Rickettsia as obligate and mycetomic bacteria - The FASEB Journal

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Rickettsia as obligate and mycetomic bacteria M. Alejandra Perotti,* Heather K. Clarke,* Bryan D. Turner,† and Henk R. Braig*,1 *School of Biological Sciences, University of Wales Bangor, Bangor, Gwynedd, UK; and †Department of Life Sciences, King’s College London, London, UK Rickettsiae are well known as intracellular pathogens of animals, humans, and plants and facultative and unorganized symbionts of invertebrates. No close relative of mitochondria has yet been associated with nutritional or developmental dependency of its host cell or organism. We have found a mycetomic Rickettsia that is a strict obligatory symbiont of the parthenogenetic booklouse Liposcelis bostrychophila (Psocoptera). These rickettsiae show an evolutionary transition from a solitary to a primary mycetomic bacterium adapted to the development of its host. These intracellular and intranuclear bacteria reside in specialized cells in several tissues. Their distribution changes markedly with the development of their host. The most advanced phenotype is a paired mycetome in the abdomen, described for the first time for Rickettsia and this host order. The mycetomic rickettsiae of two parthenogenetic book lice species are in the spotted fever group and in the basal limoniae group. While mycetomic bacteria are well known for their metabolic or light-emitting functions, these rickettsiae have an essential role in the early development of the oocyte. Removal of the Rickettsia stops egg production and reproduction in the book louse. In two phylogenetically distant psocopteran species, Rickettsia are shown to be associated with four transitional stages from free bacteria, infected cells, through single mycetocytes to organ-forming mycetomes.—Perotti, M. A., Clarke, H. K., Turner, B. D., Braig, H. R. Rickettsia as obligate and mycetomic bacteria. FASEB J. 20, E1646 –E1656 (2006)

ABSTRACT

Key Words: evolution 䡠 development 䡠 Liposcelis bostrychophila 䡠 Cerobasis guestfalica 䡠 Psocoptera Endosymbiotic bacteria are very common in invertebrates (1– 4). Some of them are reproductive parasites, some are nutritional symbionts, and the majority have unknown effects on their hosts. The interactions between intracellular symbionts and host cells are complex (5, 6). The diversity by which intracellular bacteria manipulate their hosts’ reproduction is considerable and ranges from parthenogenesis, male-killing, cytoplasmic incompatibility to functional feminization of infected females (7–10). These parasitic associations are relatively young in evolutionary terms. In contrast, nutritional associations of prokaryotes and eukaryotes are older and often show elaborate adaptations in the host for their symbiont (11). The host provides during E1646

very early development specialized cells for its intracellular bacteria, the mycetocytes or bacteriocytes. The primary endosymbiont housed in a mycetome (bacteriome, bacteriotome) is recognized as an organ-forming bacterium (2). These organs have become an integral part of the hosts’ anatomy and physiology so that they form even if the symbiont has been removed. In a few instances, the primary bacterial endosymbiont becomes replaced by a secondary symbiont, which can be a bacterium or a yeast that takes over the existing host structures. Reproductive parasites affect individual species, yet their distribution is unpredictable and speciation of parasite and host show little, if any, coevolution. Nutritional symbioses on the other hand are characterized by host clades that are infected with endosymbiotic bacteria, and both organisms exhibit far-reaching coevolution. Host clades can encompass an entire order as, for example, in cockroaches. Nutritional symbionts and reproductive parasites are not infectious. Acquisition of either is a rare event and poorly understood (12). The genus Rickettsia is well studied for its pathogenicity in humans and animals, being responsible (among others) for various forms of typhus and spotted fevers (13). Recently, several spotted fever group rickettsiae of ticks and fleas have been identified as human pathogens and emerging zoonoses (14). Rickettsia have also been implicated as plant pathogens (15). These Rickettsia use ticks, mites, lice, fleas, and leafhoppers as their vectors. Although they are transovarially transmitted to a certain degree by their vector, they may remain pathogens of their invertebrate host as well (16). Insectonly Rickettsia may act as male-killing agents in ladybird and leaf mining beetles (17–19). In the pea aphid, the rickettsial PAR symbiont might have a negative effect on host fitness depending on environmental factors (20, 21). In ticks and fleas, it is still almost impossible to discriminate between a symbiotic Rickettsia and a vectored Rickettsia. R. parkeri has recently been recognized as a human pathogen ⬎60 years after its initial isolation from ticks (14). Rickettsia infections in some leech species can lead to remarkable increases in host size (22). The effects of Rickettsia on other host arthropods such as seed weevils, whiteflies, or mites are not yet 1

Correspondence: School of Biological Sciences, University of Wales Bangor, Bangor, Gwynedd LL57 2UW, UK. E-mail: [email protected] doi: 10.1096/fj.06-5870fje 0892-6638/06/0020-1646 © FASEB

known (23–25). A new Rickettsia has recently been described from an amphizoic amoeba isolated from the gills of roach (26). Where tested, these associations of Rickettsia are facultative for the host. The aphid Rickettsia, for example, are found in the hemolymph, but recently were also localized in secondary mycetocytes and sheath cells (21, 27). The importance of mycetomes has long been realized (1, 2, 11, 28), and the developmental origin and evolution of mycetomes— especially the early steps in the interaction of host and symbiont—are becoming accessible to investigation (12, 29, 30). We have identified a Rickettsia that is obligatory for early development of the parthenogenetic booklouse Liposcelis bostrychophila and that solely occupies a primary mycetome. TABLE 1.

