Application of Roseovarius marinus in promoting the growth of Portunus trituberculatus

Abstract

The present invention provides the application of Roseovarius litoreus in promoting the growth of Portunus trituberculatus. Another aspect of the present invention provides another use of Roseovarius litoreus, which is the application in the preparation of products for promoting the growth of Portunus trituberculatus. The present invention also provides a feed for Portunus trituberculatus, and viable bacteria of Roseovarius litoreus are added to the feed. The present invention discovers that Roseovarius litoreus can significantly promote the growth of Portunus trituberculatus, further improve the weight gain rate and hepatopancreas index of crabs, and has broad application prospects in aquaculture.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial feed additives, and particularly relates to the application of Roseovarius litoreus in promoting the growth of Portunus trituberculatus. Background Art

[0002] The quality of gametes, nutrition, environmental conditions, and the interaction between the host and intestinal microorganisms are four key factors for improving the stable production of aquatic species. Relative to the great progress in the first three factors, the research on the interaction between the host and intestinal microorganisms is less. Research shows that the interaction between the host and microorganisms is of great significance to the health status, metabolism, and immunity of aquatic animals. Generally speaking, the microbial community structure of healthy animals has relatively high similarity among individuals and temporal stability. On the contrary, compared with healthy individuals, diseased animals have higher inter-individual microbial community variability and temporal changes. In recent years, the relationship between the microbiome and the survival or growth of aquatic invertebrates (such as crabs) has received extensive attention.

[0003] As one of the commercially cultured and widely consumed crustaceans in the coastal areas of China, the aquaculture production of Portunus trituberculatus is mainly limited by the unstable individual growth. Long-term research has found that even under the same aquaculture mode management, there are still significant differences in the growth performance among offspring, and the host-microbe interaction is a possible explanation. At present, the research on Portunus trituberculatus mainly focuses on the effects of diseases, growth, nutrition, and environmental stress. Although some studies have reported changes in the microbial community of Portunus trituberculatus, the correlation between the intestinal microbiota and the growth of offspring is still unclear.

[0004] The microbial metabolic functions in ecosystems are usually regulated by the interactions between species. Existing research shows that the intestinal microbiota of overgrown shrimp exhibits more synergistic effects and complex inter-species interactions, thus improving the nutrient intake efficiency. In addition, network research helps to identify key taxa from complex microbial interactions, and these taxa may make significant contributions to host growth differences. Although the key taxa are relatively low in abundance, they can provide a high degree of connectivity for the microbial network, thereby characterizing the trends and compositional changes of the microbial community. However, it is still unclear whether the key taxa will cause individual growth differences in Portunus trituberculatus. Summary of the Invention

[0005] The purpose of the present invention is to provide the application of Roseovarius litoreus in promoting the growth of Portunus trituberculatus, so as to make up for the deficiencies of the existing technology.

[0006] The present invention first provides a use of *Alteromonas roseovictrix*, which is an application in promoting the growth of *Portunus trituberculatus*;

[0007] Another aspect of the present invention provides another use of *Alteromonas roseovictrix*, which is an application in preparing a product for promoting the growth of *Portunus trituberculatus*;

[0008] As a specific record of an embodiment, the product is a microbial bacterial liquid;

[0009] The present invention also provides an application of *Alteromonas roseovictrix* in preparing a feed additive.

[0010] The present invention also provides a feed for *Portunus trituberculatus*, and live bacteria of *Alteromonas roseovictrix* are added to the feed.

[0011] Another aspect of the present invention also provides a cultivation method for *Portunus trituberculatus*. In the method, live bacteria of *Alteromonas roseovictrix* are added to the feed;

[0012] As a specific record of an embodiment, adding live bacteria of *Alteromonas roseovictrix* to the feed means adding *Ruditapes philippinarum* added with *Alteromonas roseovictrix*.

