The dye Rhodamine B (Rho B) was used to label the seminal fluid of male mosquitoes to determine paternity in mating competition assays [27]. One day old male mosquitoes from the S-Cairns, R-TL, and R-BC strains were placed in 30 × 30 × 30 cm cages (BugDorm Store, Taichung, Taiwan) and provided access to 0.1% Rho B (Sigma Aldrich, 95% dye content) (w/v) in 10% sucrose solution for 96 h to label seminal fluid. At the conclusion of the 96 h labelling period, 100% of the Rho B fed males were confirmed for labelling success by visual inspection. A separate group of males from each strain was fed a 10% sucrose only solution as an unstained control group. The Rho B and sucrose solutions were replaced every 48 h. The mating competitiveness of each strain was tested by allowing an equal number of males from two strains (one Rho B marked, one unmarked) to compete freely for females from a single strain. Reciprocal assays (swapping the Rho B stained strains) were performed to ensure that there was no labelling bias (S1 Table). The males and females of each strain were separated as pupae prior to emergence to ensure that all mosquitoes were unmated at the start of the competition assay. For each assay, 20 virgin females (3–5 d old), 10 Rho B marked virgin males, and 10 unmarked virgin males (all 5–6 d old) were placed in a cage (i.e. a 1:1 female: male ratio). Both marked and unmarked males were added to each cage before females were introduced. After 24 h, female mosquitoes were collected and knocked down by freezing at -20°C for 30 min. Individual females were dissected in 1% PBS solution to isolate the bursa and spermathecae. These were mounted on a slide and gently crushed with a coverslip to break open the spermathecae. Specimens were examined using a fluorescent microscope (Zeiss Axioskopp2) with a fluorescence illuminator (Xcite 120Q) and a Cy3 4040c fluorescence filter (531/40 nm Excitation; 593/40 nm Emission) to determine the presence of Rho B. This filter optimised visual differentiation of Rho B-stained tissues. The presence of Rho B (Fig 1A) indicated the mating success of a marked male, while the presence of an expanded bursa (Fig 1B) in the absence of Rho B indicated a successful mating event with an unmarked male. The absence of sperm within the spermathecae and no expansion of the bursa, indicated that successful mating had not occurred (Fig 1C). Three replicates were performed for each treatment. If all females were mated and both competing male groups were equally successful, we would expect 50% of mated females to contain Rho B in the bursa and spermathecae and 50% to show evidence of mating (sperm and an expanded bursa) without staining.

(A) Staining of an expanded bursa (Br), spermathecae (S) and labelled sperm (Ls), indicating a successful mating event with a male marked with Rho B. (B) No staining in the spermathecae, unlabelled sperm (Uls) or bursa but the expanded bursa indicates a successful mating event with a non-Rho B-marked male. (C) Unmated virgin female, no sperm present in the spermathecae and the bursa is not expanded.

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