bioRxiv preprint doi: https://doi.org/10.1101/579052; this version posted March 16, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 1 A draft genome sequence of the miniature parasitoid wasp, Megaphragma 2 amalphitanum 3 4 Artem V. Nedoluzhko1,2*, Fedor S. Sharko3, Brandon M. Lê4, Svetlana V. Tsygankova2, 5 Eugenia S. Boulygina2, Sergey M. Rastorguev2, Alexey S. Sokolov3, Fernando 6 Rodriguez4, Alexander M. Mazur3, Alexey A. Polilov5, Richard Benton6, Michael B. 7 Evgen'ev7, Irina R. Arkhipova4, Egor B. Prokhortchouk3,5*, Konstantin G. Skryabin2,3,5 8 *Correspondence: [email protected], [email protected] 9 10 1Nord University, Faculty of Biosciences and Aquaculture, Bodø, 8049, Norway. 11 2National Research Center “Kurchatov Institute”, Moscow, 123182, Russia 12 3Institute of Bioengineering, Research Center of Biotechnology of the Russian 13 Academy of Sciences, Moscow, 117312, Russia 14 4Josephine Bay Paul Center for Comparative Molecular Biology and Evolution, Marine 15 Biological Laboratory, Woods Hole, Massachusetts 02543 16 5Lomonosov Moscow State University, Faculty of Biology, Moscow, 119234, Russia 17 6Center for Integrative Genomics, Faculty of Biology and Medicine, Génopode 18 Building, University of Lausanne, CH-1015 Lausanne, Switzerland 19 7Institute of Molecular Biology RAS, Moscow, 119991, Russia 20 21 22 Full correspondence address: Dr. Artem V. Nedoluzhko, Nord University, Faculty of 23 Biosciences and Aquaculture, Universitetsalléen 11, 8049, Bodø, Norway. 24 Phone: +79166705595 25 bioRxiv preprint doi: https://doi.org/10.1101/579052; this version posted March 16, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 2 26 ABSTRACT 27 Body size reduction, also known as miniaturization, is an important evolutionary 28 process that affects a number of physiological and phenotypic traits and helps animals 29 to conquer new ecological niches. However, this process is poorly understood at the 30 molecular level. Here, we report genomic and transcriptomic features of arguably the 31 smallest known insect – the parasitoid wasp, Megaphragma amalphitanum 32 (Hymenoptera: Trichogrammatidae). In contrast to expectations, we find that the 33 genome and transcriptome sizes of this parasitoid wasp are comparable to other 34 members of the Chalcidoidea superfamily. Moreover, the gene content of M. 35 amalphitanum compared to other chalcid wasps is remarkably conserved. Among the 36 very rare cases of apparent gene loss is centrosomin, which encodes an important 37 centrosome component; the absence of this protein might be related to the large number 38 of anucleate neurons in M. amalphitanum. Intriguingly, we also observed significant 39 changes in M. amalphitanum transposable element dynamics over time, whereby an 40 initial burst was followed by suppression of activity, possibly due to a recent 41 reinforcement of the genome defense machinery. Thus, while the M. amalphitanum 42 genomic data reveal certain features that may be linked to the unusual biological 43 properties of this organism, miniaturization is not associated with a large decrease in 44 genome complexity. 45 bioRxiv preprint doi: https://doi.org/10.1101/579052; this version posted March 16, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 3 46 INTRODUCTION 47 Miniaturization in animals is an evolutionary process that is frequently 48 accompanied by structural simplification and size reduction of organs, tissues and cells 49 [1, 2]. The parasitoid wasp Megaphragma amalphitanum (Hymenoptera: 50 Trichogrammatidae, subfamily Oligositinae) is one of the smallest known insects, 51 whose size (250 μm adult length) is comparable with unicellular eukaryotes and even 52 some bacteria (Figure 1). Parasitoids from the genus Megaphragma parasitize 53 greenhouse thrips Heliothrips haemorrhoidalis (Thysanoptera: Thripidae) developing 54 on the shrubs Viburnum tinus (Adoxaceae) and Myrtus communis (Myrtaceae) [3], and 55 possibly Hercinothrips femoralis (Thysanoptera: Thripidae) [4]. The wasp spends most 56 of its life cycle in host eggs, while the imago stage is very short and lasts only a few 57 days [3, 4]. M. amalphitanum belongs to chalcid wasps, which represent one of the 58 largest insect superfamilies (∼23,000 described species)[5]. The higher-level taxonomic 59 relationships of Trichogrammatidae, Chalcidoidea and Hymenoptera have been 60 investigated in several recent studies [6-10] that helped to establish the placement of 61 this unique taxon that related to Mymaridae and Pteromalidae. 