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 ... -  《-》

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 《-》

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 ... -  《-》

Bacteria associated with gut lumen of Camponotus japonicus Mayr.

Camponotus ants harbor the obligate intracellular endosymbiont Blochmannia in their midgut bacteriocytes, but little is known about intestinal bacteria living in the gut lumen. In this paper we reported the results of a survey of the intestinal microflora of Camponotus japonicus Mayr based on small-subunit rRNA genes (16S rRNAs) polymerase chain reaction (PCR)-restriction fragment-length polymorphism analysis of worker guts. From 107 clones, 11 different restriction fragment-length polymorphism profiles were identified, and sequences blasting analysis found these represent four types of bacteria. Most (91.6%) of the clones were "Candidatus Blochmannia", the obligate endosymbionts of Camponotus ants, and 6.5% of the clones were "Candidatus Serratia symbiotica", a secondary endosymbiont of aphids; the remaining 2% clones were Fructobacillus fructosus and uncultured Burkholderiales bacterium, respectively. These results show that the diversity of gut bacteria in C. japonicus was low. "Candidatus Serratia symbiotica" was identified from Camponotus ants for the first time, an interesting result because Blochmannia's closest bacterial relative is also in the genus Serratia. This discovery supports the scenario that consumption of aphid honeydew or tissue provides an initial step in the evolution of an advanced symbiosis, and suggests that Camponotus ant could acquire other secondary endosymbionts from Hemiptera host through their diet. In addition, Burkholderiales bacterium also was identified from the gut of C. japonicus for the first time, and whether it is a nitrogen-recycling endosymbiont in Camponotus ants needs to be investigated further.

He H ，Chen Y ，Zhang Y ，Wei C ... -  《-》

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》