Introduction

Schistosomiasis, caused by blood fluke worms of the genus Schistosoma, is a major neglected tropical disease. Among three main schistosomes infecting human beings including Schistosoma mansoni, Schistosoma japonicum, and Schistosoma haematobium, S. japonicum is mainly endemic in the Southeast Asia, including China, the Philippines, and Indonesia (Gryseels et al. 2006). In China, after nearly 60 years of control effort, a great success in the disease control has been made with the number of infections in humans reduced by 97.9 %, from 11.61 million in 1950s (Zhou et al. 2005) to 0.24 million in 2012 (Li et al. 2013). However, the disease remains one main public concern in China today because a huge area of Oncomelania snail habitats (for example, 368,741.67 hm2 in 2012 (Li et al. 2013)) still exist or even is increasing as a result of the climate change (Zhou et al. 2008) or water development programs (McManus et al. 2010); the annual seasonal flooding along the Yangtze River causes spread of snails (Wilke et al. 2000); acute infections in humans have been frequently reported (Li et al. 2014) and the increasing number of mobile human populations (Zhou et al. 2007), if infected, would transfer the parasite (Wang et al. 2013); and about 46 mammal species serving as potential reservoirs for the parasite (He et al. 2001) make the transmission far more complicated. All these factors raise a great challenge for the control or elimination of the disease.

In Anhui Province of China, by the year 2012, among 51 counties (cities or districts) endemic with S. japonicum, the disease transmission have not been well controlled or interrupted in 23 counties (Li et al. 2013). Moreover, since the mid-1990s, S. japonicum has reemerged in previously well-controlled areas. This is particularly true in Shitai County, a hilly region southern of Anhui, China. In 1985, disease control reached “transmission control” level at the scale of county (Lin et al. 2004; for the criteria, see Chen and Zheng 1999). However, since 1995, the infected snail habitats have increased each year, and a total of 24 infected snail habitats were found in 2009 (Chen et al. 2010). Acute cases were annually reported (Lin et al. 2004), particularly in 2003, an outbreak of 10 acute cases occurred in one village (Cao and Wu 2004). A series of epidemiological investigations demonstrated that among humans, dogs, cats, and rodents infected with S. japonicum, the highest infection prevalence was in rodents with the values of 26.53 % in 2006 and 17.65 % in 2007 (Lu et al. 2010), and 12.24 % in 2011 (Liu et al. 2013). All the above put forward the rodents as the main reservoir for the local transmission. However, there are currently no effective interventions targeting such wild animals in the hilly region.

The hilly region is very close to the Yangtze River, along which S. japonicum was once seriously endemic. Three small rivers cut through the hilly region and then flow down into the river. The rapid development in land transport has resulted an influx or output of people to the hill. It is then very likely that S. japonicum could then be transferred via final host humans or other mammals to the river valley, a huge area of marshland regions. S. japonicum is capable of developing into cercariae in several intermediate host species belonging to the genus Oncomelania (Ohmae et al. 2003), and the susceptibility of Oncomelania snails to S. japonicum has been reported to differ among/within countries (Cross et al. 1984; He et al. 1991). Therefore, knowledge on the vectorial capacity of snail populations from the river valley and S. japonicum from the hilly region is of great importance in epidemiology. The main aim of this study was then to investigate the levels of snail-parasite compatibility, including infection rate, prepatent period, cercarial production, and also test if the nocturnal cercarial emergence rhythm for the hill parasite, as previously reported (Lu et al. 2009; Su et al. 2013), still appear when harbored in different geographical snail populations.

Materials and methods

Snails and schistosome, and sampling sites

Two geographical strains of marshland Oncomelania hupensis hupensis from Anhui Province were used. One was collected in Zongyang County, located on the north bank of the Yangtze River and about 120 km from Shitai. The other was sampled in Hexian County on the north bank of the river, about 270 km from the hilly region. Zongyang and Hexian are separated by approximately 240 km. In September of 2013, snail surveys were performed in three counties including Shitai, Hexian, and Zongyang, and adult field-collected O. h. hupensis were transferred to the laboratory and raised for 4 weeks at a room temperature. The snails were maintained on wet culture paper in 20 × 30 cm trays labeled with names of locations. The culture paper, sprayed with spring water daily, and changed weekly, served as both food and microhabitats for snails (Jiang et al. 1997). All snails were tested twice for schistosome infection with cercarial shedding (Mao 1990). The snails with no S. japonicum infections were used for the subsequent experiments.

