Fungal and Bacterial Endosymbionts of Eared Leafhoppers of the Subfamily Ledrinae (Hemiptera: Cicadellidae)
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Appl Entomol Zool DOI 10.1007/s13355-016-0422-7 ORIGINAL RESEARCH PAPER Fungal and bacterial endosymbionts of eared leafhoppers of the subfamily Ledrinae (Hemiptera: Cicadellidae) Takanori Nishino1,2 · Masahiko Tanahashi1 · Chung-Ping Lin3 · Ryuichi Koga1 · Takema Fukatsu1,2,4 Received: 16 March 2016 / Accepted: 18 April 2016 © The Japanese Society of Applied Entomology and Zoology 2016 Abstract In general, leafhoppers (Hemiptera: Cicadelli- even the ancient Sulcia symbiont was finally lost and taken dae) possess a pair of bacteriomes in the abdomen, which over by the fungal symbiont. Meanwhile, the possibility harbor two types of obligate bacterial symbionts: a very of multiple independent fungal acquisitions from closely ancient Sulcia symbiont and another ancient Nasuia-allied related entomoparasitic Ophiocordyceps fungi cannot be co-symbiont (or a younger Baumannia co-symbiont). How- excluded. Our finding uncovers an evolutionary process ever, when we inspected three eared leafhoppers of the sub- from a prokaryotic essential symbiosis to a eukaryotic one. family Ledrinae, namely Ledra auditura Walker, Ledrop- sis discolor (Uhler) and Tituria angulata (Matsumura), L. Keywords Ledrinae · Leafhopper · Symbiont · discolor harbored only Sulcia symbiont while L. auditura Ophiocordyceps · Sulcia and T. angulata possessed no bacterial symbionts. Instead, all the species possessed specialized cells full of yeast-like fungal symbionts within fat bodies. Molecular phyloge- Introduction netic analysis revealed that the fungal symbionts are placed within the entomoparasitic fungal genus Ophiocordyceps Diverse insects and other organisms are associated with (Ascomycota: Hypocreales: Ophiocordycipitaceae). These symbiotic microbes, which entail a variety of ecological results suggest the possibility that (1) the fungal symbiont and evolutionary consequences (Bourtzis and Miller 2003; was acquired in the evolutionary course of the Ledrinae, Buchner 1965; Moran et al. 2008; Oliver et al. 2010; Wer- (2) the original fungus was likely an entomoparasite of the ren et al. 2008). In particular, plant-sucking insects of the genus Ophiocordyceps, (3) the fungal symbiont replaced order Hemiptera are highly dependent on their microbial the Nasuia-allied symbiont in an ancestral lineage, and (4) partners and possess well-developed symbiotic systems in their specialized tissues, cells or gut regions. Since plant sap is usually deficient in proteins, vitamins and * Takema Fukatsu other nutrients, the insects require symbiont-provisioned t‑[email protected] essential amino acids, B vitamins and other nutrients for 1 National Institute of Advanced Industrial Science their growth and survival (Baumann 2005; Douglas 1998; and Technology (AIST), 1‑1‑1 Higashi, Tsukuba 305‑8566, Kikuchi 2009; Moran et al. 2008; Salem et al. 2015). Japan Leafhoppers (Hemiptera: Auchenorrhyncha: Cicadomor- 2 Graduate School of Life and Environmental Sciences, pha: Cicadellidae) consist of over 20,000 described species University of Tsukuba, 1‑1‑1 Ten‑noudai, Tsukuba 305‑8572, in the world (Forero 2008), whose growth and survival are Japan dependent on symbiotic microorganisms (Douglas 1988; 3 Department of Life Science, National Taiwan Normal Noda et al. 2012). In general, leafhoppers possess a pair University, No. 88, Sec. 4, Tingzhou Rd., Taipei 11677, Taiwan of symbiotic organs, called bacteriomes, in the abdomen, which harbor two types of obligate bacterial symbionts: 4 Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo 7‑3‑1, Bunkyo‑ku, a very ancient and highly conserved flavobacterialSulcia Tokyo 113‑0033, Japan symbiont originating from the common ancestor of the 1 3 Appl Entomol Zool Auchenorrhyncha (Moran et al. 2005) and another ancient probably representing independent symbiont-replacing betaproteobacterial Nasuia-allied co-symbiont presumably events. In some leafhoppers such as Scaphoideus titanus dating back to the common ancestor of the Cicadomorpha Ball, strikingly, neither Sulcia, Nasuia nor Baumannia was (Bennett and Moran 2013; Koga et al. 2013; Noda et al. found, but a yeast-like fungal symbiont and a facultative 2012). On the other hand, some leafhopper lineages exhibit symbiont Cardinium were detected (Sacchi et al. 2008). different symbiotic microbial associates. For example, These observations highlight repeated acquisitions, losses, in the bacteriomes of leafhopper species of the tribe Pro- replacements and diversification of symbiotic microorgan- coniini, so-called sharpshooters, their bacteriomes harbor isms in the evolutionary history of the leafhoppers (Bennett Sulcia and a gammaproteobacterial Baumannia symbiont and Moran 2013; Bennett