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Genetic diversity of Schistosoma japonicum miracidia from individual rodent hosts
International Journal for Parasitology 41 (2011) 1371–1376
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Genetic diversity of Schistosoma japonicum miracidia from individual rodent hosts Da-Bing Lu a,b,⇑, Tian-Ping Wang c, James W. Rudge b,d, Christl A. Donnelly b, Guo-Ren Fang c, Joanne P. Webster b a
Department of Epidemiology and Statistics, School of Public Health, Medical College of Soochow University, Suzhou 215123, China Department of Infectious Disease Epidemiology, School of Public Health, Faculty of Medicine, Imperial College, Norfolk Place, London W2 1PG, UK c Anhui Institute of Parasitic Diseases, Hefei 230061, China d Department of Global Health and Development, London School of Hygiene and Tropical Medicine, Bangkok, Thailand b
a r t i c l e
i n f o
Article history: Received 2 June 2011 Received in revised form 8 September 2011 Accepted 9 September 2011 Available online 29 October 2011 Keywords: Schistosoma japonicum Infrapopulation Rodents Genetic diversity
a b s t r a c t Schistosoma japonicum is an important parasite in terms of clinical, veterinary and socio-economic impacts, and rodents, a long neglected reservoir for the parasite, have recently been found to act as reservoir hosts in some endemic areas of China. Any difference in the host’s biological characteristics and/or associated living habitats among rodents may result in different environments for parasites, possibly resulting in a specific population structure of parasites within hosts. Therefore knowledge of the genetic structure of parasites within individual rodents could improve our understanding of transmission dynamics and hence our ability to develop effective control strategies. In this study, we aimed to describe a host-specific structure for S. japonicum and its potential influencing factors. The results showed a significant genetic differentiation among hosts. Two factors, including sampling seasons and the number of miracidia genotyped per host, showed an effect on the genetic diversity of an infrapopulation through a univariable analysis but not a multivariable analysis. A possible scenario of clustered infection foci and the fact of multiple definitive host species, the latter of which is unique to S. japonicum compared with other schistosomes, were proposed to explain the observed results and practical implications for control strategies are recommended. Ó 2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Schistosoma japonicum, which has been reported to infect 46 mammalian species naturally (He et al., 2001), is an important parasite in terms of clinical, veterinary and socio-economic impacts. Knowledge of the genetic structure of the parasite population would improve our understanding of transmission dynamics between hosts, as well as between regions and host species. Several studies have recently characterised differences in the population genetic structure of S. japonicum between villages (Rudge et al., 2009; Lu et al., 2010a), provinces (Shrivastava et al., 2005) and countries (Rudge et al., 2008), but little is known about the parasite population genetic structure at the level of individual host. A definitive host is, however, anticipated to be an important factor affecting the genetic structure of the parasite infrapopulation (a group of parasites of the same species within one individual host) (Caillaud et al., 2006), as any difference in the host’s biological characteristics
⇑ Corresponding author at: Department of Epidemiology and Statistics, School of Public Health, Medical College of Soochow University, Suzhou 215123, China. Tel.: +86 512 6588 0079; fax: +86 512 6588 4830. E-mail address: [email protected] (D.-B. Lu).
and/or associated living habitats may result in different environments for parasites. The biological differences between male and female hosts, in terms of sex-specific behaviour and the level of steroid hormones in males, for example, have been used to explain differential susceptibility to parasites between female and male vertebrate hosts (Klein, 2000; Christe et al., 2007). This may be translated to hostsex-specific genetic patterns in nature, as illustrated in the system Schistosoma mansoni and its natural host Rattus rattus in Guadeloupe, West Indies (Caillaud et al., 2006). After investigating the neutral genetic variability of S. mansoni within its natural murine host R. rattus, Caillaud and colleagues demonstrated that schistosomes from male hosts were genetically more diverse than from female hosts, although an experiment in mice displayed that male hosts were more resistant to S. mansoni than females (Eloi-Santos et al., 1992). Such findings suggest that male and female hosts may potentially play different roles in parasite maintenance. This could have significant implications for the control of parasites, as infection control of parasites in one gender would, in theory at least, potentially lead to a decrease of the intensity of the parasite in the other gender (Ferrari et al., 2004). Rodents have been found to act as reservoir hosts for S. japonicum in some hilly areas of China (Lu et al., 2009). Even in marshland/lake
0020-7519/$36.00 Ó 2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2011.09.002
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areas, where bovines have generally been regarded as playing a primary role in the maintenance and/or transmission of parasites, high infection prevalence levels of S. japonicum in rodents have frequently been reported (Xu et al., 1999; Zhao et al., 2009; Zhang et al., 2010) and one strain of S. japonicum parasites, associated with rodents, has been observed in a marshland village (Lu et al., 2009). The ongoing programme of replacing bovines with machines for schistosomiasis control across endemic areas of China (Wang et al., 2009) may have already reduced the role of this species in parasite transmission. Rodents, however, remain a major issue in hilly endemic areas and may even become major reservoirs in currently bovine-maintained transmission in marshland/lake areas. Therefore, data on population genetics of parasites from such host rodents will inform improvements in control strategies. Moreover, such research may provide insights into the mechanisms underlying the development of a rodent-associated parasite strain (Lu et al., 2009). In this study, we quantified within-host parasite genetic structure with six highly polymorphic microsatellite markers for S. japonicum (Shrivastava et al., 2003) and investigated the effect on this for a few potential influential factors. In addition, to test the hypothesis of the existence of host gender-specific structure (Prugnolle et al., 2003) for S. japonicum, we computed and compared statistical estimates of FIS (Wright, 1951), a measure of the extent of the departure from panmixia (an individual of a population is equally likely to mate with any other individual of that population) (Curtis and Minchella, 2000), and FST (Weir and Cockerham, 1984), a measure of the difference in allelic frequencies between populations. To the best of our knowledge, this is the first study to examine the genetic structure of S. japonicum specifically at the level of individual hosts.
2. Materials and methods 2.1. Rodent trapping and parasite sampling and genotyping The study was performed in three hilly villages in Shitai county of Anhui province of China during October/November of both 2006 and 2007. Detailed geographic and demographic information regarding S. japonicum transmission in the sampled villages, as well as the procedure for capturing rodents, sampling their faeces and performing faecal examinations, have been previously reported (Lu et al., 2010b). Briefly, in such hilly areas, farmers’ houses often form small groups within each village, sometimes separated by roads or small rivers, and their fields are often located nearby. Snails live in ditches around fields. Snail surveys were systematically conducted in all ditches during the spring of each year and the locations of infected snails were mapped accordingly for setting live traps for rodents during the following autumn. Depending on the area of an infected snail habitat, traps were set at an interval distance of 5–10 m. Small rodents were captured by setting traps in the late afternoon and checking at dawn for three consecutive days and captured rodents were kept inside overnight to obtain their faecal samples. Each captured rodent had its gender recorded and its body mass measured with an electronic balance while anaesthetized. All captured rodents belonged to Apodemus agrarius or Rattus norvegicus. After an infection with S. japonicum was identified in a rodent, individual miracidia were sampled from each host and 10–12 miracidia (if available) per host were genotyped using six microsatellite markers (Shrivastava et al., 2003). Infection intensity in individual hosts was measured as eggs plus hatched miracidia per gram of faeces (Lu et al., 2010b). The protocols on parasite larvae DNA storage, extraction, DNA multiplex amplification, genotyping of PCR products and assignment of allele sizes have been presented previously (Rudge et al., 2009; Lu et al., 2010a).
