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Symbiosis in the Dryophthoridae Weevils (Coleoptera, Curculionoidea): Morphological Variability of Symbiotic Intracellular Bacteria
Abstract
Dryophthoridae weevils are phytophagous insects thriving on a broad host-plant spectrum. Some, such as Sitophilus spp. or Cosmopolites sordidus, are major agricultural pests of cereals and bananas, respectively. Previous studies have concluded that several Dryophthoridae species harbour intracellular bacteria (endosymbionts) in specialized organs named bacteriomes. In this work, we have demonstrated the presence of intracellular symbiosis in seven out of the eight Dryophthoridae species tested, and have assessed the morphological variability of these endosymbionts. Histological analyses of the endosymbionts in either the larval bacteriome or adult female ovaries reveal two fields of variability: physiologic (length variation during host development) and morphologic (both interspecific and intraspecific). These variations are expressed to different degrees in the species studied. Indeed, Sitophilus granarius displays much less pleiomorphism than S. zeamais. Such pleiomorphism may be a consequence of both genetic polymorphism and host/bacteria interaction.
... Nardon P., Charles H., Heddi A., 1997 14. Nardon P., Charles H., Delobel B., Lefèvre C., Heddi A., 2003 19. Pascal C., Pintureau B., Charles H., Katchadourian C., Grenier S., Bolland P., Robin C., 2005. ...
These last ten years, the dazzling advances in computing and microfluidics applied to molecular biology (PCR and sequencing) caused a complet rescaling of the data sets coming from a single experiment. This essay presented for the « Habilitation à Diriger des Recherches » is devoted to the transcriptome of the intracellular symbiotic bacteria of aphids, Buchnera aphidicola. In a first part, the main methods of differential statistics and data integration will be presented as a bibliographic report. The second part details bioinformatic tools for microarrays: ROSO, a software for oligonucleotide probe optimisation, the Buchnera microarray and SITRANS, an information system for the management of microarray data. The last part characterizes the transcriptome of Buchnera in trophic stressed conditions of their aphid host. Transcription regulation is yet little-known in the reduced genomes of intracellular bacteria. This question will be first adressed in Buchnera at the level of the molecular evolution with the analysis of the relationship between gene expression and genome organisation. Secondly, the functional response of the bacteria will be analysed in an experimental design combining the effect of essential amino acid depletion and osmotic stress in the diet of the aphid host.
... In dryophtherid beetles (Coleoptera: Curculionoidea) even three lineages of Enterobactericeae exist that colonized their host in independent evolutionary steps (cf. NARDON et al. 2003). ...
Due to their world-wide distribution in marine and terrestrial (as well as freshwater)
habitats, the order Isopoda (Crustacea: Malacostraca: Peracarida) provides an excellent model for
the evolutionary ecology of terrestrialization.
(1) Terrestrial isopods (Oniscidea) harbor endosymbiotic bacteria in their midgut glands (hepatopancreas)
that are lacking in marine isopods of the suborders Valvifera and Sphaeromatidea, considered
being (part of) a sister taxon of Oniscidea. Thus, these bacterial endosymbionts seem to be
significant in the context of living in terrestrial habitats and may have been important during the
course of terrestrialization. In truly terrestrial species (Crinocheta), two different endosymbionts
have been characterized that are distantly related to known parasites and pathogens of the orders
Rickettsiales and Mycoplasmatales, respectively. Both these endosymbionts form cytoplasmic appendages
that are in contact with the host epithelium and may serve in the exchange of nutrients
and information and or serve as holdfasts. In non-crinochete terrestrial isopods (Diplocheta, Tylida,
Synocheta), hepatopancreatic bacteria belong to the genus Pseudomonas.
Both marine and freshwater Asellota also harbor bacteria in their midgut glands. The lack of bacteria
in other marine suborders (as studied so far) may be due to antibiotic agents in these isopods.
