TTX resistance of SCNA4 sodium channel
Sodium channels (NaV) are formed by an α subunit consisting of four domains (I-IV) and an optional β subunit that may alter its activity (Yu et al., 2005). Furthermore, the third whole-genome duplication of teleosts has expanded the α subunit family to eight members (Zakon et al., 2011). NaV channels are the target of several neurotoxins (Daly 1995, Fry et al., 2009) and TTX is known to bind to the outer pore of the NaV1.4 (SCN4) channel, blocking the transport of sodium ions across the pore (Hanifin 2010). As different pufferfishes have been shown to acquire TTX resistance through mutations in specific domains of NaV1.4 (Venkatesh et al., 2005, Soong and Venkatesh 2006, Jost et al., 2008), we checked if this is also the case for L. sceleratus . For this purpose, we investigated whether the sequences of voltage-gated sodium channels (SCN4AA, SCN4AB) in L. sceleratus carry the previously reported mutations associated with TTX resistance in other pufferfish. According to these studies (Venkatesh et al., 2005, Soong and Venkatesh 2006), the residues that are associated with TTX resistance are located in the same position in NaV1.4a Domain I and were mutated to Cys andAsn , in T. nigroviridis and T. rubripes , respectively (Figure 6). Surprisingly, despite the fact that the Domain I mutations lead to extensive decrease in TTX binding (Satin et al. 1992; Kaneko et al. 1997; Yotsu-Yamashita et al. 2000; Venkatesh et al. 2005) and have been associated with TTX resistance in pufferfishes and in one C. pyrrhogaster newt (Kaneko et al. 1997), we did not observe any of these reported mutations in the SCN4A_A gene of L. sceleratus (Figure 6). Similarly, no changes from the ancestral sequence of the same paralog were found in M. mola , which is also a toxin resistant species (Halstead, 1988). The absence of mutations inL. sceleratus and M. mola , in conjunction with the updated Tetraodontiformes relationships presented in Fig.3, suggest that the changes seen in T. rubripes and T. nigroviridis have arisen via convergent evolution, a hypothesis further supported by the different amino acid replacement observed in these two species. This could also suggest that the position of the replacement is more important than the amino acid change per se . Our results are also congruent with previous findings for Hapalochlaena lunulata(Geffeney et al., 2019) and Hapalochlaena maculosa (Whitelaw et al., 2020), as NaV1.4a Domain I is highly conserved in both octopod species. Furthermore, unlike the pufferfishes A. nigropunctatusand T. nigroviridis (Jost et al., 2008), the garter snakeThamnophis couchii (Feldman et al., 2012) and the octopusesHapalochlaena lunulata (Geffeney et al., 2019) andHapalochlaena maculosa (Whitelaw et al., 2020), replacements were also not observed in the NaV1.4a Domain III of L. sceleratus . Similar to L. sceleratus, mutated residues are not found inT. rubripes either. However, in NaV1.4a Domain IV all the Tetraodontiformes species have a replacement in the same position (outlined in red in Fig.7). Interestingly, a different mutation is observed in each species, except for T. rubripes and T. pardalis, which both share a change to Thr . Similar to what is discussed earlier for the changes observed in domain I, the position of the replacement may be more crucial than the specific changes observed. This could hint to a decrease in TTX binding by disturbing the conserved ancestral binding interface, as previously shown in domain I mutations in T. rubripes and T. nigrovidis (Venkatesh et al., 2005, Soong and Venkatesh 2006). Previously, these mutations forT. rubripes and T. nigroviridis had not been associated with TTX resistance (Venkatesh et al., 2005, Soong and Venkatesh 2006). At the last positions of Domain IV, there are also two replacements toHis and Ser in H. lunulata (Geffeney et al., 2019) and H. maculosa (Whitelaw et al., 2020), which may inhibit TTX binding.
The same studies (Venkatesh et al., 2005, Soong and Venkatesh 2006, Jost et al., 2008) also showed that an Asp mutation in Domain II ofT. nigroviridis NaV1.4b is highly associated with TTX resistance. A similar substitution has been reported in the soft-shelled toxin-resistant clam M. arenaria (Bricelj et al., 2005). Strikingly, the L. sceleratus and M. mola NaV1.4b sequences also lack mutations in Domain II, comparable to NaV1.4a domain I (Figure 6).
While the origin of TTX resistance remains elusive, several studies have provided insight into resistance mechanisms. Toxin tolerance may appear through mutations in sodium channels or toxin binding proteins (Ho et al., 1994, Zou 2020). A wide range of different organisms have taken advantage of mutations which confer TTX tolerance either independently or complementary. The lack of previously characterised pufferfish mutations associated with TTX resistance in L. sceleratus raises several questions about how TTX resistance has evolved. Nevertheless, the results of our analysis imply that combined effects of complex polygenic adaptations working redundantly have played a role in the evolution of this complex trait, while similar genetic changes have arisen convergently multiple times.
CONCLUSIONS
Invader fishes, such as L. sceleratus, often thrive in novel environments. Our analysis provides the first high-quality genome assembly and a comprehensive evolutionary genomic analysis of the species. We uncovered a close phylogenetic position of L. sceleratus with T. nigroviridis, untangling relationships within the pufferfish group, that were not clearly resolved in previous studies. The study gives insights into a variety of genomic signatures that may be associated with L. sceleratus invasion and colonisation effectiveness. Surprisingly, examination of voltage-gated sodium channels (NaV1.4) revealed a lack of TTX resistance associated mutations found in other pufferfishes, highlighting the complex evolution of the trait. Overall, the L. sceleratus genome will be an invaluable resource for additional studies on immune response in novel environments, osmoregulation, reconstruction of ancient chromosome rearrangements, investigation of complex TTX resistance mechanisms and population genomics and adaptation. Such studies are expected to elucidate the mechanisms behind the high invasiveness of L. sceleratus and assist the management of this invasive sprinter in the Mediterranean.