Inherited symbionts in grapevinefeeding leafhoppers and planthoppers

Among symbionts that revealed a major interest in recent years are those able to spread into insect host populations by manipulating host reproduction These microorganisms are intracellular symbionts able to enter female germ line cells and to be directly transmitted to the progeny The most known models of these manipulators of host reproduction are the a-Proteobacterium Wolbachia and the Bacteroidetes Cardinium In particular for Wolbachia a vast literature has been produced in the last few years In several arthropods these bacteria are able to manipulate host reproduction by determining cytoplas-mic incompatibility (CI) Other manipulations include feminization of genetic males, male killing, and induction of parthenogenesis (Stouthamer et al ., 1999; Bandi et al. 2001) . The capacity of these bacteria to interfere with the host reproduction offers intriguing clues for the development of strategies for biocontrol of insect population and for interfering with insect-vector competence (Zchori-Fein et al ., 2001) . CI is particularly promising, because it has been proposed to efficiently drive a desired genetic trait in an arthropod population or as a method to suppress natural populations of insect pests in a way analogous to the sterile insect technique (Zabalou et al , 2004)

Very few investigations have been performed to date on the microbiology of the insect vectors of phytoplasmas in grapevine, and several considerations can only be done from studies performed in phylogenetically related insect models . Among the Hemiptera related to S. titanus and H. obsoletus, an insect that received strong attention for microbial symbi-onts is the glassy-winged sharpshooter Homalodisca vitripennis (formerly H. coagulata), the vector of Xylella fastidiosa, the causative agent of Pierce's disease of grapevine . H. vitripennis, a Cicadellidae of the same family of S. titanus, has been shown to host two major symbionts, the y-Proteobacterium Candidatus Baumannia cicadellinicola (Moran et al ., 2003) and the Bacteroidetes Candidatus Sulcia muelleri (Moran et al ., 2005) . These two symbionts were defined as two "coprimary" symbionts due to their long-term coinheritance during the diversification of the host (Takiya et al , 2006) Based on cocladogenesis and genome size evidences and on functions deduced from the genomes, Moran (2007) proposed that Sulcia became an obligate associate of an insect host that began to feed by sucking on primitive vascular plants when they appeared on earth, i e , in the late Permian Only much later, in the tertiary, following an adaptation of the host to feed on a xylem sap diet, the second obligate symbiont Baumannia appeared . This evolutionary reconstruction and the definition of coprimary symbionts are supported by the actual function of the two symbionts of H. vit-ripennis Genome sequencing showed that these two symbionts play complementary roles for the host nutrition (Wu et al , 2006; McCutcheon and Moran, 2007) The very small, 245 kb genome of Sulcia retains pathways for the synthesis of most essential amino acids that are lacking in the xylem sap Baumannia, with its 686 kb genome, retains the amino acid synthesis pathways, lacking in the Sulcia genome, e .g., the histidine pathway, and those for biosynthesis of vitamins (Wu et al ., 2006; McCutcheon and Moran, 2007) .

Further studies have shown that besides the two primary symbionts, H. vitripennis hosts as a secondary symbiont Wolbachia, which was found to be the most frequently detected bacterium in the hemolymph of the glassy-winged sharpshooter (Takiya et al ., 2006; Cur-ley et al ., 2007) . Other studies have been performed on H. vitripennis from a more applied perspective for the control of Pierce's disease transmission . A culturable bacterium of the genus Alcaligenes that has been found associated with the insect vector of Pierce's disease and the grapevine plant has been proposed as a potential biocontrol agent for blocking the transmission of the disease through a paratransgenic approach (Bextine et al ., 2004; Miller et al , 2006)

As far as the case of FD and BN, very little is known about the microbiota associated with the insect vectors in general and about the sexual endosymbionts in particular The only sexual endosymbiont described to date associated with the vectors of phytoplasmas in grapevine is a Cardinium sp . that has been described in S. titanus (Marzorati et al ., 2006) .

Cardinium symbionts in S . titanus and H . obsoletus Using a classical community fingerprinting approach that consisted of the application of LH-PCR with primers targeting the 16S rRNA gene of bacteria, PCR-DGGE and sequencing, Marzorati et al . (2006) identified a major symbiont of S. titanus that was affiliated to the genus Cardinium. By using a specific PCR, Cardinium was identified in almost all of 103 field-collected individuals of S. titanus, with a minimal field infection rate of 94 .2% .

