Nature Communications 14권, 기사 번호: 3287(2023) 이 기사 인용
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해양 원뿔 달팽이는 모든 분야의 연구자들의 관심을 끌었지만 어린 표본에 접근하거나 양육하는 데 어려움이 있어 초기 생활 단계에서는 제한적인 관심을 받았습니다. 여기서 우리는 변태 이후의 청소년과 성체 표본 사이의 약탈적인 먹이 행동의 극적인 변화를 밝히기 위해 알에서 변태를 통해 Conus magus의 문화를 기록합니다. 성체 C. magus는 독이 있는 물고기를 묶는 데 사용되는 갈고리 모양의 치아와 결합된 마비성 독 펩타이드 세트를 사용하여 물고기를 포획합니다. 대조적으로, 초기 치어는 짧고 가시가 없는 치근상 이빨과 먹이의 활동 저하를 유발하는 뚜렷한 독 레퍼토리에 의해 촉진되는 독특한 "쏘고 줄기를 치는" 채집 행동을 사용하여 다모류 벌레만을 먹습니다. 우리의 결과는 형태학적, 행동적 및 분자적 변화가 어떻게 조정되어 C. magus에서 벌레 사냥에서 물고기 사냥으로의 전환을 촉진하는지 보여주고 어린 원뿔 달팽이가 생태학, 진화 및 생물 발견 연구를 위한 풍부하고 탐구되지 않은 새로운 독 펩타이드의 원천임을 보여줍니다.
생명의 역사를 통틀어 진화적 혁신을 통해 진화하는 계통은 생태학적 기회를 열고 많은 경우 다양성을 촉진하는 새로운 기능을 획득할 수 있었습니다1,2. 이러한 전환이 어떻게 발생했는지 이해하는 것은 어려울 수 있습니다. 관찰된 특성은 종종 복잡한 특성으로 끝나는 일련의 진화적 변화에서 발생합니다3,4. 해양 원뿔달팽이(복족류: Conidae)의 독 장치는 앞창자의 형태학적 변형을 통해 진화한 진화적 혁신의 한 예이며5, 시신세 이후 이 그룹의 광범위한 방사를 촉진하여 전 세계적으로 1,000종 이상의 현존 종이 분포되어 있습니다6. 이 포식성 복족류 그룹은 이상 생활사 내에서 진화했으며, 대부분의 종은 자유 수영 유충으로 부화하여 변태 후에 저서 육식성 청소년이 됩니다7,8. 변태 후 약탈적인 섭식은 긴 관형 독샘에서 분비되고 고도로 변형된 속이 빈 방사형 치아를 통해 주입되는 강력한 신경독(코노톡신)의 배치에 의존합니다9,10. 이 정교한 먹이 전략을 통해 느리게 움직이는 포식자가 처음에는 벌레를 먹을 수 있었고 최근에는 연체동물 및 물고기 사냥으로의 진화적 전환이 촉진되었습니다11,12.
최근의 광범위한 방사선과 그들이 생성하는 과다한 독 펩타이드로 인해 원추 달팽이는 진화 생물학자11, 약리학자13 및 독성학자14로부터 관심을 끌었지만 이러한 광범위한 관심은 초기 생활 단계에 대한 문헌의 부족과 대조됩니다. 현장에서 청소년 관찰은 미세한 크기로 인해 방해를 받았으며 관련 종 간의 높은 형태학적 유사성으로 인해 식별이 제한되는 경우가 많습니다. 반면, 원뿔 달팽이를 키우는 문제로 인해 이전 조사는 배아 및 애벌레 단계18,19,20,21의 탐사로 제한되었습니다. 이러한 제한으로 인해 어린 원뿔달팽이의 생태학과 생화학은 크게 간과되어 왔습니다. 이는 FDA 승인 진통제 Prialt®(Ω-코노톡신 MVIIA)22의 공급원인 마술사의 원뿔(Conus magus Linnaeus, 1758)과 같이 널리 연구된 종까지 확장됩니다. 해부된 야생 포획 표본을 기반으로 C. magus는 개체 발생 동안 벌레 사냥에서 물고기 사냥으로 식단 전환을 겪는 것으로 제안되었지만 초기 생활 단계에 접근하는 데 어려움이 있어 경험적 증거가 부족합니다.
여기에서 우리는 달걀 캡슐에서 부화하는 유충까지, 그리고 변태를 통해 육식성 청소년으로 Conus magus를 배양했습니다. 변태 후, 어린 C. magus는 성체가 되어 물고기 사냥으로 전환하기 전에 조상과 같은 방사상 치아와 독특한 독 레퍼토리를 사용하여 다모류 벌레만을 잡아먹는 것으로 관찰되었습니다. 실험적 접근법의 조합을 통해 우리는 개체 발생 동안 벌레 사냥에서 물고기 사냥으로의 전환이 생물학적 조직의 모든 수준에 걸쳐 일련의 조정된 변화로 표시되는 방법을 보여줍니다. 우리의 결과는 실험실에서 사육된 표본이 어떻게 비밀 생활 단계의 생태학에 대한 새로운 통찰력을 제공할 수 있는지 보여주고, 엑손 포획이나 게놈 서열 분석을 통해서만 접근할 수 있는 새로운 생체 활성 독 펩타이드의 미개발 공급원으로서 어린 원뿔 달팽이의 잠재력을 강조합니다.
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>
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