2.2. Molecular analyses 2.2.1. Genetic structure within host sexes As host sex is considered a potentially important factor influencing the population genetic structure of parasites, the population structure of S. japonicum within each host sex was characterised. The unbiased estimate of FIS (Wright, 1951) was computed over all loci and all parasite infrapopulations (an infrapopulation here refers to all miracidia sampled from one host) from one sex according to Weir and Cockerham (1984) and the 95% confidence intervals (CIs) were obtained through bootstrapping. The hypothesis that FIS equalled 0 was tested using a procedure of random permutations of alleles between individual miracidia within each sex set at 15,000 permutations. The value of FST (Weir and Cockerham, 1984) was calculated over all loci to measure the difference in allelic frequencies between infrapopulations for each host sex. Bootstrapping was used to obtain 95% CIs for each FST value. Genetic differentiation was tested using the G-based test, which takes into account the particular structure within each infrapopulation (Goudet et al., 1996), distinct from the conventional exact test based on Hardy-Weinberg (HW) expectations (Raymond and Rousset, 1995). The multiple comparisons between males and females of FIS and FST were performed with the application of Bonferroni corrections (Holm, 1979) due to the repetitive nature of the procedure. All calculations and tests were performed using FSTAT V. 2.9.3 (http:// www.unil.ch/izea/softwares/fstat.html). 2.2.2. Factors influencing genetic diversity and deviations from HW expectations of an infrapopulation The genetic diversity (HS) of a parasite infrapopulation was computed using Nei’s unbiased mean heterozygosity (Nei, 1987), and the unbiased estimate of Wright’s FIS within an infrapopulation was computed over all loci for each infected rodent. To examine any effects of host body mass, infection intensity, number of miracidia genotyped per host, sampling years and locations, together with host sex, on the HS and the deviation from HW expectations (FIS) of an infrapopulation, we conducted univariable analyses and then multivariable analyses using STATA 7 software (Stata Corporation, 702 University Drive East, College Station, TX 77840 USA). If an obvious disagreement was observed between the univariable analyses and the multivariable analyses, a collinearity diagnostic, using a regression analysis, of all test factors was performed and the indices of Tolerance and the variance inflation factor (VIF) were calculated using SPSS 11.0 software (SPSS Base 11.0 for Windows. 2002. SPSS Inc. Chicago, Illinois, USA). 3. Results 3.1. Infection and genetic structure of parasites within host sexes Out of the 49 and 51 rodents trapped in 2006 and 2007, respectively, 13 and nine were infected with S. japonicum. Non-significantly higher infection prevalence was observed in male rodents (25–30%) than in female rodents (9–21%) in both years (Table 1). We could not measure infection intensity for each rodent (Table 3), due to difficulties encountered in the field. Table 2 shows that parasites sampled from females in 2006 displayed a significant departure from HW expectations (under the null hypothesis FIS = 0), as did those from males in 2007. With the exception of the parasites from males in 2007, all parasite populations either from males or from females showed a significant genetic differentiation among individual hosts. As seen in Fig. 1, in 2007 FIS was larger in males than in females and FST was smaller in males than in females. The two 95% CIs, either FIS or FST, however, indeed overlapped. Such a different trend
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D.-B. Lu et al. / International Journal for Parasitology 41 (2011) 1371–1376 Table 1 Schistosoma japonicum infection in male and female rodents during 2006 and 2007 in Shitai county, Anhui province, China. Village/year
Male
Female
No. captured
No. infected
No. captured
No. infected
2006 Longquan Longshang Yuantou Total
11 15 4 30
4 4 1 9 (30%)
7 7 5 19
2 1 1 4 (21%)
2007 Longquan Longshang Yuantou Total
7 13 8 28
3 2 2 7 (25%)
5 9 9 23
1 1 0 2 (9%)
Table 2 Genetic structure of Schistosoma japonicum miracidia within rodent males and females. Year
Sex
FIS (95% CI)
FST (95% CI)
2006
Male
0.056 ( 0.199, 0.158) P = 0.929 0.173 (0.001, 0.467) P = 0.006 0.421 (0.023, 0.742) P < 0.001 0.121 ( 0.116,0.397) P = 0.082
0.295 (0.194,0.396) P < 0.001 0.461 (0.343,0.585) P < 0.001 0.232 ( 0.072,0.454) P = 0.337 0.404 (0.258,0.510) P = 0.003
Female 2007
Male Female
Tests for the departure from Hardy-Weinberg (HW) expectations were conducted by permutating alleles within infrapopulations 15,000 times and for population differentiation by permutating genotypes between infrapopulations 15,000 times. 95% confidence interval (CI) was obtained by a bootstrap. FIS, Inbreeding coefficient; FST, difference in allelic frequencies between populations.
(FIS versus FST) did not occur in the local hosts in 2006 with both FIS and FST in males less than in females (Fig. 1) and, after using Bonferroni corrections, no difference between sexes was observed in FIS (P = 0.163) and FST (P = 0.085).
3.2. Genetic diversity of an infrapopulation and its potential influential factors A total of 196 miracidia were genotyped from 22 rodents with an average of 8.9 larvae per host. The unbiased mean heterozygosity (HS) of an infrapopulation varied between 0.30 and 0.58 among hosts, with the exception of 0.83 when only two miracida were genotyped, and the inbreeding coefficient (FIS) ranged widely from 0.57 to 0.51 (Table 3). As can be seen in Table 4, the univariable analyses revealed that only two variables, year of sampling and number of miracidia genotyped per host, were significantly correlated with the genetic diversity (HS). Neither effect was significant in a multivariable model including both of these effects (P > 0.1 in both cases). No variables examined were observed to have a significant impact on the inbreeding coefficient FIS. Due to the difference between the two types of analyses, two new (dummy) variables were computed from the variable of Village to categorise three villages for performing collinearity diagnostics. As seen in Table 4, all of the values of Tolerance are over 0.1 and those of VIF were less than 10, suggesting that there was no collinearity among factors.
4. Discussion Of the human schistosomes, S. japonicum is the only schistosome for which zoonotic transmission is considered important (Nelson, 1972), with over 40 species of wild and domesticated animals suspected of serving as reservoir hosts for this parasite (He et al., 2001). In the studied areas previous research has, based on the infection prevalence and intensity in each species, including humans, dogs, cats, pigs, bovines and rodents (Lu et al., 2010b), and the biological traits of the parasites from local infected snails (Lu et al., 2009), demonstrated that small rodents have been suggested to play an important role in the transmission of S. japonicum to humans and other mammals in the hilly regions. In this study, we analysed the genetic structures of S. japonicum miracidia within and between rodent individuals in three hilly villages of China. The results demonstrated that a significant genetic differentiation of infrapopulations (FST values) was seen among hosts within either gender (with the exception of males in 2007), and that two factors
Table 3 Ecological and genetic characteristics of Schistosoma japonicum miracidia infrapopulations within individual rodents (HS, genetic diversity; FIS, inbreeding coefficient). Host
Year
Village
Host sex
Host body mass (g)
Infection intensity (EPG)
No. miracidia genotyped
HS
LQS02 LQS03 LQS04 LQS05 LQS15 LQS18 LSS22 LSS23 LSS25 LSS32 LSS42 YTS47 YTS49 LQM02 LQM09 LQM10 LQM13 LSM09 LSM17 LSM19 YTM08 YTM09
2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2007 2007 2007 2007 2007 2007 2007 2007 2007
Longquan Longquan Longquan Longquan Longquan Longquan Longshang Longshang Longshang Longshang Longshang Yuantou Yuantou Longquan Longquan Longquan Longquan Longshang Longshang Longshang Yuantou Yuantou
Male Male Female Male Female Male Male Male Female Male Male Male Female Male Male Female Male Male Female Male Male Male
25.0 120.0 120.0 140.0 31.2 123.1 127.0 143.1 141.0 47.5 36.4 116.4 31.0 79.4 19.8 34.0 61.6 152.1 128.0 92.7 31.6 134.0
– – – – – – 283.6 3311.7 – – 201.5 40.3 85.7 105.0 825.0 21.1 13.7 4.8 150.6 6.5 7.1 3268.3
11 12 9 10 10 11 11 10 10 11 9 11 11 12 8 4a 7a 2a 12 3a 1a 11
0.55 0.50 0.42 0.39 0.30 0.57 0.33 0.47 0.41 0.36 0.44 0.32 0.40 0.54 0.58 0.52 0.45 0.83 0.48 0.50 – 0.42
EPG, eggs per gram of faeces. a Less than eight miracidia were genotyped from each of five hosts due to small numbers of miracidia samples available.
