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Molecular detection of Kudoa septempunctata (Myxozoa: Multivalvulida) in sea water and marine invertebrates

Molecular detection of Kudoa septempunctata (Myxozoa: Multivalvulida) in sea water and marine... The exportation of cultured olive flounder (Paralichthys olivaceus) in Korea has been recently decreasing due to the infections with a myxozoan parasite Kudoa septempunctata, and there is a strong demand for strict food safety management because the food poisoning associated with consumption of raw olive flounder harbouring K. septempunctata has been frequently reported in Japan. The life cycle and infection dynamics of K. septempunctata in aquatic environment are currently unknown, which hamper establishment of effective control methods. We investigated sea water and marine invertebrates collected from olive flounder farms for detecting K. septempunctata by DNA-based analysis, to elucidate infection dynamics of K. septempunctata in aquaculture farms. In addition, live marine polychaetes were collected and maintained in well plates to find any possible actinosporean state of K. septempunctata.The levelof K. septempunctata DNA in rearing water fluctuated during the sampling period but the DNA was not detected in summer (June–July in farm A and August in farm B). K. septempunctata DNA was also detected in the polychaetes Naineris laevigata intestinal samples, showing decreased pattern of 40 to 0%. No actinosporean stage of K. septempunctata was observed in the polychaetes by microscopy. The absence of K. septempunctata DNA inrearing wateroffishfarm and the polychaetes N. laevigata intestinal samples during late spring and early summer indicate that the infection may not occur during this period. N. laevigata was suspected as the possible alternate invertebrate host of K. septempunctata, but the actinosporean stage was not found by well plate method and further studies will be necessary. This research provides important baseline information for understanding the infection dynamics of K. septempunctata in olive flounder farms and further establishment of control strategies. Keywords: Kudoa septempunctata, Myxozoa, Olive flounder, Paralichthys olivaceus, Naineris laevigata Background Adlard 2012). Most of the species are histozoic which Myxozoans belong to the group of metazoan parasites of develop symptoms of macroscopic whitish cyst or cause fish and act as a cause for several outbreaks in both fresh post-mortem myoliquefaction (Shirakashi et al. 2012). water and marine fish (Canning and Okamura 2003). However, some Kudoa species do not cause any of the Disease transmission by these myxozoan parasites can symptoms mentioned above and Kudoa septempunctata, often have disastrous economic impact in aquaculture a newly found myxosporean found in olive flounder industries, although most of them are known to have in- (Paralichthys olivaceus) is probably the most well-known significant or negligible effect in fish (Yokoyama et al. example of them (Yokoyama et al. 2004; Matsukane et 2012). The genus Kudoa comprises more than 70 species al. 2010) . reported from a broad range of fish host (Miller and Since 2011, food poisoning due to ingestion of farmed olive flounders in Japan has been reported (Kawai et al. * Correspondence: jhkim70@gwnu.ac.kr 2012). Epidemiological studies have revealed that this Department of Marine Bioscience, Gangneung-Wonju National University, outbreak is associated with the presence of K. septem- Gangneung, Gangwon 25457, South Korea punctata in the causative foods (Kawai et al. 2012) and Department of Marine Bioscience, Gangneung-Wonju National University, Gangneung, Gangwon 210-702, South Korea the food-borne outbreaks associated with consumption Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Paari et al. Fisheries and Aquatic Sciences (2017) 20:16 Page 2 of 8 of raw olive flounder harbouring K. septempunctata are septempunctata infection occurs (farm A, B). Sampling becoming a prominent public health concern in Japan. was conducted on a monthly basis during May and As the customs of consuming raw fish is spreading, the November 2014 and approximately 2 l of water was occurrence of this food-borne disease is predicted to in- collected in each sampling. Water samples were fil- crease (Harada et al. 2012). Although there have been tered through a fresh nitrocellulose membrane filter outbreaks in Japan since 2011, the question of olive (5 μm pore size; ADVENTEC, Japan) using a suction flounder in Korea acting as a host of K. septempunctata pump (DOA-P704-AA, GAST, USA), and the mem- remains unanswered (Iwashita et al. 2013). Considering brane filter were placed in an individual micro centri- the commercial value of olive flounder and the public fuge tube andstoredat −20 °C until DNA extraction. health concern, it is urgently needed to solve the nega- Acetone dissolution method was used for extracting tive impact of this parasite on public health and food DNA from the filter samples (Hallett et al. 2012). The safety, but almost nothing is known about its transmis- membrane filter in microcentrifuge tube was air dried sion biology, infection dynamics in aquatic environment. and dissolved by adding 2 ml of acetone (Cica reagent, Myxozoan parasites have been believed to be transmit- Japan). The completely solubilized filter components by ted from fish to fish until Wolf and Markiw (1984) de- repeated vortexing were then centrifuged at 3000g for monstrated that freshwater oligochaete was essential for 15 min, and the supernatant was discarded. This step the transmission of Myxobolus cerebralis and since then, was repeated twice to ensure complete dissolution of fil- many studies have confirmed that some myxozoans trate particles from the dissolved materials. To the dis- undergo two-host life cycle (Lom and Dykova 2006; solved filtrate samples, 1 ml of 95% ethanol was added Markussen et al. 2015). Currently, more than 30 fresh- and thoroughly mixed. The suspended pellet after cen- water myxozoans are known to have two-host life cycles trifugation was then air dried and used directly for (Yokoyoma et al. 2012), but only 7 marine myxozoans DNA extraction. are found to have marine invertebrates to complete their DNA was extracted by using a QIAamp DNA Mini life cycles (Karlsbakk and Køie 2012; Køie et al. 2004, Kit (QIAGEN, USA) according to the manufacturer’s 2007, 2008, 2013; Rangel et al. 2015), and neither life instructions with slight modifications. Briefly, 180 μlof cycle nor invertebrate alternate hosts have been eluci- tissue lysis buffer (Buffer AE, QIAGEN, USA) was added dated in kudoid myxozoans. to the air-dried pellet sample, and then 20 μl of Proteinase Environmental water analysis is indispensable for in- K (QIAGEN, USA) was added. Following overnight incu- vestigating epidemiology of myxozoan infections because bation, wash buffers (Buffer AW1, AW2, QIAGEN, USA) fish myxozoans occur in aquatic environment and trans- were added and eluted using elution buffer (Buffer AE, mission between two different hosts also occur in envi- QIAGEN, USA). Extracted DNA was stored at −20 °C ronmental water. Many studies revealed that the disease until used for PCR detection. transmission occurs via water in endemic area and ap- propriate water treatments were effective for the man- PCR and real-time PCR for detecting K. septempunctata in agement of several myxozoan infections (Cobcroft and rearing water Battaglene 2013; Nehring et al. 2003; Yanagida et al. PCR was carried out for detecting K. septempunctata in 2006). Thus, the environmental water analysis would be water samples using the following set of primer sets: the first step to clarify infection dynamics and develop Ksf-GTGTGTGATCAGACTTGATATG; KsR-AAGCCA further effective management strategy for K. septem- AAACTGCTGGCCATTT [25]. 