Lectin histochemistry of Kudoa septempunctata genotype ST3-infected muscle of olive flounder (Paralichthys olivaceus)

The localization of carbohydrate terminals in Kudoa septempunctata ST3-infected muscle of olive flounder (Paralichthys olivaceus) was investigated using lectin histochemistry to determine the types of carbohydrate sugar residues expressed in Kudoa spores. Twenty-one lectins were examined, i.e., N-acetylglucosamine (s-WGA, WGA, DSL-II, DSL, LEL, STL), mannose (Con A, LCA, PSA), galactose/N-acetylgalactosamine (RCA12, BSL-I, VVA, DBA, SBA, SJA, Jacalin, PNA, ECL), complex type N-glycans (PHA-E and PHA-L), and fucose (UEA-I). Spores encased by a plasmodial membrane were labeled for the majority of these lectins, with the exception of LCA, PSA, PNA, and PHA-L. Four lectins (RCA 120, BSL-I, DBA, and SJA) belonging to the galactose/N-acetylgalactosamine group, only labeled spores, but not the plasmodial membrane. This is the first confirmation that various sugar residues are present in spores and plasmodial membranes of K. septempunctata ST3.


Introduction
Kudoa septempunctata Matsukane et al., 2010 [9], a myxosporean species of the order Multivalvulida, has been identified in the trunk muscle of aquacultured olive flounder (Paralichthys olivaceus), occasionally causing food poisoning in Japan [8,9]. Kudoa septempunctata-infected raw olive flounder fillets have occasionally reached diners' tables because the infection was not grossly identified. Even though the life cycle of K. septempunctata has not been clarified within or outside of olive flounders, it has been reported that Kudoa species is maintained between oligochaete and fish [18]. After infection of fish by Kudoa species, it is suggested that they move to the tissues of preference and develop into a plasmodium [1,3].
Kudoa septempunctata spores are composed of six or seven shell valves and polar capsules [9], which are genetically classified into three groups, i.e. ST1, ST2, and ST3. Both ST1 and ST2 are common in Japan, while ST3 is dominant in the Republic of Korea [17]. Despite the distinct genetic differences among K. septempunctata, infected tissues show similar parasite presentation in that spores develop within pseudocysts in the muscle fibers of infected flat fish [11]. However, no inflammatory lesions are found either in or around infected muscle fibers.
Carbohydrate residues detected by lectin histochemistry are widely localized on epithelial cells in flat fish, where they play important roles in protecting the organism [6]. It was recently reported that carbohydrate residues are involved in protecting the organism against the environment and protecting parasites from the host as decoys for host immune cells [15]. Thus, carbohydrate residues are important factors for the interaction between host cells and parasites participating in the adhesion and penetration of parasites [13]. Carbohydrate residues on the spores of myxozoan parasites (Myxobolus cerebralis) have been characterized through lectin histochemistry [7]. A previous study using a limited number of lectins (WGA, SBA, BS-I, Con A, UEA-I, and SNA) in several myxosporean parasite infections showed that lectin reactivity revealed different binding patterns [10], which would be useful in diagnostic studies [10].
The present study was performed to evaluate the characteristics of carbohydrate residues on spores of K. septempunctata genotype ST3 in infected muscle of cultured olive flounder, an abundant genotype in Korea.

Sample collection
Olive flounder (Paralichthys olivaceus) is one of the most important aquaculture fish in Korea. Olive flounder has been cultured in 10 ton flow-through land-tanks at 22°C ± 1 under a natural photoperiod. Supplemental aerations were provided to maintain dissolved oxygen levels near 7.0 ± 0.5 ppm and the salinity was 32 ± 1 ppt. Kudoa septempunctata-infected fish were periodically screened by microscopic examination of crude suspensions of muscle tissue at 400· magnification. To screen for parasite infection, fish were anesthetized in buffered 3-aminobenzoic acid ethyl ester methanesulfonate (Sigma). Kudoa septempunctata infection in flat fish was further confirmed by histological examination, as reported previously [2].

Histological studies
Muscle samples of K. septempunctata-infected fish were fixed in 10% neutral buffered formalin and processed for embedding in paraffin. The paraffin-embedded tissues were cut into sections at a thickness of 5 lm using a rotary microtome (Leica, Nussloch, Germany). The tissue sections were stained with hematoxylin and eosin for routine histopathological examination. The histological findings were reported in our previous report [2], and some of the samples diagnosed by PCR were also used for lectin histochemistry in the present study.
Briefly, as in our previous study [12], the paraffinembedded muscles were cut into 5 lm thick sections using a microtome. The sections were mounted on glass microscope slides, the paraffin was removed, and the sections were rehydrated. Endogenous peroxidase activity was blocked using 0.3% hydrogen peroxide in methanol for 30 minutes. After three washes with phosphate-buffered saline (PBS), the sections were incubated with 1% bovine serum albumin to block nonspecific binding. The sections were rinsed with PBS and incubated with the avidin-biotin-complex (ABC) method using 21 biotinylated lectins (Table 1) from the lectin screening kits I-III (Vector Laboratories) at 4°C overnight.
Horseradish peroxidase (HRP) was developed using a diaminobenzidine substrate kit (DAB Kit; Vector Laboratories). The sections were counterstained with hematoxylin before mounting. Negative controls for the lectin histochemistry included (1) omission of primary reagent (biotinylated lectins) and (2) preincubation of the lectins with the appropriate inhibitors (0.2 M-0.5 M in Tris buffer) for 1 hour at room temperature, as shown in a previous paper [7]. The intensities of the lectin-binding patterns on the slides were arbitrarily scored blind by three researchers as follows: À negative, ± occasionally weakly positive, + some, but not all, positive, ++ moderately positive, and +++ very strongly positive.

