New genomic resources for the honey bee(Apis mellifera L.): development of a deep-coverage BAC library and a preliminary STC database

We have constructed a bacterial artificial chromosome (BAC) library for a European honey bee strain using the cloning enzyme HindIII in order to develop resources for structural genomics research. The library contains 36,864 clones (ninety-six 384-well plates). A random sampling of 247 clones indicated an average insert size of 113 kb (range = 27 to 213 kb) and 2% empty vectors. Based on an estimated genome size of 270 Mb, this library provides approximately 15 haploid genome equivalents, allowing >99% probability of recovering any specific sequence of interest. High-density colony filters were gridded robotically using a Genetix Q-BOT in a 4 x 4 double-spotted array on 22.5-cm2 filters. Screening of the library with four mapped honey bee genomic clones and two bee cDNA probes identified an average of 21 positive signals per probe, with a range of 7-38 positive signals per probe. An additional screening was performed with nine aphid gene fragments and one Drosophila gene fragment resulting in seven of the nine aphid probes and the Drosophila probe producing positive signals with a range of 1 to 122 positive signals per probe (average of 45). To evaluate the utility of the library for sequence tagged connector analysis, 1152 BAC clones were end sequenced in both forward and reverse directions, giving a total of 2061 successful reads of high quality. End sequences were queried against SWISS-PROT, insect genomic sequence GSS, insect EST, and insect transposable element databases. Results in spreadsheet format from these searches are publicly available at the Clemson University Genomics Institute (CUGI) website in a searchable format (http://www.genome.clemson.edu/projects/stc/bee/AM__Ba/).     

Influence of hydrophilicity of cationic polymers on the biophysical properties of polyelectrolyte complexes formed by self-assembly with DNA

To investigate the possibility of producing charge-neutral gene delivery complexes with extended, non-particulate structures, DNA was allowed to self-assemble with a series of hydrophilic cationic polymers containing quaternary charged trimethylammonio ethylmethacrylate (TMAEM, 5, 15, 50, 100 mol%) copolymerised with hydrophilic N-(2-hydroxypropyl)methacrylamide (HPMA, 95, 85, 50, 0 mol%, respectively). Copolymers were all able to bind DNA, assessed using ethidium bromide fluorescence, although copolymers with low TMAEM content did not expel ethidium bromide. Increasing TMAEM content of the copolymers changed the morphology of the complexes from extended (5-15 mol% TMAEM), through partially condensed particles (50 mol%) to discrete nanoparticles (100 mol% TMAEM). Complexes based on copolymers with low TMAEM content (5-50 mol%) showed less resistance to degradation by nucleases and lower surface charge (21.2+/-5.9-45.1+/-3.9 mV) than those formed using 100 mol% TMAEM (57.8+/-8.2 mV). They also showed significantly less association with phagocytic cells in vitro (human leucocytes, uptake decreased by up to 92.3%; murine peritoneal macrophages, uptake decreased by up to 69.6%), although in vivo their hepatic accumulation was only slightly decreased (maximum decrease 27.6%). Finding the appropriate balance of hydrophilicity and stability is key to development of effective vectors for gene delivery.     

Investigating the genetic architecture of conditional strategies using the environmental threshold model

The threshold expression of dichotomous phenotypes that are environmentally cued or induced comprise the vast majority of phenotypic dimorphisms in colour, morphology, behaviour and life history. Modelled as conditional strategies under the framework of evolutionary game theory, the quantitative genetic basis of these traits is a challenge to estimate. The challenge exists firstly because the phenotypic expression of the trait is dichotomous and secondly because the apparent environmental cue is separate from the biological signal pathway that induces the switch between phenotypes. It is the cryptic variation underlying the translation of cue to phenotype that we address here. With a ‘half-sib common environment’ and a ‘family-level split environment’ experiment, we examine the environmental and genetic influences that underlie male dimorphism in the earwig Forficula auricularia. From the conceptual framework of the latent environmental threshold (LET) model, we use pedigree information to dissect the genetic architecture of the threshold expression of forceps length. We investigate for the first time the strength of the correlation between observable and cryptic ‘proximate’ cues. Furthermore, in support of the environmental threshold model, we found no evidence for a genetic correlation between cue and the threshold between phenotypes. Our results show strong correlations between observable and proximate cues and less genetic variation for thresholds than previous studies have suggested. We discuss the importance of generating better estimates of the genetic variation for thresholds when investigating the genetic architecture and heritability of threshold traits. By investigating genetic architecture by means of the LET model, our study supports several key evolutionary ideas related to conditional strategies and improves our understanding of environmentally cued decisions.     