The same stored food pest liposcelid species, the parthenogenetic L. bostrychophila, and the bisexual L. entomophila, originally used by Yusuf and Turner, were reared for this work (31). An epiphytic parthenogenetic species, Cerobasis guestfalica, was collected in Wales by HKC and in greater London by BDT. All the other psocid species screened for Rickettsia were collected by HKC and MAP in Wales or received from collectors (Table 1). Elimination of rickettsiae One first instar nymph of Liposcelis bostrychophila was transferred to an individual vial (5 ml vol) and reared to adulthood in darkness at 37°C and 70 – 80% RH. Six vials were used for the elimination experiment and three vials were used to

Rickettsia in Psocopteraa

Species

Liposcelidae (Troctomorpha) Liposcelis bostrychophila L. entomophila L. brunnea L. paeta Psocomorpha Archipsocidae Archipsocus nomas Caecelidae Caecilius flavidus Kolbea quisquiliarum Ectopsocidae Ectopsocus meridionalis Elipsocidae Reuterella helvimacula Eliposocus hyalinus E. westwoodi Lachesillidae Lachesilla greeni L. pedicularia Philotarsidae Philotarsus picicornis Psocidae Amphigerontia bifasciata Metylophorus novaescotiae Loensia moesta Stenopsocidae Graphopsocus cruciatus Stenopsocus immaculatus S. stigmatus Trichopsocidae Trichopsocus acuminatus Trogiomorpha Lepidopsocidae Echmepteryx hageni Pterozanium kelloggi Psyllipsocidae Dorypteryx domestica Psyllipsocu ramramburii Trogiidae Cerobasis guestfalica Lepinotus patruelis Trogium pulsatorium a

MATERIALS AND METHODS

Reproduction

Collection

Rickettsia

Parthenogenetic Sexual Sexual Sexual

England, Wales, Czech Republic England, Czech Republic Czech Republic Czech Republic

All positive Negative Negative Negative

Sexual

USA

Negative

Sexual Sexual

Wales Wales

Negative Negative

Sexual

Wales

Negative

Sexual Sexual Sexual

Wales Wales Wales

Negative Negative Negative

Sexual Sexual

Wales Wales

Negative Negative

Sexual

Wales

Negative

Sexual Sexual Sexual

Wales Wales USA

Negative Negative Negative

Sexual Sexual Sexual

Wales Wales Wales

Negative Negative Negative

Sexual

Wales

Negative

Sexual Sexual

USA Wales

Negative Negative

Sexual Sexual

Czech Republic Wales

Negative Negative

Parthenogenetic Sexual Sexual

Wales: Gwynedd, Anglesey Wales Wales

All positive Negative Negative

Screening of Psocoptera by PCR for Rickettsia.

MYCETOMIC RICKETTSIA

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check for the elimination of rickettsiae in the produced aposymbiotic adults and eggs. The vials contained a strip of black filter paper. For 3 wk the papers were replaced every 3 days. The papers of the six vials of the experiment were kept under the same conditions to observe hatching, and the number of eggs was recorded. The eggs attached to the papers of the three extra vials were recovered; 50% were used for polymerase chain reaction (PCR) screening and 50% for microscopical screening for the presence of Rickettsia (using 4⬘,6⬘-diamnidino-2-phenylidole (DAPI) and a rickettsiae probe). At the end of each trial, adults were also dissected and examined by fluorescence microscopy. The life span of the individual specimens was also recorded. The same procedures were conducted on control specimens reared at room temperature (26°C, 70 – 80% RH), and the entire experiment was repeated three times. Dissections Specimens of L. bostrychophila and C. guestfalica were dissected for light and fluorescence microscopy. A maximum dissecting resolution of 10 –15 ␮m was obtained. The staging of the embryonic development followed the nomenclature of Goss (32). PCR and primers DNA was extracted from single psocids with a QIAGEN DNAeasyTM Tissue Kit following the protocol for Animal Tissues. PCR was performed in 20 ␮l volumes for 35 cycles in an Eppendorf Mastercycler 5331 Gradient with an initial denaturation at 95°C for 5 min. Forty-five seconds were used for each step. For the primer pair 99F-994R, the annealing temperature was set to 54°C; for all other primer combinations it was 55°C. The primers used were universal 16S rRNA primers for bacteria: 27F and 63F-1387R (33); specific primers for Wolbachia 16S rRNA, 99F-994R (34), wspA, originally Wolbachia outer surface protein: wsp81F-wsp691R (35); ftsZ: ftsZf1-ftsZr1, ftsZAdf-ftsZAdr, ftsZBf-ftsZbr (19), and ftsZHF 5⬘-CCG TAT GCC GAT TGC AGA GCT TG-3⬘, FtsZHR 5⬘-GCC ATG AGT ATT CAC TTG GCT-3⬘, which amplify a larger range of Wolbachia strains; specific primers for Cardinium hertigii 16S rRNA, originally CFB bacteria: EPS-f-EPS-r for insect-like CFB bacteria (36) and mite-like CFB bacteria CFBmite16S-FCFBmite16S-R (37). The diagnostic primers for detecting Rickettsia were designed following the sequencing of L. bostrychophila: Rick-16S-F 5⬘-ASG CGG TCA TCT GGG CTA CAA CT-3⬘ and Rick-16S-R 5⬘-CCC GCT GGC AAA TAA GAA TGA GG-3⬘ amplifying a 409 bp fragment. This pair is specific for 64 (86 allowing one mismatch) of 104 aligned Rickettsia rRNA sequences and does not recognize any of 171,148 non-Rickettsia bacteria. The universal primers for 28S rDNA, 28Sa-28Sb were used as controls (38). PCR products obtained were ligated into QUIAGEN pDrive Cloning Vectors and 15 positive colonies per ligation were sequenced in both directions. The sequences are deposited in GenBank with accession numbers listed in Fig. 1.

