Ultrabithorax is essential for bacteriocyte development Yu Matsuuraa,b,c, Yoshitomo Kikuchid, Toru Miurab, and Takema Fukatsua,c,1 aBioproduction Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba 305-8566, Japan; bGraduate School of Environmental Science, Hokkaido University, Sapporo 060-0810, Japan; cGraduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba 305-8572, Japan; and dBioproduction Research Institute, National Institute of Advanced Industrial Science and Technology, Hokkaido Center, Sapporo 062-8517, Japan Edited by Nancy A. Moran, University of Texas at Austin, Austin, TX, and approved June 19, 2015 (received for review February 18, 2015) Symbiosis often entails the emergence of novel adaptive traits in transcription factors, in particular Ultrabithorax (Ubx), are involved organisms. Microbial symbionts are indispensable for diverse insects in the development of the host cells and organs specialized for via provisioning of essential nutrients, wherein novel host cells and harboring the symbiotic bacteria. organs for harboring the microbes, called bacteriocytes and bacter- iomes, have evolved repeatedly. Molecular and developmental mech- Results and Discussion anisms underpinning the emergence of novel symbiotic cells and Bacteriocyte Development During Embryogenesis of N. plebeius. We organs comprise an unsolved question in evolutionary developmental performed a detailed description of the embryogenesis of N. ple- biology. Here, we report that a conserved homeotic gene, Ultra- beius with special focus on the dynamics of the bacteriocyte- bithorax, plays a pivotal role in the bacteriocyte differentiation in a associated gammaproteobacterial symbiont “Candidatus Schneideria hemipteran insect Nysius plebeius. During embryonic development, nysicola” (18) (Figs. 1 and 2, Table S1,andMovie S1). The sym- six pairs of aggregated presumptive bacteriocytes appear on both biotic bacteria were found as an aggregate at the anterior pole of sides of six abdominal segments, incorporate the symbiotic bacteria newly laid eggs (Fig. 1 A and B). After blastoderm formation at the stage of germband retraction, and fuse into a pair of lateral (12–24 h after oviposition; Fig. 2A) and germband elongation bacteriomes at the stage of germband flip, where bacteriocyte-asso- (24–33 h; Fig. 2 B–D), the symbionts were wrapped within ab- ciated Ultrabithorax expression coincides with the symbiont infec- dominal segments of the germband (36–60 h; Figs. 1 I and J and 2 tion process. Suppression of Ultrabithorax expression by maternal E–G). After germband retraction (∼72 h; Fig. 2H), the symbionts RNA interference results in disappearance of the bacteriocytes and migrated from the abdominal population to presumptive bacter- EVOLUTION the symbiont localization therein, suggesting that Ultrabithorax is iocytes that appeared on both sides of abdominal segments A2–A7 involved in differentiation of the host cells for symbiosis. Suppression as six pairs of clusters (72–84 h; Figs. 1 K and L and 2 I and J). of other homeotic genes abdominal-A and Antennapedia disturbs Then, during the process of drastic embryonic flip called katatrepsis, integrity and positioning of the bacteriomes, affecting the configura- the six bacteriocyte clusters on each side fused into a coherent tion of the host organs for symbiosis. Our findings unveil the molec- bacteriome located at abdominal segments A2–A4 (84–96 h; ular and developmental mechanisms underlying the bacteriocyte Figs. 1 M and N, and 2 K–M and Q–R). After the symbiont in- differentiation, which may have evolved either via cooption of the fection, the bacteriocytes accumulated red pigment and became transcription factors for inducing the novel symbiotic cells, or via re- easily recognizable (∼84 h; Figs. 1 C and 2 R–T). The red pig- vival of the developmental pathway for the bacteriocytes that had mentation provided a visible marker useful for tracing the existedintheancestralhemipterans. bacteriocytes, although it is unclear whether the red pigment was derived from the symbiont or the host. bacteriocyte | homeotic gene | transcription factor | evolution | symbiosis Significance ymbiosis is the source of novel adaptive traits, thereby con- Stributing to organismal evolution and diversification (1, 2). A Among the most fundamental questions in developmental bi- variety of insects are obligatorily dependent on microbial sym- ology is how novel cell types have emerged in the metazoan bionts via provisioning of essential nutrients lacking in their diets evolution. Among the most challenging questions in evolu- (3, 4), wherein novel host cells and organs for harboring the mi- tionary biology is how sophisticated symbiotic associations crobes, called