Based on the present findings, I propose a common (marine) ancestor of Asellota and Oniscidea
that acquired the ability to harbor bacterial endosymbionts inside the hepatopancreas. While symbiotic
relationships remained unspecific in marine Asellota, they developed towards specific primary
symbioses with bacteria that aid in digesting cellulosic and phenolic compounds, and thus, facilitate
the utilization of terrestrial food sources in semi-terrestrial and terrestrial Oniscidea and in
freshwater Asellota. I, further, hypothesize that later during early phylogeny of Crinocheta, primary
symbionts have been replaced by secondary endosymbionts that are still characteristic of recent
Crinocheta.
In contrast to previous studies, suggesting a role of hepatopancreatic bacteria in nutrition, our present
knowledge does not provide any evidence for crinochete symbionts to supply any digestive enzymes
to their isopod host. However, Pseudomonas spp. are well-known to degrade both cellulosic and phenolic
compounds. Thus, I hypothesize that, while primary symbionts of Oniscidea provide cellulases
and/or phenol oxidase, a transfer of cellulase and/or phenol oxidase genes from symbiont to host
occurred in early Crinocheta, resulting in endogenous cellulase of evolutionarily bacterial origin. Besides
(a) providing enzymes for the digestion of leaf litter, further possible contributions of hepatopancreatic
endosymbionts to their hosts physiological constitution and fitness include (b) increasing
the availability of nitrogen on a nitrogen-poor food source, (c) protecting their host from secondary
(pathogenic) infection, (d) protecting their host from predatory attack, or (e) increasing fertility, mating
success and fecundity of their host these hypotheses are briefly discussed.
(2) Terrestrial isopods interact with leaf litter-colonizing microbiota that they ingest along with their
major food source. While, however, it is well-documented that isopods gain from feeding on microbially
inoculated leaf litter, reasons for this dependence are not well understood. Possibly, (a) microbiota
serve as supplementary high-quality food source and provide essential or otherwise limiting
nutrients; (b) microbiota promote digestion of leaf litter itself, either prior to ingestion or during the
gut passage; (c) microbiota simply act as indicators of easily digestible food sources of high quality.
These explanations are not mutually exclusive, and the prevailing reason for preferentially consuming
microbially inoculated leaf litter depends on both the species and developmental stage of the isopod
and the nutritional context, i.e. the food source as such; recent results, however, indicate that cellulolytic
capabilities of litter-colonizing microbiota [see (b)] may be less significant than previously
thought, while a role of litter-colonizing microbiota in indicating high-quality food [see (c)] is supported.
The ability to digestively utilize microbial cells as supplementary food [see (a)] depends on cell
wall characteristics as indicated by gram-staining of the microbes, gram-positive bacteria being
digested more effectively than gram-negative bacteria and fungi, and being preferred as food source.
Despite numerous studies, the most recent ones using modern molecular techniques, it is still debated
whether or not terrestrial isopods harbor resident gut microbes in their hindgut. Most hindgut
bacteria that may be candidates for hindgut residents appear to belong to gram-negative bacterial
taxa, and are taxonomically related to anaerobic species. Thus, we have to assume anoxic
microhabitats in cuticular wrinkles. Further, the radial center of the hindgut is anoxic, too, allowing
for fermentative digestive processes, while the periphery of the hindgut lumen is largely oxic and
oxidizing, thus, allowing for aerobic and oxidative digestive processes. These processes are promoted
through cell compounds of ingested microbiota resulting in homeostatic maintenance of a
slightly acidic pH that is optimal for the activity of involved enzymes. Potentially harmful effects of
phenolic food compounds that are likely under such conditions are counteracted through hydrolytic
enzymes and surfactants of microbial origin.
In conclusion, our up-to-date knowledge as summarized and discussed herein strongly confirms
the assumption that (terrestrial) isopods strongly depend on microbial activity and nutrients for
their capability of digestively utilizing terrestrial leaf litter; on an evolutionary scale, this dependence
may indicate the role that microbiota played during the course of terrestrialization, although
this aspect of isopod-microbe interactions is far from being understood.
... In dryophtherid beetles (Coleoptera: Curculionoidea) even three lineages of Enterobactericeae exist that colonized their host in independent evolutionary steps (cf. Nardon et al., 2003). ...
