Isolating Spermathecae and Determining Mating Status of Drosophila suzukii: A Protocol for Tissue Dissection and Its Applications

Abstract

The spotted wing drosophila, Drosophila suzukii (Diptera: Drosophilidae), is an emerging invasive pest, which attacks a wide variety of fruits and berries. Although previous studies have focused on different aspects of D. suzukii reproductive biology, there are no protocols available for determining the mating status of D. suzukii females and drosophilids in general. In this study, a step-by-step protocol for tissue dissection, isolating spermathecae, and determining the mating status of females was developed specifically for D. suzukii. This protocol is an effective and relatively quick method for determining female mating status. It has important applications from exploring reproductive output of D. suzukii females to understanding the biology of D. suzukii winter morph, which presumably plays the main role in the overwintering of this invasive species. We demonstrated applicability of this protocol for both field collected flies and flies reared in the lab, including fly specimens stored on a long-term basis.

Keywords: Drosophila suzukii; invasive species; mating status; sperm storage; spermathecae; spotted wing drosophila; tissue dissection.

Figure 1

Scheme of Drosophila suzukii‘s reproductive system from the dorsal perspective; the seminal receptacle is not shown due to its location on the ventral side of the common oviduct. (The drawing is by Claire Mattmiller; images by Kathryn Hietala-Henschell and Alina Avanesyan).

Figure 2

Basic steps of dissecting female Drosophila suzukii flies and isolating spermathecae. Step 1 includes cutting the exoskeleton of the abdomen and pulling out the reproductive system; step 2 includes removal of the spermathecal glands and surrounding tissues and staining the spermathecae (Images by Alina Avanesyan).

Figure 3

A spermatheca of a mated Drosophila suzukii female under the compound microscope at 20× objective lens; the scale is 50 µm (Image by Claire Mattmiller).

Figure 4

A crushed spermatheca of a mated Drosophila suzukii female observed under the compound microscope at 40× objective lens. The toroidal sperm mass is indicated with arrows; the scale is 20 µm (Image by Alina Avanesyan).

Figure 5

Spermathecae isolated from field collected Drosophila suzukii females. (a) Intact spermatheca (at 20× objective lens; the scale is 50 µm); (b) Crushed spermathecae with toroidal sperm mass indicated (at 40× objective lens; the scale is 20 µm) (Images by Claire Mattmiller).