More than meets the eye: A shift in the gastropod and trematode communities in Lake Kariba, Zimbabwe


 Background: Freshwater gastropod-borne trematodes pose a great public health burden and cause major economic losses in the livestock and fish industries. Knowledge on the composition, diversity and ecology of both gastropods and trematodes communities is key to understand disease transmission dynamics and control trematodiases of economic significance. The objective of this study is to investigate the diversity and spatio-temporal ecological trends of gastropod and trematode communities on the Northern shore of Lake Kariba in Zimbabwe, the largest artificial lake in volume worldwide. Methods: A sampling campaign was undertaken at 16 sites along the lake shoreline of Kariba town. Gastropods and water samples were collected monthly during one year. All 2477 specimens were identified, counted and subjected to shedding experiments; infection status was confirmed by the use of a Rapid Diagnostic PCR (RD-PCR). To explain spatio-temporal trends in gastropod and trematode occurrence, water samples were analysed for different physico-chemical parameters including pH, temperature, nitrates and phosphates. Results: Gastropod species collected include Bulinus truncatus, Bulinus forskalii, Gyraulus sp., Pseudosuccinea columella, Radix sp, Physella acuta, Bellamya sp. and Melanoides tuberculata. Bulinus truncatus was found to be infected with trematode species belonging to the families Notocotylidae, Psilostomstidae, Paramphistomidae and Diplostomatidae. A species of the latter family was also found to infect B. forskalii. As previously reported, lymnaeid species P. columella and Radix sp. were both infected with a species belonging to the Fasciolidae family, while Radix sp. was also infected with amphistomes. Conclusions: We confirm the occurrence of new species of gastropods in Lake Kariba and the absence of the previously reported B. pfeifferi and B. globosus. This explains the absence of human schistosome species, but we report the presence of other trematode families that have not been reported in Lake Kariba or Zimbabwe before. Compared to the latest study in 2001, there is a remarkable shift in the gastropod community, which is likely driven by the introduction of exotic gastropod species that are now abundant in the lake.


Background
Some freshwater gastropod species are of high medical and or economic importance as they act as The creation of embankments such as dams and dikes may create new habitats and disrupt the ecological and limnological conditions of water systems, often increasing the risk of water-borne disease transmission (Araoye, 2002;Steinmann et al., 2006). Construction of dams in the tropical world has been known to intensify transmission of diseases such as schistosomiasis, malaria and filariases in endemic regions, as well as the creation of new transmission areas (Khalil, 1949;Farid, 1977;Hunter et al., 1982;Sokolow et al., 2017). The construction of the Kariba dam wall on the Zambezi River bordering Zimbabwe and Zambia is also an example of a man-made hydroelectric construction that led to increased transmission of schistosomiasis. After the dam construction and formation of the lake, studies reported increasing prevalence of human schistosomiasis, as well as Bulinus globosus and Biomphalaria pfeifferi, both intermediate gastropod hosts of Schistosoma species, on both the Zimbabwean (Machena, 1988;Chimbari & Chirundu, 2003) and the Zambian (Hira, 1969;Hira, 1970;Mungomba et al., 1993;Mungomba and Kalumba, 1995) sides of Lake Kariba. In more recent years, the prevalence has however decreased in Kariba town as a result of mollusc and schistosomiasis control strategies and improved water sanitation facilities (Chimbari et al., 2003).
Because most research in Lake Kariba has focused on Schistosoma species and their intermediate hosts, little is known regarding the diversity and prevalence of other trematodes and gastropod hosts present in the lake. We adopted the same sites studied by Chimbari et al. (2003) in their investigation on the prevalence of Schistosoma spp. and their respective snail hosts, and investigate the diversity and ecology of the complete gastropod and trematode fauna of Lake Kariba. This is the first study in which molecular diagnostic tools such as DNA barcoding and Rapid Diagnostic-PCR (RD-PCR) are used alongside gastropod shedding experiments and ecological analysis to investigate the gastropod and trematode community of Lake Kariba.

