Sheep Blowflies Research Articles

DIFFERENCE EQUATION FOR AUSTRALIAN SHEEP BLOWFLIES GROWTH

The population of Australian sheep blowflies, Lucilia cuprina, in Australia is of concern to many researchers because it causes several problems. These problems occur in the sheep industry where there is a term "flystrike" in the industry. Flystrike is a fly attack on sheep that causes myiasis on the sheep's skin, affecting the quality and quantity of wool. In the worst cases, the sheep may die if not treated. This issue has attracted researcher to conduct a population control study of fly growth to suppress flystrike in the Australian sheep industry. In this paper, fly growth will be approached using a difference equation to better represent the industry’s situation. This equation will be analyzed using its approximate solution that is obtained through linearization of perturbation method, Cardano’s formula, and Galois solution’s method. By studying fly growth, Australian sheep farmers may find it easier to handle and prevent fly infestations using the solution.

The Deadly Toxin Arsenal of the Tree-Dwelling Australian Funnel-Web Spiders.

Australian funnel-web spiders are amongst the most dangerous venomous animals. Their venoms induce potentially deadly symptoms, including hyper- and hypotension, tachycardia, bradycardia and pulmonary oedema. Human envenomation is more frequent with the ground-dwelling species, including the infamous Sydney funnel-web spider (Atrax robustus); although, only two tree-dwelling species induce more severe envenomation. To unravel the mechanisms that lead to this stark difference in clinical outcomes, we investigated the venom transcriptome and proteome of arboreal Hadronyche cerberea and H. formidabilis. Overall, Hadronyche venoms comprised 44 toxin superfamilies, with 12 being exclusive to tree-dwellers. Surprisingly, the major venom components were neprilysins and uncharacterized peptides, in addition to the well-known ω- and δ-hexatoxins and double-knot peptides. The insecticidal effects of Hadronyche venom on sheep blowflies were more potent than Atrax venom, and the venom of both tree- and ground-dwelling species potently modulated human voltage-gated sodium channels, particularly NaV1.2. Only the venom of tree-dwellers exhibited potent modulation of voltage-gated calcium channels. H. formidabilis appeared to be under less diversifying selection pressure compared to the newly adapted tree-dweller, H. cerberea. Thus, this study contributes to unravelling the fascinating molecular and pharmacological basis for the severe envenomation caused by the Australian tree-dwelling funnel-web spiders.

CRISPR/Cas9 Genome Editing in the New World Screwworm and Australian Sheep Blowfly.

Blowflies are of interest for medical applications (maggot therapy), forensic investigations, and for evolutionary developmental studies such as the evolution of parasitism. It is because of the latter that some blowflies such as the New World screwworm and the Australian sheep blowfly are considered major economic pests of livestock. Due to their importance, annotated assembled genomes for several species are now available. Here, we present a detailed guide for using the Streptococcus pyogenes Cas9 RNA-guided nuclease to efficiently generate both knockout and knock-in mutations in screwworm and sheep blowfly. These methods should accelerate genetic investigations in these and other closely related species and lead to a better understanding of the roles of selected genes in blowfly development and behavior.

Conditional knockdown of transformer in sheep blow fly suggests a role in repression of dosage compensation and potential for population suppression.

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.

Crouching Tiger, Hidden Protein: Searching for Insecticidal Toxins in Venom of the Red Tiger Assassin Bug (Havinthus rufovarius).

Assassin bugs are venomous insects that prey on other arthropods. Their venom has lethal, paralytic, and liquifying effects when injected into prey, but the toxins responsible for these effects are unknown. To identify bioactive assassin bug toxins, venom was harvested from the red tiger assassin bug (Havinthus rufovarius), an Australian species whose venom has not previously been characterised. The venom was fractionated using reversed-phase high-performance liquid chromatography, and four fractions were found to cause paralysis and death when injected into sheep blowflies (Lucilia cuprina). The amino acid sequences of the major proteins in two of these fractions were elucidated by comparing liquid chromatography/tandem mass spectrometry data with a translated venom-gland transcriptome. The most abundant components were identified as a solitary 12.8 kDa CUB (complement C1r/C1s, Uegf, Bmp1) domain protein and a 9.5 kDa cystatin. CUB domains are present in multidomain proteins with diverse functions, including insect proteases. Although solitary CUB domain proteins have been reported to exist in other heteropteran venoms, such as that of the bee killer assassin bug Pristhesancus plagipennis, their function is unknown, and they have not previously been reported as lethal or paralysis-inducing. Cystatins occur in the venoms of spiders and snakes, but again with an unknown function. Reduction and alkylation experiments revealed that the H. rufovarius venom cystatin featured five cysteine residues, one of which featured a free sulfhydryl group. These data suggest that solitary CUB domain proteins and/or cystatins may contribute to the insecticidal activity of assassin bug venom.

