Ant Genetics: Reproductive Physiology, Worker Morphology, and Behavior.

Many exciting studies have begun to elucidate the genetics of the morphological and physiological diversity of ants, but as yet few studies have investigated the genetics of ant behavior directly. Ant genomes are marked by extreme rates of gene turnover, especially in gene families related to olfactory communication, such as the synthesis of cuticular hydrocarbons and the perception of environmental semiochemicals. Transcriptomic and epigenetic differences are apparent between reproductive and sterile females, males and females, and workers that differ in body size. Quantitative genetic approaches suggest heritability of task performance, and population genetic studies indicate a genetic association with reproductive status in some species. Gene expression is associated with behavior including foraging, response to queens attempting to join a colony, circadian patterns of task performance, and age-related changes of task. Ant behavioral genetics needs further investigation of the feedback between individual-level physiological changes and socially mediated responses to environmental conditions.

Cellular conditions that modulate the fungicidal activity of occidiofungin.

AIMS: To identify cellular conditions that significantly alter susceptibility of Saccharomyces cerevisiae, Candida albicans and Candida glabrata to the antimicrobial peptide, occidiofungin. METHODS AND RESULTS: Genetic and pharmacological approaches were used to determine a role for calcium signalling in occidiofungin sensitivity profiles for S. cerevisiae, C. albicans and C. glabrata strains of yeast. Minimum inhibitory concentration (MIC) and drop assays found that extracellular calcium resulted in a fourfold resistance, and this was independent of an intact calmodulin-calcineurin signalling pathway. A similar resistance was found in the presence of magnesium but not other cations. Occidiofungin was found to be ineffective against cells in a quiescent state when measured by MIC, drop assay and short-term time-kill assays. A similar resistance pattern was detected for S. cerevisiae cultures pre-exposed to cycloheximide or placed in depleted media conditions. CONCLUSIONS: Extracellular calcium results in fungal tolerance to occidiofungin bioactivity outside of the calmodulin-calcineurin pathway. In addition, the resistance of quiescent cells suggests that active cellular growth is a requirement for occidiofungin's mechanism of action. SIGNIFICANCE AND IMPACT OF THE STUDY: The identification of cellular conditions that have a role in the activity of occidiofungin provided insight into potential cellular targets of this novel antifungal.

Harvester ant colonies differ in collective behavioural plasticity to regulate water loss.

Collective behavioural plasticity allows ant colonies to adjust to changing conditions. The red harvester ant (Pogonomyrmex barbatus), a desert seed-eating species, regulates foraging activity in response to water stress. Foraging ants lose water to evaporation. Reducing foraging activity in dry conditions sacrifices food intake but conserves water. Within a year, some colonies tend to reduce foraging on dry days while others do not. We examined whether these differences among colonies in collective behavioural plasticity persist from year to year. Colonies live 20-30 years with a single queen who produces successive cohorts of workers which live only a year. The humidity level at which all colonies tend to reduce foraging varies from year to year. Longitudinal observations of 95 colonies over 5 years between 2016 and 2021 showed that differences among colonies, in how they regulate foraging activity in response to day-to-day changes in humidity, persist across years. Approximately 40% of colonies consistently reduced foraging activity, year after year, on days with low daily maximum relative humidity; approximately 20% of colonies never did, foraging as much or more on dry days as on humid days. This variation among colonies may allow evolutionary rescue from drought due to climate change.

Social networks and the spread of Salmonella in a sleepy lizard population.

Although theoretical models consider social networks as pathways for disease transmission, strong empirical support, particularly for indirectly transmitted parasites, is lacking for many wildlife populations. We found multiple genetic strains of the enteric bacterium Salmonella enterica within a population of Australian sleepy lizards (Tiliqua rugosa), and we found that pairs of lizards that shared bacterial genotypes were more strongly connected in the social network than were pairs of lizards that did not. In contrast, there was no significant association between spatial proximity of lizard pairs and shared bacterial genotypes. These results provide strong correlative evidence that these bacteria are transmitted from host to host around the social network, rather than that adjacent lizards are picking up the same bacterial genotype from some common source.

Irradiated sporozoite vaccine induces HLA-B8-restricted cytotoxic T lymphocyte responses against two overlapping epitopes of the Plasmodium falciparum sporozoite surface protein 2.

