The role played by electron microscopy in advancing our understanding of Toxoplasma gondii and other apicomplexans

In many ways the history of the discovery of the life cycle of Toxoplasma gondii and the development of biological electron microscopy progressed in parallel through the 1950s and 1960s. Although Toxoplasma was discovered in 1908, it was only in the 1950s that the extent of the infection in humans and domestic animals was realised and work was undertaken to elucidate its life cycle (reviewed elsewhere in this edition). The development of ultrastructural techniques and their application to biological systems including Toxoplasma developed over the same period. This resulted in a synergistic effect with the re-classification of previously unrelated parasites within a single phylum, the Apicomplexa, which was based on the ultrastructural appearances of the infectious stages. This review will describe the central role played by electron microscopy and Toxoplasma in the developments associated with this progress.     

Spatial and behavioural measures of social discrimination by captive male zebra finches: Implications of sexual and species differences for recognition research

All bird species reproduce sexually and individuals need to correctly identify conspecifics for successful breeding. Captive zebra finches are a model system for studying the factors involved in species recognition and mate choice. However, male zebra finches’ behavioural responses in a spatial preference paradigm to a range of estrildid finch species, other than domesticated Bengalese finches, remain unknown. We investigated spatial and display responses of male zebra finch subjects to stimulus females between conspecific and four phylogeographically relevant finch species, in addition to female Bengalese finches. Surprisingly, male subjects did not show consistent spatial association with conspecific over heterospecific females. Overall, as predicted by sexual selection theory, the spatial proximity responses of males were less discriminatory compared to female zebra finches’ responses tested previously using the same paradigm. However, male subjects showed consistently more behavioural displays towards female conspecifics than heterospecifics which were positively related to the behavioural display rates of the respective female stimuli. Some male behavioural responses, other than song, also showed significant differences between the different stimulus species and consistently differed across individual test subjects, with the most individual subject variation seen in choice trials between female conspecific and Bengalese finch stimuli. The results are important for the design and interpretation of future behavioural and neurobiological experiments on species recognition systems using the zebra finch as a model species.     

Proximate and ultimate factors explaining floating behavior in long-lived birds

Avian populations are composed of reproductive individuals coexisting in space and time with young, non-breeding conspecifics or “floaters”. Despite the fact that first breeding can be delayed for years and can exert profound effects on fitness, many aspects of the behavior, ecology and physiology of young floaters remain poorly understood. By means of combining endocrine, behavioral and life history information from a population of black kites (Milvus migrans) monitored long-term, we tested several hypotheses suggesting endocrine function as a determinant of floating status. Sexual function in non-breeding males, estimated through determination of systemic testosterone and progesterone levels, was similar to that in reproductively active conspecifics. Floating females, on the contrary, displayed an endocrine pattern of circulating estrogens and progesterone that was parallel in timing but reduced in magnitude as compared to breeders. Our results suggest that floaters are not physiologically constrained to reproduce, but the cost-benefit balance of attaining complete gonadal function is sexually dependent. While young, unmated males could increase their breeding prospects by attaining sexual maturity regardless of their social environment, natural selection would favor females relying on social cues to mature. Consistent with the sexual roles of socially monogamous species, gonadal recrudescence and testosterone production would allow unmated males to access breeding resources (e.g. through male-male competition and extra-pair fertilizations). Unmated females, on the contrary, would reduce physiological costs by means of delaying ovarian maturation until establishing pair bonds with a male providing access to breeding resources.     

