Fine-scale succession patterns and assembly mechanisms of bacterial community of Litopenaeus vannamei larvae across the developmental cycle

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By: Yanting Wang, Kai Wang, Lei Huang, Pengsheng Dong, Sipeng Wang, Heping Chen, Zheng Lu, Dandi Hou y Demin Zhang *

Unveiling the assembly mechanism of shrimp microbiota can help resolve the debate on whether we could improve the success rate of shrimp culture via manipulating their microbiota. In this study, we used 16S rRNA gene amplicon sequencing to investigate the succession and assembly processes of L. vannamei larval bacterial community in a realistic aquaculture practice with sufficient biological replicates.

The most productive shrimp species in world aquaculture industry, the Pacific white shrimp (Litopenaeus vannamei) culture is mainly restricted by unstable quality of larvae and frequent outbreak of diseases.

Larval nursery, covering nauplius, zoea, mysis, and early postlarvae stages, is a crucial process that largely determines the success of Pacific white shrimp culture. The quality of larvae is closely related to the growth, development, and resistance to stress/disease of shrimps in subsequent culture stages.

At present, the Pacific white shrimp microbiome research is mainly focused on other growth stages in terms of their associations with outbreak of disease, growth, and stress-resistance.

“Unveiling the assembly mechanism of shrimp microbiota can help resolve the debate on whether we could improve the success rate of shrimp culture via manipulating their microbiota.”

Burns et al. found that the contribution of neutral processes to the assembly of zebrafish intestinal bacteria declined with host development, while the importance of neutral processes in shaping intestinal bacterial communities increases with the age of shrimp (from postlarvae to adult) in culture practice but declined with disease outbreak. However, little is known about the dynamics and taxonomic dependency of assembly processes of bacteria in larval shrimp.

In this study, we used 16S rRNA gene amplicon sequencing to investigate the succession and assembly processes of L. vannamei larval bacterial community in a realistic aquaculture practice with sufficient biological replicates.

“A high-frequency sampling strategy was applied to collect shrimp larvae (from the fertilized eggs of a pair of parents) and rearing water samples across nauplius, zoea I, zoea II, zoea III, mysis, and early postlarvae stages lasting for 15 days.”

Using multivariate analyses, the neutral model, and functional prediction we aimed to reveal the following:

  1. The dynamics of α-diversity, composition, and predicted functional potentials of larval shrimp bacterial community with host development.
  2. The taxonomic and phylogenetic succession pattern of larval bacterial community.
  3. The dynamics and taxonomic dependency in assembly processes of larval shrimp bacteria across developmental stages.
  4. To what extent the rearing water bacterio plankton can influence the assembly of larval shrimp bacteria.

Results

The bacterial   α-diversity   indices of shrimp larvae and rearing water showed dramatic variability with host development. Overall, the composition of dominant bacterial groups (at the phylum or family level) at the stages nauplius, and postlarvae were more complex than other stages.

The succession pattern of dominant bacterial OTUs (operational taxonomic units) of shrimp larvae, and 89.1% of samples could be classified into four clusters according to the developmental stage: cluster I (nauplius), cluster II (zoea I and II), cluster III (zoea III), cluster IV (mysis and postlarvae).

In general, bacterial community compositions of shrimp larvae and rearing water were both clustered according to developmental stages. The taxonomic composition of bacterial community in larvae and water showed distinct successional trajectories, while the phylogenetic turn over trajectories of two communities overlapped to some extent during the substages of zoea.

We observed overall low taxonomic similarity but high phylogenetic similarity between larval and water bacterial communities. In addition, we found that larval bacterial communities at the stages zoea I-II and mysis showed higher similarities and/or more shared OTUs with the water bacterial communities from the previous stage compared with that at the same stage.

When assuming larval bacterial metacommunity as the source community, the goodness of fit of the neutral model was largely improved compared with that when assuming bacterio plankton as the source across all stages, suggesting that exchange of bacteria among larval individuals was a more important source of larval bacterial communities.

The cumulative relative abundance and taxonomic distribution of three categories of OTUs in the neutral model varied with host development, especially between pre- and post-mouth opening stages.

The cumulative relative abundance of the OTUs above prediction was overall low across developmental stages, with little changes in the taxonomic distribution. At any stages, the assembly of larval bacteria was dominantly governed by neutral processes, and the neutral model performed better than the binomial distribution mode, suggesting that, except dispersal, ecological drift and dispersal limitation also contributed.

In general, functional potentials relevant with genetic information processing were enriched in naupliar shrimps compared with larvae at post-mouth opening stages, while many metabolismrelevant potentials (such as biosynthesis of other secondary metabolites and the metabolism of amino acids; carbohydrate; lipid; cofactors and vitamins; and terpenoids and polyketides) were enriched in larvae after the mouth opening.

However, some metabolism relevant potentials somewhat showed a decreasing trend at early postlarvae stage. As the predominant bacterial group at the stages zoea and mysis, the family Rhodobacteraceae was predicted to be the major contributor to functional potentials (including metabolism) of larval shrimp bacterial community.

Discussion

The U-shaped pattern in larval bacterial α-diversity with host development

As the larvae are too small to obtain their intestines, the larval microbiota should be mainly derived from the intestinal tract and the fraction attached to the epidermis. At nauplius stage, with the release of yolk nutrients and the enlargement of epidermis area, the larval bacterial community could be mainly originated from fertilized eggs (the inheritance of the parents and initial hatching environment) and the epidermis attachment, thus maintaining at a relatively high diversity.

