Handler: 1) Efforts decreased somewhat during this period since the research technician primarily responsible for embryo injections resigned during the summer to pursue a graduate degree. Our intention is to hire a new post-doc to take on these (and other) responsibilities, but this will not be possible until the revised budget is approved (submitted by Dr. Pelz-Stelinski) so that remaining funds are available for hiring on the UF OPS system. 2) During this quarter 1,125 Asian citrus psyllid (ACP) control uninjected eggs were set-up yielding a 25% nymphal hatch rate, while 2,719 eggs injected with transformation vector/helper plasmids hatched at a 3.2% frequency. Of the 87 newly hatched nymphs from injected eggs, 58 survived to adulthood and were backcrossed to wild (uninjected) ACP adults. 173 G1 offspring were screened with none exhibiting DsRed fluorescence indicative of potential transformation. Although the overall hatch rate from control and injected eggs had decreased from the improved rates observed in the previous quarter, this can be attributed to unusually high temperatures in the greenhouse facilities during July and August, sometimes approaching 100oF or greater. Nevertheless, the 67% survival rate of hatched nymphs to adulthood, after dusting the injected eggs and tape with potato starch, remained consistent with the improved post-hatch survival observed previously. To ameliorate ongoing seasonal difficulties in regulating temperature and humidity, resulting in decreased egg lays (in winter) and decreased nymph survival (in summer), a newly available environmental room at CMAVE will be retro-fitted with a heat pump, a regulated-humidifier and a new lighting system that we expect to improve environmental conditions. 3) To further improve embryo injections, nymphal hatching and survival to adulthood tests have been initiated to determine optimal egg desiccation times and heat shock temperature and duration. 4) Personnel were trained in quantitative PCR methods so that expression of the helper transposase gene and vector marker gene can be assessed in injected embryos. 800 eggs have been injected for each experiment and controls and processing of RNA and PCR assays are in progress. 5) In the last quarter, efforts were initiated to test CRISPR/Cas9 gene-editing techniques in ACP as a means of genetic modification. While we determined that DNA modifications did occur, the specific modification of a known gene were not observed. To improve our methodology for this powerful, though complex technique, efforts were focussed on developing successful protocols in fruit fly species that could be monitored more precisely. We have now succeeded in CRISPR/Cas9 mutagenesis (gene knock-outs) in A. suspensa and D. suzukii, and a gene insertion in D. suzukii. We will now use this knowledge and protocols to continue with gene-editing attempts in ACP.