The midgut glands (hepatopancreas) of terrestrial isopods contain bacterial symbionts. We analysed the phylogenetic diversity of hepatopancreatic bacteria in isopod species from various suborders colonizing marine, semiterrestrial, terrestrial and freshwater habitats. Hepatopancreatic bacteria were absent in the marine isopod Idotea balthica (Valvifera). The symbiotic bacteria present in the midgut glands of the freshwater isopod Asellus aquaticus (Asellota) were closely related to members of the proteobacterial genera Rhodobacter, Burkholderia, Aeromonas or Rickettsiella, but differed markedly between populations. By contrast, species of the suborder Oniscidea were consistently colonized by the same phylotypes of hepatopancreatic bacteria. While symbionts in the semiterrestrial isopod Ligia oceanica (Oniscidea) were close relatives of Pseudomonas sp. (Gammaproteobacteria), individuals of the terrestrial isopod Oniscus asellus (Oniscidea) harboured either 'Candidatus Hepatoplasma crinochetorum' (Mollicutes) or 'Candidatus Hepatincola porcellionum' (Rickettsiales), previously described as symbionts of another terrestrial isopod, Porcellio scaber. These two uncultivated bacterial taxa were consistently present in each population of six and three different species of terrestrial isopods, respectively, collected in different geographical locations. However, infection rates of individuals within a population ranged between 10% and 100%, rendering vertical transmission unlikely. Rather, feeding experiments suggest that 'Candidatus Hepatoplasma crinochetorum' is environmentally transmitted to the progeny.
Bacterial intracellular symbiosis in the weevil Sitophilus spp. was discovered in 1927 and since then has been studied by several authors through many approaches. This has led to a deep characterization of variety of symbiotic aspects in relation with morphology, histology, genetic, physiology, as well as host-symbiont molecular and cellular biology. This paper reviews the basic findings from several teams and analyzes them with respect to current knowledge on weevil symbiosis evolution and cell-to-cell molecular interactions. Sitophilus spp. intracellular symbiosis appears as a model appropriate for studying and elucidating molecular and genetic mechanisms and evolutionary features involved in the early steps of insect intracellular symbiosis establishment.
Les progrès fulgurants de ces dix dernières années réalisés dans les domaines de la microinformatique et de la microfluidique associés au génie génétique (PCR et séquençage) ont permis un changement d'échelle dans la quantité des données acquises au cours d'une même expérience. La transcriptomique est directement issue de ces avancées technologiques. Ce mémoire présenté pour l'obtention d'une Habilitation à Diriger des Recherdes porte sur l'analyse du transcriptome de la bactérie intracellulaire obligatoire des pucerons, Buchnera aphidicola. Dans la première partie, les principales méthodes d'analyses statistiques différentielles et d'intégration des données transcriptomiques sont présentées sous la forme d'une analyse bibliographique. La deuxième partie est consacrée au développement d'outils bioinformatiques : ROSO, un logiciel d'optimisation des sondes oligonucléotidiques, la puce Buchnera et SITRANS, un système d'information pour la gestion et la publication des données d'expression. Enfin, la dernière partie est consacrée à la caractérisation du transcriptome de Buchnera en condition de stress trophique de son hôte, le puceron du pois Acyrthosiphon pisum. La régulation transcriptionnelle chez les bactéries symbiotiques intracellulaires à génome réduit est encore actuellement très mal connue. Cette question sera abordée chez Buchnera tout d'abord au niveau évolutif par l'étude de la relation entre l'expression des gènes et leur organisation dans le génome, puis au niveau fonctionnel, par la caractérisation de la réponse de la bactérie à une diminution de la quantité d'acides aminés essentiels dans le substrat nutritif du puceron, combinée à un stress osmotique.
