the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
The 16S rDNA microbiome of the Arctic foraminifera Neogloboquadrina pachyderma is comprised of hydrocarbon-degrading bacteria and a diatom chloroplast store
Abstract. Neogloboquadrina pachyderma is the only true polar species of planktonic foraminifera. It therefore plays a crucial role in the calcite flux, and in reconstructions and modelling of seasonality and environmental change within the high latitudes. The rapidly changing environment of the polar regions of the North Atlantic and Arctic Oceans poses challenging conditions for this (sub)polar species in terms of temperature, sea-ice melt, calcite saturation, ocean pH and contraction of the polar ecosystem. To model the potential future for this important high latitude species, it is vital to investigate the modern ocean community structure throughout the annual cycle of the Arctic to understand the inter-dependencies of N. pachyderma. We use 16S rDNA metabarcoding and TEM to identify the microbial interactions of N. pachyderma during the summer ice-free conditions in Baffin Bay. We demonstrate that the N. pachyderma diet consists of diatoms and bacteria. The core microbiome is defined as the 16S rDNA amplicon sequencing variants (ASVs) found in 80 % of individuals investigated. This core microbiome consists of two diatom chloroplast ASVs and seven bacterial ASVs and accounts for, on average, 50 % of the total ASVs in any individual. The bacterial ASVs represent hydrocarbon-degrading bacteria, including those found routinely in the diatom phycosphere. On average the two chloroplast ASVs compose 40 % of the core microbiome. Significantly, an average of 55.7 % of all ASVs in any individual are of chloroplast origin. TEM highlights the importance of diatoms to this species, conclusively revealing that chloroplasts remain undigested in the foraminiferal cytoplasm in very high numbers, comparable to those observed in kleptoplastic benthic foraminifera. Diatoms are the major source of kleptoplasts in benthic foraminifera and other kleptoplastic groups, but this adaptation has never been observed in a planktonic foraminifer. Further work is required to understand the association between N. pachyderma, diatoms and their chloroplasts in the pelagic Arctic realm. It may confer an advantage to this species for survival in this extreme habitat, but it could also become compromised by the rapidly changing climate.
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RC1: 'Comment on egusphere-2024-497', Anonymous Referee #1, 20 May 2024
GENERAL COMMENTS
In their paper, Bird et al. investigate the prokaryotic interactions of the polar foraminifera N. pachyderma in Baffin Bay using a combined approach involving 16S metabarcoding and TEM images. While the manuscript offers valuable insights into the core microbiome of N. pachyderma, it is important to address the limitations of the dataset and consider repeating some analyses to mitigate potential biases. Moreover, the data analysis strategy requires clearer presentation and justification. Additionally, the conclusion section needs to be included.
SPECIFIC COMMENTS
At line 177 (Pg. 9), the authors report that the ASV reads were transformed into relative counts and a threshold of 0.1% abundance was used to filter out ASVs. However, amplicon data are inherently compositional, meaning the proportions of the different ASVs are not independent of each other, which can lead to incorrect estimates of similarity between samples. After removing contaminants such as mitochondria and eukaryotes, the read counts should be transformed into centred log-ratios, and the similarity of the samples should then be calculated using the Aitchison distance [1].
It is unclear which analyses were performed. In line 184 (page 9) the authors generally refer to the mvabund R package and successively mention the generalized linear model but the name of the test is not reported. (Possibly anova.manylm?)
Similarly, in line 194 the authors mention the R function adonis for performing a multivariate analysis of variance. This function performs a permutational multivariate analysis of variance (PERMANOVA) and the authors should indicate the number of permutations used in their analysis.
Both tests should be repeated using centre-log ratio transformed data and the results should be reported in a table.
The authors performed differential expression analyses using DESeq2. However they do not clarify whether they used pre-processed data or the raw read counts as input for the function, as recommended by the algorithm's developers [2], [3].
