Efficient germ-line transformation of the economically important pest species Lucilia cuprina and Lucilia sericata (Diptera, Calliphoridae)

The green blowfly species Lucilia cuprina and Lucilia sericata are economically important pests for the sheep industries of Australia and New Zealand. L. cuprina has long been considered a good target for a genetic pest management program. In addition, L. sericata maggots are used in the cleaning of wounds and necrotic tissue of patients suffering from ulcers that are difficult to treat by other methods. Development of efficient transgenesis methods would greatly facilitate the development of strains ideal for genetic control programs or could potentially improve “maggot therapy”. We have previously reported the germ-line transformation of L. cuprina and the design of a “female killing system” that could potentially be applied to this species. However, the efficiency of transformation obtained was low and transformed lines were difficult to detect due to the low expression of the EGFP marker used. Here we describe an efficient and reliable method for germ-line transformation of L. cuprina using new piggyBac vector and helper plasmids containing the strong promoter from the L. cuprina hsp83 gene to drive expression of the transposase and fluorescent protein marker gene. We also report, for the first time, the germ-line transformation of L. sericata using the new piggyBac vector/helper combination.     

Development and evaluation of male-only strains of the Australian sheep blowfly, <Emphasis Type="Italic">Lucilia cuprina</Emphasis>

The Australian sheep blowfly Lucilia cuprina (Wiedemann) is a major pest of sheep in Australia and New Zealand. From the 1960s to the 1980s there was a major effort to develop "field female killing" or FFK strains of L. cuprina that could be used for a cost-effective genetic control program. The FFK strains carried eye color mutations that were lethal to females in the field but not under conditions in the mass rearing facility. Males did not die in the field as normal copies of the eye color genes had been translocated to the Y chromosome and an autosome. Although the FFK strains showed some promise in field tests, a genetic control program in mainland Australia was never implemented for several reasons including instability of the FFK strains during mass rearing. A stable transgenic strain of L. cuprina that carried one or more dominant repressible female lethal genes offered the potential for efficient genetic control of blowfly populations. Here I review our research on tetracycline-repressible female lethal genetic systems, Lucilia germ-line transformation and sex determination genes that ultimately led to the successful development of transgenic "male-only" strains of L. cuprina. The technology developed for L. cuprina should be directly transferable to other blowfly livestock pests including L. sericata and the New World and Old World screwworm. 29     

Tribolium castaneum Transformer-2 regulates sex determination and development in both males and females

Tribolium castaneum Transformer (TcTra) is essential for female sex determination and maintenance through the regulation of sex-specific splicing of doublesex (dsx) pre-mRNA. In females, TcTra also regulates the sex-specific splicing of its own pre-mRNA to ensure continuous production of functional Tra protein. Transformer protein is absent in males and hence dsx pre-mRNA is spliced in a default mode. The mechanisms by which males inhibit the production of functional Tra protein are not known. Here, we report on functional characterization of transformer-2 (tra-2) gene (an ortholog of Drosophila transformer-2) in T. castaneum. RNA interference-mediated knockdown in the expression of gene coding for tra-2 in female pupae or adults resulted in the production of male-specific isoform of dsx and both female and male isoforms of tra suggesting that Tra-2 is essential for the female-specific splicing of tra and dsx pre-mRNAs. Interestingly, knockdown of tra-2 in males did not affect the splicing of dsx but resulted in the production of both female and male isoforms of tra suggesting that Tra-2 suppresses female-specific splicing of tra pre-mRNA in males. This dual regulation of sex-specific splicing of tra pre-mRNA ensures a tight regulation of sex determination and maintenance. These data suggest a critical role for Tra-2 in suppression of female sex determination cascade in males. In addition, RNAi studies showed that Tra-2 is also required for successful embryonic and larval development in both sexes.     

Regulation of Sex-Specific Selection of fruitless 5′ Splice Sites by transformer and transformer-2

In Drosophila melanogaster, the fruitless (fru) gene controls essentially all aspects of male courtship behavior. It does this through sex-specific alternative splicing of the fru pre-mRNA, leading to the production of male-specific fru mRNAs capable of expressing male-specific fru proteins. Sex-specific fru splicing involves the choice between alternative 5′ splice sites, one used exclusively in males and the other used only in females. Here we report that the Drosophila sex determination genes transformer (tra) and transformer-2 (tra-2) switch fru splicing from the male-specific pattern to the female-specific pattern through activation of the female-specific fru 5′ splice site. Activation of female-specific fru splicing requires cis-acting tra and tra-2 repeat elements that are part of an exonic splicing enhancer located immediately upstream of the female-specific fru 5′ splice site and are recognized by the TRA and TRA-2 proteins in vitro. This fru splicing enhancer is sufficient to promote the activation by tra and tra-2 of both a 5′ splice site and the female-specific doublesex (dsx) 3′ splice site, suggesting that the mechanisms of 5′ splice site activation and 3′ splice site activation may be similar.     

