Editor: Patricia Sobecky
Bacteria associated with Artemia spp. along the salinity gradient of the solar salterns at Eilat (Israel)
Article first published online: 16 MAY 2011
© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved
FEMS Microbiology Ecology
Volume 77, Issue 2, pages 310–321, August 2011
How to Cite
Tkavc, R., Ausec, L., Oren, A. and Gunde-Cimerman, N. (2011), Bacteria associated with Artemia spp. along the salinity gradient of the solar salterns at Eilat (Israel). FEMS Microbiology Ecology, 77: 310–321. doi: 10.1111/j.1574-6941.2011.01112.x
- Issue published online: 11 JUL 2011
- Article first published online: 16 MAY 2011
- Accepted manuscript online: 14 APR 2011 09:19AM EST
- Received 27 July 2010; revised 24 February 2011; accepted 10 April 2011., Final version published online 16 May 2011.
The crustacean genus Artemia naturally inhabits various saline and hypersaline environments and is the most frequently laboratory-hatched animal for live feed in mari- and aquaculture. Because of its high economic importance, Artemia–bacteria interactions were so far studied mostly in laboratory strains. In this study, we focused our attention on the Artemia-associated microbiota in its natural environment in the solar salterns of Eilat, Israel. We applied a culture-independent method (clone libraries) to investigate the bacterial community structure associated with Artemia in five evaporation ponds with salinities from slightly above seawater (5%) to the point of saturation (32%), in two different developmental stages: in nauplii and in the intestine of adult animals. Bacteria found in naupliar and adult stages were classified within the Proteobacteria, Bacteroidetes, Firmicutes, Actinobacteria and Cyanobacteria. The halophilic proteobacterial genera Halomonas spp. and Salinivibrio spp. dominated the Artemia microbiota in both stages in all ponds. We also analysed a clone library of entire adult animals, revealing a novel bacterial phylogenetic lineage. This is the first molecular study of bacteria associated with two developmental stages of Artemia along a salinity gradient.
The branchiopod genus Artemia (brine shrimp) inhabits various saline and hypersaline environments: saline lakes such as Great Salt Lake, UT, Lake Urmia, Iran, and others, hypersaline lagoons (e.g. Cabo Rojo, Puerto Rico), and solar salterns for the production of salt worldwide (van Stappen, 2002). Artemia spp. are continuous nonselective particle feeders (Martin, 1992). They filter the water and graze on benthic microbial mats that cover the bottom of many shallow salt lakes. Besides filtering and grazing, feeding strategies of cannibalism and coprophagy are also used (Martin, 1992).
Artemia is one of the most important live feeds in the larviculture of economically important crustaceans and fishes due to its relatively easy production and its suitable biochemical composition (Verschuere et al., 1999). In view of the economic importance of Artemia, some attention was paid to the study of potential pathogens and contaminants that may decrease cyst production and increase the mortality of adults after hatching. Vibrio parahaemolyticus is a known Artemia pathogen (Puente et al., 1992) and there are reports of Artemia infection by pathogenic spirochetes (Tyson, 1974) and by eukaryotic microorganisms such as fungi (Overton & Bland, 1981) and microsporidia (Martinez et al., 1993).
Certain bacteria have been shown to have a beneficial effect on the growth rate and survival of Artemia (D'Agostino, 1980), such as Acinetobacter and Flexibacter (Intriago & Jones, 1993). Bacteria also contribute to the nutritional value of Artemia as a major source of proteins and amino acids (Gorospe & Nakamura, 1996) and assist in the digestion of unicellular algae (Intriago & Jones, 1993). Post & Youssef (1977) showed the presence of symbiotic intracellular bacteria in the epithelial lining of Artemia salina collected from Great Salt Lake, but their taxonomic status and role remained unknown.
So far, several attempts have been made toward the elucidation of the structure of bacterial communities associated with different developmental stages of laboratory-hatched Artemia (Austin & Allen, 1982; Prieto et al., 1987; García et al., 1988; Igarashi et al., 1989; Orozco-Medina et al., 2002). All these studies have used classical, culture-dependent microbiological methods. A recent study of the microorganisms associated with Artemia nauplii reared for aquaculture, and using both culture-dependent and molecular, culture-independent techniques, yielded isolates related to the genera Vibrio, Pseudomonas and Rhizobium and others, and showed the presence of phylotypes affiliated with the genera Oleiphilus, Beggiatoa, Thioalkalivibrio and others (Høj et al., 2009). Culture-independent molecular methods have been applied to the study of Artemia-associated microbial communities in their natural environments only once. The results of this study, dealing with cysts and adults of Artemia franciscana in Great Salt Lake (UT), were only published in symposia abstracts (Riddle et al., 2007, 2008).