0.5 μM of forward and punctata infection. As knowledge about the infection reverse primer, 1 μl of template DNA was added to the dynamics of this parasite is scarce, we carried out a PCR premix tube (Bioneer, Korea) and the total volume monthly inspection of water samples to study the occur- was made to 20 μl using ultra-pure distilled water rence pattern of K. septempunctata in aquatic environ- (Invitrogen, USA). PCR cycling parameters followed ment by molecular analysis. We also investigated the the protocols of Grabner et al. (2012) with some minor prevalence of K. septempunctata in marine polychaetes modifications. PCR cycling parameters were an initial collected around the farms using both of well plate denaturation at 95 °C for 4 min, followed by 35 cycles method described by Yokoyama et al. (1991) and mo- at 95 °C for 35 s, 56 °C for 30 s and 72 °C for 30 s and lecular analysis, to speculate the possible life cycle of K. ended with a final extension at 72 °C for 7 min. septempunctata. Real-time PCR was carried out using the following sequence of primers and probe; F-CATGGGATTAGCCC Methods GGTTTA; R-ACTCTCCCCAAAGCCGAAA; P-(FAM)- Water sampling and DNA extraction TCCAGGTTGGGCCCTCAGTGAAAA (Kawai et al. Water samples were directly collected from the inlet 2012). Real-time PCR was carried out in a 0.2-ml PCR pumping units from two aquaculture farms where K. strip tube containing 2× Premix Ex Taq (Takara, Japan) Paari et al. Fisheries and Aquatic Sciences (2017) 20:16 Page 3 of 8 10 μl, primer (0.4 uM, Bioneer, Korea), probe (0.25 μM, mt COI gene were aligned and identified based on the Bioneer, Korea), ROX II reference dye (Takara, Japan), 4 μl percentage identity of nucleotide sequences previously template DNA using ABI 7500 Fast Real-time PCR system registered in NCBI. (Applied Biosystems, USA). Cycling parameters were preheating at 95 °C for 10 min, followed by 45 cycles Microscopic detection of K. septempunctata from at 95 °C for 15 s, 60 °C for 1 min and the analyses polychaetes were conducted twice. Live polychaetes collected from the outflow waterway sediments of fish tank were observed for the occurrence Collection of marine invertebrates and species of actinospores by well plate method (Yokoyama et al. identification 1991). Briefly, collected polychaetes were washed with Marine invertebrates from the sediments of fish tanks sterile sea water several times, individually placed in a and coastal areas near fish farms were collected to inves- 12-well plate and observed microscopically using an tigate the prevalence of K. septempunctata infection. For inverted microscope (Leica, Germany) every day to find collecting invertebrate samples, mud was collected from if the possible K. septempunctata actinospores were re- approximately 0.5 m depth from outflow waterway of leased. Sea water in the wells was replaced in a 2-day the same fish farms where the water samples were col- interval. Squash preparations were also made from the lected and transferred to laboratory. Sediments were isolated polychaetes samples at every 2 days interval for sieved through a mesh (0.5 mm) to separate marine observation of the actinosporeans; several posterior seg- polychaetes within a day of collection of sediment. Live ments of the intestinal region of polychaetes were polychaetes were collected, washed with sterile sea water squashed between the slide and cover slip, fixed with several times, and then maintained in a 12-well plate at methanol, stained with Giemsa solutions and examined 15 °C, for observing the possible actinosporean stages of using a light microscope (Leica, Germany). K. septempunctata. Invertebrate samples collected around the coastal re- For collecting invertebrate samples from coastal areas, gions were not maintained in well plates but imme- quadrats and dredging devices were used by trained di- diately processed for microscopic analysis using squash vers to obtain marine invertebrates near the olive floun- preparations and for PCR detection using the same der farms. Subsamples of invertebrate samples collected methodology mentioned above. from the gravel materials were washed with sterile sea water and were fixed in 70% ethanol for taxonomic iden- PCR and real-time PCR detection of K. septempunctata in tification and molecular detection. marine invertebrates Species identification for all the collected invertebrate Polychaetes collected from the outflow waterway sedi- samples was conducted by morphological observations or ments of fish tank were examined for K. septempunctata PCR amplification of the mitochondrial cytochrome c oxi- by PCR and real-time PCR. DNA was extracted from the dase subunit I (mt COI) gene as described by Maturana et whole body of polychaetes using QIAamp DNA Mini al. (2011). For molecular identification, PCR primers tar- Kit (QIAGEN, USA) following manufacturer’s instruc- geting the partial mt COI gene described by Folmer et al. tions. The PCR and real-time PCR primers and amplifi- (1994) were used. LCO1490: 5′-GGTCAACAAATCAT- cation conditions used in this analysis are mentioned AAAGATATTGG-3′; HC02198: 5′-TAAACTTCA above. Prevalence of K. septempunctata was calculated GGGTGACCAAAAAATCA-3′ DNA was extracted from as the proportion of infected invertebrate host in the the polychaete samples using the QIAamp DNA Mini Kit whole number of studied host. PCR for detecting K. sep- with the protocol described previously, and PCR was car- tempunctata was also conducted for invertebrate sam- ried out in a 20 μl of reaction volume consisted of 10 μlof ples collected from the coastal regions using the same PCR premix (Bioneer, Korea), 1 μloftemplateDNA,1 μl protocol mentioned previously. of 10 μM of each primers and 17 μl of double-distilled de- ionized water. PCR cycling parameters were an initial de- Results naturation phase at 94 °C for 1 min, followed by 35 cycles Detection of K. septempuncta in rearing water samples by at 94 °C for 30 s, 49 °C for 55 s, and 72 °C for 90 s, and a PCR and real-time PCR final extension at 72 °C for 10 min. Following amplifica- During the sampling period, positive signals for K. sep- tion, PCR products were analysed in a 2% agarose gels tempunctata were not detected in any of the water sam- and stained with ethidium bromide. PCR products of the ples from two farms by PCR (Table 1). The standard expected size were purified using PCR gel purification kit curve for real-time PCR was derived from 10-fold serial (Bioneer, Korea). Gel-purified PCR amplicons were se- dilutions of different plasmid DNA concentrations ran- 8 1 quenced in both directions utilizing the same primers ging from 1 × 10 to 1 × 10 copies/μl, as described by used for initial amplification. The obtained sequences of Kawai et al. (2012). The assays were linear with R values Paari et al. Fisheries and Aquatic Sciences (2017) 20:16 Page 4 of 8 Table 1 Detection of K. septempunctata DNA in rearing water samples from fish farms by molecular methods Sampling date PCR Real-time PCR Ct value rDNA copy number Farm A Farm B Farm A Farm B Farm A Farm B 2014. May. –– 40.9, 41.1 40.1, 43.0 9.0, 7.7 16.3, 1.9 2014. Jun. –– ND, ND 42.2, 40.4 ND, ND 3.4, 13.1 2014. Jul. –– ND, ND 36.1, 38.3 ND, ND 3.2 × 10 , 62.5 2014. Aug. –– 42.2, ND ND, ND 3.5, ND ND, ND 2014. Sept. –– 41.4, 39.3 40.7, ND 6.2. 