The results of the permutational multivariate analysis of variance presented on line 273 (page 13) show that location explains 49% of the variability in the ASV profile of N. pachyderma. This means that almost half of the observed differences in the microbiome composition among the 28 specimens can be attributed to the different sampling locations. In light of this significant effect of sampling location, the authors should refrain from including the 17 specimens with no contextual seawater samples (Stations 176, BB2, 129, 301) in the differential expression analyses.
In lines 261-266, the authors state that a differential expression analysis was repeated with a subset of foraminifera and water samples from station 101 to control for the effect of geographic location, claiming that the results are consistent with those presented in Fig. 4. The results of this control test are reported in Fig. A3 (page 30).
However, upon comparing the plots, it becomes evident that the results are dissimilar and partially contradict those reported in Fig. 4:
- The taxa Flavobacteriaceae and Crocinitomix are more expressed in the foraminifera samples in Fig. 4 but more expressed in water samples in Fig. A3.
- The taxa Chaetoceros, Bradyrhizobium, Moritella, Synedra, and Rubripirellula (among others) are not found in the differential abundance analysis presented in Fig. A3.
These inconsistencies indicate the significant influence that the selection of samples included in the differential abundance analyses can have on the results.
The same is true for the core microbiome analyses. Flavobacteriaceae found to be more abundant in the water column at station 101 (Fig. A3), are presented as part of the core microbiome of N. pachyderma. In line 503 (pg 23), the authors speculate on the potential role of this taxon as an endosymbiont.
Given the relevance of the surrounding community in determining interactions with the foraminifera (line 273, page 13), the authors should be more cautious in their interpretation of their results. A thorough ecological discussion on the prokaryotic community in the water column samples, taking into account the environmental parameters measured, should be included given the importance of geographic location before speculating on the potential role of a given taxon as part of the N. pachyderma core microbiome.
For example, the authors could consider including the time since the ice break-up in their analysis to provide ecological context to the prokaryotic community, which is dominated by Bacteroidetes and Proteobacteria in particulate organic matter in sea ice and sinking particles under the ice [4].
Line 435 The authors briefly mention the potential differences in the core microbiome or diet at different ontological stages. Since no data is presented on the size of the specimens analysed this aspect should be discussed.
Lines 422-424 (page 20): The authors do not present any evidence beyond the low ASV richness to support the classification of specimens Fm176b and Fm101e as gametogenic. Other potential explanations for this pattern should be discussed.
Lines 444-447 The authors should be cautious in interpreting the absence of evidence (i.e., absence of a given ASV) as evidence of absence, and in comparing reads across samples, given the potential impact of PCR-induced biases on compositional data. This aspect should also be discussed.
The datasets, the ASV sequences utilized for the analyses, including the code should be made accessible in public repositories to ensure the reproducibility of the results.
TECHNICAL CORRECTIONS
-Station 60 is mentioned in Table 1 but not plotted in Map in Fig. 1 nor mentioned in the text.
-Lines 243-244 should be moved to the methods section
-The conclusion section is missing
-Line 819 correct reference format
-Greco et al 2021 is cited in the text but not present in the reference list
-Greco et al 2019 reported twice in the reference list
[1] G. B. Gloor, J. M. Macklaim, V. Pawlowsky-Glahn, and J. J. Egozcue, “Microbiome Datasets Are Compositional: And This Is Not Optional,” Front. Microbiol., vol. 8, 2017, https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2017.02224
[2] M. I. Love, W. Huber, and S. Anders, “Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2,” Genome Biol., vol. 15, no. 12, pp. 1–21, 2014, doi: 10.1186/s13059-014-0550-8.
[3] K. Van den Berge et al., “Observation weights unlock bulk RNA-seq tools for zero inflation and single-cell applications,” Genome Biol., vol. 19, no. 1, p. 24, Feb. 2018, doi: 10.1186/s13059-018-1406-4.
[4] R. Amiraux et al., “Bacterial diversity and lipid biomarkers in sea ice and sinking particulate organic material during the melt season in the Canadian Arctic,” Elem. Sci. Anthr., vol. 9, no. 1, p. 040, Mar. 2021, doi: 10.1525/elementa.2019.040.