Vaccines designed to protect against malaria by inducing CD8+ cytotoxic T lymphocytes (CTL) in individuals of diverse HLA backgrounds must contain multiple conserved epitopes from various preerythrocytic-stage antigens. Plasmodium falciparum sporozoite surface protein 2 (PfSSP2) is considered an important antigen for inclusion in such vaccines, because CD8+ CTL against the P. yoelii SSP2 protect mice against malaria by eliminating infected hepatocytes. To develop PfSSP2 as a component of malaria vaccines, we investigated the presence of anti-PfSSP2 CTL in two HLA-B8+ volunteers immunized with irradiated P. falciparum sporozoites and characterized their CTL responses using PfSSP2-derived 15-amino acid peptides bearing the HLA-B8-binding motif. Peripheral blood mononuclear cells from both volunteers stimulated with recombinant vaccinia expressing PfSSP2 displayed antigen-specific, genetically restricted, CD8+ T cell-dependent CTL activity against autologous target cells expressing PfSSP2. Of the five HLA-B8 motif-bearing 15-mers identified in the PfSSP2 sequence, two peptides sharing a 10-amino acid overlap sensitized HLA-B8-matched target cells from both volunteers for lysis by peptide-stimulated effectors. The CTL activity was HLA-B8 restricted and dependent on CD8+ T cells. Analysis of the three shorter peptides representing HLA-B8 motif-bearing sequences within the two positive peptides for their ability to bind to HLA-B8 in vitro, and to sensitize target cells for lysis by effectors stimulated with the 15-mers, identified two overlapping HLA-B8-restricted CTL epitopes. Available data indicate that the sequence of one CTL epitope is conserved and the other is variant among P. falciparum isolates. Circulating activated CTL against the conserved epitope could be directly identified in one of the two volunteers. The identification of two HLA-B8-restricted CTL epitopes on PfSSP2 provides data critical to developing an epitope-based anti-liver stage malaria vaccine.

Fine-scale genetic structure and dispersal distance in the harvester ant Pogonomyrmex barbatus.

Dispersal has important genetic and evolutionary consequences. It is notoriously difficult to study in some ant species, because reproductives fly from parent nests to mating aggregations and then to new nest sites. We used genetic techniques to measure dispersal distance and characterize patterns of genetic variation in a population of the harvester ant Pogonomyrmex barbatus. This population consists of two interdependent yet genetically distinct mitochondrial lineages, each associated with specific alleles at nuclear loci. We found moderate levels of genetic structure for both lineages and a significant pattern of isolation by distance when individual colonies were the operational unit of study. Dispersal distances calculated from the slope of the regression of genetic on geographic distance were 65.3 m for J1 and 85.8 m for J2. These results are consistent with previous observations of many mating aggregations over small geographic areas. In dependent-lineage populations like our study population, females must mate with males of the opposite lineage to produce workers, and with males of the same lineage to produce female reproductives. Because lineage ratios differ from 1:1 throughout the southwestern United States, restricted dispersal between sites with different lineage ratios could have important effects on dependent-lineage population dynamics. Our results suggest that it is unlikely that many individuals disperse from areas dominated by one lineage to areas dominated by another. Short dispersal distances lead to low gene flow, giving local populations evolutionary independence.

Efficacy of murine malaria sporozoite vaccines: implications for human vaccine development.

As part of a study of potential vaccines against malaria, the protective efficacy of sporozoite subunit vaccines was determined by using the Plasmodium berghei murine malaria model. Mice were immunized with recombinant DNA-produced or synthetic peptide-carrier subunit vaccines derived from the repetitive epitopes of the Plasmodium berghei circumsporozoite gene, or with radiation-attenuated sporozoites. Immunization with subunit vaccines elicited humoral responses that were equivalent to or greater than those elicited by irradiated sporozoites, yet the protection against sporozoite challenge induced by either of the subunit vaccines was far less than that achieved by immunization with attenuated sporozoites. Passive and adoptive transfer studies demonstrated that subunit vaccines elicited predominantly antibody-mediated protection that was easily overcome whereas irradiated sporozoites induced potent cell-mediated immunity that protected against high challenge doses of sporozoites. These studies indicate that new strategies designed to induce cellular immunity will be required for efficacious sporozoite vaccines.

Immunogenicity of synthetic peptides from circumsporozoite protein of Plasmodium falciparum.

In a study of recombinant proteins that might be useful in developing a vaccine against malaria, synthetic peptides from the circumsporozoite (CS) protein of Plasmodium falciparum were found to be immunogenic for mice and rabbits. Antibody to peptides from the repeating region of the CS protein recognized native CS protein and blocked sporozoite invasion of human hepatoma cells in vitro. Antibodies to peptides from regions I and II had no biologic activity, although antibody to region I recognized processed CS protein by Western blot analysis. These data support the feasibility of developing a vaccine against the sporozoite stage of the malaria parasite by using synthetic peptides of the repeating region of the CS protein conjugated to a carrier protein.

The behavioral ecology of variation in social insects.

