Folia biologica (Krakoacutew) vol 57 (2009) No 3-4doi103409fb57_3-4131-137

Ultrastructure and Transovarial Transmission of Endosymbiotic

Microorganisms in Conomelus anceps and Metcalfa pruinosa (Insecta

Hemiptera Fulgoromorpha)

Anna MICHALIK Wsup3adyssup3awa JANKOWSKA and Teresa SZKLARZEWICZ

Accepted April 20 2009

MICHALIK A JANKOWSKA W SZKLARZEWICZ T 2009 Ultrastructure and transovarialtransmission of endosymbiotic microorganisms inConomelus anceps andMetcalfa pruinosa(Insecta Hemiptera Fulgoromorpha) Folia biol (Krakoacutew) 57 131-137Endosymbiotic microorganisms commonly occur in fulgoromorphans as in other plantsap-sucking hemipterans Large syncytial organs termed mycetomes are present in the bodycavities of Conomelus anceps (Delphacidae) and Metcalfa pruinosa (Flatidae) in the closevicinity of the ovaries The mycetomes are surrounded by a one-layered epithelium Themycetome cytoplasm is filled with yeast-like symbiotic microorganisms (YLSs) The YLSsare transovarially transmitted to the next generation The endosymbionts are released fromthe mycetomes and migrate towards the ovarioles containing vitellogenic oocytes The YLSspass through the cells of the ovariole stalk (pedicel) and enter the perivitelline space Then adeep depression is formed at the posterior pole of the oocyte The YLSs accumulate in theoocyte depression and form a characteristic symbiont ball The mycetome cytoplasm ofMetcalfa pruinosa as well as epithelial cells surrounding the mycetome contain smallrod-shaped bacteriaKey words Endosymbiotic microorganisms transovarial transmission ovarioleFulgoromorpha planthoppersAnnaMICHALIK Wsup3adyssup3awa JANKOWSKA Teresa SZKLARZEWICZ Department of SystematicZoology and Zoogeography Institute of Zoology Jagiellonian University R Ingardena 630-060 Krakoacutew PolandE-mail szklaizujedupl

Fulgoromorphans like other plant sap-suckinghemipterans harbor obligate intracellular symbi-otic microorganisms (see BUCHNER 1965 HOUKamp GRIFFITHS 1980 BAUMANN 2005 2006 forfurther details) Endosymbiotic microorganismsare housed in large cells termed mycetocytes(=bacteriocytes) Mycetocytes are usually inte-grated into discrete organs termed mycetomes(=bacteriomes) that in some hemipterans may besurrounded by a one-layered epithelium BUCH-NER (1965) on the basis of comparative studies ofall hemipteran groups distinguished two catego-ries of endosymbiotic microorganisms primaryendosymbionts (currently called P-symbionts) andaccessory endosymbionts (currently called facul-tative secondary or S-symbionts) Primary endo-symbionts are always present in all the specimenswhereas secondary endosymbionts occur in somepopulations only More recent studies have shownthat the occurrence of primary endosymbionts in

the insect body is related to a restricted diet defi-cient in some essential nutrients (see BAUMANN2005 2006 for full review) Since hemipteransfeed on phloem sap devoid of amino acids theirprimary endosymbionts are responsible for aminoacid synthesis and delivery to the host insect (egSASAKI amp ISHIKAWA 1995 WILKINSON amp ISHI-KAWA 2001) Thus the occurrence of primary en-dosymbionts is necessary for the survival andreproduction of the host insects In contrast to pri-mary endosymbionts the role of the secondary en-dosymbionts for their host insects remains unclearRecent studies revealed that aphids harboring S-symbionts may better survive heat stress (MONTL-LOR et al 2002) as well as attacks by parasitic hy-menopterans (OLIVER et al 2003) and fungalpathogens (SCARBOROUGH et al 2005) than ster-ile specimens

The symbiotic associations between primary endo-symbionts and insects are the results of ancient in-

_______________________________________Supported by Research Grant DSIZZS2008

fections with free living bacteria (BAUMANN 2006)whereas associations between secondary endosym-bionts and insects are much younger and are due tomultiple independent infections (THAO et al 2000)

Most hemipterans have prokaryotic endosymbi-onts however eukaryotic microorganisms histori-cally termed ldquoyeast-like symbiontsrdquo (YLSs) occurin some aphids scale insects leafhoppers and ful-goromorphans (see BUCHNER 1965 ISHIKAWA2003 for further details)

Both bacterial and yeast-like endosymbionts arematernally inherited by transovarial transmissionStudies on transovarial transmission of bacterialendosymbionts have shown that variable modes ofovary infection by bacteria exist in insects (macrELA-ZOWSKA amp BILINtildeSKI 1999 SZKLARZEWICZ ampMOSKAL 2001 SZKLARZEWICZ et al 2006) Theendosymbiotic microorganisms may invade younggerm cells (=cystocytes) or oocytes in the olderstage of development (ie vitellogenic or chorio-genic) The ovaries may be invaded by bacteria orby intact mycetocytes The endosymbionts maymigrate through the neighboring follicular cells(ie cells surrounding oocytes) or may enter thecytoplasm of follicular cells In contrast to bacte-rial endosymbionts the transovarial transmissionof YLSs is poorly known and there are only threepublished observations on this process (NODA1977 CHENG amp HOU 2001 SACCHI et al 2008)two of which consider members of the family Del-phacidae

The present study was undertaken to provide in-formation about the mode of transmission of YLSsin Metcalfa pruinosa a member of the Flatidaefamily and in another member of the family Del-phacidae Conomelus anceps

Material and Methods

Adult specimens of Conomelus anceps (Germar1821) were collected in July near Nowy Targ(southern Poland) Adult specimens of Metcalfapruinosa (Say 1830) were collected in July inGrabels (southern France) The abdomens and dis-sected ovaries of individuals of each species werefixed in 25 glautaraldehyde in 01 phosphatebuffer (pH 74) for three months The material wasthen rinsed in 01 M phosphate buffer (pH 74)with addition of 58 sucrose postfixed in 1 os-mium tetroxidae dehydrated in a series of alcoholand acetone and embedded in epoxy resin Epox812 (Fullam Inc Latham NY USA) Semithinsections were stained with 1 methylene blue in1 borax and photographed in a Jenalumar (ZeissJena) microscope Ultrathin sections were stainedwith lead citrate and uranyl acetate and examinedusing a JEM 100 SX electron microscope at 80 kV

Results

Gross morphology of the ovaries

The paired ovaries of Metcalfa pruinosa andConomelus anceps consist of several ovarioles oftelotrophic type (Fig 1) The individual ovariole issubdivided into a terminal filament tropharium(trophic chamber) vitellarium and ovariolar stalk(pedicel) that joins the ovariole to the lateral oviduct(Fig1)The trophariumiscomposedofsyncytial lobescontaining 3-5 trophocyte nuclei (Fig 1) Early pre-vitellogenic oocytes (termed arrested oocytes) andprefollicular cells are present at the base of the tro-pharium (Fig 1) The vitellarium in mature femalescomprises 4-5 linearly arranged oocytes that aresurrounded by a one-layered follicular epithelium(Fig 1) The vitellarial oocytes develop through

A MICHALIK et al132

Fig 1 Schematic representation of the ovariole of aplanthopper Arrows YLSs F follicular epithelium OC oocyte TF terminal filament TR tropharium VIT vitellarium P pedicel

Endosymbiotic Microorganisms in Planthoppers 133

Figs 2-8 Fig 2M pruinosa The mycetome filled with YLSs (arrowhead) in the body of the male T testis Methylene blue H570 Fig 3 M pruinosa Fragment of the epithelial cell surrounding the mycetome Arrows rod-shaped bacteria EN nucleus of the epithelial cell L lipid droplet TEM H 18 100 Fig 4 C anceps The mycetome filled with YLSs(arrowheads) in the body of the female Arrows mycetome nuclei embedded in the common cytoplasm EP epithelial cellssurrounding the mycetome L lipid droplets Methylene blue H 530 Fig 5M pruinosa Cross section through the cell of theYLS Arrow cell wall composed of two distinct layers YN nucleus NU nucleolus TEM H 16 500 Fig 6 M pruinosaFragment of the mycetome cytoplasm Arrows rod-shaped bacteria MN mycetome nuclei embedded in the commoncytoplasm TEM H 17 500 Fig 7 M pruinosa Fragment of the cell of the YLS Arrow cell wall L lipid droplet M mitochondria YN nucleus TEM H 15 100 Fig 8 C anceps The YLS reproducing by budding TEM H 9 300

three stages previtellogenesis (ie synthesis andaccumulation of RNArsquos) vitellogenesis (ie syn-thesis and accumulation of reserve substances) andchoriogenesis (ie synthesis and secretion of pre-cursors of eggshells and their deposition on the oo-cyte surface) (for a detailed description of ovariesof planthoppers see SZKLARZEWICZ et al 2007)

Ultrastructure distribution and transovarialtransmission of yeast-like endosymbiotic micro-organisms

Both in males and females of C anceps and Mpruinosa the spaces between internal organs arefilled with large structures termed mycetomes(Figs 2 4) The mycetomes have a syncytial char-acter ie they have numerous nuclei embedded in

a common cytoplasm (Figs 4 6) The mycetomesare surrounded by a one-layered epithelium (Fig 4)In young specimens epithelial cells are small izo-diametric and closely adhere to each other (notshown) while in the older specimens they becomeirregular and voluminous with large lipid dropletsin the cytoplasm (Fig 4) Numerous rod-shapedbacteria occur in epithelial cells (Fig 3) as well asin mycetome cytoplasm (Fig 6) of all examinedspecimens of M pruinosa The bacteria measure12-16 Fm in length and 03-05 Fm in diameterBoth in M pruinosa and C anceps the mycotomecytoplasm contains an enormous number ofyeast-like symbionts (YLSs) (Figs 2 4) The sizeof YLSs is 8-10 Fm in length and 30-35 Fm in di-ameter They are surrounded by a thick cell wallcomposed of two distinct layers (Figs 5 7 8) The

Figs 9-13 Figs 9 10 Longitudinal section through the posterior end of the ovariole pedicel (P) and lateral oviduct (LOV)during infection by the YLSs (arrows) Methylene blue H 1 000 Fig 9 C anceps Fig 10 M pruinosa Asterisk deepdepression of the oolemma F follicular cells OC oocyte Fig 11 C anceps Fragment of the follicular epithelium (F)during migration of YLSs FN follicular cell nucleus TEM H 6 500 Figs 12 13 C anceps Cross section through theposterior pole of the ovariole containing a symbiont ball filled with YLSs (encircled) TEM H 500 Fig 12 Oocyte duringlate vitellogenesis stage Fig 13 Full-grown oocyte F follicular epithelium OC oocyte

A MICHALIK et al134

outer layer is electron dense and has a thickness of30 nm The inner layer has lower electron densityand is 140 nm thick In the central part of the yeastcell a large spherical nucleus with a single nucleo-lus is present (Fig 5) The remaining cytoplasm isfilled with ribosomes mitochondria and large lipiddroplets (Fig 7) The YLSs reproduce by budding(Fig 8) Sexual reproduction of YLSs was not ob-served In older females (ie containing terminaloocytes in the stage of late vitellogenesis) theYLSs leave the mycetome cytoplasm They be-come released into a haemolymph and migrate to-wards the terminal oocytes (Fig 9) The endosym-bionts pass through the cells of the ovariole stalk(pedicel) (Fig 9) as well as follicular cells surround-ing the posterior pole of the oocyte (Fig 10 11)Subsequently they enter the perivitelline space(Figs 9 10) At the same time a deep depression isformed at the posterior pole of the oocyte TheYLSs accumulate in the oocyte depression andform a characteristic ldquosymbiont ballrdquo (Fig 12) Atthe end of oocyte growth the ldquosymbiont ballrdquo istightly packed with YLSs (Fig 13) Until the endof oocyte growth the YLSs are isolated from theooplasm by oolemma and do not enter the oo-plasm The endosymbionts gathered in the depres-sion of the oocytes like those harbored in themycetome undergo budding (not shown)