29.7 10.4, ND 2 3 2 2014. Oct. –– 36.3, 34.5 37.0, 37.9 2.8 × 10 , 1.1 × 10 1.6 × 10 , 87.5 2014. Nov. –– 40.0, 39.5 39.3, 39.8 18.3, 26.2 29.7, 20.4 of 0.993 (Fig. 1). Relative K. septempunctata DNA con- specimens were made with the intestinal segments of ran- centration was calculated based on the Ct value. The domly selected polychaetes, and microscopic observation amount of K. septempunctata DNA was inversely pro- was also conducted after Giesma and eosin staining, but portional to the Ct value obtained in this study, and the any actinosporean-like stage of K. septempunctata was Ct value of the highest standard (10 copies/μl) was 14.8 not found (data not shown). and the lowest standard (10 copies/μl) was 37.1. All of the polychaetes were identified using PCR. The level of rDNA copy number for all of the water Primers targeting mt COI gene amplified a PCR product samples fluctuated during the sampling period in both of size 710 bp, and the amplified sequences represent of the farms. The Ct value ranged from 36.3–42.2 in the species Naineris laevigata (Polychaeta, Orbiniidae) farm A and 36.1–42.2 in farm B (Table 1). The highest with 99.0% homology (data not shown). rDNA copy number (1.1 × 10 )of K. septempunctata in K. septempunctata DNA was detected in polychaetes by Farm A was recorded in October. In Farm B, the highest PCR and real-time PCR during the sampling period. PCR rDNA copy number was recorded 3.2 × 10 in August. detection of K. septempunctata in polychaete intestinal Interestingly, K. septempuctata DNA was not detectable sample showed the mean prevalence of 9.5% (55/578) during June–July in farm A and August in farm B. (Table 2). The highest prevalence of K. septempunctata in polychaetes by PCR (40.0%) was recorded in May of 2014, Incidence of K. septempunctata in marine invertebrate then, gradually decreased to 0% in August. samples collected from fish farms Quantitative analysis of K. septempunctata DNA in poly- Microscopic observation of the marine polychaetes col- chaetes samples revealed the parasitic DNA was detectable lected was conducted on a daily basis until they die. The only in May and June. The Ct value was 38.9–41.4 in May live polychaetes survived for 7 to 10 days in the well plate; and 35.5–38.3 in June. Although the incidence of PCR- however, no actinosporean release was observed from them during the incubation period. The squashed slide Table 2 PCR and real-time PCR results for detecting K. septempunctata in orbiniid polychaete N. laevigata isolated from outflow waterway of fish tank PCR Positive samples/ Real-time PCR tested samples (%) Ct value rDNA copy number/ body weight (g) 3 3 May 36/90 (40.0%) 38.9–41.4 2.5 × 10 –1.6 × 10 3 4 June 14/62 (22.6%) 35.5–38-3 2.0 × 10 –2.1 × 10 July 5/126 (4.0%) nd nd August 0/84 nd nd September 0/82 nd nd October 0/62 nd nd Fig. 1 Standard curve derived from 10-fold serially diluted plasmid November 0/72 nd nd DNA containing a partial 18s rDNA sequence of K. septempunctata. Ct values obtained in three technical replicates are presented as 55/578 (9.5%) mean ± standard deviations nd not detected Paari et al. Fisheries and Aquatic Sciences (2017) 20:16 Page 5 of 8 positive samples was higher in May, the rDNA copy num- However, information on the transmission biology of K. ber was higher in June (Table 2). septempunctata is still lacking as we are not aware of the alternate polychaete host to complete its life cycle or the Incidence of K. septempunctata in marine invertebrate transmission dynamics of K. septempunctata in marine samples collected from coastal area environment. In this study, we analysed rearing water Invertebrate samples collected during May to November samples from fish farms on a monthly basis for the pre- around the coastal area near olive flounder farm were sence of K. septempunctata by molecular methods. identified by microscopic observation at the lowest Since the discovery of the myxozoan life cycle by Wolf taxon level, and the results are summarized in Table 3. and Markiw (1984), a lot of fresh water myxozoans have All of the collected samples were negative for K. septem- been known to use freshwater oligochaetes as alternate punctata by PCR and not detected by real-time PCR. invertebrate hosts (Yokoyama et al. 2012). For marine myxozoans, however, polychaetes have been suggested Discussion as the best candidates for the alternative invertebrate Outbreaks due to consumption of raw olive flounder har- hosts; seven marine myxozoan life cycles have been eluci- bouring K. septempunctata have been reported in Japan dated at present time, and all of them are known to use since 2011 (Kawai et al. 2012; Harada et al. 2012). polychaetes as alternative invertebrate hosts (Karlsbakk and Køie 2012; Køie et al. 2004, 2007, 2008, 2013; Rangel et al. 2009), except for Ortholinea auratae using marine Table 3 Detection of K. septempunctata in marine invertebrates oligochaete as marine invertebrate host (Rangel et al. collected from coastal waters in this study 2015). Thus, we exclusively sampled marine invertebrates Family name Species name Number of individuals PCR from the sediment of outflow waterway of fish tank and examined around the fish farms, then investigated them by micro- Arabellidae Arabella iricola 4 – scopic observation and molecular analysis, to find the pos- Chrysopetalidae Chrysopetalum 1 – sible invertebrate hosts they use for transmission. Our occidentale investigations on myxospore infection were restricted for Cirratulidae Dodecaceria laddi 1 – few months due to adverse climate conditions. Eunicidae Eunice antennata 6 – Parasite density in aquatic environment is an impor- Lysidice collaris 2 – tant factor affecting the level of myxozoan outbreaks Euphrosinidae Euphrosine superba 3 – (Ray et al. 2012) because the transmission of actinos- Nereidae Nereis multignatha 4 – pores to teleost hosts occurs in aquatic environment. Real-time PCR has successfully detected the actinos- Nereis neoneanthes 1 – pores to measure parasite density in freshwater environ- Perinereis cultrifera 5 – ment (Hallet and Bartholomew 2009; Hallett et al. 2012; Platynereis 5 – True et al. 2009) but less frequently in marine environ- bicanaliculata ment. Alma-Bermejo et al. (2013) and Ishimaru et al. Opheliidae Polyophalmus pictus 3 – (2014) developed real-time PCR assay for detecting mar- Orbiniidae Naineris laevigata 5 – ine myxosporeans, Ceratomyxa puntazzi and Kudoa Phyllodocidae Eulalia virdis 1 – yasunagai in environmental sea water, respectively. They Notophyllum sp. 2 – found the seasonal changes in parasitic density and men- tioned that this may reflect the infection dynamics of Polynoidae Halosydna 19 – brevisetosa marine myxozoans. In a similar manner, K. septempunc- Harmothoe 3 – tata DNA was detected both in water samples of two imbricata farms examined in this study. Overall Ct value was Lepidonotus sp. 1 – 36.3–42.2 in farm A and 36.1–43.0 in farm B, corre- sponding 3.5 to 2.8 × 10 copies of K. septempunctata 18s Serpulidae Hydroides exalata 2 – rDNA from 2 l water samples in farm A and 3.0 to Spirobranchus 2 – tetraceros 3.2 × 10 copies in farm B, respectively (Table 1). These values are lower than those of K. yasunagai (Ishimaru et Syllidae Unidentified 1 11 – al. 2014) but higher than those of C. puntazzi (Alma-Ber- Unidentified 2 4 – mejo et al. 2013). These differences are thought to be due Trypanosyllis zebra 2 – to many factors including different infection dynamics be- Typosyllis 2 – tween the parasites and their hosts, different aquaculture ehlersioides systems, many physical and chemical factors in the aquatic Terebellidae Unidentified 3 – environments, as suggested by Ishimaru et al. (2014). In Paari et al. Fisheries and Aquatic Sciences (2017) 20:16 Page 6 of 8 particular, a flow-through system pumping seawater dir- plate method. Rangel et al. (2011) mentioned that the well- ectly from the open sea makes more difficult to under- plate method may not be suitable for relatively big marine stand the infection dynamics of K. septempunctata in polychaetes. Otherwise, different mechanisms may work olive flounder farms as because it has been suggested that for the release of