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Inhibitory effect of benthic diatom species on three aquaculture pathogenic vibrios
Algal Research 27 (2017) 131–139
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Inhibitory effect of benthic diatom species on three aquaculture pathogenic vibrios
MARK
Ceres A. Molina-Cárdenas, M. del Pilar Sánchez-Saavedra⁎ Centro de Investigación Científica y de Educación Superior de Ensenada (CICESE), Carretera Tijuana-Ensenada 3918, Zona Playitas, 22860 Ensenada, Baja California, Mexico
A R T I C L E I N F O
A B S T R A C T
Keywords: Benthic diatoms co-culture Vibrio inhibition, biocontrol
Diatoms can produce inhibitory compounds against bacteria, constituting an alternative to the use of chemicals to control pathogenic bacterial growth. Antibacterial activity has been detected in co-cultures of microalgaebacteria and extracts. Thus, we evaluated the ability of 6 benthic diatom species to inhibit the growth of Vibrio alginolyticus, V. campbellii, and V. harveyi, which are bacterial species that are pathogenic to mollusks, crustaceans, and fish. Triplicate cultures of benthic diatoms were inoculated with each individual Vibrio species, and the density of microalgae cells, vibrios, and heterotrophic bacteria was estimated at baseline (0 h), 24, 48, 72, 96, 168 h post-inoculation, and when the diatom cultures reached the stationary growth phase. Significant differences in the growth of each diatom species were observed, depending on the Vibrio species that was inoculated, and in all cultures, the density of Vibrio cells decreased, even to undetectable levels (< 0.01 Vibrio cells mL− 1), after 4, 7, and 16 days post-inoculation. Initial bacterial counts in the diatom cultures were on the order of 103 (Colony Forming Units) CFU mL− 1, reaching 106 to 107 CFU mL− 1 by the end. The concentration of bacteria in the diatom cultures was similar between co-cultures and control treatments, indicating that the inhibitory effects of the diatom strains were specific for Vibrio species. We conclude that the diatom species that we tested control the concentration of pathogenic vibrios, presumably through the production of antibacterial compounds. These findings should encourage the use of diatom cultures as a feeding source and to control the density of pathogenic Vibrio species in culture systems, adding diatom species to culture water as a “green water” technique.
1. Introduction Diatoms are eukaryotic microalgae that have greater diversity than other microalgae groups. These photosynthetic organisms belong to the class Bacillariophyceae and are characterized by their silica frustules, high diversity [1], and high fatty acid content, containing such compounds as eicosapentanoic acid (EPA) and docosahexanoic acid (DHA) [2,3]. Under natural conditions, diatoms interact with other organisms, especially bacterial species [4]. When these interactions are positive, the bacteria can enhance the growth of microalgae cells through the production of growth-promoting factors, such as vitamins and idole3acetic-acid, or the regeneration of inorganic nutrients [5]. In parallel, microalgae synthesize exudates that can be a source of assimilable carbon for bacteria [6]. However, microalgae can generate antibacterial compounds that inhibit the growth of bacteria, and vice versa [7,8]. The production and sensitivity of algae and bacteria to these
compounds are species-specific and can be influenced by the culture conditions [9]. Marine microalgae produce secondary metabolites that affect the growth of bacteria, fungi, viruses, and other epibionts [10–11]. Several studies have examined the inhibitory activity of microalgae on the concentration of pathogenic bacteria, such as Vibrio species [12–16]. Diatoms produce a wide variety of chemical compounds with many types of bioactivity, such as antibacterial activity. For example, Skeletonema costatum and Phaeodactylum tricornutum, generate secondary metabolites that have effects on the pathogenic bacteria Staphylococcus aureus and Vibrio anguillarum [12,17]. Infectious diseases that are caused by pathogenic Vibrio bacterial species are associated with high mortality rates in aquaculture production systems, impacting the profitability of this industry [18–19]. Vibrios can be introduced to culture ponds through live prey, such as copepods, Artemia, and microalgae that are used as feed for fish, crustaceans, and mollusks [20–21]. Strains, such as Vibrio alginolyticus, are
Abbreviations: Va, Vibrio alginolyticus; Vh, Vibrio harveyi; Vc, Vibrio campbellii ⁎ Corresponding author. E-mail address: [email protected] (M.d.P. Sánchez-Saavedra). http://dx.doi.org/10.1016/j.algal.2017.09.004 Received 8 February 2017; Received in revised form 30 August 2017; Accepted 1 September 2017 2211-9264/ © 2017 Elsevier B.V. All rights reserved.
Algal Research 27 (2017) 131–139
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culture collections: Vibrio alginolyticus (Va) ATCC 17749 (American Type Culture Collection); Vibrio campbellii (Vc) (CAIM 416, CICESE 559), obtained from the Collection of Aquacultural Importance Microrganisms (CAIM) and belonging to “Centro de Investigación en Alimentación y Desarrollo (CIAD)”; and Vibrio harveyi (Vh) DN01 (CICESE 597). All Vibrio species were cultured in Petri dishes with ZoBell medium [42] and incubated at 28 °C for 24 h. Then, colonies were removed by rinsing off the agar surface with 9 mL of sterile physiological serum, and the optical density of the suspension was adjusted to 0.05 at 600 nm on a Hach DR-4000 UV-VIS spectrophotometer. This optical density equals a concentration of 106 cells mL− 1 [22]. The Vibrio suspensions were used to inoculate the diatom cultures.
pathogens to mollusks, such as the larvae of the red abalone Haliotis rufescens, Ruditapes decussatus, Argopecten ventricosus, Nodipecten subnodosus, Mytilus galloprovincialis, and Haliotis diversicolor supertexta [22–23]. Vibrio campbellii is a potential pathogen of crustaceans [24], and Vibrio harveyi is a pathogenic species in fish [25–26]. For aquaculture activity, vibriosis is controlled most frequently with antibiotics, such as oxytetracycline, enrofloxacin, florfenicol, sarafloxacin, amoxicillin, and oxolinic acid [19,27–28]. Yet, the excessive application prophylactic antibiotics in aquaculture has led to the appearance of bacterial strains that are resistant to the compounds that are used to control them [29–30]. Other strategies, such as the disruption of quorum sensing, phage therapy, and probiotic bacteria in culture systems, are effective in limiting the density of Vibrio species in in vitro assays. However, their effects under actual culture conditions remain unknown, and none of them alone can control this problem [31–32]. New strategies and molecules that control the density of Vibrio bacteria in culture systems are urgently needed. Microalgae are a reservoir of compounds with various activities, including antimicrobial action [33–34]. Antimicrobial compounds that are produced by microalgae vary in nature and include fatty acids and their derivates [35–36], peptides [33], toxins [37], pigments [38], polysaccharides, flavonoids, and terpenoids [33]. The antibacterial activity of diatoms has been attributed to specific fatty acids in the cells [35], their capacity to compete for nutrients, such as phosphorus [9], and the synthesis of exopolysaccharides [39]. The aim of this work was to determine the ability of 6 taxa of benthic diatoms that were isolated from Pacific coastal waters to inhibit the growth of Vibrio alginolyticus, V. campbellii, and V. harveyi.