Discussion

Our observations revealed that both adult males andfemales of M pruinosa (Flatidae) as well as C anceps(Delphacidae) harbor a large number of intracellu-lar YLSs This observation shows that these endo-symbionts are essential for both sexes of examinedplanthoppers In recent years the metabolic signifi-cance of YLSs for growth and reproduction of thehost insects has been extensively studied using therice brown planthopper Nilaparvata lugens (SASAKIet al 1996 HONGOH amp ISHIKAWA 19972000WILK-INSON amp ISHIKAWA 2001) These studies revealedthat planthopper YLSs are involved in nitrogen re-cycling using uric acid as a nitrogenous resourcePlanthoppers in addition to producing uric acid asa nitrogenous waste product also synthesize it as astorage product during nitrogen deficiency Uricacid is stored in mycetomes and converted by uri-case secreted by YLSs into compounds of nutri-tional value It should be noted that CHENG andHOU (2005) demonstrated that YLSs are also en-gaged in synthesis of yolk precursors in females ofthe rice brown planthopper Nilaparvata lugens

Our studies showed that YLSs in M pruinosaand C anceps are transmitted from the mother tothe progeny by a similar route ie via cytoplasm ofcells of the pedicel as well as follicular cells sur-

rounding the posterior pole of the oocyte Since thesame situation has been observed in other mem-bers of the family Delphacidae ie Laodelphaxstriatellus (NODA 1977) and Nilaparvata lugens(CHENG amp HOU 2001) as well as in the leafhopperScaphoideus titanus (SACCHI et al 2008) it seemsprobable that all hemipterans have developed thesame mode of transmission of YLSs to the nextgeneration Thus the transmission of YLSs in in-sects is more uniform than the transmission of bac-teria (see Introduction) Moreover molecular stud-ies revealed that YLSs in hemipterans are not onlysimilarly inherited but are also phylogeneticallyclosely related to each other The analysis of 18Sribosomal DNA sequences of YLSs in delphacids(NODA et al 1995 XET-MULL et al 2004) aphids(FUKATSU amp ISHIKAWA 1996) and leafhoppers (SAC-CHI et al 2008) indicated that they belong to theclass Pyrenomycetes in the phylum AscomytinaThe YLSs in the examined rice delphacids are char-acterized by a high degree of similarity of 18S ribo-somal DNA sequences (NODA et al 1995 XET-MULL et al 2004) This finding strongly suggeststhat YLSs of delphacids are closely related (ieconstitute a monophyletic group) This implies inturn that the symbiosis of YLSs and delphacids isthe result of a single infection of the common an-cestor of present delphacids The YLSs harboredin members of the Flatidae family have so far notbeen examined by molecular methods thereforetheir systematic position remains unknown Sinceflatids and delphacids are phylogenetically distantwithin planthoppers (ie do not represent a mono-pyletic taxon) (BOURGOIN et al 1997 URBAN ampCRYAN 2007) it is unlikely that their endosymbi-onts have been acquired through a common ancestorIn this light it may be assumed that YLSs were hori-zontally transferred between flatid and delphacidlineages It is noteworthy that HONGOH and ISHI-KAWA (2000) on the basis of an analysis of uricasegene sequences of YLSs in aphids and delphacidsprovided evidence of a close relationship betweenYLSs in these phylogenetically distant groups ofinsects as a result of the horizontal transfer of micro-organismsfrom the aphid to the planthopper lineage

Both in M pruinosa and C anceps the migrationof YLSs is correlated with ovary development (theYLSs infect vitellogenic oocytes) Thus this ob-servation strongly supports the hypothesis that themovement of microorganisms is stimulated by anunknown factor released by ovaries (EBERLE ampMC LEAN 1982 macrELAZOWSKA amp BILINtildeSKI 1999SZKLARZEWICZ amp MOSKAL 2001 SZKLARZEWICZet al 2006)

We observed that apart from YLSs rod-shapedbacteria are present in the body of all specimens ofM pruinosa In contrast to YLSs bacteria havenever been found in the ovaries of M pruinosa

Endosymbiotic Microorganisms in Planthoppers 135

The absence of these bacteria in the ovaries indi-cates that they may be horizontally transmitted be-tween specimens The large number of bacteria bothin the mycetome cytoplasm as well as in its epithe-lium suggests that they may have a significant(positive or negative) influence on the host insectThese microorganisms may represent S-symbiontsof M pruinosa but may also prove to be patho-genic It should be noted that NODA and SAITO(1979) detected rod-shaped bacteria in mycetomesof the planthopper Laodelphax striatellus (Del-phacidae) but did not suggest a possible role forthem It may be also speculated that these bacteriabelong to the widespread within arthropodsricketsia-like genus Wolbachia pipientis The lastassumption is supported by PCR detection of Wol-bachia in two delphacids Laodelphax striatellusand Sogatella furcifera (NODA et al 2001) as wellas by the observation that specimens of Laodel-phax striatellus may be horizontally infected byWolbachia (KANG et al 2003) To verify these hy-potheses further studies of specimens of M prui-nosa taken from different populations are needed

Acknowledgements

We would like to express our gratitude to DrJean-Francois GERMAIN (Labolatoire National dela Protection des Vegetaux Montpellier France)and Dr Sebastian PILARCZYK (Silesian Univer-sity Katowice Poland) for collection and identifi-cation of specimens We are also grateful to DrBeata SZYMANtildeSKA (Jagiellonian University De-partment of Systematic Zoology and Zoogeogra-phy Krakoacutew Poland) and Dr Olga WOicircNICKA(Jagiellonian University Department of Cytologyand Histology Krakoacutew Poland) for their skilledtechnical assistance

References

BAUMANN P 2005 Biology of bacteriocyte-associated endo-symbionts of plant sup-sucking insects Annu Rev Micro-biol 59 155-189BAUMANN P 2006 Diversity of prokaryote-insect associa-tionswithin the Sternorrhyncha (psyllids whiteflies aphidsmealybugs) (In Insect Symbiosis vol 2 TA Miller KBourtzis ed Contemporary Topics in Entomology Series)1-24BOURGOIN T STEFFEN-CAMPBELL D CAMPBELL B C1997 Molecular phylogeny of Fulgoromorpha (InsectaHemiptera Archaeorrhyncha) The enigmatic Tettigometri-dae evolutionary affiliations and historical biogeographyCladistics 13 207-224BUCHNER P 1965 Endosymbiosis of Animals with PlantMi-croorganisms Interscience Publishers New York LondonSydneyCHENG D-J HOU R F 2001 Histological observations ontransovarial transmission of a yeast-like symbiote in Nila-

parvata lugens Stal (Homoptera Delphacidae) Tissue Cell33 273-279

CHENGD- J HOUR F 2005 Determination and distributionof a female-specific protein in the brown planthopper Nila-parvata lugens Stal (Homoptera Delphacidae) Tissue Cell37 37-45

EBERLEM W MC LEAN D L 1982 Initiation and orienta-tion of the symbiote migration in human body louse Pedicu-lus humanus L J Insect Physiol 28 417-422

FUKATSU T ISHIKAWA H 1996 Phylogenetic position ofyeast-like symbiont of Hamiltonaphis styraci (HomopteraAphididae) based on 18rDNA sequence Insect BiochemMol Biol 26 383-388HONGOH Y ISHIKAWA H 1997 Uric acid as a nitrogen re-source for the brown planthopperNilaparvata lugens stud-ies with synthetic diets and aposymbiotic insects Zool Sci

14 581-586HONGOHY ISHIKAWAH 2000 Evolutionary studies on uri-cases of fungal endosymbionts of aphids and planthoppersJ Mol Evol 51 265-277HOUKE J GRIFFITHSGW 1980 Intracellular symbiotes ofthe Homoptera Annu Rev Entomol 25 161-187ISHIKAWA H 2003 Insect Symbiosis An Introduction (InInsect Symbiosis vol 1 TA Miller K Bourtzis ed Con-temporary Topics in Entomology Series) 1-21KANG L MAX CAI L LIAO S SUN L ZHUH CHEN XSHEN D ZHAO S LI C 2003 Superinfection of Laodel-

phax striatellus with Wolbachia from Drospohila simulansHeredity 90 71-76MONTLLOR C B MAXMENA PURCELL A H 2002 Facul-tative bacterial endosymbionts benefit pea aphidsAcyrthosi-

phon pisum under heat stress Ecol Entomol 27 189-195NODAH 1977Histological and histochemical observation ofintracellular yeastlike symbiotes in the fat body o the smallerbrown planthopper Laodelphax striatellus (HomopteraDelphacidae) Appl Ent Zool 12 134-141NODA H SAITO T 1979 Effects of high temperature on thedevelopment of Laodelpax striatellus (Homoptera Delpha-cidae) and on its intracellular yeastlike symbiotesAppl EntZool 14 64-75NODA H NAKASHIMA N KOIZUMIM 1995 Phylogeneticposition of yeast-like symbiotes of rice planthoppers basedon partial 18S rDNA sequences Insect Biochem Mol Biol

25 639-646NODA H KOIZUMI Y ZHANG Q DENG K 2001 Infectiondensity of Wolbachia and incompatibilty level in two plan-thopper species Laodelpax striatellus and Sogatella furcif-

era Insect Biochem Mol Biol 31 727-737OLIVER K M RUSSEL J A MORAN N A HUNTERM S2003 Facultative bacterial symbionts in aphids confer resis-tance to parasitic wasps Proc Natl Acad Sci 1001803-1807SACCHI L GENCHIM CLEMENTI E BIGLIARDI E AVAN-ZATTI A M PAJOROI M NEGRI I MARZORATI MGONELLA E ALMA A DAFFONCHIO D BANDI C 2008Multiple symbiosis in the leafhopper Scaphoideus titanus(Hemiptera Cicadellidae) Details of transovarial transmis-sion of Cardinium sp and yeast-like endosymbionts TissueCell 40 231-242

SASAKIT ISHIKAWAH 1995 Production of essential aminoacids from glutamate by mycetocyte symbiont of the peaaphid Acyrthosiphon pisum J Insect Physiol 41 41-46SASAKIT KAWAMURAM ISHIKAWAH 1996 Nitrogen re-cycling in the brown planthopper Nilaparvata lugens in-volvement of yeast-like endosymbionts in uric acidmetabolism J Insect Physiol 42 125-129SCARBOROUGH C L FERRARI J GODFRAY H C J 2005Aphid Protected from Pathogen by Endosymbiont Science

310 1781SZKLARZEWICZ T MOSKAL A 2001 Ultrastructure distri-bution and transmission of endosymbionts in the whitefly

A MICHALIK et al136

fections with free living bacteria (BAUMANN 2006)whereas associations between secondary endosym-bionts and insects are much younger and are due tomultiple independent infections (THAO et al 2000)

