Nature Communications volume 14, número do artigo: 3287 (2023) Citar este artigo
1347 Acessos
93 Altmétrico
Detalhes das métricas
Os caracóis-cone marinhos têm atraído investigadores de todas as disciplinas, mas as fases iniciais da vida têm recebido atenção limitada devido às dificuldades de acesso ou criação de espécimes juvenis. Aqui, documentamos a cultura de Conus magus desde os ovos até a metamorfose para revelar mudanças dramáticas no comportamento alimentar predatório entre juvenis pós-metamórficos e espécimes adultos. C. magus adulto captura peixes usando um conjunto de peptídeos de veneno paralítico combinados com um dente radular em forma de gancho usado para amarrar peixes envenenados. Em contraste, os primeiros juvenis se alimentam exclusivamente de vermes poliquetas, usando um comportamento único de forrageamento de “ferrão e caule”, facilitado por dentes radulares curtos e não farpados e um repertório de veneno distinto que induz hipoatividade nas presas. Nossos resultados demonstram como mudanças morfológicas, comportamentais e moleculares coordenadas facilitam a mudança da caça aos vermes para a caça aos peixes em C. magus, e mostram os caracóis cônicos juvenis como uma fonte rica e inexplorada de novos peptídeos de veneno para estudos ecológicos, evolutivos e de biodescoberta.
Ao longo da história da vida, as inovações evolutivas permitiram que as linhagens em evolução adquirissem novas funções que abrem oportunidades ecológicas e, em muitos casos, promovem a diversificação1,2. Compreender como essas transições ocorreram pode ser desafiador, com características observadas muitas vezes decorrentes de uma série de mudanças evolutivas que eventualmente culminam em uma característica complexa3,4. O aparelho venenoso dos caramujos-cone marinhos (Gastropoda: Conidae) é um exemplo de inovação evolutiva que evoluiu através de modificações morfológicas do intestino anterior5, promovendo a extensa radiação do grupo desde o Eoceno, com mais de 1000 espécies existentes distribuídas em todo o mundo6. Este grupo de gastrópodes predadores evoluiu dentro de um ciclo de vida bifásico, com a maioria das espécies eclodindo como larvas de natação livre que se tornam juvenis carnívoros bentônicos após a metamorfose . A alimentação predatória após a metamorfose depende da implantação de potentes neurotoxinas (conotoxinas) secretadas em uma glândula de veneno tubular longo e injetadas através de dentes radulares ocos altamente modificados9,10. Esta sofisticada estratégia de alimentação permitiu que estes predadores lentos se alimentassem inicialmente de vermes e, mais recentemente, facilitou a mudança evolutiva para a caça de moluscos e peixes11,12.
Devido à sua recente e extensa radiação e à infinidade de peptídeos de veneno que produzem, os caramujos cônicos têm atraído o interesse de biólogos evolucionistas11, farmacologistas13 e toxicologistas14, mas esse amplo interesse contrasta com a escassez de literatura sobre os primeiros estágios da vida. As observações de juvenis no campo têm sido dificultadas pelo seu tamanho diminuto e a sua identificação muitas vezes limitada pela elevada semelhança morfológica entre espécies relacionadas . Por outro lado, os desafios na criação de caramujos cônicos restringiram investigações anteriores à exploração de estágios embrionários e larvais . Devido a essas limitações, a ecologia e a bioquímica dos caracóis juvenis têm sido amplamente negligenciadas. Isso se estende a espécies amplamente estudadas, como o cone do mágico (Conus magus Linnaeus, 1758), fonte do analgésico Prialt® (ω-conotoxina MVIIA) aprovado pela FDA22. Com base em espécimes dissecados capturados na natureza, sugeriu-se que C. magus passasse por uma mudança na dieta da caça de vermes para a caça de peixes durante a ontogenia23, mas faltam evidências empíricas devido aos desafios de acesso aos primeiros estágios da vida.
Aqui cultivamos Conus magus desde cápsulas de ovo até larvas em incubação e, através da metamorfose, até juvenis carnívoros. Após a metamorfose, observou-se que os juvenis de C. magus atacavam exclusivamente vermes poliquetas usando dentes radulares semelhantes aos ancestrais e um repertório de veneno único, antes de passarem a caçar peixes na idade adulta. Através de uma combinação de abordagens experimentais, demonstramos como a transição da caça aos vermes para a caça aos peixes durante a ontogenia é marcada por uma série de mudanças coordenadas que abrangem todos os níveis da organização biológica. Nossos resultados mostram como os espécimes criados em laboratório podem fornecer novos insights sobre a ecologia dos estágios secretos da vida e destacar o potencial dos caramujos cônicos juvenis como uma fonte inexplorada de novos peptídeos de veneno bioativos que, de outra forma, só seriam acessíveis através da captura de éxons ou sequenciamento do genoma.
