Descripción del centro, dependencias e instalaciones donde se

Allele size range 194 - 248 294 - 366 242 - 287 186 - 237 253 - 296 302 - 338

Ar 24 22 16 18 15 24

He 0.7927 0.9342 0.8779 0.7826 0.8383 0.8993

Ho 0.3786 0.8357 0.8071 0.7286 0.6429 0.5500

Figure 3.7. Biplot of a principal component analysis on the allele frequencies of Schistosoma bovis

cercariae from Bulinus spp. collected in the North and South of Niamey in the Niger River Valley (156 observations). PCA produced using six microsatellite loci. The cumulative proportion is 14.2 for the first two principal components (PC1: 7.4%; PC2: 6.8%). Observations are coloured by populations of S.

bovis collected from the North and South of Niamey (see Table 3.7).

d = 0.5

Figure 3.8. Distribution of Schistosoma spp. cercariae shed from Bulinus spp. collected North and

South of Niamey in the Niger River Valley.

3.3.8 Distribution of infected Bulinus and the Schistosoma species

S. haematobium and the S. haematobium group hybrids involved in human urogenital schistosomiasis infections were only detected in five villages north of Niamey (Figure 3.7). S. bovis occurred throughout the study villages in the north and south (except Yoreizé Koira and Tagabati) but was the only schistosome species found in the southern sites, where the majority (n=56) of infected snails infected were collected (Table 3.4), predominantly B. truncatus collected from two villages: Tokeye (n=23) and Dokimana (n=18) (Figure 3.1). Considerably fewer snails were involved in the transmission of human urogenital schistosomiasis (n=21) compared to those involved in the transmission of the bovid schistosomes (Table 3.6). The infected B. globosus and B. forskalii were present in low numbers in both the northern and southern sites, transmitting S. bovis.

There was no significant effect of snail habitat type (c2_{=0.53, df=4, P=0.26), Bulinus} species (c2_{=0.75, df=3, P=0.86) nor schistosome species (c}2_{=2.58, df=3, P=0.46) on the} number of cercariae MLGs found per infected Bulinus spp.

3.4 Discussion

Molecular identification of schistosome cercariae and their snail hosts has proved vital for identifying the species of schistosome being transmitted in the Niger River Valley, enabling the future mapping of human and animal schistosomiasis transmission and risk. Additionally, unravelling the relationships between the different schistosome species and their snail host

10 0 10 20 30 4 0 km No infected snails Hybrids co-infection Major river River Schistosoma bovis Schistosoma haematobium Schistosoma Schistosoma

Lata Kabia _{Zama Koira Tégui} Namaro Koutoukalé Zéno

Tagabati

Karma Bangou Koirey

Yoreizé Koira

N

Direction of water flow

Youri Kohan Garantche Doguel Kaina Tiaguirire Tokeye Gantchi Bassarou Say Dokimana 1 23 Number of Schistosoma infected Bulinus snails collected per village

species at a focal level, allows us to gain a better understanding of disease transmission and snail-schistosome epidemiology. Here we have identified the sympatric transmission of S. haematobium and S. bovis, causing human and livestock schistosomiasis respectively, in the northern area of the Niger River Valley region, and allopatric distribution of bovid schistosomes in southern sites (Figure 3.8). We also detected S. haematobium-bovis hybrids, known human pathogens, adding to the geographical range of these hybrids now reported in several countries, Senegal, Niger, Mali, Côte d’Ivoire, Malawi (Huyse et al., 2009; Léger et al., 2016; Soentjens et al., 2016; Webster et al., 2019) and also imported into Corsica, France (Moné et al., 2015). Additionally, S. haematobium-bovis- urassoni hybrid cercariae were identified, confirming the transmission of this unusual hybrid combination previously reported from humans in Niger (Léger et al., 2016).

