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

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.

Algal symbiont diversity in Acropora muricata from the extreme reef of Bouraké associated with resistance to coral bleaching.

Widespread coral bleaching has generally been linked to high water temperatures at larger geographic scales. However, the bleaching response can be highly variable among individual of the same species, between different species, and across localities; what causes this variability remains unresolved. Here, we tracked bleached and non-bleached colonies of Acropora muricata to see if they recovered or died following a stress event inside the semi-enclosed lagoon of Bouraké (New Caledonia), where corals are long-term acclimatized to extreme conditions of temperature, pH and dissolved oxygen, and at a nearby control reef where conditions are more benign. We describe Symbiodiniaceae community changes based on next-generation sequencing of the ITS2 marker, metabolic responses, and energetic reserve measures (12 physiological traits evaluated) during the La Niña warm and rainy summer in 2021. Widespread coral bleaching (score 1 and 2 on the coral colour health chart) was observed only in Bouraké, likely due to the combination of the high temperatures (up to 32°C) and heavy rain. All colonies (i.e., Bouraké and reference site) associated predominantly with Symbiodinaceae from the genera Cladocopium. Unbleached colonies in Bouraké had a specific ITS2-type profile (proxies for Symbiodiniaceae genotypes), while the bleached colonies in Bouraké had the same ITS2-type profile of the reef control colonies during the stress event. After four months, the few bleached colonies that survived in Bouraké (B2) acquired the same ITS2 type profiles of the unbleached colonies in Bouraké. In terms of physiological performances, all bleached corals showed metabolic depression (e.g., Pgross and Rdark). In contrast, unbleached colonies in Bouraké maintained higher metabolic rates and energetic reserves compared to control corals. Our study suggests that Acropora muricata enhanced their resistance to bleaching thanks to specific Symbiodiniaceae associations, while energetic reserves may increase their resilience after stress.

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

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.

Microbiota of the Rearing Water of Penaeus stylirostris Larvae Influenced by Lagoon Seawater and Specific Key Microbial Lineages of Larval Stage and Survival.

Aquacultured animals are reared in water, where they interact with microorganisms which can be involved in their development, immunity, and disease. It is therefore interesting to study the rearing water microbiota, especially in the hatcheries of the Pacific blue shrimp Penaeus stylirostris, where larval mass mortalities occur. In this study, using HiSeq sequencing of the V4 region of the 16S rRNA molecule coupled with zootechnical and chemical analyses, we investigated whether any microbial lineages could be associated with certain mortality rates at a given larval stage. Our results indicate that the active microbiota of the rearing water was highly dynamic throughout the rearing process, with distinct communities influenced by progressive water eutrophication, larval stage, and survival rate. Our data also highlighted the role of the lagoon seawater on the rearing water microbiome, as many operational taxonomic units (OTUs) specific to a given larval stage and survival rate were detected in the primary reservoir which contained the lagoon water. We also identified biomarkers specific to water eutrophication, with Alteromonadaceae, Vibrionaceae, and Methylophilaceae, respectively, linked to increases in ammonia, nitrogen, and soluble reactive phosphate, or to increases in colored dissolved organic matter in the rearing water; other biomarkers were specific to certain larval stages and survival rates. Indeed, the Marinobacteraceae were specific to the Nauplii, and the Thalassospiraceae and Saprospiraceae to the Zoea Good condition; when mortality occurred, the Litoricolaceae were specific to the Zoea Bad, Microbacteraceae to the Mysis Bad, and Methylophilaceae to the Mysis Worst condition. Thus, these biomarkers might be used as potential early warning sentinels in water storage to infer the evolution of larval rearing to improve shrimp larval rearing. IMPORTANCE In New Caledonia, rearing of P. stylirostris is one of the main economic activities; unfortunately, mass larval mortalities cause important production decreases, involving major economic losses for the farmers and the Territory. This phenomenon, which has occurred at any larval stage over the past decade, is poorly understood. The significance of our research is in the identification of biomarkers specific to larval stage and survival rate, with some of these biomarkers being already present in the lagoon water. This enhances the role of the lagoon on the active microbiota of the rearing water at various larval stages and survival rates. Together, our results help us understand which active microbial communities are present in the rearing water according to larval stage and health. This might lead to broader impacts on hatcheries by helping to develop useful tools for using the water-lagoon, reservoir, or rearing-to test for the presence of these biomarkers as an early monitoring strategy.

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

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.

Potential lineage transmission within the active microbiota of the eggs and the nauplii of the shrimp Litopenaeus stylirostris: possible influence of the rearing water and more.