The aim of the present study was to characterize the bacterial communities associated with different developmental stages of Artemia spp. along the salt gradient of a commercial solar saltern operation in Eilat, Israel (the Israel Salt Company), using molecular, culture-independent techniques, and to investigate the effect of water salinity on the prokaryotic communities associated with two developmental stages in the growth of the brine shrimp.
Materials and methods
Sampling sites and sample collection
Artemia specimens (nauplii and adults) were collected between 28 April and 5 May 2008 from the saltern evaporation ponds of the Israel Salt Company at Eilat (Fig. 1) with a planktonic mesh with 70 μm pore diameter. Information on the saltern ponds and their biota was provided by Sørensen et al. (2009) and Řehákováet al. (2009), and references therein. Animals were immediately brought to the lab in a brine from the corresponding pond in a 1-L plastic beaker. The following ponds were sampled: pond no. 63 (brine density 1.035 g mL−1, 5.5% total dissolved salt), no. 65 (density 1.071 g mL−1, 11% salt), no. 104 (density 1.089 g mL−1, 13.8% salt), no. 200 (density 1.134 g mL−1, 20.6% salt) and no. 201 (density 1.200 g mL−1, 30.7% salt). The density of the water was measured in situ using hydrometers. The water temperature at the time of sampling was around 22–25 °C. Both adult specimens and nauplii were found in all the ponds sampled, with the exception of pond no. 200, which yielded only adult brine shrimps.
For each pond sampled, 23 adult brine shrimp and 50 nauplii (except for pond 200) were rinsed for 2 min with filter-sterilized 70% ethanol, followed by three rinses with sterile ddH2O. Nauplii were transferred using a sterile pipette and adult animals using a sterile inoculation loop. Guts of 20 adults were aseptically isolated under a stereomicroscope using a pair of sterile tweezers. Pooled intestines, pooled nauplii and three individual adults were suspended in 0.12 M phosphate buffer, pH 8. Total DNA of the pooled samples was isolated using the UltraClean Soil DNA Isolation Kit (MoBio), and was additionally purified by Sephadex 200-G gel chromatography as described by Boccuzzi et al. (1998).
Preparation of clone libraries and sequence analysis
The structure of bacterial communities associated with nauplii sampled in ponds 63, 65, 104 and 201, intestine of adult Artemia sampled in the ponds 63, 65, 104, 200 and 201 and whole animals sampled in the pond 201 were analysed using clone library construction. The primer pair fD1 (5′-AGAGTTTGATCCTGGCTCAG-3′) (Brosius et al., 1981) and 1392r (5′-ACGGGCGGTGTGTAC-3′) (Ferris et al., 1996) was used to generate 16S rRNA gene fragments of approximately 1300 bp. The PCR mixture (25 μL) contained 1 μL of isolated DNA, 0.3 μL Taq polymerase (Fermentas), 1 × buffer (Fermentas), 0.77 mM MgCl2 (Fermentas), 0.08 mM of dNTP (Fermentas) and 0.2 mM of each primer. We used a protocol composed of preincubation at 94 °C for 3 min, denaturation at 94 °C for 30 s, annealing at 60–50 °C for 30 s and polymerization at 72 °C for 1 min. The annealing temperature was decreased from 60 to 54 °C for 1 °C per cycle, followed by 25 cycles with annealing temperature 50 °C. Elongation in the last cycle lasted for 3 min, followed by a final incubation at 4 °C. The PCR product was loaded onto a 1% agarose gel, and following electrophoresis, fragments of appropriate length were cut out of the gel and purified using the GenJet™ Gel Extraction Kit (Fermentas). Purified amplicons were ligated into the vector pJET1.2/blunt (Fermentas). The ligation mixtures were transformed into chemically competent Escherichia coli strain XL1-Blue MRF (Invitrogen) that were prepared using the TransformAid™ Transformation Kit (Fermentas) as described by the manufacturer. Transformants were selected on Luria–Bertani agar plates with ampicillin (100 mg mL−1). The inserts were screened using the pJET_f and pJET_r primers (Fermentas). Ninety-six clones from each library were sequenced in one-way (Macrogen Inc., Korea) and identified on the basis of an approximately 700-bp-long amplicon using the Ribosomal Database Project database (http://rdp.cme.msu.edu). A gene tree of representative sequences from the clone library from the whole adult animals that could not be identified was generated using the neighbour-joining method, implemented in mega4 (Tamura et al., 2007). The evolutionary distances were computed using the maximum composite likelihood method. All positions containing gaps and missing data were eliminated from the dataset (complete deletion option).