FIS 0.08 0.12 0.51 0.24 0.33 0.00 0.04 0.06 0.14 0.55 0.41 0.57 0.08 0.12 0.36 0.06 0.15 0.20 0.15 0.33 – 0.35
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Fig. 1. Comparison of genetic structures of Schistosoma japonicum from male and female rodents, during 2006 and 2007 in Shitai county, Anhui province, China. (A) Inbreeding coefficient (FIS) estimates with 95% confidence intervals (CIs) and (B) difference in allelic frequencies between populations (FST) estimates with 95% CIs.
including sampling seasons and number of miracidia per host, based on an univariable analysis, may have an effect on the genetic diversity (Hs) of an infrapopulation. All observed factors from a multivariable analysis, however, showed no effect on the genetic diversity or the HW expectation of a parasite infrapopulation within a host. An infra-population of parasites is exclusively formed by recruitment of external free-living infective stages, cercariae for S. japonicum, and consequently any genetic structure of the parasites at the level of individual hosts would mainly result from the recruitment histories experienced by each host, which may depend on host movement and its living environment, differing from individual to individual. Rodents are usually considered to be highly philopatric, as they are often recaptured within a short distance of their first capture location, for example within only 14– 27 m (Delattre and Louarn, 1981). It is therefore conceivable that most infected rodents may deposit their parasites or their schistosome progeny, via faeces, in their own small areas. As a consequence the parasites, in relation to each other in terms of genetic make-up, may stay in close proximity when transmitted by such a host. Moreover, the above clumped transmission could be facilitated by the unique characteristics of S. japonicum compared with other schistosomes infecting humans such as S. mansoni and Schistosoma haemotobium in terms of biochemical and immunological features (Ruppel et al., 2004). Schistosoma japonicum cercariae, for example, have shown a remarkable speed of migration through a host’s skin, partially due to the effect of more potent penetrating enzymes released from parasite larvae on the skin penetration (He et al., 2002). Another main aspect for this parasite is that the cercariae, after being shed from their intermediate host, cluster together on the water, possibly due to an unknown chemical released by the parasite which makes the surrounding water very sticky (Spear et al., 2004). Such patchy foci may be further enhanced by the amphibious snail host, Oncomelania hupensis, which prefers moist earth to water (Mao, 1990). When submerged in water, this host species will mostly immediately climb out if possible, leaving a very narrow window for the larvae to escape from the host body
into the water. It would be imagined that an irregular distribution and aggregation of parasites in terms of space would develop under this context. Therefore, the more often a rodent frequents a limited area of one focus, the larger inbreeding coefficient (FIS) of the parasite infrapopulation it may have; and for different host individuals, the higher genetic differentiation of parasites between them may occur. Such genetic differentiation between rodents could be reduced more or less if they experience a few or all of the foci. This could explain the significant genetic differentiation between hosts and the quite wide range of the inbreeding coefficients among hosts observed in our study. We also observed a possible effect of sampling years on the genetic diversity of a parasite infrapopulation from an univariable analysis. This could be partly due to the existence of differences between those two years in the numbers of infected snail habitats, in the infection profile among mammalian species (Lu et al., 2010b), probably in control measures, and in the numbers of miracidia genotyped, in the last of which both the average number of miracidia genotyped per host and the number of infected rodent females captured were smaller in 2007 than in 2006. However, no significant effect of sampling years was seen when using a multivariable analysis. The intrinsic differences between female and male hosts in physiology, immunology, behaviour and ecology can make one sex or the other a more benign environment for parasites (Zuk and McKean, 1996; Christe et al., 2007). A male bias in parasitism has been reported to occur in a wide range of mammalian species (Poulin, 1996; Klein, 2000; Moore and Wilson, 2002; Amo et al., 2005), but several exceptions were also reported (Eloi-Santos et al., 1992; Christe et al., 2007; Ci et al., 2008). A strong influence of the sex of the host on the genetic variability of their parasites was observed for the system of S. mansoni and their hosts R. rattus in a marshy forest focus, in which parasite populations of male rats were more genetically diverse than parasites of female hosts (Caillaud et al., 2006). One plausible explanation is accredited to a higher immunity-based, diversifying selection process, a synergistic effect of testosterone, which may inhibit mitochondrial respiratory chain function of the parasite larvae at the schistosomula stage, and host immune ability, acting in male hosts (Caillaud et al., 2006). To our surprise, however, in this study of S. japonicum and its definitive hosts, we observed a slight but non-significant difference in genetic structure of parasites between male and female hosts. Although rodents are limited to their own territories, males are usually more active and often disperse across wider geographic ranges than females (Dobson, 1982), and might be more likely to recruit more infective forms of larvae than females, thus possibly leading to the observed lower genetic differentiation (FST) among males than among females. Moreover, in the studied areas, apart from infected rodents, infection with the parasite was also identified in humans, dogs and cats, particularly in dogs with a comparable infection prevalence (Lu et al., 2010b). An infected dog with a longer life span, compared with a rodent with a shorter life span of 2 years on average, may recruit more external infective forms of parasites during that period and, as a consequence, coupled with an average life span of 5 years for schistosomes within their hosts (Bush et al., 2001), may excrete a well-mixed pool of parasite offspring in terms of genotypes. This may add to the complex population genetic structures observed from the local rodents only, i.e. in terms of FIS, males < females in 2006 but males > females in 2007, as the extent to which dogs have spread the parasites may vary with villages or years (Lu et al., 2010a). In our study, we observed a negative effect of the number of miracidia genotyped per host on genetic diversity of an infrapopulation from a univariable analysis, but no effect from a multivariable analysis. Sampling and genotyping parasite larvae for inferring population genetic structures, together with sample size,
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Table 4 Univariable analyses of factors influencing the genetic diversity (HS) and inbreeding coefficient (FIS) of Schistosoma japonicum miracidia infrapopulations and collinearity statistics. Variable
HS P
FIS Reg. coef.
95% CI
P
Year
0.017
0.12
0.2165,
0.0235
Village Longquan/Yuantoua Longshang/Yuantoub Host sex Host body mass Infection intensity No. miracidia genotyped per host
0.201 0.233 0.284 0.648 0.688 0.013
0.102 0.0975 0.0617 0.0003 0.00001 0.0212
0.0595, 0.2635 0.0686, 0.2636 0.0554, 0.1788 0.0009, 0.0014 0.00008, 0.00006 0.0373, 0.0051
Collinearity statistics Tolerance
VIF
0.143
Reg. coef. 0.1835
95% CI 0.4347, 0.0677
0.435
2.300
0.306 0.46 0.257 0.676 0.552 0.209
0.1957 0.1442 0.1543 0.0006 0.00004 0.0269
0.1946, 0.2572, 0.4307, 0.0033, 0.0001, 0.0701,
0.262 0.354 0.765 0.395 0.723 0.603
3.820 2.822 1.307 2.529 1.384 1.657
0.5860 0.5456 0.1221 0.0022 0.0002 0.0164
CI, confidence interval; VIF, variance inflation factor. Two new variables derived from the variable of ‘village’ for collinearity statistics.
a,b
is an inherent limitation in this sort of study, which has been detailed in a review (Steinauer et al., 2010). However, our research did find a significant genetic differentiation among hosts and two possible factors influencing the genetic diversity of the parasites. Future work based on population genetics of adult worms within and/or between rodent individuals and of gender typed larvae, if possible, produced from such worms would further our understanding of the transmission of S. japonicum maintained by this host species, as the direct comparison of population genetic structures between two stages, adult worms and miracidia, will provide deep insights into the transmission dynamics between rodents and any associated biological characteristics. In conclusion, in this study we investigated the genetic structure of parasites within rodent individuals and sexes and any potential influencing factors. We did not observe any host sexspecific genetic structure but found significant genetic differentiation between hosts and a considerable range of genetic diversity of parasites among host individuals. The results suggest that intensive density/population control for local rodents may be an effective measure for militating against transmission of this parasite, as control measures targeted at individuals contributing the most to transmission can be very efficient but, conversely, there will be a high risk of it being ineffective if any of these individuals are missed (Woolhouse, 1998), particularly for rodents inherent with sampling difficulties. Acknowledgements This work was funded by the Royal Society, UK (to JPW), Kwok Foundation, Hongkong, China (to DBL, CAD and JPW) and the Medical Research Council, UK (to JWR). The laboratory work was performed at Imperial College, London, UK, when Dabing Lu studied there as a PhD student. References Amo, L., Lopez, P., Martin, J., 2005. Prevalence and intensity of haemogregarine blood parasites and their mite vectors in the common wall lizard, Podarcis muralis. Parasitol. Res. 96, 378–381. Bush, A.O., Fernández, J.C., Esch, G.W., Seed, J.R., 2001. Parasitism: The Diversity and Ecology of Animal Parasites. Cambridge University Press, Cambridge. Caillaud, D., Prugnolle, F., Durand, P., Theron, A., de Meeus, T., 2006. Host sex and parasite genetic diversity. Microb. Infect. 8, 2477–2483. Christe, P., Glaizot, O., Evanno, G., Bruyndonckx, N., Devevey, G., Yannic, G., Patthey, P., Maeder, A., Vogel, P., Arlettaz, R., 2007. Host sex and ectoparasites choice: preference for, and higher survival on female hosts. J. Anim. Ecol. 76, 703–710. Ci, H.X., Lin, G.H., Su, J.P., Cao, Y.F., 2008. Host sex and ectoparasite infections of plateau pika (Ochotona curzoniae, Hodgson) on the Qinghai Tibetan Plateau. Pol. J. Ecol. 56, 535–539. Curtis, J., Minchella, D.J., 2000. Schistosome population genetic structure: when clumping worms is not just splitting hairs. Parasitol. Today 16, 68–71. Delattre, P., Louarn, H.L., 1981. Dynamique des populations du rat noir, Rattus rattus, en mangrove lacustre. Mammalia 45, 275–288.