[0013] The present invention discovers that *Alteromonas roseovictrix* can significantly promote the growth of *Portunus trituberculatus*, further improve the weight gain rate and hepatopancreas index of crabs, and has broad application prospects in aquaculture. Description of the Drawings

[0014] Figure 1 : Diagram of the change in intestinal bacterial community between fast-growing and slow-growing swimming crabs,

[0015] Figure 2 : Intestinal bacterial network diagram of two types of swimming crabs,

[0016] Figure 3 : Phylogenetic tree diagram of key bacteria in the network,

[0017] Figure 4 : Diagram of the effect of *Alteromonas roseovictrix* on the growth of swimming crabs. Detailed Embodiments

[0018] The present invention selects adult swimming crabs from the same parent and with the same aquaculture management, which also provides an ideal model for evaluating the influence of the intestinal microbial community on the individual growth of swimming crabs. Using 16S rRNA gene high-throughput amplicon sequencing, the intestinal bacterial community structure and bacterial association network between two types of swimming crabs (fast-growing and slow-growing swimming crabs) were studied. Thus, the connection between the intestinal bacterial community and the host weight was established, and the correlation between the intestinal bacterial community characteristics and swimming crabs was evaluated.

[0019] Example 1:

[0020] Screen and determine that Roseovarius is related to the growth of Portunus trituberculatus. The specific steps are as follows:

[0021] (1) Culturing and sampling of juvenile Portunus trituberculatus

[0022] To eliminate the interference of different genetic backgrounds in the present invention, juvenile Portunus trituberculatus from the same parent were collected and placed in three earthen ponds (≈666.7 m 2 , with a depth of 1.8 m each) in Ningbo, Zhejiang. The stocking density of Portunus trituberculatus in each earthen pond was 2,500. The seawater salinity in the earthen pond was 23 psu, the water depth was 1.2 m, and the daily water exchange volume was 5% of the total seawater volume. Other aquaculture management methods were the same. During the entire aquaculture period, no disease outbreaks occurred. After four months of aquaculture, 23 Portunus trituberculatus with a weight of 205.47 ± 20.63 g (set as fast-growing Portunus trituberculatus) and 28 Portunus trituberculatus with a weight of 73.47 ± 12.53 g (set as slow-growing Portunus trituberculatus) were collected from the three earthen ponds. There was a significant difference in weight between the two types of Portunus trituberculatus (Table 1, p < 0.001). Each Portunus trituberculatus was dissected after ice bath, and its intestinal samples were immediately stored at -80 °C for further analysis.

[0023] Table 1: Information table of experimental sampling

[0024]

[0025]

[0026]

[0027] (2) Intestinal DNA extraction and 16S rRNA gene sequencing

[0028] Total genomic DNA was extracted from 51 intestinal samples respectively, and then the V3-V4 region of the bacterial 16S rRNA gene was amplified using the specific primers 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). Finally, Magigene Biotechnology Co., Ltd. (Guangzhou, China) was commissioned to perform sequencing on the NovaSeq platform (Illumina, USA). The USEARCH process (http: / / www.drive5.com / usearch / ) was used to analyze the sequencing data, and the zero-radius operational taxonomic units (ZOTUs) were annotated at 97% similarity according to the Silva database (version 123) (https: / / www.arb-silva.de / ). All sequences were normalized to the minimum sequence of each sample to avoid interference.

[0029] (3) Identification of key bacterial taxa related to growth

[0030] The DESeq2 package was used to perform statistical analysis on the relative abundances of ZOTUs in two categories of swimming crabs (selecting those with relative abundances greater than 0.1% in at least one sample). Compared with the slowly growing swimming crabs, only the ZOTUs with Log2|fold change| > 1 and padj < 0.05 after Benjamini-Hochberg (BH) correction in the fast-growing swimming crabs were retained for analysis. The rfUtilities and rfPermute packages were used to construct a random forest regression model to analyze the contribution degrees of variables such as the differential ZOTUs between groups and network topological properties to the swimming crabs. Subsequently, the A3 package and 5000 permutations were used to evaluate the reliability of the random forest regression model.

