Dong et al. AMB Expr (2018) 8:192 https://doi.org/10.1186/s13568-018-0722-0 ORIGINAL ARTICLE Open Access Comparative analysis of the intestinal bacterial community and expression of gut immunity genes in the Chinese Mitten Crab (Eriocheir sinensis) Jing Dong1, Xiaodong Li1,2, Ruiyang Zhang1, Yingying Zhao1, Gaofeng Wu1, Jinling Liu1, Xiaochen Zhu1 and Lin Li1* Abstract Remarkably little information is available about the interaction between the gut microbiota and intestinal immunity in fsh and crustaceans. In our study, we used Illumina MiSeq sequencing and real-time quantitative PCR to compare the microbial community and immunity genes expression in the foregut, midgut and hindgut of Chinese mitten crab (Eriocheir sinensis). Our results indicated that the community richness of the midgut is higher than in the foregut or the hindgut, although the bacterial diversity in the hindgut is higher. The predominant phyla were Tenericutes and Firmicutes in the foregut, Tenericutes and Proteobacteria in the midgut and Proteobacteria, Tenericutes and Bacteroidetes in the hindgut. When compared with the midgut, the expression of antimicrobial peptides (AMPs) were signifcantly elevated in the hindgut (P < 0.05), and the gene expression of EsRelish (IMD pathway) was higher than the Toll sign- aling pathway genes. Actinobacteria and Lactobacillus had negative correlationship with the expression of AMPs, although Acinetobacter, Bacteroides, Flavobacterium can up-regulate the expression of AMP genes. Collectively, our data indicate that microbiota are site-specifc within the digestive tracts of crabs and the bacterial community and intestinal immunity have a close relationship in E. sinensis. Keywords: Eriocheir sinensis, Intestinal bacterial community, Gut immunity, Antimicrobial peptides, Gene expression Introduction and other illnesses (Abreu 2010; Olmos et al. 2010). Te connections between gut fora and host was one of Tere is a lot of information and fast growing about this the most important impactors infuence aquatic animal topic. Comparative analysis of the intestinal bacterial health (Chaiyapechara et al. 2012; Yan et al. 2012). Te community and expression of gut immunity genes is very intestinal fora is vital to the development (Shang et al. important. 2017), immunity and disease resistance of gut (Cerf- In vertebrates and invertebrates, antimicrobial pep- Bensussan and Gaboriau-Routhiau 2010). Te digestive tides (AMPs) play a crucial role in gut immunity tract is the richest part of human body in terms of num- (Destoumieux-Garzón et al. 2016; Zhang and Gallo ber and diversity of bacterial species (Quigley 2013). Te 2016) and form efective defense mechanisms against dysregulation of intestinal bacteria was closely related to multitudinous pathogens. Many studies on AMPs chronic infammatory and epithelial barrier dysfunction and invertebrate gut defense have been performed in insects, especially Drosophila melanogaster, because this species is the model organism of invertebrate *Correspondence: [email protected] (Broderick 2016; Loch et al. 2017; Rolf and Schmid- 1 Liaoning Provincial Key Laboratory of Zoonosis, College of Animal Science & Veterinary Medicine, Shenyang Agricultural University, Hempel 2016). Te key role of AMPs in gut immune Shenyang, Liaoning 110866, People’s Republic of China response is demonstrated through oral reactive oxy- Full list of author information is available at the end of the article gen species (ROS) blockers to Drosophila mutants © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat​iveco​mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Dong et al. AMB Expr (2018) 8:192 Page 2 of 12 that are defective in the NF-κB pathway, resulting in Materials and methods increased mortality (Ryu et al. 2006). Tis mortal- Crab culture and crab intestine collection ity rate decreases when a single AMP is expressed in Healthy female E. sinensis (9.5 ± 0.5) g were collected the intestines of these fies (Ryu et al. 2008). In addi- with permission from Panjin Guanghe Crab Industry Co., tion, closely regulated AMPs in the gut of Drosophila Ltd. (Panjin, Liaoning, China) in July 2016. In the labora- seem to be indispensable for maintaining the homeo- tory, crabs were fed with formula diets (Shenyang Hefeng stasis of the intestinal fora (Ryu et al. 2008). Te pro- Aquatic Feed Co., Ltd., Shenyang, Liaoning, China) in a duction of AMPs was afected by the Toll and IMD concrete recirculating aquaculture system with the fol- pathways in Drosophila hemocytes, but mainly by the lowing conditions: dissolved oxygen > 7.0 mg L−1, pH latter in gut (Garcia-Garcia et al. 2013). As the central 7.5–8.3, ammonia < 0.2 mg L−1, and nitrite < 0.01 mg L−1. components of Toll and IMD signaling pathways, e.g., Following acclimation (1 week), feeding was stopped 24 h EsTolls, EsDorsal, EsPelle, EsRelish (a novel NF-κB-like prior to handling and sampling to minimize the stress on transcription factor), these genes have been proven to E. sinensis. Because the tissue of the crab digestive tract participate in the innate immunity of Chinese mitten is very small, 24 crabs were used for the determination crab Eriocheir sinensis (Crustacea: Decapoda: Brach- of intestinal fora, the accumulation of 8 crabs is a sam- yura: Varunidae) and defense against several patho- ple and repeat three times for each sample. In addition, gens (Li et al. 2010; Ying et al. 2015; Yu et al. 2013a, b). 