Examining relations between performance on non-verbal executive function and verbal self-regulation tasks in demographically-diverse populations.

Self-regulation is a widely studied construct, generally assumed to be cognitively supported by executive functions (EFs). There is a lack of clarity and consensus over the roles of specific components of EFs in self-regulation. The current study examines the relations between performance on (a) a self-regulation task (Heads, Toes, Knees Shoulders Task) and (b) two EF tasks (Knox Cube and Beads Tasks) that measure different components of updating: working memory and short-term memory, respectively. We compared 107 8- to 13-year-old children (64 females) across demographically-diverse populations in four low and middle-income countries, including: Tanna, Vanuatu; Keningau, Malaysia; Saltpond, Ghana; and Natal, Brazil. The communities we studied vary in market integration/urbanicity as well as level of access, structure, and quality of schooling. We found that performance on the visuospatial working memory task (Knox Cube) and the visuospatial short-term memory task (Beads) are each independently associated with performance on the self-regulation task, even when controlling for schooling and location effects. These effects were robust across demographically-diverse populations of children in low-and middle-income countries. We conclude that this study found evidence supporting visuospatial working memory and visuospatial short-term memory as distinct cognitive processes which each support the development of self-regulation.

Differential toxicity of silver and titanium dioxide nanoparticles on Drosophila melanogaster development, reproductive effort, and viability: size, coatings and antioxidants matter.

Silver and titanium dioxide nanoparticles are known to induce oxidative stress in vitro and in vivo. Here we test if they impact development, mating success, and survivorship in Drosophila melanogaster, and if so, if these effects are reversible by antioxidants. Ingestion of nanotitanium dioxide during the larval stage of the life cycle showed no effects on development or survivorship, up to doses of 200 µg mL(-1). Conversely, ingestion of nanosilver had major dose, size, and coating-dependent effects on each of these aspects of life history. Each of these effects was partially or fully reversible by vitamin C. Larvae growing on nanosilver supplemented with vitamin C showed a greater than twofold increase in survivorship compared to flies reared on nanosilver alone, and a threefold increase in mating success. Vitamin C also rescued cuticular and pigmentation defects in nanosilver fed flies. Biochemical assays of superoxide dismutase and glutathione show these markers respond to nanotitanium dioxide and nanosilver induced oxidative stress, and this response is reduced by vitamin C. These results indicate that life history effects of nanosilver ingestion result from oxidative stress, and suggest antioxidants as a potential remediation for nanosilver toxicity. Conversely, the lack of nanotitanium dioxide life history toxicity shows that oxidative stress does not necessarily result in whole organism effects, and argues that nanoparticle toxicity needs to be examined at different levels of biological organization.

Tubulin evolution in insects: gene duplication and subfunctionalization provide specialized isoforms in a functionally constrained gene family.

BACKGROUND: The completion of 19 insect genome sequencing projects spanning six insect orders provides the opportunity to investigate the evolution of important gene families, here tubulins. Tubulins are a family of eukaryotic structural genes that form microtubules, fundamental components of the cytoskeleton that mediate cell division, shape, motility, and intracellular trafficking. Previous in vivo studies in Drosophila find a stringent relationship between tubulin structure and function; small, biochemically similar changes in the major alpha 1 or testis-specific beta 2 tubulin protein render each unable to generate a motile spermtail axoneme. This has evolutionary implications, not a single non-synonymous substitution is found in beta 2 among 17 species of Drosophila and Hirtodrosophila flies spanning 60 Myr of evolution. This raises an important question, How do tubulins evolve while maintaining their function? To answer, we use molecular evolutionary analyses to characterize the evolution of insect tubulins. RESULTS: Sixty-six alpha tubulins and eighty-six beta tubulin gene copies were retrieved and subjected to molecular evolutionary analyses. Four ancient clades of alpha and beta tubulins are found in insects, a major isoform clade (alpha 1, beta 1) and three minor, tissue-specific clades (alpha 2-4, beta 2-4). Based on a Homarus americanus (lobster) outgroup, these were generated through gene duplication events on major beta and alpha tubulin ancestors, followed by subfunctionalization in expression domain. Strong purifying selection acts on all tubulins, yet maximum pairwise amino acid distances between tubulin paralogs are large (0.464 substitutions/site beta tubulins, 0.707 alpha tubulins). Conversely orthologs, with the exception of reproductive tissue isoforms, show little sequence variation except in the last 15 carboxy terminus tail (CTT) residues, which serve as sites for post-translational modifications (PTMs) and interactions with microtubule-associated proteins. CTT residues overwhelming comprise the co-evolving residues between Drosophila alpha 2 and beta 3 tubulin proteins, indicating CTT specializations can be mediated at the level of the tubulin dimer. Gene duplications post-dating separation of the insect orders are unevenly distributed, most often appearing in major alpha 1 and minor beta 2 clades. More than 40 introns are found in tubulins. Their distribution among tubulins reveals that insertion and deletion events are common, surprising given their potential for disrupting tubulin coding sequence. Compensatory evolution is found in Drosophila beta 2 tubulin cis-regulation, and reveals selective pressures acting to maintain testis expression without the use of previously identified testis cis-regulatory elements. CONCLUSION: Tubulins have stringent structure/function relationships, indicated by strong purifying selection, the loss of many gene duplication products, alpha-beta co-evolution in the tubulin dimer, and compensatory evolution in beta 2 tubulin cis-regulation. They evolve through gene duplication, subfunctionalization in expression domain and divergence of duplication products, largely in CTT residues that mediate interactions with other proteins. This has resulted in the tissue-specific minor insect isoforms, and in particular the highly diverse alpha3, alpha4, and beta2 reproductive tissue-specific tubulin isoforms, illustrating that even a highly conserved protein family can participate in the adaptive process and respond to sexual selection.

