Antibacterial activity and pore-forming properties of ceratotoxins: a mechanism of action based on the barrel stave model

Ceratotoxins are alpha-helical cationic peptides isolated from the medfly Ceratitis capitata. These amphipathic peptides were found to display strong antibacterial activity and weak hemolytic activity. When reconstituted into planar lipid bilayers, ceratotoxins developed highly asymmetric I/V curves under voltage ramps and formed, in single-channel experiments, well-defined voltage-dependent ion channels according to the barrel stave model. The antibacterial activity and pore-forming properties of these molecules were well correlated. Similar experiments performed with synthesized truncated fragments showed that the C-terminal domain of ceratotoxins is strongly implicated in the formation of helical bundles in the membrane whereas the largely cationic N-terminal region is likely to anchor ceratotoxins on the lipid surface.     

The ceratotoxin gene family in the medfly Ceratitis capitata and the Natal fruit fly Ceratitis rosa (Diptera: Tephritidae)

Ceratotoxins (Ctxs) are a family of antibacterial sex-specific peptides expressed in the female reproductive accessory glands of the Mediterranean fruit fly Ceratitis capitata. As a first step in the study of molecular evolution of Ctx genes in Ceratitis, partial genomic sequences encoding four distinct Ctx precursors have been determined. In addition, anti-Escherichia coli activity very similar to that of the accessory gland secretion from C. capitata was found in the accessory gland secretion from Ceratitis (Pterandrus) rosa. SDS-PAGE analysis of the female reproductive accessory glands from C. rosa showed a band with a molecular mass (3 kDa) compatible with that of Ctx peptides, also slightly reacting with an anti-Ctx serum. Four nucleotide sequences encoding Ctx-like precursors in C. rosa were determined. Sequence and phylogenetic analyses show that Ctxs from C. rosa fall into different groups as C. capitata Ctxs. Our results suggest that the evolution of the ceratotoxin gene family might be viewed as a combination of duplication events that occurred prior to and following the split between C. capitata and C. rosa. Genomic hybridization demonstrated the presence of multiple Ctx-like sequences in C. rosa, but low-stringency Southern blot analyses failed to recover members of this gene family in other tephritid flies.     

地中海实蝇幼虫分子鉴定

通过对从秘鲁进口的葡萄中截获实蝇类幼虫进行ITS区和线粒体COⅠ,COⅡ,COⅢ、ND5基因序列的扩增和测序,并与GenBank中对应的序列进行比对,结果表明,截获样品ITS区序列和地中海实蝇Ceratitis capitata(Wiedemann)同源性为95·16%(其中ITS1为99·52%,ITS2为86·2%),线粒体COⅠ,COⅡ,COⅢ,ND5基因序列和地中海实蝇C.capitata同源性为100%,99·9%,99·5%,99·8%;基于COⅠ序列构建的系统发育树中,幼虫样品和地中海实蝇最为接近。根据序列分析和系统发育关系分析的结果,将截获的实蝇类幼虫鉴定为地中海实蝇C.capitata。     

Evolutionary conservation of Ceratitis capitata transformer gene function

Transformer functions as a binary switch gene in the sex determination and sexual differentiation of Drosophila melanogaster and Ceratitis capitata, two insect species that separated nearly 100 million years ago. The TRA protein is required for female differentiation of XX individuals, while XY individuals express smaller, presumably nonfunctional TRA peptides and consequently develop into adult males. In both species, tra confers female sexual identity through a well-conserved double-sex gene. However, unlike Drosophila tra, which is regulated by the upstream Sex-lethal gene, Ceratitis tra itself is likely to control a feedback loop that ensures the maintenance of the female sexual state. The putative CcTRA protein shares a very low degree of sequence identity with the TRA proteins from Drosophila species. However, in this study we show that a female-specific Ceratitis Cctra cDNA encoding the putative full-length CcTRA protein is able to support the female somatic and germline sexual differentiation of D. melanogaster XX; tra mutant adults. Although highly divergent, CcTRA can functionally substitute for DmTRA and induce the female-specific expression of both Dmdsx and Dmfru genes. These data demonstrate the unusual plasticity of the TRA protein that retains a conserved function despite the high evolutionary rate. We suggest that transformer plays an important role in providing a molecular basis for the variety of sex-determining systems seen among insects.     