Most hemipterans have prokaryotic endosymbi-onts however eukaryotic microorganisms histori-cally termed ldquoyeast-like symbiontsrdquo (YLSs) occurin some aphids scale insects leafhoppers and ful-goromorphans (see BUCHNER 1965 ISHIKAWA2003 for further details)

Both bacterial and yeast-like endosymbionts arematernally inherited by transovarial transmissionStudies on transovarial transmission of bacterialendosymbionts have shown that variable modes ofovary infection by bacteria exist in insects (macrELA-ZOWSKA amp BILINtildeSKI 1999 SZKLARZEWICZ ampMOSKAL 2001 SZKLARZEWICZ et al 2006) Theendosymbiotic microorganisms may invade younggerm cells (=cystocytes) or oocytes in the olderstage of development (ie vitellogenic or chorio-genic) The ovaries may be invaded by bacteria orby intact mycetocytes The endosymbionts maymigrate through the neighboring follicular cells(ie cells surrounding oocytes) or may enter thecytoplasm of follicular cells In contrast to bacte-rial endosymbionts the transovarial transmissionof YLSs is poorly known and there are only threepublished observations on this process (NODA1977 CHENG amp HOU 2001 SACCHI et al 2008)two of which consider members of the family Del-phacidae

The present study was undertaken to provide in-formation about the mode of transmission of YLSsin Metcalfa pruinosa a member of the Flatidaefamily and in another member of the family Del-phacidae Conomelus anceps

Material and Methods

Adult specimens of Conomelus anceps (Germar1821) were collected in July near Nowy Targ(southern Poland) Adult specimens of Metcalfapruinosa (Say 1830) were collected in July inGrabels (southern France) The abdomens and dis-sected ovaries of individuals of each species werefixed in 25 glautaraldehyde in 01 phosphatebuffer (pH 74) for three months The material wasthen rinsed in 01 M phosphate buffer (pH 74)with addition of 58 sucrose postfixed in 1 os-mium tetroxidae dehydrated in a series of alcoholand acetone and embedded in epoxy resin Epox812 (Fullam Inc Latham NY USA) Semithinsections were stained with 1 methylene blue in1 borax and photographed in a Jenalumar (ZeissJena) microscope Ultrathin sections were stainedwith lead citrate and uranyl acetate and examinedusing a JEM 100 SX electron microscope at 80 kV

Results

Gross morphology of the ovaries

The paired ovaries of Metcalfa pruinosa andConomelus anceps consist of several ovarioles oftelotrophic type (Fig 1) The individual ovariole issubdivided into a terminal filament tropharium(trophic chamber) vitellarium and ovariolar stalk(pedicel) that joins the ovariole to the lateral oviduct(Fig1)The trophariumiscomposedofsyncytial lobescontaining 3-5 trophocyte nuclei (Fig 1) Early pre-vitellogenic oocytes (termed arrested oocytes) andprefollicular cells are present at the base of the tro-pharium (Fig 1) The vitellarium in mature femalescomprises 4-5 linearly arranged oocytes that aresurrounded by a one-layered follicular epithelium(Fig 1) The vitellarial oocytes develop through

A MICHALIK et al132

Fig 1 Schematic representation of the ovariole of aplanthopper Arrows YLSs F follicular epithelium OC oocyte TF terminal filament TR tropharium VIT vitellarium P pedicel

Endosymbiotic Microorganisms in Planthoppers 133

Figs 2-8 Fig 2M pruinosa The mycetome filled with YLSs (arrowhead) in the body of the male T testis Methylene blue H570 Fig 3 M pruinosa Fragment of the epithelial cell surrounding the mycetome Arrows rod-shaped bacteria EN nucleus of the epithelial cell L lipid droplet TEM H 18 100 Fig 4 C anceps The mycetome filled with YLSs(arrowheads) in the body of the female Arrows mycetome nuclei embedded in the common cytoplasm EP epithelial cellssurrounding the mycetome L lipid droplets Methylene blue H 530 Fig 5M pruinosa Cross section through the cell of theYLS Arrow cell wall composed of two distinct layers YN nucleus NU nucleolus TEM H 16 500 Fig 6 M pruinosaFragment of the mycetome cytoplasm Arrows rod-shaped bacteria MN mycetome nuclei embedded in the commoncytoplasm TEM H 17 500 Fig 7 M pruinosa Fragment of the cell of the YLS Arrow cell wall L lipid droplet M mitochondria YN nucleus TEM H 15 100 Fig 8 C anceps The YLS reproducing by budding TEM H 9 300

three stages previtellogenesis (ie synthesis andaccumulation of RNArsquos) vitellogenesis (ie syn-thesis and accumulation of reserve substances) andchoriogenesis (ie synthesis and secretion of pre-cursors of eggshells and their deposition on the oo-cyte surface) (for a detailed description of ovariesof planthoppers see SZKLARZEWICZ et al 2007)

Ultrastructure distribution and transovarialtransmission of yeast-like endosymbiotic micro-organisms

Both in males and females of C anceps and Mpruinosa the spaces between internal organs arefilled with large structures termed mycetomes(Figs 2 4) The mycetomes have a syncytial char-acter ie they have numerous nuclei embedded in

a common cytoplasm (Figs 4 6) The mycetomesare surrounded by a one-layered epithelium (Fig 4)In young specimens epithelial cells are small izo-diametric and closely adhere to each other (notshown) while in the older specimens they becomeirregular and voluminous with large lipid dropletsin the cytoplasm (Fig 4) Numerous rod-shapedbacteria occur in epithelial cells (Fig 3) as well asin mycetome cytoplasm (Fig 6) of all examinedspecimens of M pruinosa The bacteria measure12-16 Fm in length and 03-05 Fm in diameterBoth in M pruinosa and C anceps the mycotomecytoplasm contains an enormous number ofyeast-like symbionts (YLSs) (Figs 2 4) The sizeof YLSs is 8-10 Fm in length and 30-35 Fm in di-ameter They are surrounded by a thick cell wallcomposed of two distinct layers (Figs 5 7 8) The

Figs 9-13 Figs 9 10 Longitudinal section through the posterior end of the ovariole pedicel (P) and lateral oviduct (LOV)during infection by the YLSs (arrows) Methylene blue H 1 000 Fig 9 C anceps Fig 10 M pruinosa Asterisk deepdepression of the oolemma F follicular cells OC oocyte Fig 11 C anceps Fragment of the follicular epithelium (F)during migration of YLSs FN follicular cell nucleus TEM H 6 500 Figs 12 13 C anceps Cross section through theposterior pole of the ovariole containing a symbiont ball filled with YLSs (encircled) TEM H 500 Fig 12 Oocyte duringlate vitellogenesis stage Fig 13 Full-grown oocyte F follicular epithelium OC oocyte

A MICHALIK et al134

outer layer is electron dense and has a thickness of30 nm The inner layer has lower electron densityand is 140 nm thick In the central part of the yeastcell a large spherical nucleus with a single nucleo-lus is present (Fig 5) The remaining cytoplasm isfilled with ribosomes mitochondria and large lipiddroplets (Fig 7) The YLSs reproduce by budding(Fig 8) Sexual reproduction of YLSs was not ob-served In older females (ie containing terminaloocytes in the stage of late vitellogenesis) theYLSs leave the mycetome cytoplasm They be-come released into a haemolymph and migrate to-wards the terminal oocytes (Fig 9) The endosym-bionts pass through the cells of the ovariole stalk(pedicel) (Fig 9) as well as follicular cells surround-ing the posterior pole of the oocyte (Fig 10 11)Subsequently they enter the perivitelline space(Figs 9 10) At the same time a deep depression isformed at the posterior pole of the oocyte TheYLSs accumulate in the oocyte depression andform a characteristic ldquosymbiont ballrdquo (Fig 12) Atthe end of oocyte growth the ldquosymbiont ballrdquo istightly packed with YLSs (Fig 13) Until the endof oocyte growth the YLSs are isolated from theooplasm by oolemma and do not enter the oo-plasm The endosymbionts gathered in the depres-sion of the oocytes like those harbored in themycetome undergo budding (not shown)

Discussion

Our observations revealed that both adult males andfemales of M pruinosa (Flatidae) as well as C anceps(Delphacidae) harbor a large number of intracellu-lar YLSs This observation shows that these endo-symbionts are essential for both sexes of examinedplanthoppers In recent years the metabolic signifi-cance of YLSs for growth and reproduction of thehost insects has been extensively studied using therice brown planthopper Nilaparvata lugens (SASAKIet al 1996 HONGOH amp ISHIKAWA 19972000WILK-INSON amp ISHIKAWA 2001) These studies revealedthat planthopper YLSs are involved in nitrogen re-cycling using uric acid as a nitrogenous resourcePlanthoppers in addition to producing uric acid asa nitrogenous waste product also synthesize it as astorage product during nitrogen deficiency Uricacid is stored in mycetomes and converted by uri-case secreted by YLSs into compounds of nutri-tional value It should be noted that CHENG andHOU (2005) demonstrated that YLSs are also en-gaged in synthesis of yolk precursors in females ofthe rice brown planthopper Nilaparvata lugens

Our studies showed that YLSs in M pruinosaand C anceps are transmitted from the mother tothe progeny by a similar route ie via cytoplasm ofcells of the pedicel as well as follicular cells sur-

rounding the posterior pole of the oocyte Since thesame situation has been observed in other mem-bers of the family Delphacidae ie Laodelphaxstriatellus (NODA 1977) and Nilaparvata lugens(CHENG amp HOU 2001) as well as in the leafhopperScaphoideus titanus (SACCHI et al 2008) it seemsprobable that all hemipterans have developed thesame mode of transmission of YLSs to the nextgeneration Thus the transmission of YLSs in in-sects is more uniform than the transmission of bac-teria (see Introduction) Moreover molecular stud-ies revealed that YLSs in hemipterans are not onlysimilarly inherited but are also phylogeneticallyclosely related to each other The analysis of 18Sribosomal DNA sequences of YLSs in delphacids(NODA et al 1995 XET-MULL et al 2004) aphids(FUKATSU amp ISHIKAWA 1996) and leafhoppers (SAC-CHI et al 2008) indicated that they belong to theclass Pyrenomycetes in the phylum AscomytinaThe YLSs in the examined rice delphacids are char-acterized by a high degree of similarity of 18S ribo-somal DNA sequences (NODA et al 1995 XET-MULL et al 2004) This finding strongly suggeststhat YLSs of delphacids are closely related (ieconstitute a monophyletic group) This implies inturn that the symbiosis of YLSs and delphacids isthe result of a single infection of the common an-cestor of present delphacids The YLSs harboredin members of the Flatidae family have so far notbeen examined by molecular methods thereforetheir systematic position remains unknown Sinceflatids and delphacids are phylogenetically distantwithin planthoppers (ie do not represent a mono-pyletic taxon) (BOURGOIN et al 1997 URBAN ampCRYAN 2007) it is unlikely that their endosymbi-onts have been acquired through a common ancestorIn this light it may be assumed that YLSs were hori-zontally transferred between flatid and delphacidlineages It is noteworthy that HONGOH and ISHI-KAWA (2000) on the basis of an analysis of uricasegene sequences of YLSs in aphids and delphacidsprovided evidence of a close relationship betweenYLSs in these phylogenetically distant groups ofinsects as a result of the horizontal transfer of micro-organismsfrom the aphid to the planthopper lineage

