Introduction
Scuticociliatosis is a globally distributed disease caused by a well-known ciliated parasite of the subclass scuticociliates (Harikrishnan et al., Reference Harikrishnan, Balasundaram and Heo2010). Scuticociliates often caused systemic tissue infection with high mortality in marine populations of fish (Iglesias et al., Reference Iglesias, Paramá, Alvarez, Leiro, Fernández and Sanmartín Durán2001), sea stars (Byrne et al., Reference Byrne, Cerra, Nishigaki and Hoshi1997) and bivalve mollusks (Karatayev et al., Reference Karatayev, Mastitsky, Burlakova, Molloy and Vezhnovets2003). Scuticociliate systemic infection in crustacean species has received comparatively more attention due to its harmful effects (Morado and Small, Reference Morado and Small1995) on American lobster, freshwater crayfish and krill (Edgerton et al., Reference Edgerton, O'Donoughe, Wingfield and Owens1996; Cawthorn, Reference Cawthorn1997; Gómez-Gutiérrez et al., Reference Gómez-Gutiérrez, Peterson, De Robertis and Brodeur2003). Recently, protistan Mesanophrys sp., a newly identified scuticociliate species, was successfully detected from DNA and other molecular studies of damaged haemocytes of cultured overwintering swimming crab (Portunus trituberculatus) (Liu et al., Reference Liu, Lei, Ren, Zhou, Qian, Yu and Wang2020). Genus Mesanophrys parasites were also reported in crab and isopods species (Armstrong et al., Reference Armstrong, Burreson and Sparks1981; Morado and Small, Reference Morado and Small1994; Wiąckowski et al., Reference Wiąckowski, Hryniewiecka-Szyfter and Babula1999), this triggers economic losses to the native farmers and affects artificial breeding of swimming crab in Eastern China (Yu et al., Reference Yu, Liu, Lei, Zhou, Jin, Qian, Xie, Yin and Wang2020).
There are some interesting studies which aimed to identify the environmental conditions such as temperature, salinity and pH conducive to the proliferation of parasites in the host that can lead to death (Oliver et al., Reference Oliver, Fisher, Ford, Calvo, Burreson, Sutton and Gandy1998; Marcogliese, Reference Marcogliese2001; Cáceres-Martínez et al., Reference Cáceres-Martínez, Ortega, Vásquez-Yeomans, García, Stokes and Carnegie2012). Among these, temperature and salinity have been considered as important environmental factors (Miller and Marcus, Reference Miller and Marcus1994), which strongly affect interactions between parasite and its host (Thomas and Blanford, Reference Thomas and Blanford2003; Arzul et al., Reference Arzul, Gagnaire, Bond, Bruno, Morga, Ferrand and Renault2009). These also control the infection rate and progression of protozoan parasites (Auzoux-Bordenave, Reference Auzoux-Bordenave1995; Audemard et al., Reference Audemard, Carnegie, Bishop, Peterson and Burreson2008; Perrigault et al., Reference Perrigault, Buggé and Allam2010). For example, the genus Perkinsus were regarded as sensitive to extreme temperatures between 15 and 35°C and salinities of 10–35 PSU (Umeda et al., Reference Umeda, Shimokawa and Yoshinaga2013). The parasite Pasteuria ramosa was also found to be temperature-sensitive and showed the highest infection rate at 15–20°C (Mitchell et al., Reference Mitchell, Rogers, Little and Read2005). The temperature of <6°C increased the prevalence of bonamiosis on oyster species (Audemard et al., Reference Audemard, Carnegie, Bishop, Peterson and Burreson2008). Furthermore, scuticociliate was found to be pathogenic with 58% infection rates in winter at temperatures <15°C (Deveney et al., Reference Deveney, Bayly, Johnston and Nowak2005). The scuticociliate rapidly grow and reproduce at an optimum temperature of 3–13°C in 30 PSU (Stickle et al., Reference Stickle, Kozloff and Story2007). Temperature and salinity also affect the development time, life cycle and swimming activities of marine ectoparasites (González and Carvajal, Reference González and Carvajal2003). For any organism, the developmental stages are the most vulnerable part of the life cycle (Bodinier et al., Reference Bodinier, Lorin-Nebel, Charmantier and Boulo2009). The environmental factors influence their life cycles (Carnegie et al., Reference Carnegie, Stokes, Audemard, Bishop, Wilbur, Alphin and Burreson2008).
