Two novel, tightly linked, and rapidly evolving genes underlie Aedes aegypti mosquito reproductive resilience during drought.

Female Aedes aegypti mosquitoes impose a severe global public health burden as vectors of multiple viral pathogens. Under optimal environmental conditions, Aedes aegypti females have access to human hosts that provide blood proteins for egg development, conspecific males that provide sperm for fertilization, and freshwater that serves as an egg-laying substrate suitable for offspring survival. As global temperatures rise, Aedes aegypti females are faced with climate challenges like intense droughts and intermittent precipitation, which create unpredictable, suboptimal conditions for egg-laying. Here, we show that under drought-like conditions simulated in the laboratory, females retain mature eggs in their ovaries for extended periods, while maintaining the viability of these eggs until they can be laid in freshwater. Using transcriptomic and proteomic profiling of Aedes aegypti ovaries, we identify two previously uncharacterized genes named tweedledee and tweedledum, each encoding a small, secreted protein that both show ovary-enriched, temporally-restricted expression during egg retention. These genes are mosquito-specific, linked within a syntenic locus, and rapidly evolving under positive selection, raising the possibility that they serve an adaptive function. CRISPR-Cas9 deletion of both tweedledee and tweedledum demonstrates that they are specifically required for extended retention of viable eggs. These results highlight an elegant example of taxon-restricted genes at the heart of an important adaptation that equips Aedes aegypti females with 'insurance' to flexibly extend their reproductive schedule without losing reproductive capacity, thus allowing this species to exploit unpredictable habitats in a changing world.

Size Quantification of Blood and Sugar Meals in Aedes aegypti Mosquitoes.

Both male and female mosquitoes consume sugar-rich nectar meals required for metabolic energy, but only females consume protein-rich blood meals, which are required for egg development. The size of each meal consumed has subsequent effects on behavior and reproduction; therefore, precise quantification is an important aspect of mosquito feeding behavior studies. This protocol describes a high-throughput, end-point assay to quantify meal volumes ingested by individual mosquitoes. The addition of a fluorescent dye to the meal allows for meal size quantification. Individual mosquitoes that have been fed this meal are homogenized in 96-well plates, and the fluorescence levels are measured with a plate reader. This protocol can also be adapted to determine if alteration of meal composition affects the ingested meal volume, if mosquito strain or genotype dictates consumption, or if meals are derived from multiple sources.

Methods to Assess Blood and Nectar Meals in Aedes aegypti Mosquitoes.

Male and female Aedes aegypti mosquitoes survive by feeding on floral nectar for metabolic energy, but females require blood protein, obtained from biting a host, for egg development. Although males exclusively derive energy from nectar sugars, females must select the meal that best matches their present metabolic and reproductive needs. In females, blood and nectar promote independent feeding behaviors with distinct sensory appendages, meal sizes, digestive tract targets, and metabolic fates. Understanding how male and female mosquitoes recognize, locate, and metabolize nutrients is essential for characterizing the survival and reproductive capabilities of this mosquito. Here, we provide an introduction to blood versus nectar feeding and methods to quantify nectar and blood meal sizes in individual Ae. aegypti mosquitoes. Precise quantification of meal size is crucial for ensuring consistency in assays that record events downstream of feeding behavior, including host attraction or fecundity.

Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes.

Females of certain mosquito species can spread diseases while biting vertebrate hosts to obtain protein-rich blood meals required for egg development. In the laboratory, researchers can deliver animal-derived and artificial blood meals to mosquitoes via membrane feeders, which allow for manipulation of meal composition. Here, we present methods for feeding blood and artificial blood meals to Aedes aegypti mosquitoes and quantifying the volume consumed by individual females. Targeted feeding and quantification of artificial/blood meals have broad uses, including testing the effects of meal components on mosquito behavior and physiology, delivering pharmacological compounds without injection, and infecting mosquitoes with specific pathogens. Adding fluorescein dye to the meal prior to feeding allows for subsequent meal size quantification. The meal volume consumed by mosquitoes can be measured either by weight, if the females are to be used later for behavioral experiments, or by homogenizing individual females in 96-well plates and measuring fluorescence levels using a plate reader as an endpoint assay. Meal size quantification can be used to determine whether changing the meal components alters the meal volume ingested or if meal consumption differs between mosquito strains. Precise meal size quantification is also critical for downstream assays, such as those measuring effects on host attraction or fecundity. The methods presented here can be further adapted to track meal digestion over the course of days or to include multiple distinguishable markers added to different meals (like nectar and blood) to quantify the consumption of each meal by a single mosquito. These methods allow researchers to singlehandedly perform high-throughput measurements to compare the meal volume consumed by hundreds of individual mosquitoes. These tools will therefore be broadly useful to the community of mosquito researchers for answering diverse biological questions.

Distinct tmRNA sequence elements facilitate RNase R engagement on rescued ribosomes for selective nonstop mRNA decay.

trans-Translation, orchestrated by SmpB and tmRNA, is the principal eubacterial pathway for resolving stalled translation complexes. RNase R, the leading nonstop mRNA surveillance factor, is recruited to stalled ribosomes in a trans-translation dependent process. To elucidate the contributions of SmpB and tmRNA to RNase R recruitment, we evaluated Escherichia coli-Francisella tularensis chimeric variants of tmRNA and SmpB. This evaluation showed that while the hybrid tmRNA supported nascent polypeptide tagging and ribosome rescue, it suffered defects in facilitating RNase R recruitment to stalled ribosomes. To gain further insights, we used established tmRNA and SmpB variants that impact distinct stages of the trans-translation process. Analysis of select tmRNA variants revealed that the sequence composition and positioning of the ultimate and penultimate codons of the tmRNA ORF play a crucial role in recruiting RNase R to rescued ribosomes. Evaluation of defined SmpB C-terminal tail variants highlighted the importance of establishing the tmRNA reading frame, and provided valuable clues into the timing of RNase R recruitment to rescued ribosomes. Taken together, these studies demonstrate that productive RNase R-ribosomes engagement requires active trans-translation, and suggest that RNase R captures the emerging nonstop mRNA at an early stage after establishment of the tmRNA ORF as the surrogate mRNA template.