When the larvae started eating at zoea I stage, their intestinal microbiota began to form, while the larvae molted, imposing the reassembly of larval bacterial community. These changes could lead to the dominance of intestinal bacteria in larval microbiota and thus decrease α-diversity.

Although a point of view has suggested that the higher microbial diversity does not necessarily correspond to a more stable and healthier ecosystem, high diversity is often considered to hold capability of maintaining the stability and ecological function of microbial community, thus being an important indicator of host health status. These studies suggest that high bacterial diversity could be a positive signal for maintaining the growth, health, and stress resistance of shrimps.

Larval bacterial community composition varied with host development

The high-frequency sampling strategy facilitated the unveiling of highly dynamic pattern of larval bacterial communities. The shrimp larvae with immature digestive system could partially rely on the assistance of bacteria for food digestion and nutrient metabolisms, which is corresponding to the enriched metabolic potentials of multiple organic matters in larval shrimp microbiota after the mouth opening.

Thus, the high variability of bacterial community composition might be due to the host’s recruitment of different functional groups for physiological needs. As the morphological and physiological properties of intestinal tract change with host development, the initial “winners” will be reorganized to form a stage specific bacterial community.

Many studies have found that host development and diet largely shape the intestinal microbiome of aquatic animals. In this study, the shifts of physiological state, nutritional intake mode, and microbial source between pre- and post-mouth opening stages likely led to dramatic differences in larval bacterial communities.

“The high variability of bacterial community composition might be due to the host’s recruitment of different functional groups for physiological needs.”

Functional prediction showed that the family Rhodobacteraceae largely contributed to the potentials in biosynthesis and the metabolism of multiple organic matters after the mouth opening of larvae, indicating that they may participate in the metabolism of organic matters in the digestive tract of larvae and/or provide essential nutrients for host growth.

The relative abundance of Rhodobacteraceae in the intestinal bacterial community of healthy L. vannamei individuals is often higher than that of diseased ones and shows an antagonistic relation ship with potential pathogens such as Vibrio.

The higher relative abundance of Rhodobacteraceae was also observed in the intestinal tract of cold resistant strain of L. vannamei relative to cold vulnerable strain. Collectively, we speculate that the dramatic enrichment of Rhodobacteraceae (including some Ruegeria taxa) after the mouth opening of larvae may play a positive role in promoting digestion, providing nutrients, and inhibiting pathogens.

Furthermore, the temporal switching among Rhodobacteraceae taxa suggests distinct assemblages of Rhodobacteraceae taxa could be recruited for maintaining certain functions such as the metabolism of different organic matters derived from the partially modified feeds at different stages.

Neutral processes dominated the assembly of larval bacteria

The larval bacterial communities mainly sourced from the larval meta community, while water bacterio plankton community only had certain contribution at zoea substages.

These results suggest that the assembly of larval bacteria overall depends on the exchanges among individuals, probably via cross feeding of feces and/or bioflocs. When larval shrimps are undergoing continuous metamorphic development with frequent molting and feed replacement, their bacterial communities are also undergoing frequent reassembly.

In addition, the compositions of OTUs neutrally distributed or deviated from neutral prediction between pre- and post-mouth opening stages were dramatically different.

“These results reveal the remarkable succession pattern and the dynamics in assembly processes of larval bacterial communities, emphasizing the importance of the mouth opening stage of larval shrimp from the ecological perspective.”

Many studies have found that the initial establishment of host microbiome can be affected by the surrounding environment. We also found rearing water as a source of larval bacterial community at three substages of zoea, which can be considered as the beginning of establishment of larval intestinal microbiota.

On the other hand, it is particularly important to ensure the microbial safety of rearing water (such as prevention of pathogenic bacteria) after the mouth opening of larvae.

“The regulation of larval microbiota by microbial management of rearing water in aquaculture practice should be launched before the mouth opening of shrimp larvae.”

Taken bacterio plankton as the source for the neutral model fitting, the relative abundance of the neutrally distributed OTUs and the migration rate gradually decreased during zoea substages, indicating that the dominant process governing the colonization of bacterio plankton into larval communities shifted from dispersal to host selection.

Knowing which bacteria are selected for and have the ability to persist in a host is vital when screening probiotic candidates. The above prediction taxa may be good candidates for potential probiotics because they have a greater chance for colonization.

Given the positive host selection for Rhodobacteraceae taxa from the rearing water during the zoea stage and its persistent dominance and large potential contribution to the metabolism of organic matters after the mouth opening of larvae, we suggest that Rhodobacteraceae could be crucial in the growth of shrimp larvae and thus be a potential source of probiotic candidates for larval nursery.

Collectively, the succession patterns and assembly mechanism of larval shrimp bacteria we revealed here highlighted the importance of the mouth opening stage from the perspective of microbial ecology, indicating the possibility and timing of microbial management of the rearing water for larval microbiota regulation and pathogen prevention in larval shrimp nursery practice.

*This is a summarized version of the original article titled “Fine-scale succession patterns and assembly mecha- nisms of bacterial community of Litopenaeus vannamei larvae across the developmental cycle” by Yanting Wang, Kai Wang, Lei Huang, Pengsheng Dong, Sipeng Wang, Heping Chen, Zheng Lu, Dandi Hou and Demin Zhang, under a creative commons license 4.0. The original versión of the article can be accessed online at: https:// doi.org/10.1186/s40168-020-00879-w

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