- Research article
- Open Access
Morphogenesis and development of midgut symbiotic organ of the stinkbug Plautia stali (Hemiptera: Pentatomidae)
© The Author(s). 2019
- Received: 22 March 2019
- Accepted: 19 May 2019
- Published: 31 May 2019
Diverse insects are intimately associated with microbial symbionts, which play a variety of biological roles in their adaptation to and survival in the natural environment. Such insects often possess specialized organs for hosting the microbial symbionts. What developmental processes and mechanisms underlie the formation of the host organs for microbial symbiosis is of fundamental biological interest but poorly understood.
Here we investigate the morphogenesis of the midgut symbiotic organ and the process of symbiont colonization therein during the developmental course of the stinkbug Plautia stali. Upon hatching, the midgut is a simple and smooth tube. Subsequently, symbiont colonization to the posterior midgut occurs, and thickening and folding of the midgut epithelium proceed during the first instar period. By the second instar, rudimentary crypts have formed, and their inner cavities are colonized by the symbiotic bacteria. From the second instar to the fourth instar, while the alimentary tract grows and the posterior midgut is established as the symbiotic organ with numerous crypts, the anterior midgut and the posterior midgut are structurally and functionally isolated by a strong constriction in the middle. By the early fifth instar, the midgut symbiotic organ attains the maximal length, but toward the mid fifth instar, the basal region of each crypt starts to constrict and narrow, which deforms the midgut symbiotic organ as a whole into a shorter, thicker and twisted shape. By the late fifth instar to adulthood, the crypts are constricted off, by which the symbiotic bacteria are confined in the crypt cavities and isolated from the midgut main tract, and concurrently, the strong midgut constriction in the middle becomes loose and open, by which the food flow from the anterior midgut to the posterior midgut recovers.
This study provides the most detailed and comprehensive descriptions ever reported on the morphogenesis of the symbiotic organ and the process of symbiont colonization in an obligatory insect-bacterium gut symbiotic system. Considering that P. stali is recently emerging as a useful model system for experimentally studying the intimate insect-microbe gut symbiosis, the knowledge obtained in this study establishes the foundation for the further development of this research field.
- Plautia stali
- Symbiotic bacteria
- Symbiotic organ
- Midgut crypt
Diverse insects and other organisms are associated with microbial symbionts, which play substantial roles in their adaptation to and survival in the natural environment. The beneficial roles of the microbial symbionts encompass assistance of food digestion [1, 2], provisioning of essential nutrients [3, 4], facilitated resistance to natural enemies [5, 6], improved tolerance to environmental stresses [7, 8], degradation of noxious chemicals [9, 10], promotion of plant adaptation [11, 12], and many others. Hence, understanding of host mechanisms that underpin the establishment and maintenance of the microbial symbiosis is of fundamental biological interest.
Some microbial symbionts are endocellular like Buchnera in aphids, where the symbiotic bacteria are harbored in specialized cells and organs for symbiosis, called the bacteriocytes and the bacteriomes, respectively [3, 13]. Meanwhile, other microbial symbionts, such as Pantoea or Burkholderia, are extracellular in a variety of stinkbugs, in which the symbiotic bacteria are harbored in the inner cavity of a specialized region of the alimentary tract, so-called the midgut symbiotic organ [14, 15].
Many plant-sucking stinkbugs are associated with symbiotic bacteria of beneficial nature in a specialized region of the posterior midgut, where numerous sac-like structures, called crypts, harbor the bacteria within the inner cavity extracellularly [14–18]. The midgut of the stinkbugs is generally differentiated into structurally distinct regions, among which the posterior end region is specialized for microbial symbiosis [19–21], but the developmental and functional aspects of the midgut regions have been poorly understood. In these stinkbugs, upon oviposition, adult females secrete a symbiont-containing excrement from the anus, and either smear it onto the eggshell, place it beside the eggs, or cover the eggs with a large amount of the excrement. Upon hatching, newborn nymphs immediately ingest the excrement and orally acquire the symbiont, thereby establishing the vertical symbiont transmission [22–28]. Symbiont removal either by sterilizing the egg surface or removing the symbiont-containing excrement results in growth retardation, elevated mortality and/or sterility of the insect host [25, 26, 29–38], highlighting the fatal importance of the gut symbiotic system in stinkbugs.
