Inherited Fungal and Bacterial Endosymbionts of a Parasitic Wasp and Its Cockroach Host

Bacterial endosymbionts of insects are increasingly being recognized as common, diverse, and integral to the biology of their hosts. Inherited fungal symbionts have been largely overlooked, however, even though insect guts appear to be a key habitat for an incredible array of fungal diversity. Like bacteria, fungal symbionts also likely play important roles in the ecology and evolution of their insect associates. The objective of this study was to lay the foundations for understanding the roles of the vertically transmitted fungal and bacterial associates of both the brownbanded cockroach, Supella longipalpa, and its parasitic wasp, Comperia merceti. We used culture-dependent and culture-independent molecular methods and phylogenetic analyses in order to identify the symbionts. Two fungal associates of brownbanded cockroaches were found. To our knowledge, this is the first record of vertically transmitted fungal symbionts in the order Blattaria. The wasp was found to house a close relative of one of the cockroach fungi but no bacterial symbionts. Finally, the brownbanded cockroaches also harbored three lineages of bacterial symbionts: Blattabacterium and two lineages of Wolbachia, indicating the number of vertically transmitted symbionts in this insect may be as many as five.     

MYCETOCYTE SYMBIOSIS IN INSECTS

1. Non-pathogenic microorganisms, known as mycetocyte symbionts, are located in specialized 'mycetocyte' cells of many insects that feed on nutritionally unbalanced or poor diets. The insects include cockroaches, Cimicidae and Lygaeidae (Heteroptera), the Homoptera, Anoplura, the Diptera Pupiparia, some formicine ants and many beetles. 2. Most mycetocyte symbionts are prokaryotes and a great diversity of forms has been described. None has been cultured in vitro and their taxonomic position is obscure. Yeasts have been reported in Cerambycidae and Anobiidae (Coleoptera) and a few planthoppers. They are culturable and those in anobiids have been assigned to the genus Torulopsis. 3. The mycetocyte cells may be associated with the gut, lie free in the abdominal haemocoel or be embedded in the fat body of the insect. The mycetocytes are large polyploid cells which rarely divide and the symbionts are restricted to their cytoplasm. 4. The mycetocyte symbionts are transmitted maternally from one insect generation to the next. In many beetles (Anobiidae, Cerambycidae, Chrysomelidae and cleonine Curculionidae), the microoganisms are smeared onto the eggs and consumed by the hatching larvae. In other insects, they are transferred from mycetocytes to oocytes in the ovary, a process known as transovarial transmission. The details of transmission in the different insect groups vary with the age of the mother (adult, larva or embryo) at which symbiont transfer to the ovary is initiated; whether isolated symbionts or intact mycetocytes are transferred; and the site of entry of symbionts to the egg (anterior, posterior or apolar). 5. Within an individual insect, the biomass of symbionts varies in a regular fashion with age, weight and sex of the insect. Suppression of symbiont growth rate and lysis of 'excess' microorganisms may contribute to the regulation of symbionts (including freshly-isolated preparations of unculturable forms) are used to investigate interactions between the partners. However, some methods to obtain aposymbiotic insects (e.g. antibiotics and lysozyme) deleteriously affect certain insects and aposymbionts may differ from the symbiont-containing stocks from which they were derived. 7. The mycetocyte symbionts have been proposed to synthesize various nutrients required by the insect. The symbionts of beetles and haematophagous insects may provide B vitamins and those in cockroaches and the Homoptera essential amino acids. The role of symbionts in the sterol nutrition of insects is equivocal. 8. Mycetocyte symbionts may have evolved from gut symbionts or guest microorganisms. The association is monophyletic in cockroaches but polyphyletic in many groups, including the sucking lice, beetles and scale insects.(ABSTRACT TRUNCATED AT 400 WORDS)     

Maternal Inheritance of Enzymes in the Mealybug PSEUDOCOCCUS OBSCURUS (Homoptera)

