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1、The locust genome provides insight into swarmformation and long-distance flight蝗虫的基因组对集群和长途飞行提供了见解Locusts are one of the worlds most destructive agricultural pests and represent a useful model system in entomology. Here we present a draft 6.5 Gb genome sequence of Locusta migratoria, which is the la
2、rgest animal genome sequenced so far. Our findings indicate that the large genome size of L. migratoriais likely to be because of transposable element proliferation combined with slow rates of loss for these elements. Methylome and transcriptome analyses reveal complex regulatory mechanisms involved
3、 in microtubule dynamic-mediated synapse plasticity during phase change. We find significant expansion of gene families associated with energy consumption and detoxification, consistent with long-distance flight capacity and phytophagy. We report hundreds of potential insecticide target genes, inclu
4、ding cys-loop ligand-gated ion channels, G-protein-coupled receptors and lethal genes. The L. migratoria genome sequence offers new insights into the biology and sustainable management of this pest species, and will promote its wide use as a model system.蝗虫是一个世界上最具破坏性的农业害虫,并且在昆虫学中代表一个有用的模式系统。在这里，我们提
5、出了东亚飞蝗6.5 Gb的基因组序列的草案，这是迄今为止序列最长的动物基因组。我们的研究结果表明，东亚飞蝗的大基因组可能是因为转座子的增加再加上这些基因缓慢的丢失速率。甲基化和转录组分析表明状态变化过程中涉及的微管动态介导突触的可塑性这一机制是一个复杂的调控机制。我们发现了具有远距离飞行能力和植食性的与能源消耗和解毒相关的基因家族的显著扩张。我们报道了很多有关潜在杀虫剂的靶基因，包括半胱氨酸型配体门控离子通道， G蛋白偶联受体和一些致死基因。东亚飞蝗基因组序列为生物学和可持续管理提供了关于这种害虫物种的新的见解，并会促进其作为一个模式系统的广泛使用。Since the dawn of agra
6、rian civilization, locust plagues have been viewed as one of the most devastating natural disasters, linked with famine, strife and dissolution of societal order as documented in the Bible, the Quran and Chinese historical records 1,2. Unfortunately, locust plagues continue to cause destruction even
7、 today. For example, in 1988, locust swarms covered an enormous area of some 29 million square kilometres, extending over or into parts of up to 60 countries, resulting in billions of dollars in economic losses 3. The primary current method for combating locust outbreaks is through intensive sprayin
8、g of chemical pesticides; however, their overall usefulness has been widely debated because of their highly negative impact on human and environmental health, and on biological diversity 2.由于农业文明的曙光，蝗灾已被视为最具破坏性的自然灾害之一，伴随着有饥荒，内乱以及社会秩序解体，这些都在圣经、古兰经和中国的历史中有所记载。不幸的是，蝗灾即使在今天也在造成不断的破坏。例如，在1988年，蝗虫群覆盖了约29万
9、平方公里的巨大区域，延伸或到达了60个国家的部分地区，造成数十亿美元的经济损失。目前防治蝗虫爆发基本的方法是通过化学农药密集喷洒的方式；然而，他们的整体实用性得到了广泛的争论，因为它们对人类和环境的健康以及对生物的多样性都有非常不利的影响。Locusts are grasshopper species with swarming and longdistance migratory behaviours 1. Locust swarms form suddenly and unpredictably through the congregation of billions of insects
10、, which can fly hundreds of kilometres each day, and even cross oceans 4. They are polyphagous and a single individual consumes its own body weight in food in 1 day; this is, proportionately,60100 times a humans daily consumption 1. Locusts are characterized by a density-dependent phase polyphenism,
11、 which involves a variety of biological and phenotypic traits, including changes in body colour, morphology, behaviour, physiology, immune responsiveness and others 59. An increase in population density triggers the transformation from a well-hidden solitarious phase to an overtly noticeable gregari
12、ous phase,which results in an extensive aggregation of individuals 1. This phase change occurs quickly and reversibly, and its speed varies among species 10. The entire transformation process of phase change pivots around a behavioural change, which has been proposed to be regulated by the periphera