The hill isolate of S. japonicum used in this current study was previously established in our laboratory from field-collected infected snails in Shitai in April of 2013 and maintained in mice (for the procedure, see Su et al. 2013). For comparison, a marshland isolate of the parasite was provided by the Jiangsu Institute of Parasitic Diseases (Wuxi, Jiangsu of China), which has been maintained in the laboratory for over 30 years. Eight-week-old ICR female mice each were infected subcutaneously with approximately 200 S. japonicum cercariae of either the hill or the marshland parasite. Six weeks postinfection, they were killed by a lethal intraperitoneal injection of sodium pentobarbital, and livers with eggs were obtained.

Snail infection experiments

The mice livers were cut into small pieces and minced and then washed with 0.85 % saline solution. The debris of livers (and eggs) with dechlorinated tap water were poured into a round-bottomed flask with a long neck. The flask was kept at a temperature of 25 °C and exposed to artificial light to allow hatching of miracidia from the eggs. Two infection trials were performed. In the first trial conducted on 30 October 2013, snails were individually infected with a single miracidium. At first, miracidia were individually captured with a micropipette under a stereomicroscope and transferred to 40-well microplates, with one miracidium per well. Following that, one snail was put into a well and kept for 2 h. In the second trial on 30 November 2013, only the Shitai schistosome was used due to logistical constraints. In addition to the challenge infection of one snail by one miracidium, a group of marshland snails from each location was collectively exposed to a batch of miracidia with a snail to miracidia ratio of 1:10. The time and groups of experiment are listed in Table 1. After the challenge infection, snails were raised in the laboratory at room temperature (without using air conditioner). Spring water was sprayed twice a day (morning and late afternoon), and the culture paper was changed weekly and dead snails were removed.

Table 1 Infection rate, prepatent period, and cercarial production of S. japonicum
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Infection rates and cercarial production

Since the development of S. japonicum within intermediate host snails is largely influenced by environmental temperature (Yang et al. 2007), under our laboratory conditions, we checked snails for schistosome infection at day 120 postinfection. The snails shedding cercariae were separated and then raised individually on a 24-well plate with wet culture paper. The left snails were again tested for cercarial shedding 1 week later. This procedure was repeated until no infected snails were identified over two successive shedding experiments. Snails that survived and shed no cercariae until the end of the observation period were crushed and verified for the presence of the parasite (i.e., sporocysts or cercariae).

Chronobiology of cercarial emergence

A shedding experiment of circadian rhythm on the above infected snails was performed. The experiment started at 7:00 am and last for a 24-h period (for the procedure, see Lu et al. 2009; Su et al. 2013). Room temperature was set at 23 °C, suitable for the activities of snails and cercarial shedding, and no artificial light was used. The intensity of the light, through a window in the laboratory, was measured at the beginning and the end of each 2-h interval. The maximum light intensity over 24 h was 2,295 lx during 11:00 am to 1:00 pm. The number of cercariae that emerged from one snail was recorded in each section (i.e., 2 h) over 24 h, and then a daily rhythm, with different time points of cercarial emergence, was obtained.

Statistical analysis

Infection rates, prepatent period, and cercarial production were compared between/among different snail/parasite combinations. Average histograms were used for the description of a cercarial emergence rhythm. The significance was tested with ANOVA, t tests, or Mann–Whitney U test using SPSS Statistics 17.0 (SPSS Inc. Chicago, IL), and results were considered statistically significant at the level of 0.05. This study was approved by the Soochow University Ethics Committee, and the care and use of experimental animals complied with the institutional standards.