Habitat preferences and carcase colonization by sheep blowflies in the northern North Island of New Zealand

Abstract. In urban Auckland, from September 1991 to May 1992, only six specimens of the Australian sheep blowfly, Lucilia cuprina, were collected in farmed parkland and in garden habitats. The common green blowfly, Lucilia sericata, was the most common calliphorid trapped in these habitats. Neither of the two Lucilia species were found in native bush remnants in urban or rural areas where Calliphora hilli was dominant. Similarly very few L.cuprina (0.3% of the total) were trapped in rural rubbish tips in January and February where the majority of blowflies were again L.sericata. These results are compared with data collected from pastoral habitats, where L.cuprina is the major cause of flystrike. Lucilia cuprina was reared from five out of ninety-nine carcases found in rural areas. Calliphora stygia and L.sericata were the most common blowflies colonizing carrion and were reared from 59% and 51% of the carcases respectively.     

Morphological identification of Lucilia sericata,Lucilia cuprina and their hybrids (Diptera,Calliphoridae)

Hybrids of Lucilia sericata and Lucilia cuprina have been shown to exist in previous studies using molecular methods, but no study has shown explicitly that these hybrids can be identified morphologically. Published morphological characters used to identify L. sericata and L. cuprina were reviewed, and then scored and tested using specimens of both species and known hybrids. Ordination by multi-dimensional scaling indicated that the species were separable, and that hybrids resembled L. cuprina, whatever their origin. Discriminant function analysis of the characters successfully separated the specimens into three unambiguous groups – L. sericata, L. cuprina and hybrids. The hybrids were morphologically similar irrespective of whether they were from an ancient introgressed lineage or more modern. This is the first evidence that hybrids of these two species can be identified from their morphology. The usefulness of the morphological characters is also discussed and photographs of several characters are included to facilitate their assessment.     

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.     

Male-specific phosphorylated SR proteins in adult flies of the Mediterranean Fruitfly <Emphasis Type="Italic">Ceratitis capitata</Emphasis>

Alternative splicing is a widely used mechanism of gene regulation in sex determination pathways of Insects. In species from orders as distant as Diptera, Hymenoptera and Coleoptera, female differentiation relies on the activities of conserved splicing regulators, TRA and TRA-2, promoting female-specific expression of the global effector doublesex (dsx). Less understood is to what extent post-translational modifications of splicing regulators plays a role in this pathway. In Drosophila melanogaster phosphorylation of TRA, TRA-2 and the general RBP1 factor by the LAMMER kinase doa (darkener of apricot) is required for proper female sex determination. To explore whether this is a general feature of the pathway we examined sex-specific differences in phosphorylation levels of SR splicing factors in the dipteran species D. melanogaster, Ceratitis capitata (Medfly) and Musca domestica (Housefly). We found a distinct and reproducible pattern of male-specific phosphorylation on protein extracts enriched for SR proteins in C. capitata suggesting that differential phosphorylation may also contribute to the regulation of sex-specific splicing in the Medfly.     

The mechanism of sex-specific splicing at the doublesex gene is different between Drosophila melanogaster and Bombyx mori

We have previously reported that Bmdsx, a homologue of the sex-determining gene, doublesex (dsx), was found to be sex-specifically expressed in various tissues at larval, pupal, and adult stages in the silkworm, Bombyx mori, and was alternatively spliced to yield male- and female-specific mRNAs. To reveal sex-specific differences in splicing patterns of Bmdsx pre-mRNA, the genomic sequence was determined and compared with male- and female-specific Bmdsx cDNA sequences. The open reading frame (ORF) consisted of five exons. Exons 3 and 4 were specifically incorporated into the female type of Bmdsx mRNA. On the other hand, exon 2 was spliced to exon 5 to produce the male type mRNA of Bmdsx. As in the case of Drosophila dsx, the OD2 domain was separated by a female-specific intron into sex-independent and sex-dependent regions. Sex-specific splicing occurred in equivalent positions in the Drosophila dsx gene. However, unlike Drosophila dsx, the female-specific introns showed no weak 3′ splice sites, and the TRA/TRA-2 binding site related sequences were not found in the female-specific exon, nor even in any other regions of the Bmdsx gene. Moreover, an in vitro splicing reaction consisting of HeLa cell nuclear extracts showed that the female-type of Bmdsx mRNA represented the default splicing. These findings suggest that the structural features of the sex-specific splicing patterns of Bmdsx pre-mRNA are similar to those of Drosophila dsx but the regulation of sex-specific alternative splicing of Bmdsx pre-mRNA is different.     