Building a transgenic sexing strain for genetic control of the Australian sheep blow fly Lucilia cuprina using two lethal effectors

BackgroundThe sterile insect technique (SIT) has been successfully used in many pest management programs worldwide. Some SIT programs release both sexes due to the lack of genetic sexing strains or efficient sex separation methods but sterile females are ineffective control agents. Transgenic sexing strains (TSS) using the tetracycline-off control system have been developed in a variety of insect pests, from which females die by either of two commonly used lethal effectors: overexpression of the transcription factor tetracycline transactivator (tTA) or ectopic expression of a proapoptotic gene, such as head involution defective (hid). The lethality from tTA overexpression is thought to be due to “transcriptional squelching”, while hid causes lethality by induction of apoptosis. This study aims to create and characterize a TSS of Lucilia cuprina, which is a major pest of sheep, by combining both lethal effectors in a single transgenic strain.ResultsHere a stable TSS of L. cuprina (DH6) that carries two lethal effectors was successfully generated, by crossing FL3#2 which carries a female-specific tTA overexpression cassette, with EF1#12 which carries a tTA-regulated LshidAla2 cassette. Females with one copy of the FL3#2 transgene are viable but up to 99.8% of homozygous females die at the pupal stage when raised on diet that lacks tetracycline. Additionally, the female lethality of FL3#2 was partially repressed by supplying tetracycline to the parental generation. With an additional LshidAla2 effector, the female lethality of DH6 is 100% dominant and cannot be repressed by maternal tetracycline. DH6 females die at the late-larval stage. Several fitness parameters important for mass rearing such as hatching rate, adult emergence and sex ratio were comparable to those of the wild type strain.ConclusionsCompared to the parental FL3#2 strain, the DH6 strain shows stronger female lethality and lethality occurs at an earlier stage of development. The combination of two tTA-dependent lethal effectors could improve strain stability under mass rearing and could reduce the risk of resistance in the field if fertile males are released. Our approach could be easily adapted for other pest species for an efficient, safe and sustainable genetic control program.

Using Moderate Transgene Expression to Improve the Genetic Sexing System of the Australian Sheep Blow Fly Lucilia cuprina.

Simple SummaryPopulations of pest insects can be suppressed through repeated mass releases of sterilized insects. This is particularly effective if only sterile males are released. We previously developed several genetically modified strains of the Australian sheep blowfly that produce only males when raised on diet that lacked tetracycline. A disadvantage of the some of the engineered strains was that females would lay few eggs unless fed a diet with a low dose of tetracycline. In this study we show that effective male-only strains can be made by combining driver/effector lines that have moderate transgene expression/activity. Furthermore, the strain does not require tetracycline in the adult diet for female fertility. This “moderate expression/activity” strategy could be more generally applied to other pests that can be genetically modified.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.

Australian funnel-web spiders evolved human-lethal δ-hexatoxins for defense against vertebrate predators

Australian funnel-web spiders are infamous for causing human fatalities, which are induced by venom peptides known as δ-hexatoxins (δ-HXTXs). Humans and other primates did not feature in the prey or predator spectrum during evolution of these spiders, and consequently the primate lethality of δ-HXTXs remains enigmatic. Funnel-web envenomations are mostly inflicted by male spiders that wander from their burrow in search of females during the mating season, which suggests a role for δ-HXTXs in self-defense since male spiders rarely feed during this period. Although 35 species of Australian funnel-web spiders have been described, only nine δ-HXTXs from four species have been characterized, resulting in a lack of understanding of the ecological roles and molecular evolution of δ-HXTXs. Here, by profiling venom-gland transcriptomes of 10 funnel-web species, we report 22 δ-HXTXs. Phylogenetic and evolutionary assessments reveal a remarkable sequence conservation of δ-HXTXs despite their deep evolutionary origin within funnel-web spiders, consistent with a defensive role. We demonstrate that δ-HXTX-Ar1a, the lethal toxin from the Sydney funnel-web spider Atrax robustus, induces pain in mice by inhibiting inactivation of voltage-gated sodium (NaV) channels involved in nociceptive signaling. δ-HXTX-Ar1a also inhibited inactivation of cockroach NaV channels and was insecticidal to sheep blowflies. Considering their algogenic effects in mice, potent insecticidal effects, and high levels of sequence conservation, we propose that the δ-HXTXs were repurposed from an initial insecticidal predatory function to a role in defending against nonhuman vertebrate predators by male spiders, with their lethal effects on humans being an unfortunate evolutionary coincidence.