Non-stop mRNA decay: a special attribute of trans-translation mediated ribosome rescue.

Decoding of aberrant mRNAs leads to unproductive ribosome stalling and sequestration of components of the translation machinery. Bacteria have evolved three seemingly independent pathways to resolve stalled translation complexes. The trans-translation process, orchestrated by the hybrid transfer-messenger RNA (tmRNA) and its essential protein co-factor, small protein B (SmpB), is the principal translation quality control system for rescuing unproductively stalled ribosomes. Two specialized alternative rescue pathways, coordinated by ArfA and ArfB, have been recently discovered. The SmpB-tmRNA mediated trans-translation pathway, in addition to re-mobilizing stalled translation complexes, co-translationally appends a degradation tag to the associated nascent polypeptides, marking them for proteolysis by various cellular proteases. Another unique feature of trans-translation, not shared by the alternative rescue pathways, is the facility to recruit ribonuclease R (RNase R) for targeted degradation of non-stop mRNAs, thus preventing further futile cycles of translation. The distinct C-terminal lysine-rich (K-rich) domain of RNase R is essential for its recruitment to stalled ribosomes. To gain new insights into the structure and function of RNase R, we investigated its global architecture, the spatial arrangement of its distinct domains, and the identities of key functional residues in its unique K-rich domain. Small-angle X-ray scattering models of RNase R reveal a tri-lobed structure with flexible N- and C-terminal domains, and suggest intimate contacts between the K-rich domain and the catalytic core of the enzyme. Alanine-scanning mutagenesis of the K-rich domain, in the region spanning residues 735 and 750, has uncovered the precise amino acid determinants required for the productive engagement of RNase R on tmRNA-rescued ribosomes. Theses analyses demonstrate that alanine substitution of conserved residues E740 and K741result in profound defects, not only in the recruitment of RNase R to rescued ribosomes but also in the targeted decay of non-stop mRNAs. Additionally, an RNase R variant with alanine substitution at residues K749 and K750 exhibits extensive defects in ribosome enrichment and non-stop mRNA decay. In contrast, alanine substitution of additional conserved residues in this region has no effect on the known functions of RNase R. In vitro RNA degradation assays demonstrate that the consequential substitutions (RNase R(E740A/K741A) and RNase R(K749A/K750A)) do not affect the ability of the enzyme to degrade structured RNAs, indicating that the observed defect is specific to the trans-translation related activities of RNase R. Taken together, these findings shed new light on the global architecture of RNase R and provide new details of how this versatile RNase effectuates non-stop mRNA decay on tmRNA-rescued ribosomes.

Structural basis for S-adenosylmethionine binding and methyltransferase activity by mitochondrial transcription factor B1.

Eukaryotic transcription factor B (TFB) proteins are homologous to KsgA/Dim1 ribosomal RNA (rRNA) methyltransferases. The mammalian TFB1, mitochondrial (TFB1M) factor is an essential protein necessary for mitochondrial gene expression. TFB1M mediates an rRNA modification in the small ribosomal subunit and thus plays a role analogous to KsgA/Dim1 proteins. This modification has been linked to mitochondrial dysfunctions leading to maternally inherited deafness, aminoglycoside sensitivity and diabetes. Here, we present the first structural characterization of the mammalian TFB1 factor. We have solved two X-ray crystallographic structures of TFB1M with (2.1 Å) and without (2.0 Å) its cofactor S-adenosyl-L-methionine. These structures reveal that TFB1M shares a conserved methyltransferase core with other KsgA/Dim1 methyltransferases and shed light on the structural basis of S-adenosyl-L-methionine binding and methyltransferase activity. Together with mutagenesis studies, these data suggest a model for substrate binding and provide insight into the mechanism of methyl transfer, clarifying the role of this factor in an essential process for mitochondrial function.

Ribosome purification approaches for studying interactions of regulatory proteins and RNAs with the ribosome.

Ribosomes are large complexes of RNA and protein that perform the essential task of protein synthesis in the cell. Ribosomes also serve as the initiation point for several translation-associated functions. To perform these tasks efficiently, ribosomes interact with a myriad of nonribosomal proteins and RNAs. Given that most of these interactions are transient, purification of the interacting factors in complex with the ribosome can be a challenging undertaking. Here, we review methods commonly used to isolate ribosomes and study ribosome-associated factors. We also discuss crucial parameters for designing and executing ribosome association studies. Finally, we present a detailed protocol for reporter based enrichment assays that are employed to selectively isolate ribosomes translating a particular message of interest. These protocols can be used to study a wide range of ribosome-associated functions.

Resource utilization and costs of age-related macular degeneration.

Data were analyzed from the 1999-2001 Medicare Beneficiary Encrypted Files for patients with age-related macular degeneration (AMD), an ophthalmic condition characterized by central vision loss. Classifying AMD subtype by International Classification of Diseases, Ninth Revision, Clinical Modifications (ICD-9-CM) (Centers for Disease Control and Prevention, 2003) code, resource utilization rates increased with disease progression. Individuals with more severe disease (wet only or wet and dry AMD) had greater costs than did those with less severe disease (drusen only or dry only). Costs among patients with wet disease increased yearly at rates exceeding inflation, possibly due in part to increased rates of treatment with photodynamic therapy among these individuals and the aging of the population.