Life-history traits are used as proxies of fitness in insects including Drosophila. Egg size is an adaptive and ecologically important trait potentially with genetic variation across different populations. However, the low throughput of manual measurement of egg size has hampered the widespread use of this trait in evolutionary biology and population genetics. We established a method for accurate and high throughput measurement of Drosophila egg size using large particle flow cytometry (LPFC). The size estimates using LPFC are accurate and highly correlated with the manual measurements. The measurement of egg size is high throughput (average of 214 eggs measured per minute) and viable eggs of a specific size can be sorted rapidly (average of 70 eggs per minute). Sorting by LPFC does not reduce the survival of eggs making it a suitable approach for sorting eggs for downstream analyses. This protocol can be applied to any organism within the detectable size range (10–1500 µm) of the large particle flow cytometers. We discuss the potential applications of this method and provide recommendations for optimizing the protocol for other organisms.

Transposable elements (TEs) are genomic parasites that proliferate within host genomes, and which can also invade new species. The P-element, a DNA-based transposable element, recently invaded two Drosophila species: Drosophila melanogaster in the 20th century, and D. simulans in the 21st. In both species, lines collected before the invasion are susceptible to ‘hybrid dysgenesis’, a syndrome of abnormal phenotypes apparently due to P-element-inflicted DNA damage. In D. melanogaster, lines collected after the invasion have evolved a maternally-acting mechanism that suppresses hybrid dysgenesis, with extensive work showing that PIWI-interacting small RNAs (piRNAs) are a key factor in this suppression. Most of these studies use lines collected many generations after the initial P-element invasion. Here, we study D. simulans collected early as well as late in the P-element invasion of this species. Like D. melanogaster, D. simulans from late in the invasion show strong resistance to hybrid dysgenesis and abundant P-element-derived piRNAs. Lines collected early in the invasion, however, show substantial variation in how much they suffer from hybrid dysgenesis, with some lines highly resistant. Surprisingly, though, these resistant lines do not show high levels of cognate maternal P-element piRNAs; in these lines, it may be that other mechanisms suppress hybrid dysgenesis.

Many life-history traits are used as proxies of fitness in Drosophila. Egg size is an adaptive trait that can be used for fitness measurement. However, the low throughput of manual measurement of egg size has hampered the widespread use of this trait in evolutionary biology. We established a method for accurate and high throughout measurement of Drosophila egg size using large particle flow cytometry which allows the automated analysis of large particles based on size and optical density. The measurement of egg size is high throughput (average 238 eggs analyzed per minute) and sorting eggs of specific size is efficient (37-75 % efficiency). Using this approach, we showed the natural variation in egg size among strains of Drosophila species. The egg size distribution determined by this approach can be used as a measure of fitness and the reproductive investment in Drosophila. Measurement and sorting by flow cytometer do not decrease the egg viability making it a suitable approach for sorting viable eggs to be used for downstream analyses such as selection experiments.