Results

Infection rates

Table 1 shows the results of the two snail infection trials. Infection rates in trial 1, in which all O. h. hupensis each were individually exposed to a single miracidium, significantly differed among four groups. The highest rate was observed in the combination of Wuxi schistosome (M, referring to a marshland region)/Zongyang snail (M), in which 26.7 % of snails were infected, and the lowest rate of 0.9 % was in the combination of Shitai schistosome (H, referring to the hilly region)/Shitai snail (H). Compared with the Shitai schistosome, the Wuxi schistosome seemed more compatible with both geographical strains of snails. However, for either parasite strains, marshland snails seemed more compatible to the hill snails.

In trial 2, two infection patterns were applied for the Shitai schistosome. The infection rates in pooled infection groups with a snail-to-miracidia ratio of 1:10 were 25.2 % in the combination of Shitai schistosome (H)/Zongyang snail (M) and 30.8 % in Shitai schistosome (H)/Hexian snail (M), significantly higher than those in the groups of snails with one miracidia per snail. When snails were individually exposed to one single miracidium, the infection rate in the Hexian snails (M) was significantly higher than that in the Shitai snails (hilly region) (χ 2 = 11.8, P = 0.001).

Prepatent period

In trial 1, the prepatent period significantly differed among four groups, with the shortest period (189.6 days) observed in the combination of Wuxi schistosome (M)/Zongyang snail (M) and the longest period (224.5 days) in Shitai schistosome (H)/Shitai snail (H). In trial 2, a significant difference in terms of prepatent period was also observed among four groups. When snails were individually exposed to one single miracidium, a longer but not significant prepatent period was observed in the combination of Shitai schistosome (H)/Shitai snail (H) than in the Shitai schistosome (H)/Hexian snail (M) (Z = 1.37, P = 0.17).

Cercarial output

The results of the total production of cercariae per infected snail over 24 h are summarized in Table 1. In trial 1, more cercariae were released in the Wuxi schistosome (M)/Zongyang snail (M) than in the Shitai schistosome (H)/Zongyang snail (M). In trial 2, when snails were individually exposed to one hill miracidium, more cercariae were released in Hexian snails than in Shitai snails. However, in both trials, no significant difference was observed between/among all compared groups (see Table 1).

Chronobiology of cercarial emergence

The patterns of daily cercarial production per snail are represented in Figs. 1, 2, and 3. A late afternoon shedding pattern was observed for the Shitai schistosome, either harbored in the hill snails or in the marshland snails and either via a single infection or multiple infections, with the highest peak around 7:00 pm–8:00 pm; whereas for the Wuxi schistosome, a morning shedding pattern was observed for both strains of snails with the highest peak around 7:00 am–9:00 am.

Fig. 1
figure 1

Emergence pattern of S. japonicum cercariae over 24 h with a single miracidial dose per snail. a Shitai schistosome (H)/Shitai snail (H). b Shitai schistosome (H)/Zongyang snail (M). c Shitai schistosome (H)/Hexian snail (M). H for the hilly region and M for the marshland

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Fig. 2
figure 2

Emergence pattern of S. japonicum cercariae over 24 h with a single miracidial dose per snail. a Wuxi schistosome (M)/Shitai snail (H). b Wuxi schistosome (M)/Zongyang snail (M). H for the hilly region and M for the marshland

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Fig. 3
figure 3

Emergence pattern of S. japonicum cercariae over 24 h with snails collectively exposed to multiple miracidia. a Shitai schistosome (H)/Zongyang snail (M). b Shitai schistosome (H)/Hexian snail (M). H for the hilly region and M for the marshland

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Discussion

The results of this study demonstrated a differential compatibility between S. japonicum and O. h. hupense from the hilly and the marshland regions. Both the hill (Shitai, Anhui) and the marshland (Wuxi, Jiangsu) strains of parasite were more infective to the marshland strains of snail (Zongyang and Hexian, Anhui) than to the hill strain of snail (Shitai, Anhui). When snails were individually exposed to one single miracidium, the longest prepatent period for cercarial development was observed in the combination of Shitai snail/Shitai schistosome in both trials. A late afternoon shedding pattern was observed for the hill parasite, regardless of the origin of the intermediate host snails.

