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.