EVOLUTIONARY INFERENCES FROM A COMPARISON OF COCKROACH NUCLEAR DNA AND DNA FROM THEIR FAT-BODY AND EGG ENDOSYMBIONTS

DNA was isolated from muscle tissue and from concentrations of the egg and fat-body endosymbionts of the cockroaches Periplaneta americana, Blatta orientalis, Blaberus giganteus, Gromphadorhina portentosa, Leucophaea maderae, Cryptocercus punctulatus, and Nyctibora lutzi. Denatured DNA from each was immobilized on nitrocellulose membranes and reassociated with labeled probe DNAs from egg endosymbionts and muscle nuclei of B. orientalis. The DNAs were compared by extent of binding and by the thermal melting profiles of the DNA duplexes. The DNAs from the endosymbionts in the eggs and fat body in both P. americana and B. orientalis were shown to be virtually identical, confirming that transovarial transmission of the bacteria does take place. The thermal stabilities of the heteroduplexes formed with the probe DNA from egg endosymbionts of B. orientalis differed from the homologous duplexes by only 1°–11°C, indicating a close relationship among the endosymbiont strains. The heteroduplexes of the nuclear DNAs differ from the homologous duplexes by 2°–7°C. Compared with known systems in bacterial and Drosophila species, these results indicate similar base-pair mismatches for host and endosymbiont DNAs. From these correlations, we deduce that the endosymbionts have probably been associated with their host cockroaches since before the latter speciated.     

Variation of clutch size and trophic egg proportion in a ladybird with and without male-killing bacterial infection

Maternally inherited bacterial endosymbionts can kill male embryos of their arthropod hosts to enhance the transmission efficiency of the endosymbionts. The resources from killed male eggs can be reallocated to infected female hatchlings as additional maternal investment. As a result, the number of offspring per patch and the maternal investment per offspring are expected to differ from the original optimal values for the host mother. Thus, in response to infection, these trait values should be adjusted to maximize the lifetime reproductive success of host females and the fitness of inherited endosymbionts as well. Here, we examined clutch size, egg size, and the proportion of trophic eggs (i.e., production of unhatched eggs, a maternal phenotype) per clutch of host mothers infected with male-killing bacteria. First, we developed a mathematical model to predict the optimal clutch size and trophic egg proportion in uninfected and infected females. Next, we experimentally compared these life-history traits in a ladybird, Harmonia yedoensis, between females infected or uninfected with male-killing Spiroplasma bacteria. Consistent with our predictions, clutch size was larger, egg size was smaller, and trophic egg proportion was lower in infected H. yedoensis females, compared with uninfected females. To our knowledge, this is the first empirical demonstration of variation in these life-history traits depending on infection with bacterial endosymbionts.     

Gill filament differentiation and experimental colonization by symbiotic bacteria in aposymbiotic juveniles of Codakia orbicularis (Bivalvia: Lucinidae)

Summary

Codakia orbicularis is a tropical lucinid harboring gill endosymbionts which are environmentally transmitted from a free living-symbiont form to the new host generation after metamorphosis. Structural changes occurring in the cellular organization from incomplete gill filaments in young aposymbiotic juveniles to full differentiated gill filaments containing bacterial endosymbionts in reared symbiotic juveniles, were analyzed for juveniles from 250 μm to 2 μm shell-length. Aposymbiotic juveniles possess differentiated gill filaments with ciliated, intermediary, and lateral zones similar to those described in wild juveniles, except for the bacteriocytes which are lacking. Granule cells, which progressively differentiate during the morphogenesis of the gill filament, do not appear as a consequence of symbiosis. Experimental colonization of aposymbiotic juveniles by the free-living symbiont form has been obtained through the addition of unsterilized sand collected from the natural habitat of C. orbicularis. Two days after exposure to crude sand, symbiosis-competent bacteria enter by endocytosis at the apical pole of undifferentiated cells which progressively differentiate into classical bacteriocytes similar to those found in the adult gill filaments. Undifferentiated cells of aposymbiotic gill filaments remain receptive to bacteria several months after metamorphosis, and become bacteriocytes when aposymbiotic juveniles get contact with the symbiont free-living form. Therefore, the environmental transmission of symbionts does not appear to be restrained to a defined period of time during post-larval development in C. orbicularis.     

