Bacterial Symbionts of the Leafhopper Evacanthus Interruptus (Linnaeus, 1758) (Insecta, Hemiptera, Cicadellidae: Evacanthinae)
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Protoplasma (2016) 253:379–391 DOI 10.1007/s00709-015-0817-2 ORIGINAL ARTICLE Bacterial symbionts of the leafhopper Evacanthus interruptus (Linnaeus, 1758) (Insecta, Hemiptera, Cicadellidae: Evacanthinae) Teresa Szklarzewicz 1 & Beata Grzywacz2 & Jacek Szwedo3 & Anna Michalik1 Received: 29 January 2015 /Accepted: 6 April 2015 /Published online: 22 April 2015 # The Author(s) 2015. This article is published with open access at Springerlink.com Abstract Plant sap-feeding hemipterans harbor obligate symbi- betaproteobacterial symbionts are transovarially transmitted from otic microorganisms which are responsible for the synthesis of one generation to the next. In the mature female, symbionts leave amino acids missing in their diet. In this study, we characterized the bacteriocytes and gather around the posterior pole of the the obligate symbionts hosted in the body of the xylem-feeding terminal oocytes. Then, they gradually pass through the cyto- leafhopper Evacanthus interruptus (Cicadellidae: Evacanthinae: plasm of follicular cells surrounding the posterior pole of the Evacanthini) by means of histological, ultrastructural and molec- oocyte and enter the space between them and the oocyte. The ular methods. We observed that E. interruptus is associated with bacteria accumulate in the deep depression of the oolemma and two types of symbiotic microorganisms: bacterium ‘Candidatus form a characteristic ‘symbiont ball’. In the light of the results Sulcia muelleri’ (Bacteroidetes) and betaproteobacterium that is obtained, the phylogenetic relationships within modern closely related to symbionts which reside in two other Cicadomorpha and some Cicadellidae subfamilies are discussed. Cicadellidae representatives: Pagaronia tredecimpunctata (Evacanthinae: Pagaronini) and Hylaius oregonensis Keywords Symbiotic microorganisms . Sulcia . (Bathysmatophorinae: Bathysmatophorini). Both symbionts are Bacteriocytes . Transovarial transmission of symbionts . harbored in their own bacteriocytes which are localized between Cicadellidae the body wall and ovaries. In E. interruptus,bothSulcia and Introduction Handling Editor: Hanns H. Kassemeyer * Anna Michalik The Hemiptera are a large group of insects with feeding habits [email protected] that range from phytophagy to predation, including Teresa Szklarzewicz ectoparasitism and hematophagy (Forero 2008). Plant feeders [email protected] suck the phloem or xylem sap, or ingest plant cell content Beata Grzywacz (Backus 1988; Campbell et al. 1994;Sorensenetal.1995). [email protected] Among the six suborders of the Hemiptera (i.e. Paleorrhyncha, Jacek Szwedo Sternorrhyncha, Fulgoromorpha, Cicadomorpha, Coleorrhyncha [email protected] and Heteroptera; Szwedo et al. 2004;GrimaldiandEngel2005; Drohojowska et al. 2013), the Cicadomorpha: Clypeata lineage (uniting extinct Hylicelloidea and extant Cicadoidea, 1 Department of Developmental Biology and Morphology of Invertebrates, Institute of Zoology, Jagiellonian University, Cercopoidea and Membracoidea sensu lato) is the only one with Gronostajowa 9, 30-387 Kraków, Poland several strong adaptations for xylem feeding (Wang et al. 2012), 2 Institute of Systematics and Evolution of Animals, Polish Academy however in some of them shifts to cell-content and back to phlo- of Sciences, Sławkowska 17, 31-016 Kraków, Poland em feeding occurred (Dietrich 2013). The diet of xylem-feeding 3 Department of Invertebrate Zoology and Parasitology, Faculty of hemipterans is extremely unbalanced, because the amount of Biology, University of Gdańsk, Wita Stwosza 59, 80- nutrients in the xylem sap is 10 times less than that in the phloem 308 Gdańsk, Poland (Andersen et al. 1989). Therefore, most plant sup-sucking 380 T. Szklarzewicz et al. hemipterans live in mutualistic associations with symbiotic mi- in Cicadomorpha and Fulgoromorpha results from a multiple, croorganisms (bacteria or yeast) which synthesize missing nutri- independent replacement of symbiotic bacteria by other mi- ents and provide them to their hosts (Wilkinson and Ishikawa croorganisms. An ancestor of Cicadomorpha and 2001;Baumann2005; Douglas 2009). The sequencing of the Fulgoromorpha has been colonized by Sulcia and genome of bacteria inhabiting the body of hemipterans has indi- betaproteobacterial symbionts (probably 270 million years cated that these microorganisms possess the biosynthetic path- ago), but during the further evolution of the hemipteran line- ways necessary for the synthesis of essential amino acids, i.e. ages the betaproteobacterial co-symbiont was replaced by oth- leucine, serine, tryptophan as well as many vitamins and other er bacteria (Moran et al. 2005;BennettandMoran2013; Koga