Gastropod sampling and cercarial shedding
Sampling was performed monthly from May 2017 to April 2018 at 16 sites on the banks of Lake Kariba near the town of Kariba, Zimbabwe as shown in Figure 1. The months were divided according to three seasons; namely the cold season (May-August), the hot and dry season (September-December) and the hot and wet season (January-April). Gastropod samples were collected by scooping the sediment and aquatic vegetation along a 40 m stretch of lake shoreline up to 1 m deep into the lake using a scooping net. A dredge was used for sampling depths up to 4 m. Gastropods were collected by two people during approximately 30 minutes per site.
Live snails were transferred to cell culture plates (pooled per five specimens of same species and collection site) filled with aged tap water and exposed to artificial light for two hours to induce cercarial shedding. Plates were checked at 30 minute intervals using a stereomicroscope. Upon shedding, pooled gastropods were individually incubated. Infected snails were stored in 1.5 ml of 98% ethanol with a subsample of the released cercariae. Non-shedding snails were kept alive in the laboratory for a week after which they underwent another shedding experiment as described above. If negative, they were fixed in 50ml Falcon tubes in 98% ethanol, pooled per species and per site.

Gastropod Morphological Identification
Morphological identification was carried out by stereomicroscope analysis and following identification keys by Mandahl-Barth (1962), Brown (1994) and Frandsen & McCullough (1980), all of which use shell morphology as well as the shape of soft parts extended out of the shell; i.e., the shape of the foot and the tentacles, head and presence or absence of an operculum. High quality stacking photography was used to photograph two specimens from each morphotype before DNA extraction.
Multiple pictures on a range of focal depths were taken and all pictures were stacked using Zerene Stacker® creating a high-resolution image of the gastropod along with a calibrated scale.

Gastropod molecular identification
For molecular identification we focused on the pulmonates as they are the main intermediate hosts of

DNA extraction
For the infection prevalence assessment, the Chelex® (Biorad™) DNA extraction method was used to extract gastropod and trematode DNA as described in Carolus et al. (2019). Gastropods were pooled per four specimens of the same morphotype, site and collection date. A maximum of 16 gastropods of the same morphotype, site and sampling date were analysed.

Multiplex RD-PCR
Two multiplex rapid diagnostic (RD)-PCR protocols were used as described by Carolus  general gastropod marker that confirms a successful PCR amplification), a trematode-specific marker and a marker that targets members of a certain trematode family of interest (i.e. Fasciola spp. in lymnaeid gastropods and Schistosoma spp. in planorbid gastropods respectively).

DNA extraction
For DNA extraction of individual cercariae, the DNeasy Blood and Tissue Kit (Qiagen™) was used. DNA was extracted from a maximum of four individual cercariae per infected gastropod. Also all snail DNA extracts that appeared positive after the Multiplex RD-PCR but not positive for the Schistosoma or Fasciola signal, were included in the simplex PCR protocol for trematode species identification.

Simplex PCR protocol
Primers used for the amplification of the 18S rDNA marker (1160 bp) were 18S_Digenea_F (5'-

Phylogenetic analysis
Consensus sequences were assembled from forward and reverse sequences using Geneious® Version 6.1.8 software; all sequences were manually trimmed and ambiguities were edited. All parts of the phylogenetic analysis described next were computed and visualized in MEGA 7 (Tamura et al., 2013).
Based on the taxonomic indications from the Basic Local Alignment Search Tool (BLAST; https://blast.ncbi.nlm.nih.gov/Blast.cgi) (i.e. order, superfamily, family and genus), the NCBI GenBank (https://www.ncbi.nlm.nih.gov/) and BOLD (http://www.boldsystems.org/index.php/) databases were mined for closely related sequences to construct a phylogenetic tree. Sequences were aligned using the MUSCLE alignment algorithm (Edgar, 2004) and trimmed homogenously for phylogenetic tree construction. The best nucleotide substitution model was calculated by Model Selection (based on the Bayesian information Criterion, BIC) in MEGA and used to compute pairwise genetic distances and construct a Maximum Likelihood (ML) tree with 500 replicates for nodal support statistics (bootstrap method). Genetic pairwise distances were calculated between cox1 sequences with 5% as threshold for intraspecific variation (Vilas et al., 2005;Lawton et al., 2015) to discriminate between species.

