**5. Succession of intestinal microbiota of a Eurasian wigeon while spending a winter**

In order to examine the stability of the intestinal bacterial communities of other birds, we examined the succession of intestinal bacterial communities in the feces of a Eurasian wigeon, which was flying to the northern part of Osaka at the beginning of the winter season, staying in the same area when spending a winter. The Eurasian wigeon (*Mareca penelope* or *Anas penelope*) is about 50 cm in length. It breeds in the northern part of the Eurasian Continent, and in winter, it crosses southern Europe, North Africa, and East/South Asia. The global population of Eurasian wigeons is estimated at 2.8–3.3 million individuals [37]. This bird lives primarily in quiet sea, estuaries, lakes, and rivers. In addition to eating plants such as grass leaves and algae, it eats aquatic insects and mollusks. The number of wigeons observed in Japan is reportedly 180,000 per year.

In December 2017, when the researchers flew to Japan, we examined the succession over time with monthly intestinal bacteria of Eurasian wigeon around the Ai River in north Osaka (**Figure 4**). Community analysis at the class level revealed that Clostridia constituted 64.7% in December, but the proportion decreased to 18.4% in April 2018 (*P* < 0.01). The proportion of Bacilli, Fusobacteria, Alphaproteobacteria, and Gammaproteobacteria significantly increased from 0.3 to 7.4%, 1.2 to 10.4%, 1.2 to 6.3%, and 4.1–29%, respectively (*P* < 0.01), while spending a winter in Japan. The intestinal bacterial community composition of Eurasian wigeon, which flew to Japan at the beginning of the winter season, significantly changed while staying in Japan for about 4 months.

We also compared the intestinal microbial communities of Eurasian wigeon in December and April at different sites and in different years. We used samples collected from different sampling sites, Lake Biwa in 2016 and Ai River in 2017, as samples in the early winter. As samples for the spring season, fecal samples were collected from Biwa Lake and Ai River between 2016 and 2018.

*Dissemination of Intestinal Microbiota by Migratory Birds across Geographical Borders DOI: http://dx.doi.org/10.5772/intechopen.82707*

#### **Figure 4.**

*Changes in the bacterial community composition of fecal samples from the Eurasian wigeon while staying in western Japan. Fecal samples were collected from around Ai River, north Osaka, Japan.*

#### **Figure 5.**

*Comparison of the bacterial community composition of fecal samples from the Eurasian wigeon in the early winter (December) and spring (April) seasons. Fecal samples were collected from around Ai River and lake Biwa, Japan in different years.*

All samples in December were dominated by Clostridia, and samples in April were dominated by Gammaproteobacteria. Regardless of the differences in the location and year of sampling, the major bacterial communities were found to be similar. PCA revealed obvious difference in the bacterial community—namely, the samples in December were distributed in gray (right panel) and the samples in April shifted to the left panel in **Figure 5**. These results revealed that the intestinal bacterial community composition of Eurasian wigeon, which flew to Japan at the beginning of winter, changed while they spend a winter in Japan.

### **6. Colistin-resistant bacteria associated with Eurasian wigeon**

The dissemination of antibiotic-resistant bacteria across borders has become an important issue in public health, and the involvement of migratory birds has been indicated as a mechanism by which resistant bacteria spread at the global level [13]. In addition, colistin is regarded as an important antimicrobial agent; it is even an

#### **Figure 6.**

*Frequency distribution of the number of colistin-resistant E. coli (a) and coliform (b) in fecal samples of Eurasian wigeon. Fecal samples were taken around Ai River.*

antibiotic used as the last resort for carbapenem-resistant Enterobacteriaceae bacteria at the World Health Organization. Thus, the succession of number of colistinresistant *E. coli* and coliform in Eurasian wigeon was investigated in this study.

The frequency distribution of the number of colistin-resistant *E. coli* per gram of the sample is shown for each count range in **Figure 6**. In December 2017, the proportion of fecal samples below the detection limit was approximately 50%, and >100 CFU/g was about 50%. In March, the proportion gradually increased to approximately 70%, about 90% at the beginning of April, and 100% at the end of April 2018. For the colistin-resistant coliform, it was also found that the proportion of samples below the detection limit increased gradually while spending a winter in Japan. These results suggest that colistin-resistant *E. coli* and coliform may have been carried over to Japan after ingestion by the Eurasian wigeon in the northern area.

## **7. DNA barcoding and diet**

DNA barcoding is a technique that allows identification of species by using a short nucleotide sequence of a specific gene region. By using a gene region that reflects the difference in species as a standard DNA barcode, it has become possible to specify species. This method can be used to identify species of plants, animals, and fungi. It also helps to discover new varieties by combining with other information.

For animals, about 650 bases in length at the 5′ end of the cytochrome C oxidase subunit I (*COI*) gene on the mitochondrial genome is regarded as a standard barcode region, but the primer used differs, depending on the research project [38, 39]. The reason that the *COI* of mitochondria was selected as a standard barcode region of DNA barcoding of animals is that the universal primers are available to cover most taxa of the animal kingdom and the region contains several mutations at the species level. With a few exceptions, the cells of all eukaryotic species contain the mitochondria. The mitochondrial genome comprises a double-stranded DNA molecule of approximately 16-kb length, accounting for 1–2% of the total DNA in mammalian cells.

*Dissemination of Intestinal Microbiota by Migratory Birds across Geographical Borders DOI: http://dx.doi.org/10.5772/intechopen.82707*


#### **Table 2.**

*Representative primers used for DNA barcoding.*

On the other hand, for plants, a few mitochondrial interspecific mutations and *COI* cannot be used for species-level identification. Therefore, researchers propose the use of the chloroplast DNA region in plants, with *rbcL* and *matK* as standard barcode regions of terrestrial plants [40].

For analyzing the intestinal contents of various types of wildlife, the DNA barcoding method described above can be used in combination with NGS [41, 42]. Representative primers used for DNA barcoding were shown in **Table 2**. Various primer sets are available from the following site (http://www.boldsystems.org/ index.php/Public\_Primer\_PrimerSearch).