Citation: https://doi.org/10.5194/egusphere-2024-497-RC1 -
AC1: 'Comment on egusphere-2024-497', Clare Bird, 26 Jun 2024
In their paper, Bird et al. investigate the prokaryotic interactions of the polar foraminifera N. pachyderma in Baffin Bay using a combined approach involving 16S metabarcoding and TEM images. While the manuscript offers valuable insights into the core microbiome of N. pachyderma, it is important to address the limitations of the dataset and consider repeating some analyses to mitigate potential biases. Moreover, the data analysis strategy requires clearer presentation and justification. Additionally, the conclusion section needs to be included.
We thank reviewer 1 for their thoughtful and constructive feedback.
In this first interactive discussion response, we wish particularly to address the following two comments:
- At line 177 (Pg. 9), the authors report that the ASV reads were transformed into relative counts and a threshold of 0.1% abundance was used to filter out ASVs. However, amplicon data are inherently compositional, meaning the proportions of the different ASVs are not independent of each other, which can lead to incorrect estimates of similarity between samples. After removing contaminants such as mitochondria and eukaryotes, the read counts should be transformed into centred log-ratios, and the similarity of the samples should then be calculated using the Aitchison distance [1].
Thank you for this input. In our original data analyses, we carried out RA normalisation and removal of 0.1%, and Bray Curtis, in line with previous literature, as cited. However, we very much appreciate reviewer 1’s invaluable experience and input here, and in line with their recommendations have carried out the following:
- In filtering ASVs we removed only those that had a total frequency count of 50 across the entire 65 samples including controls. This has replaced the removal of <0.1% after converting to relative abundance.
- Mitochondria and eukaryotic sequences have been removed, and those not assigned beyond the “kingdom” level (as previously performed).
- We used Decontam (R-package) to remove contaminants from our dataset, using the prevalence with batch method. This then gave us our final dataset for analysis.
- We have generated a robust Aitchison distance matrix after converting to robust clr, (rclr). This method works well with sparse data sets, such as our foram microbiomes, (Martino et al 2019).
- Ordination was carried out in q2-gemelli using a RPCA (robust PCA).
These steps have now been clearly documented in the manuscript. Since the number of ASVs retained by this filtering method is greater than those retained by the previous filtering method, we will re-do all our other analyses on differential abundance, and core microbiome. Therefore, we will address all other comments (beyond the below) once our analyses are complete.
- It is unclear which analyses were performed. In line 184 (page 9) the authors generally refer to the mvabund R package and successively mention the generalized linear model but the name of the test is not reported. (Possibly anova.manylm?). Similarly, in line 194 the authors mention the R function adonis for performing a multivariate analysis of variance. This function performs a permutational multivariate analysis of variance (PERMANOVA) and the authors should indicate the number of permutations used in their analysis. Both tests should be repeated using centre-log ratio transformed data and the results should be reported in a table.
The mvabund test used was anova.manyglm, our apologies for this omission. We have now carried out our PERMANOVA (Provenance, Depth and Station) in gemelli on the rclr transformed data, as requested. Number of permutations is 999 (the default setting) which will be included in the manuscript. We apologise for this omission in our previous analysis.
Citation: https://doi.org/10.5194/egusphere-2024-497-AC1 -
RC2: 'Comment on egusphere-2024-497', Anonymous Referee #2, 03 Jul 2024
As written, the rationale for this study is compelling (lines 90-94)—the species is important to the Arctic, which is a rapidly changing habitat due to global warming. That passage is a bit misleading, however, as it notes the data presented here directly impacts understanding of trophic interactions, seasonal shell geochemistry, population dynamics modeling, carbonate flux, and evolutionary pressures. While insights can be gleaned about trophic interactions and – perhaps—evolutionary pressures, the sampling was not done seasonally, so little to nothing can be asserted about carbonate geochemistry, carbonate flux, and population dynamics.