The status of the conditional evolutionarily stable strategy

The conditional evolutionarily stable strategy (ESS) has proven to be a versatile tool for understanding the production of alternative phenotypes in response to environmental cues. Hence, we would expect the theoretical basis of the conditional strategy to be robust. However, Shuster and Wade have recently criticized the conditional ESS based on Gross's 1996 proposal that most alternative reproductive tactics are conditional and have evolved by 'status-dependent selection.' We critically assess Gross's status-dependent selection model and Shuster and Wade's critique. We find shortcomings and misconceptions in both. We return to the findings of the strategic models behind the conditional ESS and demonstrate how environmental threshold models use a reaction norm approach and quantitative genetic theory to understand the evolution of conditional strategies.     

Optimization and clinical validation of a pathogen detection microarray

DNA microarrays used as 'genomic sensors' have great potential in clinical diagnostics. Biases inherent in random PCR-amplification, cross-hybridization effects, and inadequate microarray analysis, however, limit detection sensitivity and specificity. Here, we have studied the relationships between viral amplification efficiency, hybridization signal, and target-probe annealing specificity using a customized microarray platform. Novel features of this platform include the development of a robust algorithm that accurately predicts PCR bias during DNA amplification and can be used to improve PCR primer design, as well as a powerful statistical concept for inferring pathogen identity from probe recognition signatures. Compared to real-time PCR, the microarray platform identified pathogens with 94% accuracy (76% sensitivity and 100% specificity) in a panel of 36 patient specimens. Our findings show that microarrays can be used for the robust and accurate diagnosis of pathogens, and further substantiate the use of microarray technology in clinical diagnostics.     

Matters of scale: positive allometry and the evolution of male dimorphisms

The developmental independence of alternative phenotypes is key to evolutionary theories of phenotypic plasticity and the origins of diversity. Male dimorphisms associated with alternative reproductive tactics are widely cited examples of such facultative expression of divergent fitness optima. Current models for the evolution of male dimorphisms invoke a size-dependent threshold at which the phenotype is reprogrammed. We use predictions derived from allometric modeling to test for the existence of reprogramming thresholds in two species of beetle, Onthophagus taurus and Onthophagus binodis, and the European earwig Forficula auricularia. We also compare the allometry of a number of morphological traits to determine whether minor males suppress their secondary sexual traits. The intercept of the horn allometry was suppressed, but there was no evidence of reprogramming of horn growth in either beetle species. There was reprogramming in the earwig. In the beetles, the horn length in all males can be explained largely in terms of exponential horn growth following an extraordinarily steep power function. The asymptote in O. taurus can be explained by exponential growth meeting the constraint of resource exhaustion. These findings question the currently held view that beetle horn dimorphisms showcase the importance of developmental independence in the evolution of diversity.     

Genic capture and resolving the lek paradox

The genic capture hypothesis offers a resolution to the question of how genetic variation in male sexually selected traits is maintained in the face of strong female preferences. The hypothesis is that male display traits are costly to produce and hence depend upon overall condition, which itself is dependent upon genes at many loci. Few attempts have been made to test the assumptions and predictions of the genic capture hypothesis rigorously and, in particular, little attention has been paid to determining the genetic basis of condition. Such tests are crucial to our understanding of the maintenance of genetic variation and in the evaluation of recent models that propose a role for sexual selection in the maintenance of sex. Here, we review approaches to testing the link between genetically determined condition and levels of sexual trait expression and consider the probable importance of deleterious mutations.     

Phenotypic plasticity in the developmental integration of morphological trade-offs and secondary sexual trait compensation

Trait exaggeration through sexual selection will tale place alongside other changes in phenotype. Exaggerated morphology might be compensated by parallel changes in traits that support, enhance or facilitate exaggeration: 'secondary sexual trait compensation' (SSTC). Alternatively, exaggeration might be realized at the expense of other traits through morphological trade-offs. For the most part, SSTC has only been examined interspecifically. For these phenomena to be important intraspecifically, the sexual trait must be developmentally integrated with the compensatory or competing trait. We studied developmental integration in two species with different development: the holometabolous beetle Onthophagus taurus and the hemimetabolous earwig Forficula auricularia. Male-dimorphic variation in trait exaggeration was exploited to expose both trade-offs and SSTC. We found evidence for morphological trade-offs in O. taurus, but no F. auricularia, supporting the notion that trade-offs are more likely in closed developmetal systems. However, we found these trade-offs were not limited solely to traits growing close together. Developmental integration of structures involved in SSTC were detected in both species. The developmental integration of SSTC was phenotypically plastic, such that the compensation for relatively larger sexual traits was greater in the exasperated male morphs. Evidence of intraspecific SSTC demands studies of the selective, genetic and developmental architecture of phenotypic integration.