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Home , Buchnera (bacterium), Vertical transmission

Cellular mechanism for selective vertical PNAS PLUS of an obligate insect symbiont at the – embryo interface

Ryuichi Kogaa, Xian-Ying Menga, Tsutomu Tsuchidaa,b, and Takema Fukatsua,1 aBioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba 305-8566, Japan; and bFrontier Research Core for Life Sciences, University of Toyama, Toyama 930-8555, Japan AUTHOR SUMMARY

Many insects harbor in onstrated that the obligate sym- their cells and tissues that in- A B C biont is transmitted fluence the functioning of vari- from maternal to

D

Embryonic ous processes, which are termed syncytium early embryos (termed blastulae, bacterial symbionts. Facultative which are hollow spheres of bacterial symbionts, such as cells), whereas the facultative Wolbachia, are found in a di- symbiont Serratia is not trans- verse range of insects, and are mitted via the route. This in- parasitic or conditionally bene- formation was obtained from ficial. Such bacteria tend to have symbiont-manipulated strains of negative effects on their hosts, A. pisum whose infections with and infect a broad range of host Buchnera and Serratia were ex- cells and tissues (1). In compar- Fig. P1. Schematic illustrations of the processes perimentally manipulated under ison, obligate bacterial sym- of the obligate symbiont Buchnera (green) and the facultative specific host genotypes. The bionts, such as Buchnera in symbiont Serratia (orange) in the pea A. pisum (yellow). (A) outcome of this analysis indi- , are mutualistic. These Cellular localization of Buchnera in uninucleate primary bacteriocytes cates that a specific mechanism fi and Serratia in syncytial secondary bacteriocytes and tiny sheath bacteria contribute to the tness cells. (B) Transmission routes of Buchnera and Serratia to an embryo is involved in the Buchnera of their hosts, and are localized at the blastula stage. (C) Exo/endocytotic transmission processes of transmission. EM observations in specialized host cells called Buchnera and Serratia at the bacteriocyte–blastula interface. revealed a series of exo-/endo- bacteriocytes (2). In general, cytotic (i.e., cellular excreting these insect symbionts are stably and engulfing) processes oper- maintained through host generations by vertical transmission ating at the bacteriocyte–blastula interface. Namely, Buchnera from mothers to their offspring via the ovarial (i.e., reproductive) cells are exocytosed from the maternal bacteriocyte, temporarily passage. Vertical transmission is pivotal for the maintenance of released to the extracellular space, and endocytosed by the such intimate host–symbiont associations; however, our under- posterior syncytial cytoplasm of the blastula (Fig. P1C). Mean- standing of the molecular and cellular mechanisms underlying while, extracellular Serratia cells are also endocytosed by the this process remains quite limited (1–5). syncytial cytoplasm of the blastula; however, unlike Buchnera

