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ISME Communications volume 3、記事番号: 84 (2023) この記事を引用

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メトリクスの詳細

海洋微生物群集に関する研究は増えていますが、海水サンプリングプロトコルが異なるため、研究を比較するのは困難です。研究者がさまざまな海水サンプリング手法を使用した研究を相互比較し、将来のサンプリングキャンペーンを設計できるようにするために、私たちは EuroMarine Open Science Exploration イニシアチブ (EMOSE) を開発しました。 EMOSE の枠組みの中で、私たちは地中海北西部の 1 つの観測所 (Service d'Observation du Laboratoire Arago [SOLA]、バニュルスシュルメール) から 1 日で数千リットルの海水をサンプリングしました。結果として得られるデータセットには、さまざまな材料タイプのフィルター (カートリッジ膜と平膜)、3 つの異なるサイズ分別 (>0.22 µm、0.22 ～ 3 µm、3 ～ 20 µm、および >20 µm) を含む複数の海水処理アプローチが含まれています。我々は、シーケンス戦略とは無関係に、濾過される海水の量が原核生物と原生生物の多様性に重大な影響を及ぼさないことを示します。しかし、サイズ画分間、およびサイズ画分と「全水」（前分画なし）との間では、アルファおよびベータの多様性に明らかな違いがありました。全体として、異なるポアサイズのフィルターを使用するデータセットからのデータをマージする場合は注意することをお勧めしますが、フィルターの種類とボリュームがテストされたシーケンス戦略の交絡変数として機能すべきではないと考えています。私たちの知る限り、公的に利用可能なデータセットによって、サンプリング量の大規模な変動を含む幅広いプロトコルにわたる海洋マイクロバイオームの方法論的オプションの影響を効果的に解明できるのはこれが初めてです。

地球上の微生物の生態の特性評価は、学際的な関心のテーマとなっています。実際、現在では、細胞から生態系に至るまでの生命をより深く理解するには、複数の生物群系にわたる微生物生態学の知識を獲得することが重要であることが認識されています。その結果、大規模な国際共同研究プロジェクトが、ヒト [1、2]、サンゴ [3]、海草 (https://seagrassmicrobiome.org/protocols/)、または海綿動物 [4、5] に関連するマイクロバイオームに焦点を当ててきました。さらに、過去 20 年間にわたり、世界規模の海洋サンプリング (2003 ～ 2010 年) [6, 7]、国際海洋微生物センサス (ICoMM) [8]、マラスピーナ 2010 年周航遠征 [9] およびタラ号海洋遠征 (2009 年から 2012 年) [10]、地球微生物叢プログラム [11、12] およびマイクロ B3 主導の海洋サンプリングデー (OSD) [13] などの国勢調査プログラムと併せて]。地球規模の海洋微生物生態学、その関連性、将来の課題に関する進歩と展望の詳細は、他の場所で広く検討されています[14]。

世界のマイクロバイオームを研究する現在の大規模な取り組みにより、さまざまな環境や宿主組織のマイクロバイオームをサンプリングするための共通プロトコルの使用や、共通の配列決定手順の使用など、複数の標準化イニシアチブが生まれています。方法論的標準化の取り組みに関連する取り組みとしては、例えば、OSD [13]、地球微生物叢 [15]、欧州海洋オミクス生物多様性観測ネットワーク (EMO BON) [16]、ヒト腸管メタゲノミクス (MetaHIT) [17] などがあります。

自由生活および宿主関連マイクロバイオームの大規模解析は、微生物と動物 [3、5、18、19] および微生物と植物 [20] の相互作用、さらには構造、機能、および相互作用を理解する上で大きな前進となります。地球の多様な生息地における微生物群集の多様性 [21]。ただし、微生物の多様性をサンプリングし、説明し、研究するためのベストプラクティスと戦略をより適切に標準化し、調和させるには、ギャップを埋める必要があります。特に、海洋マイクロバイオームの研究では、微生物の豊富さの推定は、メタバーコーディングに使用されるマーカー遺伝子とプライマー [22]、さまざまな DNA 抽出プロトコル [23、24]、配列決定の深さとゲノム配列など、いくつかの要因に依存することが知られています。アプローチ（アンプリコン配列決定とメタゲノム配列決定）およびクラスタリング基準[25]。サンプリング戦略が微生物プランクトンの多様性の推定に影響を与えることは認識されているが[25]、海洋マイクロバイオームの多様性と分類学的構成を研究するためのサンプリング手順に対する方法論的変数の影響を体系的にテストするように設計された研究は不足している。これらの研究は、海洋微生物群集のサイズ範囲全体をサンプリングするための正確なプロトコルを設計するために重要です[26]。

0.22 µm) and of the 0.22 µm to 3 µm size fractions used 142 mm diameter polyethersulfone Express Plus membrane filters (Product Code GPWP14250, Millipore). For the 3 µm to 20 µm fractionations, 142 mm diameter polycarbonate membrane filters were used (Product Code TSTP14250, Millipore). As for the large size fractions (>20 µm), the 47 mm diameter nylon mesh filter was used instead (referred to as flat membrane from here on)./p>20 µm). Additionally, whole water cartridge membrane volumes from 1 L to 10 L were also compared for the metagenomes. Below, we consider the prokaryotes and protists results independently./p>0.22 µm) or size fractions (0.22–3 µm, 3–20 µm and >20 µm) in columns. Color distinguishes between flat and cartridge membrane filters. Within each grid unit, the prokaryotic species richness is plotted against volume, which ranges from 2.5 L to 1000 L./p>0.22 µm) and 0.22–3 µm size fraction samples presented a similar number of prokaryotic taxonomic lineages (Fig. 3a) and both presented fewer prokaryotic taxonomic lineages than the 3–20 µm size fraction (Fig. 3a). Accordingly, the statistical test indicated significant differences in the species richness obtained after > 0.22 µm, 0.22–3 µm and 3–20 µm (p < 0.05, Kruskal–Wallis), more specifically, between >0.22 µm and 3–20 µm size fractions (p < 0.05, post-hoc Dunn test). On the metagenomes side, for the same comparison, there were no appreciable differences