Comparison of traps for the control of sheep blowfly in the U.K.

The ability of three commercially available trap types to catch Lucilia (Diptera: Calliphoridae) blowflies was assessed on three sheep farms in southwest England in 2008. The aim was to evaluate their relative value for the control of ovine cutaneous myiasis (sheep blowfly strike) on farms. There was a highly significant difference between the total number of female Lucilia caught per day by the traps, with an Agrilure Trap (Agrimin Ltd, Brigg, U.K.) catching more than the other trap types (Rescue Disposable Fly Trap, Sterling International, Spokane, U.S.A.; Redtop Trap, Miller Methods, Johannesburg, South Africa). However, there was no significant difference between the traps in the numbers of female Lucilia sericata (Meigen) caught. Nevertheless, consideration of the rate at which female L. sericata were caught over time showed that the Agrilure trap did not begin catching until about 30 days after its initial deployment. It subsequently caught L. sericata at a faster rate than the other two traps. The data suggest that the freeze‐dried liver bait used in the Agrilure trap required a period of about 30 days to become fully rehydrated and decompose to the degree required to attract and catch L. sericata. Once the bait was attractive, however, the trap outperformed the other two traps in terms of the rate of L. sericata capture. The Agrilure trap would appear to be the most effective of the designs tested for use against sheep blowfly and blowfly strike in the U.K., but care would be needed to ensure that the traps were deployed in advance of the blowfly season so that the bait was suitably aged when trapping was required.     

The mechanism of sex-specific splicing at the doublesex gene is different between Drosophila melanogaster and Bombyx mori

We have previously reported that Bmdsx, a homologue of the sex-determining gene, doublesex (dsx), was found to be sex-specifically expressed in various tissues at larval, pupal, and adult stages in the silkworm, Bombyx mori, and was alternatively spliced to yield male- and female-specific mRNAs. To reveal sex-specific differences in splicing patterns of Bmdsx pre-mRNA, the genomic sequence was determined and compared with male- and female-specific Bmdsx cDNA sequences. The open reading frame (ORF) consisted of five exons. Exons 3 and 4 were specifically incorporated into the female type of Bmdsx mRNA. On the other hand, exon 2 was spliced to exon 5 to produce the male type mRNA of Bmdsx. As in the case of Drosophila dsx, the OD2 domain was separated by a female-specific intron into sex-independent and sex-dependent regions. Sex-specific splicing occurred in equivalent positions in the Drosophila dsx gene. However, unlike Drosophila dsx, the female-specific introns showed no weak 3′ splice sites, and the TRA/TRA-2 binding site related sequences were not found in the female-specific exon, nor even in any other regions of the Bmdsx gene. Moreover, an in vitro splicing reaction consisting of HeLa cell nuclear extracts showed that the female-type of Bmdsx mRNA represented the default splicing. These findings suggest that the structural features of the sex-specific splicing patterns of Bmdsx pre-mRNA are similar to those of Drosophila dsx but the regulation of sex-specific alternative splicing of Bmdsx pre-mRNA is different.     

The gene fl(2)d is needed for the sex-specific splicing of transformer pre-mRNA but not for double-sex pre-mRNA in Drosophila melanogaster

In Drosophila melanogaster, regulation of the sex determination genes throughout development occurs by sex-specific splicing of their products. The first gene is Sex-lethal(Sxl). The downstream target of Sxl is the gene transformer (tra): the Sxl protein controls the female-specific splicing of the Tra pre-mRNA. The downstream target of the gene tra is the gene double-sex (dsx): the Tra protein of females, controls the female-specific splicing of the Dsx pre-mRNA. We have identified a gene, female-lethal-2-d fl(2)d, whose function is required for the female-specific splicing of Sxl pre-mRNA. In this report we analyze whether the gene fl(2)d is also required for the sex-specific splicing of both Tra and Dsx pre-mRNAs. We found that the Sxl protein is not sufficient for the female-specific splicing of Tra pre-mRNA, the fl(2)d function also being necessary. This gene, however, is not required for the female-specific splicing of Dsx pre-mRNA.     

