Transmission and diversity of Schistosoma haematobium and S. bovis and their freshwater intermediate snail hosts Bulinus globosus and B. nasutus in the Zanzibar Archipelago, United Republic of Tanzania.

BACKGROUND: The Zanzibar Archipelago (Pemba and Unguja islands) is targeted for the elimination of human urogenital schistosomiasis caused by infection with Schistosoma haematobium where the intermediate snail host is Bulinus globosus. Following multiple studies, it has remained unclear if B. nasutus (a snail species that occupies geographically distinct regions on the Archipelago) is involved in S. haematobium transmission on Zanzibar. Additionally, S. haematobium was thought to be the only Schistosoma species present on the Zanzibar Archipelago until the sympatric transmission of S. bovis, a parasite of ruminants, was recently identified. Here we re-assess the epidemiology of schistosomiasis on Pemba and Unguja together with the role and genetic diversity of the Bulinus spp. involved in transmission. METHODOLOGY/PRINCIPAL
FINDINGS: Malacological and parasitological surveys were conducted between 2016 and 2019. In total, 11,116 Bulinus spp. snails were collected from 65 of 112 freshwater bodies surveyed. Bulinus species identification were determined using mitochondrial cox1 sequences for a representative subset of collected Bulinus (n = 504) and together with archived museum specimens (n = 6), 433 B. globosus and 77 B. nasutus were identified. Phylogenetic analysis of cox1 haplotypes revealed three distinct populations of B. globosus, two with an overlapping distribution on Pemba and one on Unguja. For B. nasutus, only a single clade with matching haplotypes was observed across the islands and included reference sequences from Kenya. Schistosoma haematobium cercariae (n = 158) were identified from 12 infected B. globosus and one B. nasutus collected between 2016 and 2019 in Pemba, and cercariae originating from 69 Bulinus spp. archived in museum collections. Schistosoma bovis cercariae (n = 21) were identified from seven additional B. globosus collected between 2016 and 2019 in Pemba. By analysing a partial mitochondrial cox1 region and the nuclear ITS (1-5.8S-2) rDNA region of Schistosoma cercariae, we identified 18 S. haematobium and three S. bovis haplotypes representing populations associated with mainland Africa and the Indian Ocean Islands (Zanzibar, Madagascar, Mauritius and Mafia).
CONCLUSIONS/SIGNIFICANCE: The individual B. nasutus on Pemba infected with S. haematobium demonstrates that B. nasutus could also play a role in the local transmission of S. haematobium. We provide preliminary evidence that intraspecific variability of S. haematobium on Pemba may increase the transmission potential of S. haematobium locally due to the expanded intermediate host range, and that the presence of S. bovis complicates the environmental surveillance of schistosome infections.

Introduction

Schistosomiasis is a snail-borne neglected tropical disease (NTD), that can cause severe morbidity and mortality in both humans and animals [1,2]. Schistosoma haematobium and S. mansoni are the two species responsible for most cases of human schistosomiasis in sub-Saharan Africa, causing urogenital and intestinal schistosomiasis, respectively. Community or school-based treatment of schistosomiasis using the only recommended preventive chemotherapeutic drug currently available, praziquantel (Merck KGaA), is the most common and effective means of alleviating disease burden [3,4]. As prevalence of the infection in humans moves towards elimination in parts of sub-Saharan Africa following large-scale multi-year treatment programmes, it is apparent that low-level transmission is continuing, facilitated by the presence of freshwater snail intermediate hosts enabling reinfection [5,6]. Monitoring schistosome infections in snails as part of control and elimination surveillance, either supplementing human/veterinary parasitology surveys or as a stand-alone measure, will aid in establishing whether Schistosoma spp. transmission is persisting, interrupted or re-established following elimination.

Unguja and Pemba islands, collectively known as Zanzibar (United Republic of Tanzania), are endemic for urogenital schistosomiasis. Zanzibar has a long history of pioneering urogenital schistosomiasis research and control dating back to the 1920s. This ranges from investigating the freshwater snails involved in disease transmission to early trials of schistosomacidal drugs and assessing disease prevalence through low cost diagnostics [7-15]. More recently, the islands have been targeted for elimination with concerted efforts within the Zanzibar Elimination of Schistosomiasis Transmission (ZEST) project (2012–2017). This trialled the additive impact of integrated interventions (such as mollusciciding against the snail intermediate host or educational measures for behavioural change) in combination with bi-annual mass drug administration (MDA) and MDA alone [16-19]. Over the course of the ZEST project, prevalence was significantly reduced across the islands, but transmission was not interrupted [18], leaving focal endemicity in hotspot areas that require new methods of surveillance and tailored interventions [19-21]

Of the four endemic snail species of Bulinus on Zanzibar (B. globosus, B. nasutus, and two B. forskalii group taxa: B. forskalii; and a taxon currently undescribed and presented previously as Bulinus sp.), it has been concluded from earlier studies investigating local intermediate host compatibility that only B. globosus is a compatible intermediate host involved in S. haematobium transmission, with B. nasutus being refractory to S. haematobium infection on Zanzibar [22-25]. Schistosoma haematobium transmission in the past couple of decades was therefore considered to be restricted across the islands to only freshwater bodies where B. globosus resided (Fig 1), with the distribution of B. globosus on the islands also being constrained by this species’ habitat preferences such as water hardness [23]. Therefore, freshwater habitats (e.g., in the southern part of Unguja) previously identified as containing only B. nasutus (a freshwater snail species closely related in the Bulinus africanus species group and morphologically overlapping with B. globosus) were considered free of S. haematobium transmission (Fig 1), despite there being evidence that B. nasutus can harbour pre-patent Schistosoma spp. on Pemba [26]. Schistosoma haematobium populations on Zanzibar are considered more genetically diverse in comparison to mainland African populations, with the presence of both Group 1 (mainland Africa) and the more diverse Group 2 (Indian Ocean Islands) strains [27,28]. However, it was not until recently that a second Schistosoma species, S. bovis which infects ruminants, was identified as being transmitted by B. globosus on Pemba [29]. Surveillance of Schistosoma transmission in Zanzibar is therefore complicated by not only the presence of two morphologically indistinguishable larval Schistosoma species shed from Bulinus snails, but also two morphologically overlapping Bulinus species.

Fig 1

Map of Pemba and Unguja islands (Zanzibar, United Republic of Tanzania) showing shehias where malacological surveys for Bulinus species were conducted in the current study and predicted distributions of Bulinus spp. and Schistosoma haematobium endemicity based on previous findings.

Bulinus spp. distribution inferred from Stothard et al. [23] and Pennance et al. [29,30]. Schistosoma haematobium infection distribution interpreted from Knopp et al. [18]. Digital shape files for Unguja and Pemba administrative regions were obtained from DIVA-GIS (https://www.diva-gis.org).

Despite several studies demonstrating the incompatibility of S. haematobium with B. nasutus on Unguja [22-25], overlapping areas of B. nasutus and urogenital schistosomiasis endemicity have been shown to exist predominantly on Pemba (Fig 1), and reports up until 1962 demonstrate high S. haematobium prevalence and only B. nasutus presence in the south of Unguja [9,10,23]. This suggests that B. nasutus could be acting as an intermediate host for S. haematobium on Pemba and in the past on Unguja, as is the case in nearby coastal regions of Kenya [31] and Tanzania [32-37], with the addition of the closely related snail species B. (nasutus) productus also identified as involved in transmission in mainland Tanzania [38].

In this study we aimed to investigate the current transmission status of urogenital and bovine schistosomiasis on Pemba and Unguja islands by monitoring the species distributions and genetic diversity of B. globosus and B. nasutus and their associated Schistosoma spp. collected between 2005 and 2019. Inferences are made on how the findings may impact schistosomiasis control, surveillance and elimination in Zanzibar.