62 63 Figure 1. Size comparison of the parasitoid wasp M. amalphitanum and 64 bacterium Thiomargarita namibiensis. (A) An adult stage of the parasitoid wasp M. 65 amalphitanum (image adapted from [5]), (B) T. namibiensis – the largest known 66 bacterium (modified from Schulz et al. 1999) [11]. 67 68 Amongst notable anatomical features of M. amalphitanum, this species has only 69 ∼4600 neurons in its brain, which is substantially fewer than in the brains of other bioRxiv preprint doi: https://doi.org/10.1101/579052; this version posted March 16, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 4 70 wasps, e.g. the parasitoid chalcid wasp Trichogramma pretiosum (Trichogrammatidae: 71 Trichogrammatinae) (∼18,000 neurons), Hemiptarsenus sp. (Chalcidoidea: Eulophidae) 72 (∼35,000 neurons), and the honey bee Apis mellifera (Apidae) (∼850,000-1,200,000 73 neurons). Moreover, by the final stage of M. amalphitanum development, up to 95 74 percent of the neurons of the central nervous system lose their nuclei [12, 13]. 75 Nevertheless, adult wasps, which have an average lifespan of 5 days, still preserve the 76 basic functional traits of hymenopteran insects including flight, mating and oviposition 77 in hosts [14]. 78 In this study, we present a draft M. amalphitanum genome, and adult 79 transcriptome, and compare these with several parasitoid wasp species of different body 80 sizes from the Chalcidoidea and Ichneumonoidea hymenopteran superfamilies. We 81 performed a comprehensive analysis of its coding potential, including both general gene 82 ontology and pathway analyses as well as specific gene categories of interest, such as 83 chemosensory receptors, venom components etc. Additionally, we investigated 84 transposable element (TE) content and dynamics across several parasitoid wasp species 85 and analyzed the major components of the genome defense machinery. As body size 86 reduction and loss of physiological or phenotypic traits is often correlated with genome 87 size diminution [15, 16] and/or gene networks reduction [17], including chromatin 88 diminution from the somatic tissues during embryogenesis[18, 19], we initially 89 anticipated that the M. amalphitanum genome would be greatly simplified during 90 miniaturization. 91 bioRxiv preprint doi: https://doi.org/10.1101/579052; this version posted March 16, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 5 92 MATERIAL AND METHODS 93 Detailed information is presented in Supplementary Information 94 Nucleic acid extraction and library construction. M. amalphitanum individuals were 95 reared in the laboratory conditions from eggs of Heliothrips haemorrhoidalis 96 (Thysanoptera: Thripidae) collected in Santa Margherita, Northern Italy. DNA was 97 extracted from ten individuals (males and females) using NucleoSpin Tissue XS kit 98 (Macherey-Nagel, Germany) for each DNA-library. Three DNA libraries (DNA- 99 library1 – whole insects; DNA-library2 – thorax and abdomen; DNA-library3 – head) 100 were constructed using Ovation Ultralow Systems V2 kit (NuGEN, USA). Limited 101 amount of biological material and low quantity of starting material (1 – 3 ng) did not 102 permit construction of mate-paired libraries. Genome was sequenced used Illumina 103 HiSeq 1500 (Illumina, USA) with 150 bp paired-end reads. RNA was extracted from 104 ten M. amalphitanum individuals (males and females) using the Trizol reagent (Thermo 105 Fisher Scientific, USA) by a standard protocol, and cDNA libraries were constructed 106 using Ovation RNA-Seq System V2 kit (NuGEN, USA) with poly(A) enrichment. 107 Genome de novo assembly. The output from Illumina sequencing of the genomic DNA 108 library (source format *.fastq) was used for de novo genome assembly. To assemble the 109 complete genome of M. amalphitanum, we used 102,188,833 paired-end reads. Genome 110 assemblies have been constructed using different assembly algorithms, and their 111 performance was compared to each other (Figure S1). Additionally, genomic DNA- 112 libraries from thorax and abdomen (DNA-library2) of M. amalphitanum (SRR5982987) 113 and from head (DNA-library3) of M. amalphitanum (SRR5982986) were prepared. 114 Totally, 79,317,970 (paired-end sequencing: 2×100 bp) and 85,409,775 (single--end bioRxiv preprint doi: https://doi.org/10.1101/579052; this version posted March 16, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No