Yin et al. BMC Genomics (2018) 19:420 https://doi.org/10.1186/s12864-018-4783-x RESEARCH ARTICLE Open Access The genomic features of parasitism, Polyembryony and immune evasion in the endoparasitic wasp Macrocentrus cingulum Chuanlin Yin1†, Meizhen Li1†, Jian Hu2*†, Kun Lang1, Qiming Chen2, Jinding Liu3,4, Dianhao Guo1,4, Kang He1, Yipei Dong2, Jiapeng Luo1, Zhenkun Song2, James R. Walters5, Wenqing Zhang2*, Fei Li1* and Xuexin Chen1 Abstract Background: Parasitoid wasps are well-known natural enemies of major agricultural pests and arthropod borne diseases. The parasitoid wasp Macrocentrus cingulum (Hymenoptera: Braconidae) has been widely used to control the notorious insect pests Ostrinia furnacalis (Asian Corn Borer) and O. nubilalis (European corn borer). One striking phenomenon exhibited by M. cingulum is polyembryony, the formation of multiple genetically identical offspring from a single zygote. Moreover, M. cingulum employs a passive parasitic strategy by preventing the host’s immune system from recognizing the embryo as a foreign body. Thus, the embryos evade the host’s immune system and are not encapsulated by host hemocytes. Unfortunately, the mechanism of both polyembryony and immune evasion remains largely unknown. Results: We report the genome of the parasitoid wasp M. cingulum. Comparative genomics analysis of M. cingulum and other 11 insects were conducted, finding some gene families with apparent expansion or contraction which might be linked to the parasitic behaviors or polyembryony of M. cingulum. Moreover, we present the evidence that the microRNA miR-14b regulates the polyembryonic development of M. cingulum by targeting the c-Myc Promoter-binding Protein 1 (MBP-1), histone-lysine N-methyltransferase 2E (KMT2E) and segmentation protein Runt. In addition, Hemomucin, an O-glycosylated transmembrane protein, protects the endoparasitoid wasp larvae from being encapsulated by host hemocytes. Motif and domain analysis showed that only the hemomucin in two endoparasitoids, M. cingulum and Venturia canescens, possessing the ability of passive immune evasion has intact mucin domain and similar O-glycosylation patterns, indicating that the hemomucin is a key factor modulating the immune evasion. Conclusions: The microRNA miR-14b participates in the regulation of polyembryonic development, and the O-glycosylation of the mucin domain in the hemomucin confers the passive immune evasion in this wasp. These key findings provide new insights into the polyembryony and immune evasion. Keywords: Macrocentrus cingulum, Genome, Polyembryony, Immune evasion, Comparative genomics * Correspondence: [email protected]; [email protected]; [email protected] †Chuanlin Yin, Meizhen Li and Jian Hu contributed equally to this work. 2State Key Laboratory of Biocontrol, Sun Yat-sen University, 135 Xingang Road West, Guangzhou 510275, China 1Ministry of Agriculture Key Lab of Molecular Biology of Crop Pathogens and Insects, Institute of Insect Science, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058, China Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Yin et al. BMC Genomics (2018) 19:420 Page 2 of 18 Background infected by other organisms [11]. However, in seeming Parasitoid wasps are a group of hymenopteran insects contrast with these distinctly antagonistic parasitic tactics, that parasitize the eggs, larvae or pupae of other arthro- M. cingulum employs a more passive parasitic strategy pods [1]. These wasps differ from other parasitic organ- (Fig. 1)[12]. Parasitism substantially retards development isms because they kill their host, and the adult wasps are of the Ostrinia host, ultimately allowing the adult wasps free-living. Because of their host specificity and lethality, to emerge and kill the host before it pupates (Fig. 1a). parasitoid wasps provide an important and effective While growing in the host hemolymph, M. cingulum em- strategy for the biological control of agricultural pests, bryos express surface features that prevent the host’sim- thus reducing the need for chemical pesticides [2, 3]. mune system from recognizing the embryo as a foreign Additionally, short generation times, ease of rearing, and body, thus the embryos evade the host’s immune system interfertile species are characteristics that make at least and are not encapsulated by host hemocytes (Fig. 1b)[12]. some parasitoid wasps highly tractable genetic model Available evidence suggests Hemomucin, an O-glycosylated