Ce travail présente une analyse histologique et moléculaire de la symbiose intracellulaire très répandue chez les Dryophthoridae. Les endocytobiotes se regroupent en trois clades distincts appartenant aux gamma-Protéobactéries. Le nom de genre Candidatus Nardonella a été proposé pour le clade montrant un fort biais AT et ayant vraisemblablement infecté l'ancêtre des Dryophthoridae, il y a 100 MA. Les deux autres clades montrent des taux de GC beaucoup plus élevés et sont vraisemblablement issus de remplacements plus récents. L'origine " pathogénique " de la symbiose chez les Sitophilus est soutenue par l'étroite parenté de leurs endocytobiotes (SOPE) avec Sodalis (endocytobiotes des glossines). L'étude comparative de leur génome (par macroarry), a permis d'appréhender l'évolution des génomes intracellulaires en réponse à des pressions sélectives différentes.
The ultrastructure of the endosymbionts of several populations of whitefly (Homoptera: Aleyrodidae) was examined using transmission electron microscopy. Consistent differences in morphology and relative number of endosymbionts were observed between species and biotypes of whitefly within the Bemisia taxon.Bemisia argentifolii (=B. tabaci B biotype) individuals from Hawaii, Florida, and Arizona contained two morphological types of microorganisms housed within the mycetocyte cells of immature whiteflies. In contrast, individuals from populations ofB. tabaci A biotype from Arizona and Mexico, andB. tabaci Jatropha biotype from Puerto Rico, consistently contained three distinct morphological types of microorganisms within their mycetocytes. Organisms fromB. tabaci A and Jatropha biotypes differed from each other in the relative frequency of each type of microorganism. These observations suggest that different whitefly biotypes may have variable combinations of micro-fauna, with some possibly unique to each group, and furthers the hypothesis that variation in whitefly endosymbionts may be associated with the development of biotypes.
Helicobacter pylori, a cause of peptic ulcer disease and certain types of gastric cancers, has usually been cultured on diverse agar-based media, resulting in a requirement for 2 to 4 days of growth at 37 degrees C. We have developed a novel broth medium consisting of a base medium supplemented with 2% newborn calf serum, Mg2+, Cu2+, Fe2+, Zn2+, Mn2+, and 1 mg of lysed human erythrocytes per ml. This medium supports rapid growth of H. pylori, with a doubling time of about 50 min. Optimal growth was obtained in a pH range higher than that supporting most other gram-negative bacteria (at pH 8.5). H. pylori cultured in this supplemented broth retains the spiral morphology seen in both histological sections and cultures from agar-based media and also retains a high urease activity. After 18 h in this broth, H. pylori transforms to a coccal form with a complete loss of urease activity. Previously these cocci have been reported to be senescent, since they could not be subcultured on agar medium. Our experiments suggest that some of the cocci can revert back to the spiral morphology with full recovery of urease activity when subcultured in fresh microaerobic broth medium.
Cell physiology in the weevil Sitophilus oryzae is coordinated by three integrated genomes: nuclear, mitochondrial, and the "S. oryzae principal endosymbiont" (SOPE). SOPE, a cytoplasmic bacterium (2 x 10(3) bacteria per specialized bacteriocyte cell and 3 x 10(6) bacteria per weevil) that belongs to the proteobacteria gamma3-subgroup, is present in all weevils studied. We discovered a fourth prokaryotic genome in somatic and germ tissues of 57% of weevil strains of three species, S. oryzae, Sitophilus zeamais, and Sitophilus granarius, distributed worldwide. We assigned this Gram-negative prokaryote to the Wolbachia group (alpha-proteobacteria), on the basis of 16S rDNA sequence and fluorescence in situ DNA-RNA hybridization (FISH). Both bacteria, SOPE and Wolbachia, were selectively eliminated by combined heat and antibiotic treatments. Study of bacteria involvement in this insect's genetics and physiology revealed that SOPE, which induces the specific differentiation of the bacteriocytes, increases mitochondrial oxidative phosphorylation through the supply of pantothenic acid and riboflavin. Elimination of this gamma3-proteobacterium impairs many physiological traits. By contrast, neither the presence nor the absence of Wolbachia significantly affects the weevil's physiology. Wolbachia, disseminated throughout the body cells, is in particularly high density in the germ cells, where it causes nucleocytoplasmic incompatibility. The coexistence of two distinct types of intracellular proteobacteria at different levels of symbiont integration in insects illustrates the genetic complexity of animal tissue. Furthermore, evolutionary timing can be inferred: first nucleocytoplasm, then mitochondria, then SOPE, and finally Wolbachia. Symbiogenesis, the genetic integration of long-term associated members of different species, in the weevil appears to be a mechanism of speciation (with Wolbachia) and provides a means for animals to acquire new genes that permit better adaptation to the environment (with SOPE).