Development of Lucilia sericata (Diptera: Calliphoridae) Under Constant Temperatures and its Significance for the Estimation of Time of Death.

Forensic entomologists usually estimate the minimum postmortem interval (PMImin) using the time required for the oldest immature insects found on the corpse to grow to its development stage and age at the time of discovery. The sheep blow fly Lucilia sericata (Meigen, 1826) is a carrion fly found nearly worldwide, and important in forensics. We studied the development time of L. sericata from egg to adult at constant temperatures of 16, 19, 22, 25, 28, 31, and 34°C, and found that the times required are 913.2 ± 19.4, 588.8 ± 35.8, 459.8 ± 15.2, 373.2 ± 15.3, 308.0 ± 9.7, 272.5 ± 9.2, and 267.5 ± 10.5 h, respectively. We established three development models to infer the age of the immature insect: isomegalen diagram, isomorphen diagram, and thermal summation model. In addition, a regression analysis was performed on the relationship between body length and total development time from hatching to dispersing. The thermal summation constant during the development of L. sericata is 6023.2 degree hours and development threshold temperature is 9.19°C. The results of this experiment provide a basis for the use of L. sericata in the estimation of PMImin.

Improved transgenic sexing strains for genetic control of the Australian sheep blow fly Lucilia cuprina using embryo-specific gene promoters.

For genetic approaches for controlling insect pests such as the sterile insect technique (SIT), it is advantageous to release only males as females are ineffective as control agents and they consume about 50% of the diet. Here we developed tetracycline-repressible Lucilia cuprina transgenic strains in which adult females were fully fertile and viable on a diet that lacked tetracycline and all of their female offspring died at the embryo stage. The transgenic strains are an improvement over the strains we developed previously, which had the disadvantage that adult females on diet without tetracycline were sterile and died prematurely. This was possibly due to the low level expression of the effector gene in ovaries. In the strains developed in this study, the early promoters from L. cuprina nullo or Cochliomyia macellaria CG14427 genes were used to drive the tetracycline transactivator (tTA) expression in the early embryo. In the absence of tetracycline, tTA activates expression of the proapoptotic gene Lshid which contains a female-specific intron. Consequently, only females produce active HID protein and die at the embryo stage. Crossing the tTA-expressing driver lines with an RFPex reporter line confirmed that there was no expression of the effector gene in the ovary. These new embryonic L. cuprina transgenic sexing strains hold great promise for genetic control programs and the system reported here might also be transferable to other major calliphorid livestock pests such as the New World screwworm, Cochliomyia hominivorax.

Volatiles from Merino fleece evoke antennal and behavioural responses in the Australian sheep blow fly Lucilia cuprina.

To identify flystrike-related volatile compounds in wool from Merino sheep, the attractiveness of wool to Lucilia cuprina Wiedmann (Diptera: Calliphoridae) was examined. First, a selection of wool samples guided by previous knowledge of sheep lines, predicted to be more susceptible or more resistant to flystrike, was tested. The attractiveness of the 10 samples selected was not associated with field susceptibility: two samples from the more resistant line were identified as most attractive and two samples from the more susceptible line were identified as least attractive, based on the behavioural assays with gravid flies. Comparison of the headspace volatiles of these samples, using solid phase microextraction and gas chromatography-mass spectrometry-electroantennographic detection, revealed octanal and nonanal to be present in the attractive wool samples that elicited responses from the fly antenna. Furthermore, the two compounds were not present in wool that was least attractive to L. cuprina. In laboratory bioassays, octanal and nonanal evoked antennal and behavioural responses in gravid L. cuprina, thus confirming their potential role as semiochemicals responsible for attracting L. cuprina to Merino sheep.

Benzoylphenyl ureas as veterinary antiparasitics. An overview and outlook with emphasis on efficacy, usage and resistance.