Water Quality Analysis
Water temperature, pH, dissolved oxygen, oxygen saturation percentage and conductivity, were measured on site using a HACH multi-meter (HQ40D HACH, USA). A EUTECH turbidity meter (TN-100, Singapore) was used to measure water transparency. Water samples were collected in cooled polythene bottles for dissolved nutrients analysis. Chemical analysis of water was conducted in the Wet Chemistry Laboratory at the University Lake Kariba Research Station (ULKRS). Samples analysed for nitrates and phosphates were first filtered using Whatman GF/C filter papers with 47 mm diameter. The filter papers were wrapped with an aluminium foil and placed in the freezer prior to chlorophyll a analysis. Nitrates, orthophosphates, total nitrogen, total phosphorous and ammonium were analysed following guidelines by USEPA (1983) and Hach Company (2007). Chlorophyll a analysis was carried out following guidelines by Aminot & Rey (2000).

Statistical data analysis
The ecological data (physico-chemical water parameters) and gastropod count data were analysed in the statistical software RStudio® Version 3.4.2. Significance threshold for all statistical tests was P ≤ 0.05. All R generated graphs were exported to Microsoft PowerPoint® for minor aesthetic adjustments using the Export package created by Wenseleers (2016). Microsoft Excel® was used to generate graphs for the visualisation of temporal trends in gastropod abundance.

Gastropod community composition
Two methods were used to visualize the relationships amongst sites based on their gastropod community structure: Nonmetric Multi-Dimensional Scaling (NMDS) and hierarchical cluster analysis.

Gastropod diversity and abundance
A total of 2,477 gastropods were collected. They were assigned to a total of 8 morphotypes including Bulinus forskalii (Ehrenberg, 1831), B. truncatus (Audouin, 1827), B. pfeifferi, Pseudosuccinea columella (Say 1817), Radix sp., Physella acuta (Draparnaud, 1805), Melanoides tuberculata (Müller, 1774) and Bellamya sp. The cox1-based BLAST results from morphotypes B. forskalii, P. columella and P. acuta were sufficiently strong (BLAST identity≥99% with query cover of 100%, sequence length of +300bp and HQ≥95%) to conclude that they represent that actual species (Supplementary Table 1; GenBank accession numbers: XXXXX). No final conclusion could be made for the B. truncatus morphotype as the BLAST hits were identical for B. truncatus and Bulinus tropicus. Based on pairwise genetic distances of all cox1 sequences, all representatives of the same morphotype belonged to the same species with p-distance < 0.1% (data not shown).
A phylogeny-based barcoding approach was used to gather more taxonomic information about the morphotypes for which BLAST results were not conclusive. The Planorbidae phylogeny ( Figure 2) shows that the morphotype B. pfeifferi does not cluster with the genus of Biomphalaria but is rather related to the genus Gyraulus. Pairwise genetic distances show that the Kariban specimen is most closely related to Gyraulus sp. from Turkey (KC495833.1) with a p-distance of 12.9% (data not shown). However, due to the lack of African gastropod sequences in Genbank we cannot conclude on this. The phylogenetic tree ( Figure 2) confirms the identity of our B. forskalii morphotype. Also the B. truncatus morphotype clusters with the B. truncatus reference sequence but shows close relationship with B. tropicus. The identification of the Radix sp and P. columella was discussed in Carolus et al.
(2019) where phylogenetic analysis identified the nearest relative to be a Radix sp. specimen from Vietnam.
The most abundant gastropod species collected was P. acuta, representing 51.91% of the total gastropods collected and being present in all sampled sites. The least abundant gastropod species was B. forskalii comprising 0.89% of the total gastropods collected. Gastropod diversity ranged from two to eight species per site. Table 2 shows the cumulative gastropod count per site and per species.