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.
In the rice weevil Sitophilus oryzae, the principal endocytobiote (SOPE) is located in a larval bacteriome and in ovaries (apical bacteriome, trophocytes and oocytes only). It was not found in other tissues and it never proliferates, except when eggs are irradiated (1,500 R), which results in oocyte and trophocyte destruction. This observation suggests a mechanism of control of the SOPE. We have demonstrated that this mechanism of control is of chromosomal origin. Firstly we have shown that it is possible to obtain, by selection, weevils with a high density of symbiotes per ovary (111,000 ± 4,000), as well as weevils with a low density (41,200 ± 3,000). Secondly, by crossing these two strains, we provide evidence of a chromosomal factor controlling the number of symbiotes per ovary. Thirdly, when males are irradiated, they induce in their female progeny a depressive effect on the number of endocytobiotes, despite the fact that they do not transmit the bacteria. These interactions between the insect and its symbiotes are necessary for the maintenance of the symbiocosme.
We have compared 38 different strains on wheat, from America, Europe, Asia and Africa, of the three Sitophilus species living on cereal grains or their by-products : S. granarius, S, oryzae and S. zeamais. Four biological characters were considered : weight of male and female, development time and fertility, and three behavioural characters : the ability of male and female to fly, and the elimination of flour and feces out of the grains by the growing larvae. We also introduced aposymbiotic strains (6) in our analysis to compare with the symbiotic ones. Multidimensional analysis placed these aposymbiotic strains, whatever their original species may be, in a same area, apart from the symbiotic strains. The three species granarius, oryzae and zeamais were clearly separated by the analysis with the used characters. Nevertheless, if we consider the identification of a particular strain of Sitophilus, the risk of error is only 4,5 % between the two sibling species oryzae and zeamais. Weight and flight were the two most important discrimination factors in this study. The variability was less important in strains being for a long time in the laboratory, than in "natural" strains. In zeamais and oryzae, we observed a negative relationship between weight and rapidity of development. It was not possible to establish a clear relation between the characteristics of strains and their geographical location. However, we observed some strains (Guadeloupe and Reunion) with intermediate characters between zeamais and oryzae.
To gain a better understanding of the occurrence of intracellular symbiosis in the Dryophthoridae family, nine species worldwide were analyzed in relation to the presence/absence and the structure of bacteriomes, the specialized organs harbouring the symbiotic bacteria. Staining protocols have indicated the presence of bacteriomes in seven species: Sitophilus rugicollis, Diocalandra frumenti, Sphenophorus striatopunctata, Sipalinus gigas, Trigonotarsus rugosus, Dynamis borassi and Rhynchophorus palmarum. Strong evidence also suggests the presence of intracellular bacteria within Sitophilus vateriae. However, Sitophilus linearis has been shown to be completely devoid of intracellular endosymbionts. In all symbiotic species, the larval bacteriome does not exhibit any diversity with regard to the form and the location, always in the anterior part of larvae. It is delimited by a thin membrane, it contains an important tracheal network and it is attached to the intestine at many sites. At least two cell types have been observed within the bacteriome: bacteriocytes, the polyploid cells harbouring bacteria and, between them, small flat cells forming the interstitial tissue. Taken together, these results argue in favor of a common origin of the symbiotic structures in the Dryophthoridae family.
