Bacteriomes of the corn leafhopper, Dalbulus maidis (DeLong & Wolcott, 1923) (Insecta, Hemiptera, Cicadellidae: Deltocephalinae) harbor Sulcia symbiont: molecular characterization, ultrastructure, and transovarial transmission

In this study, we surveyed the bacteriome-associated microbiota of the corn leafhopper Dalbulus maidis by means of histological, ultrastructural, and molecular analyses. Amplification and sequencing of 16S rDNA genes revealed that the endosymbiont “Candidatus Sulcia muelleri” (Phylum Bacteroidetes) resides in bacteriomes of D. maidis. Phylogenetic analysis showed that the sequence was closely allied to others found in representatives of the subfamily Deltocephalinae. We failed to amplify other sequences as “Candidatus Nasuia deltocephalinicola,” a co-primary symbiont frequently associated to deltocephaline leafhoppers. In addition, a metagenetic analysis carried out in order to investigate the presence of other bacteriome-associated bacteria of D. maidis showed that the sequence of Sulcia accounted for 98.56 % of all the sequences. Histological and ultrastructural observations showed that microorganisms harbored in bacteriomes (central syncytium and cytoplasm of uninucleate bacteriocytes) look like others Sulcia described in hemipteran species and they were transovarially transmitted from mother to offspring which is typical of obligate endosymbionts. The only presence of Sulcia in the bacteriomes of D. maidis was discussed.


Introduction
Most members of the hemipteran suborder Auchenorrhyncha (leafhoppers, cicadas, froghoppers, treehoppers, and planthoppers) feed mainly on plant sap (xylem or phloem), which leads to a notoriously unbalanced nutrition (Moran 1998;Sandstrom and Moran 1999).Mutualistic associations with microorganisms (bacteria or yeasts) provide them with essential amino acids and/or vitamins that complement their diet (Buchner 1965;McCutcheon and Moran 2007;McCutcheon et al. 2009).
Leafhoppers, belonging to the family Cicadellidae, establish obligate symbioses with bacteria that live in the cytoplasm of specialized host cells (bacteriocytes) that form structures known as bacteriomes (Buchner 1965;Baumann 2005;Baumann et al. 2006).Obligate symbionts living within bacteriomes widely distributed in a clade of hosts are referred to as primary symbionts (Baumann 2005) and they are vertically transmitted through infected eggs (Buchner 1965;Moran et al. 2003Moran et al. , 2005;;Michalik et al. 2009;Kobialka et al. 2015a).The mutual dependence of endosymbionts and leafhoppers and the way they are transmitted to the offspring lead to their coevolution (Moran et al. 2003;Moran et al. 2005;Takiya et al. 2006;Moran 2007;Bennett and Moran 2013).Mutualistic interactions were established a long time ago through the initial infection of an ancestor of the insect group by a free-living microorganism (Baumann 2005;Baumann et al. 2006).
An obligate endosymbiont that is widespread in Auchenorrhyncha and has been documented in hosts from Fulgoroidea, Cicadoidea, Cercopoidea, and Membracoidea (which includes leafhoppers) is BCandidatus Sulcia muelleri( Bacteroidetes) (hereafter Sulcia).This symbiont is highly conserved and exhibits a drastic genome reduction (Moran et al. 2005;McCutcheon et al. 2009).Phylogenetic relationships among Sulcia strains identified in diverse auchenorrhynchan hosts are congruent with host's phylogenies suggesting that these associations and cospeciation dated back to 260 million years ago from the time these insects emerged (Moran et al. 2005).More recently, Koga et al. (2013) mentioned that the common ancestor of Cicadomorpha and Fulgoromorpha was also infected by a betaproteobacterial symbiont.However during evolution of some hemipterans lineages, the betaproteobacterium was replaced by other bacteria, e.g., gammaproteobacterium (Baumannia) in some Cicadellinae and alphaproteobacterium (Hodgkinia) in cicadas; therefore, Sulcia can coexist, depending on the particular host, with an array of co-primary symbionts belonging to different bacterial divisions (Moran et al. 2003;Takiya et al. 2006;Urban and Cryan 2012;Bennett and Moran 2013;Ishii et al. 2013;Koga et al. 2013;Kobialka et al. 2015a).
The term co-primary symbiont has been referred to as the obligate symbionts that co-occur inside bacteriomes (Moran et al. 2003;Takiya et al. 2006).Recent genomic analysis revealed that these symbionts have the smallest known bacterial genomes, ranging between 112 and 245 kb (Wu et al. 2006;Bennett and Moran 2013;Chang et al. 2015), and interestingly, it was found that the bacteria living in bacteriomes have complementary sets of pathways (Wu et al. 2006;McCutcheon andMoran 2007, 2010;McCutcheon et al. 2009).
The advent of high-throughput next-generation sequencing (NGS) technologies brought new tools to study the genomics at their highest depths at relatively low prices.So far, this new technology was used to sequence genomes of obligate symbionts such as Sulcia and Nasuia of the Deltocephalinae Macrosteles quadrilineatus and Macrosteles quadripunctulatus (Bennett andMoran 2013 andBennett et al. 2016) and Sulcia genoma of Dalbulus maidis (Chang et al. 2015).Sequencing technologies-based techniques such as metagenetics allow the analysis of the biodiversity of complex genomic samples that might include DNA from both culturable and unculturable organisms.Amplicons of high-throughput partial 16S rDNA are particularly useful to identify unculturable organisms.Therefore the metagenetic analysis is a powerful molecular tool to identify endosymbiotic microorganisms inhabiting bacteriomes, where more than one unculturable bacterial symbiont could co-exist.
The corn leafhopper, D. maidis (DeLong & Wolcott) (Cicadellidae: Deltocephalinae), is the major pest of maize Zea mays L. in the Americas.It is widely distributed from southern USA to central areas of Argentina (Nault 1990) and becomes a serious pest mainly in subtropical areas (Giménez Pecci et al. 2002;Virla et al. 2004;Carloni et al. 2013).Not much research has been done on the characterization of endosymbionts inhabiting bacteriomes of leafhoppers from the genus Dalbulus.As far as we know, only one report described the bacteriomes in Dalbulus elimatus, based on morphophysiological analysis (Galindo Miranda 1994).
Considering that several Auchenorrhyncha have been found associated with an ancient clade of the endosymbiont Bacteroidetes and other bacterial co-primary symbionts, our hypothesis states that the corn leafhopper, D. maidis, has bacteriomes containing Bacteroidetes and/or other microorganisms.Therefore, the purpose of this study was to look for the presence of bacteriomes in specimens of D. maidis, to survey their associated microbiota and to analyze the phylogenetic relationships with other endosymbionts of sap-feeding insects.In addition, the ultrastructure of endosymbionts and their mode of transmission were examined.

