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

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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

Dynamic of active microbial diversity in rhizosphere sediments of halophytes used for bioremediation of earthen shrimp ponds

Background In New-Caledonia, at the end of each shrimp production cycle, earthen ponds are drained and dried to enhance microbial decomposition of nutrient-rich waste trapped in the sediment during the rearing. However, excessive ponds drying may not be suitable for the decomposition activities of microorganisms. Halophytes, salt tolerant plants, naturally grow at vicinity of shrimp ponds; due to their specificity, we explored whether halophytes cultivation during the pond drying period may be suitable for pond bioremediation. In addition, plants are closely associated with microorganisms, which may play a significant role in organic matter decomposition and therefore in bioremediation. Thus, in this study we aimed to determine the impact of 3 halophyte species (Suaeda australis, Sarcocornia quinqueflora and Atriplex jubata) on active sediment microbial communities and their implications on organic matter degradation. Results Drying significantly decreased the microbial diversity index compared to those of wet sediment or sediment with halophytes. Microbial profiles varied significantly over time and according to the experimental conditions (wet, dry sediment or sediment with halophyte species). Halophytes species seemed to promote putative microbial metabolism activities in the sediment. Taxa related to nitrogen removal, carbon mineralisation, sulphur reduction and sulphide oxidation were significant biomarkers in sediment harbouring halophytes and may be relevant for bioremediation. Whereas microbial communities of dry sediment were marked by soil limited-moisture taxa with no identification of microbial metabolic functions. Nitrogen reduction in sediments was evidenced in wet sediment and in sediments with halophytes cultures, along with putative microbial denitrification activities. The greatest nitrogen reduction was observed in halophytes culture. Conclusion The efficiency of sediment bioremediation by halophytes appears to be the result of both rhizosphere microbial communities and plant nutrition. Their cultures during the pond drying period may be used as aquaculture diversification by being a sustainable system

Selection and characterization of potential probiotic bacteria for Litopenaeus stylirostris shrimp hatcheries in New Caledonia

In New Caledonia, shrimp hatcheries are confronted with mass mortality in the larval stages, a phenomenon poorly understood as no specific causative agent has been identified. This has resulted in an excessive use of prophylactic antibiotics, although their adverse effects in aquaculture are notorious. The present work was thus aimed at selecting potential probiotic strains for penaeid hatcheries. From a pool of more than 400 marine bacterial isolates sampled from the local marine environment seven strains exhibited in vitro antagonistic activity towards Vibrio harveyi. These isolates were characterized both phenotypically and genotypically using a biochemical approach and 16S rDNA sequencing. Six out of these seven strains were found to belong to the genus Pseudoalteromonas, the last one belonging to the Vibrionaceae family and related to the Harveyi clade. Selected probiotic candidates were individually tested for antagonistic activities in vitro using a green fluorescent protein (GFP)-labelled transconjugant of V. harveyi and for inherent pathogenicity towards cultured shrimp larvae at two different developmental stages. This approach enabled rapid processing and selection of candidates to be tested in our experimental hatchery. Four different experiments were conducted to test candidate strains either alone or in combination. Repeated trials showed that postlarval survival was significantly improved by adding the strain NC201 individually to the rearing water compared to unchallenged controls. The analysis of immune-related gene expressions showed that the Litsty PEN3 transcript abundance of larvae was significantly increased after being reared in probiotic-containing water; however no significant difference in lysozyme gene expression was recorded in this study. Together, these results open new insights into the use of these strains as potential substitutes to antibiotherapy in shrimp larval rearing in New Caledonia

Suaeda australis and its associated rhizosphere microbiota: a comparison of the nutrient removal potential between different shrimp farm sediments in New Caledonia

Shrimp rearing generate organic waste that is trapped in the pond sediment. In excess, these wastes may impair aquaculture ecosystem and shrimps’ health. To promote the biological oxidation of accumulated organic waste, the pond is drained and dried at the end of each production cycle. However, this practice is not always conducive to maintaining microbial decomposition activities in sediments. Shrimp production in New Caledonia is no exception to this problem of pollution of pond bottoms. One promising way of treating this waste would be bioremediation, using a native halophyte plant and its microbiota. Thus, this study explored the nutrient removal potential of Suaeda australis and its microbiota on sediments from four shrimp farms. Suaeda australis was grown in an experimental greenhouse for 6 months. In order to mimic the drying out of the sediments, pots containing only sediments were left to dry in the open air without halophytes. An analysis of the chemical composition and active microbiota was carried out initially and after 6 months in the sediments of the halophyte cultures and in the dry sediments for each farm, respectively. In the initial state, the chemical parameters and the microbial diversity of the sediment varied considerably from one farm to another. Growing Suaeda australis reduced the nitrogen, phosphorus and sulfur content in all type of sediment. However, this reduction varied significantly from one sediment to another. The rhizosphere of Suaeda australis is mainly composed of micro-organisms belonging to the Alphaproteobacteria class. However, the families recruited from this class vary depending on the farm in question. Depending on the sediment, the variation in microbiota leads to different putative biochemical functions. For two of the farms, a similar reduction in nitrogen concentration was observed in both dry and cultivated sediments. This suggests that certain initial chemical characteristics of the sediments influence the nutrient removal efficiency of Suaeda australis. Our study therefore highlights the need to control the pH of sediments before cultivation or in dry sediments in order to ensure optimal microbial decomposition of organic waste and nutrient cycling

Detection of shrimp pathogen Vibrio nigripulchritudo in sediments of a New-Caledonian grow-out pond during a drying period