Intracellular endosymbiotic bacteria of Camponotus species (carpenter ants): systematics, evolution and ultrastructural characterization

Intracellular endosymbiotic bacteria inherent to ants of the genus Camponotus were characterized. The bacteria were localized in bacteriocytes, which are specialized cells of both workers and queen ants; these cells are intercalated between epithelial cells of the midgut. The bacteriocytes show a different morphology from the normal epithelial cells and carry a large number of the rod-shaped Gram-negative bacteria free in the cytoplasm. The bacteria were never observed in the neighbouring epithelial cells, but they were found intracellularly in oocytes, strongly indicating a maternal transmission of the bacteria. The 16S DNA encoding rrs loci of the endosymbionts of four species of the genus Camponotus derived either from Germany (C. herculeanus and C. ligniperdus), North America (C. floridanus) or South America (C. rufipes) were cloned after polymerase chain reaction (PCR) amplification using oligonucleotides complementary to all so far known eubacterial rrs sequences. The DNA sequences of the rrs loci of the four endosymbionts were determined, and, using various genus- and species-specific oligonucleotides derived from variable regions in the rrs sequences, the identity of the bacteria present in the bacteriocytes and the ovarian cells was confirmed by PCR and in situ hybridization techniques. Comparison of the 16S DNA sequences with the available database showed the endosymbiotic bacteria to be members of the γ-subclass of Proteobacteria. They formed a distinct taxonomic group, a sister taxon of the taxons defined by the tsetse fly and aphid endosymbionts. Within the γ-subclass, the cluster of the ant, tsetse fly and aphid endosymbionts are placed adjacent to the family of Enterobacteriaceae. The evolutionary tree of the ant endosymbionts reflects the systematic classification and geographical distribution of their host insects, indicating an early co-evolution of the symbiotic partners and a vertical transmission of the bacteria.     

Microbial community of predatory bugs of the genus Macrolophus(Hemiptera: Miridae)

Background

The predatory mirids of the genus Macrolophus are key natural enemies of various economically important agricultural pests. Both M. caliginosus and M. pygmaeus are commercially available for the augmentative biological control of arthropod pests in European greenhouses. The latter species is known to be infected with Wolbachia -inducing cytoplasmic incompatibility in its host- but the presence of other endosymbionts has not been demonstrated. In the present study, the microbial diversity was examined in various populations of M. caliginosus and M. pygmaeus by 16S rRNA sequencing and denaturing gradient gel electrophoresis.

Results

Besides Wolbachia, a co-infection of 2 Rickettsia species was detected in all M. pygmaeus populations. Based on a concatenated alignment of the 16S rRNA gene, the gltA gene and the coxA gene, the first is phylogenetically related to Rickettsia bellii, whereas the other is closely related to Rickettsia limoniae. All M. caliginosus populations were infected with the same Wolbachia and limoniae-like Rickettsia strain as M. pygmaeus, but did not harbour the bellii-like Rickettsia strain. Interestingly, individuals with a single infection were not found. A PCR assay on the ovaries of M. pygmaeus and M. caliginosus indicated that all endosymbionts are vertically transmitted. The presence of Wolbachia and Rickettsia in oocytes was confirmed by a fluorescence in situ hybridisation. A bio-assay comparing an infected and an uninfected M. pygmaeus population suggested that the endosymbionts had minor effects on nymphal development of their insect host and did not influence its fecundity.

Conclusion

Two species of the palaearctic mirid genus Macrolophus are infected with multiple endosymbionts, including Wolbachia and Rickettsia. Independent of the origin, all tested populations of both M. pygmaeus and M. caliginosus were infected with three and two endosymbionts, respectively. There was no indication that infection with endosymbiotic bacteria had a fitness cost in terms of development and fecundity of the predators.