cies of 0.0056. The first 25% of trees were discarded. The arithmetic mean of the log likelihood of best state was – 4687.3. Fluorescence in situ hybridization (FISH) Probes The Rickettsia probe RickB1 5⬘-(Cy3) CCA TCA TCC CCT ACT ACA C-3⬘, which matches with 93 (98 allowing one mismatch) of 104 known Rickettsia rRNA sequences and does not recognize any of 171,148 non-Rickettsia sequences; and the 3 helpers, RH1 5⬘-TCT AGA TTA GTA GTT TTG A-3⬘; RH2 5⬘-CAC CTC TAC ACT AGG AAT-3⬘; and RH3 5⬘-CAG TTG TAG CCC AGA TGA C-3⬘ were based on the rickettsial sequence of L. bostrychophila. The Wolbachia probe described by Heddi et al. (39) W2 5⬘-(Cy5) CTT CTG TGA GTA CCG TCA TTA TC, which recognizes 213 (228) of 247 Wolbachia rRNA sequences, zero (0) Rickettsia and zero (208) other bacteria, was used with two helpers, W2H1 5⬘-TTC CTC ACT AAA AGA GCT TT-3⬘ and W2H2 5⬘-CAC GGA GTT AGC CAG GAC T-3⬘, to increase sensitivity. In addition, a modified Wolbachia probe W1 5⬘-(Cy3) AAT CCG GCC GAA CCG ACC C-3⬘, which recognizes 40 (107) of 247 Wolbachia rRNA sequences, zero (0) Rickettsia, and zero (0) other bacteria, was successfully used with three helpers W1H1 5-CCA TGC AAC ACT T3⬘, W1H2 5⬘-TAT CCC TTC GAA TAG GTA T-3⬘, and W1H3 5⬘-ATT TTC ATG TCA AGA AGT G-3⬘. Wolbachia probes were tested with Drosophila simulans infected with the Riverside strain of Wolbachia and tetracycline-treated Wolbachia-free controls as well as Wolbachia-infected lice. The rickettsial probes were tested with Rickettsia-free bisexual psocid species. No-probe and competition suppression controls using excess unlabeled probes were performed.

RESULTS Rickettsia are primary and sole endosymbionts Using universal and specific primers, only Rickettsia sequences were amplified from the parthenogenetic booklice Liposcelis bostrychophila and Cerobasis guestfalica (Table 1 and data not shown). Wolbachia and Cardiniumspecific primers did not result in any amplified products. The two parthenogenetic species and the bisexual L. entomophila used as a negative control were studied by confocal microscopy. Wolbachia probes did not recognize any target in booklice. Specimens that were simultaneously stained with DAPI and the rickettsial probe did not show any bacterial signal that was not recognized by the specific probe. Rickettsia were the only bacteria detected in these booklice. The gut lumen contained no bacterial cells. Light microscopy showed that the gut content was dominated by live gregarines or crithidiids.

Phylogenetic analysis

Mycetomic Rickettsia have multiple origins Multiple alignments were generated by using the Clustal W method within the program DNASTAR and corrected manually. Bayesian interference was analyzed under the GTR ⫹ I ⫹ ⌫ model (General Time Reversible with a proportion of invariable sites and a gamma-shaped distribution of rates across sites estimated using four discrete categories) as implemented in version 3.1 of MrBayes. The analysis was run for 1 million generations reaching an average sd of split frequenE1648

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The 16S genes of the two booklice Rickettsia differ by ⬎6% and are associated with two very different clades (Fig. 1). The clades are well established in both lineages. The L. bostrychophila Rickettsia is closest to the spotted fever group R. felis whereas the C. guestfalica Rickettsia joins the diverse ancestral group around R. limoniae.

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PEROTTI ET AL.

Figure 1. The phylogenetic position of the mycetomic bacteria within the genus Rickettsia. Mycetomic Rickettsia are indicated in boldface on a posterior probabilities tree based on 16S rRNA sequences. GenBank accession numbers are given in brackets. The tree has been rooted with O. tsutsugamushi as an outgroup. The numbers at nodes represent clade credibility values. In cases where the rickettsiae have not been formally named, the name of the host is given. Next to the name is a column of the invertebrate host group. Conservatively, four major groups of the genus Rickettsia have been identified.

Mycetomic Rickettsia are essential for egg development in a parthenogenetic species Rickettsia-specific primers did not detect Rickettsia in 26 known bisexual species of booklice and barklice (Table 1). First instar L. bostrychophila were reared at 37°C to eliminate the rickettsiae. The egg production of adults was reduced by ⬎90% (Table 2). The eggs laid did not contain Rickettsia and did not hatch. The elimination did not affect the life span of the book lice. Since C. guestfalica is difficult to culture and manipulate in the same way as the liposcelids, we cannot determine whether Rickettsia also plays a similar essential role in this parthenogenetic psocid.

in C. guestfalica. An overview of the anatomy of L. bostrychophila is provided in supplemental Fig. 1. Early oocytes harbored clusters of rickettsiae in the first anterior quarter of the ooplasm as they were received from the nurse cells (Supplemental Fig. 2B). While these clusters advanced toward the posterior end, rickettsiae started to disperse uniformly inside the ooplasm (Video S1). They then surrounded the nucleus, staying close to yolk vacuoles. When the nucleus TABLE 2. Elimination of Rickettsia stops egg laying and developmenta B

Treatment

Egg output

Lb-01

CT:27°C HT:37°C CT:27°C HT:37°C CT:27°C HT:37°C CT:27°C HT:37°C

110.0 (⫾32.1) 10.0 (⫾4.2) 110.8 (⫾19.7) 10.2 (⫾4.1) 135.3 (⫾8.9) 8.2 (⫾2.9) 118.7 (⫾24.2) 9.6 (⫾3.6)

Rickettsia change their cellular association during development

Lb-02

The infection pattern was very reproducible in individual liposcelid specimens. The tissue distribution of Rickettsia in L. bostrychophila was studied in detail. Transovarial transmission, the distribution of infection in Malpighian tubules and the mycetome was observed

Total

MYCETOMIC RICKETTSIA

Lb-03

a

Average egg output per female exposed to 37°C to remove rickettsial bacteria (⫾SD). CT: 27°C is the control treatment; HT: 37°C is the heat treatment.