In the pea aphid , the obligate symbiont transmission, a spatially regulated exocytotic process does not Buchnera is located in specialized cells called bacteriocytes, occur in Serratia transmission (Fig. P1C). where the bacterium provides the host with essential amino acids These results indicate that selective Buchnera transmission and other nutrients that are lacking in the aphid diet of plant probably arises as a result of Buchnera-specific exocytosis by the phloem sap (2). In addition to Buchnera, the pea aphid may be maternal bacteriocyte, whereas both Buchnera and Serratia are associated with various facultative bacterial symbionts, such as nonselectively incorporated by the endocytotic activity of the Serratia, Hamiltonella, and Regiella. These bacteria are not es- posterior region of the blastula. Regarding the transmission sential, but are conditionally beneficial to the host depending on process of Buchnera to early embryos, previous histological various ecological conditions: for example, they confer toler- studies have proposed various hypotheses, such as “free symbi- ance to heat stress, resistance to parasites and , and ont infection” wherein bacterial cells circulating with the stream broadened food plant ranges (2). The pea aphid provides a model of hemolymph are trapped by a blastula (3), “membranous obligate symbiotic system for which host and symbiont genomic conduit formation” wherein bacterial cells are transported from data are available; however, the mechanisms underlying ver- a maternal bacteriocyte to a blastula through a membranous tical symbiont transmission remain poorly understood except for conduit connecting the bacteriocyte and the blastula (3, 4), and some morphological/cytological aspects (2–5). “symbiont packet fusion” wherein small membranous packets Here, we demonstrate a previously unknown cellular mecha- containing bacterial cells work as “vehicles” for transmission of nism for selective symbiont transmission operating at the bac- teriocyte–embryo interface of A. pisum. In the aphid body, the obligate symbiont Buchnera is harbored within uninucleate (i.e., Author contributions: R.K. and T.F. designed research; R.K., X.-Y.M., and T.T. performed research; R.K. analyzed data; and R.K. and T.F. wrote the paper. with a single nucleus per cell) primary bacteriocytes, whereas fl the facultative symbiont Serratia is present within syncytial (i.e., The authors declare no con ict of interest. with multiple nuclei per cell) secondary bacteriocytes and small This article is a PNAS Direct Submission. sheath cells, in addition to hemolymph (Fig. P1A). At the tip Freely available online through the PNAS open access option. region of ovarioles, Buchnera cells are transmitted from maternal 1To whom correspondence should be addressed. E-mail: [email protected]. bacteriocytes to adjacent early embryos at the blastula stage in See full research article on page E1230 of www.pnas.org. a highly coordinated manner (Fig. P1B). In this study, we dem- Cite this Author Summary as: PNAS 10.1073/pnas.1119212109.

www.pnas.org/cgi/doi/10.1073/pnas.1119212109 PNAS | May 15, 2012 | vol. 109 | no. 20 | 7597–7598 Downloaded by guest on September 30, 2021 the bacteria from a maternal bacteriocyte to a blastula (5). Our of expressed genes) and proteomic (i.e., large-scale study of study rejects these existing hypotheses, and presents an alter- ) analyses of the bacteriocyte–blastula interface. native “exo-/endocytotic transport” hypothesis (Fig. P1C) for Buchnera transmission at the bacteriocyte–blastula interface. 1. Werren JH, Baldo L, Clark ME (2008) Wolbachia: Master manipulators of invertebrate In conclusion, we discovered a sophisticated cellular mecha- biology. Nat Rev Microbiol 6:741–751. nism for the selective vertical transmission of Buchnera, which 2. Oliver KM, Degnan PH, Burke GR, Moran NA (2010) Facultative symbionts in aphids probably evolved to facilitate the obligate host–symbiont asso- and the horizontal transfer of ecologically important traits. Annu Rev Entomol 55: 247–266. ciation. We also propose an evolutionary hypothesis whereby 3. Buchner P (1965) Endosymbiosis of with Plant Microorganisms (Interscience, facultative symbionts, such as Serratia, might have coopted the New York). endocytotic component of the mechanism to enter host embryos. 4. Wilkinson TL, Fukatsu T, Ishikawa H (2003) Transmission of symbiotic bacteria Buchnera to The spatially restricted formation of the vertical transmission parthenogenetic embryos in the aphid Acyrthosiphon pisum (Hemiptera: Aphidoidea). Arthropod Struct Dev 32:241–245. apparatus indicates intricate molecular and cellular interactions 5. Miura T, et al. (2003) A comparison of parthenogenetic and sexual embryogenesis of between the maternal bacteriocyte and the early blastula em- the pea aphid Acyrthosiphon pisum (Hemiptera: Aphidoidea). J Exp Zoolog B Mol bryo, which deserves future transcriptomic (i.e., large-scale study Dev Evol 295:59–81.

7598 | www.pnas.org/cgi/doi/10.1073/pnas.1119212109 Koga et al. Downloaded by guest on September 30, 2021