Functional Conservation in the SIAMESE-RELATED Family of Cyclin-Dependent Kinase Inhibitors in Land Plants.

The best-characterized members of the plant-specific SIAMESE-RELATED (SMR) family of cyclin-dependent kinase inhibitors regulate the transition from the mitotic cell cycle to endoreplication, also known as endoreduplication, an altered version of the cell cycle in which DNA is replicated without cell division. Some other family members are implicated in cell cycle responses to biotic and abiotic stresses. However, the functions of most SMRs remain unknown, and the specific cyclin-dependent kinase complexes inhibited by SMRs are unclear. Here, we demonstrate that a diverse group of SMRs, including an SMR from the bryophyte Physcomitrella patens, can complement an Arabidopsis thaliana siamese (sim) mutant and that both Arabidopsis SIM and P. patens SMR can inhibit CDK activity in vitro. Furthermore, we show that Arabidopsis SIM can bind to and inhibit both CDKA;1 and CDKB1;1. Finally, we show that SMR2 acts to restrict cell proliferation during leaf growth in Arabidopsis and that SIM, SMR1/LGO, and SMR2 play overlapping roles in controlling the transition from cell division to endoreplication during leaf development. These results indicate that differences in SMR function in plant growth and development are primarily due to differences in transcriptional and posttranscriptional regulation, rather than to differences in fundamental biochemical function.

Experimental manipulation reveals a trade-off between weapons and testes.

Theory predicts a trade-off between sexually selected weapons used to secure mates and post-copulatory traits used to maximize fertilization success. However, individuals that have a greater capacity to acquire resources from the environment may invest more in both pre- and post-copulatory traits, and trade-offs may not be readily apparent. Here, we manipulate the phenotype of developing individuals to examine allocation trade-offs between weapons and testes in Mictis profana (Hemiptera: Coreidae), a species where the hind legs are sexually selected weapons used in contests over access to females. We experimentally prevented males from developing weapons by inducing them to autotomize their hind legs before the final moult to adulthood. We compared trait expression in this group to males where autotomy was induced in the mid-legs, which are presumably not under sexual selection to the same extent. We found males without weapons invested proportionally more in testes mass than those with their mid-legs removed. Males that developed to adulthood without weapons did not differ from the mid-leg removal group in other traits potentially under precopulatory sexual selection, other post-copulatory traits or naturally selected traits. In addition, a sample of adult males from the same population in the wild revealed a positive correlation between investment in testes and weapons. Our study presents a critical contribution to a growing body of literature suggesting the allocation of resources to pre- and post-copulatory sexual traits is influenced by a resource allocation trade-off and that this trade-off may only be revealed with experimental manipulation.

Feasibility of a text-based smoking cessation intervention in rural older adults.

Text-based interventions are effective for smoking cessation, but have not been tested in rural older adults. The purpose of this study was to compare the feasibility, acceptability and preliminary efficacy of a text-based Scheduled Gradual Reduction (SGR) program to a non-SGR text messaging support condition among rural older adults. Adults over 60 years were randomized to either: (i) the SGR program (n = 20), a text-based program to reduce smoking over 4-weeks plus text-based support messages; or (ii) control (n = 20), receipt of text-based support messages only. Participants completed surveys at baseline and end of program to assess feasibility and acceptability of the intervention, and biochemically validated 7-day point prevalence cessation was assessed at end of treatment. Most participants (81%) reported reading all the messages they received. Participants found both interventions useful in quitting smoking (SGR = 57%, Control = 63%) and would recommend it to a friend (SGR = 72%, Control = 79%). Although not statically significant, the SGR group had a higher rate of biochemically validated cessation (SGR = 15%, Control = 5%, Cohen d = 0.67). Among those still smoking, the median percent reduction in cigarettes was 33.3% for both groups. Text-based cessation interventions are feasible, acceptable and can be easily disseminated to rural older adult tobacco users.