in the number of prokaryotic taxonomic lineages (Fig. 3a) and they were not significant (p > 0.05, Kruskal–Wallis). Details on the above-mentioned statistical tests are available in Supplementary Table S7./p>0.22 µm), 0.22–3 µm and 3–20 µm size fractions for the same volume (10 L) and filter (flat membrane), for MetaB16SV4V5 (left) and metagenomes (right). Note that metagenomes didn’t include samples in 3–20 µm size fraction in (a). b Comparison for size fractions (0.22–3 µm, 3–20 µm and > 20 µm size fractions) for the same volume (100 L) and filter (flat membrane), for MetaB16SV4V5 (left) and metagenomes (right). Note that metagenomes didn’t include samples in >20 µm size fraction in (b). c Comparison for flat membrane vs cartridge membrane, for the same volume (10 L) and whole water (>0.22 µm), for MetaB16SV4V5 (left) and metagenomes (right). d Comparison between 2.5 L (single filter) and 10 L (four 2.5 L filters pooled together), using the same filter (cartridge membrane) and whole water (> 0.22 µm), for MetaB16SV4V5 (left) and metagenomes (right). All panels illustrate the species richness obtained for each sample (point). To help the reader compare the variables, we added boxplots on top of the points. Significance was determined using either Mann–Whitney test for two independent groups, or Kruskall–Wallis for more than two independent groups, followed by a post-hoc Dunn test, if needed. Significance was illustrated with the symbols: p > 0.05 (empty); p < 0.05 (*); p < 0.01 (**); and p < 0.001 (***)./p>20 µm, using the flat membrane filter, which revealed an increase in the prokaryotic species richness with increasing pore size, for both MetaB16SV4V5 and metagenomes (Fig. 3b). In fact, the median number of prokaryotic taxonomic lineages obtained by MetaB16SV4V5 increased significantly from 335 (0.22–3 µm) to 429 (3–20 µm) and 538 (>20 µm) (p < 0.05, Kruskal–Wallis, Fig. 3b), more specifically between 0.22–3 µm and > 20 µm size fractions (p < 0.05, post-hoc Dunn test). Similarly, metagenomes increased the median number of prokaryotic taxonomic lineages from 155 (0.22–3 µm) to 195 (3–20 µm) (Fig. 3b), which was also significant (p < 0.05, Mann–Whitney). Details on the above mentioned statistical tests are available at Supplementary Table S7. Please note that for metagenomes in Fig. 3b there are no samples for the >20 µm size fraction because some samples were lost due to insufficient DNA for sequencing, while some samples that were successfully sequenced were later discarded due to low number of reads (below 10 000 reads, for a list of discarded samples in the rarefaction step see Supplementary Table S4). The overview of prokaryotic species richness was overall consistent and supported by the rarefaction curves because the different size fractions had similar levels of alpha diversity, while the same did not apply for volume (Supplementary Figs. S2 and S3)./p> 0.05, Mann–Whitney). Metagenomes provided an equivalent number of prokaryotic taxonomic lineages between either filter (Fig. 3c) and the differences were not significant (p > 0.05, Mann–Whitney). Although we compared cartridge and flat membrane filters under the same volume (10 L), the cartridge membrane filters reached 10 L by pooling together four cartridge membrane filters of 2.5 L. However, the single 2.5 L cartridge membrane filter and 10 L pooled from four cartridge membrane filters of 2.5 L obtained an equivalent number of prokaryotic taxonomic lineages, without significant differences (p > 0.05, Mann–Whitney) for either sequencing approach (Fig. 3d). Details on the above mentioned statistical tests are available at Supplementary Table S7./p>20 µm size fractions. Additionally, the