Functional and Compositional Changes in the Fecal Microbiome of a Shorebird during Migratory Stopover

ABSTRACT Shorebirds migrate long distances twice annually, which requires intense physiological and morphological adaptations, including the ability to rapidly gain weight via fat deposition at stopover locations. The role of the microbiome in weight gain in avian hosts is unresolved, but there is substantial evidence to support the hypothesis that the microbiome is involved with host weight from mammalian microbiome literature. Here, we collected 100 fecal samples of Ruddy Turnstones to investigate microbiome composition and function during stopover weight gain in Delaware Bay, USA. Using 16S rRNA sequencing on 90 of these samples and metatranscriptomic sequencing on 22, we show that taxonomic composition of the microbiome shifts during weight gain, as do functional aspects of the metatranscriptome. We identified 10 genes that are associated with weight class, and polyunsaturated fatty acid biosynthesis in the microbiota is significantly increasing as birds gain weight. Our results support that the microbiome is a dynamic feature of host biology that interacts with both the host and the environment and may be involved in the rapid weight gain of shorebirds. IMPORTANCE Many animals migrate long distances annually, and these journeys require intense physiological and morphological adaptations. One such adaptation in shorebirds is the ability to rapidly gain weight at stopover locations in the middle of their migrations. The role of the microbiome in weight gain in birds is unresolved but is likely to play a role. Here, we collected 100 fecal samples from Ruddy Turnstones to investigate microbiome composition (who is there) and function (what they are doing) during stopover weight gain in Delaware Bay, USA. Using multiple molecular methods, we show that both taxonomic composition and function of the microbiome shifts during weight gain. We identified 10 genes that are associated with weight class, and polyunsaturated fatty acid biosynthesis in the microbiota is significantly increasing as birds gain weight. Our results support that the microbiome is a dynamic feature of host biology that interacts with both the host and the environment and may be involved in the rapid weight gain of shorebirds.


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Response to reviewers
Using 16S rRNA sequencing and metatranscriptomics, Grond et al. investigated variation in bacterial community composition and function across various weight classes of migratory ruddy turnstones during stopover in coastal Delaware. The authors suggest that weight gain and coincident changes in microbial communities indicate that the microbiome may play a role in the process of weight gain. A potential successional change in microbial taxa was detected, with medium-weight individuals clustering intermediately between low and high-weight individuals in a beta diversity ordination. Differentially abundant taxa were detected across each weight class, with previously known gut-associated bacteria being more abundant in light and medium weight class individuals. Ten genes (with functions related to cell growth, metabolism, tissue remodeling, etc.) were found to be differentially expressed in pairwise comparisons between weight classes. Clustering analysis detected four groups of genes with similar patterns of expression, highlighting genes that may function together in each weight class. The expression levels of several pathways related to lipid metabolism were found to increase across weight classes. This paper provides interesting insight into microbial community changes associated with the dramatic physiological process of weight gain during migration stopover. There are sections of the manuscript that would benefit from minor editing for clarity.

Minor issues
1. This study uses fecal samples as a proxy for processes occurring in the gut. Since it's known that fecal microorganism communities can be different than gut communities, it would be worth highlighting this potential discrepancy. It might also help explain some of the unexpected results (such as not seeing the expected increase in Firmicutes in the heavy weight class).
2. At the end of the introduction, outlining the expected outcomes of each of the study aims would be helpful to the reader.
3. Throughout the manuscript there are long sentences with lots of commas that are a bit difficult to read. Clarity could be improved by reorganizing and shortening these sentences. 4. The phrase "pre-migratory" is used throughout the manuscript, and it is at times unclear if the authors are referring to physiological changes occurring at the stopover sites or before the initiation of migration. Could a different term be used for each of these phases? 5. Line 28: Change "requires" to "require".
8. Paragraph at lines 85-103: Specifically mention that PUFAs are essential fatty acids. Essential fatty acids are defined, but it's unclear if the lipids present in Horseshoe crabs are essential fatty acids. 9. Line 99: Add (PUFA) after polyunsaturated fatty acids.
10. Line 117-199: The sentence that starts with "Long-distance migrations" is not worded clearly. 11. Line 456, 457: The phrasing "when they first land" and "immediate arrival" are a bit vague -is this referring to when they arrive at stopover sites? Or at the final destination of the migration?
Thank you for the comments on our manuscript; they have strengthened the final paper and we are grateful. Below are the specific changes we made to the manuscript to address reviewer comments (text from reviewer in black, our response in blue).
-Kirsten Grond, Artemis Louyakis, Sarah Hird Response To Reviewers 1. This study uses fecal samples as a proxy for processes occurring in the gut. Since it's known that fecal microorganism communities can be different than gut communities, it would be worth highlighting this potential discrepancy. It might also help explain some of the unexpected results (such as not seeing the expected increase in Firmicutes in the heavy weight class).
We added a few sentences to the discussion addressing this potential problem: "Potentially of note is that we used fecal samples in this study, which is a good, but imperfect, proxy for the avian gut microbiome [66]. This was due to our inability and unwillingness to sacrifice birds for their gut contents. In shorebirds, different gut compartments contain specific microbial communities [67] so further testing is required to determine whether the compositional changes in the gut were closer to our expectations than the fecal samples." 2. At the end of the introduction, outlining the expected outcomes of each of the study aims would be helpful to the reader. We added expected outcomes to the end of the introduction: "We expect to see changes in both taxonomic and functional microbial communities because of the bird's rapid weight gain and its associated physiological changes. In addition, we predict an increase in gene expression of pathways related to fatty acid metabolism as a mechanism to facilitate said weight gain." 3. Throughout the manuscript there are long sentences with lots of commas that are a bit difficult to read. Clarity could be improved by reorganizing and shortening these sentences.
We attempted to find difficult to read sentences but were generally happy with the readability of the manuscript and did not end up editing many sentences for this comment.
4. The phrase "pre-migratory" is used throughout the manuscript, and it is at times unclear if the authors are referring to physiological changes occurring at the stopover sites or before the initiation of migration. Could a different term be used for each of these phases? For clarity, we have changed the wording for each use of pre-migratory.
7. Line 70: Should be "extract more energy from a given amount". Thank you, corrected.
8. Paragraph at lines 85-103: Specifically mention that PUFAs are essential fatty acids. Essential fatty acids are defined, but it's unclear if the lipids present in Horseshoe crabs are essential fatty acids. Thank you, wording has been clarified.
10. Line 117-199: The sentence that starts with "Long-distance migrations" is not worded clearly.
Thank you, wording has been clarified.
11. Line 456, 457: The phrasing "when they first land" and "immediate arrival" are a bit vague -is this referring to when they arrive at stopover sites? Or at the final destination of the migration? Thank you, wording has been clarified. Your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication. For your reference, ASM Journals' address is given below. Before it can be scheduled for publication, your manuscript will be checked by the mSystems production staff to make sure that all elements meet the technical requirements for publication. They will contact you if anything needs to be revised before copyediting and production can begin. Otherwise, you will be notified when your proofs are ready to be viewed.
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