the actinospores from the invertebrate the actinosporean stages are generally fragile and easy to hosts; Køie (2002) mentioned that the actinospores are re- be destroyed by strong water flow (Kerans and Zale 2002; leased via the gonopores of polychaetes, while Rangel et al. Hoz Franco and Budy 2004; Hallet and Bartholomew (2009) described that the actinospores are released to- 2007). Nevertheless, considering the sea water volume gether with the gametes by the rupture of the host’sbody flowing into the fish tanks, the parasite density calculated wall. If this is the case, it will be necessary to exclusively in this study cannot be negligible and should be consid- make squash preparation or histological sections to ob- ered for elucidating infection dynamics of K. septempunc- serve possible actinosporean stages of K. septempunctata. tata in olive flounder farms. PCR could successfully detect K. septempunctata DNA The level of K. septempunctata DNA in rearing waters in polychaetes in this study. The percentage of PCR fluctuated during the experimental period, but K. sep- positive individuals was the highest (40.0%) in May, then tempuctata DNA was not detected in June–July (farm A) gradually decreased during the experimental periods and or August (farm B). Similar results were obtained by maintained 0% after August of 2014. Seasonal prevalence Ishimaru et al. (2014) with K. yasunagai and Alama- of actinospores in invertebrate hosts has been conducted Bermejo et al. (2013) with C. punctzaaii,suggesting the in several experiments, but the seasonal variation pat- changes in parasite density in the water may be related to terns were different depending on the species examined the water temperature. But it is not clear if the parasite (Rangel et al. 2009, 2011). These are thought to reflect DNA detected from water in this study is originated from the seasonality of vertebrate host life cycle or inverte- actinospores from invertebrate hosts or from myxospores brate host life cycle for at least some myxozoans. In case from fish hosts, which is also indicated as the major short- of N. laevigata in this study, seasonal pattern of PCR- coming of their study by the authors mentioned above. In positive rate was also observed. However, this should be addition, our data have some limitations because we ana- carefully interpreted because PCR can detect both of lysed water samples only for a half year. Seasonality in the mature and developmental stages of actinospores. In prevalence has been reported in many myxosporean infec- general, the prevalence of actinosporean infection in tions (Al-Qahtani et al. 2015; Abdel-Baki et al. 2015), thus polychaetes is estimated by microscopic observation and sentinel fish exposure to water from the endemic area known to be very low (Rangel et al. 2009, 2011). Thus, it throughout the year and discovery of actinospores with would be helpful to detect any seasonal patterns in subsequent quantification of them in seawater are thought prevalence of actinosporean infection in polychaetes to be necessary, to prove the parasite DNA in seawater hosts by molecular method, but successful microscopic came from actinosporean stage of K. septempunctata. observation should be accompanied, which is also indis- Recently, Yokoyama et al. (2015) described that the K. pensable to make a clear conclusion whether N. laevi- septempunctata predominantly invade juvenile olive gata is the alternate host of K. septempunctata. flounder in July. In our study, K. septempunctata DNA in rearing water was not detected during June and July or in August, which is also indicating the infection may occur Conclusions during summer season and can be helpful for avoiding K. Myxozoan infections in wild and farmed fish is becoming septempunctata infection of olive flounder. increasingly important as marine aquaculture expands to All of the live polychaetes from fish farm sediments were meet the resource demands and some of them actually identified as Naineris laevigata (Polychaeta, Orbiniidae) by cause economic loss in aquaculture industry by causing PCR amplification of mt COI gene and maintained in 12- considerable mortality or losing market value of them. K. well plates for approximately 2 weeks, but no actinospor- septempunctata does not belong to any of two types men- ean stage were observed. Freshwater actinospores from tioned above because it does not cause any negative effect oligochaetes have been successfully observed by well plate on host but may affect human beings. Thus, effective con- method (Yokoyama et al. 1991, 2012). However, we could trol methods for K. septempunctata infection in olive not find any actinospores released from N. laevigata main- flounder are urgently needed. Based on the knowledge of tained in well plates in this study. Most of the actinospores the transmission biology, several methods have been sug- from marine polychaetes have been observed either by col- gested for controlling myxozoan infections and some of lecting coelomic fluid of marine polychaetes with syringe them have been proved to be effective. The information needles or squash preparations (Køie et al. 2008; Rangel et obtained in this study is thought to be helpful for estab- al. 2009, 2011) and our study was the first trial to observe lishing strategies to avoid K. septempunctata infection in the release of actinospores from marine polychaetes by well olive flounder farms. Paari et al. Fisheries and Aquatic Sciences (2017) 20:16 Page 7 of 8 Abbreviations Hallet SL, Bartholomew JL. Development and application of a duplex qPCR for Ct: Threshold cycle; mt COI: Mitochondrial cytochrome c oxidase subunit 1; river water samples to monitor the myxozoan parasite Parvicapsula PCR: Polymerase chain reaction minibicornis. Dis Aquat Org. 2009;86:36–50. Hallet SL, Bartholomew JL. Effects of water flow on the infection dynamics of Acknowledgements Myxobolus cerebralis. Parasitology. 2007;135:371–84. We thank Jong-Goo Choi for his help during the experiments. Hallett SL, Ray RA, Hurst CN, Buckles GR, Atkinson SD, Bartholomew JL. Density of the waterborne parasite Ceratomyxa shasta and its biological effects on Funding salmon. Appl Environ Microbiol. 2012;78:3724–31. This work was supported by grants from the National Institute of Fisheries Harada T, Kawai T, Jinnai M, Ohnishi T, Sugita-Konishi Y, Kumeda Y. Detection of Science (R2016065). Kudoa septempunctata 18S ribosomal DNA in patient fecal samples from novel food-borne outbreaks caused by consumption of raw olive flounder Author’s contributions (Paralichthy solivaceus). J Clin Microbiol. 2012;50:2964–8. JHK conceived and designed the experiments. JHK and AP prepared the Hoz Franco E, Budy P. Linking environmental heterogeneity to the distribution manuscript. AP and CHJ carried out sample collections and analyses. JHK, AP, and prevalence of Myxobolus cerebralis: a comparison across sites in a HSC, SHJ, and CHJ interpreted the results. All authors read and approved the Northern Utah water shed. Trans Am Fish Soc. 2004;133:1176–89. final manuscript. Ishimaru K, Matsuura T, Tsunemoto K, Shirakashi S. Seasonal monitoring of Kudoa yasunagai from sea water and aquaculture water using quantitative PCR. Dis Ethics approval and consent to participate Aquat Org. 2014;108:45–52. Not applicable. Iwashita Y, Kamijo Y, Nakahashi S, Shindo A, Yokoyama K, Yamamoto A, Omori Y, Ishikura K, Fujioka M, Hatada T, Takeda T, Maruyama K, Imai H. Food Consent for publication poisoning associated with Kudoa septempunctata. J. Emerg. Med. 2013; 44: Not applicable. 943–945. Karlsbakk E, Køie M. The marine myxosporean Sigmomyxa sphaerica (Thélohan, Competing interests 1895) gen. n., comb. n. (syn. Myxidium sphaericum) from garfish (Belone The authors declare that they have no competing interests. belone (L.)) uses the polychaete Nereis pelagica L. as invertebrate host. Parasitol. Res. 2012;110:211–218. Kawai T, Sekizuka T, Yahata Y, Kuroda M, Kumeda Y, Iijima Y, Kamata Y, Sugita- Publisher’sNote Konishi Y, Ohnishi T. 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Molecular detection of Kudoa septempunctata (Myxozoa: Multivalvulida) in sea water and marine invertebrates