Cardinium associated with S. titanus had the closest 16S rRNA gene sequence identity and phylogenetic relationship with a symbiont of the tick Ixodes scapularis (98% identity) . It grouped in a phylogenetic branch with endosymbionts of several species of the genus Brevipalpus, including the feminizing symbiont of B. phoenicis (Weeks et al , 2001, 2003), and of other acarine genera such as Metaseiulus, Oppiella, and Petrobia (Jeyaprakash and Hoy, 2004; Weeks et al , 2003) A bit more distant, in a separate branch, grouped Candidatus Cardinium hertigii endosymbionts of Encarsia pergandiella (Zchori-Fein et al., 2001, 2004), and endosymbionts of Aspidiotus paranerii (Weeks et al , 2003) and Plagiomerus diaspidis (Zchori-Fein and Perlman, 2004) Recent experiments performed in our laboratories with molecular ecology and microscopy techniques indicated that Cardinium is also hosted by H. obsoletus in the female reproductive system Endosymbionts phylogenetically related to Cardinium have been observed in insects and Acarinae (both mites and ticks) (Kurtii et al ., 1996; Zchori-Fein and Perlman, 2004; Enigl and Schausberger, 2007) and recently, intracel-lular structures with the same micromorphology of Cardinium cells have been found in the femoral organs of spiders (Pekar and Sobotnik, 2007) . Endosymbionts related to Cardinium have also been identified in the plant-parasitic nematode Heterodera glycines (Noel and Ati-balentja, 2006) . By using a TEM approach endosymbiotic cells with the same morphological signatures of Cardinium were detected in several tissues of the nematode However, a phy-logenetic classification indicated that the symbiont of H. glycines is sufficiently distant from Cardinium to be attributed to a new genus named Paenicardinium (Noel and Atibalentja, 2006) All these recent observations further suggest that this Bacteroidetes group might be even more widespread and diverse than thus far supposed

The biological significance of the association between Cardinium and the different organisms has been addressed only in some cases . Cardinium has been shown to be associated with a variety of effects on the reproductive behavior (Kenyon and Hunter, 2007) and of reproductive alterations, including parthenogenesis, feminization of genetic males, and CI (Zchori-Fein et al , 2001, 2004) No data are currently available that indicate any bias in

Figure 16.3 Localization of Cardinium sp . in the organs and tissue of S. titanus. (A) A Cardinium cell in the fat body. (B) Micrograph showing Cardinium in the female reproductive system . Cardinium cells are indicated by an arrowhead in the follicular cells (FC) and by an arrow in the egg (E) (C) Magnification of Cardinium cells in the egg cytoplasm (D) A Cardinium cell in the salivary gland

Figure 16.3 Localization of Cardinium sp . in the organs and tissue of S. titanus. (A) A Cardinium cell in the fat body. (B) Micrograph showing Cardinium in the female reproductive system . Cardinium cells are indicated by an arrowhead in the follicular cells (FC) and by an arrow in the egg (E) (C) Magnification of Cardinium cells in the egg cytoplasm (D) A Cardinium cell in the salivary gland the sex ratio of S. titanus or H. obsoletus, and because the prevalence of Cardinium has been found similar in males and females, no obvious indication of interference with the sex ratio can be predicted Such a high prevalence in both sexes of S. titanus could be the result of a selective sweep caused by CI (Stouthamer et al , 1999), or of a mutualistic interaction with the host

Cardinium has been shown to be capable of colonizing several organs/tissues of S. titanus (Marzorati et al ., 2006; Bigliardi et al ., 2006; Sacchi et al ., 2008; Figure 16.3). Examination by TEM of adult females indicated the presence of numerous Cardinium cells in the fat body (Figure 16 3A), suggesting that this symbiont may have a metabolic role for the host Cardinium was also found in both the oocytes and the follicle cells of the ovary (Figure 16 3B and 16 3C), indicating that this bacterium is vertically transmitted to the offspring Another very interesting localization of Cardinium within the body of S. titanus is in the salivary glands (Figure 16 3D) This localization, besides overlapping with that of phytoplasmas, opens the question of whether this bacterium might be transmitted to the plant during feeding and from the plant to other insect individuals Horizontal transmission patterns for secondary sexual symbionts have been proposed several times based on the lack of evidence for cocladogensis between (secondary) symbionts and their hosts, but to our knowledge there are very few reports documenting horizontal transmission of obligate symbionts (Huigens et al ., 2001; Nussbaumer et al ., 2006) . For example, it has been reported that parthenogenesis-determining Wolbachia is horizontally transmitted from infected to uninfected larvae of the egg parasitoid wasp Trichogramma kaykai while feeding on the butterfly host Apodemia mormo deserti (Huigens et al 2001) . On the opposite, Matalon et al ., (2007) failed to find a horizontal transmission of Cardinium between the cactus scale Diaspis echinocacti and its parasitoids Plagiomerus diaspidis and Aphytis sp . and the hyperparasitoid Marietta leopardina. By using molecular ecology approaches, including fluorescence in situ hybridization, the authors were able to find Cardinium only in the parasitoid P. diaspis (Matalon et al ., 2007) .