2.3. Inhibition of Vibrio species in diatom culture assays Inhibition assays of Vibrio were performed with monospecific, nonaxenic cultures of each strain and in triplicate for each diatom species in 500-mL Erlenmeyer flasks with 200 mL “f” medium [43] at an initial concentration of 100,000 cells mL− 1. To quantify Vibrio cells in each experimental culture condition, aliquots of 1 mL and another of 10 mL were passed through 45-mm sterile filters (0.22-μm pore size). Filters that contained the cells were placed on thiosulfate citrate bile sucrose (TCBS) agar media. Concentrations of Vibrio of 0.01 cells mL− 1 or lower indicated the absence of this type of bacteria. The cultures of each diatom strain were inoculated with 2 mL of Vibrio bacterial suspension by monospecific addition, which equals a final concentration of 103 Vibrio cells mL− 1. Immediately after the inoculation, the initial concentration of Vibrio in each experimental culture condition was corroborated as described above. These initial filtrates corresponded to time T0 + 1, and the concentration of Vibrio bacteria on subsequent days—ie, the filtrates at 0, 24, 48, 96, and 168 h post-inoculation—and when the growth of the diatom cultures reached the exponential and stationary growth phases was measured. Filters were placed in Petri dishes with TCBS media and incubated at 28 °C for 24 h. As controls, Erlenmeyer flasks with triplicate cultures of each diatom strain without Vibrio bacteria and flasks with “f” medium that were inoculated with each Vibrio strain tested alone were maintained under the same culture conditions as in the Vibrio inhibition assays. Heterotrophic bacterial concentrations were measured by seeding 0.1 mL of serial dilutions of co-cultures or monospecific cultures of diatoms in Petri dishes with ZoBell medium at the same times and under the same conditions as the incubations for the Vibrio counts. The concentrations of this type of bacteria were expressed in colony forming units per milliliter (CFU mL− 1).
2. Materials and methods 2.1. Characteristics of diatom species and cultures We used 6 benthic diatom strains that were isolated from the coast of Baja California and Nayarit, Mexico. The diatom strains from Baja California were Nitzschia laevis Hustedt and Nitzschia frustulum var. perminuta Grunow, isolated and identified per Correa-Reyes et al. [2]. Navicula incerta Grunow was isolated by the Institute of Oceanological Research (IIO), Autonomous University of Baja California (UABC). The diatom strains from Nayarit were Navicula biskanterae Hust., Nitzschia fustulum (Kútzing) Grunow, and Navicula cf. incerta Grunow in Van Heurck, isolated from the water culture of a shrimp farm by AguilarMay [40]. The diatom species belong to the collection of the Group of Algae Cultures in the Aquaculture Department of CICESE. The diatom strains were identified per Correa-Reyes et al. [2] and López-Fuerte and Siqueiros-Beltrones [41]. Monospecific and non-axenic (concentration of associated bacteria < 103 cell mL− 1 and hereafter referred to as heterotrophic bacteria) batch cultures of the 6 benthic diatom strains were maintained in 500-mL Erlenmeyer flasks with 200 mL “f” medium [63]. All cultures were inoculated with 100,000 cells mL− 1 of each benthic diatom strain. Cultures were established in triplicate and maintained at 22 ± 1 °C, 33 ± 1‰ salinity, and 24 h of continuous light at 110 μEm− 2 s− 1, provided by cool white fluorescent lamps. To measure cell concentrations, daily counts were performed with a hemocytometer (Hausser Scientific) under a compound microscope at 40× magnification. The cell concentrations were used to calculate growth rates as Fogg and Thake [44].
2.4. Statistical analysis The initial and final concentrations of Vibrio bacteria in diatom cultures were compared by student t-test. The growth of each diatom species in the presence of Vibrio bacteria and their growth rates were compared by one-way ANOVA. Significant differences were determined by a posteriori Tukey test. Differences in heterotrophic bacteria were significant if the concentrations exceeded one order of magnitude. Statistical analysis was performed using Statistica 7.0 with α = 0.05, and Sigma Plot 10 was used to construct the graphs. 3. Results 3.1. Growth of diatom species and Vibrio species
2.2. Vibrio cultures The diatoms N. laevis and N. frustulum var. perminuta had higher cell concentrations (p < 0.05) on day 9 of culture. However, N. incerta, N. biskanterae, and N. frustulum reached similar concentrations on day 7. The strain N. cf. incerta had the lowest cell concentration of all diatom cultures on day 9 of culture (Fig. 1).
Three Vibrio species that have been reported to be pathogens of aquaculture organisms were selected, based on their associated high mortality rates in commercial aquaculture production. These Vibrio species are part of the CICESE Microorganism Collection, from various 132
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reached the stationary phase, settling at a concentration of 1.7 × 106 cells mL− 1. On day 9, N. biskanterae (Fig. 3C) halted its growth and reached a concentration of 1.6 × 106 cells mL− 1, whereas N. laevis (Fig. 3B) and N. frustulum (Fig. 3D) stopped growing on day 10. At this time, the average cell concentration was 1 × 106 for both species, and N. incerta (Fig. 3A) reached 2 × 106 cells mL− 1 after 11 days in culture. The strain Navicula cf. incerta required more time to enter the stationary growth phase (Fig. 3F), registering a concentration of 1.4 × 106 cells mL− 1 after 13 days in culture. The amount of heterotrophic bacteria in all cultures increased over time (p < 0.05). At the beginning of all assays, the concentration was 5 × 105 CFU mL− 1, increasing to 1 × 107 CFU mL− 1 at the end of the experiments. In diatom cultures with V. campbellii, bacterial inhibition was achieved on day 4 post-inoculation in nearly all treatments (Fig. 4)—with Navicula cf. incerta, the inhibitory effect was observed on day 7. In the challenge with V. campbellii, N. incerta grew to a concentration of approximately 1.5 × 106 cells mL− 1 after 11 days in culture (Fig. 4A)—the time that was needed for this microalgae species to enter the stationary growth phase. A similar response with regard to time and cell density was seen in N. laevis (Fig. 4B) and Navicula cf. incerta (Fig. 4F). The cultures with N. biskanterae (Fig. 4C) and N. frustulum (Fig. 4D) ceased their growth on day 10, when they reached concentrations of 2 × 106 cells mL− 1 and 1.7 × 106 cells mL− 1, respectively. N. frustulum var. perminuta was the slowest species to reach the stationary growth phase (Fig. 4), requiring 12 days to stop its growth and obtain an average concentration of 2.3 × 106 cells mL− 1. In these treatments, all initial counts of heterotrophic bacteria were 5 × 105 CFU mL− 1, ultimately rising to 107 CFU mL− 1 (p < 0.05). The V. harveyi strain was the most resistant bacterium to inhibition (p < 0.05), and even in cultures of N. laevis, N. frustulum, and Navicula cf. incerta, Vh remained at densities above detection limits (> 0.01 Vh cells mL− 1). However, significant differences were found between the initial and final concentrations of V. harveyi. In this assay, inhibition was observed on day 7 in cultures of N. laevis (Fig. 5B), N. biskanterae (Fig. 5C), N. frustulum var. perminuta (Fig. 5E), and Navicula cf. incerta (Fig. 5F), versus day 4 for N. incerta (Fig. 5A) and N. frustulum (Fig. 5D). On day 10 post-inoculation, cultures of N. biskanterae, N. frustulum, and N. frustulum var. perminuta reached the stationary growth phase and had average concentrations of 2.2 × 106 cells mL− 1. Navicula incerta, N. laevis, and Navicula cf. incerta stopped their growth on day 12 and had concentrations of 2 × 106, 3 × 106, and 1.5 × 106 cells mL− 1, respectively. As in the other experiments, the amount of heterotrophic bacteria in cultures that were challenged with V. harveyi had starting concentrations in the order of 105 and finished significantly higher, in the order of 107 CFU mL− 1.
Fig. 1. Mean values and standard deviation of densities of Navicula incerta (●), Nitzschia laevis (○), Navicula biskanterae (▼), Nitzschia frustulum (△), Nitzschia frustulum var. perminuta (■), and Navicula cf. incerta (□) in monospecific cultures. Letters indicate significant differences (one-way ANOVA and Tukey a posteriori test p < 0.05, a > b > c > d).
Fig. 2. Mean values and standard deviation of densities of Vibrio alginolyticus (●), Vibrio campbellii (○), and Vibrio harveyi (▼) grown in “f” medium without microalgae cells.
Based on the cell counts, Vibrio alginolyticus, V. campbellii, and V. harveyi survived in “f” medium after 14 days; the final bacterial densities for these 3 species were on the order of 103 Vibrio cells mL− 1 (Fig. 2).