13、l and central nervous system (CNS) 1113.蝗虫是具有集群和长途迁徙行为的蝗虫物种。蝗虫集群非常突然和不可预料，在数十亿的昆虫中，他们可以每天飞行几百公里，甚至横跨大洋。他们是杂食性的，并且一个单独的个体一天中消耗自己的体重来提供能量；这些能量相当于人类日常消耗量的60- 100倍。蝗虫具有密度制约的非遗传多型性特征，它拥有多样性的生物和表型性状，包括体色的变化、形态、行为、生理、免疫反应和其他一些性状。种群密度的增加触发了蝗虫从一个很好的隐蔽性独居状态转变到一个公开的明显的群居状态，从而导致了个体的大量聚集。这个状态变化发生得非常快并且是可逆的，其速度在不
14、同物种之间也有所不同。整个状态转变过程的核心是行为的改变，这已经被认为是由外周和中枢神经系统（CNS ）调控的。The migratory locust,Locusta migratoria, is the most widespread locust species and has long served as a model organism for insect morphology, behaviour and physiological research 1416. Here we provide a draft genome sequence of L. migratoria, t
15、he largest animal genome sequenced so far. We assess changes of gene families related to long-distance migration, feeding and other biological processes unique to the locust and identify genes that might serve as potential pesticide targets. Combining a set of transcriptome and methylome data from g
16、regarious and solitarious locusts, we reveal potential neuronal regulatory mechanisms underlying phase change in the locust. 飞蝗属的东亚飞蝗，是最普遍的蝗虫物种并长期作为昆虫形态学研究形态和生理的模式生物。在这里，我们提供了东亚飞蝗的基因组序列草图，是迄今最大的动物基因组序列。我们评估了基因家与长途迁徙，觅食和其他蝗虫特有的生物过程的变化和可能成为潜在的杀虫剂的目标基因，。结合一组来自群居和独居的昆虫的转录组和DNA甲基化的数据，我们揭示了蝗虫潜在的以神经调节机制为基础
17、的状态变化。ResultsGenome assembly. We sequenced the genome of a single, eight generation inbred female individual of L. migratoria, worldwide distributed agricultural pests (Supplementary Fig. S1), using the Illumina HiSeq 2000 sequencing platform. After quality control and filtering, 721 Gb of data were
18、 generated, covering 114of the 6.3 Gb L. migratoria genome size as estimated by k-mer analysis and flow cytometry (Supplementary Tables S1 and S2, and Supplementary Figs S2 and S3). We used SOAPdenovo 17 to perform denovo assembly, achieving a final assembly of 6.5 Gb with a length-weighted median (
19、N50) contig size of 9.3 kb and scaffold N50 of 320.3 kb (Supplementary Table S3). A genetic map containing 11 major linkage groups was further developed based on 8,708 markers using restriction-site-associated DNA sequencing data (Fig. 1a).结果 基因组组装。我们用Illumina公司的HiSeq2000测序平台测序了全世界分布的农业害虫（补充图S1）东亚飞蝗
20、的八次世代交配的雌性个体的基因组。经过质量监控和过滤，生成了721 GB的数据，包括通过K-MER分析和流式细胞术（补充表S1和S2，以及补充图S2和S3）所估算出的东亚飞蝗的基因组大小为1146.3 Gb。我们使用SOAPdenovo17来从头测序组装序列，得到一个6.5 Gb的最终组装序列，它是一个长度加权中值大小为9.3 kb的连续片段（N50），还有一条320.3 KB（补充表S3）的非连续N50片段。一个包含11个主要连锁群的遗传图谱基于8,708个使用了限制性内切位点相关的DNA测序数据的标记（图1a）得到进一步的开发。The total heterozygosity rate o
21、f the L. Migratoria genome, which is the portion of heterozygous single-nucleotide polymorphisms between the two haploid components in the diploid genome, was estimated to be 1.1510-3. We also observed a heterogeneous distribution of heterozygosity rates (Fig. 1a, track b), which possibly resulted f
22、rom inbreeding 18. The possible reasons are the existence of alleles, which are homozygous lethal and the variation of recombination rates between different genomic regions 19,20. We assessed the integrity and quality of the genome assembly using multiple evaluation methods (Supplementary Figs S4 an
23、d S5, and Supplementary Tables S4S7). On the basis of these analyses, our assembly provides a good representation of the L. Migratoria genome and is of suitable quality for subsequent analysis.东亚飞蝗基因组中总的杂合率，是在二倍体基因组中的2个单倍体组件之间杂合的单核苷酸多态性的一部分，估计为1.15103，我们还观察到了杂合率的不均匀分布（图1a，轨道b），其中可能由于近亲繁殖造成。可能的原因是等位基
24、因的存在，这是纯合致死和不同的基因组区域之间的重组率的变化。我们使用多种评估方法（补充图S4和S5，并补充表格中S4S7）来评估基因组组装的完整性和质量。在这些分析的基础上，我们的组装为东亚飞蝗基因组提供了一个很好的示例，并为后续进行的分析提供了适当的质量。Genome annotation and evolutionary analysis. We annotated the genome and predicted 17,307 gene models by combining denovo prediction and evidence-based searches using four
25、 sequenced insect reference gene sets (Drosophila melanogaster,Apis mellifera, Acyrthosiphon pisumand Pediculus humanus),L. migratoria expressed sequence tags (ESTs) and RNA-seq data from transcriptomes that we created previously and for this work from multiple organs and developmental stages (Suppl
