Inhibitory effect of marine microalgae used in shrimp hatcheries on Vibrio parahaemolyticus responsible for acute hepatopancreatic necrosis disease

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Microalgae play a pertinent role in nutrition of larvae and are carrier of indigenous bacteria associated with the microalgae in a microalgae– bacterial interaction. Microalgae plus bacteria combinations such as Isochrysis galbana and Alteromonas sp., Labrezia sp. and Marinobacter sp., resulted in a better total length, survival, and metamorphosis from the zoea to mysis stages of Penaeus indicus larvae.

However, microalgae–bacteria interactions are complex, as the secretion of stimulating substances by bacteria improves microalgae growth and vice versa, indicating mutualistic or antagonistic relationships. Those relationships happen generally in the phycosphere, which is a region that it is immediately surrounding a phytoplankton cell that contains organic molecules for the photosynthetic activity, which can serve as bacterial nutrients.

This takes high relevance in hatchery management because microalgae can carry bacteria that not only can be beneficial but also could act as a vector of specific pathogens that could be introduced into the hatcheries.

Acute hepatopancreatic necrosis disease (AHPND) is an emerging bacterial disease that has caused great financial loss in the shrimp industry.

Due to the importance of AHPND and the relevance of the use of microalgae in hatcheries, this study aimed to evaluate the activity of cells, as well as extracts of four marine microalgae commonly used in shrimp hatcheries, the growth of one Vp AHPND+and a non- pathogenic Vp strain, and the effect of the bacteria load on the physiological response of microalgae.

Materials And Methods

Bacterial inoculum and Microalgae Culture

Two V. parahaemolyticus strains were used, Vp M0904, a highly virulent strain causing AHPND (Vp AHPND+), and Vp M0702, a non- pathogenic strain, both previously isolated from the hepatopancreas and stomach of shrimp affected with AHPND respectively. Four marine microalgae species were used: Chaetoceros calcitrans, Tetraselmis suecica, Nannochloropsis sp. and Thalassiosira weissflogii under sterile conditions.

Inhibition assays with Vp M0904 and Vp M0702 strains were performed with batch monospecific, vibrio-free co-cultures of each microalga.

All assays had three biological replicates. Uninoculated microalgae cultures with Vp strains were used as negative controls.

Samples, total lipids and carbohydrates

To know if microalgae suffer stress by the Vp strain’s presence, with metabolism being affected, the total lipids (TL) and total carbohy- drates (TC) were considered.

The quantification of TL and TC was done according to the methods suggested by Pande et al. (1963) methodology, adapted to a 96-well microplate assay. Samples (10 ml) of each microalgae culture were centrifuged and the cellular pellet was suspended in deionized water. the solution was refrigerated. Subsequently, solutions were vortexed and centrifuged. The samples were then cooled in water, and finally, distilled water was added.

Microalgae extracts and Fluorescent labelled bacteria (FLB)

To evaluate the effect of lipid or hydrophilic compounds of micro- algae cells on Vp growth, two microalgae extracts were obtained. Three hundred millilitres of duplicate C. calcitrans was taken from the exponential growth phase.

Since V. parahaemolyticus has ability to form biofilms on organic and inorganic surfaces, Vp M0904 may have adhered to the bottom of the flask or to micro-algae cells surface during co-culture assays, causing lower CFU counts.

Statistical analysis

Bacterial and microalgae growth, total carbohydrates and total lipids at different times of experimentation were analyzed using one-way ANOVA, followed by a post hoc Tukey test to evaluate significant differences (p < 0.05) between means.

Results

Non-culturable Vibrionaceae was observed in all control treatments in coculture with microalgae throughout the experiment, but C. calcitrans, T. suecica and Nannochloropsis sp. caused different levels of bacterial inhibition compared with the growth of Vp M0702 and Vp M0904 at 0 day and 3 days p.i. C. calcitrans showed a strong inhibitory effect on both Vp strains at all times p.i. T. weissflogii was the only microalgae that did not show an inhibitory effect on Vp M0904 growth. C. calcitrans had a higher percentage (39.4%) than the rest of the evaluated microalgae, followed by T. suecica (29%), Nannochloropsis sp. (25.6%) and T. weissflogii (16.1%).

In general, non-significant differences (p > 0.05) were found compared with the control of TC in C. calcitrans, T. suecica and Nannochloropsis sp. inoculated with both Vp strains. Conversely, using the indirect method, the growth of Vp M0904 observed in the three EE concentrations was significantly lower than the positive control.

Intact viable microalgae cells and monodispersed fluorescence Vp M0904 cells were observed in the assays, while Vp M0904 FLB was mainly observed adhered to the debris of senescence microalgae cells in both microalgae assays, but not to intact microalgae cells. It was evident that FLB did not adhere to viable microalgae cells, only to dead cells.

Discussion

Regarding the growth of Vp M0904, a strain responsible for AHPND and Vp M0702 a non-pathogenic strain, a significant inhibitory effect was observed when they were co-cultured with microalgae. Regardless of the virulence of the strain or the incubation time, a higher bacteriostatic effect was observed with T. suecica and C. calcitrans.