volume did not follow any clear direction in the ordination figures (Fig. 4a, b). PERMANOVA tests were made to support the ordination figures, with similar results for MetaB16SV4V5 and metagenomes. Specifically, both volume and size fractions significantly changed the community composition (p < 0.05, PERMANOVA), but this result should be interpreted with caution, because if the same test considers the division of samples by size fraction, then community composition did not change significantly across volume (p > 0.05, PERMANOVA). Details on the PERMANOVA statistical tests for prokaryotes are available at Supplementary Table S8. The variation within size fractions, measured by distance to centroid, further supported the clustering of prokaryotic community composition by size fractions (Fig. 4c,d, Supplementary Table S9)./p>0.22 µm), 0.22–3 µm, 3–20 µm and >20 µm size fractions. Division by (a) MetaB16SV4V5 and (b) metagenomes. Additionally, boxplots represent the distance to centroids of samples within each size fraction, divided by (c) MetaB16SV4V5 and (d) metagenomes. Note that metagenomes didn’t include the >20 µm size fraction. For details on missing replicates, we refer the reader to Supplementary Table S1./p> 20 µm) using the same volume (100 L) and filter (flat membrane)./p>0.22 µm) for MetaB18SV9 showed any appreciable change in the protist species richness from 2.5 L (median = 343, IQR = 6.75, n = 4) to 10 L (median = 348, IQR = 34.8, n = 12) (Fig. 7). However, for either MetaB18SV9 and metagenomes, there was no appreciable difference in the protist species richness from 10 L to 1000 L, within any of the size fractions (Fig. 7). Comparing pore sizes, whole water (>0.22 µm), 3–20 µm and >20 µm size fractions identified more protist taxonomic lineages than 0.22–3 µm size fraction samples (Fig. 7). The number of protist taxonomic lineages obtained after each sample are available at Supplementary Table S10. The higher impact of size fraction, rather than volume, on protist species richness was further supported by rarefaction curves (Supplementary Figs. S2 and S3), even though the size fractions were not as distinct from one another as they were with the prokaryotic data./p>0.22 µm) or size fractions (0.22–3 µm, 3–20 µm and >20 µm) in columns. Color distinguishes between flat membrane and cartridge membrane filters. Within each grid unit, the protist species richness is plotted against volume. For details on missing replicates, we refer the reader to Supplementary Table S1./p>0.22 µm) or 3–20 µm size fraction (Fig. 8a). However, the range of the number of protist taxonomic lineages obtained for whole water included the range of values for both the 0.22–3 µm and 3–20 µm size fractions (Fig. 8a). More specifically, the number of protist taxonomic lineages obtained by MetaB18SV9 varied between 290 and 380 for the whole water, 289 and 338 for 0.22–3 µm size fraction, and 338 to 357 in 3–20 µm size fractions (Fig. 8a), which were not significantly different (p > 0.05, Kruskal–Wallis). The number of protist taxonomic lineages obtained by metagenomes varied between 88 and 129 for whole water, 91 and 97 for 0.22–3 µm, and 105 and 128 for 3–20 µm size fractions (Fig. 8a); these differences were also statistically non-significant (p > 0.05, Kruskal–Wallis). We note, however, the number of samples for the metagenome provide little support for the described differences in this specific comparison. Details on the above mentioned statistical tests are available in Supplementary Table S11./p>0.22 µm), 0.22–3 µm and 3–20 µm size fractions for the same volume (10 L) and filter (membrane), for MetaB18SV9 (left) and metagenomes (right). b Comparison for size