[0031] To reduce the complexity of the constructed bacterial network, the ZOTUs with relative abundances greater than 0.1% in at least one sample were retained. The WGCNA package was used to calculate the pairwise correlations of all species between ZOTUs, and the pairwise correlations of species with significant (p < 0.01 after BH correction and Pearson correlation coefficient |ρ| ≥ 0.75) were retained. Finally, Gephi 0.9.2 (https: / / gephi.org) was used to visualize the co-occurrence network and calculate the node attributes. The nodes with node connectivity > 40, closeness centrality > 0.37, and betweenness centrality < 0.35 were regarded as key taxa.

[0032] (4) Results and analysis

[0033] After quality control, a total of 5,467,137 high-quality sequences were obtained in this invention, with 107,199 ± 27,925 sequences per sample. Due to unequal sequencing depths, the sequences of each sample were normalized to 41,572, resulting in 4,778 ZOTUs in all samples. DESeq2 analysis showed that the relative abundances of 44 ZOTUs changed significantly between the two types of swimming crabs ( Figure 1 A, p < 0.05). For example, 18 ZOTUs belonging to Marinococcus, Microbacteriaceae, Rhodobacterales, Propionigenium, Synechococcus, Tenacibaculum, and Nesterenkonia were significantly enriched in the intestines of fast-growing swimming crabs ( Figure 1 B, p < 0.05). In contrast, 26 ZOTUs belonging to 19 bacterial taxa (mainly Sedimenticola, Winogradskyella, and Lewinella) were significantly enriched in the intestines of slow-growing swimming crabs ( Figure 1 B, p < 0.05). Among them, 7 ZOTUs contributed significantly to the body weight of swimming crabs. ZOTU 34 (Vibrio), ZOTU 369 (Rhodobacterales), ZOTU249 (Rhodobacteraceae), ZOTU 389 (Microbacteriaceae), and ZOTU 188 (Synechococcus) were enriched in the intestines of fast-growing swimming crabs, while ZOTU 475 (Winogradskyella) and ZOTU 559 (Sedimenticola) were enriched in the intestines of slow-growing swimming crabs ( Figure 1 C, p < 0.05). These findings indicate that the different ZOTUs between the two types of swimming crabs may be related to growth.

[0034] In this invention, co-occurrence networks of two types were constructed at the ZOTU level (relative abundance > 0.1% in at least one sample) ( Figure 2 A, B). In these two types of networks, there were 27 key taxa in the intestinal bacterial community of fast-growing swimming crabs, including 20 belonging to Rhodobacteraceae and 7 belonging to Sphingomonadales, Erythrobacteraceae, Flavobacteriaceae, and Cryomorphaceae ( Figure 2 C; Table 2). For slow-growing swimming crabs, only two key taxa belonging to Rhodobacteraceae were found ( Figure 2D; Table 2).

[0035] Table 2: Table of Key Group Attributes of Intestinal Bacteria in Swimming Crabs

[0036]

[0037]

[0038] The present invention determines the indicator bacteria according to the following three criteria: (1) The relative abundance of ZOTU must have a significant difference between groups; (2) ZOTU is at least a key group of the co-occurrence network in one group; (3) ZOTU makes a significant contribution to the growth changes of swimming crabs. Therefore, only ZOTU 249 (Rhodobacteraceae) is the intestinal indicator bacteria that meet the above criteria. DNA sequence alignment and phylogenetic tree construction analysis show that the highest sequence similarity with the V3-V4 region sequence of the 16S rRNA gene of bacterial ZOTU 249 is Roseovarius litoreus, with a sequence similarity of 99.75%. The Latin name of this species is Roseovarius litoreus, the strain preservation number is DSM28249, and the NCBI accession number is jgi.1107621( Figure 3 ). Therefore, it is determined that Roseovarius litoreus is a potentially beneficial bacterium that promotes the growth of swimming crabs.

[0039] Example 2: Verification of the Effect of Roseovarius litoreus

[0040] The present invention further proves that Roseovarius litoreus has the effect of improving the growth of swimming crabs, and the specific steps are as follows:

[0041] The present invention purchases 60 juvenile swimming crabs (about 25 g) from an aquaculture farm in Ningbo City, transports them back to the laboratory in a light-shielded and oxygenated manner, measures the initial weight, and places each swimming crab in a plastic box with small holes (320×190×170 mm), and then transfers them into 12 aquaculture ponds (1.5×1×0.6 m) respectively. Each aquaculture pond is filled with 300 L of natural seawater (pH is 8.0, salinity is 25.0 psu, and temperature is 28 °C).