Dobson, F.S., 1982. Competition for mates and predominant juvenile male dispersal in mammals. Anim. Behav. 30, 1183–1192. Eloi-Santos, S., Olsen, N.J., Correa-Oliveira, R., Colley, D.G., 1992. Schistosoma mansoni: mortality, pathophysiology, and susceptibility differences in male and female mice. Exp. Parasitol. 75, 168–175. Ferrari, N., Cattadori, I.M., Nespereira, J., Rizzoli, A., Hudson, P.J., 2004. The role of host sex in parasite dynamics: field experiments on the yellow-necked mouse Apodemus flavicollis. Ecol. Lett. 7, 88–94. Goudet, J., Raymond, M., de Meeus, T., Rousset, F., 1996. Testing differentiation in diploid populations. Genetics 144, 1933–1940. He, Y.X., Salafsky, B., Ramaswamy, K., 2001. Host–parasite relationships of Schistosoma japonicum in mammalian hosts. Trends Parasitol. 17, 320–324. He, Y.X., Chen, L., Ramaswamy, K., 2002. Schistosoma mansoni, S. haematobium, and S. japonicum: early events associated with penetration and migration of schistosomula through human skin. Exp. Parasitol. 102, 99–108. Holm, S., 1979. A simple sequentially rejective multiple test procedure. Scand. J. Statist. 6, 65–70. Klein, S.L., 2000. The effects of hormones on sex differences in infection: from genes to behavior. Neurosci. Biobehav. Rev. 24, 627–638. Lu, D.B., Wang, T.P., Rudge, J.W., Donnelly, C.A., Fang, G.R., Webster, J.P., 2009. Evolution in a multi-host parasite: chronobiological circadian rhythm and population genetics of Schistosoma japonicum cercariae indicates contrasting definitive host reservoirs by habitat. Int. J. Parasitol. 39, 1581–1588. Lu, D.B., Rudge, J.W., Wang, T.P., Donnelly, C.A., Fang, G.R., Webster, J.P., 2010a. Transmission of Schistosoma japonicum in marshland and hilly regions of China: parasite population genetic and sibship structure. PLoS Negl. Trop. Dis. 4, e781. Lu, D.B., Wang, T.P., Rudge, J.W., Donnelly, C.A., Fang, G.R., Webster, J.P., 2010b. Contrasting reservoirs for Schistosoma japonicum between marshland and hilly regions in Anhui, China – a two-year longitudinal parasitological survey. Parasitology 137, 99–110. Mao, S.P., 1990. Schistosome Biology and Control of Schistosomiasis. Publishing House of People’s Health, Beijing. Moore, S.L., Wilson, K., 2002. Parasites as a viability cost of sexual selection in natural populations of mammals. Science 297, 2015–2018. Nei, M., 1987. Molecular Evolutionary Genetics. Columbia University Press, New York. Nelson, G.S., 1972. Immunological control of schistosomiasis. Brit. Med. J. 4, 172. Poulin, R., 1996. Helminth growth in vertebrate hosts: does host sex matter? Int. J. Parasitol. 26, 1311–1315. Prugnolle, F., Durand, P., Theron, A., Chevillon, C., de Meeus, T., 2003. Sex-specific genetic structure: new trends for dioecious parasites. Trends Parasitol. 19, 171– 174. Raymond, M., Rousset, F., 1995. An exact test for population differentiation. Evolution 49, 1280–1283. Rudge, J.W., Carabin, H., Balolong, E., Tallo, V., Shrivastava, J., Lu, D.B., Basanez, M.G., Olveda, R., McGarvey, S.T., Webster, J.P., 2008. Population genetics of Schistosoma japonicum within the Philippines suggest high levels of transmission between humans and dogs. PLoS Negl. Trop. Dis. 2, e340. Rudge, J.W., Lu, D.B., Fang, G.R., Wang, T.P., Basanez, M.G., Webster, J.P., 2009. Parasite genetic differentiation by habitat type and host species: molecular epidemiology of Schistosoma japonicum in hilly and marshland areas of Anhui Province. China Mol. Ecol. 18, 2134–2147. Ruppel, A., Chlichlia, K., Bahgat, M., 2004. Invasion by schistosome cercariae: neglected aspects in Schistosoma japonicum. Trends Parasitol. 20, 397–400. Shrivastava, J., Barker, G.C., Johansen, M.V., Zhou, X.N., Aligui, G.D., 2003. Isolation and characterization of polymorphic DNA microsatellite markers from Schistosoma japonicum. Mol. Ecol. Notes 3, 406–408. Shrivastava, J., Qian, B.Z., McVean, G., Webster, J.P., 2005. An insight into the genetic variation of Schistosoma japonicum in mainland China using DNA microsatellite markers. Mol. Ecol. 14, 839–849. Spear, R.C., Seto, E., Liang, S., Birkner, M., Hubbard, A., Qiu, D., Yang, C., Zhong, B., Xu, F., Gu, X., Davis, G.M., 2004. Factors influencing the transmission of Schistosoma japonicum in the mountains of Sichuan Province of China. Am. J. Trop. Med. Hyg. 70, 48–56.
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D.-B. Lu et al. / International Journal for Parasitology 41 (2011) 1371–1376
Steinauer, M.L., Blouin, M.S., Criscione, C.D., 2010. Applying evolutionary genetics to schistosome epidemiology. Infect. Genet. Evol. 10, 433–443. Wang, L.D., Chen, H.G., Guo, J.G., Zeng, X.J., Hong, X.L., Xiong, J.J., Wu, X.H., Wang, X.H., Wang, L.Y., Xia, G., Hao, Y., Chin, D.P., Zhou, X.N., 2009. A strategy to control transmission of Schistosoma japonicum in China. New Engl. J. Med. 360, 121–128. Weir, B.S., Cockerham, C.C., 1984. Estimating F-statistics for the analysis of population structure. Evolution 38, 1358–1370. Woolhouse, M.M.E., 1998. Heterogeneities in schistosome transmission dynamics and control. Parasitology 117, 475–482. Wright, S., 1951. The genetical structure of populations. Ann. Eugen. 15, 323–354.
Xu, G.Y., Tian, J.C., Cheng, G.M., Yang, H.M., Qiu, L., Hu, H.B., Xie, Z.Y., Zhou, F., Ying, W.G., 1999. Observation on natural focal disease of schistosomiasis in Rattus norvegicus in Nanjing. Chin. J. Para. Dis. 7, 4–6. Zhang, Q.D., Wu, Y.F., Xiao, M., Li, C.N., Su, J.G., Suo, Y., Dai, J.R., 2010. A survey of field mouse density and the rate of schistosome infection in rodents in marshlands of the City of Yangzhong. J. Path. Biol. 6, 230–231. Zhao, H.M., Su, J.Y., Liu, C.H., Zhao, S.Y., Wang, F.Y., 2009. Survey of Schistosoma japonicum in rodents in Sihu region. Hubei. Chin. J. Zoon. 25, 919–920. Zuk, M., McKean, K.A., 1996. Sex differences in parasite infections: patterns and processes. Int. J. Parasitol. 26, 1009–1023.
