Active Microbiota of Penaeus stylirostris Larvae: Partially Shaped via Vertical and Horizontal Transmissions and Larval Ontogeny

Abstract

During their entire lifecycle, mariculture animals are farmed in water that contains various microorganisms with which they are in close associations. Microbial exchanges between the animals and their surrounding water can occur. However, little is known about the interactions between shrimp larvae and water, and more especially, about larval bacterial selection and microbiota modulation across ontogeny. To address this gap, using HiSeq sequencing targeting the V4 region of the 16S rRNA molecule, we investigated the active prokaryotic diversity and structure of healthy Penaeus stylirostris larvae and seawater. Comparisons between different larval stages revealed evidence of stage-specific microbiotas and biomarkers, a core microbiota common to all stages, and shared taxa between successive stages, suggesting vertical transmission of bacterial taxa. Comparisons between stage-specific microbiotas and core microbiotas with water storages highlighted that many taxa associated with the larvae were originally present in the natural seawater, underlining horizontal transmission of bacteria from water to larvae. As some of these lineages became active at specific larval stages, we suggest that larvae were able to modulate their microbiota. This study provides insight into larvae-microbiota interactions at the larval stage scale.

Keywords: active microbiota; biomarkers; larvae; shrimp; shrimp ontogeny; specific microbiota; taxa transmission.

Figure 1

Mean percent of larval survival in the 3 tanks through the 10 days of the rearing, shown in grey, compared to the reference in black. The reference corresponds to the larval survival and stages obtained for a given day. For each day, the reference was calculated using data from 10 years of successful larval rearing (Ifremer data, personal communication with Pham).

Figure 2

Alpha diversity indexes of the microbial diversity associated with the larvae. (A) Observed, (B) ACE, (C) Shannon and (D) Inverse Simpson. Turquoise stand for the egg samples, light blue for the nauplii, medium blue for the zoea and navy blue for the mysis samples. Data are available in Table S1.

Figure 3

Ordination of the microbial diversity associated with the larvae.

Figure 4

Microbial composition of the larvae throughout the rearing period and of the water storage.

Figure 5

Specific and core microbiotas of the larvae according to their larval stages and comparison with the water storages. (A) Venn diagram of commons ASVs among all the larvae samples; (B,C) Venn diagrams of commons ASVs between the water storages ResI and ResT, and (B) the eggs and (C) the nauplii collected on D0, (D–F) Venn diagrams of commons ASVs between the specific ASVs of the water storages ResI, ResT and ResNT, and (D) the nauplii collected on D1, (E) the zoea, (F) the mysis; (G) Venn diagram of the core microbiota of the larvae and the ASVs of the water storages. Colored ellipses are related to specific ASVs, in blue = eggs, turquoise = nauplii collected on D0, light red = nauplii collected on D1, light green = zoea, orange = mysis, light orange = larval core microbiota, brown ellipse = primary reservoir ResI, grey = secondary reservoir ResT and khaki = the storage reservoir ResNT. The core microbiota, made of 109 ASVs shared by all the samples, is represented by the overlapping of all the ellipses. Numbers inside the ellipses and in the overlapping represent the number of ASVs of a given condition.

Figure 6

Differentially abundant genera according to the larval stage. (A) LEfSe displaying the genera which were statistically more abundant according to each larval stage. (B) Correlogram of the biomarkers detected via the LEfSe according to the larval stage. Heatmap color gradient is linked to Spearman correlation coefficient intensity: red stands for positive correlation, while blue corresponds to negative correlation. Significant correlations are noted with an asterisk (*), with no asterisk: p > 0.05, *: p ≤ 0.05, ***: p ≤ 0.001.

Figure 7

Correlogram of putative ecological functions assigned with FAPROTAX to the core microbiotas of all the larvae. The heatmap color gradient related to Spearman correlation coefficient intensity: red for positive and blue for negative correlations. Significant correlations are denoted with an asterisk (*), no asterisk: p > 0.05, *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001.