J. Agr. Sci. Tech. (2021) Vol. 23(1): 137-148 The Crucial Role of the Endosymbiont Pantoea sp. in Morphology and Mating of the Pistachio Green Stink Bug, Brachynema germari (Hemiptera: Pentatomidae) M. Kashkouli1, Y. Fathipour1*, and M. Mehrabadi1 ABSTRACT The pistachio green stink bug, Brachynema germari Kolenati (Hemiptera: Pentatomidae), is one of the most important pests of pistachio nuts in many pistachio- growing regions in Iran. This insect harbors a gammaproteobacterial symbiont, related to the genus Pantoea, in the numerous crypts of its posterior midgut, which is vertically transmitted by infection of the egg masses and orally acquired by newborn nymphs. In the present study, the effects of the symbiont on host morphology, emergence rates, and mating frequency of B. germari were explored. For this purpose, two symbiont elimination strategies, high temperature and egg surface sterilization, were used and their effects were compared. We found external morphological changes (e.g. abnormalities in notum and wings) as well as significantly fewer emergence rates (in all stages, except for the first instar) in the surface-sterilized and heat-treated insects compared with the controls. Also, the second, third, and fourth regions of the midgut exhibited remarkable morphological changes in the aposymbiotic insects compared with the controls. Besides, less mating frequency was observed in the aposymbiotic population compared with the control. Together, these results provided a close relationship between the bacterial symbiont and B. germari and suggested the importance of the symbiont for the morphogenesis, development, and reproduction of the insect host. Keywords: Alimentary canal, Body-color, Emergence rates, Mating frequency, Morphogenesis. INTRODUCTION host. These symbionts infect their host sporadically (Sudakaran et al., 2017), and are predominantly vertically transmitted, although Downloaded from jast.modares.ac.ir at 11:05 IRST on Saturday October 2nd 2021 Mutualistic association with symbiotic microorganisms characterizes many, if not all, horizontal transmission occasionally occurs animal species (Raman, 1991). The most (Haine, 2008). These symbionts can be located important symbiotic associations are generally intra- or extracellularly, and invade various categorized into three groups. The first cells and tissues of hosts (Su et al., 2013). The category consists of obligate (or primary) third group encompasses extracellular mutualists that are housed within a special host symbionts that are localized in the gut lumen organ (bacteriome) and relayed within or gut-associated crypts or caeca (Sudakaran et transovarial transmission between the host al., 2017). These bacteria are often inherited generations (Moran et al., 2008). These vertically to newborn nymphs by egg smearing symbionts are essential for host survival and (Sudakaran et al., 2017), coprophagy reproduction (Haine, 2008). In contrast to (Buchner, 1965), or special symbiont- primary symbionts, facultative or secondary containing capsules (Fukatsu and Hosokawa, symbionts are generally not essential for their 2002), jelly (Kaiwa et al., 2014), or mucus _____________________________________________________________________________ 1Department of Entomology, Faculty of Agriculture, Tarbiat Modares University, Tehran, Islamic Republic of Iran. *Corresponding author; e-mail: [email protected] 137 _____________________________________________________________________ Kashkouli et al. (Hosokawa et al., 2012), but can also be generations per year (Ramzi and horizontally acquired (Kikuchi et al., 2012a). Hosseininaveh, 2010) and overwinters as an In general, the mutualistic symbionts adult. The bug injury leads to desiccation, significantly benefit their host by playing epicarp lesion, and kernel necrosis of nuts significant roles in digestion and detoxification resulting in the dropping of the damaged nuts of food (Su et al., 2013), provisioning of from the trees (Bagheri et al., 2010; essential nutrients (Akman et al., 2002; Engel Mehrnejad, 2001). Also, this pest has been and Moran, 2013), defense against natural suggested to be a vector for fungal pathogen enemies (Brownlie and Johnson, 2009), Nematospora coryli Peglion tolerances to environmental stresses (Saccharomycetaceae), which is the causal (Wernegreen, 2012), detoxification of noxious agent of the diseases in pistachio nuts chemicals (Kikuchi et al., 2012a), nitrogen (stigmatomycosis) (Ershad and Barkhordary, recycling (Engel and Moran, 2013), promotion 1974). Despite the agricultural importance of of plant adaptation (Frago et al., 2012), B. germari, its association with diapause modification (Rahimi-Kaldeh et al., microorganisms is poorly understood. The 2019), and many others. Although many roles previous study revealed that B. germari and effects of stinkbug symbionts have been harbors a gamma-proteobacterial symbiont, investigated in detail, understanding of their related to the Pantoea genus, in the numerous roles in host insect morphogenesis and crypts of its posterior midgut (Kashkouli et al., behavior has remained poorly understood. 