Overall, the contribution is mostly well written and mostly logical. There are, surprisingly, considerable errors in mechanics and grammar, as well as minor points, which are listed separately, below these more-substantial points.
N. pachyderma is touted as a polar / high-latitude taxon, yet lines 592-593 indicate it also occurs in the tropics. This contradiction should be explained a bit more thoroughly in the context of impact to high (or low) latitudes.
It is rather unfortunate that the samples for DNA barcoding were collected in a different year and region from those collected for cellular ultrastructural analysis (TEM). How do the authors truly know that the TEM results (collected in 2018) are representative / comparable to sequencing results from populations collected in 2017?
Given the assertion of phototrophy (line 544), a short passage should discuss light levels at the sample sites and water depths in different parts of the year, with literature citations. Are other N. pachyderma known to photosynthesize?
Lines 87-90 state that 16S rDNA is used here to determine prokaryotic biotic and trophic interactions, and potential symbiotic associations with TEM utilized to note ultrastructural attributes. It is not clear how these methods can be used to inform that these observed chloroplasts are of diatom origin. More specifically, how can ASVs be assigned to diatoms (eukaryotes) from 16S rDNA sequence data (e.g., lines 224-229, Fig. 3)? How were chloroplast ASVs assigned to diatoms (Fig 4, line 252)? While my molecular colleagues and one publication (Bonfantine et al 2021 PeerJ) more-or-less explained how this might work, it might be wise of the authors to explain more precisely how they performed these analyses.
Why doesn’t the ASV noted on line 301 align as Chaetoceros? There are other Chaetoceros in Fig. 5. Why the inconsistency? Why doesn’t Fig. 6 show Chaetoceros gelidus while it shows the other common diatom, Fragilariopsis?
Another aspect of the contribution is also confusing in that most of the Introduction, Results, and early Discussion focus on trophic aspects (i.e., food and diet) while the last part of the Discussion focuses on kleptoplasty. It was refreshing to see that TEM was performed for this contribution because it is perhaps the best way to document a putative kleptoplasty. Unfortunately, the TEM images presented were not convincing for a few reasons (noted below). More TEM images are required for review. If the authors can firmly justify this taxon is kleptoplastidic, then the results should be written in this context. If such justification is not possible, then the authors should consider altering those kleptoplasty passages to reflect phagotrophy and digestion. Further, the ultimate conclusion that N. pachyderma is a mixotroph and kleptoplastidic is rather a leap from TEM analysis of specimens collected once in the summer. At the very least, structural documentation should be obtained to show plastid degradation in winter or very early spring (to account for digestion / mixotrophy). While such collections are likely out of the question, the authors should address these shortfalls in the Discussion.
Figure 7 is adequate documentation of potential kleptoplasty ONLY if additional higher-magnification images are included. It is imperative to be able to clearly examine some individual chloroplasts to determine if they are intact or in degraded state. Also, foraminiferal viability is typically determined by status of mitochondria (e.g., LeKieffre et al 2018 Mar. Micropaleo.; Nomaki et al 2016; Bernhard et al 2010), none of which are shown in this figure / contribution. Lipids should not be black, as shown in panel d.
TEM images provided in Fig. A6 are equally too low in magnification to be very helpful in the context of documenting kleptoplasty. It appears that the images in panels f, g, and h have degraded chloroplasts (i.e., evidence of phagotrophy rather than kleptoplasty). Organelles in Panels b and c are difficult to identify at all (very low magnification).
More specifically regarding Fig. 7, there is no scale bar in b; the scale in d cannot be correct (caption states bar is 1 micron but that would make those lipids and plastids all far less than 1 um, which is highly atypical; scale in e has no length assigned; panel e should be oriented as it appears in panel c (c has box outlining panel e). Panel b mostly shows the exterior (if one believes the caption); it is not clear why that image was selected as it barely shows N. pachyderma cytology. Fig 7 caption is unclear in places (“pore shape” noted for panel a—presumably intended as “cross section of pore plugs and inner organic lining”?). Further, panel b lacks any “f” label (see caption).
