Gut microbiome of migratory shorebirds: Current status and future perspectives

Abstract Migratory shorebirds have many unique life history characteristics, such as long‐distance travel between breeding sites, stopover sites, and wintering sites. The physiological challenges for migrant energy requirement and immunity may affect their gut microbiome community. Here, we reviewed the specific features (e.g., relatively high proportion of Corynebacterium and Fusobacterium) in the gut microbiome of 18 migratory shorebirds, and the factors (e.g., diet, migration, environment, and phylogeny) affecting the gut microbiome. We discussed possible future studies of the gut microbiome in migratory shorebirds, including the composition and function of the spatial‐temporal gut microbiome, and the potential contributions made by the gut microbiome to energy requirement during migration.

lenges (e.g., body mass loss and partial atrophy of the gastrointestinal tract [McWilliams & Karasov, 2001;Piersma et al., 1999]) may affect the host-microbe interactions and lead the changes in the gut microbiome community (e.g., Cho & Lee, 2020;Grond et al., 2019;Risely et al., 2017Risely et al., , 2018. The gut microbiome is defined as a combination of all the genomes of microorganisms in a specified environment (Grond et al., 2018). The gut microbiome plays an important role in the activities of the host (Kohl, 2012). Understanding the importance of the gut microbiome to bird host physiology and health is important, and studies have focused on the formation and maintenance of the gut microbiome and its effects on host fitness (Grond et al., 2018;Rooks et al., 2014;Waite & Taylor, 2015). Thus, the current studies on the gut microbiome of the migratory shorebirds mainly focus on the composition and specific microbes associated with the extreme physiological challenges during migration. Here, we reviewed the specific features of the gut microbiome of 18 migratory shorebirds (Table 1) and discuss possible future studies of the gut microbiome in migratory shorebirds.
Thus, a high proportion of Corynebacterium in the recently arrived migrants may enable migrating shorebirds to maximize fat deposition (Risely et al., 2017(Risely et al., , 2018. This specific feature in the migrant gut microbiome may also be associated with the physiological changes during the migration or an intestinal immune response (Risely et al., 2017 2018). However, the strain level and real function of Corynebacterium in the migratory shorebird gut still not know and need to be investigated in the experiments (e.g., germ-free animal models).
Moreover, the analysis of the published 16S rRNA gene data of the migratory shorebirds showed Corynebacterium was mainly enriched in Calidris shorebirds in Australia ( Figure 2a). The abundance of Corynebacterium in the Calidris shorebirds in other regions (e.g., Arctic, Greenland, and US regions) was low ( Figure 2a). Considering Corynebacterium was rare in the living environment (e.g., sediment [Risely et al., 2017]), we speculated that the different physiological stages (e.g., recently arrived migrants vs. breeding stage) might explain this difference in the abundance of Corynebacterium among these geographic populations. However, the difference in the dietary composition and living environment would also affect the migrant gut microbiome composition and function. Thus, we still need to investigate that whether this finding was the common pattern in the recently arrived migratory shorebirds along with different geographic populations.

| A HI G H PROP ORTI ON OF FUSOBAC TERIUM IN THE G UT MI CROB I OME OF THE MI G R ATORY S HOREB IRDS P OTENTIALLY REL ATED TO THE C ARNIVOROUS DIE T
A high proportion of Fusobacteria (e.g., Fusobacterium) was identified in the gut microbiome of the migratory shorebirds in the Arctic region (Grond et al., 2019). The analysis on the published 16S rRNA gene data of the migratory shorebirds revealed another specific feature that most of the migratory shorebirds across different geographic populations (e.g., Australia, US, and Arctic regions) harbored a high proportion of Fusobacteria (mean abundance about 20 percent) ( Figure 2b). Fusobacteria is the Gram-negative and non-spore-forming anaerobes and may produce endotoxins (Tan et al., 1996;Warner et al., 1975). Fusobacterium is the common members of the gut microbiome in birds (Hird et al., 2015; The analysis of the published bacterial 16S ribosomal RNA gene data (using high-throughput sequencing) of the migratory shorebirds.  Table 1. The trimmomatic function was used for quality control on these raw datasets (Bolger et al., 2014). Operational taxonomic units (OTU) were defined as sharing >97% sequence identity by searching clean sequences against the SILVA132 database by pick-up-closed-OTU methods (Christian et al., 2013). Taxon summary was created using the OTUs table (2,000 sequences per sample) in QIIME 1

| A HI G H PROP ORTI ON OF OPP ORTUNIS TI C PATHOG EN S IN THE G UT MI CROB I OME OF THE MI G R ATORY S HOREB IRDS
Several bacteria, considered to be opportunistic pathogens, can cause bird morbidity and mortality (Kohl, 2012). The potentially pathogenic bacteria include Gram-negative Campylobacter and Helicobacter in the migratory shorebird species (e.g., Calidris species) (Grond et al., 2020;Risely et al., 2018;Ryu et al., 2014;Santos et al., 2012). The analysis uncovered that several migratory shore- and their contradictory (pathogenic potential and energy harvest), the investigation on the function of migratory shorebirds' gut microbiome should focus on the interaction between the microbes and the host-microbe interaction.

