4.1. Gastropod Diversity
A total of eight freshwater gastropod species were identified in this study; namely B. forskalii, B. truncatus/tropicus, P. acuta, Bellamya sp., P.columella, Radix sp., M. tuberculata and Gyraulus sp. This is slightly less compared to the study by Chimbari and Chirundu (2003) which reported the following species: B. pfeifferi, B. globosus, Bulinus depressus, B. tropicus, L. natalensis, P. acuta, M. tuberculata, Bellamya capillata (Frauenfeld, 1865) and Cleopatra ferruginea (Lea & Lea, 1850). The most important difference with respect to disease transmission is the absence of B. pfeifferi and B. globosus in this study. Chimbari et al. (2003) found both B. pfeifferi and B. globosus at 13 out of 16 sites, with B. globosus being generally more abundant than B. pfeifferi, although both species had a low abundance that varied between seasons. There are several factors that might explain this absence. One of these might be competitive exclusion. There are multiple invasive gastropod species present in the lake, including Radix sp., P. columella (Carolus et al., 2019), P. acuta and M. tuberculata that compete for food and space. Rondelaud et al. (2016) showed that the physid Aplexa hypnorum exerted competitive pressure on two lymnaied species, consequently lowering their abundance. There is also the possibility of gastropod predation by the invasive red claw crayfish, Cherax quadricarinatus (von Martens, 1868), which was accidentally introduced into Lake Kariba and is now at an established stage in the lake (Marufu et al., 2014; Marufu et al,. 2018). Several studies have shown that gastropods make up the diet of freshwater crayfish, such as Procambarus clarkii whose prey of first choice was shown to be Biomphalaria alexandrina, B. truncatus and L. natalensis (Khalil & Sleem, 2011) while it has also been reported to feed on B. globosus and M. tuberculata (Monde et al., 2017). Another factor that might play a role is the water level, which has been fluctuating greatly during the past decennia, which affects snail distribution (Chimbari and Chirundu, 2003), possibly in a species-specific manner. After the sudden rise in waterlevel between 1999 and 2001, B. pfeifferi and B. globosus were only found 200 m from the shoreline, at depths of four to six meters (Chimbari and Chirundu, 2003).
Another striking outcome is the absence of L. natalensis, which was the second most abundant species in 2001 (Chimbari and Chirundu, 2003). This species might have been replaced by the highly invasive P. columella and Radix sp. that currently dominate in Lake Kariba (Carolus et al., 2019). The exact species identity of the Radix sp. sampled is not known yet, but phylogenetic analyses point to a close relationship to Radix specimen from Vietnam (Carolus et al. 2019). Due to morphological similarities with L. natalensis, it cannot be ruled out that these two species were already present in 2001.
It is worth noting that the other two bulinid species reported by Chimbari & Chirundu (2003) in Kariba, namely B. tropicus and B. depressus, were not identified in this study. However, we cannot rule out that our B. truncatus morphotype is actually B. tropicus. Due to similarity in morphological characteristics of the shells of gastropods, especially in juveniles, distinction between shells of species belonging to the same genus, as well as those belonging to different genera, is often difficult and error-prone (Frandsen & McCullough, 1980; Brown,1994; Palasio et al., 2017). The BLAST results 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.
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). Bellamya capillata was previously found in the lake (Machena & Kaustky, 1988; Chimbari & Chirundu, 2003; Brodersen et al., 2010). In this study we were only able to identify our Bellamya specimen to genus level, but according the phylogenetic analysis it is not B. capillata as the BLAST ID of its cox1 sequence showed only 91% similarity with B. capillata. Future molecular studies will need to establish its exact species identification. Finally, C. ferruginea was reported in previous studies of Kautsky & Kiibus (1997) and Chimbari & Chirundu (2003), but not identified in this study. Its absence may be attributed to the absence of the macrophyte Vallisneria aethiopica, to which it was reported to be restricted (Kautsky & Kiibus, 1997). In this study a few V. aethiopica plants were only observed at Site 5 (Gundamusaira) in the month of March 2018.
