Drosophila melanogaster (fruit fly) is a model organism and its behaviours including oviposition (egg-laying) on agar substrates have been widely used for assessment of a variety of biological processes in flies. Physical and chemical properties of the substrate are the dominant factors affecting Drosophila's oviposition, but they have not been investigated precisely and parametrically with the existing manual approaches. As a result, many behavioral questions about Drosophila oviposition, such as the combined effects of the aforementioned substrate properties (e.g., exposure area, sugar content, and stiffness) on oviposition and viability, and their threshold values, are yet to be answered. In this paper, we have devised a simple, easily implementable, and novel methodology that allows for modification of physical and chemical composition of agar substrates in order to quantitatively study survival and oviposition of adult fruit flies in an accurate and repeatable manner. Agar substrates have been modified by surface patterning using single and hexagonally arrayed through-hole polydimethylsiloxane (PDMS) membranes with various diameters and interspacing, as well as by substrate stiffness and sugar content modification via alteration of chemical components. While pure PDMS substrates showed a significant lethal effect on flies, a 0.5 mm diameter through-hole access to agar was found to abruptly increase the survival of adult flies to more than 93%. Flies avoided ovipositing on pure PDMS and on top of substrates with 0.5 mm diameter agar exposure areas. At a hole diameter of 2 mm (i.e., 0.25% exposure area) or larger, eggs were observed to be laid predominately inside the through-holes and along the edges of the PDMS-agar interface, showing a trending increase in site selection with 4 mm (i.e., 1% exposure area threshold) demonstrating natural oviposition rates similar to pure agar. The surface-modified agar-PDMS hybrid devices and the threshold values reported for the substrate physical and chemical conditions affecting oviposition are novel; therefore, we advocate their use for future in-depth studies of oviposition behaviour in Drosophila melanogaster with accuracy and repeatability. The technique is also useful for development of novel assays for learning and decision-making studies as well as miniaturized devices for self-assembly of eggs and embryonic developmental investigations.

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