Rapid glucocorticoid stimulation and GABAergic inhibition of hippocampal serotonergic response: in vivo dialysis in the lizard anolis carolinensis

Central serotonin (5-HT) is activated during stressful situations and aggressive interactions in a number of species. Glucocorticoids secreted peripherally during stressful events feed back on central systems and may affect 5-HT mediation of stress-induced behavioral events. To test the neuromodulatory effect of stress hormone secretion, serotonin overflow was measured from the hippocampus of the lizard Anolis carolinensis. Microdialysis was used to collect repeated samples from anesthetized lizards, with perfusate measured by HPLC with electrochemical analysis. Following initially high levels of 5-HT, concentrations stabilized to basal levels after approximately 2 h. Intracortical infusion of 200 ng/ml corticosterone evoked transient increases in 5-HT release of approximately 400%. The effect of corticosterone on 5-HT overflow appears to be dose dependent as 20 ng/ml stimulated an increase of 200%, whereas 2 ng/ml stimulated a 50% increase. Administration of 0.1 and 1 ng/ml GABA via the dialysis probe significantly inhibited 5-HT overflow by 20 and 40%, respectively. The duration of GABA inhibition is greater than the stimulatory response for glucocorticoids. Short-lived glucocorticoid stimulation of 5-HT release suggests a possible mechanism for endocrine mediation of continuously changing social behavioral events.     

Catecholaminergic cells and fibers in the brain of the lizard Anolis carolinensis identified by traditional as well as whole-mount immunohistochemistry

Summary Using traditional as well as whole-mount immunohistochemistry, we described the location of tyrosine hydroxylase-and dopamine beta hydroxylase-positive cells and fibers in the brain of the lizard Anolis carolinensis. Major catecholaminergic cell groups were in the ependyma in certain ventricular regions, alous coeruleus, anterior hypothalamic and lateral hypothalamic areas, and in the mesencephalic tegmental region, locus coeruleus, nucleus of the solitary tract, vagal motor nucleus, and rhombencephalic reticular formation. Major catecholaminergic fibers, tracts and varicosities included tuberohypophysial, mesolimbic, nigrostriatal, isthmocortical, medullohypothalamic, and coeruleospinal systems. Although the catecholaminergic systems in A. carolinensis are similar to those in the brains of other lizards studied, there are a few species differences. Our information about A. carolinensis will be used to help localize the hypothalamic asymmetry in catecholamine metabolism previously described in this lizard.     

The importance of vegetative and sexual dispersal of Luronium natans

Luronium natans (L.) Rafin. is a very rare macrophyte even though it has the ability to grow in a wide variety of habitat types. Previous studies leave two possibilities for this pattern: the species has a poor ability to disperse and establish or it is unable to grow and develop a sustainable population after colonization.     

Sex,season, and time of day interact to affect body temperatures of the Giant Gartersnake

1.

We examined multiple hypotheses regarding differences in body temperatures of the Giant Gartersnake using temperature-sensitive radio telemetry and an information-theoretic analytical approach.     

Seasonal dynamics of entomopathogenic nematodes of the genera Steinernema and Heterorhabditis as a response to abiotic factors and abundance of insect hosts

The seasonal dynamics of entomopathogenic nematodes (EPNs) of the genus Steinernema and Heterorhabditis were studied during one season in meadow and oak wood habitats, in the vicinity of Ceské Budejovice, Czech Republic. The influences of soil temperature, moisture, and abundance of suitable hosts on EPN dynamics were investigated. The host range of these nematodes, in both habitats was also observed. A total of four EPN species were found in both habitats. Steinernema affine was the dominant species both in oak wood and in meadow. Additionally, the oak wood habitat was inhabited by S. kraussei and S. weiseri; the meadow habitat by Heterorhabditis bacteriophora. The mean abundance of total EPN community was 28,000ind./m(2) in oak wood and 11,000ind./m(2) in meadow. The seasonal dynamics of entomopathogenic nematodes in both habitats were characterized by high nematode densities in the beginning of the season, followed by a rapid decrease, and then stabilization. EPN abundances did not show any apparent correlation with soil temperature and moisture, but they were negatively correlated with the abundance of suitable insect hosts. Inter- and intraspecific competition for limited nutrients (hosts) probably played a major role in EPN seasonal dynamics. Broad host range of entomopathogenic nematodes in both habitats was predominantly represented by dipteran and coleopteran larvae. Most common hosts belonged to the families Asilidae, Bibionidae, and Empididae (Diptera), as well as Carabidae and Curculionidae (Coleoptera).     