3.3. Effects of Vibrio species on the growth of benthic diatom species The growth of diatom species differed significantly between cultures with and without Vibrio species (p < 0.05) (Fig. 6). In N. incerta cultures (Fig. 6A), the addition of V. alginolyticus and V. harveyi stimulated their growth, and higher growth rates were observed (Table 1); when co-cultured with V. campbellii however, the cell densities were lower (p < 0.05). Vibrio harveyi did not show any significant differences with regard to controlling growth in N. laevis cultures (Fig. 6B), which was, in contrast, altered by V. campbelli and V. alginolyticus (p < 0.05). Nonetheless, the growth rate for this diatom species was higher when cultured with V. campbellii (Table 1). For N. biskanterae (Fig. 6C), the highest cell densities were obtained with V. harveyi and V. campbellii and in control cultures; V. alginolyticus was the bacterial specie that inhibited the growth of N. biskanterae most extensively (p < 0.05). The growth rate of N. biskanterae peaked when co-cultured with V. campbellii (Table 1). Growth-inhibiting effects of V. alginolyticus were also observed with N. frustulum (Fig. 6D) and N. frustulum var. perminuta (Fig. 6E), which increased their concentrations
3.2. Inhibition of Vibrio species in cultures of diatom species None of the filtrates of the diatom cultures before inoculation with Vibrio species showed bacterial growth in TCBS selective medium, indicating that the diatom cultures were free of Vibrio species. In all diatom cultures, the concentrations of Vibrio-like bacteria per mL (VLB cells mL− 1) were below 0.01. In nearly all cultures of diatom species, V. alginolyticus (Va) was inhibited on day 4—with N. biskanterae, the concentrations fell below 0.01 Va cells mL− 1 on day 2 post-inoculation (Fig. 3). In all experiments, once the Vibrio was inhibited, this effect persisted until the diatom cultures reached the stationary growth phase. This response was also observed in the assays with V. campbellii (Vc) (Fig. 4) and V. harveyi (Vh) (Fig. 5). The time that was needed to reach the stationary growth phase in all diatom species that were cultured with V. alginolyticus ranged from 8 to 14 days (Fig. 3A–F). After 8 days post-inoculation, N. frustulum var. perminuta (Fig. 3E) 133
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Fig. 3. Mean values and standard deviation of densities of Vibrio alginolyticus (Va as cells mL− 1: ★) and heterotrophic bacteria (CFU mL− 1: ■) in cultures of various diatom species (cells mL− 1: ●) of Navicula incerta (A), Nitzschia laevis (B), Navicula biskanterae (C), Nitzschia frustulum (D), Nitzschia frustulum var. perminuta (E), and Navicula cf. incerta (F). Letters indicate significant differences in initial and final Vibrio densities (student t-test p < 0.05, a > b).
algynolyticus and V. campbelli were completely inhibited in all co-cultures. Despite the partial inhibition of V. harveyi, its concentration decreased significantly in co-cultures with diatoms. The antagonistic effects of diatoms on bacterial species occur naturally in marine environments [45]. The inhibition of vibrios by microalgae species has been reported by Lio-Po et al. [13], who demonstrated that Chaetoceros calcitrans and Nitzchia sp. suppresses the growth of luminescent vibrios at 24 or 48 h and that a low load of vibrios remains in microalgae-bacteria co-cultures after 7 days. Similar results were shown culturing Leptolyngbia sp. with luminescent vibrios and Makridis et al. [16], who found that Chlorella minutissima and Tetraselmis chuii reduce the load of Vibrio anguillarum. The capacity of diatom species to inhibit the growth of pathogenic
and growth rates when cultured with V. harveyi and V. campbellii, respectively (p < 0.05) (Table 1). In Navicula cf. incerta cultures (Fig. 6F), Vibrio had beneficial effects on cell concentration, with higher cell densities attained when co-cultured with all Vibrio bacteria species (p < 0.05). Conversely, the growth rate for Navicula cf. incerta was highest in cultures without Vibrio species (Table 1). 4. Discussion 4.1. Inhibition of Vibrio species in cultures of diatom species In all challenges, the concentration of Vibrio species declined when they were co-cultured with each diatom species; the bacterial strains V. 134
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Fig. 4. Mean values and standard deviation of densities of Vibrio campbellii (Vc as cells mL− 1:★) and heterotrophic bacteria (CFU mL− 1: ■) in cultures of various diatom species (cells mL− 1: ●) of Navicula incerta (A), Nitzschia laevis (B), Navicula biskanterae (C), Nitzschia frustulum (D), Nitzschia frustulum var. perminuta (E), and Navicula cf. incerta (F). Letters indicate significant differences in initial and final Vibrio densities (student t-test p < 0.05, a > b).
vibrios has been exploited in the “green water” technique—a system in which microalgae species are added to the culture water of crustaceans and fishes [46]. Several reports have confirmed that the population of pathogenic vibrios can be controlled in cultures of Penaues monodon, in which the dominant species of phytoplankton are diatoms, such as Skeletonema sp., Nitzchia sp., Nanochlorum sp., Navicula sp., Thalassiosira sp., Anabaena sp., and Chaetoceros sp. [47]. The antibacterial activities of diatom species in the Nitzschia and Navicula genera have been reported by Findlay and Patil [48], who evaluated the effects of sterols and fatty acids from Navicula delognei. This group found that compounds that were produced in Navicula
delognei were active against Staphylococcus aureus, Staphylococcus epidermis, Salmonella typhimurium, and Proteus vulgaris. Organic extracts of Nitzschia palea have been tested against Micrococcus luteus, S. aureus, and Pseudomonas aeruginosa [49]. With regard to the inhibition of Vibrio species, there is evidence that extracts from Thalassiosira rotula inhibit the growth of V. harveyi [50] and that cellular components of Odontella aurita suppress that of V. alginolyticus and P. vulagaris [51]. In most of our assays, inhibition was observed when the diatom species were in the early exponential growth phase, and low or undetectable Vibrio cell densities were maintained until the cultures reached the stationary growth phase. In the experiments with V. 135
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Fig. 5. Mean values and standard deviation of densities of Vibrio harveyi (Vh as cells mL− 1: ★) and heterotrophic bacteria (CFU mL− 1: ■) in cultures of various diatom species of Navicula incerta (A), Nitzschia laevis (B), Navicula biskanterae (C), Nitzschia frustulum (D), Nitzschia frustulum var. perminuta (E), and Navicula cf. incerta (F). Letters indicate significant differences in initial and final Vibrio densities (student t-test p < 0.05, a > b).
reactive oxygen species (ROS). These compounds can enhance the antibacterial activity of fatty acids [53], which are synthesized in high quantities during the stationary phase [54–55]. According to Van Rijssel et al. [52], synergism between ROS and fatty acids inhibits the growth of Vibrio fisheri; thus, it is possible that the inhibitory pattern in our study is attributed to the activity of several compounds that are produced and released during the exponential and stationary growth phases.
harveyi, we noted inhibition in the late exponential growth phase and even when the growth of the diatoms entered the stationary phase. More antibacterial compounds are produced when microalgae cultures have lower growth rates or are in the early stationary growth phase [33–12]. Moreover, microalgae cells synthesize various secondary metabolites—depending on the growth phase—that have species-specific effects. For example, Van Rijssel et al. [52] reported that the generation of secondary metabolites by the microalgae Fibrocapsa japonica was greater in phases in which the growth rate was high, as in the exponential phase, and that in this growth phase, F. japonica produces 136
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Fig. 6. Mean values and standard deviation of cellular density of monospecific cultures (●) of Navicula incerta (A), Nitzschia laevis (B), Navicula biskanterae (C), Nitzschia frustulum (D), Nitzschia frustulum var. perminuta (E), and Navicula cf. incerta (F) in mixed cultures with Vibrio alginolyticus (▲), V. campbellii (★), and V. harveyi (■). Letters indicate significant differences (ANOVA and Tukey a posteriori test, p < 0.05, a > b > c).