Partial 16S rRNA gene sequences obtained in this study were first matched to the GenBank nonredundant nucleotide database using the blastn algorithm. Relevant sequences longer than 1100 bp were retrieved in an automated manner using the freely available Biopython source code (Cock et al., 2009), aligned using muscle (Edgar, 2004) and analysed using operational taxonomic units (OTU)-based methods. mothur version 1.7.2 (Schloss et al., 2009) was used in order to estimate the level of phylotype redundancy to calculate various species richness indices at different levels of evolutionary distance (Table 1), to compare clone libraries and to determine the degree of similarity between them. The sequences were assigned to OTUs using the farthest-neighbour algorithm at a genetic distance of 3%, 5% and 20%, corresponding to the arbitrary and generally recognized distances for taxonomic species, genera and phyla, respectively.
|2% Evolutionary distance difference||5% Evolutionary distance difference||20% Evolutionary distance difference||2% Evolutionary distance difference||5% Evolutionary distance difference||20% Evolutionary distance difference|
Bacterial communities present in the intestine of adult Artemia and in nauplii inhabiting ponds with five different salinities were examined using a culture-independent approach, the clone library. Between 31 and 81 satisfactory sequences were obtained for the different samples analysed (Figs 2 and 3). The bacterial 16S rRNA gene sequences have been submitted to the GenBank database, under accession numbers FN823320–FN824096.
The bacterial taxa associated with the Artemia nauplii were highly diverse, and clear differences were observed between the bacterial community structures on nauplii harvested from ponds of different salinities (Fig. 2). At the lowest salinity (pond 63, 5.5% salt), the dominant types were affiliated with the proteobacterial genera Nereida, Halomonas, the family Rhodobacteraceae and a group of Gammaproteobacteria related to sequences retrieved earlier from a coastal salt pond in Massachusetts (Simmons et al., 2004), and from Victoria Harbor, Hong Kong (Zhang et al., 2007). In pond 65, in which the salinity had increased to 11%, Nereida and the unknown Gammaproteobacteria phylotype were no longer present in significant numbers; at this salinity, we found the dominance of Vibrio and Halomonas, with lower numbers of sequences associated with the genera Comamonas, Salinivibrio, Acinetobacter and Roseovarius. Salinivibrio was the most abundant organism in pond 104 (13.8% salt), with Halomonas being abundant as well. Salinivibrio and Halomonas were still present on nauplii collected from the highest salinity (over 30% in pond 201), but they were accompanied by relatively large numbers of sequences related to Acinetobacter, Comamonas, Propionibacterium and Capnocytophaga.
Analyses of the clone libraries prepared from the adult brine shrimps showed that a few OTU only dominated the intestinal bacterial community: Halomonas, Salinivibrio and a representative of the Flavobacteriaceae. These three groups together account for up to 62% of the total clones recovered. There are distinct trends in the distribution of these three taxa along the salinity gradient. The contribution of Halomonas increased with salinity, from 11% at 11% salt to 94% at 30%, which was the highest salinity at which Artemia occurred in the Eilat saltern ponds. Salinivibrio-affiliated sequences were absent from the library obtained from Artemia living at the lowest salinity; they dominated in the library at 14% salt (33% of the total clones recovered), to decrease again at increasing salt concentrations, where Halomonas increased its share of the community. Representatives of the genus Vibrio were relatively abundant in the brine shrimp intestines at the lowest salinity, to disappear from the gene library at salt concentrations of 14% and higher. The peak occurrence of a representative of the Flavobacteriaceae was found in Artemia collected from 21% salinity. The sequence obtained from this clone was not closely related to any cultured organism; however, closely related 16S rRNA gene sequences were recovered from the mat from solar salterns of Eilat (Sørensen et al., 2005) and similar salterns in Tunisia (Baati et al., 2008). Its abundance decreased at lower salinities, and its phylotype was not detected in the clone library prepared from the individual animals collected from the highest salinity ponds.
Among the phylotypes found in lower numbers, species of Roseovarius and Halochromatium were prominent in the lower salinity ponds. Phylotypes belonging to other groups of Bacteria were occasionally encountered (Fig. 3).