Materials and methods
Insects were collected during samplings performed in 2009-2010 on maize grown at BEl Manantial^(26°50′03, 41S-65°16′30, 62 W 435), an area within Chaco Subhúmedo (Tucumán province, Argentina).Insects were preserved in 96°e thanol and stored at −20°until they were dissected for molecular studies.Some adults of D. maidis collected from the field were used to establish a colony under controlled conditions (L16/D8 photoperiod; 24 ± 1 °C; 40-50 % RH) in a rearing room at División Entomología, Facultad de Ciencias Naturales y Museo, UNLP.Females at reproductive stage from the colony were used for light and electron microscopy analyses.

Genomic DNA preparation
One hundred bacteriome-like structures from 50 D. maidis females were obtained through the following procedure.Surface microorganisms were removed by sterilizing individuals with 70 % EtOH and 6 % sodium hypochlorite for 3 min, followed by three washes with sterilized water.Females were then dissected with fine needles under a stereomicroscope and bacteriome-like structures were placed into 96 % ethanol until processed later.Total genomic DNA was extracted with DNeasy Blood and Tissue Kit (QIAGEN, GmbH, Germany).Bacteriomes were crushed with a sterile iron pestle and homogenized in the buffer provided by the kit and the extraction followed the manufacturer's instructions.The quality and quantity of genomic DNA were assessed by electrophoresis in 0.7 % agarose gel stained with ethidium bromide.Gels were photographed and analyzed with an image analyzer.Extracted DNA was stored at −70 °C until analysis.