A particular insect cell morphotype with the cytoplasm filled with Cardinium was found to be present in the apical region of the ovary (Sacchi et al ., 2008) . These cells resemble bac-teriocytes, i e , cells harboring symbiotic bacteria described in a variety of insects, including cockroaches and aphids (e.g., Sacchi and Grigolo, 1989; Nardon and Nardon, 1998) . It has been proposed that the bacteriocyte-like cells play an active role in the transmission of the symbionts to the progeny (Sacchi et al , 2008), similarly to the bacteriocytes of cockroaches and the termite Mastotermes darwiniensis (Sacchi and Grigolo, 1989) . In these insects another Bacteroidetes symbiont of the genus Blattabacterium lives within bacteriocytes that infiltrate the ovarioles, ensuring bacterial transmission to the oocytes In S. titanus such a transmission pattern was supported also by the detection of (symbiotic) Cardinium cells in the initial phases of embryo development and during the third nymphal stages when bacterial cells were found in the cytoplasm of the oogonia (Sacchi et al , 2008)

When examined by TEM, Cardinium cell presents several peculiar morphological structures (Bigliardi et al ., 2006; Sacchi et al ., 2008; Figure 16 .4), including a brush-like structure that resembles the parallel roads of ancient Roman towns (Zchori-Fein et al ., 2004), i . e ., the cardi (from which the genus name derives) Cardinium cell shows a two-layered envelope (an outer cell wall and an inner plasma membrane) and presents the already mentioned brush-like array of microtubule-like structures, which have been considered a morphological signature of the genus The microtubule-like complex consists of a system of parallel microtubule elements, a fibrous electron dense plaque, and a set of electron dense structures adhering to the outer leaflet of the bacterial plasma membrane (Figure 16 4) The metabolic and physiological significance of this complex tubular structure is unknown; it might perhaps represent a membrane system where enzymatic activities occur

A yeast-like symbiont in the body of S . titanus Several Hemiptera, including aphids and planthoppers, have been shown to host, besides prokaryotes, intracellular eukaryotic microorganisms (Buchner, 1965; Noda, 1974; Chen et al ., 1981; Ishikawa, 2003). One of the most studied models for the association with yeastlike symbionts (YLS) is the Asian rice brown planthopper Nilaparvata lugens (see for example Sasaki et al ., 1996) . It has been shown that in this planthopper the YLS was affiliated to Pyrenomycetes (now Sordariomycetes; Noda et al ., 1995). In N. lugens and the other insect species thus far investigated, essential roles for the normal host development have been proposed, including recycling of nitrogen contained in the uric acid waste produced by the host by way of uricase enzymes (Chen et al , 1981, Sasaki et al , 1996; Hongoh and Ishikawa, 1997; Wilkinson and Ishikawa, 2001; Cheng and Hou, 2005) In the tobacco beetle Lasioderma serricorne a fungal gut endosymbiont detoxifies plant material ingested by the beetle (Dowd, 1989) .

Very recently Sacchi et al . (2008) used molecular methods for the analysis of the fungal community associated with S. titanus By using LH-PCR with primers targeting the fungal 18S rRNA gene they discovered that several fungal species were associated with the leafhopper Among others (e g , Cladosporium cladosporioides) that were supposed to be occasional commensal symbionts of S. titanus, sequences with 93% identity with Bio-

Figure 16.4 A particular ultrastructural morphology characterizes the cells of Cardinium sp ., including the symbionts of S. titanus. (A) The brush-like structure as seen by a longitudinal view shows numerous microtubules (ML) inserted in an electron-dense plaque (EP) laying over a regularly distributed electron-dense structure (ES) . An outer envelope (OE) that covers the cell wall (CW) over the plasma membrane (PM) is clearly visible . (B) A transversal view of the brush-like structure clearly shows that it is composed of microtubules (arrow) The outer envelope covering the cell wall and the plasma membrane could be the residue of an invagination process within the host cell membrane