Both in M pruinosa and C anceps the migrationof YLSs is correlated with ovary development (theYLSs infect vitellogenic oocytes) Thus this ob-servation strongly supports the hypothesis that themovement of microorganisms is stimulated by anunknown factor released by ovaries (EBERLE ampMC LEAN 1982 macrELAZOWSKA amp BILINtildeSKI 1999SZKLARZEWICZ amp MOSKAL 2001 SZKLARZEWICZet al 2006)

We observed that apart from YLSs rod-shapedbacteria are present in the body of all specimens ofM pruinosa In contrast to YLSs bacteria havenever been found in the ovaries of M pruinosa

Endosymbiotic Microorganisms in Planthoppers 135

The absence of these bacteria in the ovaries indi-cates that they may be horizontally transmitted be-tween specimens The large number of bacteria bothin the mycetome cytoplasm as well as in its epithe-lium suggests that they may have a significant(positive or negative) influence on the host insectThese microorganisms may represent S-symbiontsof M pruinosa but may also prove to be patho-genic It should be noted that NODA and SAITO(1979) detected rod-shaped bacteria in mycetomesof the planthopper Laodelphax striatellus (Del-phacidae) but did not suggest a possible role forthem It may be also speculated that these bacteriabelong to the widespread within arthropodsricketsia-like genus Wolbachia pipientis The lastassumption is supported by PCR detection of Wol-bachia in two delphacids Laodelphax striatellusand Sogatella furcifera (NODA et al 2001) as wellas by the observation that specimens of Laodel-phax striatellus may be horizontally infected byWolbachia (KANG et al 2003) To verify these hy-potheses further studies of specimens of M prui-nosa taken from different populations are needed

Acknowledgements

We would like to express our gratitude to DrJean-Francois GERMAIN (Labolatoire National dela Protection des Vegetaux Montpellier France)and Dr Sebastian PILARCZYK (Silesian Univer-sity Katowice Poland) for collection and identifi-cation of specimens We are also grateful to DrBeata SZYMANtildeSKA (Jagiellonian University De-partment of Systematic Zoology and Zoogeogra-phy Krakoacutew Poland) and Dr Olga WOicircNICKA(Jagiellonian University Department of Cytologyand Histology Krakoacutew Poland) for their skilledtechnical assistance

References

BAUMANN P 2005 Biology of bacteriocyte-associated endo-symbionts of plant sup-sucking insects Annu Rev Micro-biol 59 155-189BAUMANN P 2006 Diversity of prokaryote-insect associa-tionswithin the Sternorrhyncha (psyllids whiteflies aphidsmealybugs) (In Insect Symbiosis vol 2 TA Miller KBourtzis ed Contemporary Topics in Entomology Series)1-24BOURGOIN T STEFFEN-CAMPBELL D CAMPBELL B C1997 Molecular phylogeny of Fulgoromorpha (InsectaHemiptera Archaeorrhyncha) The enigmatic Tettigometri-dae evolutionary affiliations and historical biogeographyCladistics 13 207-224BUCHNER P 1965 Endosymbiosis of Animals with PlantMi-croorganisms Interscience Publishers New York LondonSydneyCHENG D-J HOU R F 2001 Histological observations ontransovarial transmission of a yeast-like symbiote in Nila-

parvata lugens Stal (Homoptera Delphacidae) Tissue Cell33 273-279

CHENGD- J HOUR F 2005 Determination and distributionof a female-specific protein in the brown planthopper Nila-parvata lugens Stal (Homoptera Delphacidae) Tissue Cell37 37-45

EBERLEM W MC LEAN D L 1982 Initiation and orienta-tion of the symbiote migration in human body louse Pedicu-lus humanus L J Insect Physiol 28 417-422

FUKATSU T ISHIKAWA H 1996 Phylogenetic position ofyeast-like symbiont of Hamiltonaphis styraci (HomopteraAphididae) based on 18rDNA sequence Insect BiochemMol Biol 26 383-388HONGOH Y ISHIKAWA H 1997 Uric acid as a nitrogen re-source for the brown planthopperNilaparvata lugens stud-ies with synthetic diets and aposymbiotic insects Zool Sci

14 581-586HONGOHY ISHIKAWAH 2000 Evolutionary studies on uri-cases of fungal endosymbionts of aphids and planthoppersJ Mol Evol 51 265-277HOUKE J GRIFFITHSGW 1980 Intracellular symbiotes ofthe Homoptera Annu Rev Entomol 25 161-187ISHIKAWA H 2003 Insect Symbiosis An Introduction (InInsect Symbiosis vol 1 TA Miller K Bourtzis ed Con-temporary Topics in Entomology Series) 1-21KANG L MAX CAI L LIAO S SUN L ZHUH CHEN XSHEN D ZHAO S LI C 2003 Superinfection of Laodel-

phax striatellus with Wolbachia from Drospohila simulansHeredity 90 71-76MONTLLOR C B MAXMENA PURCELL A H 2002 Facul-tative bacterial endosymbionts benefit pea aphidsAcyrthosi-

phon pisum under heat stress Ecol Entomol 27 189-195NODAH 1977Histological and histochemical observation ofintracellular yeastlike symbiotes in the fat body o the smallerbrown planthopper Laodelphax striatellus (HomopteraDelphacidae) Appl Ent Zool 12 134-141NODA H SAITO T 1979 Effects of high temperature on thedevelopment of Laodelpax striatellus (Homoptera Delpha-cidae) and on its intracellular yeastlike symbiotesAppl EntZool 14 64-75NODA H NAKASHIMA N KOIZUMIM 1995 Phylogeneticposition of yeast-like symbiotes of rice planthoppers basedon partial 18S rDNA sequences Insect Biochem Mol Biol

25 639-646NODA H KOIZUMI Y ZHANG Q DENG K 2001 Infectiondensity of Wolbachia and incompatibilty level in two plan-thopper species Laodelpax striatellus and Sogatella furcif-

era Insect Biochem Mol Biol 31 727-737OLIVER K M RUSSEL J A MORAN N A HUNTERM S2003 Facultative bacterial symbionts in aphids confer resis-tance to parasitic wasps Proc Natl Acad Sci 1001803-1807SACCHI L GENCHIM CLEMENTI E BIGLIARDI E AVAN-ZATTI A M PAJOROI M NEGRI I MARZORATI MGONELLA E ALMA A DAFFONCHIO D BANDI C 2008Multiple symbiosis in the leafhopper Scaphoideus titanus(Hemiptera Cicadellidae) Details of transovarial transmis-sion of Cardinium sp and yeast-like endosymbionts TissueCell 40 231-242

SASAKIT ISHIKAWAH 1995 Production of essential aminoacids from glutamate by mycetocyte symbiont of the peaaphid Acyrthosiphon pisum J Insect Physiol 41 41-46SASAKIT KAWAMURAM ISHIKAWAH 1996 Nitrogen re-cycling in the brown planthopper Nilaparvata lugens in-volvement of yeast-like endosymbionts in uric acidmetabolism J Insect Physiol 42 125-129SCARBOROUGH C L FERRARI J GODFRAY H C J 2005Aphid Protected from Pathogen by Endosymbiont Science

310 1781SZKLARZEWICZ T MOSKAL A 2001 Ultrastructure distri-bution and transmission of endosymbionts in the whitefly

A MICHALIK et al136

Endosymbiotic Microorganisms in Planthoppers 133

Figs 2-8 Fig 2M pruinosa The mycetome filled with YLSs (arrowhead) in the body of the male T testis Methylene blue H570 Fig 3 M pruinosa Fragment of the epithelial cell surrounding the mycetome Arrows rod-shaped bacteria EN nucleus of the epithelial cell L lipid droplet TEM H 18 100 Fig 4 C anceps The mycetome filled with YLSs(arrowheads) in the body of the female Arrows mycetome nuclei embedded in the common cytoplasm EP epithelial cellssurrounding the mycetome L lipid droplets Methylene blue H 530 Fig 5M pruinosa Cross section through the cell of theYLS Arrow cell wall composed of two distinct layers YN nucleus NU nucleolus TEM H 16 500 Fig 6 M pruinosaFragment of the mycetome cytoplasm Arrows rod-shaped bacteria MN mycetome nuclei embedded in the commoncytoplasm TEM H 17 500 Fig 7 M pruinosa Fragment of the cell of the YLS Arrow cell wall L lipid droplet M mitochondria YN nucleus TEM H 15 100 Fig 8 C anceps The YLS reproducing by budding TEM H 9 300

three stages previtellogenesis (ie synthesis andaccumulation of RNArsquos) vitellogenesis (ie syn-thesis and accumulation of reserve substances) andchoriogenesis (ie synthesis and secretion of pre-cursors of eggshells and their deposition on the oo-cyte surface) (for a detailed description of ovariesof planthoppers see SZKLARZEWICZ et al 2007)

Ultrastructure distribution and transovarialtransmission of yeast-like endosymbiotic micro-organisms

Both in males and females of C anceps and Mpruinosa the spaces between internal organs arefilled with large structures termed mycetomes(Figs 2 4) The mycetomes have a syncytial char-acter ie they have numerous nuclei embedded in

a common cytoplasm (Figs 4 6) The mycetomesare surrounded by a one-layered epithelium (Fig 4)In young specimens epithelial cells are small izo-diametric and closely adhere to each other (notshown) while in the older specimens they becomeirregular and voluminous with large lipid dropletsin the cytoplasm (Fig 4) Numerous rod-shapedbacteria occur in epithelial cells (Fig 3) as well asin mycetome cytoplasm (Fig 6) of all examinedspecimens of M pruinosa The bacteria measure12-16 Fm in length and 03-05 Fm in diameterBoth in M pruinosa and C anceps the mycotomecytoplasm contains an enormous number ofyeast-like symbionts (YLSs) (Figs 2 4) The sizeof YLSs is 8-10 Fm in length and 30-35 Fm in di-ameter They are surrounded by a thick cell wallcomposed of two distinct layers (Figs 5 7 8) The

Figs 9-13 Figs 9 10 Longitudinal section through the posterior end of the ovariole pedicel (P) and lateral oviduct (LOV)during infection by the YLSs (arrows) Methylene blue H 1 000 Fig 9 C anceps Fig 10 M pruinosa Asterisk deepdepression of the oolemma F follicular cells OC oocyte Fig 11 C anceps Fragment of the follicular epithelium (F)during migration of YLSs FN follicular cell nucleus TEM H 6 500 Figs 12 13 C anceps Cross section through theposterior pole of the ovariole containing a symbiont ball filled with YLSs (encircled) TEM H 500 Fig 12 Oocyte duringlate vitellogenesis stage Fig 13 Full-grown oocyte F follicular epithelium OC oocyte

A MICHALIK et al134

outer layer is electron dense and has a thickness of30 nm The inner layer has lower electron densityand is 140 nm thick In the central part of the yeastcell a large spherical nucleus with a single nucleo-lus is present (Fig 5) The remaining cytoplasm isfilled with ribosomes mitochondria and large lipiddroplets (Fig 7) The YLSs reproduce by budding(Fig 8) Sexual reproduction of YLSs was not ob-served In older females (ie containing terminaloocytes in the stage of late vitellogenesis) theYLSs leave the mycetome cytoplasm They be-come released into a haemolymph and migrate to-wards the terminal oocytes (Fig 9) The endosym-bionts pass through the cells of the ovariole stalk(pedicel) (Fig 9) as well as follicular cells surround-ing the posterior pole of the oocyte (Fig 10 11)Subsequently they enter the perivitelline space(Figs 9 10) At the same time a deep depression isformed at the posterior pole of the oocyte TheYLSs accumulate in the oocyte depression andform a characteristic ldquosymbiont ballrdquo (Fig 12) Atthe end of oocyte growth the ldquosymbiont ballrdquo istightly packed with YLSs (Fig 13) Until the endof oocyte growth the YLSs are isolated from theooplasm by oolemma and do not enter the oo-plasm The endosymbionts gathered in the depres-sion of the oocytes like those harbored in themycetome undergo budding (not shown)