What developmental processes and mechanisms underlie the formation of the insect cells and organs specialized for microbial symbiosis has been poorly understood and remains as an unanswered issue in the evolutionary developmental biology. While notable old histological descriptions, some of which were comprehensive but most of which were fragmentary snapshots, may be available (reviewed in ), recent detailed studies have been limited to only a few cases, which are represented by the pea aphid Acyrthosiphon pisum [39–41], the seed bug Nysius plebeius , and the bean bug Riptortus pedestris [43, 44].
Here, we focus on the brown-winged green stinkbug Plautia stali (Hemiptera: Pentatomidae), which has been known as a devastating pest of various fruits and crops  and recently emerging as a laboratory model for studying the insect-microbe gut symbiosis. P. stali is associated with a specific γ-proteobacterial symbiont, allied to Pantoea spp., within numerous crypts arranged in four rows along the posterior midgut [28, 29]. As a notorious agricultural pest, stable and reliable system for rearing P. stali in the laboratory has been established for decades , including the recent development of aseptic rearing procedures . While an uncultivable gut symbiont (so-called symbiont A) is fixed in the mainland Japan, the gut symbiotic microbiota is distinct and polymorphic in southwestern islands of Japan, where an uncultivable gut symbiont (symbiont B) coexists with multiple cultivable gut symbionts (symbionts C–F) in the same islands and populations, which plausibly represents an ongoing process of symbiont diversification and/or replacement in natural insect populations . For the cultivable symbionts, genetic and experimental manipulations are feasible, and for the host insect, knockdown of gene expression by RNA interference works efficiently [28, 47].
In this study, we investigated the morphogenesis of the midgut symbiotic organ and the process of symbiont colonization during the developmental course of P. stali in detail, by which we identified the first instar and the fifth instar as the important developmental stages for shaping the gut symbiotic system in the insect.
A mass-reared laboratory strain of P. stali, which had been established from adult insects collected in Tsukuba, Ibaraki, Japan, was used in this study. All populations of P. stali in the mainland Japan, including the Tsukuba population, are associated with a specific γ-proteobacterial gut symbiont, so-called symbiont A, which is phylogenetically close to Pantoea dispersa and allied environmental bacteria . The symbiont A of P. stali is uncultivable, with a degenerate genome, and is essential for normal growth and survival of the host insect [28, 29, 47]. Ovipositing adult females of P. stali smear a symbiont-containing excrement onto the egg surface, and newborn nymphs actively suck it for around 30 min to acquire the symbiont immediately after hatching . The insects maintained in mass-stock culture were reared on raw peanuts and water supplemented with 0.05% ascorbic acid (DWA) in large plastic containers at 25 ± 1 °C under a long-day regime of 16 h light and 8 h dark, as described previously . It has been reported that raw peanut is the best laboratory food for P. stali in comparison with other seeds, grains, and fruits that were tested . Egg masses were collected from the stock culture, their hatching was monitored every day, and newborn nymphs from them were reared and used for experiments. For the purpose of developmental monitoring, each newborn nymph was individually reared in a plastic Petri dish (90 mm in diameter, 20 mm in depth) with 3 raw peanuts and a cotton ball soaked with DWA, and inspected for molting or eclosion from 15:00 to 20:00 every day. For quantitative PCR (qPCR) and fluorescence in situ hybridization (FISH), about 10 insects were kept in each plastic Petri dish with 5–8 raw peanuts and a cotton ball soaked with DWA. The insects were monitored for molting or eclosion from 15:00–20:00 every day, and sorted according to the date of molting or eclosion for developmental staging. The rearing Petri dishes were kept in climatic chambers at 25 ± 1 °C under a long-day regime of 16 h light and 8 h dark. The food and DWA were renewed once a week.