fractions (0.22–3 µm, 3–20 µm and > 20 µm size fractions) for the same volume (100 L) and filter (membrane), for MetaB18SV9 (left) and metagenomes (right). c Comparison for flat membrane vs cartridge membrane, for the same volume (10 L) and whole water (>0.22 µm), for MetaB18SV9 (left) and metagenomes (right). d Comparison between 2.5 L (single filter) and 10 L (four 2.5 L filters pooled together), using the same filter (cartridge membrane) and whole water (> 0.22 µm), for MetaB18SV9 (left) and metagenomes (right). All panels illustrate the species richness obtained for each sample (point). To help the reader compare the variables, we added boxplots on top of the points. Significance was determined using either Mann–Whitney test for two independent groups, or Kruskall–Wallis for more than two independent groups, followed by a post-hoc Dunn test, if needed. Significance was illustrated with the symbols: p > 0.05 (empty); p < 0.05 (*); p < 0.01 (**); and p < 0.001 (***)./p>20 µm size fractions for the same filter (membrane) and volume (100 L), the 0.22–3 µm size fraction had fewer protist taxonomic lineages than the 3–20 µm and >20 µm size fractions (Fig. 8b), for either MetaB18SV9 and metagenomes. These differences were significant for the MetaB18SV9 (p < 0.05, Kruskal–Wallis), but not for the metagenomes (p > 0.05, Kruskal–Wallis). However, the significance of the test was not very strong and the post-hoc test for MetaB18SV9 was not significant for any combination of size fractions, after adjustment (p > 0.05, post-hoc Dunn test). Details on the above mentioned statistical tests are available in Supplementary Table S11./p>0.22 µm). The differences in the number of protist taxonomic lineages between cartridge and flat membrane filters were small (Fig. 8c) and not significant (p > 0.05, Mann–Whitney). However, the range of values was wider for the flat membrane filter than the cartridge membrane filter with the MetaB18SV9 approach (Fig. 8c). The number of protist taxonomic lineages within the replicates of flat membrane filters varied between 290 and 380 (difference of 90 taxonomic lineages), while in the cartridge membrane filters varied between 354 and 373 (difference of 19 taxonomic lineages) (Fig. 8c). For metagenomes, the values were equivalent between both types of filters (Fig. 8c). Please note that the cartridge membrane and flat membrane filters were compared at 10 L volume, but the cartridge membrane samples obtained 10 L by pooling together four cartridge membrane filters of 2.5 L together. For MetaB18SV9, the number of protist taxonomic lineages obtained after pooling four 2.5 L cartridge membrane filters was higher than using a single filter of 2.5 L (Fig. 8d), but not significant (p > 0.05, Mann–Whitney). However, this was not the same for the metagenomes, where the number of protist taxonomic lineages was equivalent and slightly higher for a single filter of 2.5 L (Fig. 8d), but also not significant (p > 0.05, Mann–Whitney). Details on the above mentioned statistical tests are available at Supplementary Table S11./p>20 µm size fractions were distant from the remaining, in either MetaB18SV9 and metagenomes (Fig. 9a, b). This was further supported by the significant results of PERMANOVA for the volume and size fractions independently (p < 0.05, PERMANOVA), but once they were considered together the effect on community composition was no longer significant (p > 0.05, PERMANOVA). Note that the variable for size fractions did not meet the homogeneity of variance pre-requisite of PERMANOVA (p > 0.05, betadisper). Details on the PERMANOVA statistical tests for protists are available in Supplementary Table S12. Additionally, a more detailed look into