cofactors (Douglas 2006;Wuetal.2006;McCutcheonand et al. 2013; Koga and Moran 2014). In some planthopper Moran 2007; McCutcheon et al. 2009). families, i.e. Flatidae and Delphacidae as well as in leafhopper The obligate symbionts may be localized both extracellu- Scaphoideus titanus (Cicadellidae: Deltocephalinae) bacterial larly, in the lumen of midgut appendages (in some symbionts have been replaced by yeast symbionts (Sacchi heteropterans), and intracellularly, in cells of the midgut epi- et al. 2008;Michaliketal.2009; Noda 1977). thelium (in some heteropterans) or in specialized cells of me- On account of their crucial role, the obligate symbionts of sodermal origin termed bacteriocytes/mycetocytes (in most hemipterans are transovarially (vertically) transmitted be- hemipterans) (see Kikuchi 2009 for further details). The latter tween generations (see Buchner 1965 for further details). microorganisms are termed ‘mycetomic symbionts’. For this reason, bacteria living in different species of insects Among hemipterans, the Sternorrhyncha (aphids, psyllids, evolved independently from one another, i.e. without gene whiteflies, scale insects) are associated with one type of obli- exchange between bacteria in different hosts. gate mycetomic symbiont (termed ‘primary symbiont’), e.g. Evacanthus interruptus (Linnaeus, 1758) is a representative aphids harbor the bacterium Buchnera aphidicola, psyllids – of leafhoppers (Cicadomorpha: Membracoidea: Cicadellidae). the bacterium Carsonella ruddii (see Baumann 2005 for further So far, the symbiotic microorganisms present in the xylem- details). Apart from obligate symbionts, sternorrhynchans, as a feeding subfamily Evacanthinae Metcalf, 1939 have been ex- rule, hold additional symbionts (termed secondary or faculta- amined only fragmentarily (Buchner 1965; Takiya et al. 2006). tive symbionts) that may play various roles for their host in- Early microscopic observations conducted by Buchner (1965) sects, e.g. may protect them from infection by fungal pathogens showed that the females of Evacanthus interruptus possess two or attack by parasitic hymenopterans (Oliver et al. 2003;Scar- types of symbiotic microorganisms, which are localized in sep- borough et al. 2005). In contrast to the situation observed in arate bacteriocytes. More recently, using molecular methods, Sternorrhyncha, in Cicadomorpha and Fulgoromorpha (both Takiya et al. (2006) indicated the occurrence of Sulcia symbi- formerly treated as Auchenorrhyncha), two or more types of ont in the Pagaronia tredecimpunctata species Ball, 1902. obligate symbionts (termed ‘coprimary symbionts’) co-occur Koga et al. (2013) detected betaproteobacterial symbionts in and all of them are engaged in the synthesis of nutrients essen- the same leafhopper species. tial to the host insect (Moran et al. 2003;Takiyaetal.2006; The objectives of this study were to examine the ultrastruc- Bressan et al. 2009; Noda et al. 2012; Urban and Cryan 2012; ture of symbionts of E. interruptus, their distribution in the host Bennett and Moran 2013;Ishiietal.2013;Kogaetal.2013; insect body, mode of transovarial transmission from one gen- Michalik et al. 2014a;Wuetal.2006). eration to the next and to determine their systematic affinity. Molecular analyses have shown that both Cicadomorpha and Fulgoromorpha usually possess the obligate Bacteroidetes bacterium ‘Candidatus Sulcia muelleri’ (hereafter referred to Material and methods as Sulcia) and one other type of coprimary symbiont, e.g. gammaproteobacterium ‘Candidatus Baumannia Insects cicadellinicola’ (hereafter Baumannia), betaproteobacterium ‘Candidatus Zinderia insecticola’ (hereafter Zinderia), Adult females of Evacanthus interruptus (Linnaeus, 1758) betaproteobacterium ‘Candidatus Vidania fulgoroideae’ were collected from herbaceous plants in Kraków (Poland) (hereafter Vidania), betaproteobacterium ‘Candidatus Nasuia and near Vorokhta (Chornohora Mountain, Eastern deltocephalinicola’ (hereafter Nasuia), alphaproteobacterium Carpathians, Ukraine), from July to September. ‘Candidatus Hodgkinia cicadicola’ (hereafter Hodgkinia) (Moran et al. 2003, 2005; Takiya et al. 2006;McCutcheon DNA analyses et al. 2009; Gonella et al. 2011;Nodaetal.2012; Urban and Cryan 2012; Ishii et al. 2013;Kogaetal.2013). The results of The dissected bacteriomes were fixed in 96 % ethanol, washed recent molecular phylogenetic analyses (Bennett and Moran twice in sterile water and homogenized in 120 μl of 0.7 M 2013;Kogaetal.2013; Bennett et al. 2014; Koga and Moran NH4OH. After 15 min of incubation at 100 °C in alkaline con- 2014) suggest that this enormous diversity of symbionts found ditions, tubes were opened and further incubated at 100 °C for Bacterial symbionts of the leafhopper Evacanthus interruptus 381 10 min. Next, the samples were centrifuged (5 min, 12 000 rpm) Molecular