Genome expansion of an obligate parthenogenesis-associated Wolbachia poses an exception to the symbiont reduction model.

BACKGROUND: Theory predicts that dependency within host-endosymbiont interactions results in endosymbiont genome size reduction. Unexpectedly, the largest Wolbachia genome was found in the obligate, parthenogenesis-associated wFol. In this study, we investigate possible processes underlying this genome expansion by comparing a re-annotated wFol genome to other Wolbachia genomes. In addition, we also search for candidate genes related to parthenogenesis induction (PI). RESULTS: Within wFol, we found five phage WO regions representing 25.4% of the complete genome, few pseudogenized genes, and an expansion of DNA-repair genes in comparison to other Wolbachia. These signs of genome conservation were mirrored in the wFol host, the springtail F. candida, which also had an expanded DNA-repair gene family and many horizontally transferred genes. Across all Wolbachia genomes, there was a strong correlation between gene numbers of Wolbachia strains and their hosts. In order to identify genes with a potential link to PI, we assembled the genome of an additional PI strain, wLcla. Comparisons between four PI Wolbachia, including wFol and wLcla, and fourteen non-PI Wolbachia yielded a small set of potential candidate genes for further investigation. CONCLUSIONS: The strong similarities in genome content of wFol and its host, as well as the correlation between host and Wolbachia gene numbers suggest that there may be some form of convergent evolution between endosymbiont and host genomes. If such convergent evolution would be strong enough to overcome the evolutionary forces causing genome reduction, it would enable expanded genomes within long-term obligate endosymbionts.

Evidence for involvement of a transformer paralogue in sex determination of the wasp Leptopilina clavipes.

Transformer (tra) is the central gear in many insect sex determination pathways and transduces a wide range of primary signals. Mediated by transformer-2 (tra2) it directs sexual development into the female or male mode. Duplications of tra have been detected in numerous Hymenoptera, but a function in sex determination has been confirmed only in Apis mellifera. We identified a tra2 orthologue (Lc-tra2), a tra orthologue (Lc-tra) and a tra paralogue (Lc-traB) in the genome of Leptopilina clavipes (Hymenoptera: Cynipidae). We compared the sequence and structural conservation of these genes between sexual (arrhenotokous) and asexual all-female producing (thelytokous) individuals. Lc-tra is sex-specifically spliced in adults consistent with its orthologous function. The male-specific regions of Lc-tra are conserved in both reproductive modes. The paralogue Lc-traB lacks the genomic region coding for male-specific exons and can only be translated into a full-length TRA-like peptide sequence. Furthermore, unlike LC-TRA, the LC-TRAB interstrain sequence variation is not differentiated into a sexual and an asexual haplotype. The LC-TRAB protein interacts with LC-TRA as well as LC-TRA2. This suggests that Lc-traB functions as a conserved element in sex determination of sexual and asexual individuals.

Collembolan Transcriptomes Highlight Molecular Evolution of Hexapods and Provide Clues on the Adaptation to Terrestrial Life.

BACKGROUND: Collembola (springtails) represent a soil-living lineage of hexapods in between insects and crustaceans. Consequently, their genomes may hold key information on the early processes leading to evolution of Hexapoda from a crustacean ancestor. METHOD: We assembled and annotated transcriptomes of the Collembola Folsomia candida and Orchesella cincta, and performed comparative analysis with protein-coding gene sequences of three crustaceans and three insects to identify adaptive signatures associated with the evolution of hexapods within the pancrustacean clade. RESULTS: Assembly of the springtail transcriptomes resulted in 37,730 transcripts with predicted open reading frames for F. candida and 32,154 for O. cincta, of which 34.2% were functionally annotated for F. candida and 38.4% for O. cincta. Subsequently, we predicted orthologous clusters among eight species and applied the branch-site test to detect episodic positive selection in the Hexapoda and Collembola lineages. A subset of 250 genes showed significant positive selection along the Hexapoda branch and 57 in the Collembola lineage. Gene Ontology categories enriched in these genes include metabolism, stress response (i.e. DNA repair, immune response), ion transport, ATP metabolism, regulation and development-related processes (i.e. eye development, neurological development). CONCLUSIONS: We suggest that the identified gene families represent processes that have played a key role in the divergence of hexapods within the pancrustacean clade that eventually evolved into the most species-rich group of all animals, the hexapods. Furthermore, some adaptive signatures in collembolans may provide valuable clues to understand evolution of hexapods on land.