Methods

Ethics statement

Ethical approval for the collection and analyses of the snail and schistosome samples collected during the ZEST project were obtained from the Zanzibar Medical Research Ethics Committee in Zanzibar, United Republic of Tanzania (ZAMREC, reference no. ZAMREC 0003/Sept/011), the “Ethikkommission beiber Basel” (EKBB) in Basel, Switzerland (reference no. 236/11) and the Institutional Review Board of the University of Georgia in Athens, Georgia, United States of America (project no. 2012-10138-0) [18]. The ZEST study is registered with the International Standard Randomized Controlled Trial Number register (ISRCTN48837681). Additional sampling and analyses of snails in Unguja and Pemba were conducted in agreement with the Neglected Diseases Program of the Zanzibar Ministry of Health and the Public Health Laboratory-Ivo de Carneri respectively. All other snail and cercariae samples used were accessioned in the Schistosomiasis Collection at the Natural History Museum [39].

Sampling of Bulinus spp. on Pemba and Unguja islands

Following oral approval to conduct surveys by local Shehas, community leaders that locally govern each area (Shehia), human-freshwater contact sites were located from previous reports or with the help of local residents. Coordinates were taken at 112 freshwater sites (109 on Pemba and three on Unguja) across 20 shehias (Fig 1) using a Garmin GPSMAP 62sc device (Garmin, Kansas City, USA) and each water body was surveyed for the presence of intermediate host snails. Between 1 and 5 surveys were conducted at each human-freshwater contact site across Pemba and Unguja in October 2016, October 2017, February, July and November 2018 and January 2019 (Table 1).

Table 1

Bulinus spp. collected in Pemba and Unguja islands (Zanzibar, United Republic of Tanzania) and samples analysed from previous collections and curated within the Schistosomiasis Collection at the Natural History Museum (SCAN).

Island Shehia	Collection Dates	No. freshwater bodies surveyed	No. Bulinus collected a	No. Bulinus molecularly identified	Bulinus spp.c of the subset molecularly identified	No. Bulinus snails infected with S. h (S. b) e
Pemba
Ukutini	21/02/2018	10	750	29	B. globosus	0
	18/07/2018	10	996	29	B. globosus	0
	19/01/2018	10	724	23	B. globosus	0
Pujini	11/10/2017	10	210	20	B. nasutus	0
	15/02/2018	10	268	17	B. nasutus	0
	22/07/2018	10	467	19	B. globosus & B. nasutus	0
	19/11/2018	10	0	0	NC b	0
Kizimbani	25/10/2016	3	199	6	B. globosus	0
	10/10/2017	3	289	9	B. globosus	2
	16/02/2018	4	283	14	B. globosus	0
	20/07/2018	4	102	12	B. globosus	0
	22/11/2018	4	75	11	B. globosus	0
Kinyasini	20/10/2016	9	463	17	B. globosus	1 (5)
	11/10/2017	11	744	27	B. globosus	0
	14/10/2018	11	627	31	B. globosus	7
	19/07/2018	11	369	25	B. globosus	0
	22/11/2018	11	241	25	B. globosus	1 (2)
Wambaa	26/10/2016	8	79	3	B. globosus	0
	08/10/2017	9	1033	24	B. globosus	0
	19/02/2018	10	267	25	B. globosus	0
	21/07/2018	11	356	18	B. globosus	0
	21/11/2018	11	348	22	B. globosus	0
Wawi	22/10/2016	2	149	3	B. globosus	0
	05/10/2017	3	183	7	B. globosus	0
	13/02/2018	3	10	4	B. globosus	0
	17/07/2018	3	1	1	B. globosus	0
	20/11/2018	3	4	3	B. globosus	0
Ole	27/10/2016	11	0	0	NC b	0
	06/10/2017	13	4	3	B. globosus	0
	12/02/2018	13	3	3	B. globosus	0
	17/07/2018	13	0	0	NC b	0
	23/11/2018	13	0	0	NC b	0
Matale	27/10/2016	10	0	0	NC b	0
	09/10/2017	11	420	16	B. globosus	0
	19/02/2018	11	0	0	NC b	0
	23/07/2018	11	212	16	B. globosus	0
	20/11/2018	11	167	14	B. globosus	0
Chambani	24/10/2016	9	291	10	B. globosus & B. nasutus	1
	07/10/2017	13	91	0	NC b	0
	09/02/2018	13	0	0	NC b	0
	24/07/2018	2	16	0	NC b	0
	19/11/2018	2	16	0	NC b	0
Uwandani	19/10/2016	9	136	10	B. nasutus	0
	05/10/2017	9	261	1	B. nasutus	0
	20/02/2018	10	18	0	NC b	0
	17/07/2018	10	28	0	NCb	0
	26/11/2018	2	12	0	NC b	0
Kangagani	22/01/2019	1	198	1	B. nasutus	1
Wingwi	-	1d	NAa	1	B. globosus	0
Wesha	22/10/16	6	0	0	NCb	0
Unguja
Jendele	-	1d	NAa	1	B. nasutus	0
Miwani	06/02/2013	1d	NAa	1	B. globosus	1
Kinyasini	10/10/2016	1d	NAa	1	B. globosus	1
Mtopepo	-	1d	NAa	1	B. nasutus	0
Chaani	06/07/2005	1d	NAa	1	B. globosus	0
Mtende	17/07/2018	1	4	4	B. nasutus	0
Kizimkazi Dimbani	23/07/2018	1	2	2	B. nasutus	0

a NA indicates that data for the total number of Bulinus snails collected during the survey were not recorded.

b NC indicates that no snails were either collected or identified during these surveys.

c Species inferred from cox1 similarity to reference sequences.

d Snails from the SCAN collections.

e S. h = S. haematobium, S. b = S. bovis inferred from cox1 and complete ITS (1–5.8S-2) rDNA region similarity to reference sequences. All Bulinus snails with patent (shedding schistosome cercariae) infections, and schistosome DNA extracted from cercariae.

At each site, snails were identified using shell morphology to their genera, with any non-Bulinus africanus group snails (namely B. forskalii group snails) being returned to the collection site. Snails were collected by hand predominantly from submerged vegetation and tree roots around the water’s edge that were in close proximity to access points to the water. Each site was surveyed for 15 minutes by three collectors, starting from access points and then searching as much of the accessible perimeter of the waterbody during this time. Snails morphologically identified as either B. globosus or B. nasutus (species differentiation on morphology alone not possible) were placed in collection pots and transported back to either the Public Health Laboratory-Ivo de Carneri (Chake Chake, Pemba) or the Neglected Diseases Program laboratory (Zanzibar Town, Unguja) where they were counted and housed in plastic trays with bottled water and covered by a glass lid overnight to acclimatise. The following morning (before 08:00), snails were rinsed with bottled water (to mitigate carry over of any cercariae between snails) and examined for cercarial shedding by placing individuals in wells of 12-well ELISA plates filled to approximately two thirds with bottled water and placed under indirect sunlight. Each well was checked using a dissection microscope after two hours and again eight hours after first sunlight to capture schistosomes with different shedding patterns [40]. An experienced microscopist distinguished furcocercous schistosome cercariae from other species using descriptions of Schistosoma spp. under a dissecting microscope [41]; a subset of any shed cercariae were individually captured and pipetted in 3.5 μl onto Whatman FTA cards (Whatman, Part of GE Healthcare, Florham Park, USA) for molecular characterisation. All snails were preserved in 100% ethanol for subsequent molecular characterisation as previously described (see [29]).