The transformer gene in Ceratitis capitata provides a genetic basis for selecting and remembering the sexual fate

The medfly Ceratitis capitata contains a gene (Cctra) with structural and functional homology to the Drosophila melanogaster sex-determining gene transformer (tra). Similar to tra in Drosophila, Cctra is regulated by alternative splicing such that only females can encode a full-length protein. In contrast to Drosophila, however, where tra is a subordinate target of Sex-lethal (Sxl), Cctra seems to initiate an autoregulatory mechanism in XX embryos that provides continuous tra female-specific function and act as a cellular memory maintaining the female pathway. Indeed, a transient interference with Cctra expression in XX embryos by RNAi treatment can cause complete sexual transformation of both germline and soma in adult flies, resulting in a fertile male XX phenotype. The male pathway seems to result when Cctra autoregulation is prevented and instead splice variants with truncated open reading frames are produced. We propose that this repression is achieved by the Y-linked male-determining factor (M).     

半滑舌鳎雌性特异AFLP标记CseF783的克隆及其在遗传性别鉴定中的应用

半滑舌鳎性别控制和全雌育种等研究领域中迫切需要一种能够快速鉴定鱼类个体遗传性别的有效方法。文章采用AFLP技术, 利用选择性引物组合(E-ACT/M-CAA)从半滑舌鳎中筛选到一条雌性特异的AFLP标记。对该标记进行二次PCR扩增、琼脂糖凝胶回收、克隆、测序。分析表明, 序列全长为791 bp, 与GenBank中的序列无同源性。以该雌性特异AFLP标记DNA序列为模板, 设计了一对特异的PCR引物, 成功地将其转化为SCAR(Sequence characterized amplified regions)标记, 并在100尾已知性别的半滑舌鳎个体(雌雄各50尾)中进行验证, 结果表明, 该SCAR标记在所有雌性个体中均扩增得到一条长度为324 bp的DNA条带, 而在49尾雄性个体中均扩增不到该DNA条带(有1尾雄性个体例外), 证明该SCAR标记是雌性特异的, 并可用于半滑舌鳎个体遗传性别鉴定。随后, 利用该SCAR标记检测了3日龄半滑舌鳎幼苗, 结果表明, 雌性个体比例为41.7%。     

Population genetics of the potentially invasive African fruit fly species, Ceratitis rosa and Ceratitis fasciventris (Diptera: Tephritidae)

A set of 10 microsatellite markers was used to survey the levels of genetic variability and to analyse the genetic aspects of the population dynamics of two potentially invasive pest fruit fly species, Ceratitis rosa and C. fasciventris, in Africa. The loci were derived from the closely related species, C. capitata. The degree of microsatellite polymorphism in C. rosa and C. fasciventris was extensive and comparable to that of C. capitata. In C. rosa, the evolution of microsatellite polymorphism in its distribution area reflects the colonization history of this species. The mainland populations are more polymorphic than the island populations. Low levels of differentiation were found within the Africa mainland area, while greater levels of differentiation affect the islands. Ceratitis fasciventris is a central-east African species. The microsatellite data over the Uganda/Kenya spatial scale suggest a recent expansion and possibly continuing gene flow within this area. The microsatellite variability data from C. rosa and C. fasciventris, together with those of C. capitata, support the hypothesis of an east African origin of the Ceratitis spp.     

Comparative analysis of microsatellite loci in four fruit fly species of the genus Ceratitis (Diptera: Tephritidae)

The possibility to cross-species amplify microsatellites in fruit flies of the genus Ceratitis was tested with the polymerase chain reaction (PCR) by analysing 23 Ceratitis capitata (Wiedemann) microsatellite markers on the genomic DNA of three other economically important, congeneric species: C. rosa (Karsch), C. fasciventris (Bezzi) and C. cosyra (Walker). Twenty-two primer pairs produced amplification products in at least one of the three species tested. The majority of the products were similar, if not identical in size to those expected in C. capitata. The structures of the repeat motifs and their flanking sequences were examined for a total of 79 alleles from the three species. Sequence analysis revealed the same repeat type as the homologous C. capitata microsatellites in the majority of the loci, suggesting their utility for population analysis across the species range. A total of seven loci were differentially present/absent in C. capitata, C. rosa, C. fasciventris and C. cosyra, suggesting that it may be possible to differentiate these four species using a simple sequence repeat-based PCR assay. It is proposed that medfly-based microsatellite markers could be utilized in the identification and tracing of the geographical origins of colonist pest populations of the four tested species and in the assessment of their risk and invasive potentials; thereby assisting regulatory authorities in implementing quarantine restrictions and other pest control measures.     