16S rDNA diagnostic PCR and sequencing
The polymerase chain reaction (PCR) for detection of symbionts of D. maidis was performed using specifics primers.The presence of Sulcia symbiont was assessed using the primers 10_CFB FF (5′-AGA GTT TGA TCA TGG CTC AGG ATG-3′) and 1515_R (5′-GTA CGG CTA CCT TGT TAC GAC TTA G-3′) based on the protocol developed by Moran et al. (2005).We also run reactions aimed at amplifying Nasuia symbiont (Betaproteobacteria) using the primers NcBeta_16S/ f1 (5′-AAG GAT AAA AGC GGG GAA AAC C-3′) and NcBeta_16S/r1 (5′-ACA CCA CTA AAA AAA ATT TTT AAC AG-3′) (Noda et al. 2012).Total DNA, extracted from bacteriomes of specimens of N. cincticeps harboring Nasuia (kindly provided by Dr. Hiroaki Noda), was included in the reactions as a positive control.Reactions were performed in 25 μl volume containing 50 ng of template DNA, 12 pmol of forward and reverse primers, 2.5 μl 10× reaction buffer (500 mM KCl; 100 mM Tris-HCl, pH 9.0 a 25 °C; 1 % Triton X-100), 1.5 mM MgCl 2 , 0.2 mM dNTPs, and 1.25 units of Taq polymerase (Inbio Highway®, Buenos Aires, Argentina).The thermocycler (PTC-0150 MiniCycler; MJ.Research.Watertown, MA, USA) was programmed as follows.(a) For the former reaction, an initial denaturation step at 94 °C for 2 min; followed by 35 cycles of a denaturing step at 94 °C for 1 min, an annealing step at 58 °C for 1 min, and an extension step at 72 °C for 2 min, followed by a final extension step at 72 °C for 6 min.(b) For the latter reaction, a denaturing step at 94 °C for 2 min followed by 35 cycles of a denaturing step at 94 °C for 30 s, an annealing step at 52 °C for 30 s, and an extension step at 72 °C for 2 min, followed by a final extension step at 72 °C for 6 min.PCR products were resolved by 1 % agarose gel electrophoresis stained with ethidium bromide and visualized by UV illumination.
The amplicons were precipitated by adding 1 vol of isopropanol and 0.1 vol of Na Ac.The DNA was sequenced by the dideoxy termination method (Sanger et al. 1977) using the BigDye Terminator Cycle Sequencing Ready Reaction kit and the automated ABI Prism 3730 DNA sequencer (Applied Biosystems, Macrogen, Seoul, Korea).

16S rDNA metagenetic analysis
An Illumina-based 16S rDNA amplicon diversity study was performed (Mr. DNA Shallowater, TX, USA).Bacterial 16S rDNA of total DNA from bacteriomes, isolated as described before, was amplified using primers 27F (5′-AGRGTTTG ATCMTGGCTCAG-3′) and ill519R (GTNTTACNGCG GCKGCTG) with barcode on the forward primer.A singlestep 30-cycle PCR using HotStarTaq Plus Master Mix Kit (Qiagen, Valencia, CA, USA) was carried out with the following cycling program: denaturation at 94 °C for 30 s, annealing at 53 °C for 40 s, and elongation at 72 °C for 1 min for 28 cycles.The PCR reaction was resolved in 2 % agarose gel electrophoresis and amplicons were purified using calibrated Ampure XP beads (Agencourt Bioscience Corporation, Danvers, MA, USA).Libraries were prepared with a TruSeq DNA library preparation kit.Sequencing was performed using an Illumina MiSeq 2000 sequencing system at MR DNA (www.mrdnalab.com,Shallowater, TX, USA).Data derived from sequencing were processed using MR DNA analysis pipeline (MR DNA, Shallowater, TX, USA).Briefly, sequences were depleted of barcodes and primers and sequences <150 bp as well as sequences with ambiguous base calls were removed.Sequences were denoised and chimeras were removed.Operational taxonomic units (OTUs) were defined by clustering at 3 % divergence (97 % similarity).Final OTUs were taxonomically classified using BLASTn against a curated database derived from GreenGenes, RDPII, and NCBI (www.ncbi.nlm.nih.gov;DeSantis et al. 2006; http://rdp.cme.msu.edu) and compiled into each taxonomic level.

Phylogenetic analysis
Phylogenetic analyses were performed using the obtained sequence of 16S rDNA with the primers describe by Moran et al. (2005) and similar sequences available at the GenBank database (Moran et al. 2005;Takiya et al. 2006;Noda et al. 2012;Wangkeeree et al. 2012;Ishii et al. 2013;Kobialka et al. 2015a).
Sequences were aligned using the ClustalW (Larkin et al. 2007) and the multiple alignment tool included in the Geneious 9.1.2package (Biomatters Ltd, Auckland, New Zealand).The alignment was automatically curated using Gblocks 0.91b (Talavera and Castresana 2007) using default settings except for the minimum length of a block which was set to 2. Best-fit model of evolution was selected with jModelTest 2.1.7(Darriba et al. 2012) and data matrices were analyzed under maximum likelihood criteria in PhyML 3.0 (Guindon and Gascuel 2003).The support of the groups within the tree was evaluated through bootstrap with 1000 replications (Felsenstein 1985).

Light and electron microscopy
Ten females at reproductive stage (4-6 days old) from the colony were fixed in glutaraldehyde 2 %, postfixed in osmium tetroxide 1 %, dehydrated in ethanol series (50-100 %), and embedded in Epoxy resin for 36 h at 35, 50, and 60 °C.Serial semithin sections (2-3 μm) were obtained by cutting them with a diamond knife in an Ultracut Reichert J-Supernova ultramicrotome.Then, samples were stained with 0.05 % O-Toluidine blue and examined with a light microscope Nikon YS2-H equipped with a digital camera Nikon D40.Ultrathin sections (60 nm) were mounted on copper grids, contrasted with uranyl acetate and lead citrate, and observed with a Jeol JEM 1200 EX II electron microscope.The micrographs were taken with a digital camera Erlangshen ES 1000W.

Bacteriome-like structures
All the specimens of D. maidis observed with the stereomicroscope presented bacteriome-like structures that were bilaterally paired and located in the lateral margins of the first and second abdominal segments (Fig. 1a).These structures were bean-shaped, small (0.4-0.6 mm long), yellow colored (Fig. 1b), and supplied with abundant tracheae.
16S rDNA and phylogenetic analysis PCR reaction using the primers 10_CFB FF and 1515_R amplified a 1368-bp-long DNA fragment that corresponded to the 16S rDNA of the putative endosymbiont of D. maidis.The sequence was annotated at the NCBI database (JX514697).A Blast analysis showed that the sequence was 99 % similar to those of the Bacteroidetes Sulcia.Additionally, PCR reactions with primers designed to amplify 16S sequences homologous to Nasuia only gave a positive result when the DNA extracted from bacteriomes of N. cincticeps was used as a control.
Phylogenetic analysis of the 16S rDNA sequences of Sulcia symbiont of various representatives of Cicadomorpha and one representative of Fulgoromorpha generated a tree showing that the sequence of Sulcia from D. maidis was closely related to Sulcia endosymbionts from leafhoppers species in the same subfamily, Deltocephalinae (Fig. 2).

16S rDNA metagenetic analysis
Since other auchenorrhynchans harbor more than one endosymbiont within bacteriomes, we made a metagenetic analysis of bacteriome's DNA using 16S rDNA primers as described in materials and methods and because of this we called it metaribosomic analysis.Cleaning raw data rendered a total number of 77.518 sequences, which were organized in 102 OTUs.The sequence of Sulcia was highly represented and accounted for 98.56 % of all sequences.The remaining 1.44 % of sequences included 101 OTUs different from Sulcia and, among these low-abundant sequences, we did not find the presence of any other reported Deltocephalinae endosymbiont nor any other closely related organism.These sequences were considered contaminants and might correspond to sequences of microorganisms that make up the microbiome of the insect and microorganisms acquired during the processing of samples (Fig. 3).