that the addition of bacteria to cultures of Thalassiosira weissflogii, Cylindrotheca fusiformis, and Nitzschia laevis stimulates the growth of these microalgae species, doubling their numbers versus cultures that are not exposed to bacteria. The antimicrobial activity in aggregates and detritus can lead to the release of nitrate, ammonia, phosphorus, and CO2, increasing and maintaining the growth of cultures, as described by several groups
4.2. Effects of Vibrio species on the growth of benthic diatom species In most of our experiments, the growth rates and cell densities of diatom species were higher when they were cultured with any Vibrio species compared with control cultures, with the exception of Navicula cf. incerta. Fukami et al. [8] demonstrated that in the presence of bacteria, axenic cultures of phytoplankton grow faster. Grossart [56] noted 137
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organic carbon that is produced by heterotrophic bacteria (1 to roughly 100 μg carbon L− 1 day− 1) constitutes < 10% to 100% of the primary production in certain cases [61,67]. Our results demonstrate that Vibrio species produce and release many compounds into the culture media, some of which can be used as nutrients or induce the synthesis of growth factors and increase the cell densities of certain diatom strains. In other cases, chemicals that are generated by Vibrio species inhibit the growth of diatom strains. Only one study has reported a rise in the cell density of Isochrysis galbana due to its interaction with V. alginolyticus, V. campbellii, and V. harveyi, noting that these Vibrio strains produced a substance that promotes microalgae growth [57]. Diatom species are used as food for mollusks, such as abalone, and crustacean larvae in aquaculture facilities. Our results should encourage the use of diatom species as a food source and as an alternative method of controlling pathogenic bacteria in culture systems, reducing the application of chemicals and antibiotics to mitigate harmful bacterial loads. Future studies should isolate and characterize the compounds that mediate the inhibition of the growth of pathogenic vibrios in aquaculture.
Table 1 Mean values and standard deviation of maximum cell densities (MCDs) and growth rates (μ, in divisions day− 1) in cultures of diatom species with various Vibrio species and control treatments (each microalgae culture without Vibrio species). Letters indicate significant differences (ANOVA and Tukey a posteriori test, p < 0.05, a > b > c). Diatom species
Vibrio species
MCDs (1 × 106 cells mL− 1)
μ
Navicula incerta
Without V. alginolyticus V. campbellii V. harveyi Without V. alginolyticus V. campbellii V. harveyi Without V. alginolyticus V. campbellii V. harveyi Without V. alginolyticus V. campbellii V. harveyi Without V. alginolyticus V. campbellii V. harveyi Without V. alginolyticus V. campbellii V. harveyi
1.7 2.0 1.4 2.1 2.4 1.6 2.0 2.4 1.7 1.5 1.7 1.6 1.8 1.5 1.7 2.2 2.1 1.7 2.3 2.1 1.2 1.4 1.6 1.5
0.25 0.32 0.25 0.33 0.27 0.23 0.31 0.26 0.20 0.20 0.25 0.23 0.21 0.16 0.27 0.29 0.31 0.20 0.25 0.32 0.39 0.15 0.27 0.30
Nitzschia laevis
Navicula biskanterae
Nitzschia frustulum
Nitzschia frustulum var. perminuta
Navicula cf. incerta
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.07 b 0.06 a 0.03 c 0.01 a 0.10 a 0.03 c 0.12 b 0.03 a 0.05 a 0.04 b 0.09 a 0.01 a 0.04b 0.09 c 0.06 bc 0.07 a 0.05 b 0.11 c 0.15 a 0.07 ab 0.04 c 0.01 b 0.07 a 0.03 a
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.03 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.03 0.01
b a b a b c a b b b a b b c a a a c b a a c b b
5. Conclusion Based on our findings, we recommend the use of diatom strain cultures for the potential biocontrol of pathogenic vibrios (V. alginolyticus, V. campbellii, and V. harveyi) in culture systems, and diatoms can be used as food for the larvae of mollusks, such as abalone. The microalgae strains that we have tested can potentially be applied to control V. alginolyticus, V. campbellii, and V. harveyi and replace or minimize the use of antibiotics in aquaculture facilities.
[56,57–59]. Park et al. [60] found that the growth of Chlorella ellipsoidea rose 0.5- to 3-fold when inoculated with bacteria of the genus Brevundimonas, an effect that they attributed in part to more efficient exchange of substrates between bacteria that were attached to phytoplankton cells. However, the relationship between bacteria and microalgae is not limited to nutrient remineralization. There is evidence of a phycosphere—a zone in which bacteria and microalgae interact in an inhibitory or stimulatory manner, depending on the extracellular compounds that are released, such as vitamins [59,61–62] and growthpromoting or -inhibiting factors [58,61,63,64]. Nonetheless, studies on the interaction between species (diatoms, Vibrio, and heterotrophic bacteria), the phycosphere, and ROS production are needed to understand the inhibitory effects of diatoms species on the Vibrio species in this work and test these hypotheses. In our experiments, the final concentrations of heterotrophic bacteria were on the order of 107 CFU mL− 1, suggesting that the antibacterial effects of diatom species are selective for Vibrio species. According to Salvesen et al. [63], cultures of Bacillariophyceae species have high bacterial concentrations, possibly due to their production of exudates. Exudates are released into the phycosphere and can include polysaccharides, which are used as a carbon source and provide favorable conditions for attaching to surfaces, acting as a substrate for the proliferation of bacteria [65–66]. Heterotrophic bacteria are more competitive with regard to nutrients than opportunistic bacterial species, such as Vibrio. Thus, although we attribute the antibacterial activity of diatoms to their production of certain compounds, competition for nutrients between heterotrophic bacteria and Vibrio species should be considered as a factor of Vibrio inhibition in future studies. We assumed that the main effect of the antibacterial activity on the Vibrio species in this report was driven by the compounds that were produced as extracellular metabolites by the various diatom species, as the biomass production of microalgae is higher than the heterotrophic bacteria. The amounts of heterotrophic bacteria, expressed in CFU mL− 1, in freshwater and marine system are approximately 106 cells mL− 1, and the bacteria cells are often smaller than 0.5 μm in diameter. The effect of heterotrophic bacteria on the growth of Vibrio species could be positive, because the
Author contributions Ceres A. Molina-Cárdenas: design and implementation of the experiments and manuscript preparation. M.P. Sánchez-Saavedra: supervision of experiments and data processing, contribution to manuscript preparation, and financial support. Conflicts of interest The authors declare that they have no conflict of interest. Acknowledgements This work was funded by Consejo Nacional de Ciencia y Tecnología, CONACyT (Grant project: SEP-CONACyT 2009-01-130074), and Centro de Investigación Científica y de Educación Superior de Ensenada, CICESE (Grant project: 623108). C.A. Molina-Cárdenas acknowledges her Master's in Science from CONACyT and CICESE. Dr. M.L. LizárragaPartida provided the Vibrio strains and laboratory facilities for the pathogen assays. Dr. D.A. Siqueiros-Beltrones and M. en C.Y. Martínez performed the taxonomic identification of the diatom species from Nayarit, Mexico. English language was edited by Blue Pencil Science services. References [1] E.V. Ambrust, The life of diatoms in the world's oceans, Nature 459 (2009) 185–192. [2] J.G. Correa-Reyes, M.P. Sánchez-Saavedra, D.A. Siqueiros-Beltrones, N. FloresAcevedo, Isolation and growth of eight strains of benthic diatoms, cultured under two light conditions, J. Shellfish Res. 20 (2) (2001) 603–610. [3] R.L. Xing, C.H. Wang, X.B. Cao, Y.Q. Chang, The potential value of different species of benthic diatoms as food for newly metamorphosed sea urchin Strongylocentrotus intermedius, Aquaculture 263 (1) (2007) 142–149. [4] S.A. Amin, M.S. Parker, V. Armbrust, Interactions between diatoms and bacteria, Microbiol. Mol. Biol. Rev. 76 (2) (2012) 667–684. [5] J.L. Fuentes, I. Garbayo, M. Cuaresma, Z. Montero, M. González-del-Valle, C. Vilchez, Impact of microalgae-bacteria interactions on the production of algal
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Inhibitory effect of benthic diatom species on three aquaculture pathogenic vibrios
MARK
Ceres A. Molina-Cárdenas, M. del Pilar Sánchez-Saavedra⁎ Centro de Investigación Científica y de Educación Superior de Ensenada (CICESE), Carretera Tijuana-Ensenada 3918, Zona Playitas, 22860 Ensenada, Baja California, Mexico
A R T I C L E I N F O
A B S T R A C T
Keywords: Benthic diatoms co-culture Vibrio inhibition, biocontrol
Diatoms can produce inhibitory compounds against bacteria, constituting an alternative to the use of chemicals to control pathogenic bacterial growth. Antibacterial activity has been detected in co-cultures of microalgaebacteria and extracts. Thus, we evaluated the ability of 6 benthic diatom species to inhibit the growth of Vibrio alginolyticus, V. campbellii, and V. harveyi, which are bacterial species that are pathogenic to mollusks, crustaceans, and fish. Triplicate cultures of benthic diatoms were inoculated with each individual Vibrio species, and the density of microalgae cells, vibrios, and heterotrophic bacteria was estimated at baseline (0 h), 24, 48, 72, 96, 168 h post-inoculation, and when the diatom cultures reached the stationary growth phase. Significant differences in the growth of each diatom species were observed, depending on the Vibrio species that was inoculated, and in all cultures, the density of Vibrio cells decreased, even to undetectable levels (< 0.01 Vibrio cells mL− 1), after 4, 7, and 16 days post-inoculation. Initial bacterial counts in the diatom cultures were on the order of 103 (Colony Forming Units) CFU mL− 1, reaching 106 to 107 CFU mL− 1 by the end. The concentration of bacteria in the diatom cultures was similar between co-cultures and control treatments, indicating that the inhibitory effects of the diatom strains were specific for Vibrio species. We conclude that the diatom species that we tested control the concentration of pathogenic vibrios, presumably through the production of antibacterial compounds. These findings should encourage the use of diatom cultures as a feeding source and to control the density of pathogenic Vibrio species in culture systems, adding diatom species to culture water as a “green water” technique.
