Comparative analysis of the composition and function of fecal‐gut bacteria in captive juvenile Crocodylus siamensis between healthy and anorexic individuals

Abstract The Siamese crocodile (Crocodylus siamensis) is a freshwater, endangered crocodile with high economic value in the farming industry. Gut microflora plays an essential role in host physiological activity, and it contributes significantly to both the health and diseased states of animals. However, thus far, no study has focused on the correlation between diseases and intestinal bacterial communities in crocodilians. Here, we first compared the composition and function of gut microbial communities in captive juvenile C. siamensis suffering from anorexia and healthy crocodile controls using deep amplicon sequencing. The gut microbial diversity of anorexic crocodiles was much lower than the healthy individuals. Obvious changes in gut microbial composition were observed between sick and healthy crocodiles, except for Cetobacterium somerae of phylum Fusobacteria. In particular, the abundance of Bacteroides luti, Clostridium disporicum, Plesiomonas shigelloides, and Odoribacter sp. in the gut flora of healthy crocodiles was distinctly higher than the diseased group. Conversely, the species Edwardsiella tarda was overrepresented in the gut of anorexic crocodiles compared to the healthy group. Furthermore, in anorexic crocodiles, the predicted microbial functions that were related to amino acid metabolism, biosynthesis of other secondary metabolites, nucleotide metabolism, replication and repair, and translation were significantly reduced, while signal transduction was significantly enriched. These findings of the present study provide a reference to enrich the field of gut microorganism studies in crocodilians and suggest that alterations in the composition and function of gut bacteria in C. siamensis juveniles may be associated with anorexia in crocodiles.


| INTRODUC TI ON
The Siamese crocodile (Crocodylus siamensis) is an endangered freshwater crocodilian that is native to most countries in Southeast Asia, including Cambodia, Laos, Indonesia, Thailand, and Vietnam (Bezuijen et al., 2013). As far back as the mid-1980s, wild crocodiles were exported from Cambodia to China as farm animals (Guo et al., 2018). In China, successful breeding of C. siamensis only occurs in a few southern provinces, such as Fujian and Hainan, because wild crocodiles have particular climate and temperature requirements for a suitable habitat (Guo et al., 2018). In addition to the requirements of the environmental conditions, bacterial diseases have also severely restricted the development of the crocodile farming industry (Camus & Hawke, 2002;Kim, Lee, & Kwak, 2016;Roh et al., 2011). The captive Siamese crocodile is universally acknowledged as "soft gold in aquatics" because it has significant economic benefits. Their skin is used in the leather industry, and their blood has potential effects in antibiotic therapy (Leelawongtawon, Siruntawineti, Chaeychomsri, & Sattaponpan, 2010). Their oil is used for medical treatment, and crocodiles are beneficial to the tourism industry (Li et al., 2012;Ryan, 1998).

The intestinal tract is an indispensable digestive organ that plays
a key role in the defense of animals' immune system, and it is where considerable amounts of microbial flora colonize (Eckburg, 2005).
Normally, the gut microflora is interdependent and interactive, which maintains the homeostasis of the internal environment, and it greatly influences the physiological activities of the host (Hooper, 2001). The composition and structure of the vertebrates' gut microbial communities are influenced by multiple factors (such as diet and environmental conditions) that also contribute to disease (Feng, Chen, & Wang, 2018;Scott, Gratz, Sheridan, Flint, & Duncan, 2013;Sharpton, 2018).
Prior studies on the gut bacterial communities of nonmammalian vertebrates have performed on birds, fish, amphibians, and reptiles (Colston & Jackson, 2016;Waite & Taylor, 2015). However, so far, the study on crocodilian's gut microbiome is still scarce, there was only one crocodilian species, American alligator (Alligator mississippiensis), which has been reported (Keenan, Engel, & Elsey, 2013). In those alligators, Fusobacteria was a unique and core flora of the gut microbiome, which is distinguished from other reptiles' gut microbiome (mainly consisted of Firmicutes and Bacteroidetes; Colston & Jackson, 2016;Keenan & Elsey, 2015). To expand the understanding of gut microflora of crocodilians, here we perform the 16S rRNA gene amplicon sequencing to compare the diversity of gut bacteria in healthy and anorexic Siamese crocodiles. The data observed from this work will elucidate the basic composition and function of the gut microbial communities in farmed crocodiles, and it will identify key bacteria that may influence the healthy growth of crocodilian.