Despite all this progress, the pattern of growth and development of crab parasite Mesanophrys sp. is still unknown. Knowledge of the different environmental factors on Mesanophrys sp. in vitro will not only improve the understanding of the ecology of the parasite but also the crab farming management. Therefore, the present study is conducted to investigate the effects of the environmental factors (temperature, salinity, pH and frequency of passage of parasite) on survival and population density, population growth rate, and body changes of Mesanophrys sp. using a 12 days incubation experiment; and to compare the same factors to identify the specific adaptation of Mesanophrys sp. This successful in vitro propagation of Mesanophrys sp. will help to understand the protozoan infection dynamics and will enable us to perform more specific accurate studies of protozoan parasites.
Materials and methods
Parasite source, isolation, identification and cultivation
Parasite Mesanophrys sp. was sampled from an aquaculture farm in Ningbo city Zhejiang Province, China (N29°37′18.14″, E121°45′21.45″). At this condition, the salinity water and water temperature ranged from (22 ± 1)‰ and 12–15°C, respectively. Mesanophrys sp. ciliates were isolated from the haemolymphs of diseased crabs by microscopic examination. The isolate was identified as Mesanophrys sp. (Liu et al., Reference Liu, Lei, Ren, Zhou, Qian, Yu and Wang2020).
Before assays, this Mesanophrys sp. ciliate were sub-cultured in a 12-well culture plate containing medium [22‰ salinity, 5% (v/v) of swimming crab soup and 10% (v/v) of fetal bovine serum (Gibco , Australlia) at 12°C for 5 days to reach the exponential growth phase. The crab soup was prepared in the laboratory following the previously reported protocol (Yu et al., Reference Yu, Liu, Lei, Zhou, Jin, Qian, Xie, Yin and Wang2020). Parasite culture was illuminated with 18 μmol m−2 s−1 using a 12 h light and 12 h dark cycles. Old ciliates were transferred to new culture plates using the ratio 1:10 after every 96 h.
Effect of temperature on survival, population growth and body size of Mesanophrys sp.
For temperature assay, the aliquots of the parasite (100 μL) were transferred into four wells (quadruplicate) of a 24-well plate containing 2 mL culture medium adjusted to the salinity (22‰) and pH (7.8). Totally, seven plates were incubated at seven (4, 8, 12, 16, 20, 24 and 26°C) different temperatures, respectively. The 20 μL parasite sample was fixed in 0.1% formaldehyde and was taken from each well and treatment (4 replicates and seven treatments = 28 samples) at each time point (every 24 h) up to 12 days. Observation and counting of ciliates were done using an inverted microscope (Olympus IX70; Olympus Optical, Tokyo, Japan). This trial was repeated in four replicates. The cultured ciliates were observed and photographed using a differential interference contrast microscope (Nikon), and total length/width was measured by using a micrometer (Julia et al., Reference Julia, Clinton, Ingo and Ian2007; Liu et al., Reference Liu, Lei, Ren, Zhou, Qian, Yu and Wang2020) (data: mean ± s.d., n = 4).
Effect of salinity on survival, population growth and body size of Mesanophrys sp.
The 5th day's cultured parasites were centrifuged at 3000 rpm for 10 min before inoculated into salinity gradients of 0, 5, 10, 20, 30, 40, 50 and 60‰. Desired salinity was prepared by adding sea salt and fresh distilled water. For each treated group, parasites were distributed in quadruplicate to a 24-well plate and incubated at 12°C. Parasites were examined every day with an inverted microscope as described above.
Effect of pH on survival, population growth and body size of Mesanophrys sp.
A pH assay was conducted in a 24-well plate, separately. For that, 10 different types of tested pH (3.5, 4.5, 5.5, 6.5, 7.5, 8.0, 8.5, 9.0, 9.5 and 10.0) were selected. Modified culture medium at different pH was prepared by using 0.1 m L−1 NaOH and HCL from the standard preparation. Aliquots of 5th-day ciliates were transferred into a 24-well plate with each tested group. Each treated group was selected with four replicates and was kept at 12°C. Data were analysed as described above.