Discussion

Our observations revealed that both adult males andfemales of M pruinosa (Flatidae) as well as C anceps(Delphacidae) harbor a large number of intracellu-lar YLSs This observation shows that these endo-symbionts are essential for both sexes of examinedplanthoppers In recent years the metabolic signifi-cance of YLSs for growth and reproduction of thehost insects has been extensively studied using therice brown planthopper Nilaparvata lugens (SASAKIet al 1996 HONGOH amp ISHIKAWA 19972000WILK-INSON amp ISHIKAWA 2001) These studies revealedthat planthopper YLSs are involved in nitrogen re-cycling using uric acid as a nitrogenous resourcePlanthoppers in addition to producing uric acid asa nitrogenous waste product also synthesize it as astorage product during nitrogen deficiency Uricacid is stored in mycetomes and converted by uri-case secreted by YLSs into compounds of nutri-tional value It should be noted that CHENG andHOU (2005) demonstrated that YLSs are also en-gaged in synthesis of yolk precursors in females ofthe rice brown planthopper Nilaparvata lugens

Our studies showed that YLSs in M pruinosaand C anceps are transmitted from the mother tothe progeny by a similar route ie via cytoplasm ofcells of the pedicel as well as follicular cells sur-

rounding the posterior pole of the oocyte Since thesame situation has been observed in other mem-bers of the family Delphacidae ie Laodelphaxstriatellus (NODA 1977) and Nilaparvata lugens(CHENG amp HOU 2001) as well as in the leafhopperScaphoideus titanus (SACCHI et al 2008) it seemsprobable that all hemipterans have developed thesame mode of transmission of YLSs to the nextgeneration Thus the transmission of YLSs in in-sects is more uniform than the transmission of bac-teria (see Introduction) Moreover molecular stud-ies revealed that YLSs in hemipterans are not onlysimilarly inherited but are also phylogeneticallyclosely related to each other The analysis of 18Sribosomal DNA sequences of YLSs in delphacids(NODA et al 1995 XET-MULL et al 2004) aphids(FUKATSU amp ISHIKAWA 1996) and leafhoppers (SAC-CHI et al 2008) indicated that they belong to theclass Pyrenomycetes in the phylum AscomytinaThe YLSs in the examined rice delphacids are char-acterized by a high degree of similarity of 18S ribo-somal DNA sequences (NODA et al 1995 XET-MULL et al 2004) This finding strongly suggeststhat YLSs of delphacids are closely related (ieconstitute a monophyletic group) This implies inturn that the symbiosis of YLSs and delphacids isthe result of a single infection of the common an-cestor of present delphacids The YLSs harboredin members of the Flatidae family have so far notbeen examined by molecular methods thereforetheir systematic position remains unknown Sinceflatids and delphacids are phylogenetically distantwithin planthoppers (ie do not represent a mono-pyletic taxon) (BOURGOIN et al 1997 URBAN ampCRYAN 2007) it is unlikely that their endosymbi-onts have been acquired through a common ancestorIn this light it may be assumed that YLSs were hori-zontally transferred between flatid and delphacidlineages It is noteworthy that HONGOH and ISHI-KAWA (2000) on the basis of an analysis of uricasegene sequences of YLSs in aphids and delphacidsprovided evidence of a close relationship betweenYLSs in these phylogenetically distant groups ofinsects as a result of the horizontal transfer of micro-organismsfrom the aphid to the planthopper lineage

Both in M pruinosa and C anceps the migrationof YLSs is correlated with ovary development (theYLSs infect vitellogenic oocytes) Thus this ob-servation strongly supports the hypothesis that themovement of microorganisms is stimulated by anunknown factor released by ovaries (EBERLE ampMC LEAN 1982 macrELAZOWSKA amp BILINtildeSKI 1999SZKLARZEWICZ amp MOSKAL 2001 SZKLARZEWICZet al 2006)

We observed that apart from YLSs rod-shapedbacteria are present in the body of all specimens ofM pruinosa In contrast to YLSs bacteria havenever been found in the ovaries of M pruinosa

Endosymbiotic Microorganisms in Planthoppers 135

The absence of these bacteria in the ovaries indi-cates that they may be horizontally transmitted be-tween specimens The large number of bacteria bothin the mycetome cytoplasm as well as in its epithe-lium suggests that they may have a significant(positive or negative) influence on the host insectThese microorganisms may represent S-symbiontsof M pruinosa but may also prove to be patho-genic It should be noted that NODA and SAITO(1979) detected rod-shaped bacteria in mycetomesof the planthopper Laodelphax striatellus (Del-phacidae) but did not suggest a possible role forthem It may be also speculated that these bacteriabelong to the widespread within arthropodsricketsia-like genus Wolbachia pipientis The lastassumption is supported by PCR detection of Wol-bachia in two delphacids Laodelphax striatellusand Sogatella furcifera (NODA et al 2001) as wellas by the observation that specimens of Laodel-phax striatellus may be horizontally infected byWolbachia (KANG et al 2003) To verify these hy-potheses further studies of specimens of M prui-nosa taken from different populations are needed

Acknowledgements

We would like to express our gratitude to DrJean-Francois GERMAIN (Labolatoire National dela Protection des Vegetaux Montpellier France)and Dr Sebastian PILARCZYK (Silesian Univer-sity Katowice Poland) for collection and identifi-cation of specimens We are also grateful to DrBeata SZYMANtildeSKA (Jagiellonian University De-partment of Systematic Zoology and Zoogeogra-phy Krakoacutew Poland) and Dr Olga WOicircNICKA(Jagiellonian University Department of Cytologyand Histology Krakoacutew Poland) for their skilledtechnical assistance

References

BAUMANN P 2005 Biology of bacteriocyte-associated endo-symbionts of plant sup-sucking insects Annu Rev Micro-biol 59 155-189BAUMANN P 2006 Diversity of prokaryote-insect associa-tionswithin the Sternorrhyncha (psyllids whiteflies aphidsmealybugs) (In Insect Symbiosis vol 2 TA Miller KBourtzis ed Contemporary Topics in Entomology Series)1-24BOURGOIN T STEFFEN-CAMPBELL D CAMPBELL B C1997 Molecular phylogeny of Fulgoromorpha (InsectaHemiptera Archaeorrhyncha) The enigmatic Tettigometri-dae evolutionary affiliations and historical biogeographyCladistics 13 207-224BUCHNER P 1965 Endosymbiosis of Animals with PlantMi-croorganisms Interscience Publishers New York LondonSydneyCHENG D-J HOU R F 2001 Histological observations ontransovarial transmission of a yeast-like symbiote in Nila-

parvata lugens Stal (Homoptera Delphacidae) Tissue Cell33 273-279

CHENGD- J HOUR F 2005 Determination and distributionof a female-specific protein in the brown planthopper Nila-parvata lugens Stal (Homoptera Delphacidae) Tissue Cell37 37-45

EBERLEM W MC LEAN D L 1982 Initiation and orienta-tion of the symbiote migration in human body louse Pedicu-lus humanus L J Insect Physiol 28 417-422

FUKATSU T ISHIKAWA H 1996 Phylogenetic position ofyeast-like symbiont of Hamiltonaphis styraci (HomopteraAphididae) based on 18rDNA sequence Insect BiochemMol Biol 26 383-388HONGOH Y ISHIKAWA H 1997 Uric acid as a nitrogen re-source for the brown planthopperNilaparvata lugens stud-ies with synthetic diets and aposymbiotic insects Zool Sci

14 581-586HONGOHY ISHIKAWAH 2000 Evolutionary studies on uri-cases of fungal endosymbionts of aphids and planthoppersJ Mol Evol 51 265-277HOUKE J GRIFFITHSGW 1980 Intracellular symbiotes ofthe Homoptera Annu Rev Entomol 25 161-187ISHIKAWA H 2003 Insect Symbiosis An Introduction (InInsect Symbiosis vol 1 TA Miller K Bourtzis ed Con-temporary Topics in Entomology Series) 1-21KANG L MAX CAI L LIAO S SUN L ZHUH CHEN XSHEN D ZHAO S LI C 2003 Superinfection of Laodel-

phax striatellus with Wolbachia from Drospohila simulansHeredity 90 71-76MONTLLOR C B MAXMENA PURCELL A H 2002 Facul-tative bacterial endosymbionts benefit pea aphidsAcyrthosi-

phon pisum under heat stress Ecol Entomol 27 189-195NODAH 1977Histological and histochemical observation ofintracellular yeastlike symbiotes in the fat body o the smallerbrown planthopper Laodelphax striatellus (HomopteraDelphacidae) Appl Ent Zool 12 134-141NODA H SAITO T 1979 Effects of high temperature on thedevelopment of Laodelpax striatellus (Homoptera Delpha-cidae) and on its intracellular yeastlike symbiotesAppl EntZool 14 64-75NODA H NAKASHIMA N KOIZUMIM 1995 Phylogeneticposition of yeast-like symbiotes of rice planthoppers basedon partial 18S rDNA sequences Insect Biochem Mol Biol

25 639-646NODA H KOIZUMI Y ZHANG Q DENG K 2001 Infectiondensity of Wolbachia and incompatibilty level in two plan-thopper species Laodelpax striatellus and Sogatella furcif-

era Insect Biochem Mol Biol 31 727-737OLIVER K M RUSSEL J A MORAN N A HUNTERM S2003 Facultative bacterial symbionts in aphids confer resis-tance to parasitic wasps Proc Natl Acad Sci 1001803-1807SACCHI L GENCHIM CLEMENTI E BIGLIARDI E AVAN-ZATTI A M PAJOROI M NEGRI I MARZORATI MGONELLA E ALMA A DAFFONCHIO D BANDI C 2008Multiple symbiosis in the leafhopper Scaphoideus titanus(Hemiptera Cicadellidae) Details of transovarial transmis-sion of Cardinium sp and yeast-like endosymbionts TissueCell 40 231-242

SASAKIT ISHIKAWAH 1995 Production of essential aminoacids from glutamate by mycetocyte symbiont of the peaaphid Acyrthosiphon pisum J Insect Physiol 41 41-46SASAKIT KAWAMURAM ISHIKAWAH 1996 Nitrogen re-cycling in the brown planthopper Nilaparvata lugens in-volvement of yeast-like endosymbionts in uric acidmetabolism J Insect Physiol 42 125-129SCARBOROUGH C L FERRARI J GODFRAY H C J 2005Aphid Protected from Pathogen by Endosymbiont Science

310 1781SZKLARZEWICZ T MOSKAL A 2001 Ultrastructure distri-bution and transmission of endosymbionts in the whitefly

A MICHALIK et al136

three stages previtellogenesis (ie synthesis andaccumulation of RNArsquos) vitellogenesis (ie syn-thesis and accumulation of reserve substances) andchoriogenesis (ie synthesis and secretion of pre-cursors of eggshells and their deposition on the oo-cyte surface) (for a detailed description of ovariesof planthoppers see SZKLARZEWICZ et al 2007)