Six benzoylphenyl ureas are currently used in formulations approved as veterinary medicines: diflubenzuron for fly control mainly on cattle, lice and blowfly strike control on sheep, and lice control on farmed salmonids; lufenuron for flea control on dogs and cats and for lice control on farmed salmonids; triflumuron for lice and blowfly strike control on sheep; fluazuron for tick control on cattle; teflubenzuron for lice control on farmed salmon; and novaluron for fly and tick control on cattle and for flea control on dogs. Resistance to diflubenzuron and triflumuron has already been reported for sheep body lice and blowflies, and to fluazuron in cattle ticks. These and other minor veterinary usages, as well as the current status of resistance, are reviewed and perspectives for future opportunities are discussed based on unexplored potentials and threats posed by future resistance development.

The Effects of Diets and Long-term Laboratory Rearing on Reproduction, Behavior, and Morphology of Lucilia cuprina (Diptera: Calliphoridae).

The Australian sheep blow fly, Lucilia cuprina (Wiedemann), is commonly reared in the laboratory for many sequential generations on simple, fixed diets, so it can be used in veterinary, medical, and forensic studies. To investigate the effect of diet and long-term laboratory rearing on L. cuprina, flies were fed with two different diets (sugar and milk-sugar) over a year and F1, F6, and F11 generations were used for comparisons based on the number of eggs, attraction to wool and liver, and wing size. The results showed that the number of eggs of gravid flies, and the attractiveness of wool and liver did not differ significantly between diets and generations, but gravid flies were more attracted to wool and liver than non-gravid flies (P < 0.05). Moreover, in the F1 generation, thorax length and wing aspect ratio were significantly longer than in the F6 and F11 generations (P < 0.05), and the wing length was significantly longer than in the F11 generation (P < 0.05). It was concluded that neither diet nor long-term laboratory rearing affect potential fecundity or the behavioral responses of L. cuprina, but the gravidity of flies affects their behavioral response, and long-term laboratory rearing significantly affects fly morphology, apparently explaining a loss in flight performance.

Dipteran toxicity assays for determining the oral insecticidal activity of venoms and toxins

The growing world population is placing an increasing demand on food production. In addition, abuse and misuse of chemical insecticides has led to the evolution of resistance in insect pests as well as environmental damage. Together, these developments have created a demand for new insecticidal compounds to facilitate global food production. Arachnid venom peptides provide an environmentally-friendly alternative as potential bioinsecticides given their advantages of being fully biodegradable, highly potent, and phyletically selective. However, the use of arachnid venom peptides as bioinsecticides has been questioned due to their presumed lack of oral toxicity. Thus, the aim of this work was to develop screens for oral insecticidal activity. Based on the high susceptibility of dipterans to venom peptides, fruit flies (Drosophila melanogaster) and sheep blowflies (Lucilia cuprina) were selected for screening 56 arachnid venoms. 71.4% of these venoms caused 50% or higher mortality in Drosophila, whereas 30.4% were lethal to blowflies at oral doses of 1 or 30 μg/fly, respectively. We used these assays to compare the oral and injection activity of four well-known spider venom peptides (Hv1a, Hv1c, Dc1a and Ta1a). Hv1c and Ta1a only showed weak or no oral activity in both species, while Hv1a and Dc1a showed higher oral activity in blowflies than Drosophila. Overall, we have established screens for oral toxicity in two dipteran insects. Our results indicate that oral insecticidal activity is more widespread in arachnid venoms than expected, and that some arachnid venoms and venom peptides exhibit phyletic differences in oral toxicity.

No blokes Is Essential for Male Viability and X Chromosome Gene Expression in the Australian Sheep Blowfly