The frequency changes of culture method on population growth of Mesanophrys sp.
In this assay, three groups were selected (group A, group B and the control group). The 96 h of parasite aliquots were centrifuged at 3000 rpm for 10 min and then resuspended into medium-containing 24-well culture plate (22‰ salinity and 7.8 pH) at 12°C.
Group A
In group A, on 3rd incubation day, the old, cultured parasite medium was halved into the new well plates, renewed the medium with the desired concentration and kept for 7 days at 12°C. Data were collected and analysed as described above. The population growth rate was calculated for each day (data: mean ± s.d., n = 4).
Group B
In group B on the 5th day, the old parasite culture medium was halved into new culture plates and renewed with a new fresh medium and kept inside the incubator at 12°C. Data were collected and analysed as described above.
Control
In control, no medium was changed; the parasites were monitored for 12 days.
Calculation of population growth rate (r)
The population growth rate was calculated according to the formula,
where N 0 represents the initial population and Nt is the final population density after a time duration ‘t’, ‘r’ is the population growth rate, and ‘t’ stands for the culture time (Dumont et al., Reference Dumont, Sarma and Ali2006).
Relationship between generation time (G) and population growth rate (r)
The relationship between generation time (G) and population growth rate (r) of Mesanophrys sp. was determined by the two-division method,
r value was obtained from the above experiment and was substituted into the formula to obtain the generation time.
Q 10 (temperature coefficient)
The Q 10 values were calculated using the following equation,
where r 2 and r 1 represent the natural population growth rate at two temperatures T 1 and T 2, respectively.
Statistical data analysis
Data were analysed using SPSS statistical software. One-way ANOVA factor, such as temperature, salinity, pH, frequency of passage of parasite, was used to compare the results of all parameters (survival rate %, population density, population growth rate, generation time and body proportions) of Mesanophrys sp. Bonferroni multiple range test was used to compare means (data: mean ± s.d., n = 4), and considered statistically significant at P < 0.05 and extremely significant at P < 0.01.
Results
Effects of different temperatures on survival, growth and body shape of Mesanophrys sp.
The observed survival rate of Mesanophrys sp. showed significant variation with tested temperatures. The survival rate significantly increased (P < 0.01) with increasing temperature, peaked at 12°C (P < 0.001) where approximately 97% of the parasite survived and then decreased (P < 0.001) with further increase in temperature. The highest and lowest survival rates were 97% (observed at 12°C) and 25% (observed at 26°C), respectively (Fig. 1).
Survival rate % of Mesanophrys sp. at different temperatures. Different superscript letters represent significant differences among seven temperature groups (post hoc Bonferroni test; P < 0.01).
The temperature also had a significant effect on the population density of Mesanophrys sp. where an optimum growth was observed from 4 to 12°C with the highest population density appeared at 12°C (P < 0.001). However, from 16 to 26°C, population density started decreasing (P < 0.001), and after 24 h, a decline in population density of Mesanophrys sp. was observed at 20–26°C (Fig. 2).
Population density of Mesanophrys sp. at different temperatures. Bar graph represents significant differences between different groups at each time point (post hoc Bonferroni test; P < 0.01).
The population growth rate of Mesanophrys sp. followed the trend of population density; increased significantly at first (P < 0.001) with increasing temperature from 4 to 16°C, peaked at 12°C (P < 0.001) and then decreased (P < 0.01) with the temperature >20°C. It was highest at 12°C and lowest at 20, 24 and 26°C. Overall, the observed population growth rate and generation time of Mesanophrys sp. were significantly higher between 4 and 16°C than 20, 24 and 26°C (P < 0.001) (Fig. 3). The calculated temperature coefficient Q 10 was 2.04 (Table 1).
Relationship between population growth rate and generation time at different temperatures. Different superscript letters represent significant differences among seven temperature groups (post hoc Bonferroni test; P < 0.01).
Temperature coefficient (Q 10) of some protistan species
In this study, we also observed that temperature strongly influenced the morphology of parasite. Parasite body proportions related to the length:width were observed considerably higher at 4, 8 and 12°C than that of 16, 20, 24 and 26°C (Fig. 4).
