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)

Mycetocyte symbiosis in insects.

'Candidatus Cardinium', a recently described bacterium from the Bacteroidetes group, is involved in diverse reproduction alterations of its arthropod hosts, including cytoplasmic incompatibility, parthenogenesis and feminization. To estimate the incidence rate of Cardinium and explore the limits of its host range, 99 insect and mite species were screened, using primers designed to amplify a portion of Cardinium 16S ribosomal DNA (rDNA). These arthropods were also screened for the presence of the better-known reproductive manipulator, Wolbachia. Six per cent of the species screened tested positive for Cardinium, compared with 24% positive for Wolbachia. Of the 85 insects screened, Cardinium was found in four parasitic wasp species and one armoured scale insect. Of the 14 mite species examined, one predatory mite was found to carry the symbiont. A phylogenetic analysis of all known Cardinium 16S rDNA sequences shows that distantly related arthropods can harbour closely related symbionts, a pattern typical of horizontal transmission. However, closely related Cardinium were found to cluster among closely related hosts, suggesting host specialization and horizontal transmission among closely related hosts. Finally, the primers used revealed the presence of a second lineage of Bacteroidetes symbionts, not related to Cardinium, in two insect species. This second symbiont lineage is closely allied with other arthropod symbionts, such as Blattabacterium, the primary symbionts of cockroaches, and male-killing symbionts of ladybird beetles. The combined data suggest the presence of a diverse assemblage of arthropod-associated Bacteroidetes bacteria that are likely to strongly influence their hosts' biology.

Distribution of the bacterial symbiont Cardinium in arthropods.

Several insect groups have obligate, vertically transmitted bacterial symbionts that provision hosts with nutrients that are limiting in the diet. Some of these bacteria have been shown to descend from ancient infections. Here we show that the large group of related insects including cicadas, leafhoppers, treehoppers, spittlebugs, and planthoppers host a distinct clade of bacterial symbionts. This newly described symbiont lineage belongs to the phylum Bacteroidetes. Analyses of 16S rRNA genes indicate that the symbiont phylogeny is completely congruent with the phylogeny of insect hosts as currently known. These results support the ancient acquisition of a symbiont by a shared ancestor of these insects, dating the original infection to at least 260 million years ago. As visualized in a species of spittlebug (Cercopoidea) and in a species of sharpshooter (Cicadellinae), the symbionts have extraordinarily large cells with an elongate shape, often more than 30 μm in length; in situ hybridizations verify that these correspond to the phylum Bacteroidetes. “Candidatus Sulcia muelleri” is proposed as the name of the new symbiont.

Symbiosis and Insect Diversification: an Ancient Symbiont of Sap-Feeding Insects from the Bacterial Phylum Bacteroidetes

The symbiotic bacterium Wolbachia pipientis has been considered unique in its ability to cause multiple reproductive anomalies in its arthropod hosts. Here we report that an undescribed bacterium is vertically transmitted and associated with thelytokous parthenogenetic reproduction in Encarsia, a genus of parasitoid wasps. Although Wolbachia was found in only one of seven parthenogenetic Encarsia populations examined, the “Encarsia bacterium” (EB) was found in the other six. Among seven sexually reproducing populations screened, EB was present in one, and none harbored Wolbachia. Antibiotic treatment did not induce male production in Encarsia pergandiella but changed the oviposition behavior of females. Cured females accepted one host type at the same rate as control females but parasitized significantly fewer of the other host type. Phylogenetic analysis based on the 16S rDNA gene sequence places the EB in a unique clade within the Cytophaga-Flexibacter-Bacteroid group and shows EB is unrelated to the Proteobacteria, where Wolbachia and most other insect symbionts are found. These results imply evolution of the induction of parthenogenesis in a lineage other than Wolbachia. Importantly, these results also suggest that EB may modify the behavior of its wasp carrier in a way that enhances its transmission.

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A newly discovered bacterium associated with parthenogenesis and a change in host selection behavior in parasitoid wasps

E ndosymbiosis has been proposed as an evolutionary innovation that underlies the success and diversification of some groups of organisms by enabling them to exploit new ecological niches (e.g., Margulis and Fester 1991, Maynard Smith and Szathmary 1995). Three kinds of information are needed to evaluate this proposal. First, it is necessary to know something of the role played by symbionts in the lives of hosts: Are their effects ones that allow use of novel ecological niches? Second, information is needed on the evolutionary history of endosymbioses: How old are they and, in particular, do endosymbiotic infections predate diversification of major host groups? Finally, the evolutionary consequences for hosts of acquiring new capabilities in the form of symbiotic associations must be evaluated: Are these novel traits stable? Do they impose constraints as well as new capabilities on hosts? Recent studies have begun to shed light on these questions for bacteriocyte-associated endosymbioses in insects. Bacteriocytes (or mycetocytes) are cells of animal hosts that are positioned at characteristic locations in the body and appear to be specialized for housing bacteria (Figure 1; Buchner 1965, Dasch et al.

