Background: Hemocyanin is the oxygen transporter of most molluscs. Thus, it is an essential protein of these animals which needs to be adapted perfectly to their environments. In Tectipleura, which is a very large and diverse gastropod group with >27,000 species living in all kinds of habitats, several hemocyanin genes have already been identified. They evolved independently from each other within different lineages due to multiple gene duplications and represent potential adaptations to different environments or lifestyles. The aim of this study is to explore the evolution of these genes by analyzing their exon-intron architectures for characteristic features indicating adaptations.

Results: We reconstructed gene architectures of ten hemocyanin genes of four species of Tectipleura: (i) Aplysia californica (ii) Lymnaea stagnalis (iii) Cornu aspersum and (iv) Helix pomatia . Their hemocyanin genes comprise 53 introns each, which is conspicuously more than in known hemocyanin genes of Cephalopoda (9-11), Vetigastropoda (15) and Caenogastropoda (28-33). The gene structures of Tectipleura hemocyanins are identical in terms of number and locations of the introns with exception of only one hemocyanin of Lymnaea stagnalis that comprises one additional intron. Deeper analyses reveal that introns which vary between gene structures of different molluscan lineages most probably evolved more recently through independent intron gains.

Conclusions: The strong conservation of the large number of introns in hemocyanin genes in Tectipleura for over 200 million years suggests a selective pressure on the gene structure. While we have not found characteristic positions or sequence motifs of introns that are conserved, it may be simply the great number of introns that offers increased possibilities of gene regulation and thus may facilitate habitat shifts, adaptive radiation and speciation. This hypothesis is supported by the increased number of introns within hemocyanin genes of Pomacea canaliculata which evolved independently from those of Tectipleura. This species belongs to Caenogastropoda, the sister group of Heterobranchia (where Tectipleura belong to) which is also very diverse and comprises species living in different habitats. Thus, our study provides first evidence that a multitude of introns may contribute to adaptive gene diversity of animals.

Animal sampling and DNA isolation

Adult snails of Cornu aspersum and Helix pomatia were obtained from a commercial dealer (Wiener Schneckenmanufaktur e.U., Vienna, Austria). Three individuals of each species were anesthetized on ice for 20 minutes and subsequently sacrificed. Hepatopancreatic tissue was isolated on an ice cooled aluminum plate. Tissue aliquots were stored in RNAlater™ (Invitrogen by Thermo Fisher Scientific, Waltham, MA, USA) at −80 °C. Samples were homogenized with a Precellys® homogenizer (Bertin Instruments, Montigny-le-Bretonneux, France). Subsequently, DNA was isolated applying the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). DNA integrity was checked on a 0.8% agarose gel (Biozym Scientific GmbH, Hessisch Oldendorf, Germany) and quantified via Nanodrop (ThermoFisher).

Compiling exon-intron structures of hemocyanin genes of Cornu aspersum and Helix pomatia

DNA samples from one adult snail of C. aspersum and H. pomatia were sent to StarSeq (Mainz, Germany) for NGS (Illumina Next Seq500) and library preparation. Bioinformatics were performed using Geneious 9.1.8. Sequencing adapters were removed and raw reads were quality trimmed. Processed genomic data were mapped to coding sequences of three known hemocyanins of C. aspersum (CaH αD: MH485355, CaH αN: MH485356, CaH β: MH485357) and H. pomatia (HpH αD: MH485358, HpH αN: MH485359, HpH β: MH485360). Sequence sections which were not covered by genomic NGS data or which were incongruous to cDNA sequences were used to separate nucleotide sequences in different parts which represent segments of different exons. Exon sequences were completed by repetitive mappings of genomic data to these sequence parts until non-cDNA sequences were assembled. At least ten base pairs of the 3’ and 5’ ends of each intron were assembled to assure that flanking sequences differ from those of neighboring cDNA sequences and therefore represent introns.

PCR confirmation of ambiguous hemocyanin sequence sections

For exon-intron borders which had a low assembly quality or which deviated from the Chambon’s / GT-AG rule (Breathnach et al., 1978; Jacob & Gallinaro, 1989) in hemocyanin genes of H. pomatia and C. aspersum, gene-specific primers (additional file 8, tab. S3 (A)) were designed (CLC main workbench, Version 6.9) and respective gene regions were confirmed via Long Distance (LD) PCR for H. pomatia. Long Fragments were PCR-amplified applying the Platinum™ SuperFi™ Green PCR Master Mix (Invitrogen by Thermo Fisher Scientific, Waltham, MA, USA) whereas shorter fragments were generated using the Advantage® 2 Polymerase Mix (Takara Bio Europe, Saint-Germain-en-Laye, France) (for PCR parameters see additional file 8, tab. S4). PCR products were visualized on a 0.8% agarose gel (Biozym, Hessisch Oldendorf, Germany). PCR products were cleaned up directly using the PCR clean-up kit (Qiagen, Hilden, Germany) or gene-specific bands were cut out and purified with the QIAquick Gel Extraction kit (Qiagen, Hilden, Germany). If possible, clean gene-specific products were sequenced directly by Microsynth (Balgach, Switzerland) using the same primers for sequencing as applied for LD PCR (additional file 8, tab. S3 (A)). Otherwise, they were cloned using the TOPO™ XL-2 Complete PCR Cloning Kit (Invitrogen by Thermo Fisher Scientific, Waltham, MA, USA) for long fragments or the TOPO® TA Cloning® Kit (Invitrogen by Thermo Fisher Scientific, Waltham, MA, USA) for shorter fragments. Three plasmids of one or two individuals were purified using the QIAprep Spin Miniprep Kit (Qiagen, Hilden, Germany) and sequenced via Sanger sequencing by Microsynth (Balgach, Switzerland) (sequencing primers in additional file 8, tab. S3 (B)).