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started moving away from the center and the nuclear membrane disappeared, filamentous chromosomes became visible. Rickettsia clusters followed the nucleus and surrounded the chromosomes (Supplemental Fig. 2C). Dense mitochondrial clouds merged between the bacterial clusters; similar close arrangements between mitochondria and Rickettsia could be seen later on in cells of the fat body and the Malpighian tubules. However, no bacteria were visible at the plane of metaphase condensation. During further successive nuclear divisions, while the egg was leaving the exit channel, the rickettsiae concentrated dorsally and posteriorly where some of the nuclei formed an oval ring shape (Supplemental Fig. 2D). In laid eggs that were ⬃ 24 h old and showing early gastric invagination, the bacteria occurred mainly in clusters at the periphery although some were still in the center. At stage J of embryo development, bacterial cells were found inside yolk droplets in the periphery of the egg. The cells of the narrow median strip of the mesoderm showed a scattered distribution of bacteria (Supplemental Fig. 2E). Heavy infection was seen in the newly invaginated germ cells (Fig. 2A, Supplemental Fig. 2F). First instar nymphs showed rickettsial aggregations in

cells lining the ventral side of the thorax. The nerve cells of the thoracic ganglia were infected. The epithelium of the anterior beginning of the midgut depicted localized cells with clusters of Rickettsia. A single heavily infected mycetocyte had migrated into the fat body on each site (Fig. 2B and Supplemental Video S2– 4). These two cells had a diameter of 5–7 ␮m (Supplemental Video S4). A few fat body cells showed individual Rickettsia. The germ line showed heavy infection. In second instar nymphs, the Rickettsia progressively spread into the posterior part of the gut epithelium. Eventually all epithelial cells of the midgut exhibited a low level of infection. Some cells showed Rickettsia inside their nuclei. The primordial ovaries showed localized infections at the anterior end just adjacent to fat body and midgut (Fig. 2C). The ovaries at each side enlarged and were heavily infected. Mycetomic Rickettsia During the third instar, small globular cells invaded the fat body and the gut epithelium. No free Rickettsia were detected in the gut lumen. The globular cells aggre-

Figure 2. Rickettsia in various developmental stages of the booklouse L. bostrychophila. Three channel confocal microscopy used with the rickettsial probe in yellow; yolk, soft tissue and cuticle in red and blue. A) Egg, stage J of development; visualization of infected invaginated germinal cells. B–G) Horizontal sections of abdomen. B) First instar: Rickettsia in germinal cells, in midgut epithelium mycetocyte, and fat body mycetocyte. Gut lumen free of bacteria. C) Second instar: Rickettsia in primordial ovaries, midgut mycetocyte and fat body mycetocyte. Two protozoans are seen inside the lumen of the midgut. D) Third instar: growing mycetomes at both sides of the body. Malpighian tubules (arrows), ovaries, and midgut epithelium infected. No bacterial signal inside rectum, fat body, or midgut lumen. E) Teneral mycetome (one side shown, arrow) and clusters of Rickettsia inside the growing oocyte. F, G) Adult female abdomen scanned at 30 ␮m intervals. The position of mycetomes is slightly modified by the growing of the oocyte. Mature oocyte presses midgut and mycetomes. F) Dorsal view, right mycetome. G) Ventral view, left mycetome. GC: germinal cells; M: midgut epithelium mycetocyte; FBM: fat body mycetocyte; GL: gut lumen; POV: primordial ovaries; P: protozoans; OV: ovaries; MT: Malpighian tubules; RT: rectum; FB: fat body; MGL: midgut lumen; OO: oocyte; RM: right mycetome; LM: left mycetome. Bars 20 ␮m.

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gated to shape a pair of structures at the posterior end of the midgut, at the ventral site, between midgut and incipient ovaries (Fig. 2D). These cell aggregates lead to the mycetomes of the adults (Supplemental Fig. 3A). In some cases the bacteria were lightly spread ventrally in the epithelium such as connecting both paired structures. The proximal portion of the Malpighian tubules started swelling under the rickettsial load. A similar pattern of localization was observed in the fourth instar; however, a pair of mycetomes with an elongated shape was now growing between the midgut and the developing ovaries (Supplemental Fig. 3B). In teneral adults (Fig. 2E), the mycetome next to the growing oocyte measured ⬃30 ␮m in length, 10 ␮m in height, and 10 ␮m in width. The mycetomes were built of 4 to 8 big and 8 to 12 small, nucleated, rhomboid mycetocytes. The large cells had a diameter of 6 – 8 ␮m; the small cells had one-fourth the size. The mycetome was held together by a thin, but dense, uniform anucleate layer. Bacterial cells were repeatedly detected in the surrounding of the mycetome unassociated with cells, which might suggest free migration in the hemolymph. A free mycetocyte was detected around the Malpighian tubules (Supplemental Fig. 3C). In adult females, the bacteria were concentrated in the two mycetomes (Fig. 2F, G, Supplemental Video S5), the four Malphigian tubules and the remaining ovarioles. Some ganglia, fat body, midgut epithelium, and ventral subepidermis cells still harbored bacteria. The two mycetomes of adults were similar in size, their mycetocytes inside became compact, globular in shape, and very crowded with slightly smaller Rickettsia bodies (Fig. 3A–C). The mycetomes shrank with age. The Rickettsia had the smallest size in the late mycetome and in the early oocyte, ranging from 700 to 1000 nm long and 350 and 450 nm wide. The number of mycetocytes remained the same but the nuclei of the mycetocytes were no longer detectable. The number of Rickettsia in young females was estimated by direct counting to be ⬃250 ⫾ 25 per big mycetocyte and around 3000 Rickettsia per mycetome. The Malpighian tubules showed the heaviest rickettsial infection in the first proximal third and only scattered infection in the remaining length. This first third developed into a very thick and strongly hypertrophic vessel (Fig. 3D). The large cells were crowded with Rickettsia. The nuclei became enormous before splitting in two. Binucleation was also seen in the uninfected sexual species (Supplemental Fig. 3D, E). The remaining length of the tubules, which exhibited only low numbers of Rickettsia, usually retained just one nucleus. In one adult specimen of L. bostrychophila we observed six Malpighian tubules instead of four. The two extra tubules were only developed to a third of the length and contained masses of Rickettsia. In one old adult of L bostrychophila, the mycetome on one side had disappeared whereas the closest region of the Malpighian tubules was even more swollen by bacteria. One teneral and one adult of L bostrychophila displayed an almost identical duplication of the mycetome MYCETOMIC RICKETTSIA