4 mm23. Additionally, the methods used for the identification of small specimens are not mentioned and the high morphological similarity between juvenile cone snails suggests the sampling could have included other species. The present study provides empirical evidence of strict vermivory in juvenile C. magus. The feeding behaviour of juveniles was initiated by extension of the proboscis which probed the surface of the worm in preparation for venom injection. After several minutes, a radular tooth held at the tip of the proboscis was stabbed into the worm and the proboscis rapidly withdrawn inside the rostrum, leaving the prey untethered. Envenomation induced hypoactivity in worm prey, characterised by the loss of normal swimming, hiding and escape behaviours. The snail then stalked its prey for several minutes before extending its rostrum and engulfing the worm whole (Supplementary Movie 2). Occasionally, worms were stung a second time. The same feeding sequence was observed in all juveniles from 10 dps, although histology and rapid shell growth between 6–10 dps suggest carnivory may have started earlier (Fig. 1d). This “sting-and-stalk” foraging behaviour was consistent with the juvenile radular tooth lacking apical barbs, blades and serrations (Fig. 4b; Supplementary Fig. 2a), as seen in wild-caught specimens23. The hooked accessory process and the basal ligament seen in the adult tooth were also absent. The juvenile radular tooth was short in absolute and relative length, measuring 69.7 ± 1.15 µm (n = 5) in length for a shell length (SL) of 1.71 ± 0.08 mm (n = 5) (4.1% of SL). It had a waist and a broad base with a wide opening, as typically seen in vermivorous species. Interestingly, similar teeth are also found in juvenile worm-27 and mollusc-hunters (Rogalski, A. et al., manuscript in preparation), indicating that this trait has been retained in early life stages across Conidae. Morphometric analyses confirmed similarity with radular teeth from vermivorous cone snails (Supplementary Fig. 3; Supplementary Data 1), and the presence of similar teeth in related conoidean lineages such as Mitromorphidae and Borsoniidae28,29 suggests this trait may be plesiomorphic within the group./p>4 kDa restricted to the adult VG (Fig. 5c; Supplementary Fig. 8a; Supplementary Data 4). Furthermore, the different MS patterns obtained from proximal and distal VG support the heterogenous distribution of conotoxins along the adult VG. While MALDI-MS is a useful technique for whole venom profiling, this approach suffers a number of limitations, including low dynamic range and ion suppression effects, preventing the detection of the full venom complexity58. To complement MALDI-MS, we additionally performed liquid chromatography-mass spectrometry (LC-MS) on the juvenile and adult C. magus VG extracts. Considering the complexity of cone snail venoms and the typical mass range of conotoxins, only monoisotopic masses between 1–10 kDa and covering ≥0.1% of relative intensity were considered to facilitate ecological interpretation (Supplementary Data 4). A total of 123 masses (104 unique) were detected in the adult VG, while 92 masses (86 unique) were found in the juvenile VG. Comparison of mass lists revealed only a single mass (1438.01 Da) was shared between both venom proteomes, supporting the differences observed by MALDI-MS. While the juvenile VG proteome was largely dominated by peptides falling into the 1–2 kDa mass range (n = 53, 57.6% of masses), the adult VG proteome contained a large proportion of 4–6 kDa peptides (n = 48, 39% of masses) compared to juveniles (n = 10, 10.9% of masses) (Fig. 4d; Supplementary Fig. 8b)./p> 10-fold the tissue volume of RNA later (Invitrogen) and stored at –80 °C until extraction. The maternal VG was dissected and divided into proximal- and distal-regions of equal sizes to investigate spatial distribution of conotoxins along the VG and RNA extracted from fresh tissue. Three segments corresponding to proximal, central and distal regions were kept in a solution of 30% acetonitrile (ACN)/1% FA for proteomics, and two small segments (proximal and distal) were placed in 2.5% glutaraldehyde and processed for histology as described above. Total RNA was extracted from all samples using TRIzol (Invitrogen) following the manufacturer’s instructions to yield 0.4–2.72 μg of purified mRNA from each sample. The RNA quality and concentration were assessed on a 2100 Bioanalyzer using the RNA 6000 Nano kit (Agilent). Complementary