Building evaluation capacity in Ontario's public health units: promising practices and strategies.

OBJECTIVES: This article presents the findings of a project focusing on building evaluation capacity in 10 Ontario public health units. The study sought to identify effective strategies that lead to increased evaluation capacity in the participating organizations. STUDY DESIGN: This study used a qualitative, multiple case research design. METHODS: An action research methodology was used to design customized evaluation capacity building (ECB) strategies for each participating organization, based on its specific context and needs. This methodological approach also enabled monitoring and assessment of each strategy, based on a common set of reporting templates. A multiple case study was used to analyze the findings from the 10 participating organizations and derive higher level findings. RESULTS: The main findings of the study show that most of the strategies used to increase evaluation capacity in public health units are promising, especially those focusing on developing the knowledge, skills, and attitudes of health unit staff and managers. Facilitators to ECB strategies were the engagement of all staff members, the support of leadership, and the existence of organizational tools and infrastructure to support evaluation. It is also essential to recognize that ECB takes time and resources to be successful. CONCLUSIONS: The design and implementation of ECB strategies should be based on organizational needs. These can be assessed using a standardized instrument, as well as interviews and staff surveys. The implementation of a multicomponent approach (i.e. several strategies implemented simultaneously) is also linked to better ECB outcomes in organizations.

Experimental evolution reveals differences between phenotypic and evolutionary responses to population density.

Group living can select for increased immunity, given the heightened risk of parasite transmission. Yet, it also may select for increased male reproductive investment, given the elevated risk of female multiple mating. Trade-offs between immunity and reproduction are well documented. Phenotypically, population density mediates both reproductive investment and immune function in the Indian meal moth, Plodia interpunctella. However, the evolutionary response of populations to these traits is unknown. We created two replicated populations of P. interpunctella, reared and mated for 14 generations under high or low population densities. These population densities cause plastic responses in immunity and reproduction: at higher numbers, both sexes invest more in one index of immunity [phenoloxidase (PO) activity] and males invest more in sperm. Interestingly, our data revealed divergence in PO and reproduction in a different direction to previously reported phenotypic responses. Males evolving at low population densities transferred more sperm, and both males and females displayed higher PO than individuals at high population densities. These positively correlated responses to selection suggest no apparent evolutionary trade-off between immunity and reproduction. We speculate that the reduced PO activity and sperm investment when evolving under high population density may be due to the reduced population fitness predicted under increased sexual conflict and/or to trade-offs between pre- and post-copulatory traits.

Genetic variation but weak genetic covariation between pre- and post-copulatory episodes of sexual selection in Drosophila melanogaster.

When females mate polyandrously, male reproductive success depends both on the male's ability to attain matings and on his ability to outcompete rival males in the fertilization of ova post-copulation. Increased investment in  ejaculate components may trade off with investment in precopulatory traits due to resource allocation. Alternatively, pre- and post-copulatory traits could be positively related if individuals can afford to invest heavily in traits advantageous at both episodes of selection. There is empirical evidence for both positive and negative associations between pre- and post-copulatory episodes, but little is known about the genetic basis of these correlations. In this study, we measured morphological, chemical and behavioural precopulatory male traits and investigated their relationship with measures of male fitness (male mating success, remating inhibition and offensive sperm competitiveness) across 40 isofemale lines of Drosophila melanogaster. We found significant variation among isofemale lines, indicating a genetic basis for most of the traits investigated. However, we found weak evidence for genetic correlations between precopulatory traits and our indices of male fitness. Moreover, pre- and post-copulatory episodes of selection were uncorrelated, suggesting selection may act independently at the different episodes to maximize male reproductive success.

Additive genetic variance in polyandry enables its evolution, but polyandry is unlikely to evolve through sexy or good sperm processes.