1. Introduction Diatoms are eukaryotic microalgae that have greater diversity than other microalgae groups. These photosynthetic organisms belong to the class Bacillariophyceae and are characterized by their silica frustules, high diversity [1], and high fatty acid content, containing such compounds as eicosapentanoic acid (EPA) and docosahexanoic acid (DHA) [2,3]. Under natural conditions, diatoms interact with other organisms, especially bacterial species [4]. When these interactions are positive, the bacteria can enhance the growth of microalgae cells through the production of growth-promoting factors, such as vitamins and idole3acetic-acid, or the regeneration of inorganic nutrients [5]. In parallel, microalgae synthesize exudates that can be a source of assimilable carbon for bacteria [6]. However, microalgae can generate antibacterial compounds that inhibit the growth of bacteria, and vice versa [7,8]. The production and sensitivity of algae and bacteria to these
compounds are species-specific and can be influenced by the culture conditions [9]. Marine microalgae produce secondary metabolites that affect the growth of bacteria, fungi, viruses, and other epibionts [10–11]. Several studies have examined the inhibitory activity of microalgae on the concentration of pathogenic bacteria, such as Vibrio species [12–16]. Diatoms produce a wide variety of chemical compounds with many types of bioactivity, such as antibacterial activity. For example, Skeletonema costatum and Phaeodactylum tricornutum, generate secondary metabolites that have effects on the pathogenic bacteria Staphylococcus aureus and Vibrio anguillarum [12,17]. Infectious diseases that are caused by pathogenic Vibrio bacterial species are associated with high mortality rates in aquaculture production systems, impacting the profitability of this industry [18–19]. Vibrios can be introduced to culture ponds through live prey, such as copepods, Artemia, and microalgae that are used as feed for fish, crustaceans, and mollusks [20–21]. Strains, such as Vibrio alginolyticus, are
Abbreviations: Va, Vibrio alginolyticus; Vh, Vibrio harveyi; Vc, Vibrio campbellii ⁎ Corresponding author. E-mail address: [email protected] (M.d.P. Sánchez-Saavedra). http://dx.doi.org/10.1016/j.algal.2017.09.004 Received 8 February 2017; Received in revised form 30 August 2017; Accepted 1 September 2017 2211-9264/ © 2017 Elsevier B.V. All rights reserved.
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culture collections: Vibrio alginolyticus (Va) ATCC 17749 (American Type Culture Collection); Vibrio campbellii (Vc) (CAIM 416, CICESE 559), obtained from the Collection of Aquacultural Importance Microrganisms (CAIM) and belonging to “Centro de Investigación en Alimentación y Desarrollo (CIAD)”; and Vibrio harveyi (Vh) DN01 (CICESE 597). All Vibrio species were cultured in Petri dishes with ZoBell medium [42] and incubated at 28 °C for 24 h. Then, colonies were removed by rinsing off the agar surface with 9 mL of sterile physiological serum, and the optical density of the suspension was adjusted to 0.05 at 600 nm on a Hach DR-4000 UV-VIS spectrophotometer. This optical density equals a concentration of 106 cells mL− 1 [22]. The Vibrio suspensions were used to inoculate the diatom cultures.
pathogens to mollusks, such as the larvae of the red abalone Haliotis rufescens, Ruditapes decussatus, Argopecten ventricosus, Nodipecten subnodosus, Mytilus galloprovincialis, and Haliotis diversicolor supertexta [22–23]. Vibrio campbellii is a potential pathogen of crustaceans [24], and Vibrio harveyi is a pathogenic species in fish [25–26]. For aquaculture activity, vibriosis is controlled most frequently with antibiotics, such as oxytetracycline, enrofloxacin, florfenicol, sarafloxacin, amoxicillin, and oxolinic acid [19,27–28]. Yet, the excessive application prophylactic antibiotics in aquaculture has led to the appearance of bacterial strains that are resistant to the compounds that are used to control them [29–30]. Other strategies, such as the disruption of quorum sensing, phage therapy, and probiotic bacteria in culture systems, are effective in limiting the density of Vibrio species in in vitro assays. However, their effects under actual culture conditions remain unknown, and none of them alone can control this problem [31–32]. New strategies and molecules that control the density of Vibrio bacteria in culture systems are urgently needed. Microalgae are a reservoir of compounds with various activities, including antimicrobial action [33–34]. Antimicrobial compounds that are produced by microalgae vary in nature and include fatty acids and their derivates [35–36], peptides [33], toxins [37], pigments [38], polysaccharides, flavonoids, and terpenoids [33]. The antibacterial activity of diatoms has been attributed to specific fatty acids in the cells [35], their capacity to compete for nutrients, such as phosphorus [9], and the synthesis of exopolysaccharides [39]. The aim of this work was to determine the ability of 6 taxa of benthic diatoms that were isolated from Pacific coastal waters to inhibit the growth of Vibrio alginolyticus, V. campbellii, and V. harveyi.