Effect of temperature on the body size of Mesanophrys sp. Different superscript letters represent significant differences among seven temperature groups (post hoc Bonferroni test; P < 0.01).
Effects of different salinities on survival, growth and body shape of Mesanophrys sp.
Results showed that the survival rate first increased significantly (P < 0.01) and then decreased (P < 0.01) with the increase of salinity. Parasite Mesanophrys sp. showed a maximum survival of 97% at 20‰ (P < 0.001) and the minimum survival rate of 42.5% at 5‰. Furthermore, the survival rates of 45.5 and 42% were concluded at 40 and 50‰, respectively. Parasite in 60‰ salinity died after 24 h; therefore, the observed survival rate was 0% (Fig. 5).
Survival rate % of Mesanophrys sp. at different salinities. Different superscript letters represent significant differences among groups (post hoc Bonferroni test; P < 0.01).
The optimum Mesanophrys sp. growth was observed at 20 and 30‰ which peaked at 20‰ on the 5th day and it differed among all salinity groups (P < 0.001). However, after the 5th day, a stationary phase went on which then followed by a decrease in growth again (P < 0.001). Compared to others, Mesanophrys sp. population growth density was significantly lower at 40–50 and 0–10‰ (P < 0.01) (Fig. 6).
Population growth density of Mesanophrys sp. at different salinities. Bar graph represents significant differences between different groups at each time point (post hoc Bonferroni test; P < 0.01).
The population growth rate and generation time of Mesanophrys sp. showed signification variation among groups (P < 0.001) with salinity. The population growth rate of Mesanophrys sp. increased with an increased salinity level in the culture media. It was peaked at 20‰ with faster generation time (P < 0.01) and then decreased significantly (P < 0.01). The highest and lowest population growth rate was at 20‰ (0.66 day−1) and 50‰ (0.33 day−1), respectively (P < 0.01) (Fig. 7).
Relationship between population growth rate and generation time of Mesanophrys sp. at different salinities. Different superscript letters represent significant differences among groups (post hoc Bonferroni test; P < 0.01).
The body size ratio/body proportions of Mesanophrys sp. at 20–50‰ showed no significant difference with each other (P > 0.01). At low salinity (5‰), the body size ratio was lower than other salinity groups (P < 0.01), whereas the high salinity had no significant effect (P > 0.01) (Fig. 8).
Effect of different salinities on the body size of Mesanophrys sp. Different superscript letters represent significant differences among groups (post hoc Bonferroni test; P < 0.01).
Effects of different pH on survival, growth and body shape of Mesanophrys sp.
Our results showed that the parasite survival rate was optimum at pH between 7.5 and 9.5 (P < 0.01). At these optimum levels, the survival rate of Mesanophrys sp. showed non-significant differences (P > 0.01). At a pH of 4.5, 5.5 and 6.5, the parasite survival rates were 57.5, 67.5 and 72.5%, respectively, and they also showed non-significant differences with one another (P > 0.01). At pH 10, Mesanophrys sp. survival rate was significantly lower (45%) (P < 0.01) compared to others (Fig. 9).
Survival rate % of Mesanophrys sp. at different pH. Different superscript letters represent significant differences among groups (post hoc Bonferroni test; P < 0.01).
In the present study, the result showed that pH between 7.5 and 9.5 had a positive impact on the population density of Mesanophrys sp. with a significantly high population density observed at pH 8 than other groups (P < 0.01). Whereas, the significantly low population density was observed at a low pH of 4.5 (P < 0.01) where after 24 h, parasite did not grow and began to decline. Similarly, at pH 10, the significantly less value was observed (P < 0.01) compared to others (Fig. 10).
Population density of Mesanophrys sp. at different pH. Bar graph represents significant differences between different groups at each time point (post hoc Bonferroni test; P < 0.01).
With the increasing pH from 3.5 to 10, the population growth rate of Mesanophrys sp. increased significantly at first (P < 0.01) and peaked at the group of pH 8 (P < 0.01), and then decreased (P < 0.01). The highest and lowest population growth rates were 0.51 day−1 (pH 8) and 0.40 day−1 (pH 10), respectively (Fig. 11).