2019b). The symbiont is vertically transmitted Recently, the morphogenesis of the midgut by infection of the egg masses and orally symbiotic organ and the process of symbiont acquired by newborn nymphs (Kashkouli et colonization were investigated in the stinkbug al., 2019b). Upon hatching, the nymphs of Plautia stali Scott (Hem.: Pentatomidae) stink bugs get into a resting status in (Oishi et al., 2019). On the other hand, the aggregation and probe the eggs to acquire the most attention among behavioral observations symbiont (Kashkouli et al., 2019b). was given to the manipulation of wasp Two approaches, i.e. egg surface sterilization behavior and alteration of the wandering and heat treatment, seem possible to eliminate behavior of stinkbugs by symbionts. The the symbiont of stinkbugs. Egg surface endosymbiotic bacterium Cardinium sp. sterilization might be helpful for symbiont makes changes in the oviposition choice of the elimination because the symbiotic bacteria are parasitoid wasp, Encarsia pergandiella present on the surface of the egg mass and Howard (Hym.: Aphelinidae) (Kenyon and newborn nymphs acquire them by probing the Downloaded from jast.modares.ac.ir at 11:05 IRST on Saturday October 2nd 2021 Hunter, 2007). Exploring the wandering eggs (Taylor et al. 2017). High temperature behavior in the stink bugs Megacopta could also be a helpful strategy for symbiont punctatissima and H. halys showed negative elimination because the symbionts are affected and positive correlations between the supply of by environmental factors, especially symbiont available and the number of temperature (Kashkouli et al., 2018). wandering nymphs, respectively (Hosokawa et Symbiont deprivations using these two al., 2008; Taylor et al., 2014). However, the elimination approaches illustrate the necessity symbiotic and aposymbiotic newborn nymphs of the symbionts for successful growth of B. of B. germari and A. heegeri showed no germari, which symbiont eliminated insects significant differences in wandering behavior suffered retarded growth, lower longevity, (Kashkouli et al., 2019a, 2019b). adult body weight, and a marked reduction in The pistachio green stink bug, Brachynema demographic parameters especially r, which is germari Kolenati (Hemiptera: Pentatomidae) indicative of population decline (Kashkouli et is one of the most important pests of pistachio al., 2018, 2019b). nuts in Iran (Bigham and Hosseininaveh, In the present study, we aimed to use these 2010; Mehrnejad, 2001) that causes qualitative symbiont elimination approaches to evaluate and quantitative damages. This pest has 3–5 the importance of the Pantoea symbiont in the 138 Symbiont Alters Host Morphology and Mating____________________________________ insect morphology, emergence rates, and constantly in controlled temperature chambers mating frequency of B. germari, and to at 25°C. compare the specific effects of these symbiont- manipulation techniques. Experimental Design MATERIALS AND METHODS The egg masses were monitored daily until all of them either reached the adult stage or Insect Rearing died. To compare the emergence rates, the insects were checked daily during the life The original population of B. germari was cycle and their growth stages were registered. received from Pistachio Research Center The emergence rate for each stage was (Rafsanjan, Iran), in which insect adults were obtained by dividing the number with collected from pistachio orchards around emergence/total eggs. The nominal emergence Rafsanjan, Kerman province, Iran (30° 21′ N, rate data were statistically analyzed using 56° 0′ E). The insects were reared on Salsola kali Fisher’s exact probability test (SPSS v. 23). L. (insect second host plant) and pistachio nuts in After the adult emergence, each female was the plastic cages (50×25×35 cm, covered with paired with a male from the same treatment. fine mesh net) for three generations before the The morphometric features of the emerged experiments. A piece of wet cotton swab was females and males were measured separately. supplied as a moisture resource to the insects. For this experiment, 34 control, 28 surface- Rearing and all experiments were performed sterilized, and 19 heat-treated females and 30 within incubators (Binder KBF 240, Germany), control, 21 surface-sterilized, and 16 heat- which were set at constant temperatures, 25 or treated males were used. The lengths and 30°C, RH