In the mealybug Pseudococcus obscurus Essig (Pseudococcidae) two esterases, a tyrosinase and a mannosephosphate isomerase, exhibited an unusual type of maternal inheritance. Electromorphs (alleles) were transmitted by both parent but segregation was delayed by one generation and full sisters always had the same phenotype. Moreover, for esterase-1, in which three alleles were present, some of the females exhibited all three alleles. Several other polymorphic loci exhibited normal transmission and segregation. This mode of inheritance can be readily explained by assuming that most or all of the enzymes coded for by these loci are produced by the mycetocytes. The mycetocytes house intracellular bacteria-like symbionts and are usually formed by the fusion ofthe polar bodies and one or more cleavage nuclei. For a locus with two alleles exhibiting this type of inheritance, the expected frequencies of the three phenotypes are p3, 3pq an equation is presented for estimating the frequency of alleles from the frequencies of the phenotypes and it is shown that for three samples from wild populations there is a good agreement between the expected and observed frequencies of the phenotypes.     

Roles of fat body trophocytes,mycetocytes and urocytes in the American cockroach,Periplaneta americana under starvation conditions: An ultrastructural study

In insects, trophocytes (adipocytes) are major cells of a storage organ, the fat body, from which stored glycogen and lipids are mobilized under starvation. However, cockroaches have 2 additional types of cell in the fat body: mycetocytes harboring an endosymbiont, Blattabacterium cuenoti, and urocytes depositing uric acid in urate vacuoles. These cells have not been investigated in terms of their roles under starvation conditions. To gain insight into the roles of trophocytes, mycetocytes and urocytes in cockroaches, structural changes were first investigated in the cells associated with starvation in the American cockroach, Periplaneta americana, by light and electron microscopy. The area of lipid droplets in trophocytes, the endosymbiont population and mitotic activity in mycetocytes, and the area of urate vacuoles in urocytes were analyzed in association with survival rates of the starved cockroaches. After 2 weeks of starvation, trophocytes lost glycogen rosettes and their area of lipid droplets decreased, but almost all cockroaches survived this period. However, further starvation did not reduce the area, but the survival rates dropped rapidly and all cockroaches died in 7 weeks. Endosymbionts were not affected in terms of population size and mitotic activity, even if the cockroaches were dying. The area of urate vacuoles rapidly decreased in a week of starvation and did not recover upon further starvation. These results indicate that starved cockroaches mobilize glycogen and lipids stored in trophocytes to survive for 2 weeks and then die after the exhaustion of nutrients in these cells. Endosymbionts are not digested for the recycling of nutrients, but uric acid is reused under starvation.     

蚜虫与体内布赫纳氏菌及其次生共生菌的相互关系

对蚜虫与其内共生细菌共生关系的研究进展进行综述。布赫纳氏菌Buchnera普遍存在于蚜虫体内特殊的菌胞中,与蚜虫形成专性共生关系,为宿主蚜虫提供多种必需氨基酸和B族维生素。蚜虫体内的菌胞数量是一个动态变量,受蚜虫体内、外环境因子的影响。在某些蚜虫体内菌胞中还发现有若干种类次生共生细菌,其功能尚不完全清楚,可能与蚜虫的生态学特征有关。对蚜虫与其体内共生菌相互关系的研究提出一些新的问题。     

Evidence for phylogenetic congruence among sulfur-oxidizing chemoautotrophic bacterial endosymbionts and their bivalve hosts