| Sample collection
In May 2016, a sudden disease occurred in captive Crocodylus siamensis juveniles (1-year-old), which was observed at the Xiamen Lonsun crocodile zoo in Fujian Province. The clinical symptom of the sick Siamese crocodiles was anorexia (apparent decrease in daily feeding activity) with no trauma. The experimental crocodiles (0.65-0.78 m, 1.04-1.37 kg) were divided into two groups: the healthy group (labeled as H, n = 3) and the diseased group (labeled as D, n = 3). Six crocodiles were fed the same diet and reared in individual feeding areas before sampling. Cloacal swabs were used for sampling the crocodile gut flora which were an acceptable source for nondestructive sampling the reptiles' intestinal microbiota (Colston, Noonan, & Jackson, 2015;Jiang et al., 2017;Johnston, Porter, Scott, Rhodes, & Webster, 2010). The cloacal samples of the H group were labeled as H1-H3, while the cloacal samples of the D group were labeled as D1-D3. Specimens were stored in liquid nitrogen and immediately transported to the laboratory for DNA extraction.

| DNA extraction
The total bacterial genomic DNA was extracted directly from each frozen sample (220 mg) using the PowerFecal ® DNA Isolation Kit (Qiagen), following the manufacturer's protocol. The quality and integrity of each DNA extraction was determined using 1% agarose gel electrophoresis before deep sequencing.

| Deep amplicon sequencing
The genomic DNA was sequenced by the Majorbio Bio-technology Company using the Illumina MiSeq PE300 platform (Illumina).

| Bioinformatic and statistical analysis
Raw amplicon sequences obtained by deep sequencing were demultiplexed, quality-filtered, and analyzed by using the software Mothur v1.35.1 (Schloss et al., 2009). All unqualified sequences, such as joint pollution, primer mismatches, low complexity, incorrect barcodes, and ambiguous bases, were discarded. Reads with a Q (base quality score) <20 and tags with less than 80% of the total base number were also removed. After the filtering and trimming procedures, all unique tags observed from each group were clustered into operational taxonomic units (OTUs) with a 3% distance level using Usearch v7.0 software (Edgar, 2010). Finally, all OTUs were classified taxonomically through the Ribosomal Database Project (RDP) Classifier, which is based on Naive Bayesian, with an 80% confidence threshold (Wang, Garrity, Tiedje, & Cole, 2007). As a result, the ACE, Chao1, Shannon, and Simpson indexes were calculated using Mothur v.1.35.1 software. The principal coordinates analysis (PCoA, weighted UniFrac distances) and bacterial taxa analysis were calculated and drawn in R v3.5.2 software. The alpha diversity indexes and relative abundance of gut microbial communities (phylum and genus level) that were identified from healthy (n = 3) and diseased (n = 3) groups were comparatively analyzed by Student's t test, and p < .05 was considered significant. Linear discriminant analysis (LDA) effect size (LEfSe) was employed to determine the key contributors of gut bacteria in healthy and anorexic crocodiles, and the LDA score threshold was 3.5 (Segata et al., 2011). Furthermore, PICRUSt analysis via the Kyoto Encyclopedia of Gene and Genomes (KEGG) database was used to predict functional profiles of gut bacteriome in healthy and diseased crocodiles (Langille et al., 2013). STAMP v2.1.3 software was used to statistically analyze the gene functions using Student's t test with Bonferroni correction (Parks, Tyson, Hugenholtz, & Beiko, 2014). A q-value (adjusted p) < .05 with an effect size >0.2 was considered significant. A brief description of the total bioinformatic analyses of crocodile gut genomic DNA is shown in Figure 1.

