incorporating multiple, validated reference genes would significantly strengthen the accuracy and credibility of the qPCR-based findings.

Response 2: We appreciate the reviewer’s important comment regarding reference gene selection for qPCR normalization. We fully agree that the use of multiple validated reference genes is considered best practice, particularly when analyzing gene expression across diverse tissues and conditions.

Although β-actin was used as the sole reference gene in our qPCR analyses, its suitability for normalization in Colossoma macropomum has been previously validated. Nascimento et al. (2016) evaluated the expression stability of several candidate reference genes and identified β-actin as the most stable, followed by 18S rRNA. Nevertheless, we recognize that optimal qPCR normalization typically requires the use of multiple reference genes validated for each experimental condition. Therefore, our current expression screening should be interpreted as qualitative, aiming to detect presence/absence and tissue-level patterns of expression rather than provide absolute quantification. Future studies will incorporate multiple reference genes and stability analysis tools (e.g., geNorm, NormFinder) to enhance the accuracy and reliability of gene expression quantification.

Furthermore, it is important to highlight that the use of a single reference gene, particularly for tissue screening analyses, is a well-established practice in the literature. For example, Gallani et al. (2021) used β-actin as the sole reference gene to evaluate the expression of cytokine- and antimicrobial peptide-coding genes in the spleen and kidney of tambaqui (Colossoma macropomum). Similarly, some studies employed β-actin as a reference gene in a tissue screening of lpl expression in zebrafish (Feng et al. 2014); red sea bream (Oku et al., 2006) and pufferfish (Kaneko et al., 2013). Wang et al. (2015) also used a single reference gene (ribosomal protein L7) to analyze the tissue-specific expression of lpl genes in medaka (Oryzias latipes). These examples reinforce that, while the use of multiple validated reference genes is ideal, employing a single reference gene—when supported by prior validation or literature precedent—remains a common and acceptable approach, especially in studies aiming to detect general tissue-specific expression patterns.

References:

●       Gallani, S. U., Michelato, M., Vargas, L. et al. (2021). Expression of immune-related genes in spleen and kidney of tambaqui (Colossoma macropomum) fed with dietary probiotics and challenged with Aeromonas hydrophila. Microbial Pathogenesis, 150:104638. https://doi.org/10.1016/j.micpath.2020.104638

●       Feng, H., Guo, S., Wang, L. et al. (2014). Molecular cloning and expression analysis of lipoprotein lipase gene in zebrafish (Danio rerio). Journal of Fish Biology, 84(3): 830–844. https://doi.org/10.1111/jfb.12423

●       Wang, Z., Zhu, Q., Yang, H. et al. (2015). Molecular cloning and expression analysis of lipoprotein lipase gene in medaka (Oryzias latipes). Fisheries Science, 81: 497–506. https://doi.org/10.1007/s12562-014-0826-7

●       Oku, H., Tsuchioka, M., Yamaguchi, K., & Ando, M. (2006). Cloning and expression of lipoprotein lipase and hepatic lipase in red sea bream (Pagrus major). Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 145(3–4), 345–355. https://doi.org/10.1016/j.cbpb.2006.06.008

●       Kaneko et al., 2013
 Kaneko, G., Yoshinaga, T., & Kondo, M. (2013). Identification of two lipoprotein lipase (LPL) genes in pufferfish and analysis of their gene expression during fasting and refeeding. General and Comparative Endocrinology, 186, 32–38. https://doi.org/10.1016/j.ygcen.2013.01.003

Comment 3: It is recommended that the third and fourth whole-genome duplication (WGD) events be visually distinguished in the phylogenetic tree using different colors. This would improve clarity and help readers more easily interpret the evolutionary context.

Response 3: We thank the reviewer for this helpful suggestion. We have now updated the phylogenetic tree by using different colors to visually distinguish the third (3R) and fourth (4R) whole-genome duplication events, thus improving the clarity and interpretability of the evolutionary context.

Comment 4: As previously mentioned, some sentences in the Results section go beyond reporting findings and begin to interpret or discuss the data. These sentences would be more appropriately placed in the Discussion section to maintain a clear separation between results and interpretation.
Thank you for your insightful comment. We appreciate your guidance on maintaining a clear distinction between the presentation of results and their interpretation. In response to your feedback, we have reviewed the Results section carefully and moved sentences that involve interpretation or discussion of the data to the appropriate Discussion section. This revision ensures that the Results section now focuses solely on presenting the findings, while the interpretation and discussion of these results are clearly separated in the Discussion section. Additionally, we have updated the "Phylogenetic Analysis" and "Synteny" sections to reflect these changes, ensuring that the structure of the manuscript is more in line with your suggestions. We hope these revisions improve the clarity and flow of the manuscript.

Comment 5: Given the large number of sequences used in the in-silico analyses, it would be helpful to include additional details in the supplementary table. Specifically, where possible, please indicate the chromosomal location of each gene. This information would provide stronger support for the hypothesis that the identified paralogs arose from WGD events.

Response 5: Thank you for the suggestion. We have updated the supplementary Figure S1 to include the chromosomal location of each gene, where available. This additional information helps strengthen the evidence for the proposed whole-genome duplication (WGD) origin of the identified paralogs.

4. Response to Comments on the Quality of English Language

Point 1: The English is fine and does not require any improvement.

Response 1:    Thank you for your feedback. We appreciate your assessment of the language quality and are glad to know that no further improvements are necessary.