A targeted malacological survey was conducted in late January 2019 to collect B. nasutus snails at one site in Kangagani on Pemba previously identified as inhabited by B. nasutus (see [26]) (Fig 1). These snails were maintained in laboratory aquaria (dimensions 45x30x30cm, 40.5L, filled to approximately two thirds full with water from the collection site and equipped with an air pump for continuous aeration) at a maximum density of 100 Bulinus individuals per aquarium. Snails were re-checked for shedding of schistosome cercariae three weeks later in an effort to capture any infections that may have been pre-patent during the initial screen. Aquarium water was replaced twice a week using water from the site of collection (Kangagani). Water was only used in aquaria after storage for at least 48 hours in transparent 15L water containers, allowing for any sediment to settle and eliminate the risk of introducing live schistosome eggs, miracidia or cercariae into the aquaria. Snails were fed on dried lettuce when all lettuce in the tank had been eaten. Dead snails were removed from the aquaria daily.

Archived samples included in the analysis

Bulinus samples, from Unguja and Pemba, accessioned within the Schistosomiasis Collection at the Natural History Museum (SCAN) [39] were included in the study providing material from areas not covered in the malacological surveys described above. Samples were only included if associated geographical information was available. Six snails were included, five from Unguja (Jendele, Miwani, Kinyasini, Mtopepo, Chaani) and one from Pemba (Wingwi) as presented in Table 1. Two of these snails from Miwani and Kinyasini on Unguja were recorded as patent with S. haematobium when they were collected, as this was later confirmed by cercarial molecular analysis as described below. The remaining four Bulinus snails from the SCAN collection were negative for patent Schistosoma infections (S1 Table). One of the infected B. globosus identified in the SCAN repository (MCF389B0F0286, S1 Table) could not be associated with its emerging S. haematobium cercariae as it was preserved together with two other B. globosus also infected with S. haematobium from the same site.

Bulinus spp. molecular characterisation

Since a significant degree of morphological overlap exists between B. globosus and B. nasutus, a molecular marker was used to unequivocally identify a subset of the Bulinus snails collected (n = 504), and those taken from archived specimens (n = 6). The shell was removed by crushing and the use of sterile forceps from the preserved sample and gDNA from whole snail tissue was extracted using either the Qiagen BioSprint 96 DNA Blood Kit following manufacturer’s instructions (Qiagen, Manchester, UK) or the Qiagen DNeasy Blood & Tissue Kit modified protocol (Qiagen, Manchester, UK) using double volumes of the lysis buffers [29]. Since not all snails could be identified using molecular characterisation due to cost and time constraints, a minimum of three non-patent snails per site per malacological survey (except for those collected on Pemba during October 2016 and from Chambani and Uwandani in 2017/2018) were randomly selected for identification. Molecular characterisation was performed for all snails with patent Schistosoma infections and snails retrieved from SCAN.

DNA was extracted from a total of 510 Bulinus spp. snails, from Unguja (11 snails collected from 8 sites) and Pemba (499 snails collected from 63 sites). A 623 bp partial region of mitochondrial cox1 DNA was amplified and Sanger sequenced following previously described methods (see [29]). Sanger sequence data were edited, manually trimmed to 463–621 bp and aligned in Sequencher v5.4.6 (GeneCodes Corp., Michigan, USA) before being collapsed into cox1 haplotype groups. Species identification were confirmed by alignment and phylogenetic analysis (see below) of Bulinus cox1 haplotypes, as presented in S2 Table, with reference data for B. globosus and B. nasutus [42].

Schistosoma spp. cercariae molecular characterisation

From each infected snail either two or six Schistosoma cercariae were processed individually for molecular identification. Six cercariae were individually processed from each infected snail collected between 2016 and 2019 (n = 20 snails) and two cercariae for snails collected as part of the ZEST study (n = 69 snails) [16-18] and made available via SCAN (S3 Table). Following elution of parasite DNA from Whatman FTA cards [43], Schistosoma species identification was confirmed by mito-nuclear genetic profiling targeting the partial mitochondrial cox1 region (956 bp) and the complete nuclear ITS (1–5.8S-2) rDNA region (967 bp) from each individual cercariae as described in [27,28]. Both mitochondrial and nuclear DNA were analysed for species identification and to identify any hybridisation [44]. The cox1 data were also used for genetic diversity and phylogenetic analyses. The sequence data were manually edited and trimmed to 750 bp for cox1, and 880bp for ITS, using Sequencher v5.4.6 (GeneCodes Corp., Michingan, USA). The cox1 species identity was confirmed by comparison to nucleotide sequences using NCBI-BLAST [45] and the ITS species ID was confirmed by comparison to reference data as described [28,29]. Species identification of each cercariae was confirmed by concordance between the cox1 and ITS genetic profiles. Cercariae of identical cox1 sequences were collapsed into cox1 haplotype groups for further phylogenetic analysis (S4 Table). ITS data were not used for phylogenetic analysis since no intra-species diversity was observed.

Phylogenetic cox1 analysis of Bulinus spp. and Schistosoma spp

The Bulinus haplotype data were imported into Geneious v11.1.4 [46] for phylogenetic analysis together with reference data for B. nasutus and B. globosus collected previously from East Africa (Zanzibar, Tanzania, Kenya, Uganda, Mafia Island; [42] and an outgroup of Biomphalaria glabrata available from GenBank (Accession: NC005439) [47]. Haplotype alignments were performed using ClustalW v2.1 [48] executed in PAUP* [49] and then an appropriate evolutionary nucleotide substitution model (HKY + I + G; -lnl 2020.2144, AIC 4052.4287) was selected in MrModelTest v2.4 [50] using the Akaike Information Criterion. Bayesian inference was performed using MrBayes v3.2.7a [51]. The burn-in was set at 3.5 million generations for consistency after confirming that the average standard deviation of split frequencies (ASDOSF) reported from MrBayes output was at least <0.01 by this point. Clades were considered to have high nodal support if Bayesian inference posterior probability was ≥0.95; tree nodes with <0.95 were collapsed in SumTrees v4.4.0 [52].

Schistosoma cercarial haplotype phylogenetic analyses were performed as above with S. curassoni (AY157210; [53]) as the outgroup. Analyses also included published S. haematobium haplotypes from Zanzibar (GU257334 –GU257360; [28]), and S. bovis from Pemba (MH014042 & MH014043; [29] and OK484569 [54]), mainland Tanzania (AY157212; [53]) and Cameroon (MH647141; [55]), with the alignment trimmed to 750 bp to maintain uniform ends. A Templeton, Crandall and Sing’s (TCS) haplotype network analysis was also conducted using PopART [56,57] using the same sequence alignment.

Statistical analysis

Within the molecular analyses S. haematobium cercariae were assigned to either the Group 1 or Group 2 cox1 haplotype group [27], and Chi-squared tests were performed in R v.4.0.0 [58] to investigate any differences in the abundance of the two groups in relation to their snail host species and geographical distribution.

Spatial distribution of Bulinus and Schistosoma species

Bulinus and Schistosoma spp. distribution data were visualised using QGIS v3.0.1 Girona (http://qgis.osgeo.org) and mapped for each site. Digital shape files for Unguja and Pemba administrative regions were obtained from DIVA-GIS (https://www.diva-gis.org).

Results

Patent schistosome infections of Bulinus

Over the six malacological surveys conducted on Pemba between 2016 and 2019, and the one survey conducted in Unguja, a total of 11,116 Bulinus spp. were collected from 65 of the 112 sites. From the subset of the snails that were identified by molecular analysis from each of these sites (n = 504), and those identified from the SCAN repository (n = 6), the majority were B. globosus (n = 433 snails), the remainder were B. nasutus (n = 77 snails). The other 10,606 Bulinus collected remain only morphologically identified as being within the Bulinus africanus species group (Bulinus genus), since morphological differentiation between B. globosus and B. nasutus is not possible. Of the 11,116 Bulinus, 0.2% (n = 20) shed Schistosoma spp. cercariae. These were collected from eight sites: four sites in Kinyasini (n = 16 snails), two in Kizimbani (n = 2 snails), one in Chambani (n = 1 snail) and one in Kangagani (n = 1 snail) as presented in Table 1. The infected snail from Kangagani was collected during the targeted malacological B. nasutus survey, in which 198 individual Bulinus spp. specimens were collected and at the time of collection were not shedding. Although having no observed patent schistosome infections during the first round of shedding, a single B. nasutus was found shedding Schistosoma cercariae 21 days later. No follow up shedding was attempted on the other snails collected in Pemba as they were preserved within 48 hours of collection.

