Int. J. Aquat. Biol. (2022) 10(4): 280-284 ISSN: 2322-5270; P-ISSN: 2383-0956 Journal homepage: www.ij-aquaticbiology.com © 2022 Iranian Society of Ichthyology Short Communication Culturable microflora of Artemia franciscana reared under laboratory conditions Haruka Kurihara, Tomoya Akagi, Hitomi Nimura, Shiro Itoi, Haruo Sugita*1 Department of Marine Science and Resources, Nihon University, Kameino 1866, Fujisawa, Kanagawa 252-0880, Japan. s Article history: Received 26 June 2022 Accepted 1 August 2022 Available online 2 5 August 2022 Keywords: Microflora Vibrionaceae Opportunistic pathogens 16S rRNA gene Abstract: Artemia is widely used as an initial food for larval and juvenile fish in aquaculture facilities around the world. However, several lines of research have strongly suggested that Artemia larvae may carry opportunistic pathogens such as Listonella anguillarum, thereby serving as a source of infection of fish. In the present study, we investigated the dynamics of the culturable microflora of Artemia reared under laboratory conditions, with the goal of understanding the risk of opportunistic infection mediated by this animal. After hatching decapsulated cysts of A. franciscana, the larvae were reared for an additional 27 days to examine, using the culture-dependent method, the culturable microflora of the rearing water and of washed Artemia. The results showed that Vibrionaceae, Flavobacteriaceae, Pseudoalteromonadaceae, Alteromonadaceae and Rhodobacteriaceae accounted for 8.3-35.8% of the rearing water isolates. In contrast, Vibrionaceae dominated in Artemia isolates, accounting for 79.2% of the flora. However, Vibrionaceae were not detected in either decapsulated or undecapsulated cysts, or in the algal concentrates used as feed, suggesting that Vibrionaceae is not indigenous to Artemia cysts and instead is derived primarily from natural seawater. These results strongly suggest that hatching and rearing live diets such as Artemia under sanitary conditions may reduce the risk of opportunistic infection. Introduction Artemia is one of the most important foods for marine fish in aquaculture facilities worldwide, as its cysts can be incubated in seawater or salt water for one to two days to produce nauplii of appropriate size. In addition, Artemia can be enriched with special nutrients such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) for the use of Artemia in the diets of captive animals that require these substances (Lavens and Sorgeloos, 1996). However, Kurosaki et al. (2021), in an examination of the gut microbiota of red seabream, Pagrus major during early development, found that Vibrio spp. in larval guts are derived primarily from rotifers Brachionus plicatilis sp. complex S-type and Artemia sp. nauplii used as the diet. Previously, Mizuki et al. (2006), in an investigation of the dynamics of the fish pathogen, Listonella anguillarum in a hatchery of Japanese flounder, Paralichthys olivaceus, suggested that L. anguillarum was being transmitted from rotifers, *Correspondence: Haruo Sugita DOI: https://doi.org/10.22034/ijab.v10i4.1637 E-mail: sugita.haruo@nihon-u.ac.jp B. plicatilis and Artemia nauplii. Similar results have been reported by many researchers (Grisez et al., 1977; Tanasomwang and Muroga, 1988; López- Torres et al., 2001; Eddy and Jones, 2002). However, to our knowledge, there are no reports on the dynamics of the microflora of Artemia during the long-term rearing of this animal. In marine fish farming, Vibrio spp. often is detected in the fish gut, and the rearing environment contains a number of opportunistic pathogens; the presence of these pathogens is an extremely important risk factor in the aquaculture industry. Therefore, in this study, we investigated the dynamics of the culturable microflora of Artemia reared for relatively long periods of time, specifically by employing a culture- dependent approach in combination with 16S rRNA gene (16S rDNA) sequencing. Materials and Methods Experiments were performed with A. franciscana 281 Int. J. Aquat. Biol. (2022) 10(4): 280-284 cysts obtained from the Great Salt Lake, Utah, USA, as supplied by OSI Marine Lab (Snowville, UT, USA). Artemia cysts were decapsulated as described by Lavens and Sorgeloos (1996). For hatching and rearing Artemia, a glass medium bottle with a capacity of 1,200 mL was used; two glass tubes with cotton filters attached were