The microsatellite data analysis showed no gene flow between the human (S. haematobium and the hybrids) and cattle (S. bovis) schistosome populations analysed (Figure 3.5). This suggests that strong reproductive barriers exist between the two populations and that the observed hybrids are not first-generation resulting from zoonotic and / or zooanthroponotic inter-species interactions. These hybrids appear to be introgressed forms, with parts of the S. bovis genome introgressed into S. haematobium, leaving two differentiated parental populations that are not panmictic or leading to hybrid speciation. This was also reported in recent studies in Senegal (Boon et al., 2019) and Niger (Platt et al., 2019), and indicates that more research is warranted to understand S. haematobium group species hybridisation, the effect of hybridisation on definitive host range (Catalano et al., 2018) and the potential human and veterinary impacts (Léger and Webster, 2017).

Mitochondrial ox1 analysis of the Bulinus snail samples identified the three species, B. trun atus, B. globosus and B. forskalii, involved in schistosome transmission in Niger. These three Bulinus species show diverse intra-species populations with no clustering by geographical region or in relation to transmission. Additionally, non-coding mitochondrial DNA was amplified in several (28) of our B. trun atus samples, the preferential sequencing of which could be attributed to unsuitable primer selection or specimen degradation. As these were non-coding, they are dissimilar to NUMTs described in other molluscs (Williams et al., 2017) and may instead be related to the high degree of polyploidy known to occur in B. trun atus/tropi us group snails (Brown and Wright, 1972; Brown, 1976; Goldman et al., 1983) promoting mitochondrial heteroplasmy.

Strong support for these non-coding sequences arising through mitochondrial heteroplasmy have been identified from mitochondrial sequences of a B. trun atus isolate from Niger sequenced on a Next Generation Sequencing (NGS) platform that is currently being analysed in collaboration with colleagues at the Natural History Museum (NHM) (Briscoe et al., unpublished). In brief, sequence data were produced on a MiSeq (600 cycles) and

unassembled reads were iteratively assembled to NHM derived Bulinus spp. ox1 sequences until resulting contigs could be circularised and annotated using MITOS (Bernt et al., 2013). When investigating a specific site of interest where a considerable 10 bp deletion had been observed in the non-coding ox1 of the B. trun atus sequenced here it was noted that in a single read of the data there was a match with these ‘alternative’ sequences. This finding provides evidence that at least within the B. trun atus species, somatic mutations can occur leading to a genetically mosaic individual. Although the mechanism by which heteroplasmic mitochondrial DNA mutations can lead to predominating tissues in comparison to the functional genes (as seems the case with the preferentially sequenced non-coding ox1 sequences of B. trun atus observed in the current study) are poorly understood, they will progressively erode mitochondrial function until a threshold is reached, which when concerning mtDNA diseases for example, would lead to the onset of symptoms (Wallace and Chalkia, 2013). This raises multiple questions considering that non-functional copies of ox1 were observed in just under half (28 of 57) of the B. trun atus analysed suggesting that these mutations are not leading to any obvious evolutionary selective pressure. It could be of interest however to compare this rate of non-coding mtDNA occurrence in schistosome infected B. trun atus with those from the same sites not harbouring patent schistosome infections, to establish if any link to schistosome susceptibility is worth investigating further. Although initially ruled out, further investigation into whether these non-coding sequences may be occurring due to the presence of NUMTs should also be performed.

The main purpose for sequencing a region of the ox1 in the current study was simply to easily obtain species identifications since it has been established mitochondrial barcoding is useful for exploring genetic diversity and relationships of Bulinus spp. (Kane et al., 2008; Zein-Eddine et al., 2014). However, the preferential sequencing of non-coding genes here highlights difficulties in using such a marker for species identification and the need for using other informative DNA regions.

B. globosus, B. forskalii and B. trun atus were confirmed as transmitting S. bovis supporting previous reports that S. bovis can utilize a wide variety of Bulinus hosts (Southgate and Knowles, 1975; Tian-Bi et al., 2019). Conversely, S. haematobium and the S. haematobium group hybrids appeared more specific and were only transmitted by B. trun atus (Table 3.3). This is consistent with historical findings from this region showing that B. globosus and B. forskalii were not compatible with S. haematobium (Vera et al., 1990) and questions the previous reports of B. forskalii snails, that could have been morphologically confused with B. senegalensis, as infected with S. haematobium (see Vera et al., 1992; Labbo et al., 2007). Proportional to the total number of snails collected (n=15,288), few had patent schistosome infections (0.56-0.90%) (see Rabone et al., 2019), however 40% (n=35) of the infected snails examined were shedding multiple (2-10) cercarial genotypes. Of these 35, 17%

had co-species infections, confirming that they had been infected multiple times by miracidia of different species/strains. The remaining 83% had mono-species infections, suggesting that they had either been infected by multiple miracidia of the same species, or that the different genotypes may have arisen through genetic mutations during clonal parasite replication from a single miracidial infection. Near identical MLG cercariae observed from individual snails has been identified previously for S. japoni um (see Shrivastava et al., 2005; Yin et al., 2008; Lu et al., 2010; Huo et al., 2016), with the conclusion that somatic mutation occurs during schistosome sporocystogenesis, resulting in genetically different cercariae originating from a single miracidium. This has also been shown for S. mansoni, with significant intra-clonal variation of cercariae resulting from single miracidial snail infections (Grevelding, 1999), and also sporocysts cultured in vitro, suggesting that mitotic recombination events occur during intramolluscan larval development (Bayne and Grevelding, 2003). For S. haematobium, intramolluscan replication of daughter sporocysts does occur and has been observed in B. trun atus (see Kechemir & Théron, 1989); however, the occurrence of mitotic recombination and / or somatic mutation during replication has not been investigated.

If our MLG data do correlate to multiple individual miracidial infections, it is clear that multiple S. bovis infections are high in these snails, suggesting that these are high transmission zones, or that S. bovis egg deposition is focally more concentrated than that of humans at transmission sites. This is also supported by the finding of all three of the snail species (B. trun atus, B. forskalii and B. globosus) being infected, however only the former two species harboured high numbers of cercarial genotypes, with B. globosus only emitting cercariae of a single genotype (Figure 3.2). This may be due to a low sample size of B. globosus (n = 4), or that this snail species is less suitable for S. bovis transmission in this region (Vera et al., 1990; Brémond et al., 1993; Labbo et al., 2003, 2007). Inter schistosome species comparisons also show that compared to S. bovis, S. haematobium / hybrids infections only showed few (£3) cercarial genotypes from individual B. trun atus (Figure 3.2). Although this again may be a consequence of sample size and the fact that far fewer snails were found shedding S. haematobium and / or the hybrids, it might also reflect biological species differences during intramolluscan development, such as variation in sporocystogenesis regulatory mechanisms (Kechemir and Théron, 1980; Jourdane, 1983; Touassem and Théron, 1986). Differences in intramolluscan replication may also be an explanation for the higher degree of diversity observed in S. bovis populations with S. haematobium showing extremely low diversity levels within and between endemic zones (Webster et al. 2012; Djuikwo-Teukeng et al. 2019). However, these differences could also be due to the transmission of S. bovis being more frequent than that of human schistosomes due to; larger numbers of parasites released in the faecal matter of infected ruminants, more frequent freshwater use of definitive hosts, and / or due to the lack of chemotherapeutic

treatment for animals, compared to that of the human populations previously reported (Ezeamama et al., 2016). The genetic variability (interpreted by Ho and He) of S. bovis populations collected South of Niamey (where no S. haematobium was collected) was lower than that of cercariae collected in the North, indicating a potentially higher degree of inbreeding within the populations South of Niamey. In the North however, Ho was higher than He at some loci, indicating a higher genetic variability than might be expected. However, this may be a direct result of sample size, since few S. bovis unique MLGs were collected in the North which would impact the accuracy of the population genetic analyses (Hale et al., 2012).