Occasional males in parthenogenetic populations of Asobara japonica (Hymenoptera: Braconidae): low Wolbachia titer or incomplete coadaptation?

Wolbachia are endosymbiotic bacteria known to manipulate the reproduction of their hosts. Some populations of the parasitoid wasp Asobara japonica are infected with Wolbachia and reproduce parthenogenetically, while other populations are not infected and reproduce sexually. Wolbachia-infected A. japonica females regularly produce small numbers of male offspring. Because all females in the field are infected and infected females are not capable of sexual reproduction, male production seems to be maladaptive. We investigated why these females nevertheless produce males. We tested three hypotheses: high rearing temperatures could result in higher offspring sex ratios (more males), low Wolbachia titer of the mother could lead to higher offspring sex ratios and/or the Wolbachia infection is of relatively recent origin and not enough time has passed to allow complete coadaptation between Wolbachia and host. In all, 33% of the Wolbachia-infected females produced males and 56% of these males were also infected with Wolbachia. Neither offspring sex ratio nor male infection frequency was significantly affected by rearing temperature or Wolbachia concentration of the mother. The mitochondrial DNA sequence of one of the uninfected populations was identical to that of two of the infected populations. Therefore, the initial Wolbachia infection of A. japonica must have occurred recently. Mitochondrial sequence variation among the infected populations suggests that the spread of Wolbachia through the host populations involved horizontal transmission. We conclude that the occasional male production by Wolbachia-infected females is most likely a maladaptive side effect of incomplete coevolution between symbiont and host in this relatively young infection.

A new case of Wolbachia dependence in the genus Asobara: evidence for parthenogenesis induction in Asobara japonica.

Wolbachia is a maternally inherited bacterium that is widely distributed among arthropods, in which it manipulates the reproduction of its hosts. Although generally facultative for its hosts, Wolbachia has recently become obligatory in Asobara tabida (Hymenoptera: Braconidae) in which it is required for the completion of oogenesis. Here, we describe a new Wolbachia strain (wAjap) that is associated with the genus Asobara and infects Asobara japonica. wAjap was detected in all female-biased populations of A. japonica found in the main islands of Japan, but not in the arrhenotokous populations from the southern islands. Using phylogenetic analyses based on multi-locus sequence typing (MLST), we show that this strain is closely related to wAtab3 (the strain required for oogenesis in A. tabida), even though they differ on Wolbachia surface protein (WSP) and WO phage sequences. Using antibiotic treatments, we show that cured thelytokous females are not dependent on Wolbachia for oogenesis. However, they produced only sons, showing that wAjap induces thelytokous parthenogenesis. Analyses of mating behavior and offspring production of individuals from Wolbachia-infected populations showed that while males were still sexually functional, females no longer attract males, making Wolbachia an obligate partner for daughter production in thelytokous populations. The fact that Wolbachia has become independently obligatory in two species of the same genus tends to show that dependence evolution can be common and swift, although no clear benefit for the parasitoid can be attributed to this dependence. Although dependence should lead to co-divergence between Wolbachia and its hosts, the very few cases of co-speciation observed in host-Wolbachia associations question the stability of these obligatory associations.

Mating system, philopatry and patterns of kinship in the cooperatively breeding subdesert mesite Monias benschi.