DNA library preparation and sequencing were performed by the Institute for Molecular Bioscience Sequencing Facility (University of Queensland). Libraries were constructed using the Illumina Stranded mRNA Prep kit. Samples were pooled in a batch of 6 and 600-cycle (2 × 300 bp) paired-end sequencing was performed on an Illumina MiSeq instrument. Raw sequencing data have been deposited in the NCBI Sequence Read Archive under BioProject accession number PRJNA943605./p>250 amino acids and with a signal region hydrophobicity score <45% were manually removed. All sequences were searched for the presence of an N-terminal signal region using the SignalP 5.071 server and sequences lacking signal regions were discarded. At this stage, nucleotide sequences were manually inspected and incomplete or aberrant sequences (internal or no stop codons, repetitions, incorrect open reading frames) were discarded. The retained contigs were annotated using blastx and blastp72 searches against the non-redundant UniprotKB/SwissProt (E-value cut off: 10–3) and Conoserver databases. The ConoPrec tool in Conoserver was then used to identify the signal-, propeptide-, mature- and post-mature regions and cysteine frameworks. Expression levels of all reads were computed in transcripts per million (TPM)73 using Kallisto 0.46.174. Expression levels were summed up for each gene superfamily and relative expression (in per cent) calculated, including a specimen from the Philippines37. We then performed a principal component analysis (PCA) to evaluate the degree of venom composition similarities between juvenile and adult C. magus using XLSTAT statistical software (Addinsoft, free trial version). For the PCA biplot, the four variables with the strongest influence on the PCs are shown. The data matrix, summary statistics, contribution of each variable (in per cent), PCA scores and loading plots can be seen in Supplementary Data 3. All peptide precursors were named according to the conventional conotoxin nomenclature (with species represented by one or two letters, cysteine framework by an Arabic numeral and, following a decimal, order of discovery by a second numeral)75, with slight modification76. The superfamily was added as a prefix and precursors differing in their propeptide regions but with conserved mature peptides were differentiated with a small roman numeral as a suffix to distinguish between minor variants. All conotoxin precursor sequences have been deposited in NCBI GenBank [https://www.ncbi.nlm.nih.gov/nuccore] under accession numbers OQ644315–OQ644445./p> 150 counts/s. The most intense isotopes were selected and fragmented with collision-induced dissociation (CID) and electron-activated dissociation (EAD) tandem mass spectrometry. MS/MS scans were collected between 50–2000 m/z over 35 ms. The dynamic collision energy setting was used, allowing collision energy to vary based on m/z and z of the precursor ion. Data were acquired using OS 3.0.0.3339 and analysed in Peakview 2.2 (both SCIEX). The CID-MS/MS spectra were searched against a database combining all translated sequences from our RNA-seq experiments and previously reported C. magus conotoxins (Supplementary Data 2) using the Paragon78 algorithm implemented in ProteinPilot 5.0 (SCIEX) with the following settings: iodoethanol (for reduced and alkylated samples), trypsin digested (for digested samples), common conotoxin post-translational modifications79, biological modifications, thorough ID. Peptides with ≥2 tryptic fragments at a confidence of 99 and a false discovery rate <1% were considered genuine. The EAD-MS/MS data were searched against the same database using Mascot 2.5.180 (Matrix Science) with the following settings: trypsin, 1 missed cleavage, carbamidomethyl as a fixed modification, oxidation of methionine and deamidation of asparagine and glutamine as variable modifications, 20 ppm peptide tolerance, 0.1 Da MS/MS tolerance, 2 + 3+ and 4+ peptide charges, with an error tolerant search included. Peptides with ≥2 tryptic fragments, individual peptide scores >60 and a significance threshold <0.05 were selected./p>3.0.CO;2-2" data-track-action="article reference" href="https://doi.org/10.1002%2F%28SICI%291522-2683%2819991201%2920%3A18%3C3551%3A%3AAID-ELPS3551%3E3.0.CO%3B2-2" aria-label="Article reference 80" data-doi="10.1002/(SICI)1522-2683(19991201)20:183.0.CO;2-2"Article CAS PubMed Google Scholar /p>
Português
English
Français
Deutsch
Español
Italiano
日本語
한국어
Русский