Inhalation method for delivery of nanoparticles to the Drosophila respiratory system for toxicity testing.

The growth of the nanotechnology industry and subsequent proliferation of nanoparticle types present the need to rapidly assess nanoparticle toxicity. We present a novel, simple and cost-effective nebulizer-based method to deliver nanoparticles to the Drosophila melanogaster respiratory system, for the purpose of toxicity testing. FluoSpheres, silver, and CdSe/ZnS nanoparticles of different sizes were effectively aerosolized, showing the system is capable of functioning with a wide range of nanoparticle types and sizes. Red fluorescent CdSe/ZnS nanoparticles were successfully delivered to the fly respiratory system, as visualized by fluorescent microscopy. Silver coated and uncoated nanoparticles were delivered in a toxicity test, and induced Hsp70 expression in flies, confirming the utility of this model in toxicity testing. This is the first method developed capable of such delivery, provides the advantage of the Drosophila health model, and can serve as a link between tissue culture and more expensive mammalian models in a tiered toxicity testing strategy.

Functional constraint underlies 60 million year stasis of Dipteran testis-specific beta-tubulin.

How do proteins evolve while maintaining their function? Previous studies find a highly stringent structure/function relationship between the Drosophila melanogaster testis-specific tubulin beta2 and the spermtail axoneme, such that small changes in the beta2 protein render it unable to generate a motile axoneme. This raises the question, how does beta2 evolve while maintaining its function? To address this question we cloned full- and partial-length beta2 sequences from 17 species of Drosophila and Hirtodrosophila flies spanning 60 Myr of evolution. Not a single amino acid difference is coded among them-beta2 maintains its function by not evolving. We also performed gene genealogical analyses to determine ortholog/paralog relationships among insect tubulins. We find that the Lepidopteran and Dipteran testis-specific beta-tubulins are likely orthologs, and surprisingly, despite functioning in the same structure, the Lepidopteran orthologs are evolving rapidly. We argue that differences in tubulin isoform use in the testes cause the Dipteran axoneme to be less evolvable than the Lepidopteran axoneme, which has facilitated the evolution of a unique amino acid synergism in Drosophila and Hirtodrosophilabeta2 that is resistant to change, contributing to its evolutionary stasis.

The best of all worlds or the best possible world? Developmental constraint in the evolution of beta-tubulin and the sperm tail axoneme.

Through evolutionary history, some features of the phenotype show little variation. Stabilizing selection could produce this result, but the possibility also exists that a feature is conserved because it is developmentally constrained--only one or a few developmental mechanisms can produce that feature. We present experimental data documenting developmental constraint in the assembly of the motile sperm tail axoneme. The 9+2 microtubule architecture of the eukaryotic axoneme has been deeply conserved. We argue that the quality of motility supported by axonemes with this morphology explains their long conservation, rather than a developmental necessity for the 9+2 architecture. However, our functional tests in Drosophila spermatogenesis reveal considerable constraint in the coevolution of testis-specific beta-tubulin and the sperm tail axoneme. The evolution of testis beta-tubulins used in insect sperm tail axonemes is highly punctuated, indicating some pressure acting on their evolution. We provide a mechanistic explanation for their punctuated evolution by testing structure-function relationships between testis beta-tubulin and the motile axoneme in D. melanogaster. We discovered that a highly conserved sequence feature of beta-tubulins used in motile axonemes is needed to specify central pair formation. Second, our data suggest that cooperativity in the function of internal beta-tubulin amino acids is needed to support the long axonemes characteristic of Drosophila sperm tails. Thus, central pair formation constrains the evolution of the axoneme motif, and intramolecular cooperativity makes the evolution of the internal residues path dependent, which slows their evolution. Our results explain why a highly specialized beta-tubulin is needed to construct the Drosophila sperm tail axoneme. We conclude that these constraints have fixed testis-specific beta-tubulin identity in Drosophila.