et al. 2016). (Moran et al. 2003; Takiya et al. 2006), suggesting replace- The subfamily Ledrinae is a small group of leafhoppers ment of the co-symbiont from Nasuia-allied to Baumannia consisting of some 38 genera and 300 species in the world in the lineage of sharpshooters (Bennett and Moran 2013, (Jones and Deitz 2009). Most members of the Ledrinae have 2015). Genomics of Sulcia, Nasuia and Baumannia sug- conspicuous ear-like projections on their head and/or tho- gested their cooperative provisioning of essential amino rax, after which they are referred to as eared leafhoppers. acids and other nutrients deficient in the plant sap diet of Notably, an early histological study reported that a Euro- the host leafhoppers (Bennett and Moran 2013; Bennett pean ledrine species, Ledra aurita (Linnaeus), possesses no et al. 2014, 2016; McCutcheon and Moran 2007; Wu et al. bacterial symbiont but a yeast-like fungal symbiont (Buch- 2006). On the other hand, Sulcia and a gammaproteobac- ner 1925), although no further study has been conducted. terial Arsenophonus symbiont were present in Macrost- Entomoparasitic fungi of the genus Cordyceps, com- eles laevis (Ribaut) (Kobialka et al. 2016), whereas Sulcia monly known as Touchu-Kasou in Japanese, Dong Chong and a gammaproteobacterial Sodalis symbiont were found Xia Cao in Chinese, and Plant Worms or Vegetable Wasps in in Cicadella viridis (Linnaeus) (Michalik et al. 2014), English, embrace over 400 described species in the world, A B C 5 mm 5 mm 5 mm D E F 20 µm 20 µm 20 µm Fig. 1 Eared leafhoppers and their fungal symbionts. Adult insects of a Ledropsis discolor, b Ledra auditura and c Tituria amgulata. Phase con- trast micrographs of fungal symbionts isolated from d L. discolor, e L. auditura and f T. angulata 1 3 Appl Entomol Zool and some species are famous for their potent use in Chinese trapping or tree sweeping and preserved in acetone (Fukatsu medicine (Kobayashi 1982; Mains 1958; Shimizu 1994). A 1999). Most of the samples were adult insects, whose abdo- recent systematic revision based on molecular phylogenetic men was detached and cut into halves in the midline, and a data divided the conventional Cordyceps into several genera half was subjected to DNA analysis while another half was including Cordyceps, Elaphocordyceps and Ophiocordyceps used for histological analysis. In addition, we obtained a L. (Sung et al. 2007). Previous studies showed that some yeast- discolor nymph and a T. angulata nymph, which were used like symbionts of aphids and planthoppers are phylogeneti- for detailed histological analysis (see Table 1). cally allied to Cordyceps/Ophiocordyceps fungi (Fukatsu and Ishikawa 1996; Noda et al. 1995; Suh et al. 2001). DNA sequencing In this study, we investigated the endosymbiotic microor- ganisms of three eared leafhopper species, Ledropsis discolor Each dissected abdominal sample was placed in a 1.5-ml (Uhler) (Fig. 1a), Ledra auditura Walker (Fig. 1b) and Titu- plastic tube, homogenized with a plastic pestle and sub- ria angulata (Matsumura) (Fig. 1c). Our results uncovered an jected to DNA extraction using the QIAamp DNA mini evolutionary transition from the ancestral bacterial co-sym- kit (QIAGEN). For L. discolor samples, freeze-thawing biosis through a bacterium-fungus dual symbiosis to a fun- and mixing with fine glass beads were also attempted gus-only symbiosis in the evolutionary course of the ledrine to facilitate disruption of the fungal cell walls. A 1.5-kb leafhoppers, where the fungal symbionts were derived from region of the fungal 18S rRNA gene was amplified by entomoparasitic fungi of the genus Ophiocordyceps. PCR with primers NS1 (5′-GTA GTC ATA TGC TTG TCT C-3′) (White et al. 1990) and FS2 (5′- TAG GNA TTC CTC GTT GAA GA-3′) (Nikoh and Fukatsu 2000) Materials and methods under a temperature profile of 94 °C for 4 min followed by 35 cycles of 94 °C for 30 s, 54 °C for 30 s and 72 °C Insects for 90 s, and a final extension at 72 °C for 5 min. A 1.5- kb region of the 16S rRNA gene of flavobacteria, includ- Samples of the eared leafhoppers used in this study are ing Sulcia symbionts of diverse hemipteran insects, was listed in Table 1. The insects were collected by either light amplified by PCR with primers 10CFBFF (5′-AGA GTT Table 1 Samples of eared leafhoppers of the subfamily Ledrinae examined in this study with nucleotide sequence accession numbers Insect species Collection locality Collection date; Sample ID for Nucleotide sequence accession number collector DNA sequences Fungal 18S rRNA Bacterial 16S Insect 28S rRNA gene rRNA gene gene Ledra auditura Oguni, Yamagata, Jul 2011; MT ogn1-ogn6 LC108751- – LC108774- Walker Japan LC108756 LC108779 Oguni, Yamagata, Jul 2014; TF ogn7-ogn8 LC108757- – LC108780- Japan LC108758 LC108781 Ledropsis discolor Tsukuba, Ibaraki, Nov 2014; MM – (Uhler)