For the malacological surveys conducted at three sites in two shehias (Mtende and Kizimbani Dimbani) on Unguja, only six B. nasutus (Table 1) were collected in total, of which none shed Schistosoma cercariae within 24 hours of their collection. No further shedding attempts were made on these snails which were preserved after the first round of shedding.

Bulinus spp. genetic diversity and distribution

From the subset of Bulinus spp. successfully sequenced, 433 out of 510 from 3 sites across Unguja and 49 sites across Pemba were B. globosus (S1 Table). The remaining 77 snails were identified as B. nasutus collected from 15 sites in Pemba and 5 in Unguja (S1 Table). Bulinus nasutus was molecularly identified only from freshwater bodies near the east coast of Pemba and the southern districts up to the central west areas of Unguja (Fig 2). Where >1 snail was molecularly identified per site, the snails were identified as either B. globosus or B. nasutus coming from the same site, except for from one waterbody on Pemba (Puj11, Fig 2B), where both species co-occurred in the same seasonal pond (S1 Table). Based on the subset of molecularly identified Bulinus, no other mixed B. globosus and B. nasutus populations were observed, however this would have to be fully confirmed with further genetic analysis of a large set of snails from each water body.

Fig 2

A: Inferred Bulinus globosus and B. nasutus distribution on Unguja and Pemba islands (Zanzibar, United Republic of Tanzania) as identified by mitochondrial cox1 sequences of a subset (n = 510) of Bulinus spp. collected. B: Highlighted South East region of Pemba, displaying human freshwater contact sites in four shehias (Matale, Pujini, Chambani, Ukutini) and the single freshwater body cohabited by B. globosus and B. nasutus (Puj11).

Digital shape files for Unguja and Pemba administrative regions were obtained from DIVA-GIS (https://www.diva-gis.org).

All 11 B. globosus and 9 out of 10 B. nasutus cox1 haplotypes were unique to either Unguja or Pemba, with one exception being B. nasutus haplotype 3, as shown in S2 Table, that was detected on both islands. A single clade of B. nasutus specimens from both Unguja and Pemba was observed, whereas the B. globosus isolates from each island fell into two distinct clades (Fig 3). All B. globosus from Pemba fell into one clade containing two sister groups, with those previously identified from Pemba [42]. The three haplotypes from Unguja fell into a another clade containing two sister groups of B. globosus from Eastern Kenya and those previously identified from Unguja (Fig 3) [42].

Fig 3

Bayesian inference of the partial mitochondrial cox1 haplotype dataset of Bulinus nasutus and B. globosus collected from Unguja and Pemba.

Reference data from East Africa (Kane et al. [42]). Tree produced using Bayesian inference using MrBayes v3.2.7A [51] under the HKY+I+G model (-lnl 2020.2144, AIC 4052.4287). Branches <0.95 posterior probability collapsed. The branch length scale bar indicates the number of substitutions per site. Text in red indicates Bulinus haplotypes generated from the current study as listed in S2 Table.

Schistosoma spp. cercariae identification

From the subset of cercariae identified from 89 of the infected Bulinus collected from Zanzibar between 2016 and 2019 (n = 20) and identified during the ZEST study (n = 69), 82 were shedding S. haematobium with 18 different haplotypes and seven were shedding S. bovis with two haplotypes (S3 and S4 Tables). Both Group 1 and 2 haplotypes of S. haematobium representing mainland African and Indian Ocean islands respectively were identified [27,28] (Figs 4 and S1). Including Schistosoma coinfections, of which there were seven determined by multiple cox1 haplotypes presented in S5 Table, the proportion of snails shedding S. haematobium Group 1 cercariae (n = 41) was similar to those shedding Group 2 cercariae (n = 45). However, the majority of Group 1 S. haematobium infections occurred in Unguja (n = 35), with significantly fewer (n = 6) from Pemba (χ2 = 10.0, df = 1, P< 0.01). In contrast, Group 2 S. haematobium cercariae were distributed evenly across the islands (Unguja n = 23 and Pemba n = 22). Most (n = 13 of 18) S. haematobium cox1 haplotypes were unique to either Pemba or Unguja, but five were present across both islands.

Fig 4

TCS haplotype network of Schistosoma spp. partial cox1 DNA sequences (750 bp).

Produced using PopArt [56]. Hatches represent SNP differences from joined nodes and size of nodes is scaled to the number of identical haplotypes listed. Schistosoma haplotype group 1 and 2 indicates whether cercariae were identified as mainland Africa (1) or Indian Ocean Island (2) haplotypes (as described in Webster et al. [27]). Schistosoma haematobium reference haplotypes (GU257334 –GU257360) from Webster et al. [28]. Schistosoma bovis reference haplotypes (OK484569, AY157212 and MH647141) from Pennance et al. [54], Lockyer et al. [53] and Djuikwo-Teukeng et al. [55], respectively. Schistosoma curassoni reference (AY157210) from Lockyer et al. [53].

Seven snails as presented in S5 Table were confirmed as shedding Schistosoma cercariae with multiple cox1 haplotypes of either S. haematobium (n = 6) or S. bovis (n = 1), indicating they had been infected by multiple miracidia. Furthermore, five of the six S. haematobium infected snails simultaneously shed both Group 1 and Group 2 S. haematobium haplotypes, whilst only one snail was identified shedding two haplotypes of Group 2.

Comparison of mitochondrial cox1 and nuclear ITS profiles from S. haematobium cercariae showed no evidence of hybridisation between S. haematobium and S. bovis. No intraspecies variation in the ITS profiles were observed, with 100% match to reference data [27].

Bulinus observed shedding Schistosoma haematobium and S. bovis

Of the 20 infected snails collected in Pemba listed in S6 Table, 12 were identified as B. globosus infected with S. haematobium and seven as B. globosus infected with S. bovis. The remaining infected Bulinus collected during the targeted survey in Kangagani was identified as B. nasutus (Haplotype 3 in S2 Table), matching that previously reported on Pemba (GenBank Accession: AM921812, see [42]). The two cercariae identified from this snail were S. haematobium of a single cox1 haplotype (Sh3). This S. haematobium haplotype has been previously identified as ‘Group 1’ (GenBank Accession: GU257343, see [28]) representing those from mainland Africa and Zanzibar (Figs 4 and S1 Fig). The infected B. globosus from Unguja from the SCAN repository was shedding ‘Group 2’ cercariae as presented in S6 Table.

Discussion

Here we update Schistosoma and Bulinus species distributions in Zanzibar as a schistosomiasis surveillance resource, while also investigating specific associations between Bulinus and Schistosoma spp. on both Pemba and Unguja islands. Of the 11,116 Bulinus spp. collected from Pemba and Unguja between 2016 and 2019, 0.2% were infected with S. haematobium group species; B. globosus shed S. haematobium (n = 12) and S. bovis (n = 7) and B. nasutus shed S. haematobium (n = 1). The latter host-parasite relationship is the first to confirm preliminary findings by Ame [26] whilst also refuting that B. nasutus is refractory to S. haematobium infection across Zanzibar [23]. The distribution of B. globosus and B. nasutus across Pemba and Unguja inferred from molecular identifications of a subset of those collected confirmed previous findings of separate distributions [23,29,30], with the exception that the two Bulinus species were present in the same waterbody at one site and in close proximity across two other neighbouring sites along the east coast of Pemba (potentially overlapping distribution) where only B. nasutus was known to be abundant from previous reports. Identification of a second compatible intermediate snail host for S. haematobium (B. nasutus) changes our understanding of the snail and Schistosoma spp. biology on Zanzibar. In addition, the confirmed presence of B. globosus infected with S. bovis almost two years following the first recording of this pathogen on the island [29] and in combination with the presence of infected cattle [54], is cause for concern since increased transmission could lead to significant animal health and economic impacts, as well as a potential risk for hybridisation with S. haematobium (see [59,60]).