In the first molecular study of a member of the threatened avian family, Mesitornithidae, we used nine polymorphic microsatellite loci to elucidate parentage, patterns of within-group kinship and occurrence of extra-group paternity in the subdesert mesite Monias benschi, of southwest Madagascar. We found this cooperatively breeding species to have a very fluid mating system. There was evidence of genetic monogamy and polygynandry: of the nine groups with multiple offspring, six contained one breeding pair with unrelated helpers and three contained multiple male and female breeders with related helpers. Although patterns of within-group kinship varied, there was a strong positive relationship between group size and relatedness, suggesting that groups form by natal philopatry. There was also a strong positive correlation between within-sex and between-sex relatedness, indicating that unlike most cooperatively breeding birds, philopatry involved both sexes. In contrast to predictions of kin selection and reproductive skew models, all monogamous groups contained unrelated individuals, while two of the three polygynandrous groups were families. Moreover, although between-group variation in seasonal reproductive success was related to within-group female relatedness, relatedness among males and between the sexes had no bearing on a group's reproductive output. While kin selection may underlie helping behaviour in females, factors such as direct long-term fitness benefits of group living probably determine helping in males. Of the 14 offspring produced by fully sampled groups, at least two were sired by males from neighbouring groups: one by a breeding male and one by a nonbreeding male, suggesting that males may augment their reproductive success through extra-group paternity.

Extra-pair paternity does not result in differential sexual selection in the mutually ornamented black swan (Cygnus atratus).

We studied patterns of parentage in 85 broods (332 cygnets) of black swans during three breeding seasons, using a set of eight polymorphic microsatellite markers. We detected both intraspecific brood parasitism (IBP; < 5% of cygnets per year) and extra-pair paternity (EPP). In these years, 10-17% (mean = 15.1%) of cygnets resulted from EPP, and 27-40% (mean 37.6%) of broods contained at least one extra-pair cygnet. Compared with levels of EPP in closely related species with similar life histories, these values are unexpectedly high. EPP in black swans appears unrelated to ecological factors (breeding density and synchrony) or genetic factors (genetic similarity between pair members or genetic quality of the offspring). We found no evidence that a mutual sexual feather ornament known to play a role in social mate choice in black swans (curled wing feathers) is involved in extra-pair mate choice. EPP does not lead to greater variance in reproductive success in males, relative to females in this species. We therefore suggest that EPP does not result in differential sexual selection on males and females, explaining why they are ornamented to the same degree.

Synthetic polysaccharide type 3-related di-, tri-, and tetrasaccharide-CRM(197) conjugates induce protection against Streptococcus pneumoniae type 3 in mice.

Di-, tri-, and tetrasaccharides, synthesized according to the chemical structure of pneumococcal polysaccharide type 3 (PS3), were coupled to the cross-reactive material (CRM(197)) of modified diphtheria toxin in different molar carbohydrate/protein ratios using the squarate coupling method. To study protective immunity, female BALB/c mice were subcutaneously immunized twice (with a 3-week interval) using the amount of conjugates corresponding to 2.5 microg of oligosaccharide per mouse. The conjugates evoked PS3 binding immunoglobulin G antibodies that lasted for at least 7 weeks after the booster. Immunogenicity was not influenced by the carbohydrate/protein ratio. All mice with PS3-specific antibodies survived the intraperitoneal challenge with Streptococcus pneumoniae type 3. Therefore, synthetic oligosaccharide-protein conjugates might have potential as vaccines.

Cross-protection by anti-core glycolipid antibodies: evidence from animal experiments.

The ability of antibodies against the core glycolipid (CGL) of endotoxin to protect experimentally infected animals against death from Gram-negative sepsis is reviewed. The limitations and confounding factors inherent to animal models of sepsis are also briefly discussed. This review considers 30 studies in mice and 12 in other animal species that investigated protection against heterologous challenge by passive immunization with anti-CGL antibodies. In 28 (67%) of the reviewed studies antibodies were found to be protective, either prophylactically (n = 17) or therapeutically (n = 11). With the possible exception of the type of antibody preparation that was used (monoclonal versus polyclonal antibodies), none of the many differences in the experimental protocols were clearly correlated with success. Convincing proof is still lacking for any of the hypothetical mechanisms of protection by anti-CGL antibodies. Moreover, the evidence that protection by these antibodies is attributable to their anti-CGL specificity is poor. The available data raise serious questions about the validity of the concept underlying the search for broadly cross-protective antibodies raised against the core region of endotoxin. However, continuing research suggests that endotoxin still is a valid target in devising new adjunctive treatment strategies to improve the outcome of serious Gram-negative infections.