2.3. Inhibition of Vibrio species in diatom culture assays Inhibition assays of Vibrio were performed with monospecific, nonaxenic cultures of each strain and in triplicate for each diatom species in 500-mL Erlenmeyer flasks with 200 mL “f” medium [43] at an initial concentration of 100,000 cells mL− 1. To quantify Vibrio cells in each experimental culture condition, aliquots of 1 mL and another of 10 mL were passed through 45-mm sterile filters (0.22-μm pore size). Filters that contained the cells were placed on thiosulfate citrate bile sucrose (TCBS) agar media. Concentrations of Vibrio of 0.01 cells mL− 1 or lower indicated the absence of this type of bacteria. The cultures of each diatom strain were inoculated with 2 mL of Vibrio bacterial suspension by monospecific addition, which equals a final concentration of 103 Vibrio cells mL− 1. Immediately after the inoculation, the initial concentration of Vibrio in each experimental culture condition was corroborated as described above. These initial filtrates corresponded to time T0 + 1, and the concentration of Vibrio bacteria on subsequent days—ie, the filtrates at 0, 24, 48, 96, and 168 h post-inoculation—and when the growth of the diatom cultures reached the exponential and stationary growth phases was measured. Filters were placed in Petri dishes with TCBS media and incubated at 28 °C for 24 h. As controls, Erlenmeyer flasks with triplicate cultures of each diatom strain without Vibrio bacteria and flasks with “f” medium that were inoculated with each Vibrio strain tested alone were maintained under the same culture conditions as in the Vibrio inhibition assays. Heterotrophic bacterial concentrations were measured by seeding 0.1 mL of serial dilutions of co-cultures or monospecific cultures of diatoms in Petri dishes with ZoBell medium at the same times and under the same conditions as the incubations for the Vibrio counts. The concentrations of this type of bacteria were expressed in colony forming units per milliliter (CFU mL− 1).
2. Materials and methods 2.1. Characteristics of diatom species and cultures We used 6 benthic diatom strains that were isolated from the coast of Baja California and Nayarit, Mexico. The diatom strains from Baja California were Nitzschia laevis Hustedt and Nitzschia frustulum var. perminuta Grunow, isolated and identified per Correa-Reyes et al. [2]. Navicula incerta Grunow was isolated by the Institute of Oceanological Research (IIO), Autonomous University of Baja California (UABC). The diatom strains from Nayarit were Navicula biskanterae Hust., Nitzschia fustulum (Kútzing) Grunow, and Navicula cf. incerta Grunow in Van Heurck, isolated from the water culture of a shrimp farm by AguilarMay [40]. The diatom species belong to the collection of the Group of Algae Cultures in the Aquaculture Department of CICESE. The diatom strains were identified per Correa-Reyes et al. [2] and López-Fuerte and Siqueiros-Beltrones [41]. Monospecific and non-axenic (concentration of associated bacteria < 103 cell mL− 1 and hereafter referred to as heterotrophic bacteria) batch cultures of the 6 benthic diatom strains were maintained in 500-mL Erlenmeyer flasks with 200 mL “f” medium [63]. All cultures were inoculated with 100,000 cells mL− 1 of each benthic diatom strain. Cultures were established in triplicate and maintained at 22 ± 1 °C, 33 ± 1‰ salinity, and 24 h of continuous light at 110 μEm− 2 s− 1, provided by cool white fluorescent lamps. To measure cell concentrations, daily counts were performed with a hemocytometer (Hausser Scientific) under a compound microscope at 40× magnification. The cell concentrations were used to calculate growth rates as Fogg and Thake [44].
2.4. Statistical analysis The initial and final concentrations of Vibrio bacteria in diatom cultures were compared by student t-test. The growth of each diatom species in the presence of Vibrio bacteria and their growth rates were compared by one-way ANOVA. Significant differences were determined by a posteriori Tukey test. Differences in heterotrophic bacteria were significant if the concentrations exceeded one order of magnitude. Statistical analysis was performed using Statistica 7.0 with α = 0.05, and Sigma Plot 10 was used to construct the graphs. 3. Results 3.1. Growth of diatom species and Vibrio species
2.2. Vibrio cultures The diatoms N. laevis and N. frustulum var. perminuta had higher cell concentrations (p < 0.05) on day 9 of culture. However, N. incerta, N. biskanterae, and N. frustulum reached similar concentrations on day 7. The strain N. cf. incerta had the lowest cell concentration of all diatom cultures on day 9 of culture (Fig. 1).
Three Vibrio species that have been reported to be pathogens of aquaculture organisms were selected, based on their associated high mortality rates in commercial aquaculture production. These Vibrio species are part of the CICESE Microorganism Collection, from various 132
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reached the stationary phase, settling at a concentration of 1.7 × 106 cells mL− 1. On day 9, N. biskanterae (Fig. 3C) halted its growth and reached a concentration of 1.6 × 106 cells mL− 1, whereas N. laevis (Fig. 3B) and N. frustulum (Fig. 3D) stopped growing on day 10. At this time, the average cell concentration was 1 × 106 for both species, and N. incerta (Fig. 3A) reached 2 × 106 cells mL− 1 after 11 days in culture. The strain Navicula cf. incerta required more time to enter the stationary growth phase (Fig. 3F), registering a concentration of 1.4 × 106 cells mL− 1 after 13 days in culture. The amount of heterotrophic bacteria in all cultures increased over time (p < 0.05). At the beginning of all assays, the concentration was 5 × 105 CFU mL− 1, increasing to 1 × 107 CFU mL− 1 at the end of the experiments. In diatom cultures with V. campbellii, bacterial inhibition was achieved on day 4 post-inoculation in nearly all treatments (Fig. 4)—with Navicula cf. incerta, the inhibitory effect was observed on day 7. In the challenge with V. campbellii, N. incerta grew to a concentration of approximately 1.5 × 106 cells mL− 1 after 11 days in culture (Fig. 4A)—the time that was needed for this microalgae species to enter the stationary growth phase. A similar response with regard to time and cell density was seen in N. laevis (Fig. 4B) and Navicula cf. incerta (Fig. 4F). The cultures with N. biskanterae (Fig. 4C) and N. frustulum (Fig. 4D) ceased their growth on day 10, when they reached concentrations of 2 × 106 cells mL− 1 and 1.7 × 106 cells mL− 1, respectively. N. frustulum var. perminuta was the slowest species to reach the stationary growth phase (Fig. 4), requiring 12 days to stop its growth and obtain an average concentration of 2.3 × 106 cells mL− 1. In these treatments, all initial counts of heterotrophic bacteria were 5 × 105 CFU mL− 1, ultimately rising to 107 CFU mL− 1 (p < 0.05). The V. harveyi strain was the most resistant bacterium to inhibition (p < 0.05), and even in cultures of N. laevis, N. frustulum, and Navicula cf. incerta, Vh remained at densities above detection limits (> 0.01 Vh cells mL− 1). However, significant differences were found between the initial and final concentrations of V. harveyi. In this assay, inhibition was observed on day 7 in cultures of N. laevis (Fig. 5B), N. biskanterae (Fig. 5C), N. frustulum var. perminuta (Fig. 5E), and Navicula cf. incerta (Fig. 5F), versus day 4 for N. incerta (Fig. 5A) and N. frustulum (Fig. 5D). On day 10 post-inoculation, cultures of N. biskanterae, N. frustulum, and N. frustulum var. perminuta reached the stationary growth phase and had average concentrations of 2.2 × 106 cells mL− 1. Navicula incerta, N. laevis, and Navicula cf. incerta stopped their growth on day 12 and had concentrations of 2 × 106, 3 × 106, and 1.5 × 106 cells mL− 1, respectively. As in the other experiments, the amount of heterotrophic bacteria in cultures that were challenged with V. harveyi had starting concentrations in the order of 105 and finished significantly higher, in the order of 107 CFU mL− 1.
Fig. 1. Mean values and standard deviation of densities of Navicula incerta (●), Nitzschia laevis (○), Navicula biskanterae (▼), Nitzschia frustulum (△), Nitzschia frustulum var. perminuta (■), and Navicula cf. incerta (□) in monospecific cultures. Letters indicate significant differences (one-way ANOVA and Tukey a posteriori test p < 0.05, a > b > c > d).
Fig. 2. Mean values and standard deviation of densities of Vibrio alginolyticus (●), Vibrio campbellii (○), and Vibrio harveyi (▼) grown in “f” medium without microalgae cells.
Based on the cell counts, Vibrio alginolyticus, V. campbellii, and V. harveyi survived in “f” medium after 14 days; the final bacterial densities for these 3 species were on the order of 103 Vibrio cells mL− 1 (Fig. 2).