Figure 3. Mycetomes and Malphigian tubule of L. bostrychophila and transovarial transmission of Rickettsia. A–C, E) Rickettsial probe yellow channel only; D, F, G) three channels, rickettsial probe in yellow channel. A–C) Mycetomes of adults. Visualization of different mycetocytes that form the mycetome. Free rickettsial cells surround mycetome. C) 3-D projection of 20 ␮ m scanning, sagittal view of panel A showing thickness of the mycetome. D) Heavy infection of Malpighian tubule and surroundings in adult. E) Duplication of the mycetome in teneral. F) Horizontal section, ventral view of teneral of picture A showing the position of mycetomes at both sides of the body (arrows), the duplicated mycetome on the left side. G) Horizontal section. Transmission of Rickettsia into growing oocyte. Visualization of infection in the posterior base (arrow) of the nurse cells where transmission of Rickettsia into the growing oocyte occurs (circle). The Rickettsia clusters commence surrounding the oocyte nucleus. Follicular cells are free of infection. Fat body contains localized infected cells and the midgut epithelium shows a uniform distribution. Gut lumen is free of bacteria. NC: nurse cells; OO: oocyte; NU: oocyte’s nucleus; FC: follicular cells; FB: fat body; MGE: midgut epithelium; GL: gut lumen. Bars 20 ␮m.

on one side of their body leading to three mycetomes (Fig. 3E, F).

DISCUSSION Mycetomic Rickettsia Rickettsia in parthenogenetic Psocoptera inhabit fully developed primary mycetomes. On a few occasions, the E1651

early literature associated Rickettsia-like organisms with mycetomes. Rickettsia-like organisms were reported from louse or hippoboscid flies and from related tsetse flies (40, 41). They turned out not to be Rickettsia. The mycetomic bacteria in Glossina are now recognized as Wigglesworthia glossinidia and belong to the Gammaproteobacteria (42). The closest intracellular bacteria to Rickettsia in these flies are Wolbachia, which have not been detected in tsetse mycetomes. Low numbers of Wolbachia have been detected in secondary bacteriocytes of aphids that carry Serratia symbiotica, previously known as PASS or R type symbiont (43, 44). These cells may also hold a facultative Rickettsia, which has a negative effect on the host fitness under laboratory conditions (21, 53). The first observation of rickettsiae in Psocoptera described a thin border of bacteria on the epithelium of the stomach of the dustlouse Psokus (54). The name Psokus was used at that time, most likely as a vernacular name for dust-associated booklice in general and not for the genus Psocus. These rickettsiae were only extracellular. The lumenal infection was stable for 3 years in a laboratory colony of the booklice. Because of a close association with lice feces and the resemblance with R. pedikuli, the transmission of these rickettsiae into the stomach of human body lice was attempted by Sikora, but failed. The rickettsiae also resembled R. melophagi, which formed a dense border of perpendicularly arranged bacteria on the surface of the stomach of the dipteran sheeplouse or ked Melophagus ovinus. It was later called Wolbachia melophagi but its 16S sequence identified it as a Bartonella species in the Rhizobiales. Cowdry was unsuccessful in finding Rickettsia in a Psocus species (Psocidae) (55). Likewise, the extracellular R. pedikuli of the louse stomach lumen might not be a Rickettsia species. The literature on psocids anatomy, physiology, and embryology failed to identify any mycetomes or mycetomic structures (32, 45–52). Troctes divinatorius and L. divergens are synonyms of L. bostrychophila. The Rickettsia and the mycetomes have been observed in disparate species of booklice that belong to opposing ends in the evolution of Psocoptera. Cerobasis guestfalica (Trogiidae) belongs to the Trogiomorpha, which represents a primitive suborder of the book and bark lice order Psocoptera; Liposcelis bostrychophila (Liposcelididae) has been associated in the past with the advanced suborder Troctomorpha but is now considered to represent a highly derived lineage in the biting and chewing lice order Phthiraptera (56 –58). This suggests that the mycetome might be an organ belonging to the ancestor of both Psocoptera and Phthiraptera. This applies only to the mycetome as an organ and not necessarily to an obligate relationship between Rickettsia or any other bacterium and the ancestral host. Mycetomes of varying structures have been well described for most members of the Phthiraptera, but are unknown for the Psocoptera (59). However, paired mycetomes in the body cavity as in the booklice are very rare in Phthiraptera. Only the male lice of elephants, E1652

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Hematomyzus elephantis (Rhynchophthirina), contain mycetomes between the testes and the ventral hypodermis in between fat body lobes; in females, the mycetome resides in the ampulla between ovarioles and oviduct (59). Booklice have a paired mycetome formed by uninuclear mycetocytes and surrounded by an anuclear membrane. Such structures have been detected in an unpaired form in the rat louse Polyplax, the dog louse, Linognathus, and in dipteran bat flies, whereas the paired form has been described in hemipteran whiteflies and the leafhopper Cicadella viridis. However, some hemipteran cicadas and scale insects carry mycetomes surrounded by an epithelium while other cicadas and treehoppers develop syncytic mycetomes (2). The exclusive feeding of lice on blood and/or epidermis has been used to explain for the presence of mycetomes. Our findings in the basal Cerobasis lineage of booklice suggest that the evolution of mycetomic provisions might have predated the acquisition of blood feeding. The advanced Liposcelis lineage might have lost the parasitic lifestyle of lice (60). The continuing presence of mycetomes and the dependence on the mycetomic Rickettsia for reproduction might underpin the antiquity of symbiotic interactions regardless of food source. Host adaptations In developing booklice, one of the first tissues to show the presence of Rickettsia is the midgut epithelium; the infection originating in the embryonic development of the anterior midgut. This is followed by a spread of a low-level, homogeneous infection over the entire length of the midgut. We assume this spread to occur from cell to cell transmission, but we have not observed it. In a third step, highly infected cells infiltrate the midgut epithelium. The Rickettsia are present in the yolk surrounding the midgut rudiment. The Rickettsia infect some of the cells destined to build the midgut epithelium but some of these putative epithelial cells will convert into mycetocytes. Such a developmental change has also been observed in several anoplurans and mallophagans. The genus Hematopinus is characterized by numerous mycetocytes that infiltrate the midgut epithelium in a clearly comparable way to that which we have detected in booklice (59). Most of the somatic tissues in booklice that harbor Rickettsia do not undergo any changes. The nerve cells in the thoracic ganglia show no signs of pathology. Transmission electron microscopy pictures of the pharynx of L. divinatorius recognized numerous Gramnegative bacteria in unaffected esophageal epithelial cells and in the subesophageal ganglion cells (61). We consider this species to be L. bostrychophila despite an original poor attempt at identification. We found high levels of infection in the ventral subepidermis or hyperdermis in the booklice. A similar pattern has also been seen for the primary endosymbionts in the cattle and pig lice Hematopinus eurysternus and H. suis (59).