[0042] Roseovarius litoreus is obtained from the Shandong Marine Microbial Strain Center and inoculated onto a 2216 solid medium containing 1 g / L sodium pyruvate. After culturing at 28 °C for 24-36 h, after selecting single colonies on the plate, they are dissolved in phosphate buffer. When the absorbance value of the bacterial solution at 600 nm under ultraviolet light is 0.7, the bacterial solution concentration is about 1×10 9 cfu / mL. Subsequently, the bacterial solution is diluted, and the final concentrations are 1×10 5 、1×10 7 and 1×10 9The bacterial suspensions with different concentrations of cfu / mL were respectively injected into the de-shelled Ruditapes philippinarum, and left standing for 5 minutes for feeding. The injection volume of the bacterial suspension for each clam was approximately 1% of the corresponding swimming crab's weight, and the weight of the clams fed to the swimming crab was 10% of its weight. In this experiment, the group without adding bacteria was set as the control group with a bacterial suspension concentration of 0 cfu / mL. Three replicates were set for each bacterial suspension concentration. The daily feeding time was from 16:00 to 18:00. Dead crabs were removed in time and residual baits were cleaned. After 6 weeks of cultivation, the final weight and wet weight of the hepatopancreas of the swimming crabs were weighed, and the weight gain rate and hepatopancreas index were calculated. The formulas are as follows:

[0043] Weight gain rate (%) = (Wt - Wo) / Wt × 100 (1)

[0044] Wt is the final weight of the swimming crab after 6 weeks of cultivation, and Wo is the initial weight of the swimming crab.

[0045] Hepatopancreas index (%) = Ht / Wt × 100 (2)

[0046] In the formula, Ht is the wet weight of the hepatopancreas of the swimming crab.

[0047] After feeding different concentrations of Chromobacterium roseum to swimming crabs for 6 weeks, the results showed that feeding with Chromobacterium roseum added could significantly promote the growth of swimming crabs (Table 3; Figure 4 , p < 0.05), but there was no significant difference in the weight gain rate of swimming crabs among these three bacterial suspension concentrations. Among them, the weight gain rate of swimming crabs was the highest (83.27 ± 14.22%) when the bacterial suspension concentration was 1×10 7 cfu / mL, which was 19.35% higher than that of the control group (the weight gain rate of the control group was 63.92 ± 10.08%). As for the hepatopancreas index, only feeding the bacterial suspension at 1×10 7 cfu / mL could significantly increase the hepatopancreas index of swimming crabs (Table 3; Figure 4 , p < 0.05).

[0048] Table 3: Table of weight gain and hepatopancreas index of swimming crabs

[0049]

[0050]

[0051]

[0052] The results in Table 3 showed that the group with Chromobacterium roseum added increased by 0.86% compared with the control group. Therefore, Chromobacterium roseum has a positive effect on promoting the growth of swimming crabs, and the optimal bacterial suspension concentration is 1×10 7 cfu / mL.

Claims

1. Use of Roseovarius marinus ( Roseovarius litoreus ), characterized in that The described use is the application in promoting the growth of swimming crab (Portunus trituberculatus), and the application is adding viable bacteria of Roseovarius litoreus to the feed.

2. Use of Roseovarius marinus, characterized in that, The described use is the application in preparing a product for promoting the growth of swimming crab (Portunus trituberculatus), and the product is a microbial bacterial liquid.

3. Application of Roseovarius litoreus in preparing a feed additive for promoting the growth of swimming crab (Portunus trituberculatus).

4. A cultivation method of swimming crab, characterized in that, The described method is adding viable bacteria of Roseovarius litoreus to the feed.

5. The method according to claim 4, wherein Adding viable bacteria of Roseovarius litoreus to the feed as described is adding viable bacteria of Roseovarius litoreus to shellfish.

6. The method according to claim 5, characterized in that, The shellfish as described is Ruditapes philippinarum.

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