3.3. Effects of Vibrio species on the growth of benthic diatom species The growth of diatom species differed significantly between cultures with and without Vibrio species (p < 0.05) (Fig. 6). In N. incerta cultures (Fig. 6A), the addition of V. alginolyticus and V. harveyi stimulated their growth, and higher growth rates were observed (Table 1); when co-cultured with V. campbellii however, the cell densities were lower (p < 0.05). Vibrio harveyi did not show any significant differences with regard to controlling growth in N. laevis cultures (Fig. 6B), which was, in contrast, altered by V. campbelli and V. alginolyticus (p < 0.05). Nonetheless, the growth rate for this diatom species was higher when cultured with V. campbellii (Table 1). For N. biskanterae (Fig. 6C), the highest cell densities were obtained with V. harveyi and V. campbellii and in control cultures; V. alginolyticus was the bacterial specie that inhibited the growth of N. biskanterae most extensively (p < 0.05). The growth rate of N. biskanterae peaked when co-cultured with V. campbellii (Table 1). Growth-inhibiting effects of V. alginolyticus were also observed with N. frustulum (Fig. 6D) and N. frustulum var. perminuta (Fig. 6E), which increased their concentrations
3.2. Inhibition of Vibrio species in cultures of diatom species None of the filtrates of the diatom cultures before inoculation with Vibrio species showed bacterial growth in TCBS selective medium, indicating that the diatom cultures were free of Vibrio species. In all diatom cultures, the concentrations of Vibrio-like bacteria per mL (VLB cells mL− 1) were below 0.01. In nearly all cultures of diatom species, V. alginolyticus (Va) was inhibited on day 4—with N. biskanterae, the concentrations fell below 0.01 Va cells mL− 1 on day 2 post-inoculation (Fig. 3). In all experiments, once the Vibrio was inhibited, this effect persisted until the diatom cultures reached the stationary growth phase. This response was also observed in the assays with V. campbellii (Vc) (Fig. 4) and V. harveyi (Vh) (Fig. 5). The time that was needed to reach the stationary growth phase in all diatom species that were cultured with V. alginolyticus ranged from 8 to 14 days (Fig. 3A–F). After 8 days post-inoculation, N. frustulum var. perminuta (Fig. 3E) 133
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Fig. 3. Mean values and standard deviation of densities of Vibrio alginolyticus (Va as cells mL− 1: ★) and heterotrophic bacteria (CFU mL− 1: ■) in cultures of various diatom species (cells mL− 1: ●) of Navicula incerta (A), Nitzschia laevis (B), Navicula biskanterae (C), Nitzschia frustulum (D), Nitzschia frustulum var. perminuta (E), and Navicula cf. incerta (F). Letters indicate significant differences in initial and final Vibrio densities (student t-test p < 0.05, a > b).
algynolyticus and V. campbelli were completely inhibited in all co-cultures. Despite the partial inhibition of V. harveyi, its concentration decreased significantly in co-cultures with diatoms. The antagonistic effects of diatoms on bacterial species occur naturally in marine environments [45]. The inhibition of vibrios by microalgae species has been reported by Lio-Po et al. [13], who demonstrated that Chaetoceros calcitrans and Nitzchia sp. suppresses the growth of luminescent vibrios at 24 or 48 h and that a low load of vibrios remains in microalgae-bacteria co-cultures after 7 days. Similar results were shown culturing Leptolyngbia sp. with luminescent vibrios and Makridis et al. [16], who found that Chlorella minutissima and Tetraselmis chuii reduce the load of Vibrio anguillarum. The capacity of diatom species to inhibit the growth of pathogenic
and growth rates when cultured with V. harveyi and V. campbellii, respectively (p < 0.05) (Table 1). In Navicula cf. incerta cultures (Fig. 6F), Vibrio had beneficial effects on cell concentration, with higher cell densities attained when co-cultured with all Vibrio bacteria species (p < 0.05). Conversely, the growth rate for Navicula cf. incerta was highest in cultures without Vibrio species (Table 1). 4. Discussion 4.1. Inhibition of Vibrio species in cultures of diatom species In all challenges, the concentration of Vibrio species declined when they were co-cultured with each diatom species; the bacterial strains V. 134
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Fig. 4. Mean values and standard deviation of densities of Vibrio campbellii (Vc as cells mL− 1:★) and heterotrophic bacteria (CFU mL− 1: ■) in cultures of various diatom species (cells mL− 1: ●) of Navicula incerta (A), Nitzschia laevis (B), Navicula biskanterae (C), Nitzschia frustulum (D), Nitzschia frustulum var. perminuta (E), and Navicula cf. incerta (F). Letters indicate significant differences in initial and final Vibrio densities (student t-test p < 0.05, a > b).
vibrios has been exploited in the “green water” technique—a system in which microalgae species are added to the culture water of crustaceans and fishes [46]. Several reports have confirmed that the population of pathogenic vibrios can be controlled in cultures of Penaues monodon, in which the dominant species of phytoplankton are diatoms, such as Skeletonema sp., Nitzchia sp., Nanochlorum sp., Navicula sp., Thalassiosira sp., Anabaena sp., and Chaetoceros sp. [47]. The antibacterial activities of diatom species in the Nitzschia and Navicula genera have been reported by Findlay and Patil [48], who evaluated the effects of sterols and fatty acids from Navicula delognei. This group found that compounds that were produced in Navicula
delognei were active against Staphylococcus aureus, Staphylococcus epidermis, Salmonella typhimurium, and Proteus vulgaris. Organic extracts of Nitzschia palea have been tested against Micrococcus luteus, S. aureus, and Pseudomonas aeruginosa [49]. With regard to the inhibition of Vibrio species, there is evidence that extracts from Thalassiosira rotula inhibit the growth of V. harveyi [50] and that cellular components of Odontella aurita suppress that of V. alginolyticus and P. vulagaris [51]. In most of our assays, inhibition was observed when the diatom species were in the early exponential growth phase, and low or undetectable Vibrio cell densities were maintained until the cultures reached the stationary growth phase. In the experiments with V. 135
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Fig. 5. Mean values and standard deviation of densities of Vibrio harveyi (Vh as cells mL− 1: ★) and heterotrophic bacteria (CFU mL− 1: ■) in cultures of various diatom species of Navicula incerta (A), Nitzschia laevis (B), Navicula biskanterae (C), Nitzschia frustulum (D), Nitzschia frustulum var. perminuta (E), and Navicula cf. incerta (F). Letters indicate significant differences in initial and final Vibrio densities (student t-test p < 0.05, a > b).
reactive oxygen species (ROS). These compounds can enhance the antibacterial activity of fatty acids [53], which are synthesized in high quantities during the stationary phase [54–55]. According to Van Rijssel et al. [52], synergism between ROS and fatty acids inhibits the growth of Vibrio fisheri; thus, it is possible that the inhibitory pattern in our study is attributed to the activity of several compounds that are produced and released during the exponential and stationary growth phases.
harveyi, we noted inhibition in the late exponential growth phase and even when the growth of the diatoms entered the stationary phase. More antibacterial compounds are produced when microalgae cultures have lower growth rates or are in the early stationary growth phase [33–12]. Moreover, microalgae cells synthesize various secondary metabolites—depending on the growth phase—that have species-specific effects. For example, Van Rijssel et al. [52] reported that the generation of secondary metabolites by the microalgae Fibrocapsa japonica was greater in phases in which the growth rate was high, as in the exponential phase, and that in this growth phase, F. japonica produces 136
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Fig. 6. Mean values and standard deviation of cellular density of monospecific cultures (●) of Navicula incerta (A), Nitzschia laevis (B), Navicula biskanterae (C), Nitzschia frustulum (D), Nitzschia frustulum var. perminuta (E), and Navicula cf. incerta (F) in mixed cultures with Vibrio alginolyticus (▲), V. campbellii (★), and V. harveyi (■). Letters indicate significant differences (ANOVA and Tukey a posteriori test, p < 0.05, a > b > c).