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International Journal for Parasitology journal homepage: www.elsevier.com/locate/ijpara
Genetic diversity of Schistosoma japonicum miracidia from individual rodent hosts Da-Bing Lu a,b,⇑, Tian-Ping Wang c, James W. Rudge b,d, Christl A. Donnelly b, Guo-Ren Fang c, Joanne P. Webster b a
Department of Epidemiology and Statistics, School of Public Health, Medical College of Soochow University, Suzhou 215123, China Department of Infectious Disease Epidemiology, School of Public Health, Faculty of Medicine, Imperial College, Norfolk Place, London W2 1PG, UK c Anhui Institute of Parasitic Diseases, Hefei 230061, China d Department of Global Health and Development, London School of Hygiene and Tropical Medicine, Bangkok, Thailand b
a r t i c l e
i n f o
Article history: Received 2 June 2011 Received in revised form 8 September 2011 Accepted 9 September 2011 Available online 29 October 2011 Keywords: Schistosoma japonicum Infrapopulation Rodents Genetic diversity
a b s t r a c t Schistosoma japonicum is an important parasite in terms of clinical, veterinary and socio-economic impacts, and rodents, a long neglected reservoir for the parasite, have recently been found to act as reservoir hosts in some endemic areas of China. Any difference in the host’s biological characteristics and/or associated living habitats among rodents may result in different environments for parasites, possibly resulting in a specific population structure of parasites within hosts. Therefore knowledge of the genetic structure of parasites within individual rodents could improve our understanding of transmission dynamics and hence our ability to develop effective control strategies. In this study, we aimed to describe a host-specific structure for S. japonicum and its potential influencing factors. The results showed a significant genetic differentiation among hosts. Two factors, including sampling seasons and the number of miracidia genotyped per host, showed an effect on the genetic diversity of an infrapopulation through a univariable analysis but not a multivariable analysis. A possible scenario of clustered infection foci and the fact of multiple definitive host species, the latter of which is unique to S. japonicum compared with other schistosomes, were proposed to explain the observed results and practical implications for control strategies are recommended. Ó 2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Schistosoma japonicum, which has been reported to infect 46 mammalian species naturally (He et al., 2001), is an important parasite in terms of clinical, veterinary and socio-economic impacts. Knowledge of the genetic structure of the parasite population would improve our understanding of transmission dynamics between hosts, as well as between regions and host species. Several studies have recently characterised differences in the population genetic structure of S. japonicum between villages (Rudge et al., 2009; Lu et al., 2010a), provinces (Shrivastava et al., 2005) and countries (Rudge et al., 2008), but little is known about the parasite population genetic structure at the level of individual host. A definitive host is, however, anticipated to be an important factor affecting the genetic structure of the parasite infrapopulation (a group of parasites of the same species within one individual host) (Caillaud et al., 2006), as any difference in the host’s biological characteristics
⇑ Corresponding author at: Department of Epidemiology and Statistics, School of Public Health, Medical College of Soochow University, Suzhou 215123, China. Tel.: +86 512 6588 0079; fax: +86 512 6588 4830. E-mail address: [email protected] (D.-B. Lu).
and/or associated living habitats may result in different environments for parasites. The biological differences between male and female hosts, in terms of sex-specific behaviour and the level of steroid hormones in males, for example, have been used to explain differential susceptibility to parasites between female and male vertebrate hosts (Klein, 2000; Christe et al., 2007). This may be translated to hostsex-specific genetic patterns in nature, as illustrated in the system Schistosoma mansoni and its natural host Rattus rattus in Guadeloupe, West Indies (Caillaud et al., 2006). After investigating the neutral genetic variability of S. mansoni within its natural murine host R. rattus, Caillaud and colleagues demonstrated that schistosomes from male hosts were genetically more diverse than from female hosts, although an experiment in mice displayed that male hosts were more resistant to S. mansoni than females (Eloi-Santos et al., 1992). Such findings suggest that male and female hosts may potentially play different roles in parasite maintenance. This could have significant implications for the control of parasites, as infection control of parasites in one gender would, in theory at least, potentially lead to a decrease of the intensity of the parasite in the other gender (Ferrari et al., 2004). Rodents have been found to act as reservoir hosts for S. japonicum in some hilly areas of China (Lu et al., 2009). Even in marshland/lake
0020-7519/$36.00 Ó 2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2011.09.002
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areas, where bovines have generally been regarded as playing a primary role in the maintenance and/or transmission of parasites, high infection prevalence levels of S. japonicum in rodents have frequently been reported (Xu et al., 1999; Zhao et al., 2009; Zhang et al., 2010) and one strain of S. japonicum parasites, associated with rodents, has been observed in a marshland village (Lu et al., 2009). The ongoing programme of replacing bovines with machines for schistosomiasis control across endemic areas of China (Wang et al., 2009) may have already reduced the role of this species in parasite transmission. Rodents, however, remain a major issue in hilly endemic areas and may even become major reservoirs in currently bovine-maintained transmission in marshland/lake areas. Therefore, data on population genetics of parasites from such host rodents will inform improvements in control strategies. Moreover, such research may provide insights into the mechanisms underlying the development of a rodent-associated parasite strain (Lu et al., 2009). In this study, we quantified within-host parasite genetic structure with six highly polymorphic microsatellite markers for S. japonicum (Shrivastava et al., 2003) and investigated the effect on this for a few potential influential factors. In addition, to test the hypothesis of the existence of host gender-specific structure (Prugnolle et al., 2003) for S. japonicum, we computed and compared statistical estimates of FIS (Wright, 1951), a measure of the extent of the departure from panmixia (an individual of a population is equally likely to mate with any other individual of that population) (Curtis and Minchella, 2000), and FST (Weir and Cockerham, 1984), a measure of the difference in allelic frequencies between populations. To the best of our knowledge, this is the first study to examine the genetic structure of S. japonicum specifically at the level of individual hosts.
2. Materials and methods 2.1. Rodent trapping and parasite sampling and genotyping The study was performed in three hilly villages in Shitai county of Anhui province of China during October/November of both 2006 and 2007. Detailed geographic and demographic information regarding S. japonicum transmission in the sampled villages, as well as the procedure for capturing rodents, sampling their faeces and performing faecal examinations, have been previously reported (Lu et al., 2010b). Briefly, in such hilly areas, farmers’ houses often form small groups within each village, sometimes separated by roads or small rivers, and their fields are often located nearby. Snails live in ditches around fields. Snail surveys were systematically conducted in all ditches during the spring of each year and the locations of infected snails were mapped accordingly for setting live traps for rodents during the following autumn. Depending on the area of an infected snail habitat, traps were set at an interval distance of 5–10 m. Small rodents were captured by setting traps in the late afternoon and checking at dawn for three consecutive days and captured rodents were kept inside overnight to obtain their faecal samples. Each captured rodent had its gender recorded and its body mass measured with an electronic balance while anaesthetized. All captured rodents belonged to Apodemus agrarius or Rattus norvegicus. After an infection with S. japonicum was identified in a rodent, individual miracidia were sampled from each host and 10–12 miracidia (if available) per host were genotyped using six microsatellite markers (Shrivastava et al., 2003). Infection intensity in individual hosts was measured as eggs plus hatched miracidia per gram of faeces (Lu et al., 2010b). The protocols on parasite larvae DNA storage, extraction, DNA multiplex amplification, genotyping of PCR products and assignment of allele sizes have been presented previously (Rudge et al., 2009; Lu et al., 2010a).