Polyandry is widespread despite its costs. The sexually selected sperm hypotheses ('sexy' and 'good' sperm) posit that sperm competition plays a role in the evolution of polyandry. Two poorly studied assumptions of these hypotheses are the presence of additive genetic variance in polyandry and sperm competitiveness. Using a quantitative genetic breeding design in a natural population of Drosophila melanogaster, we first established the potential for polyandry to respond to selection. We then investigated whether polyandry can evolve through sexually selected sperm processes. We measured lifetime polyandry and offensive sperm competitiveness (P2 ) while controlling for sampling variance due to male × male × female interactions. We also measured additive genetic variance in egg-to-adult viability and controlled for its effect on P2 estimates. Female lifetime polyandry showed significant and substantial additive genetic variance and evolvability. In contrast, we found little genetic variance or evolvability in P2 or egg-to-adult viability. Additive genetic variance in polyandry highlights its potential to respond to selection. However, the low levels of genetic variance in sperm competitiveness suggest that the evolution of polyandry may not be driven by sexy sperm or good sperm processes.

No evidence for a trade-off between sperm length and male premating weaponry.

Male ornaments and armaments that mediate success in mate acquisition and ejaculate traits influencing competitive fertilization success are under intense sexual selection. However, relative investment in these pre- and post-copulatory traits depends on the relative importance of either selection episode and on the energetic costs and fitness gains of investing in these traits. Theoretical and empirical work has improved our understanding of how precopulatory sexual traits and investments in sperm production covary in this context. It has recently also been suggested that male weapon size may trade off with sperm length as another post-copulatory sexual trait, but the theoretical framework for this suggestion remains unclear. We evaluated the relationship between precopulatory armaments and sperm length, previously reported in ungulates, in five taxa as well as meta-analytically. Within and between taxa, we found no evidence for a negative or positive relationship between sperm length and male traits that are important in male-male contest competition. It is important to consider pre- and post-copulatory sexual selection together to understand fitness, and to study investments in different reproductive traits jointly rather than separately. A trade-off between pre- and post-copulatory sexual traits may not manifest itself in sperm length but rather in sperm number or function. Particularly in large-bodied taxa such as ungulates, sperm number is more variable interspecifically and likely to be under more intense selection than sperm length. We discuss our and the previous results in this context.

Intralocus tactical conflict: genetic correlations between fighters and sneakers of the dung beetle Onthophagus taurus.

Males and females differ in their phenotypic optima for many traits, and as the majority of genes are expressed in both sexes, some alleles can be beneficial to one sex but harmful to the other (intralocus sexual conflict; ISC). ISC theory has recently been extended to intrasexual dimorphisms, where certain alleles may have opposite effects on the fitness of males of different morphs that employ alternative reproductive tactics (intralocus tactical conflict; ITC). Here, we use a half-sib breeding design to investigate the genetic basis for ISC and ITC in the dung beetle Onthophagus taurus. We found positive heritabilities and intersexual genetic correlations for almost all traits investigated. Next, we calculated the intrasexual genetic correlation between males of different morphs for horn length, a sexually selected trait, and compared it to intrasexual correlations for naturally selected traits in both sexes. Intrasexual genetic correlations did not differ significantly between the sexes or between naturally and sexually selected traits, failing to support the hypothesis that horns present a reduction of intrasexual genetic correlations due to ITC. We discuss the implications for the idea of developmental reprogramming between male morphs and emphasize the importance of genetic correlations as constraints for the evolution of dimorphisms.

BRANCHLESS TRICHOMES links cell shape and cell cycle control in Arabidopsis trichomes.