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Springer Journals
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Copyright © 2017 by The Author(s)
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Life Sciences; Fish & Wildlife Biology & Management; Marine & Freshwater Sciences; Zoology; Animal Ecology
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2234-1757
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10.1186/s41240-017-0062-z
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Abstract

The exportation of cultured olive flounder (Paralichthys olivaceus) in Korea has been recently decreasing due to the infections with a myxozoan parasite Kudoa septempunctata, and there is a strong demand for strict food safety management because the food poisoning associated with consumption of raw olive flounder harbouring K. septempunctata has been frequently reported in Japan. The life cycle and infection dynamics of K. septempunctata in aquatic environment are currently unknown, which hamper establishment of effective control methods. We investigated sea water and marine invertebrates collected from olive flounder farms for detecting K. septempunctata by DNA-based analysis, to elucidate infection dynamics of K. septempunctata in aquaculture farms. In addition, live marine polychaetes were collected and maintained in well plates to find any possible actinosporean state of K. septempunctata.The levelof K. septempunctata DNA in rearing water fluctuated during the sampling period but the DNA was not detected in summer (June–July in farm A and August in farm B). K. septempunctata DNA was also detected in the polychaetes Naineris laevigata intestinal samples, showing decreased pattern of 40 to 0%. No actinosporean stage of K. septempunctata was observed in the polychaetes by microscopy. The absence of K. septempunctata DNA inrearing wateroffishfarm and the polychaetes N. laevigata intestinal samples during late spring and early summer indicate that the infection may not occur during this period. N. laevigata was suspected as the possible alternate invertebrate host of K. septempunctata, but the actinosporean stage was not found by well plate method and further studies will be necessary. This research provides important baseline information for understanding the infection dynamics of K. septempunctata in olive flounder farms and further establishment of control strategies. Keywords: Kudoa septempunctata, Myxozoa, Olive flounder, Paralichthys olivaceus, Naineris laevigata Background Adlard 2012). Most of the species are histozoic which Myxozoans belong to the group of metazoan parasites of develop symptoms of macroscopic whitish cyst or cause fish and act as a cause for several outbreaks in both fresh post-mortem myoliquefaction (Shirakashi et al. 2012). water and marine fish (Canning and Okamura 2003). However, some Kudoa species do not cause any of the Disease transmission by these myxozoan parasites can symptoms mentioned above and Kudoa septempunctata, often have disastrous economic impact in aquaculture a newly found myxosporean found in olive flounder industries, although most of them are known to have in- (Paralichthys olivaceus) is probably the most well-known significant or negligible effect in fish (Yokoyama et al. example of them (Yokoyama et al. 2004; Matsukane et 2012). The genus Kudoa comprises more than 70 species al. 2010) . reported from a broad range of fish host (Miller and Since 2011, food poisoning due to ingestion of farmed olive flounders in Japan has been reported (Kawai et al. * Correspondence: jhkim70@gwnu.ac.kr 2012). Epidemiological studies have revealed that this Department of Marine Bioscience, Gangneung-Wonju National University, outbreak is associated with the presence of K. septem- Gangneung, Gangwon 25457, South Korea punctata in the causative foods (Kawai et al. 2012) and Department of Marine Bioscience, Gangneung-Wonju National University, Gangneung, Gangwon 210-702, South Korea the food-borne outbreaks associated with consumption Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Paari et al. Fisheries and Aquatic Sciences (2017) 20:16 Page 2 of 8 of raw olive flounder harbouring K. septempunctata are septempunctata infection occurs (farm A, B). Sampling becoming a prominent public health concern in Japan. was conducted on a monthly basis during May and As the customs of consuming raw fish is spreading, the November 2014 and approximately 2 l of water was occurrence of this food-borne disease is predicted to in- collected in each sampling. Water samples were fil- crease (Harada et al. 2012). Although there have been tered through a fresh nitrocellulose membrane filter outbreaks in Japan since 2011, the question of olive (5 μm pore size; ADVENTEC, Japan) using a suction flounder in Korea acting as a host of K. septempunctata pump (DOA-P704-AA, GAST, USA), and the mem- remains unanswered (Iwashita et al. 2013). Considering brane filter were placed in an individual micro centri- the commercial value of olive flounder and the public fuge tube andstoredat −20 °C until DNA extraction. health concern, it is urgently needed to solve the nega- Acetone dissolution method was used for extracting tive impact of this parasite on public health and food DNA from the filter samples (Hallett et al. 2012). The safety, but almost nothing is known about its transmis- membrane filter in microcentrifuge tube was air dried sion biology, infection dynamics in aquatic environment. and dissolved by adding 2 ml of acetone (Cica reagent, Myxozoan parasites have been believed to be transmit- Japan). The completely solubilized filter components by ted from fish to fish until Wolf and Markiw (1984) de- repeated vortexing were then centrifuged at 3000g for monstrated that freshwater oligochaete was essential for 15 min, and the supernatant was discarded. This step the transmission of Myxobolus cerebralis and since then, was repeated twice to ensure complete dissolution of fil- many studies have confirmed that some myxozoans trate particles from the dissolved materials. To the dis- undergo two-host life cycle (Lom and Dykova 2006; solved filtrate samples, 1 ml of 95% ethanol was added Markussen et al. 2015). Currently, more than 30 fresh- and thoroughly mixed. The suspended pellet after cen- water myxozoans are known to have two-host life cycles trifugation was then air dried and used directly for (Yokoyoma et al. 2012), but only 7 marine myxozoans DNA extraction. are found to have marine invertebrates to complete their DNA was extracted by using a QIAamp DNA Mini life cycles (Karlsbakk and Køie 2012; Køie et al. 2004, Kit (QIAGEN, USA) according to the manufacturer’s 2007, 2008, 2013; Rangel et al. 2015), and neither life instructions with slight modifications. Briefly, 180 μlof cycle nor invertebrate alternate hosts have been eluci- tissue lysis buffer (Buffer AE, QIAGEN, USA) was added dated in kudoid myxozoans. to the air-dried pellet sample, and then 20 μl of Proteinase Environmental water analysis is indispensable for in- K (QIAGEN, USA) was added. Following overnight incu- vestigating epidemiology of myxozoan infections because bation, wash buffers (Buffer AW1, AW2, QIAGEN, USA) fish myxozoans occur in aquatic environment and trans- were added and eluted using elution buffer (Buffer AE, mission between two different hosts also occur in envi- QIAGEN, USA). Extracted DNA was stored at −20 °C ronmental water. Many studies revealed that the disease until used for PCR detection. transmission occurs via water in endemic area and ap- propriate water treatments were effective for the man- PCR and real-time PCR for detecting K. septempunctata in agement of several myxozoan infections (Cobcroft and rearing water Battaglene 2013; Nehring et al. 2003; Yanagida et al. PCR was carried out for detecting K. septempunctata in 2006). Thus, the environmental water analysis would be water samples using the following set of primer sets: the first step to clarify infection dynamics and develop Ksf-GTGTGTGATCAGACTTGATATG; KsR-AAGCCA further effective management strategy for K. septem- AAACTGCTGGCCATTT [25]. 