Greco et al. (2021) is cited often (e.g., line 61, 82, 85, 396, 413, 443, 457, 481, 533, 588, 612), yet that reference does not appear in Reference list. It is rather unfair to the reviewer and reader that a paper is cited >10 times but details of publication are not given.
Minor, but important, points
The title does not indicate the report is on a planktic foraminifer.
Line 321 is arguable because the two foraminifera could have phagocytosed while at a given water depth and then migrated vertically to another layer / depth horizon.
Please use the proper term for the hard parts of foraminifera: “tests”, instead of “shells” which is used throughout the document.
Line 43: please be more specific about “within a short time frame”.
Iine 53: omit “habitat”.
Beginning of sentence spanning lines 71-73 is awkward (“Metabarcoding investigations of other taxa within the Neogloboquadrina genus…”). Why not simply “Microbiome metabarcoding of Neogloboquadrina species…”?
Line 112: a colon should replace the semicolon.
Line 116-117: what is “salt adjusted phosphate buffered saline”? First, “salt adjusted” should likely be hyphenated. Second, what salt(s) is adjusted and from what concentration to ending concentration?
Line 142: presumably need to insert comma after parenthetic.
Line 174: Shouldn’t “faith pd” be capitalized to read “Faith PD” or “Faith Phylogenetic Diversity”?
Line 193 requires comma after assemblages.
Omit comma after “differences” in line 195.
Line 203: insert comma after parenthetic.
Lines 213-217 largely are not results, but sample site information. Further, some of the statements lack literature citations.
Line 265: remove comma after “column”
Line 330: phylum should be capitalized in both instances.
Line 341 should read “Pseudoalteromonas” (missing “o”).
Sentence on Line 355 is irrelevant to TEM results (section topic).
Passage on line 390-391 discusses predicted reductions in non-spinose foraminifera yet the reader has not been informed if N. pachyderma is spinose or non-spinose.
Explain how foraminifera can “sit” (line 401)—implies existence of foraminiferal posterior and appendages.
Reference is required for statement ending on line 400.
Line 416: What is a detrital diatom?
Why are bacteria necessarily linked to the diatoms (lines ~415-418)? Bacteria and archaea can live independently from diatoms.
Line 425-426 notes twice that there are eight core ASVs in microbiome yet Table 2 lists nine “core” ASVs in microbiome (see also line 433). Why the difference? Which is correct?
It is not clear how some specimens were denoted “potentially gametogenic” (lines 462, 525-526). No data is presented on this (not in tables, etc).
Discussion of light use by foraminifera appears on lines 487 to 488 yet light levels are not reported in the data presented here.
Line 493 is oddly worded—how can microbes be clustered in a gene?
The passage from Line ~493 to 497 is confusing because it seems to be advocating that these foram populations are N2-fixing but that was not measured. Further, it is not clear that “our findings” on line 497 are supported by Fernandez-Mendez et al. (2016).
Authors must explain why ASV27 is called Aurantivirga (line 498 and perhaps elsewhere) yet lines 503 to 505 says it is a Tenacibaculum species. Which is the reader to believe? Then the authors pontificate about fish teeth and how this ASV27 provides extra calcium to the foraminifer for calcification, which is pure speculation.
Line 537: please explain what “intracellular ingestion” is. Ingestion is the act of taking something from outside the cell / body into the cell / body. Most biologists would simply say the foram phagocytoses the complete diatom (frustule + cell).
While the term kleptoplasty was used three times in the Abstract and once earlier in the contribution (line 440), it is not until section 4.4 that “kleptoplastic behaviour” is asserted for N. pachyderma. The term is never fully defined although a partial definition appears on line ~544.
Provide references for foraminifera, ciliates and dinoflagellates that are kleptoplastic (line 543).
Proper spelling is “byproduct” line 549, vs “biproduct”.