Diversity of Bacteria and Archaea from a landfill in Chandigarh,India as revealed by culture-dependent and culture-independent molecular approaches

The bacterial community structure of a municipal landfill in Chandigarh, India was analysed by culture-dependent as well as culture-independent molecular approaches, and archaeal structure by the latter method. Samples were collected in two phases from the surface and a depth of 0.91 m in June, 2004 and from 0.91 m, 1.52 m and 1.68 m in May, 2005. After serial dilutions, samples were plated onto tryptic soy agar (TSA), plate count agar (PCA), tryptic soy broth agar (TSBA) and TSBA100 (TSBA diluted 100 times and solidified with agarose), and incubated aerobically at 30 °C. The number of bacteria (CFU) on different media ranged between 9.4 × 10⁵ g⁻¹ (on PCA) and 1.9 × 10⁷ g⁻¹ (on TSA) (wet weight). The numbers of bacteria enumerated from plates incubated anaerobically (anaerobic agar and reinforced clostridial agar) were 2.1 × 10⁷ and 1.7 × 10⁶ g⁻¹, respectively. Of the 468 isolated and purified bacteria (183 in the first phase and 285 in the second phase), 135 were characterised using phenotypic characteristics as well as 16S rRNA gene sequence analysis. It was found that members of the phylum Firmicutes were overwhelmingly predominant (86.6%) in the landfill, followed by Actinobacteria (9.6%) and Proteobacteria (3.7%). Among the Firmicutes, at least 17 species from the single genus Bacillus were the most abundant inhabitants of the landfill. Detailed polyphasic characterisation of many of these isolates led to the discovery of a novel genus Paenisporosarcina (and the species P. quisquiliarum), a novel species of Microbacterium, M. immunditiarum, and reclassification of Sporosarcina macmurdoensis, Pelagibacillus goriensis, Bacillus silvestris, Bacillus insolitus, Bacillus psychrotolerans and Bacillus psychrodurans. Culture-independent analysis of two 16S rRNA gene libraries also revealed that the phylum Firmicutes was the predominant group in this community. The diversity of Archaea was found to be limited mainly to members of two orders: Methanosarcinales and Methanomicrobiales of the phylum Euryarchaeota. When these results were compared to those reported earlier on similar studies, it was found that irrespective of differences in composition of municipal solid waste (especially compostable organic matter and paper) and climate, the members of bacterial and archaeal communities in landfills of many countries remained broadly similar.     

Lectin binding patterns of Scrippsiella lachrymosa (Dinophyceae) in relation to cyst formation and nutrient conditions

In many dinoflagellates, it has been a challenging task to study the qualitative and quantitative processes leading to encystment because gametes are often not morphologically distinguishable from other vegetative cells. We examined whether sexual differentiation is accompanied by changes in cell surface glycoprotein properties that are reflected in the binding patterns of complementary lectins. Labeling percentages of nine different fluorescein isothiocyanate (FITC)-conjugated lectins were analyzed together with cell and cyst abundance in N-deplete and f/2 control cultures of Scrippsiella lachrymosa Lewis throughout an encystment experiment. Although labeling varied between lectins and treatments and considerable changes occurred through time, no direct correlation was observed between glycoconjugate properties and sexual life cycle processes. A conspicuous decrease in labeling of lectins that are complementary to amino sugars (in particular, with WGA, a lectin that is complementary to N-acetylglucosamine) was observed in the low nitrogen treatment, suggesting a link between the nutrient status of a cell and expression of surface carbohydrates. Presumably, the reduction of N-acetylglucosamine residues was an early indication of N stress in cell populations. Labeling experiments with phosphate-limited cells showed that the decrease in WGA-complementary amino-sugar residues was not specific for N stress, but appeared to be a general response to nutrient limitation. Our findings confirm that glycoconjugate composition of dinoflagellate cells can change depending on their physiological state, which has to be considered when applying lectins for taxonomic differentiation of dinoflagellate species.     