     

Identification,origin, and evolution of leaf nodulating symbionts of <Emphasis Type="Italic">Sericanthe</Emphasis> (Rubiaceae)

Bacterial leaf symbiosis is an intimate association between bacteria and plants in which endosymbionts are housed within leaf nodules. This phenomenon has been reported in three genera of Rubiaceae (Pavetta, Psychotria, and Sericanthe), but the bacterial partner has only been identified in Psychotria and Pavetta. Here we report the identification of symbiotic bacteria in two leaf nodulating Sericanthe species. Using 16S rRNA data and common housekeeping genetic markers (recA and gyrB) we studied the phylogenetic relationships of bacterial endosymbionts in Rubiaceae. Endosymbionts of leaf nodulating Rubiaceae were found to be closely related and were placed as a monophyletic group within the genus Burkholderia (β-Proteobacteria). The phylogenetic analyses revealed a pattern of strict host specificity and placed the two investigated endosymbionts at two distinct positions in the topology of the tree, suggesting at least two different evolutionary origins. The degree of sequence divergence between the Sericanthe endosymbionts and their relatives was large enough to propose the Sericanthe endosymbionts as new species (‘Candidatus Burkholderia andongensis’ and ‘Candidatus Burkholderia petitii’). In a second part of this study, the pylogenetic relationships among nodulating and non-nodulating Sericanthe species were investigated using sequence data from six chloroplast regions (rps16, trnG, trnL-trnF, petD, petA-psbJ, and atpI-atpH). Overall, genetic variation among the plastid markers was insufficient to enable phylogenetic estimation. However, our results could not rule out the possibility that bacterial leaf symbiosis originated once in a common ancestor of the Sericanthe species.     

Effects of mass releases of Trichogramma brassicae on predatory insects in maize

Abstract Females of the rove beetle genus Paederus (Coleoptera: Staphylinidae) accumulate large amounts of the defensive compound pederin produced by an endosymbiotic bacterium. Pederin is transferred in the eggs, while the endosymbionts are transmitted via the egg shell. In all three species analyzed (P. melanurus, P. riparius, and P. sabaeus), descendants of aposymbiotic matrilines (lacking both endosymbionts and biosynthetic capabilities) acquire biosynthetic capability by the ingestion of eggs with endosymbionts during larval development. Successful colonization by endosymbionts depends on the number of eggs consumed and the age of the feeding larvae. During the adult stage, however, the females cannot acquire biosynthetic capabilities. Adult females are able to sequester pederin from eggs eaten, and they transfer the substance into their own eggs. Lack of intrinsic biosynthesis after ingestion of endosymbionts indicates that these are not biosynthetically active within the gut and have to reach an unknown internal location before completion of metamorphosis. These results are discussed with regard to the benefit of intraspecific cannibalism.     

Faithful vertical transmission but ineffective horizontal transmission of bacterial endosymbionts during sexual reproduction of the black bean aphid,Aphis fabae

1. Insects are commonly infected with bacterial endosymbionts. In addition to the costs and benefits associated with harbouring these symbionts, their rates of vertical and horizontal transmission are important determinants of symbiont prevalence. 2. Aphids are cyclical parthenogens and show virtually perfect maternal transmission of endosymbionts during asexual reproduction. Less clear is the role of the annual sexual generation, during which overwintering eggs are produced. Data from pea aphids (Acyrthosiphon pisum Harris) suggest that maternal transmission failures and horizontal transmission via males may occur under sexual reproduction at least occasionally. No such data exist for other aphid species. 3. In the present study, the rates of maternal and paternal transmission of facultative endosymbionts during sexual reproduction in the black bean aphid, Aphis fabae (Scopoli) were examined. Crosses were performed between clones infected with Hamiltonella defensa, clones infected with Regiella insecticola and clones without facultative endosymbionts, and eggs were overwintered under three different conditions. 4. Only one of 205 offspring from crosses testing for maternal transmission failed to inherit the symbiont present in the maternal clone, and in crosses testing for horizontal transmission, only one of 412 offspring acquired a facultative symbiont from the father. 5. These results show that in A. fabae, maternal transmission of H. defensa and R. insecticola is extremely reliable also during sexual reproduction, indicating that maternal transmission failures are unlikely to exert a significant influence on frequencies of infection in the field. Paternal transmission of endosymbionts was exceedingly rare, suggesting that this route of horizontal transmission may be less important than hitherto assumed.     