A similar effect of bacterial inhibition has been observed in co-culture experiments with marine microalgae against Vibrio species, although the mechanism of action has not been determined in most of the studies. The above suggests an antibacterial potential of specific microalgae to be used as a biological tool against pathogenic bacteria in hatcheries. Nevertheless, several experiments with axenic microalgae demonstrated the ability of some species to produce and release compounds with potent activity against pathogenic bacteria.

“Moreover, microalgae can produce a broad spectrum of antimicrobials and secondary allelopathic metabolites including polyunsaturated fatty acids, glycolipids, alkaloids, indols, phenols, terpenoids, etc. to gain a competitive advantage in dynamic and complex communities.”

Antibacterial activity observed in diatoms, Chaetoceros and Skeletonema, has been associated with their fatty acids content, primarily those with 10 carbon atoms, which might induce lysis in bacterial protoplasts.
However, fatty acids are inside the microalgae cells, therefore only extraction methods can make them bioavailable.

Organic and aqueous extracts from marine microalgae also produce antibacterial substances against clinical bacteria and Vibrio species. Methanolic extract of C. muelleri had potent antibacterial activity against Bacillus subtilis and S. aureus.

Crude seawater extract containing all cellular material of C. calcitrans showed significant inhibition on the growth of Vp M0904 at higher concentrations.”

The above might mean that potential antibacterial compounds remain active after a lyophilization process. Nonetheless, aqueous ex- tracts did not show potential in controlling the microbial pathogens, and antibiotic activity was also dependent on the microalgae phase of growth.

On the contrary, the aqueous phases of P. tricornuturn extracts in the log growth phase were active against the marine bacteria Alteromonas communis, ARTICLE Alteromonas haloplanktis, V. parahaemo- lyticus, Vibrio fischeri, Pseudomonas marina and Alcaligenes cupidus; in contrast, during the stationary phase, there was no activity against marine bacteria.

Depending on the existence of bioactive compounds, the different organic algal extracts show a difference in their inhibition against bacteria, and despite the antimicrobial role of fatty acids, other types of compounds may exhibit similar bioactivities It should be highlighted that the method used to estimate the bacterial growth influenced the result of the antibacterial activity of C. calcitrans extracts.

“Ethanolic extract showed a significantly lower OD of Vp M0904 than control, contrary to the OD of aqueous extract.”

In contrast, the growth of bacteria in seawater extracts of C. calcitrans had significant inhibitory activity. The optical density test measures the absorbance of the solutions, including live and dead cells, debris, organic material, etc., meanwhile the viable growth estimates the real effect of the microalgae extracts of bacterial cells able to grow in these extracts. For this type of study, we recommend the use of the TVC method.

In addition to microalgae species, the presence of antibacterial compounds in the microalgae extracts is also highly dependent on the solvent used during the extraction.

In this work, total lipid metabolism was not altered for the four evaluated microalgae (C. calcitrans, T. suecica, Nannochloropsis sp. and T. weissflogii) in co-culture with both Vibrio strains compared with the control. However, C. calcitrans and T. suecica had higher percentages (39.4% and 29%, respectively), which showed higher inhibitory activity on the tested Vibrio strains, although these differences in percentages could be due to intrinsic characteristics of the microalgae strain.

“Nannochloropsis sp. and T. weissflogii slightly promoted the growth of Vp M0702 and Vp M0904, which could imply that they act as stimulant of pathogenic vibrios and, therefore, the choice of microalgae in hatcheries must consider these microalgae– pathogen interactions.”

Regarding the labelled bacteria assays, growth of Vp M0904 and Vp M0904 FLB in the water column was similar to the bottom of the flask when co-cultured with C. calcitrans as well as T. suecica, meaning that bacterial cells were not attached to the bottom of the flask.

In addition, planktonic bacterial FLB cells were mainly observed adhered to debris of senescence cells of both microalgae.

Conclusions

Bacteriostatic effects of microalgae in co-cultures with Vibrio strains were dependent on microalgae species and Vibrio strains. C. calcitrans caused a higher degree of inhibition of Vp M0904, meanwhile T. suecica inhibited Vp M0904 as well as Vp M0702. Additionally, the bacteriostatic effect of C. calcitrans on Vp M0904 was dependent on the type of extract, only the hydrophilic extract showed inhibition by the TVC method.

It should be noted that C. calcitrans and T. suecica possess antibiotic activities over Vp M0904, a highly virulent strain that causes AHPND, a devastating disease for farmed shrimp around world.

This is a summarized version developed by the editorial team of Aquaculture Magazine based on the review article titled “INHIBITORY EFFECT OF MARINE MICROALGAE USED IN SHRIMP HATCHERIES ON VIBRIO PARAHAEMOLYTICUS RESPONSIBLE FOR ACUTE HEPATOPANCREATIC NECROSIS DISEASE” developed by: SONIA ARACELI SOTO-RODRIGUEZ, PAOLA MAGALLÓN-SERVÍN, MELISSA LÓPEZ-VELA, MARIO NIEVES SOTO. The original article was published on NOVEMBER 2021, through QUACULTURE RESEARCH under the use of a creative commons open access license. The full version can be accessed freely online through this link: DOI: 10.1111/are.15668

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