Insect dissection and sample preparation
The insects were dissected in Petri dishes filled with a phosphate-buffered saline (PBS: 137 mM NaCl, 8.1 mM Na2HPO4, 2.7 mM KCl, 1.5 mM KH2PO4 [pH 7.4]) using fine scissors, forceps, razors and/or needles, and photographed under a stereomicroscope (S8APO; Leica). For comparing the symbiont titers between the symbiotic regions and the non-symbiotic regions of the alimentary tract, adult insects 1 week after eclosion were dissected, and the dissected tissue samples were individually kept in a ultracold freezer at − 80 °C. For quantifying the symbiont population dynamics during the developmental course of P. stali, nymphs and adults of each developmental stage were collected 1 day after hatching, molting or eclosion. First-, second- and third-instar nymphs were preserved in acetone . Fourth- and fifth-instar nymphs and adults were dissected in PBS and their symbiotic organs were kept in a ultracold freezer at − 80 °C. For analyzing the symbiont population dynamics after egg hatching, we performed strict developmental staging of the newborn nymphs of P. stali. We collected many egg masses, each of which consisted of around 14 eggs, and on the fifth day after oviposition, the eggs were inspected every hour to collect newborn nymphs. These strictly-staged newborn nymphs were preserved in acetone every 24 h for 5 days.
DNA preparation and quantitative PCR (qPCR)
Each insect tissue sample was homogenized in a plastic tube with a lysis buffer (100 mM NaCl, 10 mM Tris-HCl [pH 8.0], 1 mM EDTA, 0.2% [w/v] SDS), extracted with phenol-chloroform-isoamylalcohol (25:24:1), precipitated and washed with ethanol, dried in air, and dissolved in a DNA suspension buffer (10 mM Tris-HCl [pH 8.0], 0.1 mM EDTA).
We quantified the symbiotic bacteria by qPCR in terms of bacterial groEL gene copies essentially as described previously . An 80 bp region of groEL gene of the symbiont A was targeted by the primers AgroL1265F (5′-TTG CGG CGA AAA TCG CAG CT-3′) and AgroL1345R (5′-TCG CAC GCA GAG CAA CCT TA-3′). The PCR reaction mixture was composed of 375 nM each of the primers, 50 × ROX low, 2 × KAPA SYBR FAST qPCR Master Mix Universal (KAPA Biosystems), 5 μl of template DNA solution (1/20 of total DNA per insect), and water in a total of 20 μl. A standard curve was drawn using a 0.4 kb groEL gene segment as standard DNA samples, which was PCR-amplified with the primers AgroL1084F (5′-AAA GAG AAA CTG CAG GAG CG-3′) and AgroL1508R (5′-CGG GTC ACT TTG GTT GGA T-3′), purified and serially diluted. For each sample quantification, two sample replicates were measured with standard DNA samples containing 8.5 × 102, 103, 104, 105, 106 and 107 groEL gene copies, respectively.
Histology and fluorescence in situ hybridization (FISH)
FISH was conducted using a fluorochrome-labeled oligonucleotide probe SymAC89R (5′-Alexa555-GCA AGC TCT TCT GTG CTG CC-3′) that targeted bacterial 16S rRNA of the symbiont A as described . Dissected symbiotic organs were fixed in PBS containing 4% paraformaldehyde (PFA) for 3 h at room temperature, and washed with PBS containing 0.1% Tween 20 (PBST). For whole-mount FISH, the organs were washed and equilibrated with a hybridization buffer (20 mM Tris-HCl [pH 8.0], 0.9 M NaCl, 0.01% sodium dodecyl sulfate, 30% formamide) and then incubated in the hybridization buffer supplemented with 100 nM probe and 1 μg/ml 4′,6-diamidino-2-phenylindole (DAPI) overnight at room temperature in a dark box. After the incubation, the samples were washed with PBST, mounted with 80% glycerol or Slowfade Gold Antifade Mountant (Thermo Fisher), and observed under a fluorescence stereomicroscope (M165FC; Leica) or a laser confocal scanning microscope (LSM700; Zeiss). For FISH of tissue sections, after fixation with PFA, the samples were dehydrated and embedded by following the Technovit 8100 protocol (Heraeus Kulzer). Then, the embedded samples were cut into 2 μm sections on a microtome (RM2255; Leica), mounted on glass slides, and incubated with the hybridization buffer supplemented with 100 nM probe and 1 μg/ml DAPI for 2 h at room temperature in a humidified dark box. Then, the samples on the glass slides were washed with PBS, mounted with Slowfade Gold Antifade Mountant, and observed under an epifluorescence microscope (LSM700; Zeiss). In order to confirm the specificity of the fluorescent signals, we conducted no-probe control experiments, RNase-treated control experiments, and competitive suppression control experiments.