that the addition of bacteria to cultures of Thalassiosira weissflogii, Cylindrotheca fusiformis, and Nitzschia laevis stimulates the growth of these microalgae species, doubling their numbers versus cultures that are not exposed to bacteria. The antimicrobial activity in aggregates and detritus can lead to the release of nitrate, ammonia, phosphorus, and CO2, increasing and maintaining the growth of cultures, as described by several groups
4.2. Effects of Vibrio species on the growth of benthic diatom species In most of our experiments, the growth rates and cell densities of diatom species were higher when they were cultured with any Vibrio species compared with control cultures, with the exception of Navicula cf. incerta. Fukami et al. [8] demonstrated that in the presence of bacteria, axenic cultures of phytoplankton grow faster. Grossart [56] noted 137
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organic carbon that is produced by heterotrophic bacteria (1 to roughly 100 μg carbon L− 1 day− 1) constitutes < 10% to 100% of the primary production in certain cases [61,67]. Our results demonstrate that Vibrio species produce and release many compounds into the culture media, some of which can be used as nutrients or induce the synthesis of growth factors and increase the cell densities of certain diatom strains. In other cases, chemicals that are generated by Vibrio species inhibit the growth of diatom strains. Only one study has reported a rise in the cell density of Isochrysis galbana due to its interaction with V. alginolyticus, V. campbellii, and V. harveyi, noting that these Vibrio strains produced a substance that promotes microalgae growth [57]. Diatom species are used as food for mollusks, such as abalone, and crustacean larvae in aquaculture facilities. Our results should encourage the use of diatom species as a food source and as an alternative method of controlling pathogenic bacteria in culture systems, reducing the application of chemicals and antibiotics to mitigate harmful bacterial loads. Future studies should isolate and characterize the compounds that mediate the inhibition of the growth of pathogenic vibrios in aquaculture.
Table 1 Mean values and standard deviation of maximum cell densities (MCDs) and growth rates (μ, in divisions day− 1) in cultures of diatom species with various Vibrio species and control treatments (each microalgae culture without Vibrio species). Letters indicate significant differences (ANOVA and Tukey a posteriori test, p < 0.05, a > b > c). Diatom species
Vibrio species
MCDs (1 × 106 cells mL− 1)
μ
Navicula incerta
Without V. alginolyticus V. campbellii V. harveyi Without V. alginolyticus V. campbellii V. harveyi Without V. alginolyticus V. campbellii V. harveyi Without V. alginolyticus V. campbellii V. harveyi Without V. alginolyticus V. campbellii V. harveyi Without V. alginolyticus V. campbellii V. harveyi
1.7 2.0 1.4 2.1 2.4 1.6 2.0 2.4 1.7 1.5 1.7 1.6 1.8 1.5 1.7 2.2 2.1 1.7 2.3 2.1 1.2 1.4 1.6 1.5
0.25 0.32 0.25 0.33 0.27 0.23 0.31 0.26 0.20 0.20 0.25 0.23 0.21 0.16 0.27 0.29 0.31 0.20 0.25 0.32 0.39 0.15 0.27 0.30
Nitzschia laevis
Navicula biskanterae
Nitzschia frustulum
Nitzschia frustulum var. perminuta
Navicula cf. incerta
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.07 b 0.06 a 0.03 c 0.01 a 0.10 a 0.03 c 0.12 b 0.03 a 0.05 a 0.04 b 0.09 a 0.01 a 0.04b 0.09 c 0.06 bc 0.07 a 0.05 b 0.11 c 0.15 a 0.07 ab 0.04 c 0.01 b 0.07 a 0.03 a
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.03 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.03 0.01
b a b a b c a b b b a b b c a a a c b a a c b b
5. Conclusion Based on our findings, we recommend the use of diatom strain cultures for the potential biocontrol of pathogenic vibrios (V. alginolyticus, V. campbellii, and V. harveyi) in culture systems, and diatoms can be used as food for the larvae of mollusks, such as abalone. The microalgae strains that we have tested can potentially be applied to control V. alginolyticus, V. campbellii, and V. harveyi and replace or minimize the use of antibiotics in aquaculture facilities.
[56,57–59]. Park et al. [60] found that the growth of Chlorella ellipsoidea rose 0.5- to 3-fold when inoculated with bacteria of the genus Brevundimonas, an effect that they attributed in part to more efficient exchange of substrates between bacteria that were attached to phytoplankton cells. However, the relationship between bacteria and microalgae is not limited to nutrient remineralization. There is evidence of a phycosphere—a zone in which bacteria and microalgae interact in an inhibitory or stimulatory manner, depending on the extracellular compounds that are released, such as vitamins [59,61–62] and growthpromoting or -inhibiting factors [58,61,63,64]. Nonetheless, studies on the interaction between species (diatoms, Vibrio, and heterotrophic bacteria), the phycosphere, and ROS production are needed to understand the inhibitory effects of diatoms species on the Vibrio species in this work and test these hypotheses. In our experiments, the final concentrations of heterotrophic bacteria were on the order of 107 CFU mL− 1, suggesting that the antibacterial effects of diatom species are selective for Vibrio species. According to Salvesen et al. [63], cultures of Bacillariophyceae species have high bacterial concentrations, possibly due to their production of exudates. Exudates are released into the phycosphere and can include polysaccharides, which are used as a carbon source and provide favorable conditions for attaching to surfaces, acting as a substrate for the proliferation of bacteria [65–66]. Heterotrophic bacteria are more competitive with regard to nutrients than opportunistic bacterial species, such as Vibrio. Thus, although we attribute the antibacterial activity of diatoms to their production of certain compounds, competition for nutrients between heterotrophic bacteria and Vibrio species should be considered as a factor of Vibrio inhibition in future studies. We assumed that the main effect of the antibacterial activity on the Vibrio species in this report was driven by the compounds that were produced as extracellular metabolites by the various diatom species, as the biomass production of microalgae is higher than the heterotrophic bacteria. The amounts of heterotrophic bacteria, expressed in CFU mL− 1, in freshwater and marine system are approximately 106 cells mL− 1, and the bacteria cells are often smaller than 0.5 μm in diameter. The effect of heterotrophic bacteria on the growth of Vibrio species could be positive, because the
Author contributions Ceres A. Molina-Cárdenas: design and implementation of the experiments and manuscript preparation. M.P. Sánchez-Saavedra: supervision of experiments and data processing, contribution to manuscript preparation, and financial support. Conflicts of interest The authors declare that they have no conflict of interest. Acknowledgements This work was funded by Consejo Nacional de Ciencia y Tecnología, CONACyT (Grant project: SEP-CONACyT 2009-01-130074), and Centro de Investigación Científica y de Educación Superior de Ensenada, CICESE (Grant project: 623108). C.A. Molina-Cárdenas acknowledges her Master's in Science from CONACyT and CICESE. Dr. M.L. LizárragaPartida provided the Vibrio strains and laboratory facilities for the pathogen assays. Dr. D.A. Siqueiros-Beltrones and M. en C.Y. Martínez performed the taxonomic identification of the diatom species from Nayarit, Mexico. English language was edited by Blue Pencil Science services. References [1] E.V. Ambrust, The life of diatoms in the world's oceans, Nature 459 (2009) 185–192. [2] J.G. Correa-Reyes, M.P. Sánchez-Saavedra, D.A. Siqueiros-Beltrones, N. FloresAcevedo, Isolation and growth of eight strains of benthic diatoms, cultured under two light conditions, J. Shellfish Res. 20 (2) (2001) 603–610. [3] R.L. Xing, C.H. Wang, X.B. Cao, Y.Q. Chang, The potential value of different species of benthic diatoms as food for newly metamorphosed sea urchin Strongylocentrotus intermedius, Aquaculture 263 (1) (2007) 142–149. [4] S.A. Amin, M.S. Parker, V. Armbrust, Interactions between diatoms and bacteria, Microbiol. Mol. Biol. Rev. 76 (2) (2012) 667–684. [5] J.L. Fuentes, I. Garbayo, M. Cuaresma, Z. Montero, M. González-del-Valle, C. Vilchez, Impact of microalgae-bacteria interactions on the production of algal
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