Figure 16.4 A particular ultrastructural morphology characterizes the cells of Cardinium sp ., including the symbionts of S. titanus. (A) The brush-like structure as seen by a longitudinal view shows numerous microtubules (ML) inserted in an electron-dense plaque (EP) laying over a regularly distributed electron-dense structure (ES) . An outer envelope (OE) that covers the cell wall (CW) over the plasma membrane (PM) is clearly visible . (B) A transversal view of the brush-like structure clearly shows that it is composed of microtubules (arrow) The outer envelope covering the cell wall and the plasma membrane could be the residue of an invagination process within the host cell membrane nectria pityrodes, a fungus belonging to the class of Sordariomycetes, were identified. PCR amplicons related to this fungus were observed in all of 32 S. titanus wild and greenhouse-maintained individuals, including males and females, indicating that this yeast is highly prevalent in the leafhopper The presence of the symbiont was confirmed by in situ hybridization analyses that allowed the identification of the symbiont in the fat bodies of S. titanus (Sacchi et al , 2008)

S. titanus YLS appear to belong to the same phylogenetic lineage of the Ascomycotina that encompasses Sordariomycetes, even though a relatively low nucleotide identity (93%) with the closest relative in the databases has been found Despite the fact that a longer sequence should be used to more carefully infer a precise phylogeny, the S. titanus YLS seems only distantly related with the already identified fungal symbionts of insects . It has been proposed that Sordariomycetes symbionts of Hemiptera stem from within the Cordyceps clade that contains obligate insect pathogens with filamentous growth (Suh et al . 2001) . Such a consideration highlights the subtle evolutive borderline between parasite/ pathogens and symbionts

The micromorphology of S. titanus YLS as examined by TEM shows rod-shaped cells of 3 x 15 ]m in size with a two-layered cell wall composed of a first 25 nm-thick electron-dense layer and a second one 100 nm-thick and electron-clear (Sacchi et al , 2008) The YLS appeared to divide by budding, as in several cases cell protuberances typical of yeast during division process were found High concentrations of YLS cells were found both in nymphs and adults, within certain specialized cells of the fat bodies (Figure 16 5A and 16 .5B) that look like mycetocytes (Cheng and Hou, 2005) . The very high number of YLSs observed in the fat body of S. titanus suggests that this microorganism plays a metabolic role that would possibly be linked to nitrogen recycling as already observed in other plan-thoppers like N. lugens. Indeed, we performed some experiments of rearing adult individuals of S. titanus in the laboratory on a diet based on sucrose solution without any nitrogen source Many individuals were able to live in those conditions for periods of almost two months, which is equivalent to the typical adult life span in the field Based on this evidence, it would be worthwhile to investigate further the possible role of the YLS in the nitrogen metabolism. Unfortunately, this kind of investigation is complicated by the fact that S. titanus is strictly monovoltine and its eggs must spend a long (but yet undefined) winter period at low temperature for hatching in spring

In planthoppers and leafhoppers, models other than S. titanus YLS are vertically transmitted to the progeny following a transovarial route (Chen et al , 1981; Ishikawa, 2003) This is also the case of the vector of flavescence dorée (Sacchi et al ., 2008) . YLS cells could be observed by TEM in the process of infecting the ovary by passing from the hemolymph to the cells of the follicular epithelium and hence to the oocyte through an endocytotic process (Figure 16 5C and 16 5D) This pattern of localization in the ovary and the finding of YLS cells in the initial phase of the embryo development indicate the capability to be vertically transmitted (Sacchi et al ., 2008). With respect to N. lugens eggs that host "symbiote ball" with a dense population of yeasts (Cheng and Hou, 2005), S. titanus ovary and young embryos contain a lower number of YLS cells . This envisages that the vertical transmission of the YLS in S. titanus has a lower rate than in N. lugens The vertical transmission of the

Figure 16.5 Localization of YLSs in the organs and tissues of S. titanus. (A) Several YLSs (arrows) are localized in the fat body (FB) close to the ovary (OV) . (B) Micrograph of the region at the border of the fat body and the ovary (OV) showing YLS cells (arrows) in the fat body. (C) Three YLS cells in the process of passing from the hemolymph to the ovary. One cell is visible in the egg (E; arrow), a second one is in the hemolymph (asterisk), and a third (arrowhead) is in the process of entering a follicular cell (FC) . (D) Magnification of a YLS cell entering a follicular cell of the ovary.

Figure 16.5 Localization of YLSs in the organs and tissues of S. titanus. (A) Several YLSs (arrows) are localized in the fat body (FB) close to the ovary (OV) . (B) Micrograph of the region at the border of the fat body and the ovary (OV) showing YLS cells (arrows) in the fat body. (C) Three YLS cells in the process of passing from the hemolymph to the ovary. One cell is visible in the egg (E; arrow), a second one is in the hemolymph (asterisk), and a third (arrowhead) is in the process of entering a follicular cell (FC) . (D) Magnification of a YLS cell entering a follicular cell of the ovary.

YLS seems to be in some way limited, possibly due to the large size of the YLS cells or to a potential competition for the transmission with the Cardinium bacterial symbiont .

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