調整された適応が線虫からの個体発生的移行を定義する | 株式会社アーチワイヤーズグループ

Nature Communications volume 14、記事番号: 3287 (2023) この記事を引用

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海洋イモガイはあらゆる分野の研究者を魅了していますが、幼体標本へのアクセスや飼育が難しいため、初期段階への注目は限定的です。今回我々は、卵から変態までのイヌイモイモの培養を記録し、変態後の幼体と成体標本の間での捕食行動の劇的な変化を明らかにする。成体の C. magus は、麻痺性の毒ペプチドのセットと、毒を持った魚をつなぐために使用される鉤状の歯状歯を組み合わせて魚を捕獲します。対照的に、初期の幼体は、短くてとげのない歯根歯と、獲物の活動低下を誘発する独特の毒レパートリーによって促進される独特の「刺して茎を使う」採食行動を利用して、もっぱら多毛虫を食べます。私たちの結果は、形態学的、行動的、分子的変化の調和が、C. magus の虫狩りから魚狩りへの移行をどのように促進するかを実証し、イモガイの幼体が生態学、進化学、生物発見研究のための新規毒ペプチドの豊富で未開発の供給源であることを示しています。

生命の歴史を通じて、進化の革新により、進化する系統は生態学的機会を切り開き、多くの場合、多様化を促進する新しい機能を獲得することができました1,2。観察される形質は多くの場合、最終的に複雑な形質に至る一連の進化的変化から生じており、これらの移行がどのように起こったのかを理解することは困難な場合があります 3,4。海産イモガイ (腹足綱: イモ科) の毒装置は、前腸の形態的変化を通じて進化した進化の革新の一例であり 5、始新世以来このグループの広範な放散を促進し、1,000 種を超える現存種が世界中に分布しています 6。この捕食性腹足類のグループは二相性のライフサイクルの中で進化しており、ほとんどの種は自由遊泳幼生として孵化し、変態後に底生の肉食性幼生になります7,8。変態後の捕食性摂食は、長い管状毒腺から分泌され、高度に改変された中空の歯根歯を介して注入される強力な神経毒（コノトキシン）の展開に依存しています9,10。この洗練された摂食戦略により、これらの動きの遅い捕食者は最初は虫を食べることが可能になり、さらに最近では軟体動物や魚の狩猟への進化的移行が促進されました11,12。

イモガイは、最近の広範囲にわたる放射線照射と、産生する大量の毒ペプチドのせいで、進化生物学者11、薬理学者13、毒物学者14の関心を集めているが、この広範な関心は、初期の生活段階に関する文献の不足とは対照的である。野外での幼体の観察は、その小さなサイズによって妨げられており、近縁種間の形態学的類似性の高さによって識別が制限されることがよくあります15、16、17。一方で、イモガイの飼育に関する課題により、これまでの調査は胚および幼虫段階の調査に限定されていた18、19、20、21。これらの制限のため、イモガイの幼体の生態と生化学はほとんど無視されてきました。これは、FDA 承認の鎮痛剤 Prialt® (ω-コノトキシン MVIIA) の原料であるマジシャンズコーン (Conus magus Linnaeus、1758 年) など、広く研究されている種にまで及びます 22。野生で捕獲された解剖標本に基づいて、C. magus は個体発生の際に虫食いから魚狩りへ食事の変化を経験したことが示唆されています 23 が、初期の生活段階にアクセスする際の課題のため経験的証拠が不足しています。

ここでは、イヌイモを卵嚢から孵化幼虫まで、そして変態を経て肉食性の幼体まで培養しました。変態後、C. magus の幼体は、祖先のような歯状歯と独特の毒レパートリーを使って多毛虫のみを捕食し、その後、成魚になると魚狩りに切り替えることが観察されました。私たちは、実験的アプローチを組み合わせることにより、個体発生時の虫狩りから魚狩りへの移行が、生物学的組織のあらゆるレベルにわたる一連の調整された変化によってどのように特徴付けられるかを実証します。私たちの結果は、実験室で飼育された標本がどのようにして分泌型の生活段階の生態について新たな洞察をもたらすことができるかを示し、また、そうでなければエクソン捕捉またはゲノム配列決定を通じてのみアクセスできる新規の生理活性毒ペプチドの未開発の供給源としてのイモガイの幼魚の可能性を強調しています。

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>

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