0.5 μM of forward and punctata infection. As knowledge about the infection reverse primer, 1 μl of template DNA was added to the dynamics of this parasite is scarce, we carried out a PCR premix tube (Bioneer, Korea) and the total volume monthly inspection of water samples to study the occur- was made to 20 μl using ultra-pure distilled water rence pattern of K. septempunctata in aquatic environ- (Invitrogen, USA). PCR cycling parameters followed ment by molecular analysis. We also investigated the the protocols of Grabner et al. (2012) with some minor prevalence of K. septempunctata in marine polychaetes modifications. PCR cycling parameters were an initial collected around the farms using both of well plate denaturation at 95 °C for 4 min, followed by 35 cycles method described by Yokoyama et al. (1991) and mo- at 95 °C for 35 s, 56 °C for 30 s and 72 °C for 30 s and lecular analysis, to speculate the possible life cycle of K. ended with a final extension at 72 °C for 7 min. septempunctata. Real-time PCR was carried out using the following sequence of primers and probe; F-CATGGGATTAGCCC Methods GGTTTA; R-ACTCTCCCCAAAGCCGAAA; P-(FAM)- Water sampling and DNA extraction TCCAGGTTGGGCCCTCAGTGAAAA (Kawai et al. Water samples were directly collected from the inlet 2012). Real-time PCR was carried out in a 0.2-ml PCR pumping units from two aquaculture farms where K. strip tube containing 2× Premix Ex Taq (Takara, Japan) Paari et al. Fisheries and Aquatic Sciences (2017) 20:16 Page 3 of 8 10 μl, primer (0.4 uM, Bioneer, Korea), probe (0.25 μM, mt COI gene were aligned and identified based on the Bioneer, Korea), ROX II reference dye (Takara, Japan), 4 μl percentage identity of nucleotide sequences previously template DNA using ABI 7500 Fast Real-time PCR system registered in NCBI. (Applied Biosystems, USA). Cycling parameters were preheating at 95 °C for 10 min, followed by 45 cycles Microscopic detection of K. septempunctata from at 95 °C for 15 s, 60 °C for 1 min and the analyses polychaetes were conducted twice. Live polychaetes collected from the outflow waterway sediments of fish tank were observed for the occurrence Collection of marine invertebrates and species of actinospores by well plate method (Yokoyama et al. identification 1991). Briefly, collected polychaetes were washed with Marine invertebrates from the sediments of fish tanks sterile sea water several times, individually placed in a and coastal areas near fish farms were collected to inves- 12-well plate and observed microscopically using an tigate the prevalence of K. septempunctata infection. For inverted microscope (Leica, Germany) every day to find collecting invertebrate samples, mud was collected from if the possible K. septempunctata actinospores were re- approximately 0.5 m depth from outflow waterway of leased. Sea water in the wells was replaced in a 2-day the same fish farms where the water samples were col- interval. Squash preparations were also made from the lected and transferred to laboratory. Sediments were isolated polychaetes samples at every 2 days interval for sieved through a mesh (0.5 mm) to separate marine observation of the actinosporeans; several posterior seg- polychaetes within a day of collection of sediment. Live ments of the intestinal region of polychaetes were polychaetes were collected, washed with sterile sea water squashed between the slide and cover slip, fixed with several times, and then maintained in a 12-well plate at methanol, stained with Giemsa solutions and examined 15 °C, for observing the possible actinosporean stages of using a light microscope (Leica, Germany). K. septempunctata. Invertebrate samples collected around the coastal re- For collecting invertebrate samples from coastal areas, gions were not maintained in well plates but imme- quadrats and dredging devices were used by trained di- diately processed for microscopic analysis using squash vers to obtain marine invertebrates near the olive floun- preparations and for PCR detection using the same der farms. Subsamples of invertebrate samples collected methodology mentioned above. from the gravel materials were washed with sterile sea water and were fixed in 70% ethanol for taxonomic iden- PCR and real-time PCR detection of K. septempunctata in tification and molecular detection. marine invertebrates Species identification for all the collected invertebrate Polychaetes collected from the outflow waterway sedi- samples was conducted by morphological observations or ments of fish tank were examined for K. septempunctata PCR amplification of the mitochondrial cytochrome c oxi- by PCR and real-time PCR. DNA was extracted from the dase subunit I (mt COI) gene as described by Maturana et whole body of polychaetes using QIAamp DNA Mini al. (2011). For molecular identification, PCR primers tar- Kit (QIAGEN, USA) following manufacturer’s instruc- geting the partial mt COI gene described by Folmer et al. tions. The PCR and real-time PCR primers and amplifi- (1994) were used. LCO1490: 5′-GGTCAACAAATCAT- cation conditions used in this analysis are mentioned AAAGATATTGG-3′; HC02198: 5′-TAAACTTCA above. Prevalence of K. septempunctata was calculated GGGTGACCAAAAAATCA-3′ DNA was extracted from as the proportion of infected invertebrate host in the the polychaete samples using the QIAamp DNA Mini Kit whole number of studied host. PCR for detecting K. sep- with the protocol described previously, and PCR was car- tempunctata was also conducted for invertebrate sam- ried out in a 20 μl of reaction volume consisted of 10 μlof ples collected from the coastal regions using the same PCR premix (Bioneer, Korea), 1 μloftemplateDNA,1 μl protocol mentioned previously. of 10 μM of each primers and 17 μl of double-distilled de- ionized water. PCR cycling parameters were an initial de- Results naturation phase at 94 °C for 1 min, followed by 35 cycles Detection of K. septempuncta in rearing water samples by at 94 °C for 30 s, 49 °C for 55 s, and 72 °C for 90 s, and a PCR and real-time PCR final extension at 72 °C for 10 min. Following amplifica- During the sampling period, positive signals for K. sep- tion, PCR products were analysed in a 2% agarose gels tempunctata were not detected in any of the water sam- and stained with ethidium bromide. PCR products of the ples from two farms by PCR (Table 1). The standard expected size were purified using PCR gel purification kit curve for real-time PCR was derived from 10-fold serial (Bioneer, Korea). Gel-purified PCR amplicons were se- dilutions of different plasmid DNA concentrations ran- 8 1 quenced in both directions utilizing the same primers ging from 1 × 10 to 1 × 10 copies/μl, as described by used for initial amplification. The obtained sequences of Kawai et al. (2012). The assays were linear with R values Paari et al. Fisheries and Aquatic Sciences (2017) 20:16 Page 4 of 8 Table 1 Detection of K. septempunctata DNA in rearing water samples from fish farms by molecular methods Sampling date PCR Real-time PCR Ct value rDNA copy number Farm A Farm B Farm A Farm B Farm A Farm B 2014. May. –– 40.9, 41.1 40.1, 43.0 9.0, 7.7 16.3, 1.9 2014. Jun. –– ND, ND 42.2, 40.4 ND, ND 3.4, 13.1 2014. Jul. –– ND, ND 36.1, 38.3 ND, ND 3.2 × 10 , 62.5 2014. Aug. –– 42.2, ND ND, ND 3.5, ND ND, ND 2014. Sept. –– 41.4, 39.3 40.7, ND 6.2. 