Spatial trends in gastropod community composition
Based on UPGMA hierarchical clustering, the 16 sites can be divided into six distinct groups. The sites within a group cluster together based on gastropod community data. Figure

Shedding results
Shedding experiments were carried out during all twelve months of the study and they showed only 9 out of a total of 2477 gastropods to be infected (0.36 % prevalence), which shed between the months of August and November 2017. Eight of the infected gastropods were B. truncatus (from a total of 148 tested) and one was a P. columella. Six of the B. truncatus gastropods from site 16 and one from site 15 shed amphistome cercariae. One B. truncatus from site 16 shed furcocercous (fork-tailed) cercariae of the longifurcate-pharyngeate distome type (also called strigeid cercariae). The ninth shedding gastropod, a P. columella from site 15 shed gymnocephalous cercariae of which Fasciola is a type genus

Multiplex PCR results
The multiplex RD-PCR protocol was performed on 290 gastropods (Table 3), excluding M. tuberculata and Bellamya sp. Infection rates per species are given in Table 3 and the prevalence of infected gastropods per site per species is shown in Figure 6. Of all the gastropods tested, 25.17% were

Trematode diversity
The 18S rDNA showed high BLAST identity matches ranging from 97% to 100% while cox1 sequences of all trematode amplicons showed lower BLAST identity matches, never exceeding 92% identity with  were not conclusive, yielding the same similarity score for B. truncatus and B. tropicus reference sequences, while phylogeny reconstruction pointed to a closer affinity with B. truncatus. We therefore opt to assign our morphotype to the latter species.

Physico-chemical habitat differentiation of sites
Three other gastropod species identified in this study were P. acuta, Bellamya sp. and M. tuberculata.
Physa acuta is an invasive species native to North America, which has been reported in previous surveys in Kariba in the past (Chimbari & Chirundu, 2003

Gastropod Ecology and Temporal trends
The planorbid gastropods (i.e. Gyraulus sp., B. forskalii and B. truncatus) showed to be highly abundant in highly eutrophic water. These results are consistent with Watson (1958), who reported that B. truncatus prefers polluted waters near human habitations, which are usually characterised by high eutrophication, low pH and low oxygen levels. Site 14, which had the third highest abundance of B. truncatus, is highly eutrophic, as it is exposed to a wastewater stream from a nearby crocodile farm. However, B. truncatus was also common in other, less contaminated sites suggesting that it tolerates a larger range of water quality conditions. Lymnaeid abundance (Radix sp. and P. columella), followed an opposite trend to that of the planorbid gastropods, being more abundant in sites 5 and 3 respectively, where nutrient concentrations were low. Radix sp. was however observed to be present in only 7 sites, all with fairly high dissolved oxygen content while P. columella was present at 14 sites. This more narrow distribution of Radix sp. may be explained by its higher demand for oxygen compared to P. columella which is tolerant to lower oxygen concentrations as well as higher eutrophication levels (Grabner et al., 2014).
Two prosobranch species, Bellamya sp. and M. tuberculata, were found together at only 4 sites; that is sites 5, 11, 15 and 16, all of which had low nutrient concentration levels. Although low numbers of Bellamya sp. were sampled, a large number of Bellamya sp. empty shells were observed, though not quantified, at the lake shore of site 15, suggesting that it is or was a hotspot for this species relative to other sites. We observed that M. tuberculata was the second least abundant gastropod species, contrasting Kautsky & Kiibus (1997) and Chimbari & Chirundu (2003), who found it to be the most abundant gastropod in Lake Kariba. In contrast, P. acuta was the most abundant species, present in large numbers and at each of the 16 sites, while it was the least abundant species in the study of Chimbari and Chirundu (2003). The wide tolerance to physical and chemical gradients that P. acuta