Sulfur-oxidizing chemoautotrophic (thioautotrophic) bacteria are now known to occur as endosymbionts in phylogenetically diverse bivalve hosts found in a wide variety of marine environments. The evolutionary origins of these symbioses, however, have remained obscure. Comparative 16S rRNA sequence analysis was used to investigate whether thioautotrophic endosymbionts are monophyletic or polyphyletic in origin and to assess whether phylogenetic relationships inferred among these symbionts reflect those inferred among their hosts. 16S rRNA gene sequences determined for endosymbionts from nine newly examined bivalve species from three families (Vesicomyidae, Lucinidae, and Solemyidae) were compared with previously published 16S rRNA sequences of thioautotrophic symbionts and free-living bacteria. Distance and parsimony methods were used to infer phylogenetic relationships among these bacteria. All newly examined symbionts fall within the gamma subdivision of the Proteobacteria, in clusters containing previously examined symbiotic thioautotrophs. The closest free-living relatives of these symbionts are bacteria of the genus Thiomicrospira. Symbionts of the bivalve superfamily Lucinacea and the family Vesicomyidae each form distinct monophyletic lineages which are strongly supported by bootstrap analysis, demonstrating that host phylogenies inferred from morphological and fossil evidence are congruent with phylogenies inferred for their respective symbionts by molecular sequence analysis. The observed congruence between host and symbiont phylogenies indicates shared evolutionary history of hosts and symbiont lineages and suggests an ancient origin for these symbioses. Correspondence to: D.L. Distel     

Blattabacteria,the endosymbionts of cockroaches,have small genome sizes and high genome copy numbers

Blattabacteria are intracellular endosymbionts of cockroaches and primitive termites that belong to the class Flavobacteria and live only in specialized cells in the abdominal fat body of their hosts. In the present study we determined genome sizes as well as genome copy numbers for the endosymbionts of three cockroach species, Blattella germanica, Periplaneta americana and Blatta orientalis. The sole presence of blattabacteria in the fat body was demonstrated by rRNA‐targeting techniques. The genome sizes of the three blattabacteria were determined by pulsed field gel electrophoresis. The resulting total genome sizes for the three symbionts were all approximately 650 ± 15 kb. Comparison of the genome sizes with those of free‐living Bacteroidetes shows extended reduction, as occurs in other obligatory insect endosymbionts. Genome copy numbers were determined based on cell counts and determination of DNA amounts via quantitative PCR. Values between 10.2 and 18.3 and between 323 and 353 were found for the symbionts of P. americana and B. orientalis respectively. Polyploidy in intracellular bacteria may play a significant role in the genome reduction process.     

Differential consumption of baits by pest blattid and blattellid cockroaches and resulting direct and secondary effects

Consumption was measured of three commercially available cockroach gel baits (0.01 and 0.05% fipronil and 0.6% indoxacarb) by two pest blattellid (German cockroach, Blattella germanica L., and brownbanded cockroach, Supella longipalpa Fabricius) and three pest blattid [oriental cockroach, Blatta orientalis L., American cockroach, Periplaneta americana L., and smokybrown cockroach, Periplaneta fuliginosa (Serville)] species (Dictyoptera), and direct and secondary effects were quantified. All three baits were greatly preferred for consumption over dog food; however, virtually all consumption (ca. 98%) by pest blattids was gel baits containing sugar feeding stimulants and water. Pest blattid greater preference for gel baits was probably due to their greater need for nutrients in baits due to their greater cuticular water permeability and higher metabolism than the pest blattellids. Brownbanded cockroaches had lowest percentage gel bait selection. Pest blattellids consumed greater amounts of bait per g body weight than pest blattids. Cockroaches consumed more active ingredient than needed to cause mortality; however, based on bait consumption, a 30‐g tube of gel bait potentially killed from 394 to 6 966 adult cockroaches, depending on species. Mortality for all cockroach species was faster for adults (≥3 days) than for nymphs (≥7 days); however, most brownbanded cockroaches exposed to indoxacarb survived despite consuming 1.5‐ to >3‐fold more than other baits, suggesting low enzyme production by brownbanded cockroaches and consequently lower conversion of indoxacarb into its toxic form. Besides direct mortality, German cockroaches died from indirect effects: exposure to debris from other cockroaches that had direct access to the gel baits or bait contact without ingestion. Although maximization of bait consumption is important, factors that enhance secondary mortality and contact toxicity should also be considered.     

Cellulose digestion in termites and cockroaches: What role do symbionts play?