It has been hypothesized that the Drosophila 4thchromosome is derived from an ancient X chromosome [1]. In the Australian sheep blowfly, Lucilia cuprina, the heterochromatic X chromosome contains few active genes and orthologs of Drosophila X-linked genes are autosomal. Of 8 X-linked genes identified previously in L.cuprina, 6 were orthologs of Drosophila 4th-chromosome genes [2]. The X-linked genes were expressed equally in males and females. Here we identify an additional 51 X-linked genes and show that most are dosage compensated. Orthologs of 49 of the 59 X-linked genes are on the4thchromosome in D.melanogaster. Because painting of fourth (Pof) is important for expression ofDrosophila 4th-chromosome genes [3], we used Cas9 to make a loss-of-function knockin mutation in an L.cuprina Pof ortholog we call no blokes (nbl). Homozygous nbl males derived from homozygous nbl mothers die at the late pupal stage. Homozygous nbl females are viable, fertile, and live longer than heterozygous nbl females. RNA expression of most X-linked genes was reduced in homozygous nbl male pupae and to a lesser extent in nbl females compared to heterozygous siblings. The results suggest that NBL could be important for X chromosome dosage compensation in L.cuprina. NBL may also facilitate gene expression in the heterochromatic environment of the X chromosome in both sexes. This study supports the hypothesis on the origin of the Drosophila 4th chromosome and that a POF-like protein was required for normal gene expression on the ancient X chromosome.

Closing the gap between Aboriginal and non-Aboriginal health workers through story telling

Non-thermal plasma has been used in many medical applications, including treatment of living cells, blood coagulation, wound healing, and sterilization. The process uses an environmentally friendly gas (e.g., argon, helium, oxygen, nitrogen, or hydrogen) to destroy bacteria cells with no serious adverse effect on humans or animals. However, information on the effect of argon plasma on blow fly eggs is lacking. In this study, we explored the ability of cold argon plasma to destroy the eggs of the Australian sheep blow fly, Lucilia cuprina (Wiedemann, 1830); its larvae are a myiasis-producing agent in both human and animals. We tested the effect of cold argon plasma exposure for 1, 2, 3 and 5 min on L. cuprina eggs. Since the temperature of cold Ar plasma is around 30 °C, to clarify the effect of temperature on the fly eggs, hot air from an electric dryer was tested for comparison. Cold argon plasma exposure in eggs significantly reduced the survival rates of second instar larvae at all exposures tested; the effects were time dependent, with a stronger effect at longer exposure (32% survival rate after a 1-min treatment; 20%, 2 min; 20%, 3 min; and 6%, 5 min), compared to the control (86%). No significant differences were observed in larval survival rates from eggs treated with hot air (80-84%, after 1- to 5-min treatments) versus the control (86%). These results were supported by observing the treated eggshells under a scanning electron microscope (SEM), we found noticeable aberrations only in the plasma treated groups. The emission spectrum of the argon gas discharge revealed emission lines of hydroxyl radicals at 309.1 nm; these may cause the deterioration of the treated L. cuprina eggs. Our results have shown the possibility of using cold argon plasma in medical applications, in particular treating myiasis wounds.

Discovery and mode of action of a novel analgesic β-toxin from the African spider Ceratogyrus darlingi.

Spider venoms are rich sources of peptidic ion channel modulators with important therapeutical potential. We screened a panel of 60 spider venoms to find modulators of ion channels involved in pain transmission. We isolated, synthesized and pharmacologically characterized Cd1a, a novel peptide from the venom of the spider Ceratogyrus darlingi. Cd1a reversibly paralysed sheep blowflies (PD50 of 1318 pmol/g) and inhibited human Cav2.2 (IC50 2.6 μM) but not Cav1.3 or Cav3.1 (IC50 > 30 μM) in fluorimetric assays. In patch-clamp electrophysiological assays Cd1a inhibited rat Cav2.2 with similar potency (IC50 3 μM) without influencing the voltage dependence of Cav2.2 activation gating, suggesting that Cd1a doesn’t act on Cav2.2 as a classical gating modifier toxin. The Cd1a binding site on Cav2.2 did not overlap with that of the pore blocker ω-conotoxin GVIA, but its activity at Cav2.2-mutant indicated that Cd1a shares some molecular determinants with GVIA and MVIIA, localized near the pore region. Cd1a also inhibited human Nav1.1–1.2 and Nav1.7–1.8 (IC50 0.1–6.9 μM) but not Nav1.3–1.6 (IC50 > 30 μM) in fluorimetric assays. In patch-clamp assays, Cd1a strongly inhibited human Nav1.7 (IC50 16 nM) and produced a 29 mV depolarising shift in Nav1.7 voltage dependence of activation. Cd1a (400 pmol) fully reversed Nav1.7-evoked pain behaviours in mice without producing side effects. In conclusion, Cd1a inhibited two anti-nociceptive targets, appearing to interfere with Cav2.2 inactivation gating, associated with the Cav2.2 α-subunit pore, while altering the activation gating of Nav1.7. Cd1a was inactive at some of the Nav and Cav channels expressed in skeletal and cardiac muscles and nodes of Ranvier, apparently contributing to the lack of side effects at efficacious doses, and suggesting potential as a lead for development of peripheral pain treatments.