bacteriocytes and bacteriomes, have evolved repeat- have evolved through less intimate interorganismal in- edly in such insect groups as hemipterans (aphids, whiteflies, – teractions. These fundamental biological issues are crystalized mealybugs, leafhoppers, spittlebugs, etc.) (5 9), dipterans (tsetse in the evolution and development of insect’s bacteriocytes flies, bat flies, etc.) (10, 11), coleopterans (weevils, etc.) (12, 13), specialized for harboring symbiotic bacteria. Here, we report and many others (14). Despite a considerable body of embryo- that a conserved transcription factor Ultrabithorax is essential logical descriptions (14), molecular mechanisms underlying the for bacteriocyte development in an insect, thereby uncovering bacteriocyte differentiation have been a long-lasting enigma in a molecular mechanism underlying the emergence of the novel evolutionary developmental biology (15, 16). Although cellular host cells for symbiosis. Our finding highlights the importance and developmental aspects of the bacteriocyte formation have of developmental cooption of preexisting transcription factors Acyrthosiphon pisum been best documented for the pea aphid, and sheds new light on a long-lasting enigma in evolutionary Nysius plebeius (5, 15, 17), the seed bug and allied heteropteran developmental biology. bugs of the superfamily Lygaeoidea have recently emerged as a promising model system for investigating the development, evo- Author contributions: Y.M. and T.F. designed research; Y.M., Y.K., and T.M. performed lution, and origin of the bacteriocytes, on the grounds that research; Y.M. analyzed data; and Y.M. and T.F. wrote the paper. (i) heteropteran bugs are generally associated with gut symbiotic The authors declare no conflict of interest. bacteria without bacteriocytes; (ii) thus, the bacteriocytes in these This article is a PNAS Direct Submission. lygaeoid species are regarded as a novel trait whose evolution was Freely available online through the PNAS open access option. iii N. plebeius a relatively recent event (18, 19); and ( )in and allied Data deposition: The nucleotide sequences determined in this study have been deposited lygaeoid species, RNA interference (RNAi) works efficiently, in the DNA Data Bank of Japan database (accession nos. LC010622–LC010625). which enables functional analysis of genes involved in the bac- 1To whom correspondence should be addressed. Email: [email protected]. teriocyte formation (20, 21). Here we demonstrate that, by making This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. use of the emerging model insect N. plebeius, conserved homeobox 1073/pnas.1503371112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1503371112 PNAS Early Edition | 1of6 Downloaded by guest on September 30, 2021 Fig. 1. Symbiont localization and bacteriocyte differentiation during the development of N. plebeius.(A and B) Newly laid eggs. (C and D) Embryos 5 d after oviposition. (E and F) First-instar nymphs. (G and H) Adult insects. (I and J) Embryos ∼48 h after oviposition. (K and L) Embryos of 72–84 h. (M and N) Embryos of ∼96 h. (O and P) Embryos of ∼120 h. A, C, E, and G are light-microscopic images; I, K, M, and O are schematic illustrations of symbiont localization (red) in the embryos; and B, D, F, H, J, L, N,andP are fluorescence microscopic images in which blue and green signals show the host nuclei and the symbiotic bacteria, respectively. Arrowheads depict bacteriocytes, and arrows indicate aggregated symbionts within the embryos. A1, A2, and A3, first, second, and third abdominal segments, respectively; ob, ovarial bacteriocytes in adult female; T3, third thoracic segment. Bacteriocyte-Associated Expression of Ubx in Embryogenesis of abdominal segments A2–A7 after the germband retraction (Fig. 3 N. plebeius. The homeobox genes encode transcription factors B–D and Fig. S1D), which agreed with the locations of the pre- that assign segment identities and specify functional body parts sumptive bacteriocytes (Figs. 1 K and L and 2J). Meanwhile, abd-A in the development of insects and other animals (16, 22). In the was expressed broadly across the abdominal segments without embryogenesis of the pea aphid A. pisum, some homeobox gene specific association with the presumptive bacteriocytes (Fig. 3 E products—including an appendage-patterning transcription fac- and F and Fig. S1 H–J). tor Distal-less (Dll), homeotic proteins Ubx or Abdominal-A (Abd-A), and a segment polarity protein Engrailed (En)—were Disappearance of Bacteriocytes by Ubx Suppression. In heteropteran shown to localize to the bacteriocytes, although their functions in species including O. fasciatus, injection of double-stranded RNA the symbiotic cells have been