• 1.1. Termites and cockroaches are excellent models for studying the role of symbionts in cellulose digestion in insects: they eat cellulose in a variety of forms and may or may not have symbionts.
• 2.2. The wood-eating cockroach, Panesthia cribrata, can be maintained indefinitely, free of microorganisms, on a diet of crystalline cellulose. Under these conditions the RQ is 1, indicating that the cockroach is surviving on glucose produced by endogenous cellulase.
• 3.3. The in vitro rate at which glucose is produced from crystalline cellulose by gut extracts from P. cribrata and Nasutitermes walkeri is comparable to the in vivo production of CO₂ in these insects, clearly indicating that the rate of glucose production from crystalline cellulose is sufficient for their needs.
• 4.4. In all termites and cockroaches examined, cellulase activity was found in the salivary glands and predominantly in the foregut and midgut. These regions are the normal sites of secretion of digestive enzymes and are either devoid of microorganisms (salivary glands) or have very low numbers.
• 5.5. Endogeneous cellulases from termites and cockroaches consist of multiple endo-β-1,4-glucanase (EC 3.2.1.4) and β-1,4-glucosidase (EC 3.2.1.21) components. There is no evidence that an exo-β-1,4-glucanase (cellobiohydrolase) (EC 3.2.1.91) is involved in, or needed for, the production of glucose from crystalline cellulose in termites or cockroaches as the endo-β-1,4-glucanase components are active against both crystalline cellulose and carboxymethylcellulose.
• 6.6. There is no evidence that bacteria are involved in cellulose digestion in termites and cockroaches. The cellulase associated with the fungus garden of M. michaelseni is distinct from that in the midgut; there is little indication that the fungal enzymes are acquired or needed. Lower termites such as Coptotermes lacteus have Protozoa in their hindgut which produce a cellulase(s) quite distinct from that in the foregut and midgut.

     

Distribution and transmission of endosymbiotic microorganisms in the oocytes of the pig louse,Haematopinus suis (L.) (Insecta: Phthiraptera)

Summary All anoplurans live symbiotically with prokaryotic microorganisms hosted in specialized cells, termed mycetocytes. In nymphs and males mycetocytes are distributed between midgut epithelial cells. In females, besides the midgut, mycetocytes are found in the reproductive organs where they are located at the base of ovarioles in contact with lateral oviducts. The mycetocyte-associated symbionts are transmitted from one generation to the next transovarially. Here, the results of histological and ultrastructural studies on the distribution and transmission of symbiotic microorganisms within the ovaries of the anopluranHaematopinus suis are presented. Interestingly, during advanced oogenesis (i.e., choriogenesis) of this species all symbionts are localized extracellularly and form a tight mass located at the posterior pole of the oocyte just below the hydropyle. In insects studied so far, such localization of transovarially transmitted microorganisms has been reported only in the closely related speciesHaematopinus eurysternus.     

Trail-following behavior in the German cockroach (Dictyoptera: Blattellidae)

The trail-following behavior of the German cockroach, Blattella germanica (L.), was evaluated by comparing the distance between cockroach movement paths and preapplied "trails" of fecal extract. For each cockroach group tested (adult males, females, gravid females, and late instars), the mean perpendicular distance of the cockroach from the trail was significantly less than the distance from a control trail. The results indicated that the German cockroaches did exhibit trail-following behavior. Trail- following accuracy varied among the cockroach groups. The mean distance from the fecal trail ranged from 18.45 to 110.05 cm with adult males < or = adult females < or = late instars < gravid females. Very dilute fecal extract could still induce trail-following behavior in adult male cockroaches. A 5.6% concentration of fecal extract in methanol was able to induce trail-following behavior in 50% of the cockroaches. Although German cockroaches have demonstrated trail-following behavior, fecal trails are still not thought to be actively deposited. Rather, the passive distribution of fecal material within the home range results in the accumulation of trails along frequently traveled routes (i.e., between resources and the cockroach harborage).     