The population growth rate of Mesanophrys sp. at different pH. Different superscript letters represent significant differences among groups (post hoc Bonferroni test; P < 0.01).
The result also showed that pH influenced the body size ratios of Mesanophrys sp. where significantly lower values of body size ratios were observed at an extreme pH of 10 and a low pH of 4.5 (P < 0.01). The value of body size ratios reached a peak at the group of pH 8 and showed a significant difference among pH 4.5, 5.5 and 10 (P < 0.01). However, there were no significant differences when parasites were cultured at pH from 6.5 to 9.5 (P > 0.01) (Fig. 12).
Effect of pH on the body size of Mesanophrys sp. Different superscript letters represent significant differences among groups (post hoc Bonferroni test; P < 0.01).
Effect of frequency changes method and time on the population density of Mesanophrys sp.
The result showed the same trend for the population growth of Mesanophrys sp. in the first 3 days (P > 0.01). The population growth in group A significantly increased on the 4th and 5th day and peaked on 7th day (P < 0.001) when the old culture parasites were transferred into the new culture plates on 3 days post-incubation and showed significant variation between group B and the control group. In addition, on the 6th day, population growth in group B was significantly higher than that of the other two groups (group A and control), peaked on the 10th day after the transfer of old culture parasites into the new one on 5th day. In the case of control, the population growth reached a peak on the 5th day and then significantly decreased (P < 0.01), no parasite survived after/on the 12th day (Fig. 13).
Effect of frequency changes method and time on the population density of Mesanophrys sp. Bar graph represents significant differences between different groups at each time point (post hoc Bonferroni test; P < 0.01).
The population growth rate of Mesanophrys sp. showed no significant differences among the three groups in the first 3 days. However, on the 4th and 5th day, group A showed significant differences (P < 0.01) from group B and control, whereas, the control and group B showed no significant differences (P > 0.01) among themselves. On the 6th to 12th day, the population growth rate was higher in group B than that of the other two groups (P < 0.01). Overall, the population growth rate in control was significantly different compared to the group A and B, no parasites survived at the end of the experiment, some colour changes observed with the acidic pH of the medium, and coinciding with a decrease in parasite population growth rate (Fig. 14).
Effect of frequency changes method and time on the population growth rate of Mesanophrys sp. Bar graph represents significant differences between different groups at each time point (post hoc Bonferroni test; P < 0.01).
Discussion
Mesanophrys sp. is a new scuticociliate species identified in Eastern China during 2017 and 2018. This parasite caused endoparasitosis in swimming crab. At present, the pattern of growth and development of Mesanophrys sp. is not known. Marine parasites usually rely on the host's tissues for their survival and spread (van Banning, Reference van Banning1991). Environmental factors may influence their survival and transmission in the host (Arzul et al., Reference Arzul, Gagnaire, Bond, Bruno, Morga, Ferrand and Renault2009). The persistence of parasitic disease in swimming crab has motivated us to investigate the environmental effects (temperature, salinity, pH and frequency of time) on Mesanophrys sp. life cycle. Previous studies reported temperature and salinity as influential factors for parasite diseases in the brackish and estuary ecosystem (Zander, Reference Zander1998; Thieltges et al., Reference Thieltges, Dolch, Krakau and Poulin2010). However, the environmental tolerance of parasites and their hosts can vary, and therefore can influence their interactions. According to Möller (Reference Möller1978), the distribution of endoparasites can be limited by host environmental tolerance, as some marine parasites are more tolerant than their hosts. Crustacean is the poikilothermic animal that is mostly affected by environmental factors (Briffa et al., Reference Briffa, Bridger and Biro2013). Moreover, they have adaptive mechanisms that allow them to adjust their body homoeostasis accordingly (Péqueux, Reference Péqueux1995).