Visualization of food passage
The experimental insects were provided with DWA containing 0.05% Brilliant Blue FCF as drinking water. For facilitating water uptake, we started the experiment using the insects that had been kept without drinking water for a day. After the treatment for 2 days, the insects were dissected, and their alimentary tracts were observed and photographed under a stereomicroscope (S8APO; Leica).
Midgut symbiotic organ of P. stali
Developmental stages of P. stali
Population dynamics of symbiotic bacteria during development of P. stali
Development of midgut symbiotic organ and colonization process of symbiotic bacteria in P. stali
The dissected alimentary tracts representing the defined developmental stages were subjected to morphological observations (Fig. 4b–g) and FISH visualization of the symbiotic bacteria (Fig. 4h-m). In the first instar nymphs, crypts were structurally not evident and symbiont signals were found only sporadically on the inner surface of the gut tube (Fig. 4b and h), which were in contrast to the well-developed crypts full of the symbiotic bacteria at the second to fifth instar stages (Fig. 4c–g and i-m). The midgut crypts arranged in four rows were already evident at the second instar stage (Fig. 4c). These observations suggested that crypt formation in the midgut M4 region must occur during the first instar period.
Morphogenesis of midgut symbiotic organ at early developmental stages of P. stali
Morphogenesis of midgut symbiotic organ at late developmental stages of P. stali
Re-organization of antero-posterior midgut connection at late developmental stages of P. stali
We found that the alimentary tract of newborn nymphs of P. stali is initially a simple tube, but soon a series of morphogenetic events occur in their gut epithelium, and the rudimentary crypts are formed and colonized by the symbiotic bacteria by the second instar stage (Figs. 4 and 5). By performing RNA sequencing of the posterior midgut at this developmental stage, identifying specifically-expressed and/or up-regulated genes therein, and knocking-down these genes by RNA interference, we would be able to understand the molecular mechanisms underpinning the intricate morphogenesis of the midgut symbiotic organ, which entails formation of the numerous sac-like crypts arranged in four rows along the midgut main tract (see Fig. 1). Furthermore, by performing such analyses on symbiotic and aposymbiotic newborn nymphs in a comparative manner, we will be able to gain insight into how and to what extent the host and the symbiont respectively contribute to the morphogenetic processes of the midgut symbiotic organ.
We found that, during the period from the fifth instar to adulthood, despite the increasing body size, the midgut symbiotic organ becomes shorter, thicker and twisted (Fig. 4a). Close histological and cytological inspections revealed that the midgut symbiotic organ experiences characteristic structural re-organization during the fifth instar period, in which the basal region of each crypt is constricted off, each crypt cavity is isolated from the midgut main tract, and consequently the symbiotic bacteria are isolated and retained within the numerous crypt cavities along the midgut symbiotic region (Fig. 6). We propose the hypothesis that the tensile force collectively generated by the basal constriction of numerous crypts is responsible for the shortening, broadening and twisting of the midgut symbiotic organ during the fifth instar period. Plausibly, the crypt constriction may entail accumulation of cytoskeletal elements at the crypt bases, involvement of contractile machineries like actomyosin system and/or tubulin-dynein system, recruitment of programmed cell death or apoptosis, etc. In the future, we are planning to investigate these molecular and cellular processes by utilizing a variety of marker molecules, antibodies, RNA sequencing and RNA interference approaches. How these morphogenetic processes are affected by the symbiotic bacteria is of interest, but it is practically difficult to obtain aposymbiotic fifth instar nymphs of P. stali due to high mortality of aposymbiotic insects.
Strikingly, we found that, concurrent with the confinement of the symbiotic bacteria within the crypt cavities and consequent isolation from the midgut main tract during the fifth instar period, the constricted M3-M4b junction, which functionally separates the anterior midgut region for food digestion and absorption from the posterior midgut region for hosting the symbiotic bacteria, becomes thick and loose, thereby restoring the food flow across the anterior and posterior midgut regions (Fig. 7). By contrast, a previous detailed study on the bean bug R. pedestris revealed that the anterior midgut region is functionally isolated from the posterior midgut region without food flow at the M3-M4b junction throughout the nymphal and adult stages . What factors are relevant to the difference between R. pedestris and P. stali?