Distribution and diversity of Bulinus globosus and B. nasutus

Co-occurrence of B. globosus and B. nasutus was observed at just one site on Pemba (Puj11), generally supporting previous observations that species distribution in freshwater bodies is dictated by species specific ecological factors (such as water conductivity) determined by the geological zones of Zanzibar (see [61,62]). However, it is noteworthy that during the current study it was only feasible to identify a proportion (n = 510 of 11,116) of the B. globosus and B. nasutus accurately through partial cox1 sequencing. Therefore, it is possible that other sites containing both B. globosus and B. nasutus may be identified on the Zanzibar Archipelago in future species identification. The development of a cheap, easily interpreted, rapid diagnostic assay to distinguish between B. globosus and B. nasutus, such as is available for the differentiation of S. haematobium and S. bovis [63], would provide a much needed solution for identifying large numbers of field collected specimens.

The 11 B. globosus cox1 haplotypes identified from the snails collected across Zanzibar fell into two distinct clades representing Unguja and Pemba taxa [24]. The B. globosus from Unguja were more closely related to those previously identified from East Kenya [42], whilst those from Pemba form an independent group distinct from the other East African isolates, suggesting independent origins. This agrees with our current understanding of Zanzibar’s geological formation, whereby Pemba island separated from mainland Africa earlier, during at least the early Pliocene compared to Unguja island during the Pleistocene [61,64]. In contrast, there was no phylogenetic distinction between the B. nasutus from Unguja, Pemba and mainland East Africa, with samples from each region all forming a single clade of multiple haplotypes. As recorded previously, B. globosus is more genetically diverse than B. nasutus (see [65]). Identical haplotypes of B. nasutus were also present on Unguja and Pemba. At this stage of investigation, we postulate that since B. nasutus infected with S. haematobium observed here in Pemba matched haplotypes of B. nasutus in the south of Unguja (see below), it might serve as an intermediate host of urogenital schistosomiasis across the Archipelago, as is the case in Kenya [31,66] and Tanzania [32-37].

A ‘new’ intermediate host of Schistosoma haematobium on Pemba: Bulinus nasutus

The finding of B. nasutus in one locality naturally infected with S. haematobium on Pemba contradicts previous results suggesting that this snail species was not involved with the transmission of urogenital schistosomiasis on Zanzibar [22-24]. Indeed, pre-patent Schistosoma spp. infections previously observed in B. nasutus from Pemba might have been S. haematobium (see [26]) but infections did not appear to result in cercarial production [25].

Schistosoma haematobium cercariae shed from the intermediate snail hosts, analysed here, fall into the two known S. haematobium haplotype groups (1 and 2) previously identified from miracidia collected from infected humans [27,28]. The cox1 haplotypes of cercariae shed from B. nasutus were identified as Group 1, a group predominantly associated with African mainland S. haematobium populations. Possibly only Group 1 S. haematobium is compatible with Pemba B. nasutus, since this same snail species acts as an intermediate host in East Africa [31-37], however more samples would be needed to test this hypothesis. Schistosoma strain specific interactions/compatibilities with intermediate host snails based on differential immune responses, have been well studied and established in strains of S. mansoni and B. glabrata (as summarised in [67]). However, relatively little is understood regarding co-evolution of host/parasite compatibility between S. haematobium group strains and Bulinus species, save some studies investigating geographical isolates (see [68-72]). Such strain dependency or local adaptation reflects the patchy compatibility of Bulinus snail hosts of Schistosoma generally [73], discussed in a previous study in Tanzania, where experimental infections of B. nasutus using a local strain of S. haematobium that usually infects B. globosus were unsuccessful [74]. As demonstrated again here, B. globosus remains the primary host of S. haematobium on Zanzibar, but endemic B. nasutus may also play a minor role in transmission involving specific S. haematobium strains, complicating future monitoring. This hypothesis also provides some explanation to how ‘intermittent’ and/or ‘unstable’ transmission was historically maintained in areas of Zanzibar (such as south Unguja) where only B. nasutus is present currently [7,9,10,75,76].

Study limitations and future work

Several limitations are apparent in the current study. The time and costs required to generate cox1 sequence data limited the number of snail intermediate hosts that could be identified by this means. Development and testing of a rapid diagnostic assay, or alternative, to provide rapid high throughput identification of snails would greatly support future studies. Additionally, few Bulinus specimens were available for analysis from Unguja, so further malacological surveys with molecular sub-sampling here would be beneficial to confirm snail species distributions across the island. Also, since the majority of infected Bulinus spp. collected during the ZEST studies were not accessioned with their associated Schistosoma spp., complete inferences on snail-Schistosoma relationships were not possible. Finally, although cercariae identification was used here to identify Schistosoma species, it would have been of interest to identify a greater number of cercariae per snail infection, as this may have significantly increased the number of coinfections observed from these Bulinus spp. and allow for the potentially immune modulated interactions of Group 1 and Group 2 S. haematobium coinfections, observed in five snails here, to be explored further. In a future study, it would also be of interest to use PCR based methods to identify pre-patent S. haematobium and S. bovis infections in snails to further assess the total number of snails that have been exposed to schistosomes but are not currently contributing to transmission [77].

Implications for future monitoring of schistosomiasis on Zanzibar

The findings discussed here provide implications for future control and elimination efforts of urogenital schistosomiasis on Zanzibar [18,19]. First, it is suggested here that the Ministry of Health, Social Welfare, Elderly, Gender and Children Zanzibar should conduct periodic surveys in areas associated with B. nasutus distribution on Unguja and Pemba. These surveys should include both malacological collections and combined human urine collections with questionnaires (including questions on freshwater usage locally and elsewhere in Zanzibar), followed by targeted treatment of infected individuals and focal snail control, to reduce any ongoing transmission. Second, a monitoring system to check the identity of schistosome cercariae shed from Bulinus spp. snails from Zanzibar to differentiate bovine and human schistosomiasis would enable accurate mapping of both schistosome species, appropriate targeting of urogenital schistosomiasis control interventions, and also monitor any potential hybridization events between S. haematobium and S. bovis that may be occurring in cattle or humans [60].

Nearly a century has passed since the first reports of widespread human urogenital schistosomiasis on Zanzibar, during which time there have been great achievements towards urogenital schistosomiasis elimination. As the battle to eliminate schistosomiasis from the Zanzibar Archipelago continues, our findings emphasize the need to carefully plan future surveillance strategies of transmission on the islands, taking into consideration the presence of bovine Schistosoma species and the capacity for an expanded intermediate host, and therefore geographical, range of S. haematobium.

Associated Bulinus spp. specimen data.

Detailed information on Bulinus spp. specimens used in current study, including species identification determined through molecular analysis (cox1), collection site name including latitude and longitude, schistosome species patency and cox1 haplotype.

(XLSX)

Click here for additional data file.

Bulinus spp. cox1 haplotypes observed from Pemba and Unguja, and associated Schistosoma spp. infecting each snail haplotype.

a Schistosoma haplotype group indicates whether cercariae were identified as mainland Africa (1) or Indian Ocean Island (2) haplotypes (as described in Webster et al. [27]). b It was not possible to associate this snail haplotype with its S. haematobium cercariae cox1 haplotype(s) as it was preserved with two other S. haematobium infected B. globosus.