Gammaproteobacteria occurrence and microdiversity in Tyrrhenian Sea sediments as revealed by cultivation-dependent and -independent approaches

Bacterial diversity in Tyrrhenian Sea sediments was assessed using cultivation-dependent and -independent approaches. Samples collected from the different sediment layers (up to 30 cm) relative to four seamount and non-seamount stations, at depths from 3425 to 3580 m, were subjected to DNA extraction and 16S rRNA amplification targeting the V3 region. Denaturing gradient gel electrophoresis (DGGE) showed several heterogeneous profiles and 27 single bands were excised and sequenced. Sequence analysis revealed the presence of Firmicutes, Actinobacteria and Chloroflexi in 26% of the DGGE bands and a predominance of sequences affiliated to cultivable and uncultivable clones of Gammaproteobacteria (55%). To corroborate these findings, cultivation attempts were performed that allowed the isolation of 87 strains assigned to the proteobacterial classes. Identification was achieved by means of automated ribosomal intergenic spacer analysis (ARISA) and by 16S rDNA sequencing. The isolates were related to the gamma, alpha and beta subclasses of Proteobacteria with respective percentages of 77, 17 and 6%. The most predominant Gammaproteobacteria isolates, assigned to the Psychrobacter marincola and P. submarinus clade (n = 53) and to Halomonas aquamarina (n = 14), showed a huge intraspecific diversity with 29 distinct ARISA haplotypes. The detection by both approaches of these psychrophilic and moderately halophilic species and their extensive microdiversity indicated their predominance in Tyrrhenian Sea sediments where they constituted the indigenous microflora.     

Babesia bigemina sexual stages are induced in vitro and are specifically recognized by antibodies in the midgut of infected Boophilus microplus ticks

Babesia bigemina, a causative agent of bovine babesiosis, is transmitted from one bovine to another only by infected ticks. The life cycle of B. bigemina includes a sexual phase in the tick host; however, molecules from sexual stages of any Babesia species have not been characterized. This is the first report of the induction of sexual stages of any Babesia species in vitro, free of tick antigens. Intraerythrocytic parasites were cultured in vitro for 20h using an induction medium. Extraerythrocytic parasites were first seen 3h post induction; elongated stages with long projections appeared at 6h post induction and by 9h they paired and fused to form larger stages. Round zygotes appeared 20h post induction. Moreover, by using Percoll gradients, sexual stages were purified free of contaminating intraerythrocytic stages. Purified parasites were used to generate polyclonal antibodies, which specifically bound to antigens expressed in sexual stages induced in vitro, but not to antigens expressed in intraerythrocytic stages. Importantly, these antibodies specifically identified sexual stages from midguts of female Boophilus microplus ticks fed on infected cattle.     

The evolutionary origin of signa in female Lepidoptera: natural and sexual selection hypotheses

Signa are structures of the inner wall of the female corpus bursae (structure where males deposit a spermatophore during copulation) of many Lepidoptera that assist in tearing open spermatophores. In this paper, three hypotheses on the evolutionary origin of signa are proposed. The first hypothesis considers natural selection pressures arising from ecological changes that favor an increase in oviposition rate as the force behind the evolution of signa. The other two hypotheses involve sexual selection. The second hypothesis proposes that sexually antagonistic coevolution is responsible of the evolution of signa: According to this hypothesis, the inverse relation between the length of the female's refractory period and the amount of ejaculate remaining in her corpus bursae, observed in most Lepidoptera studied, selects in males a decreased rate of spermatophore digestion (e.g. a thicker spermatophore envelope or a higher chitin content) that increases the length of the refractory period beyond the female's optimum; in response, females evolved signa as a counteradaptation to restore the female's optimum by increasing the rate of spermatophore digestion. The last hypothesis considers that signa may have evolved as a female device for cryptic choice of males based on the ability of these to influence the length of post-copulatory female refractory period. The different hypotheses make different predictions of the sequence of appearance of specific ecological factors and novel phenotypic traits through evolutionary time. Therefore, testing the relative importance of the hypotheses requires a formal comparative analysis.     