Experimental infections together with epidemiological studies have shown that pathogenic and non‐pathogenic isolates of V. nigripulchritudo co‐existed in shrimp farm environment (2, 3). Moreover, obtained results also demonstrated that the “summer syndrome”was caused by a single, possibly emerging, cluster of virulent strains. Consequently, it was hypothesized that pathogenic strains of V. nigripulchritudo may persist from one year to the next in the shrimp farm environment and re‐develop inside the grow‐out system at the following rearing cycle (3). This study was therefore aimed at determining whether V. nigripulchritudo isolates may survive, or not, in a shrimp pond bottom soil during a 18‐week drying period. To this end, V.nigripulchritudo mapping was performed with recently developed molecular tools and classical culture‐dependent techniques

Bases des connaissances sur l'épidémiologie de Vibrio nigripulchritudo, agent étiologique du « Syndrome d'été » chez les crevettes d’élevage en Nouvelle-Calédonie

A new disease has been affecting the shrimp aquaculture industry of New Caledonia since 1998. This pathology leads to mass mortalities in summer and were therefore called « Summer Syndrome ». Though Vibrio nigripulchritudo, the putative etiological agent, can be isolated from almost any farming site, it causes severe mass mortalities in only one farm. This report is aimed at compiling the current knowledge on geographical distribution of this pathogen in New Caledonia, his epidemiology with regards to the « summer syndrome » and presents data on pathogenicity studies of a few selected strains. A few research perspectives are summarized at the end of the report.L’aquaculture de crevettes en Nouvelle-Calédonie rencontre depuis 1998 une nouvelle pathologie. Celle-ci se traduit par une mortalité au caractère estival marqué qui lui a valu le nom de « syndrome d’été ». Bien que l’agent pathogène identifié, la bactérie Vibrio nigripulchritudo, puisse être retrouvé sur la majorité des sites d’élevage, une seule ferme apparaît réellement affectée par cette maladie. Ce rapport fait le point des connaissances sur la répartition géographique de ce pathogène dans les sites d’élevage de crevettes, son épidémiologie dans le contexte du « Syndrome d’été » et présente des données sur l’étude du pouvoir pathogène de quelques souches. Il s’efforce ensuite de lister quelques pistes de recherche sur ce « Syndrome d’été »

Pathotyping of Vibrio Isolates by Multiplex PCR Reveals a Risk of Virulent Strain Spreading in New Caledonian Shrimp Farms

International audienceTwo recurring syndromes threaten the viability of the shrimp industry in New Caledonia, which represents the second largest export business. The "Syndrome 93" is a cold season disease due to Vibrio penaeicida affecting all shrimp farms, while the "Summer Syndrome" is a geographically restricted vibriosis caused by a virulent lineage of Vibrio nigripulchritudo. Microbiological procedures for diagnosis of these diseases are time-consuming and do not have the ability to discriminate the range of virulence potentials of V. nigripulchritudo. In this study, we developed a multiplex PCR method to simultaneously detect these two bacterial species and allow for pathotype discrimination. The detection limits of this assay, that includes an internal amplification control to eliminate any false-negative results, were determined at 10 pg purified DNA and 200 cfu/ml. After confirming the effectiveness of our method using experimentally infected animals, its accuracy was compared to standard biochemical methods during a field survey using 94 samples collected over 3 years from shrimp farms encountering mortality events. The multiplex PCR showed very high specificity for the detection of V. penaeicida and V. nigripulchritudo (inclusivity and exclusivity 100%) and allowed us to detect the spreading of highly pathogenic isolates of V. nigripulchritudo to a farm adjoining the "Summer Syndrome area." This assay represents a simple, rapid, and cost-effective diagnostic tool for implementing timely risk management decisions but also understanding the seasonal and geographical distribution of these pathogens

Survival improvement conferred by the Pseudoalteromonas sp. NC201 probiotic in Litopenaeus stylirostris exposed to Vibrio nigripulchritudo infection and salinity stress

This study aimed to investigate the potential protection conferred by the probiotic strain NC201 against biotic and abiotic stresses in Pacific blue shrimp Litopenaeus stylirostris that had received the probiotic throughout their lives. The presence of NC201 in shrimp hemolymph was investigated over the course of 24 h, before exposure to bacterial or physical stress. Results showed that NC201 had invaded the shrimp hemolymph 2 h following administration, but had completely disappeared by 48 h. NC201 identification through morphotype observation was confirmed by MALDI-TOF biotyping and results also indicated that NC201 and Pseudoalteromonas piscicida are closely related. A challenge by immersion was carried out on subadults using Vibrio nigripulchritudo at 105 CFU/ml. Cumulative mortality was two-fold lower in the treated group (24%) than in the control group (48%) at 144 h post infection. The probiotic in the shrimp hemolymph was diminished in infectious conditions compared with non-infectious ones and V. nigripulchritudo prevalence was simultaneously lower in animals treated with NC201. The relative expression of genes coding lysozyme and penaeidin 3 was evaluated 24 h post infection and their transcript numbers were found to be lower in probiotic animals than in control animals for both genes. Hyposaline stress was also used to evaluate the benefits of NC201 treatment on early juveniles and subadults. At low salinities, animals showed an increased survival rate when treated with NC201, by 10 and 17.5% at 48 h post stress, respectively. Moreover, in subadults treated with the probiotic, a better recovery of the plasmatic osmolality was observed. All these results confirm that NC201 is a good candidate probiotic for shrimp aquaculture

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

International audienceThe 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 112.3 kDa. 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