Gene expression analysis of the endosymbiont-bearing midgut tissue during ontogeny of the carpenter ant Camponotus floridanus.

Insects have frequently evolved mutualistic relationships with extracellular and/or intracellular bacterial endosymbionts. Infection with endosymbionts seems to affect several cellular functions of the host such as immune pathways, oxidative stress regulation and autophagy. Our current knowledge about specific host factors leading to endosymbiont tolerance and/or control is still scarce and is based on very few associations between insect hosts and bacteria only. Camponotus floridanus ants harbour the obligate intracellular bacterium Blochmannia floridanus within specialized midgut cells called bacteriocytes. The number of Blochmannia endosymbionts within the midgut tissue increases strongly during host development and reaches a maximum at the late pupal stage, where the entire midgut is transformed into a symbiotic organ. After eclosion of workers the number of Blochmannia strongly decreases again. We chose 15 candidate genes from C. floridanus likely to be involved in host-symbiont interactions based on their significant homology to previously investigated symbiosis-relevant genes from other insects. We determined the expression of these genes in the endosymbiont-bearing midgut tissue in comparison to the residual body tissue at different developmental stages of C. floridanus in order to reveal changes in gene expression correlating with changes in endosymbiont number per host. Strikingly, two pattern recognition receptors (amidase PGRP-LB and PGRP-SC2) were highly expressed in the midgut tissue at the pupal stage, potentially down-modulating the IMD pathway to enable endosymbiont tolerance. Moreover, we investigated the immune gene expression in response to bacterial challenge at the pupal stage. Results showed that the midgut tissue differs in expression pattern in contrast to the residual body. Our results support a key role for amidase PGRPs, especially PGRP-LB, in regulation of the immune response towards endosymbionts in C. floridanus and suggest an involvement of the lysosomal system in control of Blochmannia endosymbionts.

Ratzka C ，Gross R ，Feldhaar H 《-》

Bacteriocyte dynamics during development of a holometabolous insect, the carpenter ant Camponotus floridanus.

The carpenter ant Camponotus floridanus harbors obligate intracellular mutualistic bacteria (Blochmannia floridanus) in specialized cells, the bacteriocytes, intercalated in their midgut tissue. The diffuse distribution of bacteriocytes over the midgut tissue is in contrast to many other insects carrying endosymbionts in specialized tissues which are often connected to the midgut but form a distinct organ, the bacteriome. C. floridanus is a holometabolous insect which undergoes a complete metamorphosis. During pupal stages a complete restructuring of the inner organs including the digestive tract takes place. So far, nothing was known about maintenance of endosymbionts during this life stage of a holometabolous insect. It was shown previously that the number of Blochmannia increases strongly during metamorphosis. This implicates an important function of Blochmannia in this developmental phase during which the animals are metabolically very active but do not have access to external food resources. Previous experiments have shown a nutritional contribution of the bacteria to host metabolism by production of essential amino acids and urease-mediated nitrogen recycling. In adult hosts the symbiosis appears to degenerate with increasing age of the animals. We investigated the distribution and dynamics of endosymbiotic bacteria and bacteriocytes at different stages during development of the animals from larva to imago by confocal laser scanning microscopy. The number of bacteriocytes in relation to symbiont-free midgut cells varied strongly over different developmental stages. Especially during metamorphosis the relative number of bacteria-filled bacteriocytes increased strongly when the larval midgut epithelium is shed. During this developmental stage the midgut itself became a huge symbiotic organ consisting almost exclusively of cells harboring bacteria. In fact, during this phase some bacteria were also found in midgut cells other than bacteriocytes indicating a cell-invasive capacity of Blochmannia. In adult animals the number of bacteriocytes generally decreased. During the life cycle of the animals the distribution of bacteriocytes and of Blochmannia endosymbionts is remarkably dynamic. Our data show how the endosymbiont is retained within the midgut tissue during metamorphosis thereby ensuring the maintenance of the intracellular endosymbiosis despite a massive reorganization of the midgut tissue. The transformation of the entire midgut into a symbiotic organ during pupal stages underscores the important role of Blochmannia for its host in particular during metamorphosis.