Intraspecific shape variation in horseshoe crabs: The importance of sexual and natural selection for local adaptation

A morphometric analysis of the body shape of three species of horseshoe crabs was undertaken in order to infer the importance of natural and sexual selection. It was expected that natural selection would be most intense, leading to highest regional differentiation, in the American species Limulus polyphemus, which has the largest climatic differences between different populations. Local adaptation driven by sexual selection was expected in males but not females because horseshoe crab mating behaviour leads to competition between males, but not between females. Three hundred fifty-nine horseshoe crabs from nine populations, representing three species, were analyzed using a digitizer to position sixty morphometric landmarks in a three-dimensional space. Discriminant analysis revealed strong regional structuring within a species, which suggests strong philopatry, and showed the existence of geographically-based intraspecific variation. An admixture analysis showed regional intraspecific differentiation for males and females of L. polyphemus and males of the Asian horseshoe crab Carcinoscorpius rotundicauda, but not for females of C. rotundicauda and another Asian horseshoe crab, Tachypleus gigas. Differences in shape variation between sexes were tested with F-tests, which showed lower intrapopulation morphometric variation in males than females. These results indicate a lower degree of local adaptation on body shape in C. rotundicauda and T. gigas than in L. polyphemus and a lower degree of local adaptation in females than in males.     

Juvenile hormone titre and egg production in Tenebrio molitor infected by Hymenolepis diminuta: effect of male and/or female infection,male age and mating

Infection of Tenebrio molitor with Hymenolepis diminuta induces curtailment of female fertility. We examined ovulation and oviposition, and associated titres of juvenile hormone (JH), in relation to parasitism and mating. Oviposition was significantly increased in infected mated and virgin beetles by days 6 and 9 post-emergence. Ovulation was not changed by infection; by the end of the 18-day experiment, the total number of laid eggs was not significantly altered. On day 6, JH levels were significantly higher in virgin infected insects, compared to non-infected controls (236+/-37.7 and 107+/-9.62 pg/g wet weight). Oviposition increased after mating, but total eggs ovulated remained the same. JH levels were higher in mated females on days 12 and 18 post-emergence, for infected and control insects. Previous studies suggested that male reproductive potential might rise following infection, because uninfected females lay more eggs when mated to infected males. We tested whether this caused an increase in female JH. Males were mated on days 5 or 12, when significant changes in their reproductive physiology begin to be observed, and are maximal, respectively. However, male age was of greater significance in promoting JH levels in females (p=0.001), than infection status of either partner (p=0.33).     

Individual variation in testosterone and parental care in a female songbird; The dark-eyed junco (Junco hyemalis)

When competition for sex-specific resources overlaps in time with offspring production and care, trade-offs can occur. Steroid hormones, particularly testosterone (T), play a crucial role in mediating such trade-offs in males, often increasing competitive behaviors while decreasing paternal behavior. Recent research has shown that females also face such trade-offs; however, we know little about the role of T in mediating female phenotypes in general, and the role of T in mediating trade-offs in females in particular. Here we examine the relationship between individual variation in maternal effort and endogenous T in the dark-eyed junco, a common songbird. Specifically, we measure circulating T before and after a physiological challenge (injection of gonadotropin releasing hormone, GnRH), and determine whether either measure is related to provisioning, brooding, or the amount of T sequestered in egg yolk. We found that females producing more T in response to a challenge spent less time brooding nestlings, but provisioned nestlings more frequently, and deposited more T in their eggs. These findings suggest that, while T is likely important in mediating maternal phenotypes and female life history tradeoffs, the direction of the relationships between T and phenotype may differ from what is generally observed in males, and that high levels of endogenous T are not necessarily as costly as previous work might suggest.