| Sequence survey
A total of 258,748 valid reads were gathered from cloacal samples from 6 individual crocodiles. The number of effective unique tags ranged from 38,125 to 48,216 per sample, resulting in 168 OTUs with same sequence similarity values of 97% (Table 1). The number of OTUs obtained from each sample ranged from 51 to 88. In this study, 99.98%-99.99% coverage of species was obtained in all samples, which demonstrates that the majority of the bacterial phylotypes present in the specimens were identified.

| Alpha and beta diversity
The rarefaction curve of the D group quickly reached the saturation plateau under 97% similarity values, which indicates lower species richness compared to the H group ( Figure 2a). The Shannon indexes of the gut communities (Table 1; Figure 2b) in the H group were significantly higher (p = .021) than in the D group, and the Simpson indexes (Table 1)    Step 6 OTU cluster

| Taxonomic composition and comparison
Step 5

Data filtering
Step 4 Sequencing Step 3

DNA extraction & PCR amplification
Step 2

Sampling
Step 1

| Significant alterations of the gut microbial community in healthy and diseased Siamese crocodiles
In this study, LEfSe analysis was employed to identify any key contributors that have a statistically significant role in the H and D groups. These data were calculated and analyzed by the nonpara-

| Comparison of the functional profiles of gut flora from healthy and diseased Siamese crocodiles
The present study used PICRUSt analysis to predict the major gene functions to determine the functional profiles of the gut microflora in the H and D groups. The identified genes that were predicted in all specimens, which were primarily involved in the