Large and small strains of the rice weevil Sitophilus oryza (L.) ( Calandra oryzae (L.) of authors following European usage) have been reported from several localities (Birch, 1944 in Australia; Kiritani, 1956 in Japan; Kono, 1955 in Nepal; Richards 1944 in Britain; and Utida, 1958, in Canada, Japan and U.S.A.). Birch and Richards both seem to have considered it probable that the two strains were separate species: but they did not make the separation. Floyd and Newsom (1960), after careful work on both strains found in the U.S. and reared in the laboratory on a variety of materials, decided that the small strain could be validly made a separate species, thus agreeing with Takahashi who, according to Floyd and Newsom (1960), had elevated this strain to species rank as Calandra sasakii . In conformity with modern North American usage Floyd and Newsom refer to this small species as Sitophilus sasakii (Tak.). In addition to differences in size, they found differences in the male and female genitalia, interstrain sterility and various ecological differences. There were also differences in pronotal markings as had been, according to Kono (1953), noted by Takahashi (1928). Kono states “…the adult of the large strain has pits dotted densely on the central part of the pronotum, the small strain does not, and, furthermore the shape and size of the pits differ distinctly”.
Weevils posses procaryotic symbiotes harbored in specialized organ-like structures, mycetomes. Inability to culture these bacterial symbiotes outside of the insect-host has hindered efforts to determine teeir taxonomic affiliation with respect to other eubacteria. In this report, molecular phylogenetic analysis of 16S ribosomal RNAs indicates that symbiotes from taxonomically divergent weevils are descendents of distinctly separate bacterial lineages. The intracellular symbiotes of two rhyncophorine weevels, Sitophilus oryzae L. and Sitophilus zeamais Mots., descended from the same hypothetical ancestor and share a common lineage with the secondary symbiote of the pea aphid, Acyrthosiphon pisum (Harris). These symbiotes are members of the Enterobacteriaceae in the γ-3 subgroup of the Proteobacteria. The symbiotes of two cleonine weevils, Bangasternus orientalis (Capiomont) and Rhinocyllus conicus (Froelich), are phylogenetically distinct from the symbiotes found in the rhyncophorines and are members of a branch of the α-subdivision which include the cytoplasmic incompatibility microorganisms of insects (CIMS), Ehrlichia and Anaplasma. The implication of genetic divergence of bacterial symbiotes to weevil speciation is discussed.
Development and growth of the cereal weevils of Sitophilus zeamais, S. oryzae, S. granarius and of S. linearis on non-cereal foods (acorn, chestnut, buckwheat, tamarind and split pea) are compared with performances on soft wheat. It shows the adaptability of cereal weevils to different seeds, and the high performances on chestnuts and acorns. Tamarind weevil which has no symbiotes did not breed on cereals. The alimentary spectrum is compared with that of the whole genus Sitophilus and discussed. The cross influence of food quality and symbiote number is investigated, showing the influence of food on development and the high variability of symbiote number (from 40,000 to 120,000 per ovary).
In this study we have investigated the rates and spatial patterns of chromosome replication and cell elongation during the growth phase of wild-type facultatively prey-independent mutant strains of Bdellovibrio bacteriovorus. For the facultatively prey-independent mutants, the total DNA content of synchronously growing cultures was found to increase exponentially, as the multiple chromosomes within each filamentous cell replicated simultaneously. Cell mass, measured as total cellular protein, also increased exponentially during this period, apparently by means of multiple elongation sites along the filament wall. The relative rates of DNA and protein synthesis were unbalanced during growth, however, with the cellular concentration of DNA increasing slightly faster than that of protein. The original cellular DNA: protein ratio was restored in the progeny cells by continued protein synthesis during the septation period that follows the termination of DNA replication. Because of technical problems, these experiments could not be conducted on the wild-type cells, but similar results are assumed. This unusual pattern of unbalanced growth may represent an adaptation by bdellovibrios to maximize their progeny yield from the determinate amount of substrate available within a given prey cell.
The essential cell division protein FtsZ forms a dynamic ring structure at the future division site. This Z-ring contracts during cell division while maintaining a position at the leading edge of the invaginating septum. Using immunofluorescence microscopy we have characterized two situations in which non-ring FtsZ structures are formed. In ftsZ26 (temperature sensitive, Ts) mutant cells, FtsZ-spirals are formed and lead to formation of spirally invaginating septa, which in turn cause morphological abnormalities. In rodAoul mutant cells, which grow as spheres instead of rods, FtsZ-arcs are formed where asymmetric septal invaginations are initiated. The FtsZ-arcs later mature into complete FtsZ-rings. Our data show that Z-spirals and Z-arcs can contract and that in doing so, they determine the shape of the invaginating septa. These results also strongly suggest that in normal cell division, FtsZ is positioned to a single nucleation site on the inner membrane, from which it polymerizes bidirectionally around the cell circumference to form the Z-ring.