The citation of LeKieffre et al. (2018) on line 554 should be the LeKieffre et al. 2018 paper in Marine Micropaleontolgy, not the one in Mar. Biol. The citation on line 583 is properly cited as the 2018 Mar. Biol. paper.
Powers et al. (2022, Frontiers in Mar. Sci.) should be cited on line 575, with Gomaa et al., as these two papers describe some of the functions of the N. stella kleptoplasts via gene expression (transcriptomics).
Regarding mixotrophy and kleptoplasty in foraminifera (line 583), I believe Cedhagen (1991) documented this long ago for Nonionellina labradorica. That paper should be cited somewhere in paragraph spanning lines 598 to 604.
Authors are reminded that many (perhaps most) forams do not have carbonate tests (shells), so that should be noted in passage about geochemistry (~ line 625).
What exactly is “metabolic C” (line 628)? An internet search reveals it is a type of health supplement of Vitamin C.
Line 630: The phrase “sediment assemblage” typically refers to the benthos (organisms that live on or in the seafloor). Perhaps authors intend “fossil record” or “sedimentological record” instead?
Line 633 to 634 should add “calcareous” to read “…throughout the lifetime of calcareous foraminifera (Spindler…”.
Statement on line 641 should change as photosynthesis was not shown, only inferred.
Line 731 should be italicized (see same genus on prior page).
Mechanics (also important)
The tense of the document is a mix of past and present, often in the same sentence; most scientific papers are in past tense. Please change lines 17 to “used”, line 19 to “was”, line 20 “consisted of”, lines 217 - 219 should be past tense, etc.
Readers who are red-green color blind will be significantly challenged while reading Figures 3, 4, 5, A1, A2, A3, and A4. The authors should consider changing their color palettes, datum point shapes, etc.
More reference strings should begin with “e.g.,” because the cited papers are only examples and not an exhaustive list: lines 52, 440, 557, 634. For line 557-558, it is surprising that Bernhard and Bowser 1999 Earth Sci. Reviews is not cited as it compiled all known foraminifera kleptoplasty cases to that time.
Remove colloquial wording (e.g., line 49 “…will find itself spatially… should be “will be spatially…”; line 171: “…was carried out…” should be “…was executed…” or similar; lines 272-273 “a degree of clustering” should read “shows clustering…”; line 305 “six out of the seven” should be “six of seven”; line 464: “break down” should read “degrade”; line 466: “carry out” should read “ perform”. Line 669 to 670 is obtuse (“There is a degree of clustering…”).
Lines 115, 772: Ship names are always italicized.
For Figure 1: suggest using different color or shape for symbols in the two different sampling years. Resolution will be, presumably, better than that provided for review.
Methods seem a bit too detailed (e.g., passage from lines 143-148).
Please fix font style and size (“t” in “taxa” on, e.g., lines 279, 329; 521).
Line 473 “sp.” should not be italicized, ever.
Suggest authors comb the text for errors in punctuation, etc. For example, see lines 512, 551, 561 (remove comma before parenthetic).
The Conclusions section is blank / non-existent.
The order of the key in Figs. A1 and A2 is peculiar because it goes from 0 to 100, to 150 to 200 to 50. Most would arrange from 0 to 50 to 100 to 150 to 200. The gray background in those figures is inappropriate (waste of ink / electrons).
References were not proofread by authors. Issues are (a) at least 46 species names have not been italicized; (b) at least six entries lack the journal name; (c) some entries are gibberish (e.g., Bell & Mitchell, Brummer et al – see penultimate author name); (d) lack of URL for software packages (Lahti); (e) lack of publisher in at least 2 references (Eegeesiak et al., Schiebel and Hemleben); (f) font color is inconsistent; (g) missing spaces between words, (h) inconsistent presentation of journals (some names abbreviated, others not), (i) lack of superscripting isotopes, (j) inconsistent line spacing.
Citation: https://doi.org/10.5194/egusphere-2024-497-RC2
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