Variation in morphological characters and habitat requirements in Carex capitata and Carex arctogena (Cyperaceae) in Norway and Sweden

Norwegian and Swedish populations of Carex capitata s. lat. were studied for variation in morphological characters. Canonical variates analysis (CVA) arranges populations in two discrete groups, corresponding to C. capitata s. str. and C. arctogena , respectively. In principal components analysis (PCA) of the total material a small overlap could be seen. However, separate PCA: s for the two regions in Scandinavia where the two taxa are sympatric show perfect separation. Student's t-test revealed significant differences in all morphological characters studied. Studies of companion plants, analysed with detrended correspondence analysis (DCA), indicate that the taxa have rather similar habitat demands in Oppland, Central Norway, although C. capitata s. str. seems to prefer wetter, and C. arctogena drier sites, respectively. Since the two taxa are distinct in morphology, they are best treated as separate species. The name Carex capitata is lectotypified.     

Estimates of gene flow from rare alleles in natural populations of medfly Ceratitis capitata (Diptera: Tephritidae)

Gene flow based on the spatial distribution of rare alleles at 25 gene loci was estimated in 15 populations of Ceratitis capitata (Wiedemann) from different parts of the world. Estimates of Nm, the number of migrants exchanged per generation among populations in different regions of the world, appeared to be quite similar, ranging from 3.36 in tropical Africa to 2.94 in the New World and 2.72 in Mediterranean basin populations. This suggests that gene flow among neighbouring populations of medfly is quite extensive. The genetic differentiation in American, Mediterranean and African populations was related to major climatic differences between North and South. These differences arise mainly from five loci that showed gene frequency patterns suggestive of latitudinal clines in allele frequencies. The clinal variation was such that tropical-subtropical populations were more heterozygous than temperate populations. It was concluded that gene flow, counteracting the forces of natural selection and genetic drift, determines the extent to which geographical populations of C. capitata are differentiated.     

Mechanisms of resistance to malathion in the medfly Ceratitis capitata

Target site insensitivity and metabolic resistance mediated by esterases have been previously suggested to be involved in resistance to malathion in a field-derived strain (W) of Ceratitis capitata. In the present study, we have obtained the coding sequence for acetylcholinesterase (AChE) gene (Ccace) of C. capitata. An allele of Ccace carrying only a point mutation Gly328Ala (Torpedo numbering) adjacent to the glutamate of the catalytic triad was found in individuals of the W strain. Adult flies homozygotes for this mutant allele showed reduced AChE activity and less sensitivity to inhibition by malaoxon, showing that target site insensitivity is one of the factors of malathion resistance. In addition, all individuals from the resistant W strain showed reduced aliesterase activity, which has been associated with specific malathion resistance in higher Diptera. However, the alphaE7 gene (CcalphaE7), sequenced in susceptible and resistant individuals, did not carry any of the mutations associated with organophosphorus insecticide resistance in other Diptera. Another esterase mechanism, perhaps a carboxylesterase selective for malathion, in addition to mutant AChE, thus contributes to malathion resistance in C. capitata.     

The Drosophila SR protein RBP1 contributes to the regulation of doublesex alternative splicing by recognizing RBP1 RNA target sequences.

The SR proteins represent a family of splicing factors several of which have been implicated in the regulation of sex-specific alternative splicing of doublesex (dsx) pre-mRNA in Drosophila. The dsx gene is involved in Drosophila sex determination. We have identified two RNA target sequence motifs recognized by the SR protein RBP1 from Drosophila using an in vitro selection approach. Several copies of these RBP1 target sequences were found within two regions of the dsx pre-mRNA which are important for the regulation of dsx alternative splicing, the repeat region and the purine-rich polypyrimidine tract of the regulated female-specific 3' splice site. We show that RBP1 target sequences within the dsx repeat region are required for the efficient splicing of dsx pre-mRNA. Moreover, our studies reveal that RBP1 contributes to the activation of female-specific dsx splicing in vivo by recognizing the RBP1 target sequences within the purine-rich polypyrimidine tract of the female-specific 3' splice site.     

Bmdsx基因在家蚕田岛嵌合体卵巢中的表达分析

根据雌特异性和雄特异性Bmdsx基因cDNA的共有序列,设计一对引物,以RT-PCR方法对家蚕田岛嵌合体蛹期卵巢中Bmdsx基因的表达情况进行了研究.结果表明,在家蚕田岛嵌合体蛹期卵巢中,在475bp附近和226bp附近分别扩增得到了一条条带,这两条条带大小与预期雌雄性别特异性Bmdsx基因cDNA片段大小相吻合.其中,雄特异性cDNA片段经DNA序列分析后得到了确认.     