Functionally antagonistic sequences are required for normal autoregulation of Drosophila tra-2 pre-mRNA splicing

Expression of functional TRA-2 protein in the male germline of Drosophila is regulated through a negative feedback mechanism in which a specific TRA-2 isoform represses splicing of the M1 intron in the TRA-2 pre-mRNA. We have previously shown that the mechanism of M1 splicing repression is conserved between distantly related Drosophila species. Using transgenic fly strains, we have examined the effects on regulation of mutations in two conserved features of the M1 intron. Our results show that TRA-2-dependent repression of M1 splicing depends on the presence of a suboptimal non-consensus 3′ splice site. Substitution of this 3′ splice site with a strong splice site resulted in TRA-2 independent splicing, while substitution with an unrelated weak 3′ splice site was compatible with repression, implying that reduced basal splicing efficiency is important for regulation. A second conserved element internal to the intron was found to be essential for efficient M1 splicing in the soma where the intron is not normally retained. We show that the role of this element is to enhance splicing and overcome the reduction in efficiency caused by the intron’s suboptimal 3′ splice site. Our results indicate that antagonistic elements in the M1 intron act together to establish a context that is permissive for repression of splicing by TRA-2 while allowing efficient splicing in the absence of a repressor.     

In vitro antibacterial activity and physicochemical properties of a crude methanol extract of the larvae of the blow fly Lucilia cuprina

The emergence of multidrug‐resistant bacterial strains has prompted the reintroduction of maggot therapy in the treatment of chronic, infected wounds. Many previous studies have demonstrated the potent antibacterial activity of larval excretions/secretions of the blowfly Lucilia sericata (Meigen) (Diptera:Calliphoridae) against bacteria. However, the antibacterial activity of its sibling species, Lucilia cuprina (Wiedemann) (Diptera:Calliphoridae) against a wide range of pathogenic bacteria has never been determined. The aim of this study was to develop a new procedure to produce whole body extract of larvae of L. cuprina via methanol extraction as well as to demonstrate the in vitro antibacterial activity of this extract against seven selected wound pathogens (Staphylococcus aureus, methicillin‐resistant S. aureus, S. epidermidis, Streptococcus pyogenes, Klebsiella pneumoniae, Pseudomonas aeruginosa and Escherichia coli). The turbidimetric assay demonstrated that L. cuprina larval extract was significantly potent against all bacteria tested (P < 0.001). Additionally, colony‐forming unit (CFU), agar well diffusion and minimum inhibitory concentration assays have confirmed the apparent potency of larval extract against P. aeruginosa. The reconstituted larval extract was highly robust and thermally stable. These observations substantiated the feasibility of the methanol extraction method in the production of larval extract.     

The Orthologue of the Fruitfly Sex Behaviour Gene Fruitless in the Mosquito Aedes aegypti: Evolution of Genomic Organisation and Alternative Splicing

In Drosophila melanogaster the doublesex (dsx) and fruitless (fru) regulatory genes act at the bottom of the somatic sex determination pathway. Both are regulated via alternative splicing by an upstream female-specific TRA/TRA-2 complex, recognizing a common cis element. dsx controls somatic sexual differentiation of non-neural as well as of neural tissues. fru, on the other hand, expresses male-specific functions only in neural system where it is required to built the neural circuits underlying proper courtship behaviour. In the mosquito Aedes aegypti sex determination is different from Drosophila. The key male determiner M, which is located on one of a pair of homomorphic sex chromosomes, controls sex-specific splicing of the mosquito dsx orthologue. In this study we report the genomic organization and expression of the fru homologue in Ae. aegypti (Aeafru). We found that it is sex-specifically spliced suggesting that it is also under the control of the sex determination pathway. Comparative analyses between the Aeafru and Anopheles gambiae fru (Angfru) genomic loci revealed partial conservation of exon organization and extensive divergence of intron lengths. We find that Aeadsx and Aeafru share novel cis splicing regulatory elements conserved in the alternatively spliced regions. We propose that in Aedes aegypti sex-specific splicing of dsx and fru is most likely under the control of splicing regulatory factors which are different from TRA and TRA-2 found in other dipteran insects and discuss the potential use of fru and dsx for developing new genetic strategies in vector control.