(XLSX)

Click here for additional data file.

Associated Schistosoma spp. specimen data.

Detailed information on Schistosoma spp. specimens used in current study, including species identification determined through molecular analysis (cox1 and ITS1-5.8S-ITS2), collection site name including latitude and longitude and Schistosoma cox1 haplotype.

(XLSX)

Click here for additional data file.

Schistosoma cox1 haplotypes identified from cercariae from Unguja and Pemba.

a Schistosoma haplotype group indicates whether cercariae were identified as mainland Africa (1) or Indian Ocean Island (2) cox1 haplotypes (as described in Webster et al. [27]). b Island; U = Unguja, P = Pemba.

(XLSX)

Click here for additional data file.

Trematode coinfections of Bulinus spp. from Unguja and Pemba.

a Schistosoma haplotype group indicates whether cercariae were identified as mainland Africa (1) or Indian Ocean Island (2) cox1 haplotypes (as described in Webster et al. [27]). b Coinfection indicates the Schistosoma cox1 haplotype group (as described in [27]) infection profile of each Bulinus spp. Sh1 = Schistosoma haematobium mainland African haplotype group 1, Sh2 = S. haematobium Indian Ocean haplotype group 2, Sb1 = S. bovis haplotype 1, Sb2 = S. bovis haplotype 2.

(XLSX)

Click here for additional data file.

Bulinus spp. infected with Schistosoma spp. identified from Unguja and Pemba.

a Schistosoma haplotype group indicates whether cercariae were identified as mainland Africa (1) or Indian Ocean Island (2) haplotypes (as described in Webster et al. [27]).

(XLSX)

Click here for additional data file.

Bayesian inference of the partial mitochondrial cox1 haplotype dataset of Schistosoma haematobium and S. bovis collected from Unguja and Pemba.

Phylogenetic tree constructed using Bayesian inference in MrBayes v3.2.7a [51] under the HKY + I model (-lnL = 1809.8890, AIC 3629.7781, ASDOSF < 0.01 at 1,791,000 generations). Branches with <0.95 posterior probability are collapsed. The branch length scale bar indicates the number of substitutions per site. Text in red indicates Schistosoma haplotypes generated in the current study.

(TIF)

Click here for additional data file.

6 Mar 2022

Dear Dr. Pennance,

Thank you very much for submitting your manuscript "Transmission and diversity of Schistosoma haematobium and S. bovis and their freshwater intermediate snail hosts Bulinus globosus and B. nasutus in Zanzibar" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

All three reviewers found the submitted paper useful and addressing an important topic. Nevertheless, they have raised some significant points, particularly with regard to identification of the snails and presentation of some of the data, which should be the focus for preparation of any resubmission.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

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Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

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Associate Editor

PLOS Neglected Tropical Diseases

Simone Haeberlein, PhD

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: See below

Reviewer #2: Overall, the methods employed seem reasonably sound. However I have some serious concerns regarding the number of snails and cercariae examined in the study.

My principal concern relates to the identification of the snails. Table 1 shows that a very large number of snails have been collected in the study but that only a very small number of these snails have been identified using molecular methods. As I initially read the methods section I assumed (as it turns out incorrectly) that all snails collected in the study had been identified (presumably using morphological methods) with molecular methods used to confirm identifications for just a small number of individuals. Such confirmation of identifications would have been perfectly acceptable but it turns out that the authors never actually bothered to identify the vast majority of snails they collected. Instead, we are told at the start of the results that the snails in a population were 'retrospectively' assigned as B. globosus or B nasutus based on the very limited number of samples for which molecular identifications were undertaken. So for the first site shown in Table 1 (Ukutine 21/2/2018) it is implied that 750 B. globosus snails were collected at that site, yet only 29 snails were actually identified as B. globosus. We see the same issue at all other sites. There would seem to be little point collecting such large numbers of snails from a site if you don't bother to look at them (to identify them) and it is most definitely not appropriate to 'retrospectively' identify snails in this way and in effect mislead the reader by implying that considerably more work has been undertaken than has actually been done.

The authors should therefore only present the data that they have. So for the site Ukutine in 21/2/2018, they collected 29 B. globosus specimens and B. globosus made up 100% of the snails identified at that site. Indeed this is exactly how most field studies are undertaken. We sample 20-30 individuals from a site which we identify and based on the proportions in our sample we determine the percentage of each species present. We don't imply that we have data for 750 snails when we do not.

Unfortunately for the authors, while sites like Ukutine with 29 identified specimens have been sampled reasonably well, other sites have not. Kizimbani 2016 has just 6 samples, Wawi 2016 has just 3 samples. The fact that you collected 199 snails from Kizimbani in 2016 and 149 snails from Wawi in 2016 is completely irrelevant as you didn't bother to look at them. Unfortunately there are many other sites with such pitiful numbers of identified snails.

The next question is does this matter i.e. is this likely to impact the results. The short answer is yes it does matter. Unfortunately, it is not the case that you only ever find monomorphic populations (with just a single species at a site) so sampling only a handful of individuals and inferring that this applies to the site as a whole is not enough. The authors own data in Table 1 shows that two sites have mixed populations of B. globosus and B. nasutus (Pujini 22/7/2018 and Chambani 24/10/2016). So for a site like Wawi with just 3 individuals identified and a site like Kizimbai with 6 individuals I have no confidence whatsoever that these populations are purely B. globosus and I wonder how many additional mixed populations might have been found if proper sample identification had been undertaken. I would suggest that a minimum of 20 individuals per site per time point would need to be identified for confidence.

As well as my concerns re snail identifications, I also have some concerns over the number of cercariae samples. Rather than tell us in the methods that a subset were sampled please be clear and tell us exactly how many cercariae were sampled. I do also wonder why the authors have used cercarial shedding to determine infection rate rather than use PCR based methods of infection detection. Such PCR based methods are much simpler and avoid inaccuracy due to missed pre-patent individuals (though having said this the authors do make an effort to screen for cercaria on more than one occasion; at least for the majority of populations).

Reviewer #3: (See below)

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: See below

Reviewer #2: My principal criticism of the results relates to the 'retrospective identification' of snail samples (or rather guessing the identification of the population at large based on a very small inadequate number of samples identified). Please see my comments above in the methods section of this review. I will not repeat these comments here but in essence the poor sampling undertaken (very few snails identified for some sites) renders all conclusions re presence or absence of B. globosus/ B. nasutus unsound.

Paraphyletic would seem inappropriate when referring to the clades in the tree. Remove and just refer to them as clades.

Please also note that the results text does not seem to match the data in Table 1. In the text we are told that just 1 site has a mixed population of B. globosus and B. nasutus. In Table 1, two sites are shown to have a mixed population of B. globosus and B. nasutus. I would also like to see some number in Table 1 for these 2 sites. How many of the individuals identified were B. globosus and how many B. nasutus.

Reviewer #3: (See below)

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: See below

Reviewer #2: Certainly some of the conclusions are not supported by the data presented. I have no confidence in conclusions relating to the presence/absence of B.globosus/B. nasutus at collection sites due to the inadequate number of snails identified at many of these sites.

However, I have confidence in the conclusion that B. nasutus acts as an intermediate host for S.haemotobium and is thus an important consideration in schistosomiasis control in humans.

Reviewer #3: (See below)

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: See below

Reviewer #2: It is stated in the manuscript that Schistosomiasis transmission on Zanzibar was believed to involve a single schistosome species (Schistosoma haematobium) transmitted via a single intermediate host species (Bulinus globosus). Statements to ths effect are not accurate and should be removed from the manuscript. I do appreciate that the authors are keen to enhance the impact of their work in this paper but S.bovis has previously been reported on Zanzibar by the authors of this paper in a previous paper from 2018. The discovery of S. bovis on Zanzibar is thus not a discovery new to this paper and should not be presented as such.