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Uninucleate mycetocytes of mesodermal origin infiltrate the fat body in cockroaches and chewing lice. Extraordinarily this relies on just two cells in booklice, and these cells behave in a symmetrical way. We suspect that Rickettsia are leaving these mycetocytes in a similar matter as they seem to leave the mycetomes later on. Such free Rickettsia might lead to the sporadic infection of fat body cells. Rickettsiae sensu stricto are not well known for infecting the fat body of arthropods. However, experimental infections of the tick Dermacentor reticulatus with R. sibirica lead to replication and accumulation of Rickettsia in the fat body (62). The preferred sites for the spotted fever group of rickettsiae in their tick vectors are salivary glands, Malpighian tubules, midgut epithelium, and ovaries if transovarially transmitted (63). We did not detect any Rickettsia in the salivary glands as might have been expected for Rickettsia that are vectored or horizontally transmitted. Treatment with high temperature confirmed that the Rickettsia in the parthenogenetic booklice are obligate for development of oocytes and egg laying. Egg laying can also be interrupted by antibiotic treatment but it is very difficult to reach a high penetrance with antibiotics (31). The presence of Rickettsia in Malpighian tubules in the literature is scattered. Most reports about Rickettsia in Malpighian tubules rely on morphological identification of the bacteria but make no attempt to exclude additional endosymbiotic bacteria. Much of the work has been done on ticks. We assume that all ticks harbor endosymbionts. Rickettsia have been described for many ticks, yet when Noda and colleagues studied the Malpighian tubules by PCR separately, none of four wellstudied species had Rickettsia (64). Many Rickettsia ascribed to Malpighian tubules on morphological grounds might not be Rickettsia in a taxonomic sense. The molecular identification of Rickettsia in the Malpighian tubules might indicate physiological functions of these Rickettsia in their invertebrate vectors. With the infection of Malpighian tubules, there is a clear host specialization in the first third of the tubules with hypertrophy of the vessels and the formation of giant cells occupied by masses of Rickettsia. In Ixodid ticks, varying zones are settled with endosymbionts and thickened; in Rhipicephalus and Dermacentor ticks, only the fourth section of the two vessels might hold endosymbionts; in Donacia leaf beetles, only two of the six tubules contain endosymbionts; in Coccotrypes bark beetles, four of six are infected. The most radical reshaping occurs in Erythrapion miniatum and related weevils, where two tubules are likewise used exclusively in the service of symbiosis and are transformed into little club-shaped structures in which the lumen is now limited to a short pedicle and which otherwise consist essentially of infected giant cells (2). This transformation of the tubules is already occurring at the stage of the evagination of the ectodermal proctodeum. This seems to exclude any interpretation of these Malpighian structures as pathological host reactions and might suggest that the Malpighian tubules are one of MYCETOMIC RICKETTSIA

the major locations at which the endosymbionts fulfill their physiological duty. Do the booklice Rickettsia contribute to the nitrogen metabolism in the wreath cells or nephrocytes of their hosts? Wolbachia endosymbionts causing cytoplasmic incompatibility and having no nutritional role in their Drosophila host infect Malpighian tubules in large numbers too (65). Genome analyses of obligate intracellular bacteria support a probable metabolic function (66 – 68). However, the curing of the parthenogenetic booklouse had no effect on life span. To secure a complete removal of Rickettsia, it was essential that the treatment started with first instar nymphs. The only observed phenomenological difference of the loss of Rickettsia is the complete cessation of egg development. Intranuclear Rickettsia With the exception of ciliates, intra- or endonuclear bacteria are quite unusual. A Gram-negative bacterium has been seen dividing within the nuclei of two termite species (69). Rickettsia belonging to the spotted fever group and Orientia tsutsugamushi are well known to invade the nucleus of both their invertebrate vector as well as their vertebrate host, but this is a rare phenomenon (63, 70 –73). Booklice rickettsiae are detected more often inside the nucleus, a behavior that seems to be similar to that of R. bellii (74). Only the nuclei of the mycetomes disappear over time, but not those of the free mycetocytes. The loss of nuclei has also been reported for other mycetomes, although in most cases a molecular identification of the endosymbionts is still outstanding. The intranuclear presence of Rickettsia and the disintegration of mycetome nuclei might not be directly related. Diversity of Rickettsia Rickettsia-like organisms with coccoid and bacilliform morphology and swollen coccoid granules have been detected in the ovaries and Malpighian tubules of the booklouse Dorypteryx pallida (Trogiomorpha: Psyllipsocidae) (75). The transmission via eggs of these bacteria had been observed. However, studies of the cytology of psocids did not detect any intracellular bacteria (52, 76). The first modern description by EM of L. bostrychophila describes endosymbionts in the ovaries, oocytes and abdominal subepidermal tissues (31, 77). This Wolbachia-like endosymbiont is molecularly recognized as a Rickettsia species. In the phylogenetic analysis, the Liposcelis Rickettsia cluster with a newly discovered Rickettsia also associated with parthenogenesis in the parasitoid wasp Neochrysocharis formosa (Hymenoptera: Eulophidae) (78) and are most closely related to R. felis. Although R. felis (ELB agent, R. azadi) is grouped with the spotted fever rickettsiae, its biology and genome separate it from the spotted fever group proper. Similarly, R. akari and R. australis break away from the other group members. The genome of R. felis carries an unexpectedly high E1653

number of ankyrsin motif-containing genes, which is much more characteristic for endosymbiotic Wolbachia than for Rickettsia (79, 80). The spotted fever group separates from the bellii group that assembles aphid, leafhopper, mite and tick Rickettsia. While many members of the spotted fever group barely differ from each other in 16S sequence, the Cerobasis Rickettsia form part of the most diverse grouping at the base of the genus. It assembles the crane fly Rickettsia, R. limoniae, with a Rickettsia associated with the amphizoic amoeba Nuclearia pattersoni of the gills of roaches and secondary and facultative Rickettsia found in leeches. The structure of this group is unresolved. Its members differ more between themselves than other groups do between each other. The Liposcelis bacteria are the first Rickettsia that function as primary endosymbionts and have become obligatory for oogenesis.