Characterization of extrachromosomal replicons present in the extended host range Rhizobium sp. LPU83

In several rhizobia, bacteria that inhabit the soil in free-living conditions and associate in symbiosis with the root of legumes as nitrogen-fixing organisms, plasmid DNA can constitute a high percentage of the genome. We have characterized acid-tolerant isolates of rhizobia-here represented by the strain Rhizobium sp. LPU83-that have an extended nodulation-host range including alfalfa, the common bean, and Leucena leucocephala. In this study we analyzed the plasmids of R. sp. LPU83 in order to characterize their role in the evolution of Medicago symbionts and their involvement in symbiotic behavior. The pLPU83a plasmid was found to be transmissible with no associated phenotypic traits. The symbiotic plasmid pLPU83b could be transferred at very low frequencies under laboratory conditions only when pLPU83a was present; could restore nodulation to a strain cured of its symbiotic plasmid, S. meliloti A818; but could not restore the full nitrogen fixation associated with alfalfa.     

Cloning of PaAtg8 and roles of autophagy in adaptation to starvation with respect to the fat body and midgut of the Americana cockroach,Periplaneta americana

Starvation, in particular amino acid deprivation, induces autophagy in trophocytes (adipocytes), the major component of the fat body cell types, in the larvae of Drosophila melanogaster. However, the fat body of cockroach has two additional cell types: urocytes depositing uric acid in urate vacuoles as a nitrogen resource and mycetocytes harboring an endosymbiont, Blattabacterium cuenoti, which can synthesize amino acids from the metabolites of the stored uric acid. These cells might complement the roles of autophagy in recycling amino acids in the fat body or other organs of cockroaches under starvation. We investigate the presence of autophagy in tissues such as the fat body and midgut of the American cockroach, Periplaneta americana, under starvation by immunoblotting with antibody against Atg8, a ubiquitin-like protein required for the formation of autophagosomes and by electron microscopy. Corresponding changes in acid phosphatase activity were also investigated as representing lysosome activity. Starvation increased the level of an autophagic marker, Atg8-II, in both the tissues, extensively stimulating the formation of autophagic compartments in trophocytes of the fat body and columnar cells of the midgut for over 2 weeks. Acid phosphatase showed no significant increase in the fat body of the starved cockroaches but was higher in the midgut of the continuously fed animals. Thus, a distinct autophagic mechanism operates in these tissues under starvation of 2 weeks and longer. The late induction of autophagy implies exhaustion of the stored uric acid in the fat body. High activity of acid phosphatase in the midgut of the fed cockroaches might represent enhanced assimilation and not an autophagy-related function.     

Parasitoid wasp affects metabolism of cockroach host to favor food preservation for its offspring

Unlike predators, which immediately consume their prey, parasitoid wasps incapacitate their prey to provide a food supply for their offspring. We have examined the effects of the venom of the parasitoid wasp Ampulex compressa on the metabolism of its cockroach prey. This wasp stings into the brain of the cockroach causing hypokinesia. We first established that larval development, from egg laying to pupation, lasts about 8 days. During this period, the metabolism of the stung cockroach slows down, as measured by a decrease in oxygen consumption. Similar decreases in oxygen consumption occurred after pharmacologically induced paralysis or after removing descending input from the head ganglia by severing the neck connectives. However, neither of these two groups of cockroaches survived more than six days, while 90% of stung cockroaches survived at least this long. In addition, cockroaches with severed neck connectives lost significantly more body mass, mainly due to dehydration. Hence, the sting of A. compressa not only renders the cockroach prey helplessly submissive, but also changes its metabolism to sustain more nutrients for the developing larva. This metabolic manipulation is subtler than the complete removal of descending input from the head ganglia, since it leaves some physiological processes, such as water retention, intact.     

Some aspects of intracellular symbiosis during embryo development of Mastotermes darwiniensis (Isoptera: Mastotermitidae).