The mothur software package (Schloss et al., 2009) was used in order to estimate the level of phylotype redundancy (rarefaction curves) and to calculate various species richness indices at different levels of evolutionary distance (Table 1). We have considered three levels of OTUs defined at evolutionary distances of 2%, 5% and 20% as rough approximations to the species, genus and phylum levels. Rarefaction curves are presented in Fig. 4.
Based on the steepness and shape of the rarefaction curves (Fig. 4) and Chao 1 and ACE (abundance-based coverage estimator) diversity indices (Table 1), the bacterial diversity in the intestine of adults was the highest at the lowest amount of salt (5.5%) and decreased with increasing salinity. The diversity of bacteria associated with nauplii was slightly higher in comparison with the diversity of intestinal bacteria in all ponds, with the exception of pond 65 with 11% salt. Decreasing diversity with increasing salinity was also observed in nauplii, except in pond 104, where high variability was observed and thus large confidence intervals of the ACE and Chao 1 diversity estimates were documented. The diversity at the genus (set here at 95% genetic distance according to general convention) and species level (97% genetic distance) was much higher compared with the phylum level (80% genetic distance). At the genus and species levels, the plateau was observed only in the rarefaction curves of the ponds with the highest salinity (Fig. 4), but at the phylum level, a plateau was observed in all ponds. The coverage of individual libraries was significantly different in each sample (P<0.0001) as calculated using ∫-libshuff with the Bonferroni correction.
For adults sampled in all five ponds, we initially analysed the contents of the intestines only, assuming that most bacteria associated with the brine shrimp are associated with the gut. Because we did not have any information on the relative contribution of the intestinal bacteria to the total bacterial DNA obtained in either case, we constructed another clone library of whole adult animals sampled in the pond 201 with the highest salinity. Surprisingly, the clone library of the entire adult animals showed that only one clone out of 88 could be identified to the level of genus, and it was classified within the genus Rhizobium (100% identity), while the remaining 87 clones could not be assigned to any as yet cultured phylum (Fig. 5).
The first studies reporting on bacteria associated with the brine shrimp Artemia were published in the 1970s by Tyson, who described spirochete-like bacteria in maxillary glands (Tyson, 1970) and distinctive renal lesions (Tyson, 1974), and by Post & Youssef (1977), who described prokaryotic intracellular symbionts. Later publications focused mainly on bacterial contaminants in cyst production and in hatcheries (e.g. Dehasque et al., 1991). Few studies concentrated on the indigenous bacterial microbiota present in different developmental stages of Artemia (Straub & Dixon, 1993; Riddle et al., 2007, 2008), although Boyle & Mitchell (1978) claimed that microorganisms are absent in the intestine of crustaceans.
The results obtained in our study indicate that only a limited number of phyla are associated with Artemia, that the number of OTU decreases with increasing salinity in nauplii and in the intestine of adult animals (Table 1, Figs 2–4) and that representatives of a probably new phylum are associated with whole animals from pond 201 with salinity 30.6% (Fig. 5). Besides this novel phylogenetic lineage, the most abundant phylum detected in association with nauplii and intestines of adult animals was Proteobacteria, followed by Actinobacteria, Bacteroidetes and Firmicutes. A few clones belonging to Cyanobacteria were also identified. According to the literature, Proteobacteria were the most frequently isolated bacteria from all developmental stages of laboratory-hatched Artemia (Austin & Allen, 1982; Prieto et al., 1987; García et al., 1988; Igarashi et al., 1989; Høj et al., 2009) and from animals caught in their natural environment (Straub & Dixon, 1993; Riddle et al., 2007, 2008).
Analysis of the clone libraries revealed that Halomonas was associated with both developmental stages sampled in all ponds, and its abundance was higher compared with most OTUs (Figs 2 and 3). Species of the genus Halomonas are metabolically very diverse and can grow between 1% and 26% salt, with optimum growth around 9% salt (Mata et al., 2002; Arahal et al., 2007). They were previously isolated from various saline and hypersaline environments, such as evaporation ponds (Baati et al., 2008), saline wetland (Martínez-Cánovas et al., 2004), soil (Quillaguaman et al., 2004) and soda lake (Duckworth et al., 2000), but also from Antarctic lakes (Franzmann et al., 1987), paintings (Heyrman et al., 2002), sea ascidians (Romanenko et al., 2002) and a sea anemone (Xiao et al., 2009). Their presence has also been confirmed in association with Artemia from the Great Salt Lake (UT) by clone library analysis, but only in association with adult animals and not with encysted embryos (Riddle et al., 2007, 2008).