Reviewer #3: (See below)

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: The paper is interesting and useful. There are some details that need to be attended to before publication.

My main point of confusion concerns the number of infected snails. In the abstract and in line 364, 89 snails were said to be infected. This probably matches the number in Table S2, but I haven’t checked in detail. In other places in the paper, it is stated that 20 snails were infected (line 307). In Table 1 and Table S1, I can identify 22 infected snails. In Table 5, there seem to be 21 snails. I assume that the 69 unidentified snails are those referred to in line 251. These numbers need to be clarified.

There are some points of English expression and other details that also caught my attention.

Line 33. “… allopatric for S. haematobium…” What does that mean?

Line 48 and elsewhere. Which islands are regarded as being members of the Indian Ocean Islands?

Line 52. “…evidence that intraspecific variability may increase the transmission potential…”. Th evidence is not very convincing to me.

Lines 81-83. Low-level transmission maintained through exposure to cercariae released from infected snails. This seems to be stating the obvious. What point is being made?

Line 171 and many other places. I would not personally use “represents”. Maybe “indicates”?

Lines 181-182. “…searching as much of the perimeter of the water body as possible…”?

Lines 207-209. This sentence should be rephrased or split.

Lines 238-241. As far as I can tell, you sequenced DNA from 510 snails, not the 1009 snails as implied here. Maybe clarify that here

Line 248. The cercariae were apparently processed individually, not pooled. Perhaps make that clear.

Lines 324, 418 and maybe elsewhere. Don’t start a sentence with an abbreviated genus name.

Line 336. Paraphyletic with respect to what? In each case, they look like sister clades to me. Please check that the rest of this description of the tree in Fig. 3 is accurate.

Line 368. “co-infections” here, with a hyphen. Without a hyphen elsewhere.

Fig. 2. At least in the review pdf, the symbols on figures are rather small and hard to read. The coloured circles in Fig. 2 could be made larger for a start. In Fig. 4, the lettering is too small.

Is Fig 5 necessary, given Fig. 4? Also, inclusion of both species separated by one very long branch has the effect of rendering the branches within S. haematobium (in particular) too short to be visualised properly. Indeed, Group 1 and Group 2 haplotypes seem to be thoroughly intermingled in this tree.

Line 459. Two distinct clades – I agree. But each of these seems to consist of two distinct subclades. Might that have some significance?

Line 494. Local strain of…

Reviewer #2: There is some good data here and the finding that B.nasutus acts as an intermediate host of S. haematobium is of particular interest. Unfortunately, this very interesting finding is overshadowed by the inadequate number of snails identified at many of the collection sites. It is simply not safe to conclude that just a single species of snail is present at a site when only a handful of snails have been identified. The findings relating to the presence/absence of B.globosus/B.nasutus at sites on Zanzibar therefore cannot be trusted. I therefore do not consider that the paper can be published in its present form.

However, I do note that the authors have undertaken excellent sample collections from Zanzibar with large numbers of snails collected from many sites. I would therefore encourage the authors to revisit their data and to undertake identifications of an adequate number of snails (minimum 20 per site per time point) and resubmit.

Reviewer #3: (See below)

--------------------

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes

Reviewer #3: Comments

This manuscript is a sequel to previous articles and a project named Zanzibar

Elimination of Schistosomiasis Transmission (ZEST) project (2012-2017) that aimed at

eliminating schistosomiasis from the Zanzibar Archipelago. The authors collected three

species of Bulinus snails from the Zanzibar Archipelago in the United Republic of

Tanzania between 2016 and 2019. Snail were exposed to cercarial emergence to identify

Schistosoma in these islands. Snails were infected with two Schistosoma species: S.

haematobium (n=82) and S. bovis (n=7). Phylogenetic analysis of the mitochondrial

COI on 433 individuals of Bulinus globosus and 77 individuals of Bulinus nasutus

revealed three distinct haplotypes of B. globosus and one haplotype of B. nasutus. The

authors also analyzed a partial mitochondrial COI region and the nuclear ITS rDNA

region of 179 Schistosoma cercariae, identifying 18 haplotypes of S. haematobium and

three haplotypes of S. bovis. I found this manuscript original and relevant to the

parasitological and malacological field since it provides more precise information aimed

at a better understanding of the risk of schistosomiasis in Tanzania.

I found the manuscript very interesting for those with closely related research interests:

parasitologists studying zoonotic pathogens in Tanzania and other Africa countries, for

instance. The methodology is appropriate. I am not a specialist in schistosomiasis nor in

Bulinus snails but I think that references are fine. I found one major issue, however, that

should be ameliorated to make the manuscript more attractive and easier to follow by

the reader. Results should be shorten and presented more straightforward to make the

manuscript clearer. This issue can be easily achieved with some rewriting. The way the

authors present the results (text, figures and tables) is confusing or too complicated (see

below). I would reduce the number of figures and tables. I would delete some tables and

figures and modify others (see below). I am not sure if the journal allow supplementary

material. If it does, I would move some figures to supplementary material (see comment

below). There are also some confusing sentences or incorrect assumptions. For

example, I think that these facts should not be related (see below): “Although S.

haematobium on Zanzibar is genetically more diverse in comparison to mainland

Africa, with the presence of both Group 1 (mainland Africa) and the more diverse

Group 2 (Indian Ocean Islands) strains, it remained the only Schistosoma species

identified on the islands”.

There are two other major issues that should be emended. First, the authors say the

manuscript demonstrates that the snail species B. nasutus could play a minor role in

transmitting Schistosoma in Zanzibar because they found one individual infected with

the parasite. However, I think that the fact that suggest that this snail species could be

playing a role in transmitting the parasite is not having found one infected snail but,

instead, the fact that this snail species transmits the disease in Kenya and Tanzania.

There is no reason to believe, I think, that if the snail species is present in the

archipelago, it would not transmit the disease. This fact should clarify because it is very

confusing as it is.

Second, the authors infer that all the snail individuals found in a given site belong to the

species identified by sequencing 1-29 individuals. In my view, this is a very risky (and

wrong) assumption and should be avoided. It is very likely that some of the sampled

sites have more than one Bulinus species. In fact, the authors found two species in one

of the sites. The authors should clarify the number of snail individuals that have been

identified at the species level. They could say that XX individuals have been identify at

the species level while XXX individuals remain only identified at the genus level. Theycould mention in the Discussion section that communities with more than one Bulinus species are common.

Here I made some other comments:

Title:

o It should better specify the geographic area studied. Include “Zanzibar

Archipelago, Tanzania” in the title.

Abstracts

o Delete the following phrase “The involvement of B. nasutus (a snail

species that occupies geographically distinct regions on the Archipelago)

in S. haematobium transmission has previously been debated.”. It is the

second phrase in the abstract and is very confusing. It does not saying

anything as it is.

o Line 52:host or parasite intraspecific variability?

o Line 53: “intraspecific variability may increase the transmission potential

of S. haeamtobium due to the expanded intermediate host range”. I think

that both facts are not necessarily linked.

o The total number of collected snails and the number of sampled sites

should be present in the abstracts since it is one of the highlights of the

manuscript. There is a great sample effort and that should be

emphasized.