In the present study, the parasite showed maximum survival, population growth rate and significantly high length to width ratio throughout the experiment at 12°C. Mesanophrys sp. showed a maximum % survival rate between 4 and 16°C, but the temperature from 16 to 26°C was found to be unfavourable for their survival. In particular, dying parasites were turned into a rounded, oval shape and became smaller in body size which is known as cell apoptosis (Nasirudeen et al., Reference Nasirudeen, Tan, Singh and Yap2001). Previously, Arzul et al. (Reference Arzul, Gagnaire, Bond, Bruno, Morga, Ferrand and Renault2009) found the low survival of parasite Bonamia ostreae at 25°C compared to 4 and 15°C. In addition, seasonal fluctuation in the prevalence of protozoan B. ostreae was also reported that peaked in the late winter and autumn (Culloty and Mulcahy, Reference Culloty and Mulcahy1996). The prevalence of B. ostreae was found to be high at 10°C than 20°C which confirmed that low temperatures may favour the protozoan parasite to infect the oyster (Carnegie et al., Reference Carnegie, Stokes, Audemard, Bishop, Wilbur, Alphin and Burreson2008). In our experiment, at 12°C, the parasite grows well with a faster generation time where the peaked population density and exponential growth rate of 0.83 day−1 were observed. However, when the temperature increased from 20 to 26°C, the population growth rate of Mesanophrys sp. did not increase. Furthermore, it was also observed that the final population growth density was generally lower between 20 and 26°C than 4 and 16°C. Hence, our study confirms that 4–16°C is the optimum temperature range for the growth of Mesanophrys sp.
The influence of temperature on the growth of parasites was also reported previously, for example, the growth rate of Plagiopyla nasuta was found to increase when the temperature increased from 8 to 18°C and remained constant at 18–24°C (0.22 day−1) (Massana et al., Reference Massana, Stumm and Pedrós-Alió1994). However, in another study, the growth of P. nasuta was found to be optimum between 15 and 20°C (Goosen et al., Reference Goosen, Horemans, Hillebrand, Stumm and Vogels1988). The population growth of parasites Paranophrys magna also showed significant differences with temperature changes (Zhang and Song, Reference Zhang and Song2000). The growth rate of ciliates Urotricha furcuta and Strobilidium lacustris was found to be 0.46–1.72 day−1 and 0.43–1.42 day−1 at different temperatures (5.5–21.5°C), respectively (Müller and Geller, Reference Müller and Geller1993). The rapid population growth rate with a high density of scuticociliate Orchitophrya stellarum was found at 3–13°C in 30 PSU (Stickle et al., Reference Stickle, Kozloff and Story2007).
Interestingly, in the present study, we showed that at 12°C parasites grow and divide rapidly with a short generation time. Our study also agrees with Liu et al.'s (Reference Liu, Lei, Ren, Zhou, Qian, Yu and Wang2020) report that the parasitosis outbreak occurred with many parasites in haemocytes of dying swimming crab where the water temperature was 12–15°C. Therefore, we hypothesized that wintering temperature probably may facilitate this Mesanophrys sp. parasite multiplication and was found to be pathogenic in swimming crab. Deveney et al. (Reference Deveney, Bayly, Johnston and Nowak2005) also found that scuticociliate shows pathogenicity to host in winter at temperatures <15°C. Hence, temperature appeared to be a major parameter influencing this parasite's growth as well as its reproductive cycle. At high temperatures, this parasite did not complete its life cycle where a sharp decline and slow growth of parasites were observed in our study. Our finding corroborates to Small et al. (Reference Small, Neil, Taylor, Bateman and Coombs2005) who also observed the infection of genus Mesanophrys at lower temperatures of 4–17°C.
This study also investigated an organism's sensitivity called temperature coefficient (Q 10), which reflects modifications associated with the enzyme and physiological needs of energy when the temperature rises (Kim et al., Reference Kim, Yoon, Kim, Gil and Lee2005). At 10°C, the temperature coefficient Q 10 was found to be 2.04. This is in agreement with a previously reported value of Q 10 = 2.0 for anaerobic freshwater ciliates at high temperatures (18–24°C) where the growth was affected (Massana et al., Reference Massana, Stumm and Pedrós-Alió1994). The reason might be that protozoan has a simple structure, and the biological index of Q 10 indicates its growth rate/metabolic rate. Our result also corroborates with the finding of Fenchel (Reference Fenchel1968) where the temperature coefficient of 2.3–3.5 was observed for aerobic marine ciliates.