Endoreplication, also called endoreduplication, is a modified cell cycle in which DNA is repeatedly replicated without subsequent cell division. Endoreplication is often associated with increased cell size and specialized cell shapes, but the mechanism coordinating DNA content with shape and size remains obscure. Here we identify the product of the BRANCHLESS TRICHOMES (BLT) gene, a protein of hitherto unknown function that has been conserved throughout angiosperm evolution, as a link in coordinating cell shape and nuclear DNA content in endoreplicated Arabidopsis trichomes. Loss-of-function mutations in BLT were found to enhance the multicellular trichome phenotype of mutants in the SIAMESE (SIM) gene, which encodes a repressor of endoreplication. Epistasis and overexpression experiments revealed that BLT encodes a key regulator of trichome branching. Additional experiments showed that BLT interacts both genetically and physically with STICHEL, another key regulator of trichome branching. Although blt mutants have normal trichome DNA content, overexpression of BLT results in an additional round of endoreplication, and blt mutants uncouple DNA content from morphogenesis in mutants with increased trichome branching, further emphasizing its role in linking cell shape and endoreplication.

Females suffer a reduction in the viability of stored sperm following an immune challenge.

Despite the ubiquitous nature of sperm storage in invertebrates, relatively little is known about its costs, or the impact that immune activation can have on a female's ability to maintain viable sperm stores. We explored the effects of an immune challenge on sperm storage under food-limited and ad libitum conditions in the field cricket, Teleogryllus oceanicus, by injecting mated adult females with either a LD5 dose of live bacteria or a nonpathogenic immune elicitor [lipopolysaccharide (LPS)] and then scoring the viability of their stored sperm. Females that were infected with bacteria showed a reduction in the viability of stored sperm 48 h after infection; interestingly, this pattern was not evident when females were injected with LPS. Reduction in sperm viability post-infection may reflect a reproductive trade-off between immune function and sperm store maintenance, as only females injected with bacteria showed an elevated antibacterial immune (lytic) response. Alternatively, bacteria may act directly on sperm quality. Dietary manipulations showed that lytic activity in females is condition dependent, irrespective of their immune challenge treatment. Diet affected the ability of females to maintain the viability of stored sperm, suggesting that sperm storage is condition dependent. That bacterial infection associated with a reduction in stored sperm quality has potentially important implications for the outcomes of sperm competition in T. oceanicus and in other species in which females store sperm between matings.

The effect of maternal and paternal immune challenge on offspring immunity and reproduction in a cricket.

Trans-generational immune priming is the transmission of enhanced immunity to offspring following a parental immune challenge. Although within-generation increased investment into immunity demonstrates clear costs on reproductive investment in a number of taxa, the potential for immune priming to impact on offspring reproductive investment has not been thoroughly investigated. We explored the reproductive costs of immune priming in a field cricket, Teleogryllus oceanicus. To assess the relative importance of maternal and paternal immune status, mothers and fathers were immune-challenged with live bacteria or a control solution and assigned to one of four treatments in which one parent, neither or both parents were immune-challenged. Families of offspring were reared to adulthood under a food-restricted diet, and approximately 10 offspring in each family were assayed for two measures of immunocompetence. We additionally quantified offspring reproductive investment using sperm viability for males and ovary mass for females. We demonstrate that parental immune challenge has significant consequences for the immunocompetence and, in turn, reproductive investment of their male offspring. A complex interaction between maternal and paternal immune status increased the antibacterial immune response of male offspring. This increased immune response was associated with a reduction in son's sperm viability, implicating a trans-generational resource trade-off between investment into immunocompetence and reproduction. Our data also show that these costs are sexually dimorphic, as daughters did not demonstrate a similar increase in immunity, despite showing a reduction in ovary mass.

Female effects, but no intrinsic male effects on paternity outcome in crickets.

Competitive fertilization success can depend on the relative abilities of competing males to fertilize available ova, and on mechanisms of cryptic female choice that moderate paternity. Competitive fertilization success is thus an emergent property of competing male genotypes, female genotype and their interactions. Accurate estimates of intrinsic male effects on competitive fertilization success are therefore problematic. We used a cross-classified nonbreeding design in which rival male family background was standardized to partition variation in competitive fertilization success among male and female family backgrounds in the field cricket Teleogryllus oceanicus. Male effects were close to zero, supporting previous quantitative genetic designs in which male competitors were assigned at random. In contrast, some 22% of the variance in competitive fertilization success was explained by female effects, suggesting that paternity in this species is influenced strongly by cryptic female choice.