Results and discussion
Histological examination and PCR genotyping Muscles of olive flounder infected with Kudoa spp. showed sarcoplasmic infection with formation of pseudocysts.
The infected muscle fibers were hypertrophied as shown in our previous report [2]. PCR analysis of the two mitochondrial genes cox 1 and rnl of the K. septempunctata resulted in amplification of 751 bp (Fig. 1, lane 2-4) and 817 bp fragments (Fig. 1, lane 8-10), respectively, matching with the results of histopathology. The obtained gene sequences were subjected to multiple sequence alignment using ClustalW (http://www.clustal.org). Aligned fragments showed high sequence similarity (100%) with the type strains LC014799 and AB915832, which revealed that the isolated K. septempunctata belonged to the ST3 genotype [2].
In the present study, we used a more diverse range of lectins (Table 1). Lectin reactivity was scored on spores and plasmodial membranes, but specific discrimination of polar capsules and valves of spores was not performed because of the limitations of identification by light microscopy. In addition, we did not discriminate between mature type spores and immature sporoblasts because both types were present in the same pseudocyst.
In K. septempunctata-infected muscles, the majority of lectins, except LCA, PSA, PNA, and PHA-L, were positive in spores, while lectin reactivity on the plasmodial membrane encasing clusters of spores mostly matched those of spores with varying intensities (Figs. 2-4). These findings suggested that spores contain a variety of carbohydrate groups on their surfaces, including N-acetylglucosamine (Fig. 2), mannose (Fig. 2), galactose/N-acetylgalactosamine (Figs. 3 and 4), and fucose groups (Fig. 4), with different intensities of each lectin ( Table 2). Due to the specificity of RCA 120 , DSL-I, DBA, and SJA on spores, these lectins may be candidates for Kudoa markers, at least for K. septempunctata.
Even though Con A, LCA, and PSA belong to the same mannose group, we found that Con A (Fig. 2G), but not LCA (Fig. 2H) and PSA (Fig. 2I), labeled on spores (Table 1). We postulate that a minor difference in preferred oligosaccharide 4GlcNAc of Con A would be one of the factors because 4(Fuca 1,6)GlcNAc is a preferred oligosaccharide in LCA and PSA (see Table 1 in this report and Table 1 [7]). With regard to the reactivity of oligosaccharides, PHA-E, but not    PHA-L, labeled spores, the plasmodial membrane, and the sarcoplasm, suggesting that the same oligosaccharide group shows distinct patterns in K. septempunctata-infected tissues. We postulate that some lectins with the same sugar specificities label different carbohydrate residues.
With regard to the differences in lectin reactivity on spores and the plasmodial membrane in this study, we postulate that carbohydrates on the plasmodial membrane originated largely from spores, while some were modified in the plasmodial membrane because lectin reactivity of DBA and SJA was not found in the plasmodial membrane despite their presence on spores. We do not exclude the possibility that some carbohydrate residues are used for penetration of the parasite into the host because carbohydrate residues are known to act as linkers between host cells and parasites [13,14].
It has been reported that the addition of glucose to the culture medium plays an important role in the release of K. septempunctata sporoplasm [16], suggesting that glucose mediates disruption of the sporoplasm. However, it is unclear which factors are involved in human diarrhea, because spores do not induce diarrhea in adult mice [2]. We cannot rule out the possibility that sporoplasm of Kudoa spp. may disturb the intestinal microorganisms in some human subjects.
For the sarcoplasm, we compared lectin reactivity of infected muscle fibers with non-infected fibers in the same tissue sections. The majority of the sarcoplasm in non-infected fibers was negative for lectins, except DSL, Con A, Jacalin, ECL, and PHA-E. We postulate that hypertrophy in spore-infected muscle fibers is not directly related to carbohydrate residues. Some lectins, i.e., WGA, DSL, LEL, STL, Con A, LCA, PSA, Jacalin, ECL, and PHA-E, were found to be positive on the endomysium, while others were not positive in this study. We postulate that interstitial connective tissues are not changed after K. septempunctata infection.
The epidermis also showed reactivity for the majority of lectins, except BSL-II, DBA, and SJA ( Table 2), suggesting that a variety of carbohydrate residues cover flat fish skin. Even in the absence of BSL-II, DBA, and SJA reactivity in the epidermis, they were positive in spores. Conversely, LCA, PAS, and PNA were negative on spores but positive on the epidermis. In a limited examination of lectin binding in flat fish [6], it was found that each lectin labels some epithelial cells and mucus cells in flat fish with varying intensities, suggesting that carbohydrate residues are present, but no examination of the epidermis was performed. In the present study, we found that a variety of lectin labelings were localized on the epidermis, suggesting that all types of carbohydrates (N-acetylglucosamine, mannose, galactose/N-acetylgalactosamine, complex type N-glycans oligosaccharides (PHA-E and PHA-L), and fucose groups) exist on the skin of flat fish. We postulate that a variety of carbohydrate residues are constitutively expressed on the epidermis, possibly contributing to protection against environmental stimuli in this species.