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Transitional stages in evolution The interaction between Rickettsia and booklice is characterized by a multitude of transitional stages. The oocyte is infected through both the germline and the nurse cells (Fig. 3G). Reproductive parasites are mainly transmitted through the germline whereas primary endosymbionts are transmitted via secondary tissues like nurse cells. Rickettsia are found in three different types of tissue. Bacteria are found in differentiated tissues, in single cell mycetocytes, and in an organ forming mycetome. Occasional duplication of one of the mycetomes and the appearance of additional Malpighian tubules might represent another transitional stage. Fluctuating asymmetry is quite common in insects; however, organ duplications are very rare in the ontology of insects. The duplication of the mycetomes might suggest that the establishment of the new organ is still experiencing genetically some instability. In “old” mycetomic associations, the mycetomic endosymbiont does not infect other tissues; in booklice it does. In most cases with centralized Malpighian infections, the nonadapted part of tubules is free from infections; in booklice it is not. The booklice Rickettsia are both intra- and extracellular, and are found in the nuclei. This suggests that we are witnessing the evolutionary transition from parasite to obligate nutritional or developmental symbiont. Rickettsia very recently described in aphids might be in a similar transitional stage (21). This Rickettsia affects the fitness of its host but is also found in secondary mycetocytes in large numbers. M.A.P. and H.R.B. were supported by funding from the Natural Environment Research Council (GR3/13199). We thank Andrew Davies, Bangor, for the calibration of the multiphoton laser and Ing. Zuzana Kucerova, Prague, and Prof. Edward L. Mockford, Normal, IL, USA, for booklice samples. E1654

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Received for publication February 24, 2006. Accepted for publication June 12, 2006.

PEROTTI ET AL.

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Rickettsia as obligate and mycetomic bacteria M. Alejandra Perotti,* Heather K. Clarke,* Bryan D. Turner,† and Henk R. Braig*,1 *School of Biological Sciences, University of Wales Bangor, Bangor, Gwynedd, UK; and †Department of Life Sciences, King’s College London, London, UK To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-5870fje

SPECIFIC AIMS We investigated the diversity of endosymbionts and their relationship to host development in parthenogenetic book lice. The unexpected identity of the symbiont led us to examine the complexity of the evolution of symbiotic and pathogenic interactions. We questioned whether we could observe transitional stages of adaptation in the ontogeny of a single individual host between extracellular bacteria and organ-forming bacteria. PRINCIPAL FINDINGS 1. Rickettsia are primary and sole endosymbionts Only Rickettsia sequences were amplified from Liposcelis bostrychophila and Cerobasis guestfalica. These two parthenogenetic species and a sexual species as a control, were studied by confocal microscopy. 2. Mycetomic Rickettsia have multiple origins The 16S genes of the two Rickettsia differ by ⬎6% and are associated with two very different clades. The Liposcelis Rickettsia are closest to the spotted fever group whereas the Cerobasis Rickettsia join a diverse ancestral group around R. limoniae. 3. Mycetomic Rickettsia are essential for egg development in parthenogenetic species Rickettsia-specific primers did not detect Rickettsia in 26 sexual species of booklice and barklice. L. bostrychophila were reared at 37°C to eliminate the rickettsiae. The egg production was reduced by ⬎90%. The eggs laid did not contain Rickettsia and did not hatch. The curing did not affect the life span. 4 Rickettsia change their cellular association during development Early oocytes harbored clusters of rickettsiae in the first anterior quarter of the ooplasm as they were received from the nurse cells. Heavy infection was seen in the newly invaginated germ cells. 2372

First instar nymphs showed rickettsial loads in cells lining the ventral side of the thorax. A single heavily infected mycetocyte had migrated into the fat body on each side. In second instar nymphs, the Rickettsia progressively spread into the posterior part of the gut epithelium. Some cells showed Rickettsia inside their nuclei. The primordial ovaries showed localized infections at the anterior end just adjacent to fat body and midgut. 5. Mycetomic Rickettsia During the third instar, small globular mycetocytes aggregated to shape a pair of structures at the posterior end of the midgut, at the ventral site, between midgut and incipient ovaries. These aggregates lead to the mycetomes of the adults. In the fourth instar, a pair of mycetomes with an elongated shape was now growing between the midgut and the developing ovaries. In tenerals, the mycetomes were built of 4 to 8 big and 8 to 12 small, nucleated rhomboid mycetocytes. Bacterial cells were repeatedly detected in the surrounding of the mycetome unassociated with cells, which might suggest free migration in the hemolymph. The bacteria were concentrated in the two mycetomes: the four Malphigian tubules and the remaining ovarioles. The two mycetomes of adults were similar in size; their mycetocytes inside became compact, globular in shape, and crowded with slightly smaller Rickettsia bodies (Fig. 1A–C). The Rickettsia had the smallest size in the late mycetome and in the early oocyte; size ranged between 700 and 1000 nm long and 350 and 450 nm wide. The Malpighian tubules showed the heaviest rickettsial infection in the first third from the gut and only scattered infection in the remaining length. This first third developed into a thick and strongly hypertrophic vessel (Fig. 1D). In one adult specimen of L. bostrychophila we observed six Malpighian tubules instead of four. The two extra tubules were only devel1