Parasites are generally best at infecting hosts from the same endemic area (Cross et al. 1984; Ibikounle et al. 2012; Manning et al. 1995), as selection is predicted to favor local adaptation (Gandon et al. 1996). However, a meta-analysis on the S. mansoni-Biomphalaria alexandrina system in Egypt revealed no evidence of local adaptation for the parasite (Abou-El-Naga 2014). Here in our study, the hill strain of S. japonicum showed lack of adaptation to its local intermediate host, as the infection rates with a single miracidial dose per snail were 0.9 % in trial 1 and 5.7 % in trial 2, significantly lower than that in the corresponding hill parasite-marshland snail. Such low infection rates in the laboratory also correspond to the previous malacological surveys which revealed lower infection rates of 0.15–1.70 % (Lu et al. 2010) and of 0.78 % (Shi et al. 2014) in O. h. hupensis in the field. The lack of adaptation for the hill parasite is also indicated from the longest prepatent period observed, as discussed later.

An introduction of the field parasite into a laboratory often reduces the compatibility in the parasite to its original host population, and even a single generation of passage of parasite (through a different strain or isolate of snails) was reported to have a large effect on the results of compatibility trials (Theron et al. 2008). However, to our surprise, in this study, the highest infection rate (up to 26.7 %) and the shortest prepatent period between O. h. hupense snails from Zongyang, Anhui (marshland region), and S. japonicum from Wuxi, Jiangsu (marshland region), indicated a highly compatible intermediate host-parasite relationship, although this parasite strain has been maintained in the laboratory for over 30 years. In addition, the susceptibility of this parasite to the hill snails is also comparable with that of the hill parasite.

The prepatent period of schistosome within snails positively correlated with environment temperature. The value of 15.3 °C was, in theory, considered the lowest developing temperature for S. japonicum, and the prepatent period of 210 days was obtained at 18 °C (Yang et al. 2007). Under our laboratory conditions (with no air conditioner or artificial light), the prepatent period varied between 189.6 and 224.5 days in the first trial started from 30 October 2013, and between 194.7 and 186.1 days in the second trial from 30 November 2013. Considering the average temperature of lower than 10 °C during December of 2013 to February of 2014 and of higher than 20 °C during June to July of 2014, the results here confirmed or, at least, were comparable with the above estimate. The difference in the prepatent period varied with schistosome strains (marshland < hilly region) or snail strains (marshland < hilly region), indicative of a possible differential compatibility.

The analysis of our cercarial shedding data presented no significant difference in the cercarial output per snail over 24 h between/among groups of snails in either trial. It is reported that the subspecies 0. h. quadrasi exposed to one single miracidia released more cercariae than to those exposed to two to five miracidia (Pesigan et al. 1958). Research on other schistosomes did not show a consistence in cercarial production between single infection and multiple infections (Mouahid and Combes 1987; Norton et al. 2008; Theron et al. 1997). The influential factors may involve background of snails (Lepesant et al. 2013; Zavodna et al. 2008), male or female parasites (Boissier et al. 1999), schistosome sex ratio (Claveria and Etges 1987), or possible environmental effects (Steinauer 2009).

The chronobiology experiments demonstrated two shedding patterns, a late afternoon emergence associated with the hill strain of parasite and a morning emergence with the marshland strain, which corroborate our previous research (Lu et al. 2009; Su et al. 2013). In addition, the same or typical circadian rhythm for the hill parasite harbored in two geographical strains of snails further verified that cercarial emergence rhythms are genetically controlled, as confirmed on other schistosomes (Theron 1989; Theron and Combes 1988). This indicates that cercarial emergence could be used as a biological marker to identify the hill parasite once the parasite is introduced into the Yangtze River valley.

In conclusion, our results on the infection rate, prepatent period, and cercarial emergence demonstrated a high compatibility between the marshland strains of snail and both strains of parasite. This would have practical implications. On one hand, the hill strain of parasite would, if transferred into marshland regions, be successfully established in the local intermediate host snails and cause a possible transmission of the disease. On the other, the introduction of the marshland snails, with an inverse water flow due to flooding in the Yangtze River, into the hilly region may worsen disease transmission, especially if immigrated snails either reproduce themselves or outcross with local host snails. The present study also raises the question whether the lower compatible relationship between the hill parasite and its local intermediate hosts is because of the snail, of the parasite, of the lab environment, or of their interplay. More experiments or molecular studies would be needed.