29.7 10.4, ND 2 3 2 2014. Oct. –– 36.3, 34.5 37.0, 37.9 2.8 × 10 , 1.1 × 10 1.6 × 10 , 87.5 2014. Nov. –– 40.0, 39.5 39.3, 39.8 18.3, 26.2 29.7, 20.4 of 0.993 (Fig. 1). Relative K. septempunctata DNA con- specimens were made with the intestinal segments of ran- centration was calculated based on the Ct value. The domly selected polychaetes, and microscopic observation amount of K. septempunctata DNA was inversely pro- was also conducted after Giesma and eosin staining, but portional to the Ct value obtained in this study, and the any actinosporean-like stage of K. septempunctata was Ct value of the highest standard (10 copies/μl) was 14.8 not found (data not shown). and the lowest standard (10 copies/μl) was 37.1. All of the polychaetes were identified using PCR. The level of rDNA copy number for all of the water Primers targeting mt COI gene amplified a PCR product samples fluctuated during the sampling period in both of size 710 bp, and the amplified sequences represent of the farms. The Ct value ranged from 36.3–42.2 in the species Naineris laevigata (Polychaeta, Orbiniidae) farm A and 36.1–42.2 in farm B (Table 1). The highest with 99.0% homology (data not shown). rDNA copy number (1.1 × 10 )of K. septempunctata in K. septempunctata DNA was detected in polychaetes by Farm A was recorded in October. In Farm B, the highest PCR and real-time PCR during the sampling period. PCR rDNA copy number was recorded 3.2 × 10 in August. detection of K. septempunctata in polychaete intestinal Interestingly, K. septempuctata DNA was not detectable sample showed the mean prevalence of 9.5% (55/578) during June–July in farm A and August in farm B. (Table 2). The highest prevalence of K. septempunctata in polychaetes by PCR (40.0%) was recorded in May of 2014, Incidence of K. septempunctata in marine invertebrate then, gradually decreased to 0% in August. samples collected from fish farms Quantitative analysis of K. septempunctata DNA in poly- Microscopic observation of the marine polychaetes col- chaetes samples revealed the parasitic DNA was detectable lected was conducted on a daily basis until they die. The only in May and June. The Ct value was 38.9–41.4 in May live polychaetes survived for 7 to 10 days in the well plate; and 35.5–38.3 in June. Although the incidence of PCR- however, no actinosporean release was observed from them during the incubation period. The squashed slide Table 2 PCR and real-time PCR results for detecting K. septempunctata in orbiniid polychaete N. laevigata isolated from outflow waterway of fish tank PCR Positive samples/ Real-time PCR tested samples (%) Ct value rDNA copy number/ body weight (g) 3 3 May 36/90 (40.0%) 38.9–41.4 2.5 × 10 –1.6 × 10 3 4 June 14/62 (22.6%) 35.5–38-3 2.0 × 10 –2.1 × 10 July 5/126 (4.0%) nd nd August 0/84 nd nd September 0/82 nd nd October 0/62 nd nd Fig. 1 Standard curve derived from 10-fold serially diluted plasmid November 0/72 nd nd DNA containing a partial 18s rDNA sequence of K. septempunctata. Ct values obtained in three technical replicates are presented as 55/578 (9.5%) mean ± standard deviations nd not detected Paari et al. Fisheries and Aquatic Sciences (2017) 20:16 Page 5 of 8 positive samples was higher in May, the rDNA copy num- However, information on the transmission biology of K. ber was higher in June (Table 2). septempunctata is still lacking as we are not aware of the alternate polychaete host to complete its life cycle or the Incidence of K. septempunctata in marine invertebrate transmission dynamics of K. septempunctata in marine samples collected from coastal area environment. In this study, we analysed rearing water Invertebrate samples collected during May to November samples from fish farms on a monthly basis for the pre- around the coastal area near olive flounder farm were sence of K. septempunctata by molecular methods. identified by microscopic observation at the lowest Since the discovery of the myxozoan life cycle by Wolf taxon level, and the results are summarized in Table 3. and Markiw (1984), a lot of fresh water myxozoans have All of the collected samples were negative for K. septem- been known to use freshwater oligochaetes as alternate punctata by PCR and not detected by real-time PCR. invertebrate hosts (Yokoyama et al. 2012). For marine myxozoans, however, polychaetes have been suggested Discussion as the best candidates for the alternative invertebrate Outbreaks due to consumption of raw olive flounder har- hosts; seven marine myxozoan life cycles have been eluci- bouring K. septempunctata have been reported in Japan dated at present time, and all of them are known to use since 2011 (Kawai et al. 2012; Harada et al. 2012). polychaetes as alternative invertebrate hosts (Karlsbakk and Køie 2012; Køie et al. 2004, 2007, 2008, 2013; Rangel et al. 2009), except for Ortholinea auratae using marine Table 3 Detection of K. septempunctata in marine invertebrates oligochaete as marine invertebrate host (Rangel et al. collected from coastal waters in this study 2015). Thus, we exclusively sampled marine invertebrates Family name Species name Number of individuals PCR from the sediment of outflow waterway of fish tank and examined around the fish farms, then investigated them by micro- Arabellidae Arabella iricola 4 – scopic observation and molecular analysis, to find the pos- Chrysopetalidae Chrysopetalum 1 – sible invertebrate hosts they use for transmission. Our occidentale investigations on myxospore infection were restricted for Cirratulidae Dodecaceria laddi 1 – few months due to adverse climate conditions. Eunicidae Eunice antennata 6 – Parasite density in aquatic environment is an impor- Lysidice collaris 2 – tant factor affecting the level of myxozoan outbreaks Euphrosinidae Euphrosine superba 3 – (Ray et al. 2012) because the transmission of actinos- Nereidae Nereis multignatha 4 – pores to teleost hosts occurs in aquatic environment. Real-time PCR has successfully detected the actinos- Nereis neoneanthes 1 – pores to measure parasite density in freshwater environ- Perinereis cultrifera 5 – ment (Hallet and Bartholomew 2009; Hallett et al. 2012; Platynereis 5 – True et al. 2009) but less frequently in marine environ- bicanaliculata ment. Alma-Bermejo et al. (2013) and Ishimaru et al. Opheliidae Polyophalmus pictus 3 – (2014) developed real-time PCR assay for detecting mar- Orbiniidae Naineris laevigata 5 – ine myxosporeans, Ceratomyxa puntazzi and Kudoa Phyllodocidae Eulalia virdis 1 – yasunagai in environmental sea water, respectively. They Notophyllum sp. 2 – found the seasonal changes in parasitic density and men- tioned that this may reflect the infection dynamics of Polynoidae Halosydna 19 – brevisetosa marine myxozoans. In a similar manner, K. septempunc- Harmothoe 3 – tata DNA was detected both in water samples of two imbricata farms examined in this study. Overall Ct value was Lepidonotus sp. 1 – 36.3–42.2 in farm A and 36.1–43.0 in farm B, corre- sponding 3.5 to 2.8 × 10 copies of K. septempunctata 18s Serpulidae Hydroides exalata 2 – rDNA from 2 l water samples in farm A and 3.0 to Spirobranchus 2 – tetraceros 3.2 × 10 copies in farm B, respectively (Table 1). These values are lower than those of K. yasunagai (Ishimaru et Syllidae Unidentified 1 11 – al. 2014) but higher than those of C. puntazzi (Alma-Ber- Unidentified 2 4 – mejo et al. 2013). These differences are thought to be due Trypanosyllis zebra 2 – to many factors including different infection dynamics be- Typosyllis 2 – tween the parasites and their