Correspondence: School of Biological Sciences, University of Wales Bangor, Bangor, Gwynedd LL57 2UW, UK. E-mail: [email protected] doi: 10.1096/fj.06-5870fje 0892-6638/06/0020-2372 © FASEB

ends in the evolution of Psocoptera. Cerobasis guestfalica (Trogiidae) belongs to the Trogiomorpha, which represents a primitive suborder of the book- and barklice order Psocoptera; Liposcelis bostrychophila (Liposcelididae) has been associated with the advanced suborder Troctomorpha, but is now considered to represent a highly derived lineage in the biting and chewing lice order Phthiraptera. This suggests that the mycetome might be an organ belonging to the ancestor of Psocoptera and Phthiraptera. Mycetomes of varying structures have been well described for most members of the Phthiraptera but have been unknown for the Psocoptera. The exclusive feeding of lice on blood and/or epidermis has been used to explain the presence of mycetomes. Our findings in the basal Cerobasis lineage in the booklice suggest that the evolution of mycetomic provisions might have predated the acquisition of blood feeding. The advanced Liposcelis lineage might have lost the parasitic lifestyle of lice. The continuing presence of mycetomes and the dependence on the mycetomic Rickettsia for reproduction might underpin the antiquity of symbiotic interactions regardless of food source. Host adaptations Figure 1. Mycetomes and Malphigian tubule of L. bostrychophila and transovarial transmission of Rickettsia. A–C) Mycetomes of adults. Visualization of different mycetocytes that form the mycetome. Free rickettsial cells surround mycetome. C) 3D projection of 20 ␮m scanning, sagittal view of panel A showing thickness of the mycetome. D) Heavy infection of Malpighian tubule and surroundings in adult. E) Duplication of the mycetome in teneral. F) Horizontal section, ventral view of teneral of picture A showing the position of mycetomes at both sides of the body (arrows), the duplicated mycetome on the left side. G) Horizontal section. Transmission of Rickettsia into growing oocyte. Visualization of infection in the posterior base (arrow) of the nurse cells where transmission of Rickettsia into the growing oocyte occurs (circle). The Rickettsia clusters commence surrounding the oocyte nucleus. Follicular cells are free of infection. Fat body contains localized infected cells and the midgut epithelium shows a uniform distribution. Gut lumen is free of bacteria. NC, nurse cells; NU, oocyte’s nucleus; FC, follicular cells; MGE, midgut epithelium; GL, gut lumen; OO, oocyte. Bars 20 ␮m.

oped to a third of the length and contained masses of Rickettsia. One teneral and one adult of L bostrychophila displayed an almost identical duplication of the mycetome on one side of their body leading to three mycetomes (Fig. 1E, F). CONCLUSIONS AND SIGNIFICANCE Mycetomic Rickettsia The Rickettsia and the mycetomes have been observed in disparate species of booklice that belong to opposing MYCETOMIC RICKETTSIA

We did not detect any Rickettsia in the salivary glands as might have been expected for Rickettsia that are vectored or horizontally transmitted. Treatment with high temperature confirmed that the Rickettsia in parthenogenetic L. bostrychophila is obligate for development of oocytes and egg laying. With the infection of Malpighian tubules there is a clear host specialization in the first third of the tubules, with hypertrophy of the vessels and the formation of giant cells occupied by masses of Rickettsia. Transformation of the tubules occurs at the stage of the evagination of the ectodermal proctodeum, which seems to exclude any interpretation of the Malpighian structures as pathological host reactions and may suggest that the Malpighian tubules are one major location at which the endosymbionts fulfill their physiological duty. However, the curing of the parthenogenetic booklouse had no effect on life span. The only observed phenomenological difference of the loss of Rickettsia is complete cessation of egg development. Intranuclear Rickettsia With the exception of ciliates, intra- or endonuclear bacteria are quite unusual. Rickettsia belonging to the spotted fever group and Orientia tsutsugamushi are known to invade the nucleus of both their invertebrate vector and their vertebrate host but this is rare. Booklice rickettsiae are much more often detected inside the nucleus. Only the nuclei of the mycetomes disappear over time but not those of the mycetocytes. 2373

Diversity of Rickettsia In phylogenetic analysis, the Liposcelis Rickettsia cluster with a newly discovered Rickettsia associated with parthenogenesis in the parasitoid wasp Neochrysocharis formosa and are most closely related to R. felis. Although R. felis is grouped with the spotted fever rickettsiae, its biology and genome separate it from the spotted fever group proper. R. felis carries an unexpectedly high number of ankyrin motif-containing genes, which is more characteristic for endosymbiotic Wolbachia than for Rickettsia. The spotted fever group separates from the bellii group that assembles aphid, leafhopper, mite, and tick Rickettsia. The Cerobasis Rickettsia form part of the most divers grouping at the base of the genus. It assembles the crane fly Rickettsia, R. limoniae, with a Rickettsia associated with the amphizoic amoeba Nuclearia pattersoni of the gills of roaches and secondary and facultative Rickettsia found in leeches. The Liposcelis bacteria are the first Rickettsia that function as primary endosymbionts and have become obligatory for oogenesis. Transitional stages in evolution The interaction between Rickettsia and booklice is characterized by a multitude of transitional stages (Fig. 2). The oocyte is infected through the germline and nurse cells (Fig. 1G). Reproductive parasites are mainly transmitted through the germline and primary endosymbionts are transmitted via secondary tissues like nurse cells. Rickettsia are found in differentiated tissues, single

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Figure 2. Overlapping evolutionary stages of host-symbiont interaction for an obligate Rickettsia in a single parthenogenetic psocid host.

cell mycetocytes, and an organ-forming mycetome. Fluctuating asymmetry is quite common in insects, but organ duplications are rare in the ontology of insects. In “old” mycetomic associations, the mycetomic endosymbiont does not infect other tissues. In most cases with centralized Malpighian infections, the nonadapted part of tubules is free from infections; in booklice it is not. The Rickettsia are both intra- and extracellular and are found in nuclei, which suggests we are witnessing the evolutionary transition from parasite to obligate nutritional or developmental symbiont.

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PEROTTI ET AL.