Evaluation of protection by two endotoxin-neutralizing IgM monoclonal antibodies in different peritonitis models.

Two anti-core glycolipid (CGL) IgM monoclonal antibodies (mAbs 8-2 and 26-20), previously shown to display cross-reactivity with heterologous lipopolysaccharide (LPS) in vitro and to provide cross-protectivity against endotoxin challenge in vivo, were evaluated for their potential to protect mice against death from peritonitis caused by heterologous bacterial challenge. Without concurrent antibiotic treatment neither antibody was protective. Compared with a control mAb, prophylactic treatment with mAb 8-2 significantly increased the survival of gentamicin-treated mice challenged with the rough strain Salmonella minnesota Re595. Both mAb 8-2 and a control mAb, in combination with a suboptimal dose of ceftazidime, increased survival following challenge with the clinical isolate Escherichia coli O7:K1. In a model of mucin-enhanced peritonitis, neither mAb was protective against challenge with inocula of E. coli O7:K1, ranging from 10(2) to 10(4) bacteria. We conclude that protection of mice by anti-CGL mAb 8-2 against heterologous challenge is vitally dependent on concurrent treatment with antibiotics and that protection may not be attributable to the anti-CGL specificity of these antibodies.

Granulocyte colony-stimulating factor enhances protection by anti-K1 capsular IgM antibody in murine Escherichia coli sepsis.

Combined prophylactic treatment with recombinant murine granulocyte colony-stimulating factor (G-CSF) and a suboptimal dose of anti-K1 capsular IgM monoclonal antibody (MAb) significantly enhanced survival in an experimental mouse Escherichia coli O7:K1 peritonitis model compared with untreated animals (67% vs. 11% survival; P < 0.001) and with either treatment alone (67 vs. 29% and 27% survival, respectively; P < 0.01), which suggests synergism between these agents. Enhanced survival by combined treatment was associated with increased neutrophil counts in blood and peritoneal lavage fluid, lower systemic and higher levels of local tumour necrosis factor (TNF) and lower bacterial counts in blood cultures. Mouse neutrophils treated with G-CSF but not infected with E. coli showed enhanced phagocytic and respiratory burst capacity, down-regulation of L-selectin receptors and enhanced expression of Fc RII-III receptors but not of complement receptors.

In vivo TNF induction by culture supernatants of antibiotic-treated Escherichia coli 07:K1. Role of antibiotic class and concentration.

Antibiotics may cause an excess release of lipopolysaccharide (LPS) from bacteria and thereby promote the production of tumour necrosis factor (TNF). TNF was measured in the serum of Swiss mice challenged with filtered supernatant of Escherichia coli O7:K1 that had been exposed to various antibiotics in vitro. Expressed as a function of a standardized number of cells remaining after 6 h of exposure to gentamicin, ceftazidime, ciprofloxacin or imipenem, TNF leves associated with antibiotic exposure always exceeded those of controls. However, if differences in the remaining number of bacteria were not taken into account, TNF induction by supernatant of control untreated cultures was greater than that elicited by supernatant from any of the antibiotic-treated cultures. With the exception of imipenem, low-dose antibiotic exposure (0.5 x MIC) invariably induced higher TNF levels than did high-dose exposure (10 x MIC). Considerable antibiotic class- and concentration-related differences were noted. LAL equivalent amounts of LPS released by different antibiotics may diverge in their capacity to induce TNF. Our results do not support the notion that the use of rapidly bactericidal and lytic antibiotics should be avoided.

Effect of imipenem on monoclonal antibody-mediated protection against Escherichia coli O18K5.

