Fig 4. The impact of Spiroplasma infection on male Gff reproductive fitness.

(A) Relative expression of sperm-specific dynein intermediate chain (sdic) in sperm located within the reproductive tract of Gff^Spi+ and Gff^Spi- males or within the female spermatheca following copulation. Sdic expression in each sample was normalized relative to tsetse’s constitutively expressed pgrp-la gene. Each dot represents one biological replicate, and bars indicate median values. Statistical significance was determined via one-way ANOVA followed by Tukey’s HSD post-hoc analysis. (B) Quantification of sperm within the spermatheca of female flies that mated with either Spi⁺ or Spi^- males. Measurements were made using a Neubauer counting chamber. Each dot represents one spermatheca, and bars indicate median values. Statistical significance was determined via student’s t-test. (C) Spermathecal fill of female flies that mated with either Spi⁺ or Spi^- males. Spermatheca fill data were analyzed using Kruskal-Wallis rank test using R companion and FSA software packages [75,76]. (D). Motility, as a measure of flagellar beat frequency in hertz (Hz), of sperm within the spermatheca of female flies that mated with either Spi⁺ or Spi^- males. Video recordings of sperm were acquired at a rate of 50 frames per second, and beat frequency was analyzed using FIJI and the ImageJ plugin SpermQ. Each dot on the graph represents the mean beat frequency of two sperm tails from each spermatheca. Statistical significance was determined via student’s t-test.