Bulinus tropicus shell

Hammoud, Cyril, Kayenbergh, Annelies, Tumusiime, Julius, Verschuren, Dirk, Albrecht, Christian, Huyse, Tine & Bocxlaer, Bert Van, 2022, Trematode infection affects shell shape and size in Bulinus tropicus, International Journal for Parasitology: Parasites and Wildlife 18, pp. 300-311 : 308-309

4.2. Factors influencing Bulinus tropicus shell phenotype

We assessed the effect of trematode infections on B. tropicus shell phenotype while controlling for the influence of snail genotype and environmental factors by sampling a single homogeneous lake habitat. Barcoding of the B. tropicus population from Lake Kasenda revealed overall low genotypic diversity (p-distances ranging from 0.002 to 0.008, Supplementary Table 3), with most of the 227 analysed snails belonging to two genetically similar haplotypes. One of these haplotypes (H2) had been detected in Lake Kasenda by Tumwebaze et al. (2019). However, Nalugwa et al. (2010) earlier reported three B. tropicus haplotypes from Lake Kasenda which all differ substantially from those we found (p-distance ranging from 0.012 to 0.018, Supplementary Table 3). Those differences suggest that Nalugwa and colleagues have sampled a different neighbouring system, as confusion exists in lake nomenclature in the Ndali-Kasenda crater lake cluster (obs. pers.), and the provided geographic coordinates map to the shore of the Kazinga Channel. Alternatively, and much less likely, the B. tropicus population in Lake Kasenda may have been completely replaced during the ~10 years separating both studies. In any case, our genetic data indicate a

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homogenous population of B. tropicus in Lake Kasenda with limited genotypic diversity. Furthermore, snail phenotype and the probability of trematode infection do not differ significantly among snail haplotypes.

Shell size and shape in B. tropicus differed significantly depending on trematode infections. More specifically, compared to uninfected specimens, infected snails (by all trematode taxa collectively) had larger shells, slightly more protuberant apices, and an inward-folded outer apertural lip. In pulmonate snails, the association between increased body size and trematode infection had previously mostly been documented in laboratory experiments (e.g., B. truncatus and B. senegalensis artificially infected with S. haematobium ; Fryer et al., 1990), and it has only been reported in wild populations of a few species such as Lymnaea stagnalis (Zbikowska ˙and Zbikowski ˙, 2005) and Galba truncatula ( Chapuis, 2009) . Multiple mechanisms have been proposed to explain the higher incidence of trematode infections in larger snails. The most frequent explanations (see Sorensen and Minchella, 2001 for a comprehensive review) are 1) castration of the snail due to destruction of gonadal tissue by asexual stages of trematodes may reallocate metabolic resources from reproduction to growth (gigantism, Sousa, 1983) or 2) larger snails may be older and, therewith, would have been exposed longer to parasites, increasing the likelihood of infection (Sousa, 1983; Sorensen and Minchella, 2001). Examining the effect of specific trematode infections on body size in B. tropicus we found that snails infected by Petasiger sp. 5 , Echinoparyphium sp. or Austrodiplostomum sp. 2 had larger shells than uninfected snails, whereas those infected by Plagiorchiida sp. did not. Secondly, comparing ‘early’ (<25 days, <45% of reads attributed to the infection) and ‘late’ infections (> 25 days,> 45% of reads) with Petasiger sp. 5 and Plagiorchiida sp., we found that snails with early infections have sizes intermediate to uninfected snails and snails with ‘late’ infections. As our results indicate specific relationships between shell size and species-specific trematode infections, as well as to the developmental stage of infection, the hypothesis that size changes are caused by trematode infections is more likely in our study system. Sorensen and Minchella (2001) argued that infections by trematode species that produce rediae (an asexual stage with a mouth, feeding directly on host tissue) cause gigantism more often than species producing sporocysts (an asexual stage without mouth that feeds by absorbing nutrients through the body wall), whereas Probst and Kube (1999) proposed the opposite. Petasiger sp. 5 and Echinoparyphium sp. likely produce rediae ( King and Van As, 2000; Huffman and Fried, 2012) whereas Austrodiplostomum sp. 2 probably produces sporocysts ( Cribb et al., 2003). The identification of Plagiorchiida sp. is taxonomically too coarse to deduce the type of asexual stage. The seven other encountered trematodes were too rare to assess their influence on shell size. In any case, the type of asexual stage does not appear to solely explain why infection by the three above-mentioned trematode species may have led to larger shell size whereas infection by Plagiorchiida sp. did not.