6 crabs were used for the determination of intestinal Te above genes could function as an adapter protein immune gene expression, the accumulation of 2 crabs is in Toll and IMD signaling and regulate the production a sample and repeat three times for each sample. First, of crab AMP genes in hemocytes (Mu et al. 2011; Ying washed the body surface with sterile water. Second, dis- et al. 2015; Zhang et al. 2010). Ten there were AMP infected the outside of the body with 75% ethanol for genes in hemocytes have various biological functions 2 min. Tird, dissected the crab in the aseptic state to in E. sinensis (Destoumieux-Garzón et al. 2016). Hence, remove the digestive tract and were then divided into these AMPs can help the screening of therapeutic or three segments (foregut, midgut, hindgut). Samples were preventive agents for healthy breeding of crabs. How- put into sterile EP tubes and stored at − 80 °C for analysis ever, the function of these very important genes in the of gut microbiota and expression of gut immune gene. crab intestine is not clear. Te study of AMPs in gut immunity of crustaceans is rare and it is unclear which DNA isolation, PCR amplifcation and Illumina MiSeq signaling pathways regulate the production of AMPs. sequencing Te E. sinensis is widely cultivated in China and other Te digestive tract samples were taken for DNA extrac- countries because of rich nutritional value and high eco- tion (0.25–0.3 g wet weight) using the E.Z.N.A.® Soil nomic value (Chen and Zhang 2007). Te culture of E. DNA Kit (OMEGA Bio-tek, Norcross, GA, US). We used sinensis has increased dramatically in the past decade fol- PCR to amplify the V3–V4 regions of the bacterial 16S lowing a rise in demand. Current production in China is rRNA gene. Te primers used in the present study were 820,000 tons with an output value of more than 50 bil- 338F (5′-barcode-ACT CCT ACG GGA GGC AGC AG-3′) lion in 2015 (China Fishery Statistics Yearbook 2016). In and 806R (5′-GGA CTA CHVGGG TWT CTAAT-3′) recent years, frequent outbreaks of diseases have resulted (amplicon length: 468 bp), 20 μL of PCR mixture con- the decreased production and economic losses (Ding taining 5× FastPfu bufer (4 μL), 2.5 mM dNTPs (2 μL), et al. 2017). Accordingly, considerable efort has been 5 μM forward primer (0.8 μL), 5 μM revese primer expended on intestinal health research of E. sinensis. In (0.8 μL), FastPfu Polymerase (0.4 μL), and template DNA the present study, we hypothesized that diferent regions (10 ng). 16S rRNA sequencing were conducted on Illu- of the digestive tract in E. sinensis have diference in the mina MiSeq platform. Paired-end sequencing 2 × 300 bp intestinal epithelium bacterial communities and that may was performed to sequence all libraries using an Illumina be related to the diferent expression of digestive tract MiSeq platform according to standard protocols. immune genes. Te goal of our research was to explore the bacterial diversity, gut immunity genes expression Illumina MiSeq sequencing and data processing and the relationship between the two in the various We used the QIIME (version 1.17 http://qiime .org/) regions of the E. sinensis digestive tract. Our results sug- package to conduct sequences processing. UPARSE (ver- gest a role for the dominant bacteria in each region of the sion 7.1 http://drive 5.com/upars e/) and UCHIME were digestive tract and provide novel insight into the bacterial used respectively to cluster operational taxonomic units community and gut immunity of E. sinensis. Tese data (OTUs) with a 97% similarity cutof and chimera identi- will be helpful to understand the relationships between fcation. Te diversity and richness of bacterial commu- symbiotic bacteria and the host. nity were identifed by the ACE, Chao1, Shannon and Dong et al. AMB Expr (2018) 8:192 Page 3 of 12 Simpson, which were conducted utilizing the Mothur those of Es-β-Actin, and the 2−∆∆Ct method was adopted program (version v.1.30.1 http://www.mothu r.org/wiki/ to data analysis using the light cycler 480 software.