A conceivable factor is the transmission mode of the symbiotic bacteria. Since R. pedestris acquires the Burkholderia symbiont from the environment at an early nymphal stage every generation [50, 51], the contaminant/symbiont ratio in the microbial inoculum ingested by the nymphs must be very high, which implies that exclusion of non-symbiotic microbe is very important for R. pedestris. On the other hand, adult females of P. stali smear an excrement containing the Pantoea-allied symbiont onto the eggshell upon oviposition [28, 47], and thus the symbiont inoculum ingested by the newborn nymphs from the eggshell contains the symbiotic bacteria of high purity, which may be relevant to the finding that the M3-M4b junction becomes less exclusive against foreign microbes in P. stali than in R. pedestris.
Another conceivable factor is the high reproductive activity of P. stali. Under a favorable rearing condition in the laboratory, adult females of P. stali start to lay eggs within 5 days after eclosion, and then continue to produce an egg mass consisting of about 14 eggs every 1 or 2 days for several weeks to months, which often amount to over 300–500 eggs in total (Oishi, unpublished data). In order to support the high level of reproductive activity in P. stali, a large amount of food must be efficiently ingested, digested and absorbed to acquire a sufficient quantity of nutrients by adult insects, which may be the reason why the food flow in the midgut of P. stali restores prior to the adult stage.
For many plant-sucking stinkbugs including P. stali, the gut symbiotic bacteria are essential for normal growth and survival [22–38], and thus stable vertical symbiont transmission is expected to be strongly selected for [14, 15, 52]. In this study, however, we observed that vertical symbiont transmission via maternal smearing onto the eggshell occasionally fails, which may result in symbiosis-deficient insects suffering growth defect and mortality (Fig. 3d; Fig. 5o). The incidence of the transmission failure may be an artifact under the laboratory rearing condition, or may be relevant to inbreeding of the host insect strain that had been maintained in the laboratory for years. Anyway, these observations highlight the importance of vertical symbiont transmission in stinkbugs.
In addition to P. stali (Pentatomidae) and R. pedestris (Alydidae), the functionally discontinuous midgut at the M3-M4b junction has been found in diverse plant-sucking stinkbugs representing the families Coreidae, Rhyparochromidae, Scutelleridae, Dinidridae, Cydnidae, Acanthosomatidae, and Plataspidae . For understanding the diversity and evolution of the midgut symbiotic organ, similar morphological and histological studies on the development of the alimentary tract in diverse stinkbugs are anticipated, with particular emphasis on the morphogenetic, developmental, transcriptomic and functional aspects of the midgut crypts and the M3-M4b junction under a comparative perspective.
In conclusion, we have described the morphogenesis and development of the midgut symbiotic organ and the process of symbiont colonization in an obligatory insect-bacterium gut symbiotic system in an unprecedented detail. Besides some old histological descriptions, there have been few comprehensive studies on the morphogenesis of the midgut symbiotic organ and the process of symbiont colonization in stinkbugs (reviewed in ). Considering that P. stali is recently emerging as a useful model system for experimentally studying the intimate insect-microbe gut symbiotic association [28, 47, 53], the knowledge obtained in this study is expected to establish the foundation for the further development of this research field.
We thank Yoshitomo Kikuchi, Takahiro Hosokawa and Ryo Futahashi for comments on the manuscript and Tomoko Matsushita, Setsuko Kimura, Ryoko Konishi and Wakana Kikuchi for technical and secretarial assistance.
This study was supported by the JSPS KAKENHI grant nos. JP25221107 and JP17H06388 to T.F.
SO, MM and RK performed experimental works. SO, MM, RK and TF designed the research. SO and TF wrote the paper. All authors read and approved the final manuscript.
Ethics approval and consent to participate
Consent for publication
The authors declare that they have no competing interests.
Open AccessThis 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.
