No abstract

Background An important function of the fat body in adult female mosquitoes is the conversion of blood meal derived amino acids (AA) into massive amounts of yolk protein precursors. A highly efficient transport mechanism for AAs across the plasma membrane of the fat body trophocytes is essential in order to deliver building blocks for the rapid synthesis of large amounts of these proteins. This mechanism consists in part of AA transporter proteins from the solute carrier family. These transporters have dual function; they function as transporters and participate in the nutrient signal transduction pathway that is activated in the fat body after a blood meal. In this study we focused on the solute carrier 7 family (SLC7), a family of AA transporters present in all metazoans that includes members with strong substrate specificity for cationic AAs. Methodology/Principal Findings We identified eleven putative SLC7 transporters in the genome sequence of Aedes aegypti. Phylogenetic analysis puts five of these in the cationic AA transporter subfamily (CAT) and six in the heterodimeric AA transporter (HAT) subfamily. All eleven Aedes aegypti SLC7 genes are expressed in adult females. Expression profiles are dynamic after a blood meal. We knocked down six fat body-expressed SLC7 transporters using RNAi and found that these ‘knockdowns’ reduced AA-induced TOR signaling. We also determined the effect these knockdowns had on the number of eggs deposited following a blood meal. Conclusions/Significance Our analysis stresses the importance of SLC7 transporters in TOR signaling pathway and mosquito reproduction.

Insect yolk protein precursor gene expression is regulated by nutritional and endocrine signals. A surge of amino acids in the hemolymph of blood-fed female mosquitoes activates a nutrient signaling system in the fat bodies, which subsequently derepresses yolk protein precursor genes and makes them responsive to activation by steroid hormones. Orphan transporters of the SLC7 family were identified as essential upstream components of the nutrient signaling system in the fat body of fruit flies and the yellow fever mosquito, Aedes aegypti. However, the transport function of these proteins was unknown. We report expression and functional characterization of AaCAT1, cloned from the fat body of A. aegypti. Expression of AaCAT1 transcript and protein undergoes dynamic changes during postembryonic development of the mosquito. Transcript expression was especially high in the third and fourth larval stages; however, the AaCAT1 protein was detected only in pupa and adult stages. Functional expression and analysis of AaCAT1 in Xenopus oocytes revealed that it acts as a sodium-independent cationic amino acid transporter, with unique selectivity to L-histidine at neutral pH (K 0.5 L-His ‫؍‬ 0.34 ؎ 0.07 mM, pH 7.2). Acidification to pH 6.2 dramatically increases AaCAT1-specific His ؉ -induced current. RNAi-mediated silencing of AaCAT1 reduces egg yield of subsequent ovipositions. Our data show that AaCAT1 has notable differences in its transport mechanism when compared with related mammalian cationic amino acid transporters. It may execute histidine-specific transport and signaling in mosquito tissues.

This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion rate. This method can be used to obtain a better understanding of the process of diuresis in insects and is especially useful in the study of diuresis in blood-feeding arthropods that are able to take up huge amounts of liquid in a single blood meal. This RNAi-mediated gene knockdown combined with an in vivo diuresis assay was developed by the Hansen lab to study the effects of RNAi-mediated knockdown of aquaporin genes on Aedes aegypti mosquito diuresis. The protocol is setup in two parts: the first demonstration illustrates how to construct a simple mosquito injection device and how to prepare and inject dsRNA into the thorax of mosquitoes for RNAi-mediated gene knockdown. The second demonstration illustrates how to determine excretion rates in mosquitoes using an in vivo bioassay.

The National Assessment of Educational Progress (NAEP) in Reading aims to measure reading comprehension for students in the United States and to monitor progress in our education system. NAEP Reading is developed based on an assessment framework document that is periodically revised to reflect the latest understandings about reading comprehension and its assessment. A key goal of the Visioning Panel (VP) and the Development Panel (DP) charged with updating the NAEP Reading assessment framework for 2026 was to lay the foundation for an assessment that made progress toward greater fairness, equity, and validity. In this essay, we discuss how the framework development process unfolded and its results. We document the unusual ways the National Assessment Governing Board (“the Board”) shaped the development process. We provide evidence that a small group of dissenting Board members aimed to preserve the status quo in reading assessment by downplaying reliance on expertise and authoritative sources of research on reading, learning, and assessment and by removing attention to equity in NAEP Reading. We also discuss both successful (i.e., approved by the Board) and unsuccessful (i.e., rejected by the Board) recommendations for changes to the 2026 Framework that initially were proposed by the DP. We end by considering how, despite the efforts of the small group of dissenting Board members, we as a literacy field can improve the nature and impact of our large-scale reading assessments, and NAEP in particular.