The Active Microbiota of the Eggs and the Nauplii of the Pacific Blue Shrimp Litopenaeus stylirostris Partially Shaped by a Potential Vertical Transmission

Abstract

The many ecological niches present in an organism harbor distinct microorganisms called microbiota. Different factors can influence the establishment of these commensal microbial communities. In a previous article, we have concluded that some bacterial lineages associated with the early larval stages of the Pacific blue shrimp Litopenaeus stylirostris could be acquired from the breeders via a potential vertical transmission. The present study was conducted in order to investigate this hypothesis. Using HiSeq sequencing of the V4 region of 16S rRNA gene, we analyzed the active microbiota associated with the eggs and the nauplii of L. stylirsotris as well as with the reproductive organs of their breeders. Microbial communities associated with the rearing water were also considered to discriminate environmental microbial lineages. Using these analyses, we highlight a set of core bacterial families present in all samples and composed of members of Colwelliaceae, Alteromonadaceae, Pseudoalteromonadaceae, Saccharospirillaceae, Oceanospirillaceae, Vibrionaceae, Burkholderiaceae, Rhodobacteraceae, Flavobacteraceae, and Corynebacteriaceae; showing the importance of the environment in the establishment of the larval microbiota. We also present specific bacteria affiliated to the Arcobacteraceae, Rhodobacteraceae, Comamonadaceae, and Colwelliaceae families, which were only found in the breeders and their offspring strengthening the hypothesis of a potential vertical transmission shaping the active microbiota of the eggs and the nauplii of L. stylirostris.

Keywords: active microbiota; core microbiota; eggs; microbial colonization; nauplii; shrimps; vertical transmission.

Figure 1

Sample collection. Sample collection was performed using an eight-steps technique. First, water was sampled from the hatchery tanks after ethylenediaminetetraacetic acid (EDTA) addition. Then, on the day the tanks were filled and sampled, artificial inseminations were performed. In order to consider two-parent families only, one male was used to inseminate one female for all the artificial inseminations. After artificial inseminations, males were dissected in order to sample their reproductive organ and females were transferred into the hatchery tanks at a rate of 1 individual per tank and were left to spawn. After spawning, females were retrieved to dissect their ovarian tissue, and eggs were sampled from all the hatchery tanks. To allow larval hatching, eggs were left until the next day in the hatchery tanks. Before transfer into rearing tanks, the nauplii were sampled from each tank. A total of three two-parent families were considered for this study.

Figure 2

Clustering and total microbial composition of all the samples. (A) Hierarchical clustering based on Bray–Curtis dissimilarity and Ward method. Clusters were defined using a 1.5 threshold depicted by the red dotted line. All the tank water and the nauplius (nii) samples are gathered in Cluster 1, in light gray. All the female, the male and the egg samples are clustered in Cluster 2, in dark gray. (B) Bacterial compositions of the tank water, the nauplius (nii), the egg, the female and the male samples. Major bacterial classes were defined as the bacterial classes with a total relative abundance higher than 1%.

Figure 3

Global Venn diagrams of shared and specific ASVs among sample types. (A) Venn diagram of specific and shared ASVs among the tank water, the egg and the nauplius (nii) samples. The blue ellipse represents the ASVs shared by all the hatchery tank water samples. The red ellipse represents the ASVs shared by all the egg samples. The yellow ellipse represents the ASVs shared by all the nauplius (nii) samples. (B) Venn diagram of specific and shared ASVs among the breeders (female and male), the egg and the nauplius (nii) samples. The green ellipse represents the ASVs shared by all the female reproductive organ samples. The purple ellipse represents the ASVs shared by all the male reproductive organ samples. The red ellipse represents the ASVs shared by all the egg samples. The yellow ellipse represents the ASVs shared by all the nauplius (nii) samples. In both Venn diagrams, numbers noted in the overlapping areas correspond to the number of shared ASVs among sample types, while numbers noted outside of the overlapping areas correspond to the numbers of specific ASVs associated with each sample type.

Figure 4

Shared bacterial lineages among the eggs, the nauplii, the tank waters, and the reproductive organs of the breeders. Top bacterial families with a total relative abundance higher than 1% shared among (A) the tank water, the egg, and the nauplius (nii) samples (268 ASVs) and (B) the female and the male reproductive organs, the egg and the nauplius (nii) samples (172 ASVs). The bacterial families in blue are those shared between the two histograms which were constructed by averaging the triplicates for each sample type.

Figure 5

Shared bacterial lineages among the eggs, the nauplii, and the breeders. Top bacterial families with a total relative abundance higher than 1% shared among (A) the male, the egg and the nauplius (nii) samples (22 ASVs) and (B) the female, the egg and the nauplius (nii) samples (37 ASVs). The bacterial families in blue are the bacterial families shared between the two histograms which were constructed by averaging the triplicates for each sample type.

Figure 6

Specific and shared bacterial lineages among the eggs and the nauplii. Top bacterial families with a total relative abundance higher than 1% (A) specifically found in the eggs in Venn1 (27 ASVs) and (B) in Venn2 (22 ASVs), (C) shared between the egg and the nauplius (nii) samples in Venn1 (35 ASVs) and (D) in Venn 2 (72 ASVs), (E) specifically associated with the nauplii (nii) in Venn1 (512 ASVs), and (F) in Venn2 (639 ASVs). The bacterial families in blue are the bacterial families shared between histograms which were constructed by averaging the triplicates for each sample type.

Figure 7

Schematic representation of the factors influencing the microbiota of the Pacific blue shrimp at all developmental stages. The egg, nauplii, zoea, and mysis stages belong to the larval stages.