Ultrastructure distribution and transovarialtransmission of yeast-like endosymbiotic micro-organisms

Both in males and females of C anceps and Mpruinosa the spaces between internal organs arefilled with large structures termed mycetomes(Figs 2 4) The mycetomes have a syncytial char-acter ie they have numerous nuclei embedded in

a common cytoplasm (Figs 4 6) The mycetomesare surrounded by a one-layered epithelium (Fig 4)In young specimens epithelial cells are small izo-diametric and closely adhere to each other (notshown) while in the older specimens they becomeirregular and voluminous with large lipid dropletsin the cytoplasm (Fig 4) Numerous rod-shapedbacteria occur in epithelial cells (Fig 3) as well asin mycetome cytoplasm (Fig 6) of all examinedspecimens of M pruinosa The bacteria measure12-16 Fm in length and 03-05 Fm in diameterBoth in M pruinosa and C anceps the mycotomecytoplasm contains an enormous number ofyeast-like symbionts (YLSs) (Figs 2 4) The sizeof YLSs is 8-10 Fm in length and 30-35 Fm in di-ameter They are surrounded by a thick cell wallcomposed of two distinct layers (Figs 5 7 8) The

Figs 9-13 Figs 9 10 Longitudinal section through the posterior end of the ovariole pedicel (P) and lateral oviduct (LOV)during infection by the YLSs (arrows) Methylene blue H 1 000 Fig 9 C anceps Fig 10 M pruinosa Asterisk deepdepression of the oolemma F follicular cells OC oocyte Fig 11 C anceps Fragment of the follicular epithelium (F)during migration of YLSs FN follicular cell nucleus TEM H 6 500 Figs 12 13 C anceps Cross section through theposterior pole of the ovariole containing a symbiont ball filled with YLSs (encircled) TEM H 500 Fig 12 Oocyte duringlate vitellogenesis stage Fig 13 Full-grown oocyte F follicular epithelium OC oocyte

A MICHALIK et al134

outer layer is electron dense and has a thickness of30 nm The inner layer has lower electron densityand is 140 nm thick In the central part of the yeastcell a large spherical nucleus with a single nucleo-lus is present (Fig 5) The remaining cytoplasm isfilled with ribosomes mitochondria and large lipiddroplets (Fig 7) The YLSs reproduce by budding(Fig 8) Sexual reproduction of YLSs was not ob-served In older females (ie containing terminaloocytes in the stage of late vitellogenesis) theYLSs leave the mycetome cytoplasm They be-come released into a haemolymph and migrate to-wards the terminal oocytes (Fig 9) The endosym-bionts pass through the cells of the ovariole stalk(pedicel) (Fig 9) as well as follicular cells surround-ing the posterior pole of the oocyte (Fig 10 11)Subsequently they enter the perivitelline space(Figs 9 10) At the same time a deep depression isformed at the posterior pole of the oocyte TheYLSs accumulate in the oocyte depression andform a characteristic ldquosymbiont ballrdquo (Fig 12) Atthe end of oocyte growth the ldquosymbiont ballrdquo istightly packed with YLSs (Fig 13) Until the endof oocyte growth the YLSs are isolated from theooplasm by oolemma and do not enter the oo-plasm The endosymbionts gathered in the depres-sion of the oocytes like those harbored in themycetome undergo budding (not shown)

Discussion

Our observations revealed that both adult males andfemales of M pruinosa (Flatidae) as well as C anceps(Delphacidae) harbor a large number of intracellu-lar YLSs This observation shows that these endo-symbionts are essential for both sexes of examinedplanthoppers In recent years the metabolic signifi-cance of YLSs for growth and reproduction of thehost insects has been extensively studied using therice brown planthopper Nilaparvata lugens (SASAKIet al 1996 HONGOH amp ISHIKAWA 19972000WILK-INSON amp ISHIKAWA 2001) These studies revealedthat planthopper YLSs are involved in nitrogen re-cycling using uric acid as a nitrogenous resourcePlanthoppers in addition to producing uric acid asa nitrogenous waste product also synthesize it as astorage product during nitrogen deficiency Uricacid is stored in mycetomes and converted by uri-case secreted by YLSs into compounds of nutri-tional value It should be noted that CHENG andHOU (2005) demonstrated that YLSs are also en-gaged in synthesis of yolk precursors in females ofthe rice brown planthopper Nilaparvata lugens

Our studies showed that YLSs in M pruinosaand C anceps are transmitted from the mother tothe progeny by a similar route ie via cytoplasm ofcells of the pedicel as well as follicular cells sur-

rounding the posterior pole of the oocyte Since thesame situation has been observed in other mem-bers of the family Delphacidae ie Laodelphaxstriatellus (NODA 1977) and Nilaparvata lugens(CHENG amp HOU 2001) as well as in the leafhopperScaphoideus titanus (SACCHI et al 2008) it seemsprobable that all hemipterans have developed thesame mode of transmission of YLSs to the nextgeneration Thus the transmission of YLSs in in-sects is more uniform than the transmission of bac-teria (see Introduction) Moreover molecular stud-ies revealed that YLSs in hemipterans are not onlysimilarly inherited but are also phylogeneticallyclosely related to each other The analysis of 18Sribosomal DNA sequences of YLSs in delphacids(NODA et al 1995 XET-MULL et al 2004) aphids(FUKATSU amp ISHIKAWA 1996) and leafhoppers (SAC-CHI et al 2008) indicated that they belong to theclass Pyrenomycetes in the phylum AscomytinaThe YLSs in the examined rice delphacids are char-acterized by a high degree of similarity of 18S ribo-somal DNA sequences (NODA et al 1995 XET-MULL et al 2004) This finding strongly suggeststhat YLSs of delphacids are closely related (ieconstitute a monophyletic group) This implies inturn that the symbiosis of YLSs and delphacids isthe result of a single infection of the common an-cestor of present delphacids The YLSs harboredin members of the Flatidae family have so far notbeen examined by molecular methods thereforetheir systematic position remains unknown Sinceflatids and delphacids are phylogenetically distantwithin planthoppers (ie do not represent a mono-pyletic taxon) (BOURGOIN et al 1997 URBAN ampCRYAN 2007) it is unlikely that their endosymbi-onts have been acquired through a common ancestorIn this light it may be assumed that YLSs were hori-zontally transferred between flatid and delphacidlineages It is noteworthy that HONGOH and ISHI-KAWA (2000) on the basis of an analysis of uricasegene sequences of YLSs in aphids and delphacidsprovided evidence of a close relationship betweenYLSs in these phylogenetically distant groups ofinsects as a result of the horizontal transfer of micro-organismsfrom the aphid to the planthopper lineage

Both in M pruinosa and C anceps the migrationof YLSs is correlated with ovary development (theYLSs infect vitellogenic oocytes) Thus this ob-servation strongly supports the hypothesis that themovement of microorganisms is stimulated by anunknown factor released by ovaries (EBERLE ampMC LEAN 1982 macrELAZOWSKA amp BILINtildeSKI 1999SZKLARZEWICZ amp MOSKAL 2001 SZKLARZEWICZet al 2006)

We observed that apart from YLSs rod-shapedbacteria are present in the body of all specimens ofM pruinosa In contrast to YLSs bacteria havenever been found in the ovaries of M pruinosa

Endosymbiotic Microorganisms in Planthoppers 135

The absence of these bacteria in the ovaries indi-cates that they may be horizontally transmitted be-tween specimens The large number of bacteria bothin the mycetome cytoplasm as well as in its epithe-lium suggests that they may have a significant(positive or negative) influence on the host insectThese microorganisms may represent S-symbiontsof M pruinosa but may also prove to be patho-genic It should be noted that NODA and SAITO(1979) detected rod-shaped bacteria in mycetomesof the planthopper Laodelphax striatellus (Del-phacidae) but did not suggest a possible role forthem It may be also speculated that these bacteriabelong to the widespread within arthropodsricketsia-like genus Wolbachia pipientis The lastassumption is supported by PCR detection of Wol-bachia in two delphacids Laodelphax striatellusand Sogatella furcifera (NODA et al 2001) as wellas by the observation that specimens of Laodel-phax striatellus may be horizontally infected byWolbachia (KANG et al 2003) To verify these hy-potheses further studies of specimens of M prui-nosa taken from different populations are needed

Acknowledgements

We would like to express our gratitude to DrJean-Francois GERMAIN (Labolatoire National dela Protection des Vegetaux Montpellier France)and Dr Sebastian PILARCZYK (Silesian Univer-sity Katowice Poland) for collection and identifi-cation of specimens We are also grateful to DrBeata SZYMANtildeSKA (Jagiellonian University De-partment of Systematic Zoology and Zoogeogra-phy Krakoacutew Poland) and Dr Olga WOicircNICKA(Jagiellonian University Department of Cytologyand Histology Krakoacutew Poland) for their skilledtechnical assistance

References

BAUMANN P 2005 Biology of bacteriocyte-associated endo-symbionts of plant sup-sucking insects Annu Rev Micro-biol 59 155-189BAUMANN P 2006 Diversity of prokaryote-insect associa-tionswithin the Sternorrhyncha (psyllids whiteflies aphidsmealybugs) (In Insect Symbiosis vol 2 TA Miller KBourtzis ed Contemporary Topics in Entomology Series)1-24BOURGOIN T STEFFEN-CAMPBELL D CAMPBELL B C1997 Molecular phylogeny of Fulgoromorpha (InsectaHemiptera Archaeorrhyncha) The enigmatic Tettigometri-dae evolutionary affiliations and historical biogeographyCladistics 13 207-224BUCHNER P 1965 Endosymbiosis of Animals with PlantMi-croorganisms Interscience Publishers New York LondonSydneyCHENG D-J HOU R F 2001 Histological observations ontransovarial transmission of a yeast-like symbiote in Nila-

parvata lugens Stal (Homoptera Delphacidae) Tissue Cell33 273-279

CHENGD- J HOUR F 2005 Determination and distributionof a female-specific protein in the brown planthopper Nila-parvata lugens Stal (Homoptera Delphacidae) Tissue Cell37 37-45

EBERLEM W MC LEAN D L 1982 Initiation and orienta-tion of the symbiote migration in human body louse Pedicu-lus humanus L J Insect Physiol 28 417-422

FUKATSU T ISHIKAWA H 1996 Phylogenetic position ofyeast-like symbiont of Hamiltonaphis styraci (HomopteraAphididae) based on 18rDNA sequence Insect BiochemMol Biol 26 383-388HONGOH Y ISHIKAWA H 1997 Uric acid as a nitrogen re-source for the brown planthopperNilaparvata lugens stud-ies with synthetic diets and aposymbiotic insects Zool Sci

