Comparative Analysis Between Paramecium Strains with Different Syngens Using the RAPD Method

Paramecium spp. are a genus of free-living protists that live mainly in freshwater environments. They are ciliates with high motility and phagocytosis and have been used to analyze cell motility and as a host model for pathogens. Besides such biological characteristics, apart from the usual morphological and genetic classification of species, the existence of taxonomies (such as syngens) and mating types related to Paramecium’s unique reproduction is known. In this study, we attempted to develop a simple method to identify Paramecium strains, which are difficult to distinguish morphologically, using random amplified polymorphic DNA (RAPD) analysis. Consequently, we can observe strain-specific band patterns. We also confirm that the presence of endosymbiotic Chlorella cells affects the band pattern of P. bursaria. Furthermore, the results of the RAPD analysis using several P. caudatum strains with different syngens show that it is possible to detect a band specific to a certain syngen. By improving the reaction conditions and random primers, based on the results of this study, RAPD analysis can be applied to the identification of Paramecium strains and their syngen confirmation tests.


Introduction
Paramecium spp., single-celled and free-living protists, are general ciliates found in freshwaters, such as ponds, lakes, and rivers. They are well studied in various fields as ideal model organisms since they are easy to deal with [1][2][3]. The unique and complex reproduction process of Paramecium, in which asexual binary fission and several types of sexual processes like conjugation and autogamy are involved, has already been revealed [4][5][6][7][8]. Conjugation can occur only among different mating types of the same syngen. Probably each Paramecium has some syngens, and in some cases, the type of syngen has been recognized as a cryptic species. For example, P. aurelia and P. jenningsi have been considered to comprise a group of 16 and 3 cryptic species [9,10]. Each syngen is genetically isolated from other syngens, but it has been impossible to distinguish them morphologically. These different syngen groups are now defined as separate species within the P. aurelia complex of sibling species [11][12][13][14].
In our previous studies, we have referred to the possibility that Paramecium can be a natural host for pathogenic bacteria in the environment [15][16][17]. We have also analyzed the relationship between the host competency of Paramecium and its species or strains and reported that Legionella pneumophila can establish intracellular symbiotic relationships with a large majority of strains of P. caudatum; however, such stable relationships have not been established in specific strains of P. caudatum, P. bursaria, or P. multimicronucleatum [15,18]. As it is currently unknown what factors in Paramecium determine their ability to act as hosts for pathogenic bacteria, the possibility that the syngen is an important factor related to this cannot be ruled out. From the perspective of controlling infectious diseases, understanding Sonoko Matsumoto and Kenta Watanabe contributed equally to this work. the natural host is of great public health significance. In this regard, it is essential to establish a system to quickly understand the characteristics of the Paramecium strains, including syngens, and to evaluate whether they can be risk factors for infection. However, classical mating tests are generally used to discriminate between syngens; thus, there is a major implementation problem, in that we need to maintain various strains whose syngens have been identified with certainty.
Random amplified polymorphic DNA (RAPD) analysis is one of the molecular biological methods for determining genetic diversity by analyzing DNA sequence homology or polymorphisms [19][20][21]. In this assay, the genomic DNA of the target organism is used as a template, and DNA fragments are amplified by PCR using primers with random sequences. By comparing the appearance pattern of the DNA fragments by electrophoresis, genetic diversity can be determined. The major advantages of this method are that it is quick and easy to obtain results and it does not require information on the genomic DNA of the target organism. Because of these advantages, this analysis has been applied to the identification and discrimination of microbial and plant species [22][23][24], phylogenetic analysis, and epidemiological fields [25][26][27]. The identification and comparative analysis of Paramecium strains using RAPD analysis have also been reported [28][29][30][31][32]. RAPD analysis is a useful tool for species identification and classification in Paramecium, which is difficult to distinguish morphologically.
In this study, we prepared several strains of various Paramecium spp. with confirmed syngen and mating type, by obtaining them from the National BioResource Project (NBRP), and attempted to apply RAPD analysis to construct a method for identifying these strains and their syngens in particular.

Isolation of Genomic DNA from Paramecium
The genomic DNA was isolated from 3 mL of Paramecium cell culture (including approximately 3000 cells) using a DNA extraction kit (QIAGEN, Venlo, Netherlands),

RAPD Analysis
RAPD analysis was performed as described previously [29], with some modifications. Briefly, RAPD-PCR was conducted in a 10-µL reaction mixture comprising 2.8 μL of PCR master mix (KOD-Plus-Neo, TOYOBO, Osaka, Japan), 1 μL of primer (10 μM), 1 μL of template DNA (adjusted to 10 ng/μL), and 5.2 μL of nuclease-free water. The PCR program consisted of first, four cycles at a denaturation temperature of 94 °C for 5 min, followed by primer annealing at 35 °C for 5 min, and finally, an elongation at 72 °C for 2 min. The subsequent 36 cycles consisted of denaturation at 94 °C for 1 min, primer annealing at 40 °C for 1 min, and elongation at 72 °C for 2 min. A final elongation step was extended to 5 min at 72 °C. The fragments were separated by electrophoresing at 100 V for 30 min on 1.8% agarose gel with DNA ladder markers (Kapa Biosystems, Bath, UK). The gels were stained with ethidium bromide and visualized using a gel imaging system (ATTO, Tokyo, Japan). All RAPD-PCR were repeated at least three times to confirm the reproducibility of the band patterns. Table 2 lists the primers used in this study.