2.2. Molecular analyses 2.2.1. Genetic structure within host sexes As host sex is considered a potentially important factor influencing the population genetic structure of parasites, the population structure of S. japonicum within each host sex was characterised. The unbiased estimate of FIS (Wright, 1951) was computed over all loci and all parasite infrapopulations (an infrapopulation here refers to all miracidia sampled from one host) from one sex according to Weir and Cockerham (1984) and the 95% confidence intervals (CIs) were obtained through bootstrapping. The hypothesis that FIS equalled 0 was tested using a procedure of random permutations of alleles between individual miracidia within each sex set at 15,000 permutations. The value of FST (Weir and Cockerham, 1984) was calculated over all loci to measure the difference in allelic frequencies between infrapopulations for each host sex. Bootstrapping was used to obtain 95% CIs for each FST value. Genetic differentiation was tested using the G-based test, which takes into account the particular structure within each infrapopulation (Goudet et al., 1996), distinct from the conventional exact test based on Hardy-Weinberg (HW) expectations (Raymond and Rousset, 1995). The multiple comparisons between males and females of FIS and FST were performed with the application of Bonferroni corrections (Holm, 1979) due to the repetitive nature of the procedure. All calculations and tests were performed using FSTAT V. 2.9.3 (http:// www.unil.ch/izea/softwares/fstat.html). 2.2.2. Factors influencing genetic diversity and deviations from HW expectations of an infrapopulation The genetic diversity (HS) of a parasite infrapopulation was computed using Nei’s unbiased mean heterozygosity (Nei, 1987), and the unbiased estimate of Wright’s FIS within an infrapopulation was computed over all loci for each infected rodent. To examine any effects of host body mass, infection intensity, number of miracidia genotyped per host, sampling years and locations, together with host sex, on the HS and the deviation from HW expectations (FIS) of an infrapopulation, we conducted univariable analyses and then multivariable analyses using STATA 7 software (Stata Corporation, 702 University Drive East, College Station, TX 77840 USA). If an obvious disagreement was observed between the univariable analyses and the multivariable analyses, a collinearity diagnostic, using a regression analysis, of all test factors was performed and the indices of Tolerance and the variance inflation factor (VIF) were calculated using SPSS 11.0 software (SPSS Base 11.0 for Windows. 2002. SPSS Inc. Chicago, Illinois, USA). 3. Results 3.1. Infection and genetic structure of parasites within host sexes Out of the 49 and 51 rodents trapped in 2006 and 2007, respectively, 13 and nine were infected with S. japonicum. Non-significantly higher infection prevalence was observed in male rodents (25–30%) than in female rodents (9–21%) in both years (Table 1). We could not measure infection intensity for each rodent (Table 3), due to difficulties encountered in the field. Table 2 shows that parasites sampled from females in 2006 displayed a significant departure from HW expectations (under the null hypothesis FIS = 0), as did those from males in 2007. With the exception of the parasites from males in 2007, all parasite populations either from males or from females showed a significant genetic differentiation among individual hosts. As seen in Fig. 1, in 2007 FIS was larger in males than in females and FST was smaller in males than in females. The two 95% CIs, either FIS or FST, however, indeed overlapped. Such a different trend
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D.-B. Lu et al. / International Journal for Parasitology 41 (2011) 1371–1376 Table 1 Schistosoma japonicum infection in male and female rodents during 2006 and 2007 in Shitai county, Anhui province, China. Village/year
Male
Female
No. captured
No. infected
No. captured
No. infected
2006 Longquan Longshang Yuantou Total
11 15 4 30
4 4 1 9 (30%)
7 7 5 19
2 1 1 4 (21%)
2007 Longquan Longshang Yuantou Total
7 13 8 28
3 2 2 7 (25%)
5 9 9 23
1 1 0 2 (9%)
Table 2 Genetic structure of Schistosoma japonicum miracidia within rodent males and females. Year
Sex
FIS (95% CI)
FST (95% CI)
2006
Male
0.056 ( 0.199, 0.158) P = 0.929 0.173 (0.001, 0.467) P = 0.006 0.421 (0.023, 0.742) P < 0.001 0.121 ( 0.116,0.397) P = 0.082
0.295 (0.194,0.396) P < 0.001 0.461 (0.343,0.585) P < 0.001 0.232 ( 0.072,0.454) P = 0.337 0.404 (0.258,0.510) P = 0.003
Female 2007
Male Female
Tests for the departure from Hardy-Weinberg (HW) expectations were conducted by permutating alleles within infrapopulations 15,000 times and for population differentiation by permutating genotypes between infrapopulations 15,000 times. 95% confidence interval (CI) was obtained by a bootstrap. FIS, Inbreeding coefficient; FST, difference in allelic frequencies between populations.
(FIS versus FST) did not occur in the local hosts in 2006 with both FIS and FST in males less than in females (Fig. 1) and, after using Bonferroni corrections, no difference between sexes was observed in FIS (P = 0.163) and FST (P = 0.085).
3.2. Genetic diversity of an infrapopulation and its potential influential factors A total of 196 miracidia were genotyped from 22 rodents with an average of 8.9 larvae per host. The unbiased mean heterozygosity (HS) of an infrapopulation varied between 0.30 and 0.58 among hosts, with the exception of 0.83 when only two miracida were genotyped, and the inbreeding coefficient (FIS) ranged widely from 0.57 to 0.51 (Table 3). As can be seen in Table 4, the univariable analyses revealed that only two variables, year of sampling and number of miracidia genotyped per host, were significantly correlated with the genetic diversity (HS). Neither effect was significant in a multivariable model including both of these effects (P > 0.1 in both cases). No variables examined were observed to have a significant impact on the inbreeding coefficient FIS. Due to the difference between the two types of analyses, two new (dummy) variables were computed from the variable of Village to categorise three villages for performing collinearity diagnostics. As seen in Table 4, all of the values of Tolerance are over 0.1 and those of VIF were less than 10, suggesting that there was no collinearity among factors.
4. Discussion Of the human schistosomes, S. japonicum is the only schistosome for which zoonotic transmission is considered important (Nelson, 1972), with over 40 species of wild and domesticated animals suspected of serving as reservoir hosts for this parasite (He et al., 2001). In the studied areas previous research has, based on the infection prevalence and intensity in each species, including humans, dogs, cats, pigs, bovines and rodents (Lu et al., 2010b), and the biological traits of the parasites from local infected snails (Lu et al., 2009), demonstrated that small rodents have been suggested to play an important role in the transmission of S. japonicum to humans and other mammals in the hilly regions. In this study, we analysed the genetic structures of S. japonicum miracidia within and between rodent individuals in three hilly villages of China. The results demonstrated that a significant genetic differentiation of infrapopulations (FST values) was seen among hosts within either gender (with the exception of males in 2007), and that two factors
Table 3 Ecological and genetic characteristics of Schistosoma japonicum miracidia infrapopulations within individual rodents (HS, genetic diversity; FIS, inbreeding coefficient). Host
Year
Village
Host sex
Host body mass (g)
Infection intensity (EPG)
No. miracidia genotyped
HS
LQS02 LQS03 LQS04 LQS05 LQS15 LQS18 LSS22 LSS23 LSS25 LSS32 LSS42 YTS47 YTS49 LQM02 LQM09 LQM10 LQM13 LSM09 LSM17 LSM19 YTM08 YTM09
2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2007 2007 2007 2007 2007 2007 2007 2007 2007
Longquan Longquan Longquan Longquan Longquan Longquan Longshang Longshang Longshang Longshang Longshang Yuantou Yuantou Longquan Longquan Longquan Longquan Longshang Longshang Longshang Yuantou Yuantou
Male Male Female Male Female Male Male Male Female Male Male Male Female Male Male Female Male Male Female Male Male Male
25.0 120.0 120.0 140.0 31.2 123.1 127.0 143.1 141.0 47.5 36.4 116.4 31.0 79.4 19.8 34.0 61.6 152.1 128.0 92.7 31.6 134.0
– – – – – – 283.6 3311.7 – – 201.5 40.3 85.7 105.0 825.0 21.1 13.7 4.8 150.6 6.5 7.1 3268.3
11 12 9 10 10 11 11 10 10 11 9 11 11 12 8 4a 7a 2a 12 3a 1a 11
0.55 0.50 0.42 0.39 0.30 0.57 0.33 0.47 0.41 0.36 0.44 0.32 0.40 0.54 0.58 0.52 0.45 0.83 0.48 0.50 – 0.42
EPG, eggs per gram of faeces. a Less than eight miracidia were genotyped from each of five hosts due to small numbers of miracidia samples available.