Secondary Endosymbionts of Psyllids Have Been Acquired Multiple Times

Previous studies have established that psyllids (Hemiptera, Psylloidea) contain primary endosymbionts, designated as Carsonella ruddii, which cospeciate with the psyllid host. This association appears to be the consequence of a single infection of a psyllid ancestor with a bacterium. Some psyllids may have additional secondary (S-) endosymbionts. We have cloned and sequenced the 16S–23S ribosomal RNA genes of seven representative psyllid S-endosymbionts. Comparison of the S-endosymbiont phylogenetic trees with those of C. ruddii indicates a lack of congruence, a finding consistent with multiple infections of psyllids with different precursors of the S-endosymbionts and/or possible horizontal transmission. Additional comparisons indicate that the S-endosymbionts are related to members of the Enterobacteriaceae as well as to several other endosymbionts and insect-associated bacteria. Received: 2 May 2000 / Accepted: 8 June 2000     

Bacterial symbionts as mediators of ecologically important traits of insect hosts

1. Bacterial symbionts play a prominent role in insect nutritional ecology by aiding in digestion of food or providing nutrients that are limited or lacking in the diet. Thereby, endosymbionts open niches to their insect host that would otherwise be unavailable. 2. Currently, several other ecologically relevant traits mediated by endosymbionts are being investigated, including enhanced parasite resistance, enhanced heat tolerance, and influences on insect–plant interactions such as manipulation of plant physiology to the benefit of the insect. 3. Traits mediated by endosymbionts are often identified by correlative studies where traits are found to be altered in the presence of a particular symbiont. Recent developments in genomic tools offer the opportunity for studying the impact of bacteria–insect symbioses under natural conditions in a population and community ecology context. In vivo experiments specifically testing putative functions of endosymbionts in parallel to population‐level studies on the prevalence of endosymbionts allow disentangling host versus symbiont contribution to phenotypic variability observed in individuals. Effects of symbionts on host phenotype are often large and relevant to host fitness, e.g. by significantly enhancing survival or fecundity in a context‐dependent manner. 4. Predominantly vertically transmitted endosymbionts contribute to the heritable genetic variation present in a host species. Phenotypic variation on which selection can act may be due to differences either among host genomes, symbiont genomes, or genotype × genotype interactions. Therefore the holobiont, i.e. the host including all symbionts, should be regarded as the unit of selection as the association between host and symbionts may affect the fitness of the holobiont depending on the environment.     

Endosymbiont metacommunities,mtDNA diversity and the evolution of the Bemisia tabaci (Hemiptera: Aleyrodidae) species complex

Bemisia tabaci, an invasive pest that causes crop damage worldwide, is a highly differentiated species complex, divided into biotypes that have mainly been defined based on mitochondrial DNA sequences. Although endosymbionts can potentially induce population differentiation, specialization and indirect selection on mtDNA, studies have largely ignored these influential passengers in B. tabaci, despite as many as seven bacterial endosymbionts have been identified. Here, we investigate the composition of the whole bacterial community in worldwide populations of B. tabaci, together with host genetic differentiation, focusing on the invasive B and Q biotypes. Among 653 individuals studied, more than 95% of them harbour at least one secondary endosymbiont, and multiple infections are very common. In addition, sequence analyses reveal a very high diversity of facultative endosymbionts in B. tabaci, with some bacterial genus being represented by more than one strain. In the B and Q biotypes, nine different strains of bacteria have been identified. The mtDNA‐based phylogeny of B. tabaci also reveals a very high nucleotide diversity that partitions the two ITS clades (B and Q) into six CO1 genetic groups. Each genetic group is in linkage disequilibrium with a specific combination of endosymbionts. All together, our results demonstrate the rapid dynamics of the bacterial endosymbiont–host associations at a small evolutionary scale, questioning the role of endosymbiotic communities in the evolution of the Bemisia tabaci species complex and strengthening the need to develop a metacommunity theory of inherited endosymbionts.     