Flow cytometry revealed that the binding of immunoglobulin M monoclonal antibodies (MAbs) to Escherichia coli O18K5 was modulated by exposure of the bacteria to subinhibitory concentrations of imipenem. The binding of anti-K5 MAb was decreased, while the binding of anti-O18 MAb was increased. In addition, anti-lipid A MAbs bound only to imipenem-treated bacteria. The biological effect of MAb binding was investigated in BALB/c mice by determination of the levels of bacteremia, tumor necrosis factor (TNF) in serum and survival after intraperitoneal challenge with bacteria preincubated with MAb. Neither MAb alone (150 micrograms per animal) proved to be protective against untreated bacteria. Anti-lipid A MAb on its own, in contrast to anti-K5 and anti-O18 MAbs, was not protective against imipenem-treated bacteria. Only combinations which included anti-O18 MAb and anti-K5 MAb exerted in mice enhanced protection against smooth E. coli O18K5 as well as imipenem-treated E. coli O18K5. This was reflected by reduced TNF levels in serum and increased survival. The addition of anti-lipid A MAb to the combination of anti-K5 MAb and anti-O18 MAb reduced serum TNF levels in mice, but not significantly.

Enhanced protection by use of a combination of anticapsule and antilipopolysaccharide monoclonal antibodies against lethal Escherichia coli O18K5 infection of mice.

To study antibody-mediated protection against Escherichia coli peritonitis in BALB/c mice, monoclonal antibodies (MAbs) were generated against the capsule (K5) and the lipopolysaccharide (O18) of E. coli. Flow cytometric analysis with two selected immunoglobulin M MAbs revealed that bacteria were antigenically heterogeneous. Arbitrarily, three subpopulations in E. coli O18K5 cultures could be distinguished by double immunofluorescence. A subpopulation bound only the anti-K5 MAb, and another subpopulation bound only the anti-O18 MAb. An intermediate subpopulation, however, bound both MAbs. In agreement with this result, combinations of both MAbs enhanced phagocytosis of fluorescein isothiocyanate-labeled bacteria by human polymorphonuclear leukocytes and mouse macrophage J774 cells as well. In protection experiments, combinations of both MAbs, preincubated with 3 50% lethal doses of E. coli O18K5, protected all mice upon intraperitoneal challenge. Relatively high doses of either MAb alone proved to be not fully protective in this infection model. Protection of mice by the combination of MAbs was associated with significantly lower (P < 0.02) tumor necrosis factor levels in serum 90 min after challenge compared with any other treatment group. Similarly, prophylactic administration of MAbs yielded significantly lower (P < 0.01) tumor necrosis factor levels in mice that received the combination of MAbs than in any other treatment group.

Escherichia coli in bacteremia: O-acetylated K1 strains appear to be more virulent than non-O-acetylated K1 strains.

A total of 174 blood isolates of Escherichia coli, collected during a 5-year period at the University Hospital Utrecht, were serotyped with rabbit sera against 171 O antigens and 73 capsule (K) antigens. The four most prevalent O-antigen serotypes were O6 (n = 22), O18 (n = 19), O1 (n = 19), and O2 (n = 15). Thirty-one strains were not typeable with any of the O-antigen-typing sera. Of the 148 strains that were subjected to K-antigen serotyping, 34 strains lacked a K antigen and 41 were not typeable with the K-antigen-specific antisera used in the study. K1 was by far the most frequently found K-antigen serotype; this was followed by K2, K53, K5, K13, K7, K(A)28, and K15. Strains possessing a K1 antigen were further classified as either O-acetyl-positive (n = 12) or O-acetyl-negative (n = 21) strains. Retrospective analysis of patients infected with different E. coli isolates--nonencapsulated (n = 23), O-acetylated K1 (n = 12), and non-O-acetylated K1 (n = 21)--revealed clinical differences. More patients suffered from sepsis (94% versus 74%), and a higher rate of mortality was found in the group infected with K1 isolates (18 versus 9%) than in the group infected with nonencapsulated isolates. More patients with severe sepsis (25 versus 10%) and a higher mortality (33 versus 10%) were found in the group infected with O-acetylated K1 isolates than in the group infected with non-O-acetylated isolated. Also, the hospitalization of these patients was prolonged. Thus, O-acetylated E. coli K1 strains seem to be more virulent than non-O-acetylated K1 strains.