Understanding the ecological relevance of variation within and between colonies has been an important and recurring theme in social insect research. Recent research addresses the genomic and physiological factors and fitness effects associated with behavioral variation, within and among colonies, in regulation of activity, cognitive abilities, and aggression. Behavioral variation among colonies has consequences for survival and reproductive success that are the basis for evolutionary change.

The ecological role of bacteriocins in bacterial competition.

Bacteriocins are an abundant class of antimicrobial molecules that appear to mediate population dynamics within species. The bacteriocins of Escherichia coli have served as a model for exploring the ecological role of these potent toxins. Studies suggest that colicins provide a competitive edge in nutrient-poor environments and that there might be a trade-off between the costs and benefits of colicin production.

Fluctuation analysis: the probability distribution of the number of mutants under different conditions.

In the 47 years since fluctuation analysis was introduced by Luria and Delbrück, it has been widely used to calculate mutation rates. Up to now, in spite of the importance of such calculations, the probability distribution of the number of mutants that will appear in a fluctuation experiment has been known only under the restrictive, and possibly unrealistic, assumptions: (1) that the mutation rate is exactly proportional to the growth rate and (2) that all mutants grow at a rate that is a constant multiple of the growth rate of the original cells. In this paper, we approach the distribution of the number of mutants from a new point of view that will enable researchers to calculate the distribution to be expected using assumptions that they believe to be closer to biological reality. The new idea is to classify mutations according to the number of observable mutants that derive from the mutation when the culture is selectively plated. This approach also simplifies the calculations in situations where two, or many, kinds of mutation may occur in a single culture.

Mechanisms of mitochondrial protein import.

Mitochondria import most of their proteins from the cytosol. Precursor forms of most matrix proteins as well as some IM and IMS proteins are synthesized on cytoplasmic ribosomes with N-terminal cleavable signal sequences. Many other mitochondrial proteins including IM carrier proteins contain internal targeting sequences. Three multisubunit translocases, one in the OM and two in the IM, participate in the import process. These translocases co-operate with cytosolic chaperones, chaperone-like soluble proteins in the IMS as well as chaperones in the matrix. Insertion of carrier proteins into the IM only requires a membrane potential. On the other hand, translocation of preproteins across the IM into the matrix requires (i) a membrane potential, (ii) GTP hydrolysis, which occurs at the outer side of the IM, and (iii) ATP-dependent interactions occurring at the matrix side. Following import, the cleavable signal sequence of most preproteins is removed in one step by the MPP. In some cases, removal of the signal sequence is achieved in two steps; first by MPP and second by either mitochondrial intermediate peptidase or by IM peptidases. Imported proteins must be folded properly to perform their functions.

New World monkey efficacy trials for malaria vaccine development: critical path or detour?

Neither GMP malaria antigens nor GMP vaccines have been compared for efficacy in monkeys and humans. It is too risky to base categorical (go/no go) development decisions on results obtained using partially characterized (non-GMP) antigens, adjuvants that are too toxic for human use or unvalidated primate models. Such practices will lead to serious errors (e.g. failure to identify and stop flawed efforts, rejection of effective vaccine strategies) and unjustifiable delays. Successful malaria vaccine development will emphasize definitive field trials in populations at risk of malaria to define and improve vaccine efficacy.

Genetic relationships among rhizopine-producing Rhizobium strains.

Chromosomal and symbiosis-related genotypes of rhizopine-producing and non-producing isolates of Rhizobium meliloti and Rhizobium leguminosarum were examined by multilocus enzyme electrophoresis and RFLP. The distribution of rhizopine production in both species was found to be independent of host genotype. Conversely, rhizopine production was associated with particular symbiotic plasmid types. This association may explain the observed distribution of rhizopine production in R. leguminosarum and R. meliloti. Rhizopine synthesis (mos) genes showed greater sequence divergence than rhizopine catabolism (moc) genes in both R. meliloti and R. leguminosarum. Furthermore, mos and moc genes were less divergent in R. leguminosarum than R. meliloti, suggesting a more recent evolution in the former species.

Autologous lymphoblastoid cell lines stably transfected with Plasmodium falciparum circumsporozoite protein as targets in cytotoxic T-lymphocyte assays.

To produce cell lines that can be used as a continuous source of antigen presenting cells for stimulating T-cell lines and clones and as targets in cytotoxic T-lymphocyte (CTL) assays, we used a retroviral vector with a simian virus (SV40) early promotor to transfer a Plasmodium falciparum circumporozoite (PfCSP) gene into human EBV transformed B-lymphoblastoid cell lines (B-LCL). We herein report successful, stable transfection and cell surface expression of this gene, as confirmed by PCR, Western blot analysis and immunoelectron microscopy. One of three successfully transfected autologous cell lines expressed PfCSP on the cell surface and was lysed by CD8+ T-cell dependent CTL from a donor volunteer who had been immunized with irradiated P. falciparum sporozoites. Such cell lines should provide excellent tools for characterizing human CD8+ T-cell responses against Plasmodium sp. proteins.

