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Cochliomyia hominivorax and Lucilia cuprina are major pests of livestock. Their larvae infest warm-blooded vertebrates and feed on host’s tissues, resulting in severe industry losses. As they are serious pests, considerable effort has been made to develop genomic resources and functional tools aiming to improve their management and control. Here, we report a significant addition to the pool of genome manipulation tools through the establishment of efficient CRISPR/Cas9 protocols for the generation of directed and inheritable modifications in the genome of these flies. Site-directed mutations were introduced in the C . hominivorax and L . cuprina yellow genes ( ChY and LcY ) producing lightly pigmented adults. High rates of somatic mosaicism were induced when embryos were injected with Cas9 ribonucleoprotein complexes (RNPs) pre-assembled with guide RNAs (sgRNAs) at high concentrations. Adult flies carrying disrupted yellow alleles lacked normal pigmentation ( brown body phenotype) and efficiently transmitted the mutated alleles to the subsequent generation, allowing the rapid creation of homozygous strains for reverse genetics of candidate loci. We next used our established CRISPR protocol to disrupt the C . hominivorax transformer gene ( Chtra ). Surviving females carrying mutations in the Chtra locus developed mosaic phenotypes of transformed ovipositors with characteristics of male genitalia while exhibiting abnormal reproductive tissues. The CRISPR protocol described here is a significant improvement on the existing toolkit of molecular methods in calliphorids. Our results also suggest that Cas9-based systems targeting Chtra and Lctra could be an effective means for controlling natural populations of these important pests.

Two studies separated effects of dietary ergot alkaloids from effects of feed intake or ambient temperature on respiration rate (RR), heart rate (HR), surface temperature (ST), rectal temperature (RT), blood pressure (BP), serum hormone, and plasma metabolite concentrations in beef steers. The balanced, single reversal design for each experiment used 8 beef steers fed tall fescue seed [2.5 g/kg body weight (BW)] with (E+) or without (E−) ergot alkaloids as part of a 60:40 switchgrass hay: supplement diet. Periods were 35 days with 21 days of preliminary phase and 14 days of feeding fescue seed once daily. Measures of dependent variables were collected on d 20, 25, 29, and 35 of each period at 0730 (before feeding), 1230 and 1530. In Experiment 1 steers weighed 286 kg, gained 0.61 kg BW/day, E+ supplied 2.72 mg ergot alkaloids including 1.60 mg ergovaline per steer daily, and mean minimum and maximum daily ambient temperatures were 23.6 and 32.3°C. In Experiment 2 steers weighed 348 kg, gained 1.03 kg BW/day, E+ supplied 3.06 mg ergot alkaloids including 2.00 mg ergovaline daily, and mean minimum and maximum daily ambient temperatures were 11.9 and 17.4°C. Dry matter intake was not affected by fescue seed treatment (P < 0.20) in either experiment. In both experiments, E+ reduced HR (P < 0.01) and increased insulin (P = 0.07). Systolic BP minus diastolic BP decreased (P < 0.05) for E+ in both experiments, due to increased diastolic BP in Experiment 1 (P < 0.03) and decreased systolic BP in Experiment 2 (P < 0.07). In Experiment 1, above the thermoneutral zone, E+ increased (P < 0.05) RR, RT, and left side ST in comparison to E−, but in Experiment 2, within the thermoneutral zone, E+ and E− did not differ (P < 0.18). Ergot alkaloids from fescue seed affect the cardiovascular system of steers separately from effects of feed intake or environmental temperature. Ergot alkaloids interact with ambient temperatures above the steers' thermoneutral zone to exacerbate the symptoms of hyperthermic stress.

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Salt intake may cause conflict for the cardiovascular system as it attempts to simultaneously maintain blood pressure (BP) and temperature homeostasis. Our objective was to determine the effect of a salt and water load vs. a water load upon rectal temperature (Tre) in healthy volunteers. Twenty-two healthy, non-hypertensive Caucasian men enrolled in two trials in which they ingested either salt and body temperature water (SALT), or body temperature water (WATER). BP, Tre, cardiac index, peripheral resistance and urine output were monitored one, 2 and 3 h post-baseline. Changes in the dependent variables were compared between those subjects who were salt sensitive (SS) and those who were salt resistant (SR) at the same time intervals. The percentage change reduction in Tre was greater following SALT compared with WATER at +120 min (À1.1±0.7 vs. À0.6 ± 0.5%, P¼0.009) and at +180 min (À1.3 ± 0.8 vs. À0.7 ± 0.6%, P¼0.003). The percentage change reduction in Tre was greater in the SR group compared with the SS group at +180 min (À1.6 ± 0.9 vs. À0.9 ± 0.5%, P¼0.043). SALT decreased Tre more than WATER. SS individuals maintained temperature homeostasis more effectively than SR individuals following SALT. These results may explain why some individuals are SS while others are SR. If these results are generalizable, it would be possible to account for the role of sodium chloride in the development of SS hypertension.

The sterile insect technique (SIT) is a promising strategy to control the Australian sheep blow fly Lucilia cuprina, a major pest of sheep. We have previously developed a transgenic embryonic sexing system (TESS) for this pest to facilitate the potential SIT application. TESS carry two transgenes, a tetracycline transactivator (tTA) driver and a tTA-activated pro-apoptotic effector. TESS females die at the embryonic stage unless tetracycline is supplied in the diet. However, undesired female sterility was observed in some TESS strains without tetracycline due to expression of tTA in ovaries. Here we investigate if TESS that combine transgenes with relatively low/moderate expression/activity improves the fertility of TESS females. tTA driver lines were evaluated for tTA expression by quantitative real time PCR and/or by crossing with a tTA-activated RFPex effector line. Fertility and lethality tests showed that a TESS strain containing a driver line with moderate tTA expression and an effector line showing moderate pro-apoptotic activity could recover the fertility of parental females and eliminated all female offspring at the embryonic stage. Consequently, such a strain could be further evaluated for an SIT program for L. cuprina, and such a “moderate strategy” could be considered for the TESS development in other pest species.

The transformer (tra) gene is essential for female development in many insect species, including the Australian sheep blow fly, Lucilia cuprina. Sex-specific tra RNA splicing is controlled by Sex lethal (Sxl) in Drosophila melanogaster but is auto-regulated in L. cuprina. Sxl also represses X chromosome dosage compensation in female D. melanogaster. We have developed conditional Lctra RNAi knockdown strains using the tet-off system. Four strains did not produce females on diet without tetracycline and could potentially be used for genetic control of L. cuprina. In one strain, which showed both maternal and zygotic tTA expression, most XX transformed males died at the pupal stage. RNAseq and qRT-PCR analyses of mid-stage pupae showed increased expression of X-linked genes in XX individuals. These results suggest that Lctra promotes somatic sexual differentiation and inhibits X chromosome dosage compensation in female L. cuprina. However, XX flies homozygous for a loss-of-function Lctra knockin mutation were fully transformed and showed high pupal eclosion. Two of five X-linked genes examined showed a significant increase in mRNA levels in XX males. The stronger phenotype in the RNAi knockdown strain could indicate that maternal Lctra expression may be essential for initiation of dosage compensation suppression in female embryos.