Allozyme differentiation between the lowland Carex capitata and the alpine Carex arctogena (Cyperaceae) in Scandinavia

Within Scandinavia, Carex capitata s.l, is usually divided into two taxa: the lowland to lowalpine C. capitata s.s., and the alpine C. arctogena. The systematic status of these taxa is uncertain, reflected by their taxonomic treatment in different floras. In this study, allozymes were used to assess the degree of genetic differentiation between and within taxa. Ten populations of Carex capitata s.s. and eight of C. arctogena, from Norway and Sweden, were included in the study. Sixteen loci in eleven different enzyme systems were analysed: five were variable within or between taxa, three could be interpreted in all plants. In all, 20 different alleles were found. Four of these alleles were confined to C. capitata s.s., and two to C. arctogena. In C. capitata s.s., three loci (18%) were polymorphic, while all loci were monomorphic in C. arctogena. The number of alleles per polymorphic locus was 2.2 in C. capitata s.l. The taxa had completely different alleles at two loci {Pgi-2, Pgm-1). Nei's genetic identity was 0.86 between taxa, and ranged between 0.95 and 1 in pairwise comparisons of populations of Carex capitata s.s., while it was 1 in all comparisons between populations of C. arctogena. The distinction of C. capitata s.s. and C. arctogena as separate species agrees well with the amount of differentiation found between other congeneric species. It is suggested that the low genetic variation observed in both taxa is due to ancient founder events. The difference in genetic variability between taxa may be due to differences in immigration history: whereas C. capitata s.s. may have colonized Scandinavia from several close refugia after the last glaciation, C. arctogena may have reached Scandinavia after long-distance dispersal of a limited number of propagules.     

Effects of adhesive powders on the mating and flight behavior of Mediterranean fruit fly (Diptera: Tephritidae)

Powders that adhere to insect cuticle can be used as carrier particles for synthetic insecticides, entomopathogens, or pheromones in insect control systems, and insects can be lured into contact with such powder mixtures by using attractants. Secondary transfer of adhesive powders to conspecifics during social interactions has been reported; however, this transfer relies on insects leaving the source of powder and continuing normal behavior when contaminated. We examined the ability of the Mediterranean fruit fly, Ceratitis capitata Wiedemann (Diptera: Tephritidae), to fly and mate after being contaminated with one of two adhesive powders: an electrostatic wax powder, Entostat, and a proprietary metallic powder, Entomag. During continuous observations for 1 h in a flight tunnel, male C. capitata made significantly more flights than females. Treating C. capitata with either powder significantly suppressed the flight activity of male C. capitata compared with untreated controls, whereas powder treatment had a negligible effect on female flight activity. Within 1 h, male C. capitata treated with Entomag recovered normal flight activity, but Entostat-treated males were not fully recovered. Virgin male C. capitata treated with either Entostat or Entomag were able to mate with virgin female C. capitata, but the onset of mating was delayed compared with control C. capitata by approximately 1 h. Even though the effect of powder uptake on behavior seemed to be temporary, scanning electron micrograph images of treated C. capitata showed that both powders were retained for > 24 h on most body parts. The adhesive powders showed potential for use as carrier particles for pesticides, entomopathogens, or pheromones in novel C. capitata control systems.     

Potential for areawide integrated management of Mediterranean fruit fly (Diptera: Tephritidae) with a braconid parasitoid and a novel bait spray

The braconid wasp, Fopius arisanus (Sonan), a biological control agent for Mediterranean fruit fly, Ceratitis capitata (Wiedemann), was studied in coffee, Coffea arabica L. Fopius arisanus, comprised 79.3% of the total parasitoids (7,014) recovered from fruits collected at three small coffee farms. Data from seasonal host/parasitoid studies at a large coffee plantation also suggested that the most effective natural enemy of C. capitata in coffee may now reside in Hawaii. The original parasitoids introduced into Hawaii for C. capitata control (Diachasmimorpha tryoni (Cameron), Tetrastichus giffardianus Silvestri, and Dirhinus giffardii Silvestri) are now rare. Abundance of F. arisanus with respect to other parasitoids collected was influenced by elevation (274, 457, 610 m). Fopius arisanus was the dominant parasitoid at all three elevations, Diachasmimorpha longicaudata (Ashmead) occurred consistently, and T. giffardianus was abundant only at low elevation. The impacts on C. capitata and F. arisanus populations of bait sprays containing malathion, spinosad, or phloxine B applied to coffee were also evaluated. All three bait sprays suppressed C. capitata populations. Spinosad and phloxine B bait sprays appeared less harmful to the wasp than malathion. Fopius arisanus offers the potential for areawide management of C. capitata that includes biological control and integration with more environmentally safe chemical controls such as spinosad and phloxine B bait sprays.