| D ISCUSS I ON
Gut microbiota is generally recognized as an indivisible "organ" of the host that is closely related to various diseases in animals ( found to be the core gut communities of most amphibians and reptiles (Bletz et al., 2016;Colston et al., 2015;Jiang et al., 2017;Kohl et al., 2016). However, the Fusobacteria were dominated in the gut microbiota of freshwater American alligators, which is consistent with the current study (Keenan et al., 2013). As a result, there was a substantial alteration in the gut microbial composition between anorexic crocodiles and healthy crocodiles. An exception was the species C. somerae because it was the most dominant bacterium identified in both the healthy and diseased groups. C. somerae was first isolated from human feces, and it has been universally identified in the gut of freshwater fish (Bledsoe, Peterson, Swanson, & Small, 2016;Larsen, Mohammed, & Arias, 2014;Lin et al., 2019). It has been found to produce vitamin B12 and acetic acid, which are beneficial for the host (Finegold et al., 2003;Tsuchiya, Sakata, & Sugita, 2008).
However, C. somerae do not seem to be strongly associated with healthy Siamese crocodiles.
The proportion of Bacteroidetes and Firmicutes in the healthy group was higher than it was in the diseased group. Among the Bacteroidetes, Bacteroides was the most abundant genera in the healthy group, and the species B. luti was significantly enriched compared to the diseased group. Bacteroides sp. is a component in the gut flora of various vertebrates, including carps, cottonmouth snakes, and crocodile lizards, and B. luti was first isolated from methanogenic sludge (Colston et al., 2015;Hatamoto, Kaneshige, Nakamura, & Yamaguchi, 2014;Jiang et al., 2017;Li et al., 2015).
However, the effect of B. luti on the host-bacteria ecosystem is vastly underexplored. Odoribacter was also a core genus of the Bacteroidetes phylum observed in this study, and it demonstrated a positive correlation with crocodile health. This was closely related to the human gut microflora and may improve host metabolism (Kulagina et al., 2012;Lim et al., 2017). Firmicutes was the third-most dominant phylum identified in healthy crocodile cloacal samples, which mainly consisted of the genus Clostridium. According to previous studies, most Clostridium sp. can produce a type of fatty acid called butyrate, which provides many benefits to the health of the host gut (Hamer et al., 2009;Pryde, Duncan, Hold, Stewart, & Flint, 2002). The Clostridium organisms observed in the healthy group had the highest bacterial diversity, which included 9 different kinds of Clostridium species (all mean relative abundance >0.1%). However, only C. disporicum reached statistical significance when compared to the diseased group. C. disporicum is an uncommon, fermentative, and anaerobic gut bacterium, and it is primarily found in mammal feces, such as rats and pigs (Horn, 1987;Su, Yao, Perez-Gutierrez, Smidt, & Zhu, 2008). It has been found to be related to the degradation of complex organic macromolecules (Vilajeliu-Pons et al., 2015).
Moreover, we found that the rarely known T. petrolearius was also a major component in the gut of healthy crocodiles' group. T. petrolearius was first isolated from oilfields, and this is the first report that suggests this microbe is associated with animal gut flora (Deng et al., 2015). Interestingly, the present study found that the human pathogen P. shigelloides in the phyla Proteobacteria was also significantly enriched in the cloacal samples of healthy crocodiles (Chen et al., 2013). This appears to be a normal component of the gut flora of aquatic animals (Johnston et al., 2010;Larsen et al., 2014;Silva, Brito, Farias, & Nicoli, 2005). In contrast, the intestinal microflora of diseased Siamese crocodiles was significantly enriched in E. tarda, which belongs to the phyla Proteobacteria. E. tarda is widely known as a zoonotic pathogen, and it is generally found The linear discriminant analysis (LDA) effect size (LEfSe) analysis that shows the significant differences in gut flora between healthy and diseased groups. (a) Cladogram plot demonstrating the significant gut bacteria in both healthy and diseased groups. (b) Highly significant bacterial species with an LDA score >3.5 in animals of an aquatic environment, such as bullfrogs, alligators, and eels (Johnston et al., 2010;Lin et al., 2019;Mauel, Miller, & Frazier, 2002). In addition, E. tarda can infect a broad range of hosts and cause various diseases, which most frequently present as gastroenteritis and septicemia (Leung, Siame, Tenkink, Noort, & Mok, 2012;Miyazawa et al., 2018;Wang et al., 2005). related to higher energy consumption, which is required to fulfill the normal growth of the host. When some of the diseased crocodiles suffered from anorexia, there was a significant decrease in several metabolic functions of the gut microbes which were compared to the healthy controls. Besides, some functional pathways of cellular processes (including replication, repair, and translation) were also significantly reduced in the gut of anorexic crocodiles. Based on the prior work, it has been shown that the signal transduction system contributed to antibiotic resistance, biofilm formation, environmental persistence, virulence protein, and pathogenicity in E. tarda (Lv et al., 2012). Therefore, a high abundance of E. tarda may be strongly associated with an increase in the signal transduction pathway, and this may contribute to the negative effect observed in sick crocodiles. Thus, we hypothesized that during the growth stage of the captive Siamese crocodiles, some juvenile individuals might have been infected with some pathogens such as E. tarda, and then abnormal alterations of composition and function of healthy crocodiles' gut bacteria might be related to the disease by causing anorexia.
In conclusion, the present study is the first to report the compo-

ACK N OWLED G M ENTS
This study was financially supported by Regional Demonstration of Marine Economy Innovative Development Project (No.
16PZY002SF18), Natural Science Foundation of Fujian Province (No. 2019J01695), and National Natural Science Foundation of China (No. 31202030 and No. 31272669).

CO N FLI C T O F I NTE R E S T S
None declared. Writing-original draft: CXZ (equal) and ML (equal). All authors reviewed and edited the manuscript, and gave the final approval for publication.

E TH I C S S TATEM ENT
This study complied to the guidelines for the kindly care and use

DATA AVA I L A B I L I T Y S TAT E M E N T
All raw sequencing data obtained in this study were deposited in the Sequence Read Archive of the National Center for Biotechnology Information (NCBI), and the SRA submission data are SRX4396839-SRX4396844.