Replicated evolutionary divergence in the cuticular hydrocarbon profile of male crickets associated with the loss of song in the Hawaiian archipelago.

Female choice based on male secondary sexual traits is well documented, although the extent to which this selection can drive an evolutionary divergence in male traits among populations is less clear. Male field crickets Teleogryllus oceanicus attract females using a calling song and once contacted switch to courtship song to persuade them to mate. These crickets also secrete onto their cuticle a cocktail of long-chained fatty acids or cuticular hydrocarbons (CHCs). Females choose among potential mates based on the structure of male acoustic signals and on the composition of male CHC profiles. Here, we utilize two naturally occurring mutations that have arisen independently on two Hawaiian islands and render the male silent to ask whether the evolutionary loss of acoustic signalling can drive an evolutionary divergence in the alternative signalling modality, male CHC profiles. QST -FST comparisons revealed strong patterns of CHC divergence among three populations of crickets on the islands of Hawaii, Oahu and Kauai. Contrasts between wild-type and flatwing males on the islands of Oahu and Kauai indicated that variation in male CHC profiles within populations is associated with the loss of acoustic signalling; flatwing males had a relatively low abundance of long-chained CHCs relative to the short-chained CHCs that females find attractive. Given their dual functions in desiccation resistance and sexual signalling, insect CHCs may be particularly important traits for reproductive isolation and ultimately speciation.

Estimating relatedness and inbreeding using molecular markers and pedigrees: the effect of demographic history.

Estimates of inbreeding and relatedness are commonly calculated using molecular markers, although the accuracy of such estimates has been questioned. As a further complication, in many situations, such estimates are required in populations with reduced genetic diversity, which is likely to affect their accuracy. We investigated the correlation between microsatellite- and pedigree-based coefficients of inbreeding and relatedness in laboratory populations of Drosophila melanogaster that had passed through bottlenecks to manipulate their genetic diversity. We also used simulations to predict expected correlations between marker- and pedigree-based estimates and to investigate the influence of linkage between loci and null alleles. Our empirical data showed lower correlations between marker- and pedigree-based estimates in our control (nonbottleneck) population than were predicted by our simulations or those found in similar studies. Correlations were weaker in bottleneck populations, confirming that extreme reductions in diversity can compromise the ability of molecular estimates to detect recent inbreeding events. However, this result was highly dependent on the strength of the bottleneck and we did not observe or predict any reduction in correlations in our population that went through a relatively severe bottleneck of N = 10 for one generation. Our results are therefore encouraging, as molecular estimates appeared robust to quite severe reductions in genetic diversity. It should also be remembered that pedigree-based estimates may not capture realized identity-by-decent and that marker-based estimates may actually be more useful in certain situations.

Sperm and seminal fluid proteomes of the field cricket Teleogryllus oceanicus: identification of novel proteins transferred to females at mating.

Reproductive proteins are amongst the most evolutionarily divergent proteins known, and research on genetically well-characterized species suggests that postcopulatory sexual selection might be important in their evolution; however, we lack the taxonomic breadth of information on reproductive proteins that is required to determine the general importance of sexual selection for their evolution. We used transcriptome sequencing and proteomics to characterize the sperm and seminal fluid proteins of a cricket, Teleogryllus oceanicus, that has been widely used in the study of postcopulatory sexual selection. We identified 57 proteins from the sperm of these crickets. Many of these had predicted function in glycolysis and metabolism, or were structural, and had sequence similarity to sperm proteins found across taxa ranging from flies to humans. We identified 21 seminal fluid proteins, some of which resemble those found to be involved in postmating changes to female reproduction in other species. Some 27% of sperm proteins and 48% of seminal fluid proteins were of unknown function. The characterization of seminal fluid proteins in this species will allow us to explore their adaptive significance, and to contribute comparative data that will facilitate a general appreciation of the evolution of reproductive proteins within and among animal taxa.