Salinity has been considered an important factor that affects the growth rate of marine and estuary organisms (Xie et al., Reference Xie, Zhao and Yang2013). In our study, we found that Mesanophrys sp. survived better at salinities between 20 and 30‰, which is consistent with the previous findings (Cheng et al., Reference Cheng, Kâ, Kumar, Kuo and Hwang2011). However, at 40–60‰ and 0–10‰, the population growth rate of Mesanophrys sp. was lower. This shows that a low level of salinities (0–10‰) strongly inhibits the growth of Mesanophrys sp. because low salinities change the internal solute concentration, which then has a negative impact on some physiological condition and survival of an organism (Heuch et al., Reference Heuch, Knutsen, Knutsen and Schram2002). Similar results have been reported previously (Oltra and Todolf, Reference Oltra and Todolf1997; Sarma et al., Reference Sarma, Elguea-Sánchez and Nandini2002) where in vitro studies showed that protozoa genus Perkinsus is sensitive to extreme salinities and temperature conditions (Queiroga et al., Reference Queiroga, Marques-Santos, De Medeiros and Da Silva2016; Umeda et al., Reference Umeda, Yang, Waki, Yoshinaga and Itoh2020).
In this study, we also demonstrated the influence of pH on the survival and growth of Mesanophrys sp. In the aquatic environment, pH is the main ecological limiting factor of protozoa. It has various forms of electrical and migration processes that indirectly affect the behaviour, growth and development of protozoa and other aquatic organisms. Our study shows that a pH of 7.5–8.5 is favourable but Mesanophrys sp. also survived even at an extreme pH of 9.5. This is similar to the previous finding where better growth and survival of marine ciliate P. magna were observed at a pH of 7–9 (Zhang et al., Reference Zhang, Ma and Song2001). We also found that all the parasites died within 24 h when cultured at a low pH of 3.5. This may be due to a high level of toxicity in the culture medium (Paquin et al., Reference Paquin, Santore, Wu, Kavvadas and Toro2000). Previously, a high mortality rate of Paramecium caudatum was found at the lowest pH of 4 (Heydarnejad, Reference Heydarnejad2008).
Furthermore, this study suggests that the survival and growth of Mesanophrys sp. mostly depend on an optimized dilution timing technique. Application of the optimized dilution timing with fresh medium and sub-cultured enabled a continuous culture of parasites in relatively short generation time. Parasite's survival and growth thorough out the experiment were well preserved to achieve the highest parasite number and exponential growth rate than the control group. Therefore, this study speculated that when parasites were cultured in a non-looping system/enclosed system (culture plates), the parasite dies with an increasing population. Therefore, the high parasite survival was observed in frequency change culture when the number of parasites was continuously reduced and halved in other culture plates. So, it is hypothesized that, in a culture tank with several crabs, the parasites can survive/multiply by spreading from one crab to others.
Conclusion
The effect of environmental factors on the survival and growth of the parasite Mesanophrys sp. has been demonstrated for the first time. Our study shows that the parasite Mesanophrys sp. tolerates a broad range of environmental conditions. The combination of salinity 20‰, pH between 8 and 8.5, and temperatures between 8 and 12°C resulted in better survival and growth of Mesanophrys sp. in vitro. In addition, our results suggest that these environmental factors data provide an important basis for controlling the frequency, the strength of systemic infection and decrease the ciliate prevalence by interruption of its life cycle in the swimming crab farming areas. Our present study was done in vitro on Mesanophrys sp. and thus further studies on their host–parasite interaction with the environment are imperative.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0031182020002127
Acknowledgement
We thank Dr M. Tariq Sajjad, Assistant professor at London South Bank University for his valuable assistance in checking language.
Financial support
This work was supported by the Public Welfare Project of Science and Technology Bureau of Ningbo City (No. 202002N3045), Zhejiang key R & D plan (No. 2020C02020, development of efficient aquaculture breeding facilities and Research on Intelligent Control Technology), the Talent Training Base of Agricultural Science and Education Cooperation, Ningbo University – Zhejiang Hongye Seafood Co., Ltd. (202000305), and K.C. Wong Magna Fund in Ningbo University.
Conflict of interest
None.
Ethical standards
During this research, all experiments were approved by the Institutional Animal Care and Use Committee of the Ningbo University.