Trematode diversity
Barcoding and phylogeny reconstruction showed six different families of trematodes infecting four species of gastropods ( Figure 5). The parasite fauna of both Radix sp. and P. columella in Kariba has already been described and discussed in detail by Carolus et al., (2019). Radix sp. was infected by an unknown amphistome, which according to pairwise genetic distances (Table 4)  Based on pairwise genetic distances of cox1 (Table 4), we know the Kariba species is not C. microbothrium. However, distances show a divergence of 11% between the Kariban haplotype and Bulinus forskalii was also shown to be infected by a species of the Diplostomatidae family, which typically infect lymnaeids as their first intermediate host, and fish (Smyth, 1976) and amphibians (Horák et al., 2014) as their second intermediate host. Our barcode sequence shows the highest similarity with Alaria mustelae with a p-distance of 12.9% (Table 4) Alaria species in humans is an area that is still being explored, therefore further investigation is needed to confirm the presence of Alaria sp. in Kariba and to identify its species identity.
We did not find any sign of Schistosoma infection in the collected gastropod species, as confirmed by the Schistosoma RD-PCR (Schols et al. 2019; described below). The absence of Schistosoma infections may be mainly attributed to the absence of the previously reported Schistosoma host B. globosus and B. pfeifferi from this study. We did, however, identify B. truncatus, which is known to be able to host both S. haematobium (Mohammed et al., 2016) and S. bovis (Akinwale et al., 2011) in some parts of Africa. However, more sequencing data for this gastropod species is needed to confirm its exact species status.
Due to the lack of cox1 reference sequences from the GenBank and BOLD database, the identification of trematodes in this study was only possible up to family level. Therefore further investigation into the trematode species sampled in Lake Kariba is required. We suggest the use of the variable internal transcribed spacer (ITS) marker that is more represented on GenBank compared to 18S rDNA or cox1 markers, combined with extensive phylogenetic analysis (Nolan et al., 2005;Vilas et al., 2005).
However, the major bottleneck is the knowledge gap of African trematodes and the associated lack of reference sequences in all major databases. Although we were only able to identify the trematode family and sometimes genus level, it is clear that Lake Kariba harbours trematodes with the potential to affect Kariba's public health and its tourism and fishing industries by causing economic losses through mortality of wild animals and fish, as well as lowering the quality of fish.

Infection prevalence
There was a sharp contrast between shedding and multiplex RD-PCR with regard to infection Our overall infection prevalence based on shedding was lower but not so different from a previous study in Kariba that reported 3.7% of the planorbid snails to be infected with mammalian-type of schistosome cercariae (Chimbari et al., 2003). Figures are however much higher for Zambia (Phiri et al., 2007) and Tanzania (Loker et al., 1981), with shedding rates of 13.7% and 14.9% respectively, accounting for all trematode species from both planorbids and lymnaeids. The infection prevalence of Schistosoma species found in this study. However, a drastic decrease was already reported in the prevalence of schistosomiasis in humans (Chimbari et al., 2003;Chimbari & Chirundu, 2003) as well as in their gastropod hosts (Chimbari et al., 2003) due to improved sanitary facilities and control strategies including mollusciciding and chemotherapy (Chimbari et al., 2003;Chimbari, 2012).

Conclusion
Through this study we gained new fundamental insight into the composition of the gastropod community as well as gastropod-trematode associations along the northern shore of Lake Kariba.
Regardless of the differences between methods used in previous studies and this study, a remarkable shift in the gastropod communities was detected. This shift is potentially driven by the spread of exotic gastropod species, now present in high numbers in the lake. While we were unable to identify the trematodes to species level due to the lack of reference sequences in genetic databases, it is possible that they have the potential to affect Kariba's public health and wildlife, as well its fishing industry, which make up the backbone of Kariba's economic structure. One of the most remarkable findings is the absence of schistosome species in the tested planorbid population, as well as the absence of their previously reported gastropod host species B. globosus and B. pfeifferi. The findings of this study show the need for further investigation into the complete trematode fauna of Lake Kariba as well as the implications it has on wildlife, aquaculture and the public health in surrounding communities in Kariba.

Availability of data and materials
The sequences generated in this study are deposited to GenBank under accession numbers XXXX.
The alignments used for the phylogenetic analysis are available from the corresponding author on reasonable request

Competing interests
The authors declare that they have no competing interests

Authors' contributions
TH and MB conceived the study and helped with fieldwork and correcting the manuscript. KM led and conducted the year round field collection, wrote the manuscript. HC led and conducted the molecular analysis and data analysis, helped with fieldwork and wrote the manuscript. CH helped with molecular data analysis and writing. RS helped with molecular work and correcting the manuscript   11  9  14  18  2  3  30   12  15  15  27  2  1  71   13  22  3  1  0  1  40   14  10  0  50  19  9  24   15  76  0  0  22  1  120   16  93  0  2  66  6  110   Table 2: The cumulative abundance of gastropods over 12 months per site per species. The column labelled "Species Richness" shows the species richness at each site. The column "Site" shows the specific sites of gastropod collection as numbers, the corresponding site names are shown in Table 1.   Map of the sampling sites along the shore of Lake Kariba.