Interestingly, no co-infections were found between S. haematobium and S. bovis or between S. bovis and the hybrids, although a co-infection involving S. haematobium and a hybrid were found as has been previously reported in Côte d’Ivoire (Tian-Bi et al., 2019). Considering the identified sympatric distribution of these S histosoma species at the village level in the north of Niamey and the absence of S. haematobium in the seven villages south (Figure 3.8), this observation raises further questions worth exploring. The lack of S. haematobium and S. bovis co-infections may be due to the transmission sites of human and cattle schistosomiasis in this region of the Niger River Valley being for the most part separated, since cattle and human schistosomes were only found together at two transmission sites.

Alternatively, there could be more complex intramolluscan mechanisms that inhibit the occurrence of this co-infection, such as the antagonism/relationship that has been observed between S. mansoni and S. haematobium with Cali ophoron spp. trematode parasites (Southgate et al., 1989; Laidemitt et al., 2019), or the induced immunoregulation and adaptive immunity of the snail during multiple schistosome infection challenges (Sire et al., 1998; Portela et al., 2013; Pinaud et al., 2016, 2019), including those of the same S histosoma species. The role that hybridisation plays in relation to these co-infections adds an extra element of complexity, with S. haematobium group hybrids being observed to co-infect with both S. bovis (Tian-Bi et al., 2019) and S. haematobium, signifying again the expanded range of compatibility between schistosome hybrids and their intermediate snail hosts warranting further investigation.

Several limitations of the current study are worth considering while making conclusions. First of all, just over a third of infected Bulinus spp. and their associated schistosomes collected in the field could not be included due to sample degradation reducing the samples available for this study. The inclusion of these collections in the present study under better circumstances may have revealed other host-parasite relationships not considered here. Second, for several of the Bulinus spp. included, ox1 sequences to distinguish snail species could not be analysed due to amplification of non-coding DNA or poor sequencing results. Although morphological observations of the shell were attributed to B. trun atus and B. forskalii species, there may be hidden genetic diversity within these

species currently undetermined. In addition, the two snails morphologically identified as B. forskalii may be those of a closely related species such as B. senegalensis reported as present in Niger (Vera et al., 1992). Thirdly, it should be reiterated that the inferences made in the current study regarding multiple schistosome miracidia infections per Bulinus spp. were established primarily using microsatellite markers alone and are therefore in need of further support. As aforementioned, we need to conduct controlled experimental infections of Bulinus spp. with S histosoma spp. to gage how these microsatellite loci may be affected during intramolluscan replication when somatic mutations may occur. During such infection experiments, microsatellite markers should be analysed alongside other variable regions of schistosome DNA (as was demonstrated for one snail in the current study where different ox1 sequences haplotypes from each MLG confirmed multiple infections) to give further support for the occurrence of multiple miracidia infections and/or somatic mutation in field collected intermediate hosts.

Detailed snail and schistosome sampling coupled with molecular analyses has advanced our understanding of human and bovid schistosomiasis transmission in the Niger River Valley region. Schistosomes found to infect humans (S. haematobium and S. haematobium hybrids) were restricted to the north sites but were much less abundant than those causing veterinary schistosomiasis (S. bovis) across the region. No genetic overlap was observed between human and bovine schistosomes, supporting population structure and division. B. trun atus, the most abundant snail species (Rabone et al., 2019), was involved in transmission of all schistosomes, whilst the less abundant B. forskalii and B. globosus were only involved in the transmission of S. bovis. The data suggest that species-specific biological traits may exist in relation to co-infections, snail-schistosome compatibility and intramolluscan schistosome development which might affect transmission dynamics and genetic outcomes of the different schistosome populations. The scarcity of human infecting schistosomes to the comparatively abundant livestock schistosomes, even in highly endemic settings such as this, shows the necessity from a public health view to identify species accurately to assess the presence and level of human schistosomiasis transmission.