Our significant correlation between shell shape and centroid size indicates that shape changes with growth, i.e., we observe substantial allometric changes, as is common in many organisms (Zelditch et al., 2004). Therefore, we included allometry into our Procrustes models to assess the relative importance of shape changes explained by other variables than shell size (Outomuro and Johansson, 2017). As verification, we have recuperated the residuals from a regression of shell shape to size to retest all Procrustes models constructed with the residuals (instead of raw shape variables) as response variable, which produced highly similar results (not shown). Similar to the abovementioned differences in shell size we found significant differences in shell shape between uninfected and infected snails and the effect of infection on shell shape was found to be parasite-specific. We also observed that in snails infected by Petasiger sp. 5 and Plagiorchiida sp., snails with ‘early’ infections are on average of intermediate shape to those of uninfected specimens and those with ‘late’ infections. Upon examining the effects of ‘early’ and ‘late’ infections on shell size and shape, a first important constraint is that this distinction decreased sampling size and therewith the power of statistical test. Nevertheless, ‘early’ infections were always found to be intermediate to uninfected snails and snails with ‘late’ infections, which is consistent with the hypothesis that prolonged exposure to parasite infections results in accumulated changes in shell size and shape. Whereas our study is mainly correlative in nature, the differences between various infection stages are consistent with expectations under a causal link. Our results on the species-specific effects of infections on shell morphology are congruent with the findings of Zbikowska ˙and Zbikowski ˙(2005) who showed that infections by different trematode species correlated with differences in shell height/width ratio in Lymnaea stagnalis populations from Poland. The shape changes observed under Petasiger sp. 5 infections were substantial and of similar magnitude to those documented for Littorina saxatilis infected by Microphallus piriformes ( McCarthy et al., 2004) , Lymnaea stagnalis infected by various trematode species (Zbikowska ˙and Zbikowski ˙, 2005) or Cominella glandiformis infected by Curtuteria australis (Thieltges et al., 2009) . We report (to our knowledge) the first changes in the shape of Bulinus shells related to trematode infection. The effect of infection on shell phenotype differed among trematode species, which strongly suggests that these trematodes exploit their host differently. These differences may either be the by-products of the pathology caused by infection or manipulative changes allowing the parasite to increase its reproductive potential ( Hay et al., 2005). For example, McCarthy et al. (2004) showed that morphological changes in the shell of the marine snail Littorina saxatilis caused by infection with Microphallus piriformes increased the space within the shell, without increasing the volume of snail tissues and organs, i.e., the extra space is used for asexual reproduction of the parasite. Thus, these authors suggested that this phenotypic alteration may be triggered by the parasite to improve its reproductive potential without affecting the viability of its host. We suspect that the elongation of the apex of B. tropicus snails infected by Petasiger sp. 5 may play a similar role. Our results corroborate the hypothesis that trematode infections alter shell size and shape in their intermediate host and that the patterns are highly specific to trematode species. Further work, including experimental infections, is required to assess the physiological link between trematode infection and intermediate host morphological alterations in more detail.

In conclusion, we used state-of-the-art methods to reliably characterize trematode (co-)infections and to document shell phenotype, which allowed us to accurately document how trematode infections affect B. tropicus shell size and shape under natural conditions. Interestingly, effects are highly trematode-specific and accumulate with the time since infection. This level of complexity may explain the contrasting results in the variety of studies on the impact of trematode-snail interactions on snail phenotype (see above). As new molecular methods enable detailed documentation of the link between parasites and intermediate hosts, it becomes possible to decipher the variety of interactions and mechanisms at play. Given the high complexity of multiple interactions, our results incline us to anticipate large variability in shell phenotypic changes linked to co-infections, which can only be disentangled with larger sampling sizes.