Campylobacter jejuni strains demonstrate a variety of growth phase-linked distinct morphological forms when grown in liquid culture. The typical spiral form of the organism, evident during logarithmic phase, undergoes elongation during stationary phase before becoming coccoid via the formation of membrane blebs and budded forms in decline phase. Cellular elongation and coccoid formation occurred despite the inhibition of protein synthesis and without a detectable change in the protein components of the inner and outer cell membranes.
Symbiobacterium thermophilum is a strictly symbiotic thermophile, the growth of which is dependent on the coexistence of an associating thermophilic Bacillus sp., strain S. S. thermophilum grows only in mixed culture with the Bacillus strain in liquid media, and does not form visible colonies on solid media. To measure the growth of this symbiotic bacterium and to analyze its growth requirements, we developed a quantitative PCR method by using its specific sequences in a putative membrane translocator gene tnaT as primers. According to this method, independent growth of S. thermophilum was first confirmed in a dialyzing culture physically separated from Bacillus strain S with a cellulose membrane. Independent growth of S. thermophilum was also managed by adding conditioned medium prepared from the culture filtrate of the Bacillus strain, but the growth in the conditioned medium stopped at a very limited extent with appearance of filamentous cells, suggesting the uncoupling of cellular growth and cell division. Formation of micro-colonies of S. thermophilum was observed on the conditioned agar medium under both aerobic and anaerobic conditions, but the colony-forming efficiencies remained below 1%. Several other bacterial species, such as Bacillus stearothermophilus, Bacillus subtilis, Thermus thermophilus, and even Escherichia coli, were also found to support the growth of S. thermophilum. These results indicate that S. thermophilum essentially requires some ubiquitous metabolite(s) of low molecular weight produced by various bacterial species as growth factor(s) but coexistence of the living partner cells is still required, probably to maintain an effective level of the putative factor(s) in the medium.
Buchnera, endosymbiotic bacteria of aphids possess many genomic copies per cell. In this study, we estimated genomic copy number per Buchnera cell from host insects at various developmental stages and of two different morphs, apterae and alatae, by fluorimetry and real-time quantitative PCR. The results indicated that the genomic copy number of Buchnera increased during postembryonic development of insects to adulthood, and that it decreased during the host's ageing. In Buchnera from alatae, the genomic copy number per cell was about twice as many as in those from apterae. DAPI-staining showed that the distribution of the genomic DNA in the Buchnera cells from old insects tended to aggregate, suggesting that intracellular structure of the genomic DNA of Buchnera varies in response to the physiological conditions of their host.
In the Curculionid beetle Sitophilus oryzae, the fat body is composed of one type of adipocyte, interstitial cells and oenocytes. Synthesis and storage of tyrosine-rich-protein granules (TRPG) in adipocytes are observed during all the larval and prepupal stages (except the first larval instar which has not been studied). They appear first in the posterior part of the fat body, around the nucleus of adipocytes. They progressively invade the cytoplasm. In the young pupa, TRPG are present in every part of the body, including the head and the appendages in formation. TRPG grow in size by fusing together. Their mean diameter is 6 microm, but some of them reach up to 50 microm. They present a basic core and an acidic periphery. Their charge in tyrosine increases until the prepupa. They are APS and lipid negative and contain no RNA. During metamorphosis they take on a reticulated structure, evoking a golf ball, and disintegrate into small granules, the tyrosine content of which diminishes drastically, especially in contact with epidermal cells, whence tyrosine is probably transferred. TRPG in S. oryzae contain 16 different insoluble proteins. Five of them are tyrostaurins characterized by their very high content in tyrosine (up to 27%) and their strict insolubility in aqueous solution. Arylphorin-like proteins have not been detected in S. oryzae granules.