26、ementary Methods and Supplementary Table S8). We found that 93.8% of gene models showed expression (reads per kb per million mapped reads 41 in at least one sample). Of the inferred proteins, 74.9% matched entries in the NR, SWISS-PROT, InterPro or TrEMBL databases (Supplementary Table S9). 基因组注释和进化
27、分析。我们注释了基因组，并通过结合denovo测序和基于证据的调查，这个调查是使用了四个已经测好序的昆虫的对照基因组（果蝇，意大利蜜蜂，蚜虱）和东亚飞蝗的表达序列标签（EST），以及我们从多个器官和在发育阶段之前为这项工作制成的转录组中的RNA序列发生的数据（RNA定序数据的补充方法和补充表S8），从而预测出了17,307个基因模型。我们发现93.8的基因模型，出现了表达（一个样品中每kb，每百万标记中至少能读出41个）。推断出来的蛋白质，在NR，SWISS-PROT，InterPro或TrEMBL等数据库（补充表S9）中有74.9匹配条目。We identified over 2,639 r
28、epeat families using Repeat Modeler; however, the top ten repeat families only represented 10% of the total genome sequences, indicating there were no dominant families in the L. migratoria genome (Supplementary Tables S10 and S11). The LINE RTE/BovB family, which has been documented in many species
29、 as having been acquired through an ancient lateral gene transfer event 21, is the most prevalent repeat family (244 Mb, 4.05%) in the L. Migratoria genome, and it may be still active as indicated by the presence of many fulllength copies and intact protein domains 22.我们证实了超过2,639个重复的家族采用了重复建模;不过，前十
30、个重复家族只表达了全部基因组序列的大约10，这表明在东亚飞蝗的基因组中并没有优势家族（补充表S10和S11）。LINE RTE/BovB家庭，这已经被记录在许多通过古老的横向基因转移事件而获得的物种中，是东亚飞蝗基因组中最普遍的重复家族（244 MB，4.05）。正如许多全长的克隆和完整的蛋白结构域的存在，它或许仍然是活跃的。The proliferation of a diverse range of repetitive elements is the main reason for the large size of the L. migratoria genome. Repetitiv
31、e elements constituted 60% of the assembled genome, of which DNA transposons (24%) and LINE retrotransposons(17%) were the most abundant elements (Fig. 1a, track c,Supplementary Note 1 and Supplementary Table S10). Among all transposons, DNA transposon exhibits the lowest divergent copies (Fig. 1a,
32、track d), which reflects their most recent invasions in the locust genome. On the basis of homologous regions of a conserved and syntenic Osiris gene family among insect orders 23, we compared genomic components of L. migratoria with that of D. melanogaster(Supplementary Fig. S6). The length of codi
33、ng region has no difference, but the length of intronic, intergenic regions and repetitive elements in the L. migratoriagenome is much larger than that of D. melanogaster(Fig. 1c). 重复元素的多元化的扩散是东亚飞蝗的基因组比较大的主要原因。重复元素组成大约60%的组装基因组，其中DNA转座子（大约24）和LINE反转录转座子（17）是最丰富的元素（图1a，跟踪C，补充说明1和补充表S10）。在所有转座子中， DNA转
34、座子表现出最低的克隆分歧（图1a，跟踪D），这反映了他们最近入侵的蝗虫基因组。在昆虫目中保守的同一染色体上的奥西里斯基因家族的同源区域的基础上，我们比较了东亚飞蝗和黑腹果蝇的基因组成分（补充图六）。编码区的长度没有任何区别，但是内含子，基因间隔区和重复序列的长度在东亚飞蝗基因组中比黑腹果蝇中要大得多（图1c）。We compared the L. migratoria DNA deletion rates with that of five other insect genomes (D. melanogaster, Anopheles gambiae, Bombyx mori, A. pis
35、umand Aedes aegypti) focusing on long terminal repeat retrotransposons, because these have neutral decay rates and easily identified complete structures24.We observed a positive correlation between DNA deletion and divergence of long terminal repeat copies (Po0.01, Pearsons correlation tests). The L
36、. Migratoria genome exhibits the lowest rate of DNA deletions relative to the other insects (Po0.01, Wilcoxon tests; Fig. 1d). These results are consistent with the previous reports and support the hypothesis that slow DNA loss contributes to genomic gigantism in animals 25.我们比较了东亚飞蝗与五个其他昆虫基因组的DNA缺失
37、率（黑腹果蝇，冈比亚按蚊，家蚕，埃及伊蚊）专注于长末端重复序列反转录转座子，因为这些具有中性衰减率并且完整结构容易被鉴定。我们观察到DNA缺失和长末端重复序列的发散呈正相关关系（Po0.01，Pearson相关性检验）。东亚飞蝗基因组与其他昆虫相比呈现出最低的DNA缺失率（Po0.01，威尔科克森检验;图1d）。这些结果与之前的报道一致，并支持了低的DNA缺失率会导致动物基因巨大症的假设。The average intron length in the L. migratoria genome was 11,159 bp, which is 10 times longer than the a