Metabolic stasis in an ancient symbiosis: genome-scale metabolic networks from two Blattabacterium cuenoti strains,primary endosymbionts of cockroaches

Background

Cockroaches are terrestrial insects that strikingly eliminate waste nitrogen as ammonia instead of uric acid. Blattabacterium cuenoti (Mercier 1906) strains Bge and Pam are the obligate primary endosymbionts of the cockroaches Blattella germanica and Periplaneta americana, respectively. The genomes of both bacterial endosymbionts have recently been sequenced, making possible a genome-scale constraint-based reconstruction of their metabolic networks. The mathematical expression of a metabolic network and the subsequent quantitative studies of phenotypic features by Flux Balance Analysis (FBA) represent an efficient functional approach to these uncultivable bacteria.

Results

We report the metabolic models of Blattabacterium strains Bge (iCG238) and Pam (iCG230), comprising 296 and 289 biochemical reactions, associated with 238 and 230 genes, and 364 and 358 metabolites, respectively. Both models reflect both the striking similarities and the singularities of these microorganisms. FBA was used to analyze the properties, potential and limits of the models, assuming some environmental constraints such as aerobic conditions and the net production of ammonia from these bacterial systems, as has been experimentally observed. In addition, in silico simulations with the iCG238 model have enabled a set of carbon and nitrogen sources to be defined, which would also support a viable phenotype in terms of biomass production in the strain Pam, which lacks the first three steps of the tricarboxylic acid cycle. FBA reveals a metabolic condition that renders these enzymatic steps dispensable, thus offering a possible evolutionary explanation for their elimination. We also confirm, by computational simulations, the fragility of the metabolic networks and their host dependence.

Conclusions

The minimized Blattabacterium metabolic networks are surprisingly similar in strains Bge and Pam, after 140 million years of evolution of these endosymbionts in separate cockroach lineages. FBA performed on the reconstructed networks from the two bacteria helps to refine the functional analysis of the genomes enabling us to postulate how slightly different host metabolic contexts drove their parallel evolution.

     

Multiple endosymbionts in populations of the ant <Emphasis Type="Italic">Formica cinerea</Emphasis>

Background  

Many insects, including ants, are infected by maternally inherited Wolbachia endosymbiotic bacteria though other secondary endosymbionts have not been reported in ants. It has been suggested that the ability of Wolbachia to invade and remain in an ant population depends on the number of coexisting queens in a colony. We study the genetic and social structure of populations in the ant Formica cinerea which is known to have populations with either monogynous or polygynous colonies. We screen populations for several endosymbiotic bacteria to evaluate the presence of different endosymbionts, possible association between their prevalence and the social structure, and the association between endosymbiont prevalence and genetic differentiation of ant populations.     

Evidence for Multiple Acquisition of <Emphasis Type="Italic">Arsenophonus</Emphasis> by Whitefly Species (Sternorrhyncha: Aleyrodidae)

Whiteflies contain primary prokaryotic endosymbionts located within specialized host cells. This endosymbiotic association is the result of a single infection of the host followed by vertical transmission of the endosymbiont to the progeny. Whiteflies may also be associated with other bacteria called secondary (S-) endosymbionts. The nucleotide sequence of the 16S–23S ribosomal DNA from S-endosymbionts of 13 whitefly species was determined. A phylogenetic analysis of these sequences indicated their grouping into two major clusters, one consisting of two S-endosymbionts related to previously described T-type endosymbionts. The second cluster contained the 16S–23S rDNA sequence of the type strain of Arsenophonus nasoniae as well as sequences of S-endosymbionts from 11 whitefly species. This Arsenophonus cluster contained four S-endosymbionts with intervening sequences of 70–184 nucleotides in their 23S rDNAs. The phylogenetic tree of the Arsenophonus cluster differed greatly from the phylogenetic tree of the primary endosymbionts. These results suggest that, unlike the primary endosymbiont, Arsenophonus may infect whiteflies multiple times and may also be horizontally transmitted.     