All examined species of cockroaches have been shown to harbour intracellular bacteria in specialized cells (bacteriocytes) of the fat body. In termites, bacteria in specialized cells have been observed only in Mastotermes darwiniensis (Isoptera: Mastotermitidae). All of these bacteria have been assigned to the same eubacterial lineage, with the bacteria of M. darwiniensis as the sister group to the cockroach bacteria. While the main steps of the life cycle of cockroach bacteria have been described, little is known about the bacteria of M. darwiniensis. More specifically, no data are available on their behaviour during the development of this termite. Using both optical and electron microscopy methods, we examined embryos of M. darwiniensis at different developmental stages. Our results show that the integration of bacteria during the development of M. darwiniensis is implemented in the same way as in cockroaches. In particular, we observed the aggregation of a large amount of bacteria in a single mass in the yolk sac, with vitellophage-associated bacterial lysis. In cockroaches, a similar process has been described in detail for Periplaneta americana (Blattaria: Blattidae), where the bacterial mass is referred to as the transitory mycetome. The formation of a transitory mycetome could thus be regarded as an ancestral condition for cockroaches and termites.     

Pollination efficiency of cockroaches and other floral visitors of Clusia blattophila

• Cockroaches have rarely been documented as pollinators. In this paper we examine whether this is because they might be inefficient at pollination compared to other pollinators. Clusia blattophila, a dioecious shrub growing on isolated rocky outcrops in French Guiana, is pollinated by Amazonina platystylata cockroaches and provides a valuable system for the study of cockroach pollination efficiency.
• We examined the species composition of the visitor guild and visitation rates by means of camcorder recordings and visitor sampling. Then, we investigated the capacity for pollen transfer of principal visitors and found correlations between visitation rates and pollen loads on stigmas. In an exclusion experiment we determined the contributions of individual species to pollination success.
• Amazonina platystylata, crickets and two species of Diptera transferred pollen, but the number of transferred pollen grains was only related to visitation rates in the case of cockroaches. Crickets visited and rarely carried pollen. Dipterans were as frequent as cockroaches, carried similar pollen loads, but transferred much less pollen. An estimated 41% and 17% of ovules were pollinated by cockroaches and dipterans, respectively. The remaining ovules were not pollinated. There was no spatial variation in pollinator guild composition, but cockroaches visited flowers less frequently at the smaller study site.
• We demonstrate that cockroaches pollinate a large proportion of ovules. Their pollination service is not confined to one study site and, unlike that provided by dipterans, is not limited to certain years. We suggest that cockroach pollination has been overlooked and that cockroach‐pollinated plants, which share certain floral features, possess adaptations to pollination by cockroaches.

     

Endosymbiotic microbiota of the bamboo pseudococcid Antonina crawii (Insecta, Homoptera)

We characterized the intracellular symbiotic microbiota of the bamboo pseudococcid Antonina crawii by performing a molecular phylogenetic analysis in combination with in situ hybridization. Almost the entire length of the bacterial 16S rRNA gene was amplified and cloned from A. crawii whole DNA. Restriction fragment length polymorphism analysis revealed that the clones obtained included three distinct types of sequences. Nucleotide sequences of the three types were determined and subjected to a molecular phylogenetic analysis. The first sequence was a member of the gamma subdivision of the division Proteobacteria (gamma-Proteobacteria) to which no sequences in the database were closely related, although the sequences of endosymbionts of other homopterans, such as psyllids and aphids, were distantly related. The second sequence was a beta-Proteobacteria sequence and formed a monophyletic group with the sequences of endosymbionts from other pseudococcids. The third sequence exhibited a high level of similarity to sequences of Spiroplasma spp. from ladybird beetles and a tick. Localization of the endosymbionts was determined by using tissue sections of A. crawii and in situ hybridization with specific oligonucleotide probes. The gamma- and beta-Proteobacteria symbionts were packed in the cytoplasm of the same mycetocytes (or bacteriocytes) and formed a large mycetome (or bacteriome) in the abdomen. The spiroplasma symbionts were also present intracellularly in various tissues at a low density. We observed that the anterior poles of developing eggs in the ovaries were infected by the gamma- and beta-Proteobacteria symbionts in a systematic way, which ensured vertical transmission. Five representative pseudococcids were examined by performing diagnostic PCR experiments with specific primers; the beta-Proteobacteria symbiont was detected in all five pseudococcids, the gamma-Proteobacteria symbiont was found in three, and the spiroplasma symbiont was detected only in A. crawii.     