Introduction:

o Line 105: Snail habitat preferences?

o Line 106-109: “Although S. haematobium on Zanzibar is genetically

more diverse in comparison to mainland Africa, with the presence of

both Group 1 (mainland Africa) and the more diverse Group 2 (Indian

Ocean Islands) strains, it remained the only Schistosoma species

identified on the islands”. I think that both facts are independent. In fact,

the authors say latter that this is not true: S. haematobium is genetically

diverse in the islands and it is not the only Schistosoma species present

in this area.

o Figure 1: I think this figure is relevant but I would midify it and include

it in the Result section. A figure that compares previous findings by

Stothard et al. [23], Pennance et al. [28,30] and Knopp et al. [18] with

the results from this manuscript would be more useful. The reader would

better visualize which are the inputs of this manuscript. The authors

could highlight the regions where the snail and parasite species have

been found and put dots in the localities from this study where the

authors have found snail and parasite. The manuscript would be become

clearer and the figure would be more helpful to the reader.

Material and methods

o Table 1: this table is very extensive for the main text. I would include it

in Supp. Mat. In the main text, the authors should include, however, an

abstract of this table mention the total number of sampled snails, the total

number analyzed from the museum collection, the total number of

molecularly identified snails, etc. It is very difficult to find in the

manuscript this information? How many snails have been collected?

How many snails have molecularly analyzed?•

•

o Line 178: “snails were identified using shell morphology to their

genera”. The authors should clarify that species cannot be

morphologically identified at species level. And that thus why molecular

analysis are needed.

o Line 205: “after being stored for at least 48 hours in transparent 15L

water containers (allowing for any sediment to settle and eliminate the

risk of introducing live schistosome eggs, miracidia or cercariae into the

aquaria)”. I am not an expert in Schistosoma. Cannot eggs live more than

48H?

o Line 242: “before being collapsed into cox1 haplotype groups.” Which

software was used for identifying haplotypes?

o Line 247: Again, the total number of individuals analyzed is not clear. It

is very difficult to infer how many cercariae per snail have been

analyzed? DNA was extracted individually? Or DNA from cercariae

belonging to one infected snail were collectively extracted?

Results

o Line 302: I think that this assumption is incorrect (see comment above):

“from that site were restrospectively assigned as B. globosus or B.

nasutus based on these identifications.”.

o Line 301: “a total of 11,110 B. globosus and B. nasutus were collected

from ...” What about the third undescribed species?

o Line 324. Replace “B. nasutus” by “Bulinus nasutus”.

o Line 326: Again I think that this assumption in not correct: “Where >1

snail was identified per site, each site was either inhabited solely by B.

globosus or B. nasutus”.

o Figure 2: I would combine Figures 1 and 2. Se comment above.

o I would move Tables 2, 3, 4 and 5 to Supp. Mat. I would present instead

a single table that resume the results of the manuscript. These tables are

very descriptive as they are but I think the reader would need some table

that resume and illustrate the results of the manuscript.

o I would move Figures 3 and 5 to Supp. Mat.

o Line 366: Replace “S2 Table” by “Table S2”.

o Figure 4 is very informative. I really like it.

Discussion

o Replace 0.18% by 0.2%. Decimals in prevalence should be only used

when prevalence is lower than 1% and higher than 99%. See

http://adc.bmj.com/content/100/7/608

o Please, explain the following idea mentioned in the Abstract:

“preliminary evidence that intraspecific variability may increase the

transmission potential of S. haematobium”. I found it relevant and it

should be better explained in the Discussion section. Why the authors

believe that intraspecific variability may increase the transmission of a

disease?

Figures and tables:

This version of the manuscript has 5 figures and 5 tables. I think it too much. I

think that two figures (one showing a map with previous and current results and

other showing the parasite haplotypes) and a table resuming the manuscript

results are more than enough for such a manuscript.•

Avoid using symbols (*, a , b ...) in the caption section. It is confusing and it

takes time to understand what the authors meant. Try to include these remarks in

the caption. For instance, in Figure 1, the sentences “* Bulinus spp. distribution

inferred from Stothard et al. [23] and Pennance et al. [28,30].” and “φ

Schistosoma haematobium infection distribution interpreted from Knopp et al.

[18].” could directly appear in the caption without including the symbols.

###### End of Reviewer 3 comments.

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4 May 2022

Submitted filename: PNTD-D-21-01740_Rebuttal_FINAL.docx

Click here for additional data file.

3 Jun 2022

Dear Dr. Pennance,

Thank you very much for submitting your manuscript "Transmission and diversity of Schistosoma haematobium and S. bovis and their freshwater intermediate snail hosts Bulinus globosus and B. nasutus in the Zanzibar Archipelago, United Republic of Tanzania" for consideration at PLOS Neglected Tropical Diseases.

We thank you for revising the manuscript. There were some more points raised by one reviewer that will further enhance the impact and readability of your work. We are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

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Sincerely,

Stephen W. Attwood, BSc,MSc,PhD

Associate Editor

PLOS Neglected Tropical Diseases

Simone Haeberlein, PhD

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

Reviewer's Responses to Questions

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: The paper is getting closer to being acceptable and will be a useful addition to the literature. I have few quibbles with the data but would like to appeal for greater clarity in the writing.

Line 33. My original question about “allopatric” still stands. In my opinion, the term is simply mis-used by the authors. Surely they mean that until recently S. haematobium was thought to be the only Schistosoma species on the archipelago, but that transmission of S. bovis has now been demonstrated there.

Line 52. Again, Indian-Ocean Islands. I agree that there are many of these, but presumably the authors know which ones they mean. Are these the Zanzibar Archipelago and Mafia? Or does that number include Mauritius and Madagascar? Anywhere else? Clarity please.

Lines 88-89. Just say that low-level transmission is continuing. Many more words than required are used here.

Line 116. Maybe an apostrophe after “species”?

Line 117. Clumsy phrasing “freshwater waterbodies”. Maybe “freshwater habitats” or something else.

Line 119. “overlapping with B. globosus”.

Line 149. “…between 2011 and 2019”. But 2016 is the year given elsewhere.

Sentence starting on line 320. Rephrase to remove the repetition of “remain(ing)”.

Line 343. Are you implying that, where >1 snail was identified at a site, all other snails from that site were regarded as belonging to that species? The phrasing is poor.

Line 347. This seems to be a place where “cohabiting” could be replaced with “sympatric”.

Lines 379-382. This sentence should be split and rephrased for clarity. As it stands, the subject of the sentence seems to be cercariae, but the authors are then clearly referring to snails.

Lines 398-399. Maybe “…variants other than those previously sequenced...“

Line 428. Where are the risk maps?

Line 434. “negating” is the wrong word. Maybe “refuting”.

Line 452. Spelling of “conductivity”.

Line 456. Be consistent in use (or not) of am initial capital letter in “archipelago”.

Lines 506-509. Use fewer words. “ the cost and time required to generate cox1 sequence data limited the number of the snails that could be identified by this means”. Or something similar.

Reviewer #3: I would like to thanks the authors for considering my comments and those of my colleagues. I found that the manuscript is much clearer and easier to follow as it is now presented.

--------------------

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

References

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article's retracted status in the References list and also include a citation and full reference for the retraction notice.

6 Jun 2022

Submitted filename: PNTD-D-21-01740_Rebuttal_2.docx

Click here for additional data file.

14 Jun 2022

Dear Dr. Pennance,

We are pleased to inform you that your manuscript 'Transmission and diversity of Schistosoma haematobium and S. bovis and their freshwater intermediate snail hosts Bulinus globosus and B. nasutus in the Zanzibar Archipelago, United Republic of Tanzania' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Stephen W. Attwood, BSc,MSc,PhD

Associate Editor

PLOS Neglected Tropical Diseases

Simone Haeberlein, PhD

Deputy Editor

PLOS Neglected Tropical Diseases

***********************************************************

30 Jun 2022

Dear Dr. Pennance,

We are delighted to inform you that your manuscript, "Transmission and diversity of Schistosoma haematobium and S. bovis and their freshwater intermediate snail hosts Bulinus globosus and B. nasutus in the Zanzibar Archipelago, United Republic of Tanzania," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

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co-Editor-in-Chief

PLOS Neglected Tropical Diseases

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases