An Unusual Symbiotic System in Elymana kozhevnikovi ( Zachvatkin , 1938 ) and Elymana sulphurella ( Zetterstedt , 1828 ) ( Insecta , Hemiptera , Cicadellidae : Deltocephalinae )

Morphological and molecular analyses revealed that the Deltocephalinae leafhoppers Elymana kozhevnikovi and E. sulphurella are host to four bacteriocyte-associated microorganisms: Sulcia (phylum Bacteroidetes), Nasuia (phylum Proteobacteria, class Betaproteobacteria), Arsenophonus (phylum Proteobacteria, class Gammaproteobacteria) and Sodalis-like bacteria (phylum Proteobacteria, class Gammaproteobacteria). Ultrastructural observations showed that in some bacteriocytes, apart from Sulcia, small elongated, rod-shaped bacteria are likewise present. The use of fluorescence in situ hybridization (FISH) revealed the occurrence of Sodalis-like bacteria in these bacteriocytes. Sodalis-like bacteria were also distributed in some cells of the bacteriome sheath. Nasuia and Arsenophonus co-existed in the same bacteriocytes. Moreover, Arsenophonus bacteria were dispersed in fat body cells. Wolbachia and Rickettsia were also detected alongside bacteriocyte-associated symbionts in E. kozhevnikovi and E. sulphurella. Sulcia, Nasuia, Arsenophonus and Sodalis-like bacteria are transovarially transmitted from one generation to the next.

Deltocephalinae leafhoppers, as other plant sapsucking hemipterans, live in mutualistic relations with microorganisms including bacteria and/or yeast-like symbionts.The presence of these associates is connected with the restricted diet of host insects, poor in essential nutrients (mainly amino acids) (reviewed e.g. in BUCHNER 1965;DOUG-LAS 1998;BAUMANN 2005).It is generally accepted that the occurrence of obligate symbionts is a result of an ancient acquisition of microorganisms by the ancestor of these insects resulting in the presence of microorganisms in all the descendants.As a consequence of the long-term coevolution between host insects and their symbionts, neither can survive as separate entities (i.e.host insects devoid of microorganisms cannot properly develop or reproduce, microorganisms cannot be cultivated on laboratory media).On ac-count of this mutualistic relationship, BUCHNER (1965) termed the obligate microorganisms "primary symbionts".BUCHNER (1965) also distinguished "accessory symbionts" (later termed "facultative symbionts" or "secondary symbionts") which may occur in some populations only.The presence of the latter in host insects is a consequence of, as a rule, their more recent acquisition.Secondary symbionts may fulfill different functions, e.g. they may protect host insects against parasites or heat stress (MONTLLOR et al. 2002;OLIVER et al. 2003;£UKASIK et al. 2013).Recent genomic analyses have shown that they may also be engaged in the synthesis of amino acids or other factors in metabolic pathways in different groups of hemipterans (TAKIYA et al. 2006;LAMELAS et al. 2011;SLOAN & MORAN 2012;LUAN et al. 2015;HUSNIK & MCCUTCHEON 2016).On ac-count on the metabolic complementarity of symbionts residing in auchenorrhynchous hemipterans and their mutualistic association with host insects, TAKIYA and co-workers ( 2006) termed these symbionts "coprimary symbionts".

Light and electron microscopy
The dissected abdomens of adult females of all species examined were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) at 4°C for three months.Next, the material was rinsed in the phosphate buffer with the addition of sucrose (5.8 g/100 ml) and postfixed in 1% osmium tetroxide in the same buffer.Then, the material was dehydrated in a graded series of ethanol and acetone and embedded in epoxy resin Epon 812 (Serva, Heidelberg, Germany).The semithin sections (1 µm thick) obtained from about twenty five females of E. kozhevnikovi and about twenty five females of E. sulphurella were stained with 1% methylene blue in 1% borax and photographed using a Nikon Eclipse 80i light microscope (LM).The ultrathin sections (90 nm thick) were contrasted with uranyl acetate and lead citrate and examined using a Jeol JEM 2100 electron transmission microscope (TEM) at 80 kV.

DNA analyses
DNA was extracted individually from the dissected abdomens of ten females preserved in 100% ethanol.DNA extraction was conducted using the Sherlock AX extraction kit (A&A Biotechnology) according to the manufacturer's protocol and next DNA was stored at 4 o C for further analyses.Molecular identification of bacteria associated with examined species was performed based on their 16S rDNA sequences which were obtained by amplifications with symbiont-specific primers (listed in Table 1).PCR reactions were run in a total volume of 20 ìl made up of 10 ìl of the PCR Mix Plus HGC mixture (A&A Biotechnology), 8 ìl of water, 0.5 ìl of each of the primers (10 ìM) and 1 ìl of the DNA template (1 ìg/ìl) under the following protocol: an initial denaturation step at 94 o C for a duration of 3 min, followed by 33 cycles at 94 o C for 30 s, 54-56 o C for 40 s (see Table 1), 70 o C for 1 min 40 s and a final extension step of 5 min at 72 o C. The PCR products were visualized by electrophoresis in 1.5% agarose gel stained with Midori Green (Nippon Genetics Europe).The positive PCR products were sent to an external company (Genomed) for DNA sequencing.The GenBank accession numbers of sequences obtained are listed in Table 2.

Phylogenetic analyses
The phylogenetic analyses were performed based on sequences of 16S rDNA of symbionts of E. kozhevnikovi and E. sulphurella and selected symbionts of Deltocephalinae leafhoppers deposited in the GenBank database.The sequences were then edited using BioEdit Sequence Alignment Editor 5.0.9 (HALL 1999), and following this, the sequence alignments were generated using Clus-talX 1.8 (THOMPSON et al. 1997).The phylogenetic analyses were conducted using MrBayes 3.2.2software (HUELSENBECK & RONQUIST 2001).In this analysis four incremental Metropolis-coupled MCMC chains (3 heated and 1 cold) were run for ten million generations with sampling every 1000 generations.The convergence of analyses was validated using Tracer software (RAMBAUT & DRUMMOND 2007) and the first 25 % of trees were discarded as 'burn-in'.The results of the Bayesian analysis were visualized using FigTree 1.4.0 software (RAMBAUT 2009).
Next, the slides were washed in PBS three times for 10 minutes, dried and covered with ProLong Gold Antifade Reagent (Life Technologies).The hybridized slides were then examined using a confocal laser scanning microscope Zeiss Axio Observer LSM 710.

Molecular identification of symbiotic microorganisms
Analysis of the 16S rDNA sequences of symbionts associated with Elymana kozhevnikovi and Elymana sulphurella indicated that the examined species of deltocephalinae leafhoppers are host to six kinds of bacteria: Sulcia, Nasuia, Arsenophonus, Sodalis,

Ultrastructure and distribution of symbiotic microorganisms
Histological observations revealed that paired bacteriomes occur in the females of Elymana kozhevnikovi and E. sulphurella (Fig. 3).Each bacteriome is composed of large bacteriocytes (Fig. 3).Two easily recognizable zones can be distinguished in the bacteriomes: a peripheral zone (Fig. 3) containing bacteriocytes with large, pleomorphic bacteria (Fig. 4) and a central zone (Fig. 3) with bacteriocytes containing large, lobated bacteria (Figs 7, 9) and large, elongated bacteria (Figs 7,8,8 insert,9).Fluorescence in situ hybridization of the bacteriocyte-associated symbionts identified the pleomorphic microorganisms residing in peripheral bacteriocytes as Sulcia bacteria (Fig. 13), and the lobated microorganisms as Nasuia bacteria (Fig. 13).Sulcia stain more intensely with methylene blue (Figs 3,8) and are more electrondense under an electron transmission microscope (Figs 4, 5) compared to Nasuia (Figs 7, 9).In all the examined individuals of E. kozhevnikovi and E. sulphurella, in the cytoplasm and in the nuclei of some bacteriocytes with bacteria Sulcia (Fig. 5) and in some cells of the bacteriome sheath (Fig. 6) small, elongated, rod-shaped microorganisms occur.These microorganisms measure about 0.4 µm in diameter.FISH experiments using specific probes showed that Sodalis-like bacteria are present in bacteriocytes with Sulcia bacteria as well as in cells of the bacteriome sheath (Fig. 13 insert).In all the bacteriocytes with Nasuia large, elongated microorganisms also occur (Figs 7, 8, 8 insert, 9).The latter measure 1-1.2 µm in diameter.The use of the FISH technique identified these microorganisms as Arsenophonus bacteria (Fig. 14).It was observed that both in the younger females and in older (i.e.reproductive) females in some bacteriocytes Arsenophonus bacteria undergo degeneration (Figs 8,10).In consequence, in these bacteriocytes numerous fagosomes and lamellar bodies appear (Fig. 10).Ultrastructural observations (Figs 11,12) as well as FISH identification (Fig. 14 insert) revealed that both in E. kozhevnikovi and E. sulphurella, Arsenophonus bacteria are also present in fat body cells.Some Arsenophonus bacteria residing in fat body cells likewise undergo degeneration (Fig. 12).

Transovarial transmission of symbionts
In reproductive females the bacteria leave the bacteriomes and invade ovaries.Ovaries of leafhoppers consist of seven elongated tubes called ovarioles.In each ovariole several linearly arranged oocytes are present.The oocytes are surrounded with a single layer of follicular cells (for further details concerning the organization of insect ovaries and course of oogenesis, see BÜNING 1994;BILIÑSKI 1998).The bacteria are released from the bacteriocytes and begin to invade follicular cells surrounding the posterior pole of the terminal oocytes which are at the stage of late vitellogenesis (Fig. 15).Sulcia, Nasuia, Arsenophonus and Sodalis-like bacteria enter the cytoplasm of follicular cells (Figs 16,17).After passing through the follicular epithelium, bacteria accumulate in the space between the former and the oocyte surface (termed the perivitelline space), finally forming a "symbiont ball" (Fig. 18).The bacteria residing inside the "symbiont ball" closely adhere to each other (Figs 19,20).

Discussion
Our morphological and molecular analyses revealed that in two species of Deltocephalinae leafhoppers, Elymana kozhevnikovi and E. sulphurella, an unusual combination of four microorganisms, Sulcia, Nasuia, Arsenophonus and Sodalis-like bacteria, occurs.To our knowledge, the co-existence of both ancient symbionts (i.e.Sulcia and betaproteobacteria) and more recently acquired Arsenophonus and Sodalis-like bacteria has not been observed in any other auchenorrhynchous hemipteran.Moreover, even the co-residence of three bacterial associates such as Sulcia, Nasuia and novel Arsenophonus/Sodalis-like bacteria is a very rare phenomenon within these insects.Both ancestral symbionts co-residing with Arsenophonus bacteria have only been found in the Deltocephalinae leafhopper Macrosteles laevis (KOBIA£KA et al. 2016), whereas these symbionts co-residing with Sodalis-like bacteria have only been observed in the spittlebug, Aphrophora quadrinotata (Cicadomorpha, Cercopoidea: Aphrophoridae) (KOGA et al.The situation observed in E. kozhevnikovi and E. sulphurella is of special interest.To our knowledge, the co-existence of Nasuia and Arsenophonus bacteria in the same bacteriocytes has never been reported for auchenorrhynchous hemipter- ans.Moreover, ultrastructural observations clearly indicate that in all the examined females of E. kozhevnikovi and E. sulphurella, both in bacteriocytes and in fat body cells, numerous Arsenophonus bacteria undergo degeneration (see Figs 8,10,12).Based on morphological observations, it is very difficult to comment on this phenomenon.Additionally, it cannot be excluded that some of the lamellar bodies in the bacteriocytes may represent remnants of Nasuia bacteria.It may be speculated that Arsenophonus, as a novel symbiont of E. kozhevnikovi and E. sulphurella, may be neutralized by the host insect.This, in turn, corresponds well with the above-mentioned hypothesis of the intermediate state between the Sulcia and Nasuia system and the Sulcia and Arsenophonus system.Thus, in both of these leafhoppers, the ancestral Nasuia still exists and functions, but newly acquired Arsenophonus bacteria have already begun the long process of elimination of this symbiont.It seems that the occurrence of Arsenophonus both in the specialized bacteriocytes and in the fat body cells confirms the intermediate state of symbiosis in Elymana.The verification of this hypothesis will be the subject of further work with the use of genomic analyses.Our PCR analyses revealed that Sodalis-like bacteria occur within all of the examined individuals of E. kozhevnikovi and E. sulphurella.The combination of results of ultrastructural observations and FISH method indicates that the microorganisms present in bacteriocytes with Sulcia and in the cells of the bacteriome sheath represent Sodalis-like bacteria.However, based on these results, we are unable to determine whether these bacteria are present both in the cytoplasm and in the cell nuclei.Taking into account the fact that E. kozhevnikovi and E. sulphurella are host to Rickettsia and Wolbachia which may occur intracellularly (ARNEODO et al. 2008;SCHULZ & HORN 2015; KOBIA£KA in preparation), it cannot be excluded that Sodalis-like bacteria are distributed in the cytoplasm, whereas Rickettsia or Wolbachia may reside inside the nucleus.To resolve this question, further detailed studies are required.The role of Sodalis-like bacteria in the biology of E. kozhevnikovi and E. sulphurella remains unclear.Several facts such as:

2013) and in the planthopper
(1) the occurrence of these bacteria in all the examined individuals detected by means of ultrastructural and molecular methods, (2) their transovarial transmission between generations and (3) lack of any symptoms of negative influence on host insects, suggest that Sodalis-like bacteria may be beneficial to their host insects.This, in turn, leads to the conclusion that these bacteria may represent the most recently acquired symbionts.
Using PCR diagnostics we detected that apart from Sulcia, Nasuia, Arsenophonus and Sodalis-like symbionts, E. kozhevnikovi and E. sulphurella may harbor Wolbachia and Rickettsia bacteria widely distributed among arthropods.It seems probable that both these bacterial associates are dispersed in different tissues of these insects.However, to determine the detailed distribution of Wolbachia and Rickettsia, their role in the biology of the host insects and mode of transmission between generations, further comprehensive molecular and ultrastructural analyses are needed.
Observations of the course of transmission of symbionts from the mother to offspring in different species of Deltocephalinae leafhoppers indicate that this process is uniform in this group of hemipterans (MÜLLER 1962;BUCHNER 1965;KOBIA£KA et al. 2015, 2016, 2017;BRENTASSI et al. 2017).In all the hitherto examined Deltocephalinae leafhoppers, the symbiotic microorganisms (both the ancient symbionts and novel associates -bacteria or yeast-like symbionts) invade the posterior pole of the ovariole.The symbionts individually migrate through the cytoplasm of follicular cells surrounding the terminal oocytes and next they gather in the perivitelline space in the deep invagination of oolemma in the form of a "symbiont ball".The single exception is M. laevis in which the novel associate Arsenophonus does not infect the ovarioles individually, but is transported inside cells of Sulcia.Thus, this atypical behavior of Arsenophonus is probably connected with the young condition of association between this microorganism and M. laevis.Our ultrastructural observations have shown that in E. kozhevnikovi and E. sulphurella, Arsenophonus and Sodalis-like bacteria are individually transmitted to the ovariole.This, in turn, indicates that both species of Elymana have already developed a stable system of novel symbiont transmission.
Wolbachia and Rickettsia.Sulcia, Nasuia, Arsenophonus and Sodalis were detected in all the examined individuals.The 16S rDNA sequences of Sulcia, Nasuia and Arsenophonus symbionts of both Elymana species were identical.Sequences of Sulcia and Nasuia show a high similarity (99%) to 16S rDNA sequences of Sulcia and Nasuia occurring in other representatives of Deltocephalinae, whereas 16S rDNA sequences of Arsenophonus symbionts are similar to those in Arsenophonus bacteria detected in the bat fly Basilia boardmani [KC597734] and aphid Aphis melosae [KF824532].In turn, the sequences of 16S rDNA of Sodalis-like bacteria of E. kozhevnikovi and E. sulphurella are almost identical (99% identity) and display 99% similarity to the 16S rDNA of bacteria Sodalis praecaptivus [CP006569] and the Sodalis symbiont of the clown stink bug Poecilocoris lewisi [AB915782].In some of the examined individuals, bacteria belonging to the genera Wolbachia (E.kozhevnikovi 2/6; E. sulphurella 2/8) and Rickettsia (E.kozhevnikovi 3/7; E. sulphurella 3/7) were also detected.Phylogenetic analyses of the obtained 16S rDNA sequences of Sulcia and Nasuia symbionts confirmed their systematic affiliation (Figs 1, 2).The topologies resulting from the Bayesian inference of the Sulcia and Nasuia symbionts are shown in Figs 1 and 2, respectively.

Fig. 1 .
Fig. 1.Phylogenetic tree showing the relationships of Sulcia symbionts of the examined Elymana kozhevnikovi and E. sulphurella leafhoppers and other representatives of the subfamily Deltocephalinae, based on 16S RNA gene sequences.The numbers associated with the branches indicate the Bayesian posterior probability values.The accession numbers of the sequences used in the phylogenetic analysis have been put in brackets.For outgroups, Sulcia symbionts of the planthopper Oliarus intermedicus (Fulgoromorpha) and leafhopper Evacanthus interruptus (Cicadellidae) were used.

Fig. 2 .
Fig. 2. Phylogenetic tree based on 16S rRNA sequences of Nasuia symbiont of the examined Elymana kozhevnikovi and E. sulphurella leafhoppers and other representatives of the subfamily Deltocephalinae, based on 16S RNA gene sequences.The numbers associated with the branches indicate Bayesian posterior probability values.The accession numbers of the sequences used in the phylogenetic analysis have been put in brackets.The Vidania symbiont of the planthopper Oliarus fulicicola was used as an outgroup.
Ommatidiotus dissimilis(MICHALIK et al. 2018a).According to KOGA and co-workers (2013), the three-symbiont association in A. quadrinotata may be a transitional situation in which the novel symbiont Sodalis did not yet eliminate the ancestral betaproteobacterial symbiont Zinderia.The similarity in the organization of the symbiotic systems in A. quadrinotata and Deltocephalinae leafhoppers E. kozhevnikovi and E. sulphurella indicates a similar evolutionary scenario occurring in these hemipterans.

Figs
Figs 13-14.Fluorescence in situ identification of symbionts of Elymana sulphurella.Fig. 13.Bacteriocytes with Sulcia (shown in green) and Nasuia (shown in red) bacteria.Fig. 13 insert.Sodalis-like bacteria (shown in green) residing in bacteriocytes (marked with a white, dashed line) and in cells of the bacteriome sheath (marked with a white, dotted line).Nasuia is shown in red.Fig. 14.Arsenophonus bacteria residing in bacteriocytes (shown in red).Fig. 14 insert.Arsenophonus residing in fat body cells (shown in red).Confocal microscope, scale bar = 25ìm.bn -bacteriocyte nucleus stained with DAPI.

Table 2
List of investigated symbiotic microorganisms with the accession numbers of the sequences Elymana kozhevnikovi(Zachvatkin, 1938)