systems [4]. This is exemplified by Nasonia vitripennis, transmembrane protein on the egg and embryo’ssurfaceof which was the first parasitoid wasp genome to be se- M. cingulum, may protect the endoparasitoid wasp from quenced, laying the foundation for genomic research in being encapsulated by host hemocytes [13, 14]. When the this economically and ecologically significant group of expression of hemomucin was decreased by RNAi, more insects [5]. embryos were encapsulated relative to the control [13]. One striking phenomenon exhibited by numerous Similar results were observed after the embryos were parasitoid wasp species is polyembryony, the formation digested by O-glycosidase, which may specifically digest of multiple genetically identical offspring from a single β-gal (1 → 3) linkages between GalNAc and Ser/Thr of the zygote. Polyembryony appears in only some parasitic mucin domain in hemomucin, indicating the important species within four families of Hymenoptera and one role of the mucin domain in hemomucin [13]. Despite species of Strepsiptera in insects, and is believed to have these initial insights, the mechanism of immune evasion re- evolved independently at least four times among parasit- mains largely unknown. oid wasps [6]. This includes the endoparasitic wasp With the aim of further investigations of polyembry- Macrocentrus cingulum, for which some details of ony and immune evasion, along with other studies of polyembryonic development have been described [6, 7]. wasp biology, we present the draft genome of M. M. cingulum usually deposits egg(s) into the larval cingulum. Comparative genomic analyses indicated that hemocoel of corn borer, Ostrinia furnacalis or O. nubi- parasitic behavior has shaped the endoparasitoid wasp lalis. Subsequently, the egg cleaves into several dozens genome and induced significant gene gain and loss asso- of primary embryonic cells, one of which may further ciated with parasitism. RNA-Seq analysis of the primary develop into a morula containing dozens of secondary and secondary embryonic cells revealed that microRNA embryonic embryos (SEE). SEE may develop into an em- (miRNA) miR-14b was abundant in the primary embry- bryo, which developed into a larva, or a pseudogerm, onic cell. Target prediction and dual-luciferase assay val- which was finally consumed by hatched larvae [8]. Pro- idation suggested that miR-14b plays critical roles in liferation of embryos are mainly related to the egg cleav- polyembryonic development. Comparative analyses of age and the formation of morula. Notably, these wasp hemomucin genes found that the presence of a meticulous physical observations of polyembryonic de- mucin domain and its glycosylation was positively asso- velopment have not yet been complemented with mo- ciated with immune evasion in parasitoid wasps. lecular analyses. Thus, there remains tremendous opportunities for investigating the molecular mecha- Results nisms underlying the developmental complexity of Genome sequencing, assembly and annotation polyembryony. We sequenced the genome of M. cingulum from ~ 1000 Beyond polyembryony, parasitoid wasps exhibit a range male wasps of an inbred strain which was maintained by of other distinct and noteworthy traits that evolved as sibling mating for five generations. Three paired-end li- strategies to manipulate their host. For instance, many braries with different insert sizes (170 bp, 500 bp, species introduce various parasitoid-associated factors 800 bp) and one mate-pair libraries with insert size 8 Kb such as venom and polydnaviruses (PDV) into the host (Additional file 1: Table S1), were constructed and se- during oviposition to block the host immune re- quenced using Illumina HiSeq 2000 platform, yielding sponses [9, 10]. Another example is the production of 103.67 Gb of raw data. After removing the low-quality teratocytes from cellular membranes after the eggs reads, 93.24 Gb of clean data were obtained, covering ~ hatched. The teratocytes are released into the hemolymph 690 X of M. cingulum genome which was estimated to be of the host to inhibit melanization and to produce 135 Mb by 17 K-mer analysis (Additional file 1:FigureS1, anti-microbial peptides to protect the host from being Additional file 1: Table S2) and flow cytometry Yin et al. BMC Genomics (2018) 19:420 Page 3 of 18 Fig. 1 The life cycle and living strategies of M. cingulum. a The wasp M. cingulum parasitizes the larvae of Asian corn borer Ostrinia