- Currie CR. A community of ants, fungi, and bacteria: a multilateral approach to studying symbiosis. Annu Rev Microbiol. 2001;55:357–80.View ArticleGoogle Scholar
- Brune A. Symbiotic digestion of lignocellulose in termite guts. Nat Rev Microbiol. 2014;12:168–80.View ArticleGoogle Scholar
- Moran NA, Nakabachi A, McCutcheon JP. Genomics and evolution of heritable bacterial symbionts. Annu Rev Genet. 2008;42:165–90.View ArticleGoogle Scholar
- Douglas AE. The microbial dimension in insect nutritional ecology. Funct Ecol. 2009;23:38–47.View ArticleGoogle Scholar
- Oliver KM, Smith AH, Russell JA. Defensive symbiosis in the real world – advancing ecological studies of heritable, protective bacteria in aphids and beyond. Funct Ecol. 2014;28:341–55.View ArticleGoogle Scholar
- Florez LV, Biedermann PHW, Engl T, Kaltenpoth M. Defensive symbioses of animals with prokaryotic and eukaryotic microorganisms. Nat Prod Rep. 2015;32:904–36.View ArticleGoogle Scholar
- Montllor CB, Maxmen A, Purcell AH. Facultative bacterial endosymbionts benefit pea aphids Acyrthosiphon pisum under heat stress. Ecol Entomol. 2002;27:189–95.View ArticleGoogle Scholar
- Dunbar HE, Wilson ACC, Ferguson NR, Moran NA. Aphid thermal tolerance is governed by a point mutation in bacterial symbionts. PLoS Biol. 2007;5:e96.View ArticleGoogle Scholar
- Kikuchi Y, Hayatsu M, Hosokawa T, Nagayama A, Tago K, Fukatsu T. Symbiont-mediated insecticide resistance. Proc Natl Acad Sci U S A. 2012;109:8618–22.View ArticleGoogle Scholar
- Henrik HDFL, Schiøtt M, Rogowska-Wrzesinska A, Nygaard S, Roepstorff P, Boomsma JJ. Laccase detoxification mediates the nutritional alliance between leaf-cutting ants and fungus-garden symbionts. Proc Natl Acad Sci U S A. 2013;110:583–7.View ArticleGoogle Scholar
- Tsuchida T, Koga R, Fukatsu T. Host plant specialization governed by facultative symbiont. Science. 2004;303:1989.View ArticleGoogle Scholar
- Hosokawa T, Kikuchi Y, Shimada M, Fukatsu T. Obligate symbiont involved in pest status of host insect. Proc R Soc B. 2007;274:1979–84.View ArticleGoogle Scholar
- Baumann P. Biology of bacteriocyte-associated endosymbionts of plant sap-sucking insects. Annu Rev Microbiol. 2005;59:155–89.View ArticleGoogle Scholar
- Salem H, Florez L, Gerardo N, Kaltenpoth M. An out-of-body experience: the extracellular dimension for the transmission of mutualistic bacteria in insects. Proc R Soc B. 2015;282:20142957.View ArticleGoogle Scholar
- Takeshita K, Kikuchi Y. Riptortus pedestris and Burkholderia symbiont: an ideal model system for insect–microbe symbiotic associations. Res Microbiol. 2017;168:175–87.View ArticleGoogle Scholar
- Buchner P. Endosymbiosis of animals with plant microorganisms. New York: Interscience; 1965.Google Scholar
- Hosokawa T, Kaiwa N, Matsuura Y, Kikuchi Y, Fukatsu T. Infection prevalence of Sodalis symbionts among stinkbugs. Zoological Lett. 2015;1:5.View ArticleGoogle Scholar
- Hosokawa T, Matsuura Y, Kikuchi Y, Fukatsu T. Recurrent evolution of gut symbiotic bacteria in pentatomid stinkbugs. Zoological Lett. 2016;2:34.View ArticleGoogle Scholar
- Glasgow H. The gastric caeca and the caecal bacteria of the Heteroptera. Biol Bull. 1914;3:101–71.View ArticleGoogle Scholar
- Miyamoto S. Comparative morphology of alimentary organs of Heteroptera, with the phylogenetic consideration. Sieboldia. 1961;2:197–259.Google Scholar