Accelerated evolutionary rate in sulfur-oxidizing endosymbiotic bacteria associated with the mode of symbiont transmission

The nearly neutral theory of molecular evolution predicts that the rate of nucleotide substitution should accelerate in small populations at sites under low selective constraint. We examined these predictions with respect to the relative population sizes for three bacterial life histories within chemolithoautotrophic sulfur-oxidizing bacteria: (1) free-living bacteria, (2) environmentally captured symbionts, and (3) maternally transmitted symbionts. Both relative rates of nucleotide substitution and relative ratios of loop, stem, and domain substitutions from 1,165 nt of the small-subunit 16S rDNA were consistent with expectations of the nearly neutral theory. Relative to free-living sulfur-oxidizing autotrophic bacteria, the maternally transmitted symbionts have faster substitution rates overall and also in low-constraint domains of 16S rDNA. Nucleotide substitition rates also differ between loop and stem positions. All of these findings are consistent with the predictions that these symbionts have relatively small effective population sizes. In contrast, the rates of nucleotide substitution in environmentally captured symbionts are slower, particularly in high-constraint domains, than in free-living bacteria.     

Ultrastructure, distribution, and transmission of endosymbionts in the whiteflyAleurochiton aceris Modeer (Insecta, Hemiptera, Aleyrodinea)

Summary The body of the whiteflyAleurochiton aceris contains specialized cells, termed mycetocytes, that enclose endosymbiotic microorganisms. The endosymbionts are transmitted from one generation to the next transovarially. In contrast to other insects, in whiteflies whole intact mycetocytes migrate into the ovaries, traverse the follicular epithelium, and reach the oocyte surface (i.e., perivitellin space). The migration of mycetocytes begins in the last instar, called puparium, from which imagines emerge. During this stage the cytoplasm of mycetocytes is tightly packed with pleomorphic bacteria and less numerous coccoid microorganisms. In adult females the mycetocytes gather extracellularly in the depression of the vitellarial oocyte. Till the end of oogenesis neither pleomorphic nor coccoid microorganisms are released from mycetocytes into the oocyte.     

R-body-producing bacteria.

Until 10 years ago, R bodies were known only as diagnostic features by which endosymbionts of paramecia were identified as kappa particles. They were thought to be limited to the cytoplasm of two species in the Paramecium aurelia species complex. Now, R bodies have been found in free-living bacteria and other Paramecium species. The organisms now known to form R bodies include the cytoplasmic kappa endosymbionts of P. biaurelia and P. tetraurelia, the macronuclear kappa endosymbionts of P. caudatum, Pseudomonas avenae (a free-living plant pathogen), Pseudomonas taeniospiralis (a hydrogen-oxidizing soil microorganism), Rhodospirillum centenum (a photosynthetic bacterium), and a soil bacterium, EPS-5028, which is probably a pseudomonad. R bodies themselves fall into five distinct groups, distinguished by size, the morphology of the R-body ribbons, and the unrolling behavior of wound R bodies. In recent years, the inherent difficulties in studying the organization and assembly of R bodies by the obligate endosymbiont kappa, have been alleviated by cloning and expressing genetic determinants for these R bodies (type 51) in Escherichia coli. Type 51 R-body synthesis requires three low-molecular-mass polypeptides. One of these is modified posttranslationally, giving rise to 12 polypeptide species, which are the major structural subunits of the R body. R bodies are encoded in kappa species by extrachromosomal elements. Type 51 R bodies, produced in Caedibacter taeniospiralis, are encoded by a plasmid, whereas bacteriophage genomes probably control R-body synthesis in other kappa species. However, there is no evidence that either bacteriophages or plasmids are present in P. avenae or P. taeniospiralis. No sequence homology was detected between type 51 R-body-encoding DNA and DNA from any R-body-producing species, except C. varicaedens 1038. The evolutionary relatedness of different types of R bodies remains unknown.