Stoll S ，Feldhaar H ，Fraunholz MJ ，Gross R ... -  《BMC MICROBIOLOGY》

Immune response of the ant Camponotus floridanus against pathogens and its obligate mutualistic endosymbiont.

Numerous insect species harbor mutualistic endosymbionts that play a role in nutrient cycling or confer other fitness benefits to their hosts. Insect hosts face the problem of having to maintain such mutualistic bacteria while staging an immune response towards pathogens upon infection. In addition, hosts may regulate the number of endosymbionts present in their tissues via the innate immune system. Camponotus floridanus ants harbor the obligate endosymbiont Blochmannia floridanus in specialized midgut cells and ovaries. We identified genes transcriptionally induced in response to septic injury by suppression subtractive hybridization (SSH). Among these were genes involved in pathogen recognition (e.g. GNBP), signal transduction (e.g. MAPK-kinase), antimicrobial activity (e.g. defensin and hymenoptaecin), or general stress response (e.g. heat shock protein). A quantitative analysis of immune-gene expression revealed different expression kinetics of individual factors and also characteristic expression profiles after injection of gram-negative and gram-positive bacteria. Likewise, B. floridanus injected into the hemocoel elicited a comparable immune response of its host C. floridanus. Thus, the host immune system may contribute to controlling the endosymbiont population.

Ratzka C ，Liang C ，Dandekar T ，Gross R ，Feldhaar H ... -  《-》

Distribution of the obligate endosymbiont Blochmannia floridanus and expression analysis of putative immune genes in ovaries of the carpenter ant Camponotus floridanus.

The bacterial endosymbiont Blochmannia floridanus of the carpenter ant Camponotus floridanus contributes to its hosts' ontogeny via nutritional upgrading during metamorphosis. This primary endosymbiosis is essential for both partners and vertical transmission of the endosymbionts is guaranteed by bacterial infestation of oocytes. Here we present a detailed analysis of the presence and localisation of B. floridanus in the ants' ovaries obtained by FISH and TEM analyses. The most apical part of the germarium harbouring germ-line stem cells (GSCs) is not infected by the bacteria. The bacteria are detectable for the first time in lower parts of the germarium when cystocytes undergo the 4th and 5th division and B. floridanus infects somatic cells lying under the basal lamina surrounding the ovarioles. With the beginning of cystocyte differentiation, the endosymbionts are exclusively transported from follicle cells into the growing oocytes. This infestation of the oocytes by bacteria very likely involves exocytosis-endocytosis processes between follicle cells and the oocytes. Nurse cells were never found to harbour the endosymbionts. Furthermore we present first gene expression data in C. floridanus ovaries. These data indicate a modulation of immune gene expression which may facilitate tolerance towards the endosymbionts and thus may contribute to their transovarial transmission.

Kupper M ，Stigloher C ，Feldhaar H ，Gross R ... -  《-》

Relevance of the endosymbiosis of Blochmannia floridanus and carpenter ants at different stages of the life cycle of the host.

Expression of several genes possibly involved in the symbiotic relationship between the obligate intracellular endosymbiont Blochmannia floridanus and its ant host Camponotus floridanus was investigated at different developmental stages of the host by real-time quantitative PCR. These included a set of genes related to nitrogen metabolism (ureC, ureF, glnA, and speB) as well as genes involved in the synthesis of the aromatic amino acid tyrosine (tyrA, aspC, and hisC). The overall transcriptional activity of Blochmannia was found to be quite low during early developmental stages and to increase steadily with host age. However, a concerted peak of gene expression related to nitrogen recycling could be detected around the entire process of pupation, while expression of biosynthesis pathways for aromatic amino acids was elevated only during a short phase in pupation. These data suggest an important role of certain metabolic functions for the symbiotic interactions of the bacteria and an individual host organism in early phases of development. General relevance of Blochmannia for its ant host was tested in fostering experiments with worker groups of Camponotus floridanus, and their success in raising pupae from first-instar larvae was used as a fitness measure. Groups treated with antibiotics had a significantly reduced success in raising the brood in comparison to untreated control groups, indicating that the symbiosis is relevant for the development of the entire colony.

Zientz E ，Beyaert I ，Gross R ，Feldhaar H ... -  《-》