inserted into the lid of the bottle, and the air was delivered from an air pump into one of the tubes. The tip of another glass tube was placed above the water surface, permitting exhaust of spent air into the atmosphere. After the apparatus without an air pump was autoclaved at 121°C for 15 min and allowed to cool to room temperature, 1,000 mL of unsterilized natural seawater and 0.01 g of decapsulated cysts were placed in the bottle; the culture then was incubated at 25°C with aeration at a flow rate of 2.9 mL/sec and exposed to constant incandescent light. The natural seawater collected on Enoshima Island, Fujisawa, Kanagawa was used. Artemia larvae were fed an algal concentrate (Chlorella Industry Co., Tokyo, Japan) once every two days for an additional 27 days. Surface bacteria loosely attached to the Artemia body were removed according to the method of Austin and Allen (1982). Specifically, sterile seawater was added to ten washed Artemia larvae to make a total volume 100-500 μL depending on Artemia size, which then was homogenized to generate a washed sample. Artemia homogenates and rearing water sampled on days 0, 1, 7, 14, 21, and 28 were diluted serially with sterile seawater and inoculated onto 1/20 PYBG agar medium, which contains the following (per 1000 mL of aged seawater): Trypticase peptone (Beckton Dickinson, Franklin Lakes, NJ, USA), 0.5 g; Phytone peptone (Beckton Dickinson), 0.25 g; Bacto-yeast extract (Beckton Dickinson), 0.1 g; Lab Lemco powder (Thermo Fisher Scientific, Waltham, MA, USA), 0.1 g; glucose, 0.1 g; and agar, 15 g; the resulting solution is adjusted to pH 7.5 (Sugita et al., 2005). The inoculated agar plates were incubated at 25°C for 7 days under aerobic conditions. Additionally, homogenates of both decapsulated and undecapsulated cysts, as well as natural seawater and an algal concentrate, were processed similarly. After incubation, the viable counts (colony-forming units (CFUs) per mL or nauplius) were determined, and 19- 20 colonies per sample were chosen and purified by streaking to a fresh solid medium. 16S rDNA of each isolate was amplified and sequenced according to Hiraishi (1992), with some modifications. Briefly, 16S rDNA was amplified by polymerase chain reaction (PCR) using a primer set consisting of 20F (5'-AGA GTT TGA TCC TGG CTC AG-3') and r2L (5'-CATCGTTTACGGCGTGGAC- 3'), and the resulting fragments were partially sequenced using the BigDye Terminator v3.1 Cycle Sequencing Ready Reaction Kit with a Model ABI 3130xl automated sequencer (Applied Biosystems, Foster City, CA, USA). The final sequence (approximately 690 bp) was determined from overlapping sequence data using the AutoAssembler ver. 2.1 computer program (Applied Biosystems). The isolates were identified using EZBioCloud (Yoon et al., 2017) on the basis of their 16S rDNA sequences. Representative sequences from this study have been deposited into the DDBJ/GenBank/EMBL databases (Accession Numbers: LC13330-LC13337). Results and Discussions A total of 299 bacteria were isolated, and all were classified into 22 families based on 16S rDNA sequences. Vibrionaceae (140 isolates), Pseudoalteromonas (34), Flavobacteriaceae (26), Moraxellaceae (16), Alteromonadaceae (15), Stappiaceae (12), and Rhodobacteriaceae (12) were the major families of isolates obtained in this study. Notably, no bacteria were detected in the decapsulated cysts. Table 1 shows the composition of 16 families in bacterial isolates from the undecaspulated cysts, from an algal concentrate, and from natural seawater, which exhibited viable counts of 1.2×106 CFU/g, 6.5×105 CFU/mL, and 6.6×103 CFU/mL, respectively. Moraxellaceae and Planococcaceae accounted for 25.0-60.0% of the isolates from undecapsulated cysts; Microbacteriaceae, Bacilli, and Moraxellaceae accounted for 20.0-30.0% of the isolates from the algal concentrate. However, bacteria belonging to Vibrionaceae were not isolated from either of those 282 Kurihara et al./ Microflora of Artemia reared under laboratory conditions samples. Nine families were isolated from the natural seawater, with Microbulbiferaceae and Stappiaceae accounting for 21.1-36.8% and Vibrionaceae accounting for 10.5% of those isolates. Ten families were isolated from the Artemia rearing water, with viable counts of 5.0×104-2.6×107 CFU/mL (Table 2). Vibrionaceae, Flavobacteriaceae, Pseudoalteromonadaceae, Alteromonadaceae, and Rhodobacteriaceae