Microbial biomarker detection in shrimp larvae rearing water as putative bio-surveillance proxies in shrimp aquaculture

Abstract

Background: Aquacultured animals are reared in water hosting various microorganisms with which they are in close relationships during their whole lifecycle as some of these microorganisms can be involved in their host's health or physiology. In aquaculture hatcheries, understanding the interactions existing between the natural seawater microbiota, the rearing water microbiota, the larval stage and the larval health status, may allow the establishment of microbial proxies to monitor the rearing ecosystems. Indeed, these proxies could help to define the optimal microbiota for shrimp larval development and could ultimately help microbial management.

Methods: In this context, we monitored the daily composition of the active microbiota of the rearing water in a hatchery of the Pacific blue shrimp Penaeus stylirostris. Two distinct rearing conditions were analyzed; one with antibiotics added to the rearing water and one without antibiotics. During this rearing, healthy larvae with a high survival rate and unhealthy larvae with a high mortality rate were observed. Using HiSeq sequencing of the V4 region of the 16S rRNA gene of the water microbiota, coupled with zootechnical and statistical analysis, we aimed to distinguish the microbial taxa related to high mortality rates at a given larval stage.

Results: We highlight that the active microbiota of the rearing water is highly dynamic whatever the larval survival rate. A clear distinction of the microbial composition is shown between the water harboring heathy larvae reared with antibiotics versus the unhealthy larvae reared without antibiotics. However, it is hard to untangle the effects of the antibiotic addition and of the larval death on the active microbiota of the rearing water. Various active taxa of the rearing water are specific to a given larval stage and survival rate except for the zoea with a good survival rate. Comparing these communities to those of the lagoon, it appears that many taxa were originally detected in the natural seawater. This highlights the great importance of the microbial composition of the lagoon on the rearing water microbiota. Considering the larval stage and larval survival we highlight that several genera: Nautella, Leisingera, Ruegerira, Alconivorax, Marinobacter and Tenacibaculum, could be beneficial for the larval survival and may, in the rearing water, overcome the r-strategist microorganisms and/or putative pathogens. Members of these genera might also act as probiotics for the larvae. Marivita, Aestuariicocccus, HIMB11 and Nioella, appeared to be unfavorable for the larval survival and could be associated with upcoming and occurring larval mortalities. All these specific biomarkers of healthy or unhealthy larvae, could be used as early routine detection proxies in the natural seawater and then during the first days of larval rearing, and might help to manage the rearing water microbiota and to select beneficial microorganisms for the larvae.

Keywords: Bio-surveillance proxies; Healthy larvae; Lagoon microbiota; Microbial biomarker; Rearing water microbiota; Shrimp larvae; Unhealthy larvae.

Figure 1. Evolution of the larval survival during the experiment.

Evolution of the larval survival during the experiment compared to the reference in turquoise (e.g., usual survival rate obtained for a specific day, calculated for each day using data of 10 years of successful rearing; Ifremer data; D. Pham, comm. pers., 2008–2018). Without-A, Without-B, Without-C, correspond to the rearing water without antibiotic in the tanks A, B and C; and With-A, With-B, With-C, stand for the rearing water with antibiotic in the tanks A, B and C. D0 to D9 correspond to the day of the rearing.

Figure 2. Ordination of the water samples based on the PCoA method and a Bray-Curtis dissimilarity matrix.

PCoA of the rearing water samples. The ellipses were constructed using a confidence level for a multivariate t-distribution set at 80%. For each cluster or samples outside the clusters, the larval stage and health (Good for high survival rate and Bad for high mortality rate) are noted. Each color corresponds to a sampling day in the figure and the corresponding sampling day is noted in the same color in the caption on the right side of the PCoA.

Figure 3. Microbial composition of the water samples.

Relative abundance of the main prokaryotic families. The relative abundance is represented in terms of percentage of the total prokaryotic sequences per sample. Only families representing more than 1% of the overall abundance in at least three samples are displayed on the barplot. ResI stands for the primary reservoir sample, ResII for the secondary reservoir sample, Control stands for the control water without larvae, antibiotic nor food; without antibiotic for the rearing water without antibiotic, with antibiotic for the rearing water supplemented with antibiotic. D0 to D8 correspond to the sampling day. Sample day are followed by A, B or C which correspond to the replicate tanks for the rearing water with or without antibiotic.

Figure 4. Microbial communities associated with the specific and core microbiomes of the whole rearing experiment in the rearing water and the water storages.

(A) Venn diagram of shared ASVs among all the rearing water samples. Coloured ellipses correspond to group-specific ASVs of the rearing water hosting in: red = nauplii, khaki = zoea with a good survival rate, green = zoea with a bad survival rate, blue = mysis with a good survival rate, purple = mysis with a bad survival rate. The overlapping area between all the ellipses, corresponds to the core microbiome composed by the 137 ASVs common to all the samples. The numbers inside the ellipses and in the overlapping zones correspond to the total number of ASVs present in the condition. (B to F) Venn diagram of shared ASVs between the specific ASVs of the water storage (yellow ellipse = group-specific ASVs of the primary reservoir and maroon = group-specific ASVs of the secondary reservoir) and (B) with the nauplii (red ellipse), (C) with the zoea with a bad survival rate (khaki ellipse), (D) with the mysis with a good survival rate (blue ellipse), (E) with the mysis with a bad survival rate (purple ellipse). (F) Venn diagram of the core microbiota of the rearing water and the ASVs of the water storages: in light yellow the primary reservoir and in beige the secondary reservoir.

Figure 5. Differentially abundant genera according to the larval growth and survival status.

(A) LEfSe, linear discriminant analysis (LDA) effect size, exhibiting the genera significantly more abundant in the rearing water A) hosting zoea that stayed healthy during the zoea stage (NTA0 Zoea G g ood), hosting zoea that were healthy at the beginning of the zoea stage and unhealthy at the end (NTSA Zoea Good, corresponding to the rearing day 2 and 3) and hosting zoea with high mortality rate at the end of the zoea stage (NTSA Zoea Bad, corresponding to the rearing day 4 to 6); (B) hosting healthy mysis (Mysis Good) and unhealthy mysis (Mysis Bad). Genera wrote in blue are biomarkers enriched in the rearing water hosting both healthy zoea and mysis. Genera wrote in red are enriched in the rearing water hosting both unhealthy zoea and mysis. Genera wrote in purple were detected as biomarkers of the unhealthy zoea and healthy mysis. Genus wrote in brown was enriched in the rearing water hosting zoea that were healthy at the beginning of the zoea stage and unhealthy at the end and in the rearing water with unhealthy mysis.