Malaria vaccines.

Malaria continues to be a major worldwide problem. Recent advances in our understanding of the parasite and the immune response to malarial infections has resulted in major advances in the progress toward an effective malaria vaccine. Owing to the complexity of the parasite's life cycle, an effective vaccine will most assuredly contain components selected to stimulate potent immune mechanisms directed at various points in the parasite's life cycle. Considering the increasing incidence of drug resistance, combined with the high case-fatality rate, most research efforts have focused on developing a vaccine against Plasmodium falciparum. A successful vaccine against P. falciparum would be a significant advance in medical science. One must not, however, forget the severe morbidity associated with the other three human malarial species. As information is gained in the effort against P. falciparum, it is rapidly incorporated into efforts against P. vivax, P. malariae, and P. ovale.

Differences in susceptibility among mouse strains to infection with Plasmodium berghei (ANKA clone) sporozoites and its relationship to protection by gamma-irradiated sporozoites.

Three inbred mouse strains, C57BL/6 (H-2b), A/J (H-2a), and BALB/c (H-2d), and 1 outbred strain, CD-1, demonstrated differences in susceptibility to iv challenge with the ANKA clone of Plasmodium berghei. Mice were challenged with 100, 1,000, or 10,000 sporozoites, then evaluated daily beginning on day 4 for patency. CD-1 mice were further evaluated at challenge doses of 12,500, 25,000, and 50,000 sporozoites. C57BL/6 mice were the easiest to infect, with 90% becoming infected with 100 sporozoites. The outbred strain CD-1 was the most difficult to infect, requiring a challenge dose of 25,000 sporozoites/mouse in order to achieve a 100% infection rate. Mouse strains also demonstrated differences in their ability to be protected by intravenous immunization with gamma-irradiated sporozoites. A/J mice needed a minimum of 3 doses of irradiated sporozoites for protection against a challenge with 10,000 sporozoites. In contrast, BALB/c mice immunized with a single dose of 1,000 irradiated sporozoites are protected against a 10,000 sporozoite challenge. These data suggest that both infectivity and protection are genetically restricted and that susceptibility to infection may be inversely related to protection.

Characteristics of natural antibody responses to the circumsporozoite protein of Plasmodium vivax.

The antibody response to the prototype circumsporozoite (CS) protein of Plasmodium vivax (CSPV) was studied in Thai soldiers experiencing occupational malaria. Seventy-four (65%) of 114 men followed during assignment to a malaria transmission area developed blood-stage infection with P. vivax. IgG antibodies against the central repeat region of the CSPV protein were quantitated by ELISA using the recombinant protein, NS181V20, as the capture antigen. One quarter of the subjects had detectable anti-CSPV antibodies at the beginning of the study. CSPV antibody seroconversion was documented in 16 of 26 subjects assessed during their first observed episodes of vivax malaria. This antibody response was of moderate magnitude, fell off after the first week post-diagnosis and appeared, at the low levels observed, to be unassociated with protection. Continued assessment of anti-CSPV antibody after subjects left the transmission area found no increase associated with release of P. vivax. These findings indicate that CS antibody responses to P. vivax during occupational malaria share many characteristics with responses to P. falciparum.

Characterization of naturally acquired antibodies to the non-repetitive flanking regions of the circumsporozoite protein of Plasmodium falciparum.

Antibody responses to the circumsporozoite (CS) protein of Plasmodium falciparum have previously been reported against the central repeating tetrapeptides of this protein. Segments of the protein flanking the repeat region also contain B-cell epitopes, but specific antibody responses have not been previously characterized. Longitudinal serum sets from 16 Thai adults who developed acute falciparum malaria were selected to represent a spectrum of antibody response to the repeat region (R32). These sera were assayed by enzyme-linked immunosorbent assay using as capture antigen a recombinant fusion protein, NS1(81)RLF, which contains both flanking regions, but lacks the NANP and NVDP repeats of the P. falciparum CS protein. Antibody responses to the repeatless flanking (RLF) regions were observed in all subjects, including five individuals who lacked detectable anti-R32 antibody responses. Anti-RLF antibody responses induced by natural infection appear to be short-lived and of low-to-moderate magnitude. Thus, if anti-RLF antibodies prove to be protective, derived vaccine candidates may require presentation of these epitopes with adjuvants or delivery systems that enhance immunogenicity.