FIS 0.08 0.12 0.51 0.24 0.33 0.00 0.04 0.06 0.14 0.55 0.41 0.57 0.08 0.12 0.36 0.06 0.15 0.20 0.15 0.33 – 0.35
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Fig. 1. Comparison of genetic structures of Schistosoma japonicum from male and female rodents, during 2006 and 2007 in Shitai county, Anhui province, China. (A) Inbreeding coefficient (FIS) estimates with 95% confidence intervals (CIs) and (B) difference in allelic frequencies between populations (FST) estimates with 95% CIs.
including sampling seasons and number of miracidia per host, based on an univariable analysis, may have an effect on the genetic diversity (Hs) of an infrapopulation. All observed factors from a multivariable analysis, however, showed no effect on the genetic diversity or the HW expectation of a parasite infrapopulation within a host. An infra-population of parasites is exclusively formed by recruitment of external free-living infective stages, cercariae for S. japonicum, and consequently any genetic structure of the parasites at the level of individual hosts would mainly result from the recruitment histories experienced by each host, which may depend on host movement and its living environment, differing from individual to individual. Rodents are usually considered to be highly philopatric, as they are often recaptured within a short distance of their first capture location, for example within only 14– 27 m (Delattre and Louarn, 1981). It is therefore conceivable that most infected rodents may deposit their parasites or their schistosome progeny, via faeces, in their own small areas. As a consequence the parasites, in relation to each other in terms of genetic make-up, may stay in close proximity when transmitted by such a host. Moreover, the above clumped transmission could be facilitated by the unique characteristics of S. japonicum compared with other schistosomes infecting humans such as S. mansoni and Schistosoma haemotobium in terms of biochemical and immunological features (Ruppel et al., 2004). Schistosoma japonicum cercariae, for example, have shown a remarkable speed of migration through a host’s skin, partially due to the effect of more potent penetrating enzymes released from parasite larvae on the skin penetration (He et al., 2002). Another main aspect for this parasite is that the cercariae, after being shed from their intermediate host, cluster together on the water, possibly due to an unknown chemical released by the parasite which makes the surrounding water very sticky (Spear et al., 2004). Such patchy foci may be further enhanced by the amphibious snail host, Oncomelania hupensis, which prefers moist earth to water (Mao, 1990). When submerged in water, this host species will mostly immediately climb out if possible, leaving a very narrow window for the larvae to escape from the host body
into the water. It would be imagined that an irregular distribution and aggregation of parasites in terms of space would develop under this context. Therefore, the more often a rodent frequents a limited area of one focus, the larger inbreeding coefficient (FIS) of the parasite infrapopulation it may have; and for different host individuals, the higher genetic differentiation of parasites between them may occur. Such genetic differentiation between rodents could be reduced more or less if they experience a few or all of the foci. This could explain the significant genetic differentiation between hosts and the quite wide range of the inbreeding coefficients among hosts observed in our study. We also observed a possible effect of sampling years on the genetic diversity of a parasite infrapopulation from an univariable analysis. This could be partly due to the existence of differences between those two years in the numbers of infected snail habitats, in the infection profile among mammalian species (Lu et al., 2010b), probably in control measures, and in the numbers of miracidia genotyped, in the last of which both the average number of miracidia genotyped per host and the number of infected rodent females captured were smaller in 2007 than in 2006. However, no significant effect of sampling years was seen when using a multivariable analysis. The intrinsic differences between female and male hosts in physiology, immunology, behaviour and ecology can make one sex or the other a more benign environment for parasites (Zuk and McKean, 1996; Christe et al., 2007). A male bias in parasitism has been reported to occur in a wide range of mammalian species (Poulin, 1996; Klein, 2000; Moore and Wilson, 2002; Amo et al., 2005), but several exceptions were also reported (Eloi-Santos et al., 1992; Christe et al., 2007; Ci et al., 2008). A strong influence of the sex of the host on the genetic variability of their parasites was observed for the system of S. mansoni and their hosts R. rattus in a marshy forest focus, in which parasite populations of male rats were more genetically diverse than parasites of female hosts (Caillaud et al., 2006). One plausible explanation is accredited to a higher immunity-based, diversifying selection process, a synergistic effect of testosterone, which may inhibit mitochondrial respiratory chain function of the parasite larvae at the schistosomula stage, and host immune ability, acting in male hosts (Caillaud et al., 2006). To our surprise, however, in this study of S. japonicum and its definitive hosts, we observed a slight but non-significant difference in genetic structure of parasites between male and female hosts. Although rodents are limited to their own territories, males are usually more active and often disperse across wider geographic ranges than females (Dobson, 1982), and might be more likely to recruit more infective forms of larvae than females, thus possibly leading to the observed lower genetic differentiation (FST) among males than among females. Moreover, in the studied areas, apart from infected rodents, infection with the parasite was also identified in humans, dogs and cats, particularly in dogs with a comparable infection prevalence (Lu et al., 2010b). An infected dog with a longer life span, compared with a rodent with a shorter life span of 2 years on average, may recruit more external infective forms of parasites during that period and, as a consequence, coupled with an average life span of 5 years for schistosomes within their hosts (Bush et al., 2001), may excrete a well-mixed pool of parasite offspring in terms of genotypes. This may add to the complex population genetic structures observed from the local rodents only, i.e. in terms of FIS, males < females in 2006 but males > females in 2007, as the extent to which dogs have spread the parasites may vary with villages or years (Lu et al., 2010a). In our study, we observed a negative effect of the number of miracidia genotyped per host on genetic diversity of an infrapopulation from a univariable analysis, but no effect from a multivariable analysis. Sampling and genotyping parasite larvae for inferring population genetic structures, together with sample size,
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Table 4 Univariable analyses of factors influencing the genetic diversity (HS) and inbreeding coefficient (FIS) of Schistosoma japonicum miracidia infrapopulations and collinearity statistics. Variable
HS P
FIS Reg. coef.
95% CI
P
Year
0.017
0.12
0.2165,
0.0235
Village Longquan/Yuantoua Longshang/Yuantoub Host sex Host body mass Infection intensity No. miracidia genotyped per host
0.201 0.233 0.284 0.648 0.688 0.013
0.102 0.0975 0.0617 0.0003 0.00001 0.0212
0.0595, 0.2635 0.0686, 0.2636 0.0554, 0.1788 0.0009, 0.0014 0.00008, 0.00006 0.0373, 0.0051
Collinearity statistics Tolerance
VIF
0.143
Reg. coef. 0.1835
95% CI 0.4347, 0.0677
0.435
2.300
0.306 0.46 0.257 0.676 0.552 0.209
0.1957 0.1442 0.1543 0.0006 0.00004 0.0269
0.1946, 0.2572, 0.4307, 0.0033, 0.0001, 0.0701,
0.262 0.354 0.765 0.395 0.723 0.603
3.820 2.822 1.307 2.529 1.384 1.657
0.5860 0.5456 0.1221 0.0022 0.0002 0.0164
CI, confidence interval; VIF, variance inflation factor. Two new variables derived from the variable of ‘village’ for collinearity statistics.
a,b
is an inherent limitation in this sort of study, which has been detailed in a review (Steinauer et al., 2010). However, our research did find a significant genetic differentiation among hosts and two possible factors influencing the genetic diversity of the parasites. Future work based on population genetics of adult worms within and/or between rodent individuals and of gender typed larvae, if possible, produced from such worms would further our understanding of the transmission of S. japonicum maintained by this host species, as the direct comparison of population genetic structures between two stages, adult worms and miracidia, will provide deep insights into the transmission dynamics between rodents and any associated biological characteristics. In conclusion, in this study we investigated the genetic structure of parasites within rodent individuals and sexes and any potential influencing factors. We did not observe any host sexspecific genetic structure but found significant genetic differentiation between hosts and a considerable range of genetic diversity of parasites among host individuals. The results suggest that intensive density/population control for local rodents may be an effective measure for militating against transmission of this parasite, as control measures targeted at individuals contributing the most to transmission can be very efficient but, conversely, there will be a high risk of it being ineffective if any of these individuals are missed (Woolhouse, 1998), particularly for rodents inherent with sampling difficulties. Acknowledgements This work was funded by the Royal Society, UK (to JPW), Kwok Foundation, Hongkong, China (to DBL, CAD and JPW) and the Medical Research Council, UK (to JWR). The laboratory work was performed at Imperial College, London, UK, when Dabing Lu studied there as a PhD student. References Amo, L., Lopez, P., Martin, J., 2005. Prevalence and intensity of haemogregarine blood parasites and their mite vectors in the common wall lizard, Podarcis muralis. Parasitol. Res. 96, 378–381. Bush, A.O., Fernández, J.C., Esch, G.W., Seed, J.R., 2001. Parasitism: The Diversity and Ecology of Animal Parasites. Cambridge University Press, Cambridge. Caillaud, D., Prugnolle, F., Durand, P., Theron, A., de Meeus, T., 2006. Host sex and parasite genetic diversity. Microb. Infect. 8, 2477–2483. Christe, P., Glaizot, O., Evanno, G., Bruyndonckx, N., Devevey, G., Yannic, G., Patthey, P., Maeder, A., Vogel, P., Arlettaz, R., 2007. Host sex and ectoparasites choice: preference for, and higher survival on female hosts. J. Anim. Ecol. 76, 703–710. Ci, H.X., Lin, G.H., Su, J.P., Cao, Y.F., 2008. Host sex and ectoparasite infections of plateau pika (Ochotona curzoniae, Hodgson) on the Qinghai Tibetan Plateau. Pol. J. Ecol. 56, 535–539. Curtis, J., Minchella, D.J., 2000. Schistosome population genetic structure: when clumping worms is not just splitting hairs. Parasitol. Today 16, 68–71. Delattre, P., Louarn, H.L., 1981. Dynamique des populations du rat noir, Rattus rattus, en mangrove lacustre. Mammalia 45, 275–288.
