Creation of Knock-In Alleles of Insulin Receptor Tagged by Fluorescent Proteins mCherry or EYFP in Fruit Fly Drosophila melanogaster.

The insulin/insulin-like growth factor-like signaling (IIS) pathway is highly conserved across metazoans and regulates numerous physiological functions, including development, metabolism, fecundity, and lifespan. The insulin receptor (InR), a crucial membrane receptor in the IIS pathway, is known to be ubiquitously expressed in various tissues, albeit at generally low levels, and its subcellular localization remains incompletely characterized. In this study, we employed CRISPR-mediated mutagenesis in the fruit fly Drosophila to create knock-in alleles of InR tagged with fluorescent proteins (InR::mCherry or InR::EYFP). By inserting the coding sequence of the fluorescent proteins mCherry or EYFP near the end of the coding sequence of the endogenous InR gene, we could trace the natural InR protein through their fluorescence. As an example, we investigated epithelial cells of the male accessory gland (AG), an internal reproductive organ, and identified two distinct patterns of InR::mCherry localization. In young AG, InR::mCherry accumulated on the basal plasma membrane between cells, whereas in mature AG, it exhibited intracellular localization as multiple puncta, indicating endocytic recycling of InR during cell growth. In the AG senescence accelerated by the mutation of Diuretic hormone 31 (Dh31), the presence of InR::mCherry puncta was more pronounced compared to the wild type. These findings raise expectations for the utility of the newly created InR::mCherry/EYFP alleles for studying the precise expression levels and subcellular localization of InR. Furthermore, this fluorescently tagged allele approach can be extended to investigate other membrane receptors with low abundance, facilitating the direct examination of their true expression and localization.

Differences in energy source storage in eye stalks between two species of stalk-eyed flies, Sphyracephala detrahens and Cyrtodiopsis dalmanni.

Some diopsid flies have sexually dimorphic eye stalks that are assumed to require considerable nutrition for growth but are advantageous in competition and courtship. According to the handicap theory, the eye span in some dimorphic species serves as a reliable signal of individual quality to an opponent. However, it is not well understood how well eye span represents energy source storage. In this study, we focused on two species: Sphyracephala detrahens, which has weak dimorphism, and Cyrtodiopsis dalmanni, which has moderate dimorphism. We found that the eye stalks of the former species contained more fat bodies than those of the latter species. When the flies were starved, the fat body cells in the eye stalks underwent autophagy. A strong positive correlation was consistently found between eye span and starvation tolerance for S. detrahens, while a weak correlation was found for C. dalmanni. Furthermore, starvation decreased the contest winning rate between S. detrahens pairs with similar eye spans. These findings suggest that the presentation of resource holding potential may be larger than the actual storage ability and that the fidelity of nutritional storage signaling varies; the signal presented by S. detrahens is more reliable than that presented by C. dalmanni.