| HOS T PHYLOG ENY AND P OTENTIAL SO CIAL CONTAC T AFFEC TING THE G UT MI CROB I OME COMMUNIT Y OF THE MIG R ATORY S HOREB IRDS DURING SYMPATRIC CLUS TERING
Most of the current research of the bird gut microbiome focus on nonmigratory birds, and in these species, host system development is the main driving force for the diversity of intestinal microbes (Grond et al., 2018;Waite & Taylor, 2015). The microbiome is a trait of the host species, and the evolutionary relationship between them and the potential shorebird phylogeny is assessed by comparative phylogenetic methods (Capunitan et al., 2020). The gut microbiome community may differ among different species of migratory shorebirds. Here, we re-analyzed the raw dataset of eight migratory shorebird species in the Arctic region (Grond et al., 2019) and found a significant difference in the abundance of some gut microbiome among these species (Figure 3a). For example, Actinobacteria was enriched in Phalaropus lobatus, and Proteobacteria was enriched in Calidris melanotos. However, NDMS analysis using unweighted UniFrac distance displayed the mixed pattern in the gut microbiome community among these eight species (Figure 3b). These findings indicated the partial or weak effect on the gut microbiome by host phylogeny, and other factors might also affect the gut microbiome community.
The impact of shared habitat on the microbiota may be manifested in the sympatric clusters of migratory birds (Lewis et al., 2017). Social contact among intraspecies and interspecies may lead to the gut microbiome transmission and affect the gut bacterial communities in individual animals (Antwis et al., 2018;Archie & Tung, 2015;Grond et al., 2014;Kwong & Moran, 2016;Ryu et al., 2014). Different species of migratory shorebirds have direct or indirect contact because they share habitat and food during their migrations or breeding (Cho & Lee, 2020;Grond et al., 2019;Risely et al., 2017Risely et al., , 2018Ryu et al., 2014;Santos et al., 2012). Therefore, we speculated that the effects of social contact on the gut microbiome community of the migratory shorebirds might also modify the effects contributed by host phylogeny.

F I G U R E 3
The re-analysis on the gut microbiome community of eight migratory shorebirds (Grond et al., 2019).  Risely et al. (2017) found red-necked stints (Calidris ruficollis) acquired little environmental microbiome (about 0.1% of gut microbiome putatively from foraging sediment) (Risely et al., 2017). This indicated that these migratory shorebirds did not acquire microbes from the sediment during migration (Risely et al., 2017). This migratory shorebird species may resist the invasion of environmental microorganisms, which allows them to maintain a stable gut microbiome during migration from the breeding area to wintering sites (Risely et al., 2017). This study raised an interesting question on the gut microbiome transmission between the living environment (e.g.,  (Grond et al., 2019). The migratory shorebirds, during migration, would face different environments (e.g., winter sites, stopover sites, and breeding sites) (Gill et al., 2009). Currently, only one pilot study has tried to evaluate the contribution of the sedimentsource microbiome to the gut microbiome of Calidris ruficollis in winter sites (Risely et al., 2017). Thus, the relationship between the potential dynamics of migratory shorebirds' gut microbiome during long-distance migration and the environment-source microbial contribution is still unknown.

| CON CLUS ION
Here, we reviewed that the current studies mainly reveal the community in the migratory shorebird gut microbiome, which was potentially associated with the physiological challenges and energy requirement during migration. Thus, the next main scientific question related to common patterns and the function of the gut microbiome will be important and necessary.

| Spatial-temporal patterns of migratory bird gut microbiomes
Currently, it is hard to conclude which factors were mainly lead to the difference in the gut microbiome community of the migratory shorebirds during migration due to the differences in sampling time  Table 1 changes in the composition of migratory shorebird gut microbiome and their living environment microbiome during migration, including premigration, migration, and end of the migration. This will be an international project, and it will face many challenges. The study will provide information on the relationship between the changes in the specific gut microbiota and migration. Studying the spatial-temporal changes of the gut microbiome during migration is significant for understanding the response of the dramatic physiological challenges and the survival status of migratory shorebird populations.

| The function of the gut microbiome in migratory shorebirds (e.g., energy requirements)
The function of the gut microbiome is particularly important for migratory shorebirds, as they face extraordinary metabolic, nutritional, and immune challenges during migration (Altizer et al., 2011;Grond et al., 2019;Risely et al., 2018;Wikelski et al., 2003). Before migration, dunlin and semipalmated sandpipers will quickly gain weight and deposit fat (Grond et al., 2020). The trade-off between microbial colonization and migratory bird energy requirement remains to be explored. Moreover, the function of gut microbiome (e.g., significant changes in some microbial group during migration) in the migratory shorebird gut needs to be deeply investigated in the experiments (e.g., germ-free animals).

ACK N OWLED G M ENTS
This study is supported by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

CO N FLI C T O F I NTE R E S T
The authors declared no conflicts of interest relevant to this manuscript.