4.2. 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 displays (Stoll et al., 2013), allows its successful colonisation of all sites despite their marked differences in water chemistry. Additionally, P. acuta lacks any association with completely submerged aquatic vegetation, but thrive on the roots of floating E. crassipes (De Kock & Wolmarans, 2007), as also found in this sampling campaign. Hence, the observed shift in abundance of P. acuta may partly be attributed to the fact that, while there is less aquatic vegetative cover in Lake Kariba than there was a decade ago (Barson, personal observation), E. crassipes is well established in the lake (Carolus et al., 2019). Therefore, while other gastropod species decline in abundance due to lack of habitats, P. acuta competes successfully against other gastropods for habitat on E. crassipes.
Seasonality in the overall abundance of gastropods was observed during the course of the year, with the highest overall abundance of gastropods collected in the hot-dry season, between the months of September and December 2017 and the lowest in the hot-wet season between January and April 2018. The peak abundance of all gastropods also occurred in the hot-dry season, with the exception of P. acuta and M. tuberculata which both peaked in the cold season. Physa acuta was the most abundant gastropod during all seasons but also present at all sites, demonstrating that the proliferation of this invasive pulmonate is widespread in Lake Kariba. Tchakonté et al. (2014) reported similar trends in the proliferation of P. acuta in urban and suburban streams in Cameroon.
Bulinus truncatus reached its peak abundance in October during the hot-dry season, which is contrary to the peak density reported by Chimbari et al. (2003) for B. globosus to have occurred in July 2001. However, the seasonal distribution of Gyraulus sp. which reached peak abundance in September 2017, was similar to that reported by Chimbari et al. (2003) for B. pfeifferi which also reached peak density in September. It was also observed that, while P. columella was present every month, Radix sp. was absent from all sites in the months of May and June 2017, as well as February and March 2018. This difference in seasonal variation between the two species was as expected, since P. columella is more tolerant to a wider range of abiotic conditions than Radix spp. and is adapted to surviving longer periods of hot and dry weather (Prepelitchi et al. 2011).
4.3. 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) is related to the amphistome Calicophoron microbothrium (p-distance of 15.2%). Both Radix sp. and especially P. columella populations were highly infected by an unknown Fasciola species. Due to its close phylogenetic affinity to F. gigantica and F. hepatica (Carolus et al., 2019), it may infect similar final host species, including wild and domesticated ruminants as well as humans (Figure 5). The most common ruminants found in Kariba are hippopotami (Hippopotamus amphibious, Linnaeus 1758) and the African buffalo (Syncerus caffer, Sparrman, 1779) (Ramberg et al., 2013), with hippopotami being a natural host of F. nyanzae and buffalo being a natural final host of F. gigantica Cobbold 1855 (Bindernagel 1972). Hippopotami are also susceptible to cattle-borne diseases as they can be naturally infected with amphistomes (Sey and Graber 1979). Currently, there are no documented investigations into the extent to which trematodes contribute to the mortality of hippopotami or African buffalo in Kariba.
Bulinus truncatus was shown to be infected by the most diverse trematode community, with species belonging to the Notocotylidae, Gastrothylacidae and Psilostomstidae family. This observation is in line with other studies that reported that B. truncatus could host up to four different trematode species, including amphistomes (Chu et al., 1962; Ahmed et al., 2006; Farahnak et al., 2008). One trematode species we found in B. truncatus (type I; Figure 5) belongs to the family Gastrothylacidae, one of the most speciose ruminant infecting amphistome families in Africa (Laidemitt et al., 2017). Amphistomes present a huge economic burden in the livestock industry worldwide (Toledo et al., 2014). In Zimbabwean cattle, Carmyerius sp. has been the only recorded Gastrothylacidae species so far. Different Carmyerius species also infect African wild ruminants including African buffalo and hippopotami (Sey and Graber, 1979). Bulinus truncatus and B. tropicus have been implicated as hosts of Carmyerius microbothrium in northern and southern Africa, respectively (Pfukenyi et al., 2005). 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 Carmyerius mancepatus, sampled in Kenyan cattle (Laidemitt et al., 2017), which suggests that the species found in Kariba might be a different, potentially unknown representative of the Carmeyerius genus.