Effect of cold argon plasma on eggs of the blow fly, Lucilia cuprina (Diptera: Calliphoridae)

Non-thermal plasma has been used in many medical applications, including treatment of living cells, blood coagulation, wound healing, and sterilization. The process uses an environmentally friendly gas (e.g., argon, helium, oxygen, nitrogen, or hydrogen) to destroy bacteria cells with no serious adverse effect on humans or animals. However, information on the effect of argon plasma on blow fly eggs is lacking. In this study, we explored the ability of cold argon plasma to destroy the eggs of the Australian sheep blow fly, Lucilia cuprina (Wiedemann, 1830); its larvae are a myiasis-producing agent in both human and animals. We tested the effect of cold argon plasma exposure for 1, 2, 3 and 5min on L. cuprina eggs. Since the temperature of cold Ar plasma is around 30°C, to clarify the effect of temperature on the fly eggs, hot air from an electric dryer was tested for comparison. Cold argon plasma exposure in eggs significantly reduced the survival rates of second instar larvae at all exposures tested; the effects were time dependent, with a stronger effect at longer exposure (32% survival rate after a 1-min treatment; 20%, 2min; 20%, 3min; and 6%, 5min), compared to the control (86%). No significant differences were observed in larval survival rates from eggs treated with hot air (80-84%, after 1- to 5-min treatments) versus the control (86%). These results were supported by observing the treated eggshells under a scanning electron microscope (SEM), we found noticeable aberrations only in the plasma treated groups. The emission spectrum of the argon gas discharge revealed emission lines of hydroxyl radicals at 309.1nm; these may cause the deterioration of the treated L. cuprina eggs. Our results have shown the possibility of using cold argon plasma in medical applications, in particular treating myiasis wounds.

Building early-larval sexing systems for genetic control of the Australian sheep blow fly Lucilia cuprina using two constitutive promoters

Transgenic sexing strains (TSS) that carry conditional female lethal genes are advantageous for genetic control programs based on the sterile insect technique (SIT). It is desirable if females die early in development as larval diet is a major cost for mass production facilities. This can be achieved by using a gene promoter that is only active in embryos to drive expression of the tetracycline transactivator (tTA), the transcription factor commonly used in two-component TSS. While an embryo-specific promoter is ideal it may not be essential for assembling an effective TSS as tTA can be repressed by addition of tetracycline to the diet at larval and/or adult stages. Here we have investigated this idea by isolating and employing the promoters from the Lucilia spitting image and actin 5C genes to drive tTA expression in embryos and later stages. L. cuprina TSS with the tTA drivers and tTA-regulated tetO-Lshid effectors produced only females when raised on a limited tetracycline diet. The Lshid transgene contains a sex-specific intron and as a consequence only females produce LsHID protein. TSS females died at early larval stages, which makes the lines advantageous for an SIT program.

Isolation of two insecticidal toxins from venom of the Australian theraphosid spider Coremiocnemis tropix

Sheep flystrike is caused by parasitic flies laying eggs on soiled wool or open wounds, after which the hatched maggots feed on the sheep flesh and often cause large lesions. It is a significant economic problem for the livestock industry as infestations are difficult to control due to ongoing cycles of larval development into flies followed by further egg laying. We therefore screened venom fractions from the Australian theraphosid spider Coremiocnemis tropix to identify toxins active against the sheep blowfly Lucilia cuprina, which is the primary cause of flystrike in Australia. This screen led to isolation of two insecticidal peptides, Ct1a and Ct1b, that are lethal to blowflies within 24 h of injection. The primary structure of these peptides was determined using a combination of Edman degradation and sequencing of a C. tropix venom-gland transcriptome. Ct1a and Ct1b contain 39 and 38 amino acid residues, respectively, including six cysteine residues that form three disulfide bonds. Recombinant production in bacteria (Escherichia coli) resulted in low yields of Ct1a whereas solid-phase peptide synthesis using native chemical ligation produced sufficient quantities of Ct1a for functional analyses. Synthetic Ct1a had no effect on voltage-gated sodium channels from the American cockroach Periplanata americana or the German cockroach Blattella germanica, but it was lethal to sheep blowflies with an LD50 of 1687 pmol/g.