Dobson, F.S., 1982. Competition for mates and predominant juvenile male dispersal in mammals. Anim. Behav. 30, 1183–1192. Eloi-Santos, S., Olsen, N.J., Correa-Oliveira, R., Colley, D.G., 1992. Schistosoma mansoni: mortality, pathophysiology, and susceptibility differences in male and female mice. Exp. Parasitol. 75, 168–175. Ferrari, N., Cattadori, I.M., Nespereira, J., Rizzoli, A., Hudson, P.J., 2004. The role of host sex in parasite dynamics: field experiments on the yellow-necked mouse Apodemus flavicollis. Ecol. Lett. 7, 88–94. Goudet, J., Raymond, M., de Meeus, T., Rousset, F., 1996. Testing differentiation in diploid populations. Genetics 144, 1933–1940. He, Y.X., Salafsky, B., Ramaswamy, K., 2001. Host–parasite relationships of Schistosoma japonicum in mammalian hosts. Trends Parasitol. 17, 320–324. He, Y.X., Chen, L., Ramaswamy, K., 2002. Schistosoma mansoni, S. haematobium, and S. japonicum: early events associated with penetration and migration of schistosomula through human skin. Exp. Parasitol. 102, 99–108. Holm, S., 1979. A simple sequentially rejective multiple test procedure. Scand. J. Statist. 6, 65–70. Klein, S.L., 2000. The effects of hormones on sex differences in infection: from genes to behavior. Neurosci. Biobehav. Rev. 24, 627–638. Lu, D.B., Wang, T.P., Rudge, J.W., Donnelly, C.A., Fang, G.R., Webster, J.P., 2009. Evolution in a multi-host parasite: chronobiological circadian rhythm and population genetics of Schistosoma japonicum cercariae indicates contrasting definitive host reservoirs by habitat. Int. J. Parasitol. 39, 1581–1588. Lu, D.B., Rudge, J.W., Wang, T.P., Donnelly, C.A., Fang, G.R., Webster, J.P., 2010a. Transmission of Schistosoma japonicum in marshland and hilly regions of China: parasite population genetic and sibship structure. PLoS Negl. Trop. Dis. 4, e781. Lu, D.B., Wang, T.P., Rudge, J.W., Donnelly, C.A., Fang, G.R., Webster, J.P., 2010b. Contrasting reservoirs for Schistosoma japonicum between marshland and hilly regions in Anhui, China – a two-year longitudinal parasitological survey. Parasitology 137, 99–110. Mao, S.P., 1990. Schistosome Biology and Control of Schistosomiasis. Publishing House of People’s Health, Beijing. Moore, S.L., Wilson, K., 2002. Parasites as a viability cost of sexual selection in natural populations of mammals. Science 297, 2015–2018. Nei, M., 1987. Molecular Evolutionary Genetics. Columbia University Press, New York. Nelson, G.S., 1972. Immunological control of schistosomiasis. Brit. Med. J. 4, 172. Poulin, R., 1996. Helminth growth in vertebrate hosts: does host sex matter? Int. J. Parasitol. 26, 1311–1315. Prugnolle, F., Durand, P., Theron, A., Chevillon, C., de Meeus, T., 2003. Sex-specific genetic structure: new trends for dioecious parasites. Trends Parasitol. 19, 171– 174. Raymond, M., Rousset, F., 1995. An exact test for population differentiation. Evolution 49, 1280–1283. Rudge, J.W., Carabin, H., Balolong, E., Tallo, V., Shrivastava, J., Lu, D.B., Basanez, M.G., Olveda, R., McGarvey, S.T., Webster, J.P., 2008. Population genetics of Schistosoma japonicum within the Philippines suggest high levels of transmission between humans and dogs. PLoS Negl. Trop. Dis. 2, e340. Rudge, J.W., Lu, D.B., Fang, G.R., Wang, T.P., Basanez, M.G., Webster, J.P., 2009. Parasite genetic differentiation by habitat type and host species: molecular epidemiology of Schistosoma japonicum in hilly and marshland areas of Anhui Province. China Mol. Ecol. 18, 2134–2147. Ruppel, A., Chlichlia, K., Bahgat, M., 2004. Invasion by schistosome cercariae: neglected aspects in Schistosoma japonicum. Trends Parasitol. 20, 397–400. Shrivastava, J., Barker, G.C., Johansen, M.V., Zhou, X.N., Aligui, G.D., 2003. Isolation and characterization of polymorphic DNA microsatellite markers from Schistosoma japonicum. Mol. Ecol. Notes 3, 406–408. Shrivastava, J., Qian, B.Z., McVean, G., Webster, J.P., 2005. An insight into the genetic variation of Schistosoma japonicum in mainland China using DNA microsatellite markers. Mol. Ecol. 14, 839–849. Spear, R.C., Seto, E., Liang, S., Birkner, M., Hubbard, A., Qiu, D., Yang, C., Zhong, B., Xu, F., Gu, X., Davis, G.M., 2004. Factors influencing the transmission of Schistosoma japonicum in the mountains of Sichuan Province of China. Am. J. Trop. Med. Hyg. 70, 48–56.
1376
D.-B. Lu et al. / International Journal for Parasitology 41 (2011) 1371–1376
Steinauer, M.L., Blouin, M.S., Criscione, C.D., 2010. Applying evolutionary genetics to schistosome epidemiology. Infect. Genet. Evol. 10, 433–443. Wang, L.D., Chen, H.G., Guo, J.G., Zeng, X.J., Hong, X.L., Xiong, J.J., Wu, X.H., Wang, X.H., Wang, L.Y., Xia, G., Hao, Y., Chin, D.P., Zhou, X.N., 2009. A strategy to control transmission of Schistosoma japonicum in China. New Engl. J. Med. 360, 121–128. Weir, B.S., Cockerham, C.C., 1984. Estimating F-statistics for the analysis of population structure. Evolution 38, 1358–1370. Woolhouse, M.M.E., 1998. Heterogeneities in schistosome transmission dynamics and control. Parasitology 117, 475–482. Wright, S., 1951. The genetical structure of populations. Ann. Eugen. 15, 323–354.
Xu, G.Y., Tian, J.C., Cheng, G.M., Yang, H.M., Qiu, L., Hu, H.B., Xie, Z.Y., Zhou, F., Ying, W.G., 1999. Observation on natural focal disease of schistosomiasis in Rattus norvegicus in Nanjing. Chin. J. Para. Dis. 7, 4–6. Zhang, Q.D., Wu, Y.F., Xiao, M., Li, C.N., Su, J.G., Suo, Y., Dai, J.R., 2010. A survey of field mouse density and the rate of schistosome infection in rodents in marshlands of the City of Yangzhong. J. Path. Biol. 6, 230–231. Zhao, H.M., Su, J.Y., Liu, C.H., Zhao, S.Y., Wang, F.Y., 2009. Survey of Schistosoma japonicum in rodents in Sihu region. Hubei. Chin. J. Zoon. 25, 919–920. Zuk, M., McKean, K.A., 1996. Sex differences in parasite infections: patterns and processes. Int. J. Parasitol. 26, 1009–1023.