Female crickets assess relatedness during mate guarding and bias storage of sperm towards unrelated males.

Recent evidence shows that females exert a post-copulatory fertilization bias in favour of unrelated males to avoid the genetic incompatibilities derived from inbreeding. One of the mechanisms suggested for fertilization biases in insects is female control over transport of sperm to the sperm-storage organs. We investigated post-copulatory inbreeding-avoidance mechanisms in females of the cricket Teleogryllus oceanicus. We assessed the relative contribution of related and unrelated males to the sperm stores of double-mated females. To demonstrate unequivocally that biased sperm storage results from female control rather than cryptic male choice, we manipulated the relatedness of mated males and of males performing post-copulatory mate guarding. Our results show that when guarded by a related male, females store less sperm from their actual mate, irrespective of the relatedness of the mating male. Our data support the notion that inhibition of sperm storage by female crickets can act as a form of cryptic female choice to avoid the severe negative effects of inbreeding.

Genetic variation underlying the expression of a polyphenism.

Polyphenic traits are widespread and represent a conditional strategy sensitive to environmental cues. The environmentally cued threshold (ET) model considers the switchpoint between alternative phenotypes as a polygenic quantitative trait with normally distributed variation. However, the genetic variation for switchpoints has rarely been explored empirically. Here, we used inbred lines to investigate the genetic variation for the switchpoint in the mite Rhizoglyphus echinopus, in which males are either fighters or scramblers. The conditionality of male dimorphism varied among inbred lines, indicating that there was genetic variation for switchpoints in the base population, as predicted by the ET model. Our results also suggest a mixture between canalized and conditional strategists in R. echinopus. We propose that major genes that canalize morph expression and affect the extent to which a trait can be conditionally expressed could be a feature of the genetic architecture of threshold traits in other taxa.

Assortative mating for relatedness in a large naturally occurring population of Drosophila melanogaster.

New theoretical work on kin selection and inclusive fitness benefits predicts that individuals will sometimes choose close or intermediate relatives as mates to maximize their fitness. However, empirical examples supporting such predictions are rare. In this study, we look for such evidence in a natural population of Drosophila melanogaster. We compared mating and nonmating individuals to test whether mating was nonrandom with respect to relatedness. Consistent with optimal inbreeding, males were more closely related to their mate than to randomly sampled females. However, all individuals collected mating showed higher relatedness and males were not significantly more related to their mate than to other mating females. We also found a negative relationship between relatedness and fecundity. Our results are consistent with the hypothesis that inclusive fitness benefits may drive inbreeding tolerance despite direct costs to fitness; however, an experimental approach is needed to investigate the link between mate preference and relatedness.

The genetics of primary and secondary sexual character trade-offs in a horned beetle.

When structures compete for shared resources, this may lead to acquisition and allocation trade-offs so that the enlargement of one structure occurs at the expense of another. Among the studies of morphological trade-offs, their importance has been demonstrated primarily through experimental manipulations and comparative analyses. Relatively, a few studies have investigated the underlying genetic basis of phenotypic patterns. Here, we use a half-sibling breeding design to determine the genetic underpinnings of the phenotypic trade-off between head horns and the male copulatory organ or aedeagus that has been found in the dung beetle Onthophagus taurus. Instead of the predicted negative genetic covariance among characters that trade-off, we find positive genetic covariance between absolute horn and aedeagus length and zero genetic covariance between relative horn and aedeagus length. Therefore, although the genetic covariance between absolute horn and aedeagus length would constrain the independent evolution of primary and secondary sexual characters in this population, there was no evidence of a trade-off. We discuss alternative hypotheses for the observed patterns of genetic correlation between traits that compete for resources and the implications that these have for selection and the evolution of such traits.