Light and electron microscopy analyses
Bacteriomes of D. maidis were composed of a monolayered epithelium with large translucent cells with large nucleus and nucleolus, a syncytium, and an aggregate of uninucleate cells (Fig. 4a-c).The organisms localized in the syncytium stained intensely with methylene blue.Ultrastructural studies revealed large (3-4 μm wide and 8-12 μm long), irregular in shape, electron-dense, pleomorphic organisms that are found in close vicinity to mitochondria (Fig. 5a-c).Organisms quite different in shape and in staining behavior (relative to the cytoplasm and the nucleus) filled the cytoplasm of uninucleate cells (Fig. 5c); these organisms were surrounded by membranes and presented an electron-dense body in their cytoplasm (Fig. 5d).
In semithin sections of mature females, we observed that bacteriomes were located close to the ovaries.Ovaries of D. maidis are composed by six ovarioles (for further details concerning morphology of the reproductive system of D. maidis, see Tsai and Perrier 1996).In the terminal part of each ovariole, there was an oocyte at the stage of advanced vitellogenesis (Fig. 6a).In addition, different stages of transovarial transmission of symbiotic bacteria were observed.Bacteria gathered at the terminal syncytial zone of the bacteriome, leave the cytoplasm of the syncytia (Fig. 6a, b), and invade the posterior pole of oocytes through the follicular cells (Fig. 6c).

Discussion
This is the first study describing bacteriome-associated endosymbionts of D. maidis, a leafhopper vector of three important phytopathogens: mollicutes Spiroplasma kunkelli, Maize bushy stunt phytoplasma, and Maize rayado fino virus that either alone or in combination are the causative agents of Bcorn stunt,^a disease complex that has become a limiting factor for maize production in tropical and subtropical America (Nault and Ammar 1989;Oliveira et al. 1998;Summers et al. 2004;Virla et al. 2004;Carloni et al. 2013).
The morphology and localization of the structures described within all specimens of D. maidis as well as their similar appearance to others described for deltocephaline leafhoppers such as Helochara communis (Cicadellidae) (Chang and Musgrave 1972), D. elimatus (Galindo Miranda 1994), N. cincticeps (Noda et al. 2012), and Macrosteles laevis (Kobialka et al. 2015a) lead us to conclude that the structures observed in D. maidis are typical of bacteriomes that host endosymbiotic organisms.
Amplification and sequencing of the 16S rDNA using bacteriomes DNA as a template showed that bacteriomes host Sulcia and the sequence was 99 % similar to other Bacteroidetes.Furthermore, the Sulcia sequence of D. maidis belongs to the same clade that includes Sulcia from other Deltocephalinae leafhoppers such as Matsumuratettix hiroglyphicus, N. cincticeps, Macrosteles sp., and Macrosteles laevis reported by Wangkeeree et al. (2012), Noda et al. (2012), Ishii et al. (2013), andKobialka et al. (2015a), respectively, reflecting the host-symbiont phylogenetic concordance.Molecular studies of symbionts associated with the three genera of the deltocephalines mentioned above showed that bacteriomes harbor bacteroidetes Sulcia and the betaproteobacterial Nasuia as well.Besides, other minor sequences of bacteria have been found (Noda et al. 2012;Ishii et al. 2013;Kobialka et al. 2015a, b).We unsuccessfully tried to amplify through PCR the 16S rDNA of Nasuia.Accordingly, Morphology is additionally an important feature to characterize endosymbionts.In general, primary symbionts are distinguished by their unusually large cell size and pleomorphic shape (Baumann 2005;Takiya et al. 2006).Bacteria within bacteriomes of D. maidis had morphological characteristics typical of obligate endosymbionts and particularly they were similar to other Sulcia symbionts already described in the deltocephalines N. cincticeps (Noda et al. 2012), Macrosteles laevis (Kobialka et al. 2015a), and Deltocephalus pulicaris (Kobialka et al. 2015b).The morphology of the bacteria inhabiting bacteriomes of D. maidis was also similar to the Ba-symbiont^present in the syncytium of the mycetome of H. communis described earlier by Chang and Musgrave (1972) and Graphocephala coccinea (Hemiptera: Jassidae) by Kaiser (1980).In species of Cicadellidae, co-primary symbionts were found residing in different bacteriocytes (Noda et al. 2012;Ishii et al. 2013;Szklarzewicz et al. 2016) or co-residing in the same bacteriocyte (Michalik et al. 2014).In agreement with our molecular results, we found that the syncytial zone of bacteriomes of D. maidis and the cytoplasm of uninucleate bacteriocytes harbored bacteria that look like Sulcia.Furthermore, the fact that symbionts appearing in micrographs as if they were leaving the syncytial zone of bacteriomes of D. maidis, apparently migrating to the posterior pole of oocytes and entering through follicular cells, suggests that they are vertically transmitted from mother to offspring, which is the typical mode of transmission of obligate symbionts (Buchner 1965;Moran et al. 2003Moran et al. , 2005;;Sacchi et al. 2008;Michalik et al. 2009;Szklarzewicz et al. 2016).
Although Sulcia has frequently been found coexisting with a co-primary symbiont in Deltocephalinae species, this study suggest that it might be the only symbiont-associated bacteriome in D. maidis.The following can be regarded as evidences of this outcome.Firstly, Bennett and Moran (2013) mentioned that replacements and losses of symbionts sometimes occurred in phloem-feeding lineages.This is the case of the deltocephaline Scaphoideus titanus, a species lacking both Sulcia and Nasuia symbionts but possessing transovarially transmitted Cardinium (Bacteroidetes) and yeast-like symbionts instead, which are related to those found in certain lineages of planthoppers and aphids (Sacchi et al. 2008).Secondly, we failed to amplify any endosymbionts other than Sulcia.Lastly, the metaribosomic approach proved that Sulcia is likely the only microorganism inhabiting bacteriomes of D. maidis.Thus, we suggest that the dual symbiotic BSulcia-betaproteobacteria^system might not be a rule for Deltocephalinae leafhoppers.2015) identified the complete genome sequence of Sulcia ML strain from whole D. maidis specimens from Brazil.Since we found that Sulcia appears to be the only symbiont within bacteriomes of this species, we are currently planning to sequence its genome in order to carry out a comparative genetic analysis with other organisms including Sulcia found in D. maidis from Brazil by Chang et al. (2015).Furthermore, larger samples of D. maidis populations as well as other species of Dalbulus genus should be obtained in order to identify if other symbionts coexist with the primary symbiont Sulcia.

Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of interest.

Fig. 1
Fig. 1 Bacteriome-like structures in D. maidis.a Habitus of an adult female showing the localization on each side of the abdomen (arrows).Scale bar = 1 mm; b yellow-colored dissected bacteriome-like structures.Scale bar = 0.6 mm

Fig. 2
Fig. 2 Maximum likelihood (ML) tree based on 16S rDNA gene sequences of the Bacteroidetes symbiont BCandidatus Sulcia muelleriô btained from bacteriomes of D. maidis (in bold) and others selected sequences of auchenorrhynchan species.The name of the host species and family and subfamily (in brackets) are shown as each taxon label.GenBank accession numbers of sequences of 16S rRNA for symbionts are given in brackets too.Outgroups included a secondary symbiont of Melanococcus albizziae (Maskell, 1892) (Hemiptera: Pseudococcidae) (AF476106) and Aphalaroidea inermis Crawford, 1914 (Hemiptera: Psyllidae) (AF263556) and BCandidatus Blochmannia herculeanus^endosymbiont of Camponotus herculeanus (Linnaeus, 1758) (Formicidae) (AJ250715).Bootstrap values (%) were obtained from a search with 1000 replicates.Numbers above nodes indicate bootstrap values with more than 50 % support

Fig. 4
Fig. 4 Light micrographs of a bacteriome-like structure of D. maidis.a Localization in the abdomen and general appearance showing the syncytial tissue and the uninucleate cells.Scale bar = 0.25 mm; b detail of the monolayered epithelium and the syncytial tissue; c detail of irregular and large size organism in the syncytial zone (arrows) and uninucleate cells.Scale bar = 25 μm.A, abdomen; T, thorax; E, epithelium; Syn, syncytial tissue; Uc, uninucleate cells

Fig. 3
Fig. 3 Frequency of operational taxonomic units (OTUs) obtained by a 16S rDNA metagenetic analysis from DNA obtained from bacteriomes of D. maidis.Others refer to contaminants microorganisms

Fig. 6
Fig. 6 Light micrographs showing different stages of the transovarial transmission of symbiotic bacteria in D. maidis (a) symbionts (arrows) in the terminal syncytial zone of the bacteriome.Scale bar = 50 μm; b detail of the symbionts in the terminal zone of the bacteriome and near the follicular cells of the terminal oocyte.Scale bar = 25 μm; c symbionts (arrows) enter the posterior pole of the oocyte through follicular cells.Scale bar = 50 μm.F, follicular cells, Syn, syncytium