38、verage intron size of other insects and twice that of the average size in Homo sapiens (Supplementary Table S8 and Supplementary Fig. S7). Pairwise comparison of intron sizes in nine insect species using one-to-one orthologous genes provided further support for this average size difference (Suppleme
39、ntary Fig. S8 and Supplementary Table S12). We also carried out a genome-wide comparison of intron and genome size of 73 sequenced animal species and found that there was a positive correlation between average intron size and genome size (Po0.01, Pearsons correlation tests; Supplementary Fig. S9). T
40、he increased intron size in the L. Migratoria genome compared with the other insects may partly be attributed to transposable element invasion (Supplementary Fig. S10). Relative to this, we compared several other intron characteristics of the L. migratoria genome with other animal species (Supplemen
41、tary Note 1) and found that the U12-type intron (minor-class intron) number and the ratio of ratcheting point sites of L. Migratoria are similar to those in vertebrates rather than insects (Supplementary Figs S11 and S12). Previous reports have indicated that most insects have an enrichment of ratch
42、eting point sites to allow for efficient splicing of long introns, whereas vertebrates use repetitive elements to aid in splicing long introns 26. Our data here indicate that there may be convergent evolution of the splicing mechanisms associated with genome size expansion in animals.在东亚飞蝗基因组中平均内含子长
43、度为11,159 bp，比其他昆虫的平均内含子长10倍多，是智人平均内含子长度的两倍（补充表S8和补充图七）。通过对9种昆虫内含子一对一的同源基因进行两两比较，为内含子平均大小差异提供了进一步的支持（补充图。 S8和补充表S12），。我们还进行了内含子和73个序列的动物基因组大小的全基因组比较，发现内含子的平均大小和基因组大小（ Po0.01 ， Pearson相关性检验;补充图S9）呈正相关。东亚飞蝗基因与其他昆虫相比内含子大小有所增加，这可以部分归因于转座元件的插入（补充图S10）。相对于这一点，我们比较了东亚飞蝗基因组与其他动物物种的一些其它内含子特征（补充说明1），发现东亚飞蝗中U12
44、型内含子（次要级的内含子）数目和棘轮位点的比率类似于脊椎动物，而不是昆虫（补充图S11和S12 ）。之前的报告表明，大多数昆虫有丰富的棘轮位点，以便于长的内含子的高效拼接，而脊椎动物使用重复序列来帮助拼接长的内含子。在这里我们的数据表明，剪接机制的趋同进化可能与动物基因组大小扩大有关。We used a maximum likelihood method for genome-scale phylogenetic analysis using 122 single-copy genes from 10 sequenced arthropod genomes (Fig. 1b and Suppl
45、ementary Fig. S13). The phylogenetic analysis revealed that the locust is the basal taxon for the other insects sequenced so far, and supports the paraphyletic status of the hemimetabolous insect species. Given the distinct developmental differences between hemimetabolous and holometabolous insects,
46、 we identified metamorphosis-specific gene sets, of which a large number of genes were related to the regulation of developmental processes(Supplementary Table S13). A gene gain/loss analysis of these insect genomes showed a gain of about 55 new gene families in the lineage leading to L. migratoria(
47、Supplementary Fig. S13).Twenty-five significantly expanded gene families in the L. migratoria genome were mainly involved in detoxification,chemoreception, chromosome activity and nutritional metabolism, indicating unique adaptation features of the L. migratoria genome (Supplementary Table S14).我们使用
48、了最大似然法用122个单基因克隆从10个序列的节肢动物基因组来测序基因组进行系统发育分析（图1b和补充图S13 ）。系统发育分析表明，蝗虫是迄今测序的昆虫中基础的类群，并支持了蝗虫是半变态昆虫的并系种。鉴于半变态和完全变态的昆虫之间的明显的发展性的差异，我们确定了基因组的特异性变态，其中大量基因均与发育过程的调节有关（补充表S13）。这些昆虫基因组的基因增加/缺失分析显示约55个新基因家族的增加导致了东亚飞蝗的发生（补充图13）。东亚飞蝗基因组中二十五个显著扩增的基因家族主要有解毒，化学感受，染色体的活性和营养代谢的功能，表明东亚飞蝗基因组独特的适应性特征（补充表S14） 。Phase cha
49、nge analysis. Phase change is the defining characteristic of locust biology because of its critical roles in swarm formation (Fig. 2a) 5. As a fascinating type of phenotypic plasticity, phase change is required to achieve biological complexity at all levels of biological organization, from molecules to large aggregation. Recent studies have highlighted the importance of DNA methylation for understanding insect phenotypic plasticity and biological complexity 2729. To investigate the potential involvement