A second trematode species (type III; Figure 5) found in B. truncatus belongs to the family Notocotylidae, a family of bird and mammalian herbivore parasites closely related to the Paramphistomoidae (Olson et al., 2003; Ma et al., 2016). Notocotylids have a two - host life cycle, with adults residing in the caeca and hindgut of birds and the intestine of mammals (Kearn 1998). It could be hypothesised that bulinid gastropods can function as hosts of Notocotylidae since some bulinid species are known to be hosts of the Paramphistomoidae (Eduardo 1987; Laidemitt et al. 2017; Phiri et al. 2007). However, this hypothesis needs further investigation by experimental infection of bulinids with species of the family Notocotylidae to determine their compatibility. Both notocotylids and paramphistomids infect ruminants as their definitive host and can cause major economic losses by infecting cattle and ovine livestock. An example of a notocotylid group known to occur in Africa is the genus Ogmocotyle. Members of this genus infect hippopotami in Southern Africa (Junker et al., 2015). This is the first report of Notocotylidae in Lake Kariba in scientific literature.
A third trematode species found in B. truncatus (type IV, Figure 5) belongs to the family Psilostomatidae. This family comprises gastro-intestinal parasites of birds and mammals that closely resemble the Echinostomatidae in their morphology (Atopkin, 2011). They are known to infect amphibians, waterfowl and mammals (Wilson et al., 2005; Mazeika et al., 2009). In Zimbabwe, the only genus of Psilostomidae reported is Ribeiroia, of which the species Ribeiroia congolensis infects the great egret (Ardrea alba Linnaeus, 1758) and the giant kingfisher (Megaceryle maxima Pallas, 1769) in Kariba (Johnson et al., 2004). The life cycle of Ribeiroia spp. involves the infection of amphibians as their second intermediate hosts, in which it causes limb deformities that are thought to increase the chance of predation by the definitive hosts (i.e. carnivorous birds; Johnson et al., 2004). Some members of the Psilostomidae have been implicated in human infections in Asia (Chai et al., 2009). Barcoding of the cercariae isolated from B. truncatus also identified two species belonging to the Diplostomidae family.
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). There is little known about alariosis and the various Alaria species that cause it (Wasiluk 2013). However, Alaria species have a wide range of hosts including fish (Locke et al. 2011), birds, amphibians, reptiles, and mammals, as well as humans (Möhl et al., 2009). Humans can act as paratenic host (which harbours the parasite without any further development before it is transmitted to its intermediate host) in the three - host life cycle of some Alaria species, and are infected through ingesting meat infected with Alaria mesocercaria (Freeman & Stuart, 1976; Möhl et al., 2009; Toledo et al., 2014). The pathogenicity of 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.
4.4. Infection prevalence
There was a sharp contrast between shedding and multiplex RD-PCR with regard to infection prevalence. The overall amount of shedding gastropods indicated an infection prevalence of 0.36% whereas the multiplex RD-PCR resulted in a prevalence of 25.17%. According to the shedding results, only eight out of 148 (5.4%) B. truncatus and one out of 456 (0.22%) P. columella were infected, whereas multiplex RD-PCR showed four species to be infected; namely P. columella, B. truncatus, B. forskalii and Radix sp., with respective infection prevalence of 59.60%, 13.43%, 16.67% and 6.82%.
It has been shown before that shedding underestimates the prevalence of trematode infections in gastropods. Born-Torrijos et al. (2014) found underestimations of up to 66% of single infections and 80% of double infections when comparing PCR-based diagnostics to classical shedding. Various explanations may be put forward like the presence of immature (pre-patent) infections and/or gastropod mortality during the shedding experiments. Also, cercarial shedding of mature infections is affected by various conditions including temperature (Rondelaud et al., 2013) and light intensity (Wagenbach and Alldredge 1974), which differ according to the trematode species and may affect the success of shedding experiments.
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 B. truncatus, as revealed by shedding, was significantly lower than the 14.9% reported in B. truncatus by Ahmed et al. (2006).
Based on the RD-PCR results, 59.60% of all lymnaeid gastropods were infected. This is slightly higher than infection prevalence based on shedding reported by Chingwena et al. (2002) and Phiri et al. (2007): 38.2% in Zimbabwe and 42.8% in the Kafue wetlands (in the vicinity of Lake Kariba) of Zambia respectively. The infection prevalence of lymnaeid gastropods is discussed in detail by Carolus et al. (2019). Despite a report by Chimbari et al. (2003) describing 3.33% prevalence of S. haematobium in B. globosus and 4.76% prevalence of S. mansoni in B. pfeifferi, there were no 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).