Comparative Analysis Among Paramecium Species Using the RAPD Method
Several studies about RAPD analysis for Paramecium species have been reported [29,36,37]. In this study, we first applied the reported primers and methods of RAPD analysis for some Paramecium strains obtained from NBRP.
Although a previous study reported that only one primer (Ro-460-04; primer-4 in this study) gave robust band patterns [29], we confirmed that all random primers work for various Paramecium strains and that specific band patterns dependent on each strain are observed in four Paramecium strains (two strains of P. caudatum, one strain of P. tetraurelia, and one strain of P. bursaria) (Fig. 1). Comparing the results between P. caudatum RB-1 and TAZ0462, explicit bands or band patterns that define the species of Paramecium were not identified. However, some primers such as primer-2, -3, or -9 showed a very clearly and distinctly different pattern of bands in all strains used in this study and might be used for rough identification of Paramecium species. Next, we performed the same RAPD analysis using DNA samples isolated only in the culture medium of Paramecium, with bacteria that are used as feeds for Paramecium, since it is impossible to deny the genomic contamination of the bacteria. As expected, some PCR products were observed in all experiments using the 10 random primers, although the band patterns were different from those of the Paramecium DNA samples. Similarly, P. bursaria is well known to maintain Chlorella cells as an endosymbiont [38][39][40]. Thus, the effect of the existence of Chlorella DNA should be considered in this assay, using a sample of P. bursaria. We have prepared a pair of two P. bursaria strains, YDS1 and HA1, which either maintain Chlorella cells (YDS1g and HA1g) or do not (YDS1w and HA1w), and evaluated the changing of band patterns. Consequently, it was found that the band patterns changed depending on the presence or absence of Chlorella, although there was a difference in degree depending on the primer used. Furthermore, the sharpness of the bands was attenuated, and the appearance of these bands tended to become smear-like in the presence of Chlorella (YDS1g and HA1g), compared with strains without Chlorella (YDS1w and HA1w) (Fig. 2).

Comparative Analysis Between the Syngen 6 and 12 of P. caudatum Strains Using the RAPD Method
Next, we applied this RAPD system to investigate a simplified method for identifying syngens of Paramecium strains. Since multiple strains were available for each syngen in NBRP, P. caudatum was adopted for this examination. A total of 10 P. caudatum strains (five strains of syngen 6 and five strains of syngen 12) were compared. Ten different random primers (1-10) were used, as in the above experiment. Consequently, it was reconfirmed that the band patterns were not completely consistent among the strains of the same species, similar to the results observed between RB-1 and TAZ0462 strains in Fig. 1 (Fig. 3). When these results were grouped by the primers used, there were cases where (1) all 10 strains showed almost similar patterns (primer-3, primer-4, primer-7, and primer-8), (2) all 10 strains showed almost disparate patterns (primer-1, primer-9, and primer-10), and (3) each syngen showed a somewhat coherent pattern (primer-2, primer-5, and primer-6).

Searching for Specific Bands That Can Be Used as Criteria for Syngen Decisions for P. caudatum
The bands surrounded by a white box in the results of primer-5 (Fig. 3) were clearly visible as single bands in all five strains of syngen 12 but did not exist as distinct common bands in any of the five strains of syngen 6. Thus, we conducted a comparative analysis for other strains of P. caudatum (syngens 1, 3, and 4) by the RAPD method using primer-5. This target band that was specifically observed in strains of syngen 12 was also observed in the Ai51 strain (syngen 1) but not in other syngen strains. The other band patterns of My43C3d and Ai51 were also almost identical.
Although we additionally compared one strain each of the mating types O and E, there was no clear trend or specific band depending on the mating type.