- Goodchild AJP. Evolution of the alimentary canal in the Hemiptera. Biol Rev. 1966;41:97–140.View ArticleGoogle Scholar
- Fukatsu T, Hosokawa T. Capsule-transmitted gut symbiotic bacterium of the Japanese common plataspid stinkbug, Megacopta punctatissima. Appl Environ Microbiol. 2002;68:389–96.View ArticleGoogle Scholar
- Hosokawa T, Kikuchi Y, Fukatsu T. How many symbionts are provided by mothers, acquired by offspring, and needed for successful vertical transmission in an obligate insect-bacterium mutualism? Mol Ecol. 2007;16:5316–25.View ArticleGoogle Scholar
- Hosokawa T, Hironaka M, Mukai H, Inadomi K, Suzuki N, Fukatsu T. Mothers never miss the moment: a fine-tuned mechanism for vertical symbiont transmission in a subsocial insect. Anim Behav. 2012;83:293–300.View ArticleGoogle Scholar
- Hosokawa T, Hironaka M, Inadomi K, Mukai H, Nikoh N, Fukatsu T. Diverse strategies for vertical symbiont transmission among subsocial stinkbugs. PLoS One. 2013;8:e65081.View ArticleGoogle Scholar
- Kaiwa N, Hosokawa T, Nikoh N, Tanahashi M, Moriyama M, Meng XY, Maeda T, Yamaguchi K, Shigenobu S, Ito M, Fukatsu T. Symbiont-supplemented maternal investment underpinning host's ecological adaptation. Curr Biol. 2014;24:2465–70.View ArticleGoogle Scholar
- Hayashi T, Hosokawa T, Meng XY, Koga R, Fukatsu T. Female-specific specialization of a posterior end region of the midgut symbiotic organ in Plautia splendens and allied stinkbugs. Appl Environ Microbiol. 2015;81:2603–11.View ArticleGoogle Scholar
- Hosokawa T, Ishii Y, Nikoh N, Fujie M, Satoh N, Fukatsu T. Obligate bacterial mutualists evolving from environmental bacteria in natural insect populations. Nat Microbiol. 2016;1:15011.View ArticleGoogle Scholar
- Abe Y, Mishiro K, Takanashi M. Symbiont of brown-winged green bug, Plautia stali Scott. Jpn J Appl Entomol Zool. 1995;39:109–15.View ArticleGoogle Scholar
- Hosokawa T, Kikuchi Y, Nikoh N, Shimada M, Fukatsu T. Strict host-symbiont cospeciation and reductive genome evolution in insect gut bacteria. PLoS Biol. 2006;4:e337.View ArticleGoogle Scholar
- Prado SS, Rubinoff D, Almeida RPP. Vertical transmission of a pentatomid caeca-associated symbiont. Ann Entomol Soc Am. 2006;99:577–85.View ArticleGoogle Scholar
- Kikuchi Y, Hosokawa T, Nikoh N, Meng XY, Kamagata Y, Fukatsu T. Host-symbiont co-speciation and reductive genome evolution in gut symbiotic bacteria of acanthosomatid stinkbugs. BMC Biol. 2009;7:2.View ArticleGoogle Scholar
- Tada A, Kikuchi Y, Hosokawa T, Musolin DL, Fujisaki K, Fukatsu T. Obligate association with gut bacterial symbiont in Japanese populations of southern green stinkbug Nezara viridula (Heteroptera: Pentatomidae). Appl Entomol Zool. 2011;46:483–8.View ArticleGoogle Scholar
- Kikuchi Y, Hosokawa T, Nikoh N, Fukatsu T. Gut symbiotic bacteria in the cabbage bugs Eurydema rugosa and Eurydema dominulus (Heteroptera: Pentatomidae). Appl Entomol Zool. 2012;47:1–8.View ArticleGoogle Scholar
- Taylor CM, Coffey PL, DeLay BD, Dively GP. The importance of gut symbionts in the development of the brown marmorated stink bug, Halyomorpha halys (Stål). PLoS One. 2014;9:e90312.View ArticleGoogle Scholar
- Bistolas KSI, Sakamoto RI, Fernandes JAM, Goffredi SK. Symbiont polyphyly, co-evolution, and necessity in pentatomid stinkbugs from Costa Rica. Front Microbiol. 2014;5:349.View ArticleGoogle Scholar