The tryptophan biosynthetic pathway of aphid endosymbionts (Buchnera): Genetics and evolution of plasmid-associated anthranilate synthase (trpEG) within the aphididae

The bacterial endosymbionts (Buchnera) from the aphids Rhopalosiphum padi, R. maidis, Schizaphis graminum, and Acyrthosiphon pisum contain the genes for anthranilate synthase (trpEG) on plasmids made up of one or more 3.6-kb units. Anthranilate synthase is the first as well as the rate-limiting enzyme in the tryptophan biosynthetic pathway. The amplification of trpEG on plasmids may result in an increase of enzyme protein and overproduction of this essential amino acid, which is required by the aphid host. The nucleotide sequence of trpEG from endosymbionts of different species of aphids is highly conserved, as is an approximately 500-bp upstream DNA segment which has the characteristics of an origin of replication. Phylogenetic analyses were performed using trpE and trpG from the endosymbionts of these four aphids as well as from the endosymbiont of Schlechtendalia chinensis, in which trpEG occurs on the chromosome. The resulting phylogeny was congruent with trees derived from sequences of two chromosome-located bacterial genes (part of trpB and 16S ribosomal DNA). In turn, trees obtained from plasmid-borne and bacterial chromosome-borne sequences were congruent with the tree resulting from phylogenetic analysis of three aphid mitochondrial regions (portions of the small and large ribosomal DNA subunits, as well as cytochrome oxidase II). Congruence of trees based on genes from host mitochondria and from bacteria adds to previous support for exclusively vertical transmission of the endosymbionts within aphid lineages. Congruence with trees based on plasmid-borne genes supports the origin of the plasmid-borne trpEG from the chromosomal genes of the same lineage and the absence of subsequent plasmid exchange among endosymbionts of different species of aphids. Received: 22 August 1995 / Accepted: 6 September 1995     

Biodiversity of Wolbachia and of their effects in Trichogramma (Hymenoptera: Trichogrammatidae)

Hymenopteran egg parasitoids belonging to the Trichogramma genus are infected by endosymbionts of the Wolbachia genus distributed into 2 supergroups and 4 groups according to the sequence of the wsp gene. The symbionts are variously distributed according to host populations and species and induce, in most cases, thelytokous parthenogenetic reproduction and, sometimes strongly, increase fecundity. Nevertheless, after an interspecific artificial transfer, no effect on reproduction has so far been recorded. Wolbachia often have a negative effect, or no effect at all, on host fitness. Positive effects have, however, been recorded especially in completely infected species. Symbionts show a variable efficiency of vertical transmission and a variable sensitivity to high temperature in relation to host species.     

Use of the Verrucomicrobia-Specific Probe EUB338-III and Fluorescent In Situ Hybridization for Detection of “Candidatus Xiphinematobacter” Cells in Nematode Hosts

Fluorescent in situ hybridization with a 16S rRNA probe specific for Verrucomicrobia was used to (i) confirm the division-level identity of and (ii) study the behavior of the obligate intracellular verrucomicrobium “Candidatus Xiphinematobacter” in its nematode hosts. Endosymbionts in the egg move to the pole where the gut primordium arises; hence, they populate the intestinal epithelia of juvenile worms. During the host's molt to adult female, the endosymbionts concentrate around the developing ovaries to occupy the ovarian wall. Some bacteria are enclosed in the ripening oocytes for vertical transmission. Verrucomicrobia in males stay outside the testes because the tiny spermatozoids are not suitable for transmission of cytoplasmic bacteria.     

Assessment of bacterial endosymbiont diversity in Otiorhynchusspp. (Coleoptera: Curculionidae) larvae using a multitag 454 pyrosequencing approach

Background

Weevils of the genus Otiorhynchus are regarded as devastating pests in a wide variety of horticultural crops worldwide. So far, little is known on the presence of endosymbionts in Otiorhynchus spp.. Investigation of endosymbiosis in this genus may help to understand the evolution of different reproductive strategies in these weevils (parthenogenesis or sexual reproduction), host-symbiont interactions, and may provide a future basis for novel pest management strategy development. Here, we used a multitag 454 pyrosequencing approach to assess the bacterial endosymbiont diversity in larvae of four economically important Otiorhynchus species.

