**8. Migratory bird diet**

We focused on the DNA barcoding method to examine the relationship between diet and intestinal microbial communities in migratory birds. It can be hypothesized that diet—based on foods available in the area—has an influence, leading to change in the intestinal bacterial community of Eurasian wigeon and the number of colistin-resistant *E. coli* while spending a winter in Japan. Therefore, we performed NGS analysis on the intestinal contents of the Eurasian wigeon using eukaryotic *COI* gene as the target (**Figure 7**).

The *Orthocladiinae* sp. belonging to Arthropoda were found to be abundant at any time period, although there were differences in their proportion depending on the season. In December, *Demodex folliculorum* and *Orthocladiinae* sp., which belonged to Arthropoda, and *Adineta vaga*, which belonged to Rotifera, were abundant. Rhodophyta and Ochrophyta were observed only in the February samples. In the April samples, *Cricotopus annulator,* which belonged to Arthropoda, and *Adineta vaga*, which belonged to Rotifera, were abundant.

Pearson's correlation coefficients did not show any strong relationships between the number of both colistin-resistant *E. coli* and coliform and the diet. The abundance of Actinobacteria and Bacilli showed a significant but week correlation with abundance of *Scaptomyza montana* (*r* = 0.340, *P* < 0.05 and *r* = 0.417, *P* < 0.01, respectively). The abundance of Clostridia was negatively related with *Orthocladiinae* sp. (*r* = −0.372, *P* < 0.05) and *Scaptomyza montana* (*r* = −0.327, *P* < 0.05) and positively related with abundance of *Talaromyces pinophilus* (*r* = 0.409, *P* < 0.01).

**Figure 7.**

*Relative proportions of phylotypes of diet in the Eurasian wigeon.*

The abundance of Gammaproteobacteria was positively related with abundance of *Abrochtha sonneborni* (*r* = 0.354 *P* < 0.05) and *Orthocladiinae* sp. (*r* = 0.393, *P* < 0.05, respectively). All correlation coefficients were not strong, and thus other parameters or multiple parameters might affect the intestinal microbial communities.

Although the Eurasian wigeon mainly ingests terrestrial plants, *COI* sequences of terrestrial plants were less detected in this study. For organisms that feed on plants, it seems better to use the sequences of *rbcL* and *matK* on chloroplast DNA. In order to investigate the relationship between the intestinal microbial community and diet in wild birds, it is necessary to select appropriate primers suitable for particular types of living organisms that are consumed as part of the diet.