hosts, different aquaculture ehlersioides systems, many physical and chemical factors in the aquatic Terebellidae Unidentified 3 – environments, as suggested by Ishimaru et al. (2014). In Paari et al. Fisheries and Aquatic Sciences (2017) 20:16 Page 6 of 8 particular, a flow-through system pumping seawater dir- plate method. Rangel et al. (2011) mentioned that the well- ectly from the open sea makes more difficult to under- plate method may not be suitable for relatively big marine stand the infection dynamics of K. septempunctata in polychaetes. Otherwise, different mechanisms may work olive flounder farms as because it has been suggested that for the release of the actinospores from the invertebrate the actinosporean stages are generally fragile and easy to hosts; Køie (2002) mentioned that the actinospores are re- be destroyed by strong water flow (Kerans and Zale 2002; leased via the gonopores of polychaetes, while Rangel et al. Hoz Franco and Budy 2004; Hallet and Bartholomew (2009) described that the actinospores are released to- 2007). Nevertheless, considering the sea water volume gether with the gametes by the rupture of the host’sbody flowing into the fish tanks, the parasite density calculated wall. If this is the case, it will be necessary to exclusively in this study cannot be negligible and should be consid- make squash preparation or histological sections to ob- ered for elucidating infection dynamics of K. septempunc- serve possible actinosporean stages of K. septempunctata. tata in olive flounder farms. PCR could successfully detect K. septempunctata DNA The level of K. septempunctata DNA in rearing waters in polychaetes in this study. The percentage of PCR fluctuated during the experimental period, but K. sep- positive individuals was the highest (40.0%) in May, then tempuctata DNA was not detected in June–July (farm A) gradually decreased during the experimental periods and or August (farm B). Similar results were obtained by maintained 0% after August of 2014. Seasonal prevalence Ishimaru et al. (2014) with K. yasunagai and Alama- of actinospores in invertebrate hosts has been conducted Bermejo et al. (2013) with C. punctzaaii,suggesting the in several experiments, but the seasonal variation pat- changes in parasite density in the water may be related to terns were different depending on the species examined the water temperature. But it is not clear if the parasite (Rangel et al. 2009, 2011). These are thought to reflect DNA detected from water in this study is originated from the seasonality of vertebrate host life cycle or inverte- actinospores from invertebrate hosts or from myxospores brate host life cycle for at least some myxozoans. In case from fish hosts, which is also indicated as the major short- of N. laevigata in this study, seasonal pattern of PCR- coming of their study by the authors mentioned above. In positive rate was also observed. However, this should be addition, our data have some limitations because we ana- carefully interpreted because PCR can detect both of lysed water samples only for a half year. Seasonality in the mature and developmental stages of actinospores. In prevalence has been reported in many myxosporean infec- general, the prevalence of actinosporean infection in tions (Al-Qahtani et al. 2015; Abdel-Baki et al. 2015), thus polychaetes is estimated by microscopic observation and sentinel fish exposure to water from the endemic area known to be very low (Rangel et al. 2009, 2011). Thus, it throughout the year and discovery of actinospores with would be helpful to detect any seasonal patterns in subsequent quantification of them in seawater are thought prevalence of actinosporean infection in polychaetes to be necessary, to prove the parasite DNA in seawater hosts by molecular method, but successful microscopic came from actinosporean stage of K. septempunctata. observation should be accompanied, which is also indis- Recently, Yokoyama et al. (2015) described that the K. pensable to make a clear conclusion whether N. laevi- septempunctata predominantly invade juvenile olive gata is the alternate host of K. septempunctata. flounder in July. In our study, K. septempunctata DNA in rearing water was not detected during June and July or in August, which is also indicating the infection may occur Conclusions during summer season and can be helpful for avoiding K. Myxozoan infections in wild and farmed fish is becoming septempunctata infection of olive flounder. increasingly important as marine aquaculture expands to All of the live polychaetes from fish farm sediments were meet the resource demands and some of them actually identified as Naineris laevigata (Polychaeta, Orbiniidae) by cause economic loss in aquaculture industry by causing PCR amplification of mt COI gene and maintained in 12- considerable mortality or losing market value of them. K. well plates for approximately 2 weeks, but no actinospor- septempunctata does not belong to any of two types men- ean stage were observed. Freshwater actinospores from tioned above because it does not cause any negative effect oligochaetes have been successfully observed by well plate on host but may affect human beings. Thus, effective con- method (Yokoyama et al. 1991, 2012). However, we could trol methods for K. septempunctata infection in olive not find any actinospores released from N. laevigata main- flounder are urgently needed. Based on the knowledge of tained in well plates in this study. Most of the actinospores the transmission biology, several methods have been sug- from marine polychaetes have been observed either by col- gested for controlling myxozoan infections and some of lecting coelomic fluid of marine polychaetes with syringe them have been proved to be effective. The information needles or squash preparations (Køie et al. 2008; Rangel et obtained in this study is thought to be helpful for estab- al. 2009, 2011) and our study was the first trial to observe lishing strategies to avoid K. septempunctata infection in the release of actinospores from marine polychaetes by well olive flounder farms. Paari et al. Fisheries and Aquatic Sciences (2017) 20:16 Page 7 of 8 Abbreviations Hallet SL, Bartholomew JL. Development and application of a duplex qPCR for Ct: Threshold cycle; mt COI: Mitochondrial cytochrome c oxidase subunit 1; river water samples to monitor the myxozoan parasite Parvicapsula PCR: Polymerase chain reaction minibicornis. Dis Aquat Org. 2009;86:36–50. Hallet SL, Bartholomew JL. Effects of water flow on the infection dynamics of Acknowledgements Myxobolus cerebralis. Parasitology. 2007;135:371–84. We thank Jong-Goo Choi for his help during the experiments. Hallett SL, Ray RA, Hurst CN, Buckles GR, Atkinson SD, Bartholomew JL. Density of the waterborne parasite Ceratomyxa shasta and its biological effects on Funding salmon. Appl Environ Microbiol. 2012;78:3724–31. This work was supported by grants from the National Institute of Fisheries Harada T, Kawai T, Jinnai M, Ohnishi T, Sugita-Konishi Y, Kumeda Y. Detection of Science (R2016065). Kudoa septempunctata 18S ribosomal DNA in patient fecal samples from novel food-borne outbreaks caused by consumption of raw olive flounder Author’s contributions (Paralichthy solivaceus). J Clin Microbiol. 2012;50:2964–8. JHK conceived and designed the experiments. JHK and AP prepared the Hoz Franco E, Budy P. Linking environmental heterogeneity to the distribution manuscript. AP and CHJ carried out sample collections and analyses. JHK, AP, and prevalence of Myxobolus cerebralis: a comparison across sites in a HSC, SHJ, and CHJ interpreted the results. All authors read and approved the Northern Utah water shed. Trans Am Fish Soc. 2004;133:1176–89. final manuscript. 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