- Karamipour N, Mehrabadi M, Fathipour Y. Gammaproteobacteria as essential primary symbionts in the striped shield bug, Graphosoma lineatum (Hemiptera: Pentatomidae). Sci Rep. 2016;6:33168.View ArticleGoogle Scholar
- Itoh H, Matsuura Y, Hosokawa T, Fukatsu T, Kikuchi Y. Obligate gut symbiotic association in the sloe bug Dolycoris baccarum (Hemiptera: Pentatomidae). Appl Entomol Zool. 2017;52:51–9.View ArticleGoogle Scholar
- Braendle C, Miura T, Bickel R, Shingleton AW, Kambhampati S, Stern DL. Developmental origin and evolution of bacteriocytes in the aphid-Buchnera symbiosis. PLoS Biol. 2003;1:E21.View ArticleGoogle Scholar
- Miura T, Braendle C, Shingleton A, Sisk G, Kambhampati S, Stern DL. A comparison of parthenogenetic and sexual embryogenesis of the pea aphid Acyrthosiphon pisum (Hemiptera: Aphidoidea). J Exp Zool B. 2003;295:59–81.View ArticleGoogle Scholar
- Koga R, Meng XY, Tsuchida T, Fukatsu T. Cellular mechanism for selective vertical transmission of an obligate insect symbiont at the bacteriocyte-embryo interface. Proc Natl Acad Sci U S A. 2012;109:E1230–7.View ArticleGoogle Scholar
- Matsuura Y, Kikuchi Y, Miura T, Fukatsu T. Ultrabithorax is essential for bacteriocyte development. Proc Natl Acad Sci U S A. 2015;112:9376–81.View ArticleGoogle Scholar
- Kikuchi Y, Fukatsu T. Live imaging of symbiosis: spatiotemporal infection dynamics of a GFP-labeled Burkholderia symbiont in the bean bug Riptortus pedestris. Mol Ecol. 2014;23:1445–56.View ArticleGoogle Scholar
- Ohbayashi T, Takeshita K, Kitagawa W, Nikoh N, Koga R, Meng XY, Tago K, Hori T, Hayatsu M, Asano K, Kamagata Y, Lee BL, Fukatsu T, Kikuchi Y. Insect's intestinal organ for symbiont sorting. Proc Natl Acad Sci U S A. 2015;112:E5179–88.View ArticleGoogle Scholar
- Schaefer CW, Panizzi AR. Heteroptera of economic importance. Boca Raton: CRC Press; 2000.View ArticleGoogle Scholar
- Kotaki T, Hata K, Gunji M, Yagi S. Rearing of the brown-winged green bug, Plautia stali Scott (Hemiptera: Pentatomidae) on several diets. Jpn J Appl Entomol Zool. 1983;27:63–8.View ArticleGoogle Scholar
- Nishide Y, Onodera-Tanifuji N, Tanahashi M, Moriyama M, Fukatsu T, Koga R. Aseptic rearing procedure for the stinkbug Plautia stali (Hemiptera: Pentatomidae) by sterilizing food-derived bacterial contaminants. Appl Entomol Zool. 2017;53:407–15.View ArticleGoogle Scholar
- Fukatsu T. Acetone preservation: a practical technique for molecular analysis. Mol Ecol. 1999;8:1935–45.View ArticleGoogle Scholar
- Koga R, Tsuchida T, Fukatsu T. Quenching autofluorescence of insect tissues for in situ detection of endosymbionts. Appl Entomol Zool. 2009;44:281–91.View ArticleGoogle Scholar
- Kikuchi Y, Hosokawa T, Fukatsu T. Insect-microbe mutualism without vertical transmission: a stinkbug acquires a beneficial symbiont from the environment every generation. Appl Environ Microbiol. 2007;73:4308–16.View ArticleGoogle Scholar
- Kikuchi Y, Hosokawa T, Fukatsu T. Specific developmental window for establishment of an insect-microbe gut symbiosis. Appl Environ Microbiol. 2011;77:4075–81.View ArticleGoogle Scholar
- Bright M, Bulgheresi S. A complex journey: transmission of microbial symbionts. Nat Rev Microbiol. 2010;8:218–0.View ArticleGoogle Scholar
- Nishide Y, Kageyama D, Yokoi K, Tanaka H, Futahashi R, Fukatsu T. Functional crosstalk across IMD and toll pathways: insight into the evolution of incomplete immune cascades. Proc R Soc B. 2019;286:20182207.View ArticleGoogle Scholar