Discussion
Many species of Paramecium have been reported to be widely isolated from aquatic environments worldwide. Several syngens and different mating types are also known to exist in each species [41][42][43]. Although the identification of species or syngens is one of the most fundamental tasks in understanding the distribution and ecology of Paramecium in the environment, it is extremely difficult to distinguish them easily and rigorously via morphological methods.
Although genomic information, such as 18S rRNA and COI mtDNA genes, has also been used for their classification [44,45], such genetic methods are limited, since the small number of species and strains have undergone whole-genome analysis because of the lack of basic information about them, such as their number of chromosomes. Given this situation, RAPD analysis is best suited for the identification of species or strains in Paramecium because it does not require detailed genomic information of the target organism. In fact, attempts to use RAPD analysis to identify Paramecium species and compare them by their strains have already been reported [29,36,37]. In this study, we also performed RAPD analysis using the reported random primers and confirmed different band patterns (number of visualized bands and their sizes) between strains (Fig. 1). These results suggest that it is feasible to determine the identity of Paramecium strains by using multiple results of the RAPD analysis with different random primers in combination.
One of the disadvantages of RAPD analysis is that it requires highly standardized experimental procedures because of its sensitivity to the reaction conditions of PCR. Additionally, RAPD analysis generally requires well-purified, high-molecular-weight DNA isolated from the target organism. This means that precautions must be heeded to avoid the contamination of the DNA sample because short random primers can amplify DNA fragments from various organisms. When cultured and maintained strains of Paramecium are used in RAPD analysis, it is not possible to avoid DNA contamination from the bacteria (such as Klebsiella or Enterobacter) that are fed as food. In the present experiment, several clear bands were still observed, using any of the random primers, when a DNA sample purified from only a culture medium containing bacteria (E. aerogenes) was applied as a template (Fig. 1). This may be due in large part to the use of DNA purified from a sample that intentionally contains large amounts of bacteria. In the case of using Paramecium strains isolated from the environment, it is also necessary to consider the possibility of DNA contamination from diverse microorganisms. Paramecium has been found to be potential hosts for various parasitic symbionts. Holospora and Legionella have already been reported as their intracellular parasites [15,16,[46][47][48]. Although centrifugation and antibiotic treatment may remove the majority of these bacteria from the host Paramecium, it is difficult to completely eliminate DNA contamination. Furthermore, unidentified or unisolated symbiotic bacteria may be present in some form within the host Paramecium, and it is necessary to assume the effects of contamination with such secret DNA. In the case of P. bursaria, the presence or absence of its intracellular symbiont, Chlorella, resulted in a change in band patterns (Fig. 2). This is an expected and natural result, because Chlorella-derived DNA is mixed in the extracted DNA sample from host P. bursaria, and this cannot be removed. The application of the RAPD analysis in Paramecium will require a strong understanding of the biology of Paramecium, which serves as a host for various symbionts, and the consideration of the effects of the presence of DNA from nonseparable symbionts other than the host Paramecium. It is important to construct a modified RAPD analysis system that is less affected by the presence of nontarget DNA, but such attempts have not been studied thus far. The investigation and improvement of the conditions of RAPD analysis to cope with such DNA contamination are issues for future study.
There have already been several reports on genes involved in determining mating types in Paramecium [49][50][51][52][53][54]. Hence, it is possible to determine the mating type of Paramecium strains via genetic analysis targeting these genes. Conversely, there remains insufficient genetic information on the syngens of Paramecium, and the general method of identifying syngens remains deeply dependent on actual mating tests. Additionally, there have been reports of cases where different syngens are crossed and conjugated [55]. Thus, the confirmation process and its complexity are obstacles to Paramecium syngen identification. These problems can be avoided if syngens can be easily identified using RAPD analysis. Also, when a strain suspected to be a novel syngen is isolated, its identification can be conducted smoothly and reliably. In the present study, we focused on the syngen 12 strain of P. caudatum and found a band that may be specifically observed among syngen 12 strains (Fig. 3). This approach is not complete as an identification of syngen markers or as a discriminant method for syngens at this point, given that the band also appears in a particular syngen 1 strain 1 3 (P. caudatum Ai51) (Fig. 4). Previous reports suggested the genetic differences among collection localities are greater than those among syngens [13,56]. My43C3d and Ai51 were collected from the same place (Table 1), although their syngens and mating types were different. Since the band patterns of the two strains shown in Fig. 4 are very similar, it is highly possible that such regional factors have a stronger influence on the results of our RAPD method than syngen, which should be taken into consideration in future studies. However, it shows the potential of this method for use in narrowing down syngen groups and in screening tests. For practical use, it is necessary to increase the number of samples of Paramecium strains used for analysis and accumulate more data. For future research, factors or genes involved in the determination of syngens of Paramecium should be investigated, and syngen markers should be searched via a detailed analysis of amplified PCR products, especially sequencing analysis.

Code Availability (Software Application or Custom Code)
Not applicable. Funding This study was supported in part by the Japan Society for the Promotion of Science Grant-in-Aid for Scientific Research (C) and (B), under grant numbers 19K06383 and 17H03914 to K.W. and M.W, respectively.

Availability of Data and Material
All relevant data are within this paper.

Declarations
Ethics Approval Not applicable.

Consent to Participate Not applicable.
Consent for publication Not applicable.

Conflict of Interest
The authors declare no competing interests.