BACKGROUND: Microbial communities associated with animals are known to be key elements in the development of their hosts. In marine environments, these communities are largely under the influence of the surrounding water. In aquaculture, understanding the interactions existing between the microbiotas of farmed species and their rearing environment could help establish precise bacterial management. METHOD: In light of these facts, we studied the active microbial communities associated with the eggs and the nauplii of the Pacific blue shrimp (Litopenaeus stylirostris) and their rearing water. All samples were collected in September 2018, November 2018 and February 2019. After RNA extractions, two distinct Illumina HiSeq sequencings were performed. Due to different sequencing depths and in order to compare samples, data were normalized using the Count Per Million method. RESULTS: We found a core microbiota made of taxa related to Aestuariibacter, Alteromonas, Vibrio, SAR11, HIMB11, AEGEAN 169 marine group and Candidatus Endobugula associated with all the samples indicating that these bacterial communities could be transferred from the water to the animals. We also highlighted specific bacterial taxa in the eggs and the nauplii affiliated to Pseudomonas, Corynebacterium, Acinetobacter, Labrenzia, Rothia, Thalassolituus, Marinobacter, Aureispira, Oleiphilus, Profundimonas and Marinobacterium genera suggesting a possible prokaryotic vertical transmission from the breeders to their offspring. This study is the first to focus on the active microbiota associated with early developmental stages of a farmed shrimp species and could serve as a basis to comprehend the microbial interactions involved throughout the whole rearing process.

Whole-Genome Sequence of Pseudoalteromonas sp. NC201, a Probiotic Strain for Litopenaeus stylirostris Hatcheries in New Caledonia.

The marine bacterium Pseudoalteromonas sp. strain NC201 has shown probiotic potential in Litopenaeus stylirostris rearing. In this study, the complete genome of NC201 was sequenced. This genome consists of a chromosome (4.13 Mb) and a chromid (1.24 Mb). The genome contains gene clusters coding for antibacterial peptides and secondary metabolites.

Ontogeny of osmoregulation in the Pacific blue shrimp, Litopenaeus stylirostris (Decapoda, Penaeidae): Deciphering the role of the Na(+)/K(+)-ATPase.

The role of the main ion transporting enzyme Na+/K+-ATPase in osmoregulation processes was investigated in Litopenaeus stylirostris. The development and localization of the osmoregulation sites were studied during ontogenesis by immunodetection of Na(+)K(+)-ATPase using monoclonal antibodies and transmission electron microscopy (TEM). Osmoregulation sites were identified as the pleurae and branchiostegites in the zoeae and mysis stages. In the subsequent post-metamorphic stages the osmoregulatory function was mainly located in the epipodites and branchiostegites and osmotic regulation was later detected in the gills. The presence of ionocytes and microvilli in these tissues confirmed their role in ionic processes. The complete open reading frame of the mRNA coding for the α-subunit of Na+K+-ATPase was characterized in L. stylirostris. The resulting 3092-bp cDNA (LsNKA) encodes a putative 1011-amino-acid protein with a predicted molecular mass of 112.3kDa. The inferred amino acid sequence revealed that the putative protein possesses the main structural characteristics of the Na+K+-ATPase α-subunits. Quantitative RT-PCR analyses indicated that LsNKA transcripts did not significantly vary between the different developmental stages. The number of transcripts was about 2.5-fold higher in the epipodites and gills than in any other tissues tested in juveniles. A reverse genetic approach was finally implemented to study the role of LsNKA in vivo. Knockdown of LsNKA expression by gene-specific dsRNA injection led to an increase of shrimp mortality following an abrupt salinity change compared to control animals. These data strongly suggest that LsNKA plays an important role in osmoregulation when the shrimp are challenged by changing salinities.

Effect of probiotic Pediococcus acidilactici on antioxidant defences and oxidative stress of Litopenaeus stylirostris under Vibrio nigripulchritudo challenge.

Antioxidant defences and induced oxidative stress tissue damage of the blue shrimp Litopenaeus stylirostris, under challenge with Vibrio nigripulchritudo, were investigated for a 72-h period. For this purpose, L. stylirostris were first infected by immersion with pathogenic V. nigripulchritudo strain SFn1 and then antioxidant defences: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (Gpx), Total antioxidant status (TAS), glutathiones and induced tissue damage (MDA and carbonyl proteins) were determined in the digestive gland at 0, 12, 24, 48 and 72 h post-infection (h.p.i.). In the meantime, TAS was also measured in the blood. Infection level of the shrimps during the challenge was followed by determining V. nigripulchritudo prevalence and load in the haemolymph of the shrimps. Changes in all these parameters during the 72-h.p.i. period were recorded for control shrimps and shrimps previously fed for one month with probiotic Pediococcus acidilactici MA18/5M at 10(7) CFU g(-1) of feed. Our results showed that immersion with V. nigripulchritudo led to maximal infection level in the haemolymph at 24 h.p.i. preceding the mortality peak recorded at 48 h.p.i. Significant decreases in the antioxidant defences were detected from 24 h.p.i. and beyond that time infection leaded to increases in oxidative stress level and tissue damage. Compared to control group, shrimps fed the probiotic diet showed lower infection (20% instead of 45% at 24 h.p.i. in the control group) and mortality (25% instead of 41.7% in the control group) levels. Moreover, infected shrimp fed the probiotic compared to uninfected control shrimps exhibited very similar antioxidant status and oxidative stress level. Compared to the infected control group, shrimps fed the probiotic sustained higher antioxidant defences and lower oxidative stress level. This study shows that bacterial infection leads to oxidative stress in L. stylirostris and highlighted a beneficial effect of P. acidilactici, suggesting both a competitive exclusion effect leading to a reduction of the infection level and/or an enhancement of the antioxidant status of the shrimps.