The second most abundant group of OTUs belonged to the family Vibrionaceae (Figs 2 and 3). Species of the genus Vibrio appear to be the most successful colonizers of Artemia intestines at the lower salinity range, whereas OTUs affiliated to the genus Salinivibrio were the most abundant in ponds with higher salinities. Previous isolations of bacteria from Artemia (Austin & Allen, 1982; Prieto et al., 1987; García et al., 1988; Igarashi et al., 1989; Straub & Dixon, 1993; Høj et al., 2009) yielded Vibrio spp. as the most abundant isolates. Different species of the genus Vibrio have also been confirmed with clone library analysis as the predominant bacteria in the intestine of adult Artemias from Great Salt Lake (Riddle et al., 2007, 2008). Few species, such as V. parahaemolyticus and Vibrio hispanicus, appeared to be pathogenic to Artemia (Gomez-Gil et al., 2004; Defoirdt et al., 2006). On the other hand, Salinivibrio is known as a nonpathogenic, moderately halophilic bacterium, which grows between 3% and 15% salt, optimally in media containing 10% salt (Mellado et al., 1996). It has been isolated previously from brine of different salterns (Yeon et al., 2005) and is known to be associated with dinoflagellates (Seibold et al., 2001) and sea anemone (Xiao et al., 2009). Riddle et al. (2007, 2008) reported Salinivibrio as the most abundant genus in cysts of Artemia from Great Salt Lake. Nauplii were not examined in these studies.
When Boyle & Mitchell (1978) published scanning electromicrographs indicating that microorganisms are absent in the intestine of crustaceans, they consequently opened a discussion about autochthonous microbiota in crustaceans. Artemia is a filter-feeding organism and a grazer, which receives nutrients from particulate and dissolved organic matter. Except for the caeca, there is no other separate gut area where bacteria could form an autochthonous microbiota, because the foregut and the midgut are of ectodermal origin and therefore they regularly moult. For this reason, microbial communities in the intestine are strongly influenced by the microbial communities in the food ingested (Olsen et al., 2000).
Probiotics are defined as live microbial feed supplements that beneficially affect the host animal by improving its intestinal balance (Fuller, 1989). Few studies have thus far been performed indicating the potential beneficial influence of certain bacteria on Artemia. Flexibacter, for example, can not only act as a food source but also assists in the digestion of the alga Tetraselmis spp. (Intriago & Jones, 1993), and nine bacterial isolates from Artemia showed a positive effect on the body length, biomass and survival of hatched nauplii when the water was previously inoculated with them, and Vibrio proteolyticus, Pseudomonas fluorescens and Vibrio alginolyticus had a significant negative effect on the parameters mentioned (Verschuere et al., 1999). It was also previously shown that food influences the structure of the microbiota (Olsen et al., 2000) and that a short-term incubation of live food organisms in a bacterial suspension consisting of one or several probiotic strains is a possible approach to replace opportunists with other less aggressive bacteria (Makridis et al., 2000).
Autochthonous microbial communities can act as probiotics with providing digestive enzymes. Salinivibrio, as one of most important genera associated with Artemia, is a known producer of chitinase (Aunpad & Panbangred, 2003), extracellular zinc-metalloproteases (Lama et al., 2005; Karbalaei-Heidari et al., 2007a, b, 2008; Amoozegar et al., 2008b), thermohalophilic lipase (Amoozegar et al., 2008a) and cellulase (Wang et al., 2009), while Halomonas spp. are known to produce extracellular α-amylase (Coronado et al., 2000; Rohban et al., 2009) and may have high lipolytic and DNase activity, moderate proteolytic, xylanolytic and inulinolytic activity and low pullulanolytic, cellulolytic (CMCase) and pectinolytic activity (Rohban et al., 2009). The high lipolytic activity is most probably due to the intracellular esterases with a preference for short-chain fatty acids (Hinrichsen et al., 1994).
This study provides the first molecular insight into the changes in the bacterial communities associated with the different developmental stages of the brine shrimp Artemia along a salinity gradient. It was demonstrated that the structure of the bacterial communities in Artemia varies between developmental stages and is strongly influenced by its environment. Clone library analysis of entire adult animals revealed a novel, as yet undescribed phylogenetic lineage of bacteria.
We thank the Israel Salt Company in Eilat, Israel, for allowing access to the salterns, the staff of the Interuniversity Institute for Marine Sciences of Eilat for logistic support, Assist. Prof. Lejla Pašić and the Slovenian Research Agency (ARRS) for a Young Researcher Grant to R.T.
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