(A) A male fertility assay was conducted at 29°C using wildtype (WT) females crossed to WT, knockout (KO), or heterozygous (‘Het’) Arp53D KO males (eight females to two males) and adult progeny were counted. We find that homozygous male KOs have much higher fertility than WT males, and that heterozygotes have lower fertility than homozygous KOs. We conclude that the presence of WT Arp53D may have a dosage-dependent detrimental effect on male fertility. (B) Targeted sequencing of two possible integration sites (chromosomal sites 30B and 40D) of an RNAi hairpin in the VDRC RNAi KK stock (Dietzl et al., 2007) I.D. 108369. The 30B site is preferable as the 40D site gives non-specific phenotypes (Green et al., 2014). The actual and expected PCR product sizes with the diagnostic method used in Green et al., 2014 are denoted with triangles. The sizes of the PCR products indicate the RNAi hairpin integrated at the 30B site, whereas the 40D site is empty. (C) Flies encoding the RNAi hairpin targeting Arp53D (VDRC KK line 108369 in B) were crossed to flies encoding the RNAi inducer topi-Gal4 (Raychaudhuri et al., 2012), which is expressed late in spermatogenesis. Flies were maintained at 25°C. RNA was extracted from males encoding both the RNAi hairpin and topi-Gal4, and males encoding only the RNAi hairpin were used as a control. RNA was reverse transcribed, and RT-PCRs (25 cycles) were done to assess expression of Arp53D. Rp49 was used to compare amounts between samples, and template without reverse transcriptase was used to verify absence of genomic DNA. Arp53D expression is mildly reduced in the presence of the topi-Gal4 inducer. (D) A male fertility assay at 29°C comparing the Arp53D-knockdown male to a control male encoding only the RNAi hairpin. Mating and embryo laying took place over 9 days, and adult progeny were counted. Even partial knockdown of Arp53D leads to an increase in male fertility. (E) Meiotic cysts from Oregon-R and isogenized Arp53D-KO whole testes were dissected and stained with DAPI (DNA) and anti-spectrin (fusome). The fusome appears similar between the two genotypes. (F, G) WT and Arp53D-KO virgin males were aged 3 days at 29°C, and seminal vesicles were then dissected, fixed, and stained for DNA to compare the quantity of mature sperm between the two genotypes. The area of the seminal vesicle was measured; KOs appeared to have similarly sized seminal vesicles as WT, suggesting comparable amounts of mature sperm. (H) WT Arp53D and 0.5 kbp of the upstream intergenic region (including its endogenous promoter) was inserted into the attP site of the Arp53D-KOs previously generated (see Figure 5—figure supplement 1A). This rescue transgene was tracked with sfGFP under the control of an eye-specific promoter. Presence of the Arp53D transgene was verified by PCR as shown in the DNA gel below the schematic (‘Rescue’), whereas the transgene was absent in the original Arp53D-KO. The PCR product was sequence verified. (I) RT-PCRs with primers targeting Arp53D were conducted with RNA extracted from testes of WT (Oregon-R) flies, Arp53D-KOs, and the modified Arp53D-KOs encoding the Arp53D rescue transgene (‘Rescue’). KOs show no expression of Arp53D, whereas the Arp53D transgene is expressed in ‘Rescue,’ albeit more weakly compared to endogenous Arp53D in Oregon-R flies. RT-PCRs with primers targeting rp49 indicate comparable amounts of cDNA among the genotypes. Samples not treated with reverse transcriptase exhibit no bands, indicating lack of genomic DNA contamination during RNA extraction.