14 581-586HONGOHY ISHIKAWAH 2000 Evolutionary studies on uri-cases of fungal endosymbionts of aphids and planthoppersJ Mol Evol 51 265-277HOUKE J GRIFFITHSGW 1980 Intracellular symbiotes ofthe Homoptera Annu Rev Entomol 25 161-187ISHIKAWA H 2003 Insect Symbiosis An Introduction (InInsect Symbiosis vol 1 TA Miller K Bourtzis ed Con-temporary Topics in Entomology Series) 1-21KANG L MAX CAI L LIAO S SUN L ZHUH CHEN XSHEN D ZHAO S LI C 2003 Superinfection of Laodel-

phax striatellus with Wolbachia from Drospohila simulansHeredity 90 71-76MONTLLOR C B MAXMENA PURCELL A H 2002 Facul-tative bacterial endosymbionts benefit pea aphidsAcyrthosi-

phon pisum under heat stress Ecol Entomol 27 189-195NODAH 1977Histological and histochemical observation ofintracellular yeastlike symbiotes in the fat body o the smallerbrown planthopper Laodelphax striatellus (HomopteraDelphacidae) Appl Ent Zool 12 134-141NODA H SAITO T 1979 Effects of high temperature on thedevelopment of Laodelpax striatellus (Homoptera Delpha-cidae) and on its intracellular yeastlike symbiotesAppl EntZool 14 64-75NODA H NAKASHIMA N KOIZUMIM 1995 Phylogeneticposition of yeast-like symbiotes of rice planthoppers basedon partial 18S rDNA sequences Insect Biochem Mol Biol

25 639-646NODA H KOIZUMI Y ZHANG Q DENG K 2001 Infectiondensity of Wolbachia and incompatibilty level in two plan-thopper species Laodelpax striatellus and Sogatella furcif-

era Insect Biochem Mol Biol 31 727-737OLIVER K M RUSSEL J A MORAN N A HUNTERM S2003 Facultative bacterial symbionts in aphids confer resis-tance to parasitic wasps Proc Natl Acad Sci 1001803-1807SACCHI L GENCHIM CLEMENTI E BIGLIARDI E AVAN-ZATTI A M PAJOROI M NEGRI I MARZORATI MGONELLA E ALMA A DAFFONCHIO D BANDI C 2008Multiple symbiosis in the leafhopper Scaphoideus titanus(Hemiptera Cicadellidae) Details of transovarial transmis-sion of Cardinium sp and yeast-like endosymbionts TissueCell 40 231-242

SASAKIT ISHIKAWAH 1995 Production of essential aminoacids from glutamate by mycetocyte symbiont of the peaaphid Acyrthosiphon pisum J Insect Physiol 41 41-46SASAKIT KAWAMURAM ISHIKAWAH 1996 Nitrogen re-cycling in the brown planthopper Nilaparvata lugens in-volvement of yeast-like endosymbionts in uric acidmetabolism J Insect Physiol 42 125-129SCARBOROUGH C L FERRARI J GODFRAY H C J 2005Aphid Protected from Pathogen by Endosymbiont Science

310 1781SZKLARZEWICZ T MOSKAL A 2001 Ultrastructure distri-bution and transmission of endosymbionts in the whitefly

A MICHALIK et al136

outer layer is electron dense and has a thickness of30 nm The inner layer has lower electron densityand is 140 nm thick In the central part of the yeastcell a large spherical nucleus with a single nucleo-lus is present (Fig 5) The remaining cytoplasm isfilled with ribosomes mitochondria and large lipiddroplets (Fig 7) The YLSs reproduce by budding(Fig 8) Sexual reproduction of YLSs was not ob-served In older females (ie containing terminaloocytes in the stage of late vitellogenesis) theYLSs leave the mycetome cytoplasm They be-come released into a haemolymph and migrate to-wards the terminal oocytes (Fig 9) The endosym-bionts pass through the cells of the ovariole stalk(pedicel) (Fig 9) as well as follicular cells surround-ing the posterior pole of the oocyte (Fig 10 11)Subsequently they enter the perivitelline space(Figs 9 10) At the same time a deep depression isformed at the posterior pole of the oocyte TheYLSs accumulate in the oocyte depression andform a characteristic ldquosymbiont ballrdquo (Fig 12) Atthe end of oocyte growth the ldquosymbiont ballrdquo istightly packed with YLSs (Fig 13) Until the endof oocyte growth the YLSs are isolated from theooplasm by oolemma and do not enter the oo-plasm The endosymbionts gathered in the depres-sion of the oocytes like those harbored in themycetome undergo budding (not shown)

Discussion

Our observations revealed that both adult males andfemales of M pruinosa (Flatidae) as well as C anceps(Delphacidae) harbor a large number of intracellu-lar YLSs This observation shows that these endo-symbionts are essential for both sexes of examinedplanthoppers In recent years the metabolic signifi-cance of YLSs for growth and reproduction of thehost insects has been extensively studied using therice brown planthopper Nilaparvata lugens (SASAKIet al 1996 HONGOH amp ISHIKAWA 19972000WILK-INSON amp ISHIKAWA 2001) These studies revealedthat planthopper YLSs are involved in nitrogen re-cycling using uric acid as a nitrogenous resourcePlanthoppers in addition to producing uric acid asa nitrogenous waste product also synthesize it as astorage product during nitrogen deficiency Uricacid is stored in mycetomes and converted by uri-case secreted by YLSs into compounds of nutri-tional value It should be noted that CHENG andHOU (2005) demonstrated that YLSs are also en-gaged in synthesis of yolk precursors in females ofthe rice brown planthopper Nilaparvata lugens

Our studies showed that YLSs in M pruinosaand C anceps are transmitted from the mother tothe progeny by a similar route ie via cytoplasm ofcells of the pedicel as well as follicular cells sur-

rounding the posterior pole of the oocyte Since thesame situation has been observed in other mem-bers of the family Delphacidae ie Laodelphaxstriatellus (NODA 1977) and Nilaparvata lugens(CHENG amp HOU 2001) as well as in the leafhopperScaphoideus titanus (SACCHI et al 2008) it seemsprobable that all hemipterans have developed thesame mode of transmission of YLSs to the nextgeneration Thus the transmission of YLSs in in-sects is more uniform than the transmission of bac-teria (see Introduction) Moreover molecular stud-ies revealed that YLSs in hemipterans are not onlysimilarly inherited but are also phylogeneticallyclosely related to each other The analysis of 18Sribosomal DNA sequences of YLSs in delphacids(NODA et al 1995 XET-MULL et al 2004) aphids(FUKATSU amp ISHIKAWA 1996) and leafhoppers (SAC-CHI et al 2008) indicated that they belong to theclass Pyrenomycetes in the phylum AscomytinaThe YLSs in the examined rice delphacids are char-acterized by a high degree of similarity of 18S ribo-somal DNA sequences (NODA et al 1995 XET-MULL et al 2004) This finding strongly suggeststhat YLSs of delphacids are closely related (ieconstitute a monophyletic group) This implies inturn that the symbiosis of YLSs and delphacids isthe result of a single infection of the common an-cestor of present delphacids The YLSs harboredin members of the Flatidae family have so far notbeen examined by molecular methods thereforetheir systematic position remains unknown Sinceflatids and delphacids are phylogenetically distantwithin planthoppers (ie do not represent a mono-pyletic taxon) (BOURGOIN et al 1997 URBAN ampCRYAN 2007) it is unlikely that their endosymbi-onts have been acquired through a common ancestorIn this light it may be assumed that YLSs were hori-zontally transferred between flatid and delphacidlineages It is noteworthy that HONGOH and ISHI-KAWA (2000) on the basis of an analysis of uricasegene sequences of YLSs in aphids and delphacidsprovided evidence of a close relationship betweenYLSs in these phylogenetically distant groups ofinsects as a result of the horizontal transfer of micro-organismsfrom the aphid to the planthopper lineage

Both in M pruinosa and C anceps the migrationof YLSs is correlated with ovary development (theYLSs infect vitellogenic oocytes) Thus this ob-servation strongly supports the hypothesis that themovement of microorganisms is stimulated by anunknown factor released by ovaries (EBERLE ampMC LEAN 1982 macrELAZOWSKA amp BILINtildeSKI 1999SZKLARZEWICZ amp MOSKAL 2001 SZKLARZEWICZet al 2006)

We observed that apart from YLSs rod-shapedbacteria are present in the body of all specimens ofM pruinosa In contrast to YLSs bacteria havenever been found in the ovaries of M pruinosa

Endosymbiotic Microorganisms in Planthoppers 135

The absence of these bacteria in the ovaries indi-cates that they may be horizontally transmitted be-tween specimens The large number of bacteria bothin the mycetome cytoplasm as well as in its epithe-lium suggests that they may have a significant(positive or negative) influence on the host insectThese microorganisms may represent S-symbiontsof M pruinosa but may also prove to be patho-genic It should be noted that NODA and SAITO(1979) detected rod-shaped bacteria in mycetomesof the planthopper Laodelphax striatellus (Del-phacidae) but did not suggest a possible role forthem It may be also speculated that these bacteriabelong to the widespread within arthropodsricketsia-like genus Wolbachia pipientis The lastassumption is supported by PCR detection of Wol-bachia in two delphacids Laodelphax striatellusand Sogatella furcifera (NODA et al 2001) as wellas by the observation that specimens of Laodel-phax striatellus may be horizontally infected byWolbachia (KANG et al 2003) To verify these hy-potheses further studies of specimens of M prui-nosa taken from different populations are needed

Acknowledgements

We would like to express our gratitude to DrJean-Francois GERMAIN (Labolatoire National dela Protection des Vegetaux Montpellier France)and Dr Sebastian PILARCZYK (Silesian Univer-sity Katowice Poland) for collection and identifi-cation of specimens We are also grateful to DrBeata SZYMANtildeSKA (Jagiellonian University De-partment of Systematic Zoology and Zoogeogra-phy Krakoacutew Poland) and Dr Olga WOicircNICKA(Jagiellonian University Department of Cytologyand Histology Krakoacutew Poland) for their skilledtechnical assistance

References

BAUMANN P 2005 Biology of bacteriocyte-associated endo-symbionts of plant sup-sucking insects Annu Rev Micro-biol 59 155-189BAUMANN P 2006 Diversity of prokaryote-insect associa-tionswithin the Sternorrhyncha (psyllids whiteflies aphidsmealybugs) (In Insect Symbiosis vol 2 TA Miller KBourtzis ed Contemporary Topics in Entomology Series)1-24BOURGOIN T STEFFEN-CAMPBELL D CAMPBELL B C1997 Molecular phylogeny of Fulgoromorpha (InsectaHemiptera Archaeorrhyncha) The enigmatic Tettigometri-dae evolutionary affiliations and historical biogeographyCladistics 13 207-224BUCHNER P 1965 Endosymbiosis of Animals with PlantMi-croorganisms Interscience Publishers New York LondonSydneyCHENG D-J HOU R F 2001 Histological observations ontransovarial transmission of a yeast-like symbiote in Nila-

parvata lugens Stal (Homoptera Delphacidae) Tissue Cell33 273-279

CHENGD- J HOUR F 2005 Determination and distributionof a female-specific protein in the brown planthopper Nila-parvata lugens Stal (Homoptera Delphacidae) Tissue Cell37 37-45

EBERLEM W MC LEAN D L 1982 Initiation and orienta-tion of the symbiote migration in human body louse Pedicu-lus humanus L J Insect Physiol 28 417-422

FUKATSU T ISHIKAWA H 1996 Phylogenetic position ofyeast-like symbiont of Hamiltonaphis styraci (HomopteraAphididae) based on 18rDNA sequence Insect BiochemMol Biol 26 383-388HONGOH Y ISHIKAWA H 1997 Uric acid as a nitrogen re-source for the brown planthopperNilaparvata lugens stud-ies with synthetic diets and aposymbiotic insects Zool Sci