accounted for 8.3-35.8% of the rearing water isolates. Bacteria isolated from washed Artemia ranged in viable counts from 1.4×104-1.1×107 CFU/nauplius and consisted of six families that Vibrionaceae and Pseudoalteromonadaceae were the top-two-most-dominant bacteria, accounting for 79.2 and 14.2%, respectively. Viable counts of bacteria in rearing water and washed Artemia during the rearing period varied by as much as three orders of magnitude, and the families of bacteria detected varied widely, indicating that the densities of the dominant culturable bacteria in each sample also varied greatly. Table 3 shows the species composition of Vibrionaceae in isolates from the natural seawater, rearing water, and washed A. franciscana. Photobacterium consisted of one species, and Vibrio of seven species (Table 3). Among the Vibrio, V. parahaemolyticus (BBQD01000032) and V. tubiashii (CP009354) are known to act as pathogenic bacteria in marine animals. V. azureus (BATL01000140), V. hyugaensis (LC004912), and V. neocaledonicus (JQ934828) were detected in 50.0- 66.7% of the rearing water samples and 83.3-100% of the washed Artemia samples. These results suggested that bacteria of the genus Vibrio have a high affinity for Artemia because these bacteria are easily taken up into the gut of Artemia or adhere to these animals’ body surfaces. Vibrio spp. are predominantly found in the gut and on the body surface of copepods, and V. cholerae and V. parahaemolyticus adhere to copepod shells, using this matrix as a nutrient source and as a cryoprotective environment (Kaneko and Colwell, 1975; Sochard et al., 1979; Shimodori et al., 1989). However, the differences among the Vibrio species in their frequencies and percentage of the bacteria may reflect the compatibility of each bacterial species with Artemia and the rearing environment. The large fluctuations in Vibrio representation in Closest family Undecapsulated cysts Algal concentrate Natural seawater Bacillaceae 1 6 0 Cellulomonadaceae 0 1 0 Erythrobacteraceae 0 0 1 Flavobacteriaceae 0 0 1 Gordoniaceae 0 1 0 Halomonadaceae 2 0 1 Intrasporangiaceae 0 1 0 Maricaulaceae 0 0 1 Microbacteriaceae 0 6 0 Microbulbiferaceae 0 0 7 Moraxellaceae 12 4 0 Nocardiaceae 0 1 0 Planococcaceae 5 0 0 Rhodobacteraceae 0 0 1 Salinisphaeraceae 0 0 1 Stappiaceae 0 0 4 Vibrionaceae 0 0 2 Viable counts (CFU/g, mL) 1.2×106 6.5×105 6.6×103 Table 1. Composition of bacterial families in isolates derived from the undecapsulated cysts, an algal concentrate, and natural seawater. 283 Int. J. Aquat. Biol. (2022) 10(4): 280-284 rearing water and among Artemia-associated bacteria during the Artemia rearing period may be related to the effects of Artemia shell molting and other factors; further research will be needed to clarify these patterns. Since no bacteria were detected in decapsulated cysts, the Vibrio spp. detected in Artemia and its rearing water presumably existed at low densities in natural seawater and/or in the algal concentrate, subsequently multiplying and becoming dominant in the Artemia-rearing environment. We hypothesize, by extension, that many opportunistic pathogens, including L. anguillarum, are not cyst-derived, but instead derive from the live diets and/or the seawater environments in which cysts are hatched and reared for a period of time (Mizuki et al., 2006). López-Torres et al. (2001) reported that Artemia larvae are vectors of Vibrio spp. in aquaculture activities. However, the present work suggests that these Vibrio spp.. Instead, result from the manipulation of Artemia hatchery tanks and are not derived from the cysts themselves. In other words, Vibrio spp. are ubiquitous in aquaculture facilities, but keeping Artemia in an environment free of opportunistic pathogens may be an effective way to prevent opportunistic infections in the early developmental stages of marine fish being fed Artemia. These previous results, viewed in the context of the present study, lead us to recommend that Artemia cysts be hatched in seawater and containers that both have been disinfected, and be reared on foods and food additives that are confirmed to be free of opportunistic pathogens. We propose that similar studies also should be performed on rotifers to facilitate further effective control of opportunistic infections in seed production for aquaculture. Acknowledgment This study was supported, in part, by the Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for Scientific Research (C) (25450263). References Austin B., Allen D.A. (1982). Microbiology of laboratory- Table 2. Composition of bacterial families in isolates derived from rearing water and washed Artemia. Closest family No. of isolates from rearing water No. of isolates from washed Artemia Day 0 Day 1 Day 7 Day 14 Day 21 Day 28 Day 0 Day 1 Day 7 Day 14 Day 21 Day 28 Alteromonadaceae 0 1 0 9 0 2 0 0 0 3 0 0 Bacillaceae 0 0 0 0 0 1 0 0 0 0 0 0 Cyclobacteriaceae 0 0 0 0 0 2 0 0 0 0 0 0 Enterobacteriaceae 0 0 0 1 0 0 0 0 0 0 0 0 Erythrobacteraceae 0 0 0 1 0 1 0 0 0 0 0 0 Flavobacteriaceae 0 0 13 0 4 8 0 0 0 0 0 0 Oceanospirillaceae 0 0 0 0 0 0 3 0 0 0 0 0 Pseudoalteromonadaceae 6 10 1 0 0 0 10 0 0 5 2 0 Rhodobacteraceae 0 0 1 3 2 4 0 0 0 1 0 0 Stappiaceae 0 0 0 5 0 2 0 0 0 1 0 0 Vibrionaceae 14 9 5 1 14 0 7 20 20 10 18 20 Viable counts (CFU/mL, nauplius) 1.8× 106 2.6× 107 1.2x 106 4.3× 105 5.0× 104 1.3× 106 1.4× 104 7.5× 105 4.9× 105 2.4× 105 1.4× 106 1.1× 107 284 Kurihara et al./ Microflora of Artemia reared under laboratory conditions hatched brine shrimp (Artemia). Aquaculture, 26: 369- 383. Eddy S.D., Jones S.H. (2002). Microbiology of summer flounder Paralichthys dentatus fingerling production at a marine fish hatchery. Aquaculture, 211: 9-28. Grisez L., Reyniers J., Verdonck L., Swings J., Ollevier F. (1977). Dominant intestinal microflora of sea bream and sea bass larvae, from two hatcheries, during larval development. Aquaculture, 155: 387-399. Hiraishi A. (1992). Direct automated sequencing of 16S rDNA amplified by polymerase chain reaction from bacterial cultures without DNA purification. Letters in Applied Microbiology, 15: 210-213. Kaneko T., Colwell R.R. (1975). Adsorption of Vibrio parahaemolyticus onto chitin and copepods. Applied Microbiology, 29: 269-274. Kurosaki M., Kunimoto M., Akiyama N., Itoi S., Sugita H. (2021). Predominant gut microbiota in the early life stages of red seabream (Pagrus major) raised in indoor tanks. International Aquatic Research, 13: 219-226. Lavens P., Sorgeloos P. (1996) Manual on the production and use of live food for aquaculture. FAO Fisheries Technical Paper No. 361. FAO. 295 p. López-Torres M.A., Lizárrage-Partida M.L. (2001) Bacteria isolated on TCBS media associated with hatched Artemia cysts of commercial brands. Aquaculture, 194: 11-20. Mizuki H., Washio S., Morita T., Itoi S., Sugita H. (2006). Distribution of a fish pathogen Listonella anguillarum in the Japanese flounder Paralichthys olivaceus hatchery. Aquaculture, 261: 26-32. Shimodori S., Moriya T., Kohashi O., Faming D., Amako K. (1989). Extraction from prawn shells of substances cryoprotective for Vibrio cholerae. Applied and Environmental Microbiology, 55: 2726-2728. Sochard M.R., Wilson D.F., Austin B., Colwell R.R. (1979). Bacteria associated with the surface and gut of marine copepods. Applied and Environmental Microbiology, 37: 750-759. Sugita H., Kurosaki M., Okamura T., Yamamoto S., Tsuchiya C. (2005). The culturability of intestinal bacteria of Japanese coastal fish. Fisheries Science, 71: 956-958. Tanasomwang V., Muroga K. (1988). Intestinal microflora of larval and juvenile stages in Japanese flounder (Paralichthys olivaceus). Fish Pathology, 23: 77-83. Yoon S.H., Ha S.M., Kwon S., Lim J., Kim Y., Seo H., Chun J. (2017). Introducing EzBioCloud: A taxonomically united database of 16S rRNA and whole genome assemblies. International Journal of Systematic and Evolutionary Microbiology, 67: 1613-1617. Table 3. Composition of bacterial species belonging to Vibrionaceae among isolates derived from natural seawater, rearing water, and washed Artemia. Closest species (accession no.; identity, %) Natural seawater No. of isolates from rearing water No. of isolates from washed Artemia Day 0 Day 1 Day 7 Day 14 Day 21 Day 28 Day 0 Day 1 Day 7 Day 14 Day 21 Day 28 Photobacterium salinisoli (KP054474; 97.7) 0 0 0 0 0 0 0 0 1 0 0 0 0 Vibrio azureus (BATL01000140; 98.8-100) 1 4 4 0 1 5 0 2 9 7 7 15 12 Vibrio hyugaensis (LC004912; 98.8-99.9) 0 1 2 0 0 8 0 2 9 9 0 2 6 Vibrio japonicus (LC143378; 99.4-99.6) 0 0 0 0 0 0 0 2 1 0 0 0 0 Vibrio natriegens (ATWU01000093; 100) 0 1 0 0 0 0 0 0 0 0 0 0 0 Vibrio neocaledonicus (JQ934828; 98.6-100) 0 7 3 5 0 1 0 1 0 4 3 1 1 Vibrio parahaemolyticus (BBQD01000032; 98.4-99.3) 0 1 0 0 0 0 0 0 0 0 0 0 1 Vibrio tubiashii (CP009354; 99.2) 1 0 0 0 0 0 0 0 0 0 0 0 0