Bacteriocyte-Associated Endosymbionts of Insects

Mycetomes in Helochara communis are paired syncytia each enclosed by an epithelium. Adjacent are loose aggregates of mycetocytes. Mycetomes harboured 3 kinds of inclusions:companion, a - and t -symbiotes according to Buchner9s classification of homopteran symbiotes. In the mycetocytes, a fourth kind of inclusion was found and named g -symbiotes. All inclusions were Feulgen and Gram negative. Nucleoids were revealed in companion symbiotes by osmium-HCL-Giemsa; and, in some g - and t -, but not in a -, symbiotes by RNase-papain Giemsa. DNase digestion caused loss of basophilia in g - and t -, but not in a -, symbiotes. By electron microscopy, using Kellenberger9s fixative, companion, g - and t - symbiotes had lucid areas with reticular strands 4 nm thick; a -symbiotes were in general, homogeneously densely granular. Companion symbiotes had Gram-negative profiles with additional surface material; a - and t -symbiotes had 3, g -symbiotes 2, peripheral, trilaminar membranes. Parallel striations and regularly arranged granules occurred in companion symbiotes; crystalline inclusions in g - and t - symbiotes; and fibrillar elements and dense bodies in a -symbiotes. Companion symbiotes may be Gram-negative bacteria or rickettsiae; g - and t -symbiotes, L-phase bacteria; but a -symbiotes are anomalous, apparently lacking DNA, and probably being derivatives of t -symbiotes, thus requiring that Muller9s hypothesis of homopteran evolutionary pathways be rephrased.

Multiple Symbiosis in a Leafhopper,Helochara Communis Fitch (Cicadellidae: Homoptera): Envelopes, Nucleoids and Inclusions of the Symbiotes

SUMMARY The mycetomal micro-organisms of Sitophilus zea-mais give evidence in electron micrographs of having two peripheral unit membranes, an intermediate layer, intracytoplasmic membranes and DNA-like material. They appear to disintegrate, at times, with the concomitant formation of membrane masses. It is suggested that these are visible with the phasemicroscope and that they are probably myelin figures.

Membranes associated with the disintegration of mycetomal micro-organisms in Sitophilus zea-mais (Mots.) (Coleoptera).

The peripheral membranes of the micro-organisms of the mycetocytes of adult midgut caecae and of larval mycetomes of Sitophilus granarius (L.), GG strain, have been examined with an electron microscope. The majority of the mycetocytes were depleted of intracellular organelles but contained large numbers of mycetomal micro-organisms, most of which exhibited only one peripheral membrane. Some mycetocytes, however, had well-developed ultrastructure and harboured mycetomal micro-organisms which showed two peripheral membranes, namely a cell wall and plasma membrane. Intermediate conditions also occurred. It is suggested that the absence of host-provided membranes around the micro-organisms categorizes them as obligate symbiotes.

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Ultrastructure and Peripheral Membranes of the Mycetomal Microorganisms of Sitophilus granarius (L.) (Coleoptera)

SUMMARY Cytochemical studies of the outer membranes and chromatinic material in the inherited, supposedly symbiotic mycetomal micro-organisms of Sitophilus granarius have revealed the presence of considerable amounts of DNA occurring as either consolidated dots or a spongy network. There is also evidence that, at times, the organisms are surrounded by cell walls.

A. J. Musgrave

Papers

Multiple Symbiosis in a Leafhopper,Helochara Communis Fitch (Cicadellidae: Homoptera): Envelopes, Nucleoids and Inclusions of the Symbiotes

Membranes associated with the disintegration of mycetomal micro-organisms in Sitophilus zea-mais (Mots.) (Coleoptera).

Ultrastructure and Peripheral Membranes of the Mycetomal Microorganisms of Sitophilus granarius (L.) (Coleoptera)

Some Studies on the Chromatin and Cell Wall of the Mycetomal Micro-Organisms of Sitophilus granarius (L.) (Coleoptera)