14 581-586HONGOHY ISHIKAWAH 2000 Evolutionary studies on uri-cases of fungal endosymbionts of aphids and planthoppersJ Mol Evol 51 265-277HOUKE J GRIFFITHSGW 1980 Intracellular symbiotes ofthe Homoptera Annu Rev Entomol 25 161-187ISHIKAWA H 2003 Insect Symbiosis An Introduction (InInsect Symbiosis vol 1 TA Miller K Bourtzis ed Con-temporary Topics in Entomology Series) 1-21KANG L MAX CAI L LIAO S SUN L ZHUH CHEN XSHEN D ZHAO S LI C 2003 Superinfection of Laodel-

phax striatellus with Wolbachia from Drospohila simulansHeredity 90 71-76MONTLLOR C B MAXMENA PURCELL A H 2002 Facul-tative bacterial endosymbionts benefit pea aphidsAcyrthosi-

phon pisum under heat stress Ecol Entomol 27 189-195NODAH 1977Histological and histochemical observation ofintracellular yeastlike symbiotes in the fat body o the smallerbrown planthopper Laodelphax striatellus (HomopteraDelphacidae) Appl Ent Zool 12 134-141NODA H SAITO T 1979 Effects of high temperature on thedevelopment of Laodelpax striatellus (Homoptera Delpha-cidae) and on its intracellular yeastlike symbiotesAppl EntZool 14 64-75NODA H NAKASHIMA N KOIZUMIM 1995 Phylogeneticposition of yeast-like symbiotes of rice planthoppers basedon partial 18S rDNA sequences Insect Biochem Mol Biol

25 639-646NODA H KOIZUMI Y ZHANG Q DENG K 2001 Infectiondensity of Wolbachia and incompatibilty level in two plan-thopper species Laodelpax striatellus and Sogatella furcif-

era Insect Biochem Mol Biol 31 727-737OLIVER K M RUSSEL J A MORAN N A HUNTERM S2003 Facultative bacterial symbionts in aphids confer resis-tance to parasitic wasps Proc Natl Acad Sci 1001803-1807SACCHI L GENCHIM CLEMENTI E BIGLIARDI E AVAN-ZATTI A M PAJOROI M NEGRI I MARZORATI MGONELLA E ALMA A DAFFONCHIO D BANDI C 2008Multiple symbiosis in the leafhopper Scaphoideus titanus(Hemiptera Cicadellidae) Details of transovarial transmis-sion of Cardinium sp and yeast-like endosymbionts TissueCell 40 231-242

SASAKIT ISHIKAWAH 1995 Production of essential aminoacids from glutamate by mycetocyte symbiont of the peaaphid Acyrthosiphon pisum J Insect Physiol 41 41-46SASAKIT KAWAMURAM ISHIKAWAH 1996 Nitrogen re-cycling in the brown planthopper Nilaparvata lugens in-volvement of yeast-like endosymbionts in uric acidmetabolism J Insect Physiol 42 125-129SCARBOROUGH C L FERRARI J GODFRAY H C J 2005Aphid Protected from Pathogen by Endosymbiont Science

310 1781SZKLARZEWICZ T MOSKAL A 2001 Ultrastructure distri-bution and transmission of endosymbionts in the whitefly

A MICHALIK et al136

The absence of these bacteria in the ovaries indi-cates that they may be horizontally transmitted be-tween specimens The large number of bacteria bothin the mycetome cytoplasm as well as in its epithe-lium suggests that they may have a significant(positive or negative) influence on the host insectThese microorganisms may represent S-symbiontsof M pruinosa but may also prove to be patho-genic It should be noted that NODA and SAITO(1979) detected rod-shaped bacteria in mycetomesof the planthopper Laodelphax striatellus (Del-phacidae) but did not suggest a possible role forthem It may be also speculated that these bacteriabelong to the widespread within arthropodsricketsia-like genus Wolbachia pipientis The lastassumption is supported by PCR detection of Wol-bachia in two delphacids Laodelphax striatellusand Sogatella furcifera (NODA et al 2001) as wellas by the observation that specimens of Laodel-phax striatellus may be horizontally infected byWolbachia (KANG et al 2003) To verify these hy-potheses further studies of specimens of M prui-nosa taken from different populations are needed

Acknowledgements

We would like to express our gratitude to DrJean-Francois GERMAIN (Labolatoire National dela Protection des Vegetaux Montpellier France)and Dr Sebastian PILARCZYK (Silesian Univer-sity Katowice Poland) for collection and identifi-cation of specimens We are also grateful to DrBeata SZYMANtildeSKA (Jagiellonian University De-partment of Systematic Zoology and Zoogeogra-phy Krakoacutew Poland) and Dr Olga WOicircNICKA(Jagiellonian University Department of Cytologyand Histology Krakoacutew Poland) for their skilledtechnical assistance

References

BAUMANN P 2005 Biology of bacteriocyte-associated endo-symbionts of plant sup-sucking insects Annu Rev Micro-biol 59 155-189BAUMANN P 2006 Diversity of prokaryote-insect associa-tionswithin the Sternorrhyncha (psyllids whiteflies aphidsmealybugs) (In Insect Symbiosis vol 2 TA Miller KBourtzis ed Contemporary Topics in Entomology Series)1-24BOURGOIN T STEFFEN-CAMPBELL D CAMPBELL B C1997 Molecular phylogeny of Fulgoromorpha (InsectaHemiptera Archaeorrhyncha) The enigmatic Tettigometri-dae evolutionary affiliations and historical biogeographyCladistics 13 207-224BUCHNER P 1965 Endosymbiosis of Animals with PlantMi-croorganisms Interscience Publishers New York LondonSydneyCHENG D-J HOU R F 2001 Histological observations ontransovarial transmission of a yeast-like symbiote in Nila-

parvata lugens Stal (Homoptera Delphacidae) Tissue Cell33 273-279

CHENGD- J HOUR F 2005 Determination and distributionof a female-specific protein in the brown planthopper Nila-parvata lugens Stal (Homoptera Delphacidae) Tissue Cell37 37-45

EBERLEM W MC LEAN D L 1982 Initiation and orienta-tion of the symbiote migration in human body louse Pedicu-lus humanus L J Insect Physiol 28 417-422

FUKATSU T ISHIKAWA H 1996 Phylogenetic position ofyeast-like symbiont of Hamiltonaphis styraci (HomopteraAphididae) based on 18rDNA sequence Insect BiochemMol Biol 26 383-388HONGOH Y ISHIKAWA H 1997 Uric acid as a nitrogen re-source for the brown planthopperNilaparvata lugens stud-ies with synthetic diets and aposymbiotic insects Zool Sci

14 581-586HONGOHY ISHIKAWAH 2000 Evolutionary studies on uri-cases of fungal endosymbionts of aphids and planthoppersJ Mol Evol 51 265-277HOUKE J GRIFFITHSGW 1980 Intracellular symbiotes ofthe Homoptera Annu Rev Entomol 25 161-187ISHIKAWA H 2003 Insect Symbiosis An Introduction (InInsect Symbiosis vol 1 TA Miller K Bourtzis ed Con-temporary Topics in Entomology Series) 1-21KANG L MAX CAI L LIAO S SUN L ZHUH CHEN XSHEN D ZHAO S LI C 2003 Superinfection of Laodel-

phax striatellus with Wolbachia from Drospohila simulansHeredity 90 71-76MONTLLOR C B MAXMENA PURCELL A H 2002 Facul-tative bacterial endosymbionts benefit pea aphidsAcyrthosi-

phon pisum under heat stress Ecol Entomol 27 189-195NODAH 1977Histological and histochemical observation ofintracellular yeastlike symbiotes in the fat body o the smallerbrown planthopper Laodelphax striatellus (HomopteraDelphacidae) Appl Ent Zool 12 134-141NODA H SAITO T 1979 Effects of high temperature on thedevelopment of Laodelpax striatellus (Homoptera Delpha-cidae) and on its intracellular yeastlike symbiotesAppl EntZool 14 64-75NODA H NAKASHIMA N KOIZUMIM 1995 Phylogeneticposition of yeast-like symbiotes of rice planthoppers basedon partial 18S rDNA sequences Insect Biochem Mol Biol

25 639-646NODA H KOIZUMI Y ZHANG Q DENG K 2001 Infectiondensity of Wolbachia and incompatibilty level in two plan-thopper species Laodelpax striatellus and Sogatella furcif-

era Insect Biochem Mol Biol 31 727-737OLIVER K M RUSSEL J A MORAN N A HUNTERM S2003 Facultative bacterial symbionts in aphids confer resis-tance to parasitic wasps Proc Natl Acad Sci 1001803-1807SACCHI L GENCHIM CLEMENTI E BIGLIARDI E AVAN-ZATTI A M PAJOROI M NEGRI I MARZORATI MGONELLA E ALMA A DAFFONCHIO D BANDI C 2008Multiple symbiosis in the leafhopper Scaphoideus titanus(Hemiptera Cicadellidae) Details of transovarial transmis-sion of Cardinium sp and yeast-like endosymbionts TissueCell 40 231-242

SASAKIT ISHIKAWAH 1995 Production of essential aminoacids from glutamate by mycetocyte symbiont of the peaaphid Acyrthosiphon pisum J Insect Physiol 41 41-46SASAKIT KAWAMURAM ISHIKAWAH 1996 Nitrogen re-cycling in the brown planthopper Nilaparvata lugens in-volvement of yeast-like endosymbionts in uric acidmetabolism J Insect Physiol 42 125-129SCARBOROUGH C L FERRARI J GODFRAY H C J 2005Aphid Protected from Pathogen by Endosymbiont Science

310 1781SZKLARZEWICZ T MOSKAL A 2001 Ultrastructure distri-bution and transmission of endosymbionts in the whitefly

A MICHALIK et al136

Aleurochiton aceris Modeer (Insecta Hemiptera Aleyrodi-nea) Protoplasma 218 45-53SZKLARZEWICZ T KEcircDRA K NImacrNIK S 2006 Ultrastruc-ture and transovarial transmission of endosymbiotic micro-organisms in Palaeococcus fuscipennis (Burmeister)(Insecta Hemiptera Coccinea Monophlebidae) Folia biol(Krakoacutew) 54 69-74SZKLARZEWICZ T JANKOWSKAW poundUKASIEWICZK SZY-MANtildeSKA B 2007 Structure of the ovaries and oogenesis inCixius nervosus (Cixiidae) Javesella pellucida andConomelus anceps (Delphacidae) (Insecta Hemiptera Ful-goromorpha)Arthr Struct Dev 36 199-207

THAOM L CLARC M A BAUMANN L BRENNAN E BMORAN N A BAUMANN P 2000 Secondary endosymbi-onts of psyllids have been aquired multiple times Curr Mi-crobiol 41 300-304

URBAN J M CRYAN J R 2007 Evolution of planthoppers(Insecta Hemiptera Fulgoroidea) Mol Phylogenet Evol42 556-572

WILKINSON T L ISHIKAWA H 2001 On the functional sig-nificance of symbiotic microorganisms in the Homoptera acomparative study of Acyrthosiphon pisum and Nilaparvatalugens Physiol Entomol 26 86-93

XET-MULLAM QUESADA T ESPINOZA AM 2004 Phy-logenetic position of the yeast-like symbiotes of Tagosodesorizicolus (Homoptera Delphacidae) based on 18S ribo-somalDNApartial sequencesRevBiol Trop 52 777-785

macrELAZOWSKA M BILINtildeSKI S M 1999 Distribution andtransmission of endosymbiotic microorganisms in the oo-cytes of the pig louse Haematopinus suis (L) (InsectaPhthiraptera) Protoplasma 209 207-213

Endosymbiotic Microorganisms in Planthoppers 137