the betadisper results, i.e., a measure of distance to the centroid of samples within each size fraction, revealed that samples were very consistent within size fractions (Fig. 9c,d and Supplementary Table S13)./p>0.22 µm), 0.22–3 µm, 3–20 µm and >20 µm size fractions divided by (a) MetaB18SV9 and (b) metagenomes. Additionally, boxplots represent the distance to centroids of samples within each size fraction, divided by (c) MetaB16SV4V5 and (d) metagenomes./p>20 µm size fraction consistently identified more taxonomic lineages, independently of the volume, for example, Dinophyceae, Bacillariophyceae and Foraminifera (Fig. 10). In contrast, other groups were more prevalent in the 3–20 µm size fraction, like Cercozoa, Hacrobia and Haptophyta (Fig. 10). Several groups did not seem to favor any specific size fraction, like Excavata or Syndinales (Fig. 10). In the metagenomes, from 10 L to 1000 L, some groups had more protist taxonomic lineages in the >20 µm size fraction, like Bacillariophyceae, or fewer, like Hacrobia (Supplementary Fig. S5). Additionally, the metagenomes did not reveal any specific taxonomic group that increased the number of protist taxonomic lineages with increasing volume (Supplementary Fig. S5)./p>20 µm size fraction consistently had more taxonomic lineages, indicating that several taxonomic lineages were specifically found in that size fraction. One possible explanation for the identification of taxonomic lineages specific to the >20 µm size fraction is that those prokaryotes were attached to particles, or to the filter material itself. Considering that the turbidity of the water was very low, the only particles plausible for the prokaryotes to attach to would be the protists or other cell debris, including aggregates. Thus, we suggest that the prokaryotic taxonomic lineages specific to the large size fraction could be prokaryotes associated with microeukaryotes, colonial bacteria and/or specialized in colonizing larger particles. Given the presence of prokaryotes on > 20 µm size fractions and protists on 0.22–3 µm size fractions, we cannot rule out the possibility that extracellular DNA, besides actual cells, is retained in the filters, for example, by sorption [64]. However, the general picture is that free-living prokaryotes are identically identified in whole water (> 0.22 µm) and 0.22–3 µm size fraction, while particle-attached prokaryotes can be retained within larger pore size fractions (3–20 µm and >20 µm). This is consistent with previous studies that account for the effect of pre-filtration on prokaryotic diversity with 16 S rRNA gene sequencing [65]. Protists also follow the same general picture described in previous studies [40], with contamination between smaller size fractions, for example, because of cell fragments. In this study, either biological group was most unique in composition at >20 µm size fraction. Notwithstanding, we highlight that it was unexpected to find more prokaryotic and protist taxonomic lineages in the > 20 µm size fraction than in whole water, which cannot be fully explained by our experimental design and should be addressed in future work./p>0.22 µm) was generally equivalent to the 0.22–3 µm size fraction. This metabarcoding and metagenomic comparison of sampling protocols can help researchers to design their own sampling campaigns and to compare studies using different protocols. Even though we did a tremendous effort to address many different variables in protocols used by different campaigns, there is more to be tested and compared for the purpose of standardization of protocols in the future, for example, DNA extraction protocols./p>

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