Results

High-throughput tag-encoded FLX amplicon pyrosequencing of a bacterial 16S rDNA fragment was used to characterise bacterial communities associated with different Otiorhynchus spp. larvae. By sequencing a total of ~48,000 PCR amplicons, we identified 49 different operational taxonomic units (OTUs) as bacterial endosymbionts in the four studied Otiorhynchus species. More than 90% of all sequence reads belonged either to the genus Rickettsia or showed homology to the phylogenetic group of “Candidatus Blochmannia” and to endosymbionts of the lice Pedicinus obtusus and P. badii. By using specific primers for the genera Rickettsia and “Candidatus Blochmannia”, we identified a new phylogenetic clade of Rickettsia as well as “Candidatus Nardonella” endosymbionts in Otiorhynchus spp. which are closely related to “Candidatus Blochmannia” bacteria.

Conclusions

Here, we used multitag 454 pyrosequencing for assessment of insect endosymbiotic communities in weevils. As 454 pyrosequencing generates only quite short sequences, results of such studies can be regarded as a first step towards identifying respective endosymbiotic species in insects. In the second step of our study, we analysed sequences of specific gene regions for a more detailed phylogeny of selected endosymbiont genera. As a result we identified the presence of Rickettsia and “Candidatus Nardonella” endosymbionts in Otiorhynchus spp.. This knowledge is an important step in exploring bacteria-insect associations for potential use in insect pest control.

     

The virulent Wolbachia strain wMelPop increases the frequency of apoptosis in the female germline cells of Drosophila melanogaster

Background

Wolbachia are bacterial endosymbionts of many arthropod species in which they manipulate reproductive functions. The distribution of these bacteria in the Drosophila ovarian cells at different stages of oogenesis has been amply described. The pathways along which Wolbachia influences Drosophila oogenesis have been, so far, little studied. It is known that Wolbachia are abundant in the somatic stem cell niche of the Drosophila germarium. A checkpoint, where programmed cell death, or apoptosis, can occur, is located in region 2a/2b of the germarium, which comprises niche cells. Here we address the question whether or not the presence of Wolbachia in germarium cells can affect the frequency of cyst apoptosis in the checkpoint.

Results

Our current fluorescent microscopic observations showed that the wMel and wMelPop strains had different effects on female germline cells of D. melanogaster. The Wolbachia strain wMel did not affect the frequency of apoptosis in cells of the germarium. The presence of the Wolbachia strain wMelPop in the D. melanogaster^w1118 ovaries increased the number of germaria where cells underwent apoptosis in the checkpoint. Based on the appearance in the electron microscope, there was no difference in morphological features of apoptotic cystocytes between Wolbachia-infected and uninfected flies. Bacteria with normal ultrastructure and large numbers of degenerating bacteria were found in the dying cyst cells.

Conclusions

Our current study demonstrated that the Wolbachia strain wMelPop affects the egg chamber formation in the D. melanogaster ovaries. This led to an increase in the number of germaria containing apoptotic cells. It is suggested that Wolbachia can adversely interfere either with the cystocyte differentiation into the oocyte or with the division of somatic stem cells giving rise to follicle cells and, as a consequence, to improper ratio of germline cells to follicle cells and, ultimately, to apoptosis of cysts. There was no similar adverse effect in D. melanogaster Canton S infected with the Wolbachia strain wMel. This was taken to mean that the observed increase in frequency of apoptosis was not the general effect of Wolbachia on germline cells of D. melanogaster, it was rather induced by the virulent Wolbachia strain wMelPop.

     

Monoxenic culture of the anaerobic ciliate Trimyema compressum Lackey

Sapropelic ciliates from anoxic mud samples were enriched and cultivated in monoculture together with natural food bacteria growing on cellulose. The ciliates lacked cytochrome oxidase and contained bluish fluorescent endosymbionts. One of the anaerobic ciliates, Trimyema compressum, contained methanogenic bacteria as was shown by methane formation. During continued cultivation, T. compressum gradually lost its endosymbionts. With SEM microscopy no episymbiotic bacteria could be detected.From enrichment cultures of T. compressum, anaerobic bacteria were isolated in pure culture. One of the strains, a Bacteroides spec., proved capable of serving as food bacteria, thus allowing establishment of monoxenic T. compressum cultures. These cultures exhibited a requirement for sterols as growth factors. The doubling time of this ciliate was 13 h at 28°C. The highest yield obtained was 2100 cells/ml.Dedicated to Holger W. Jannasch on the occasion of his 60th birthday