Choriogenesis in the medfly Ceratitis capitata (Wiedermann) (Diptera : Tephritidae)

Four chorionic stages (11, 12, 13, 14) can be discerned in the medfly Ceratitis capitata (Diptera : Tephritidae) by light microscope. More detailed staging (stages 11A, 11B, 12A, 12B, 12C, 13A, 13B, 13C, 14A and 14B) is possible only by electron microscope. Throughout these stages, the first chorionic layer (wax layer) is formed at stage 11A, followed by the formation of the innermost chorionic layer (stage 12A), the inner endochorion, the pillars and the cavities of the first trabecular layer (stage 12), creation of the second trabecular layer and the chorionic network (stage 13), and finally the secretion of the exochorionic layers (stage 14). Pulse-chase autoradiography has revealed that the follicle cells are responsible for the synthesis of all proteinaceous layers. No extensive regional complexity is observed besides the existence of the micropylar apparatus and aeropyles in the anterior pole. Biochemical analysis has revealed several eggshell proteins and their stage-specific synthesis: two intermediate molecular-weight proteins are the major chorion proteins; in addition, there are 2 more groups comprising 6 proteins, which might be characteristic for the different chorion layers as can be deduced by their stage-electrophoretic pattern.     

Sexual behavior of mutant strains of the medfly Ceratitis capitata (Diptera: Tephritidae)

Males of the mutant strains (blind, vestigal-winged) of the Mediterranean fruit fly (medfly), Ceratits capitata (Wiedmann) showed differences in behavior compared with control (mass-reared) males. Mutant males made fewer mating attempts and achieved fewer matings than control males. Vestigal-winged females copulated less frequently with both mutants. Blind males climbed rather than jumped onto females and copulated in very low numbers compared with control and vestigal males. Blind females copulated normally with control, males and in very low numbers with both types of mutant males.     

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.     

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.     

Acrosome formation in Ceratitis capitata (Diptera, Tephritidae)

The stages of morphogenesis of the acrosome of Ceratitis capitata are well defined. This organelle is formed by the Golgi complex and, as it matures, takes up a position laterally in relation to the anterior region of the sperm nucleus. An interstitial membrane marks the area of contact between nucleus and acrosome in the spermatid, and is found even in mature sperm cells. The acrosome contains hydrolytic enzymes, as detected by acid phosphatase reaction.     

Stage-specific regulation of programmed cell death during oogenesis of the medfly Ceratitis capitata (Diptera, Tephritidae)

In the present study, we describe novel features of programmed cell death in developing egg chambers occurring during mid- and late-oogenesis of the medfly Ceratitis capitata. During mid-oogenesis, the spontaneously degenerated egg chambers exhibit typical characteristics of apoptotic cell death. Their nurse cells contain fragmented DNA and fragmented actin, as revealed by TUNEL assay and immunolabelling, respectively. In vitro caspase activity assays and immunostaining procedures demonstrated that the atretic egg chambers acquired high levels of caspase activity. Distinct features of autophagic cell death were also observed during C. capitata mid-oogenesis, as revealed by the monodansylcadaverine staining approach and ultrastructural examination performed by transmission electron microscopy. Additionally, atretic egg chambers exhibit an upregulation of lysosomal proteases, as demonstrated by a procathepsin L immunolabelling procedure. At the late stages of C. capitata oogenesis, apoptosis and autophagy coexist, manifesting cell death features that are similar to the ones mentioned above, being also chaperoned by the involvement of an altered cytochrome c conformational display. We propose that apoptosis and autophagy operate synergistically during C. capitata oogenesis for a more efficient elimination of the degenerated nurse cells and abnormal egg chambers.     

Conservation of nucleolar structure in polytene tissues of Ceratitis capitata (Diptera: Tephritidae)

Nucleolar structure was studied in mitotic and three polytene tissues of the Mediterranean fruit fly, Ceratitis capitata using in situ hybridization with a tritium-labelled rDNA probe and silver staining. In mitotic metaphase chromosomes nucleolar organiser regions were localised in the short arms of both sex chromosomes. In polytene nuclei of trichogen cells, salivary glands and fat body rDNA was detected within nucleoli. Nucleoli in these tissues have a similar structure with rDNA labelling concentrated in a central core. Silver staining resulted in very heavy staining of polytene nucleoli and interphase nucleoli in diploid cells. Silver staining of nucleolar organisers in metaphase chromosomes is weak or absent although the X chromosome has numerous dark silver bands in other locations. The results suggest that nucleolar structure is conserved in polytene tissues contrasting with the variability of autosomal banding patterns and sex chromosome structure. They also indicate that silver staining is not necessarily specific for nucleolar regions.     

Improving male mating competitiveness and survival in the field for medfly,Ceratitis capitata (Diptera: Tephritidae) SIT programs

The success of the sterile insect technique (SIT) depends critically upon mating between released sterilized males and wild females. In Hawaii, improvements in the efficiency of sterile males were attempted on two separate fronts – mating enhancement and survival improvement. In the former, two methods have been investigated – selective breeding and aromatherapy. In the latter, flies which survived in field cages for several days were selected and bred to produce progeny with enhanced survival ability compared to control flies. Regarding mating selection, standard laboratory-reared males that successfully mated with wild females in field cages were allowed to breed. F1 offspring were inbred, then the selection procedure was repeated for four additional cycles. In the aromatherapy procedure, laboratory-reared males were exposed to ginger root oil for several hours 1 day prior to testing in field cages. Compared to controls, the selected flies improved the mating competitiveness of male flies ca. 3-fold, irradiation reduced this increase to ca. 2.5-fold. Exposing the selected, hybrid strain raised the fitness of the lab males to ca. 9-fold that of wild males. In the ongoing survival selection study, we have obtained lines in which the selected males survived ca. 2-fold better than laboratory control males over several days in an outdoor field cage, with food and water provided. The goal is to combine the traits of higher survival and mating ability into a single strain for SIT release.     

Fluorescent sperm marking to improve the fight against the pest insect Ceratitis capitata (Wiedemann; Diptera: Tephritidae)

The Sterile Insect Technique (SIT) involving area-wide release of mass-reared and sterilized pest insects has proven successful to reduce, control and eradicate economically important pest species, such as the Mediterranean fruit fly (medfly). For the efficient application, effective monitoring to assess the number and mating success of the released medflies is essential. Here, we report sperm-specific marking systems based on the spermatogenesis-specific Ceratitis capitata beta2-tubulin (Ccbeta2t) promoter. Fluorescent sperm can be isolated from testes or spermathecae. The marking does not cause general disadvantages in preliminary laboratory competitiveness assays. Therefore, transgenic sperm marking could serve as a major improvement for monitoring medfly SIT programs. The use of such harmless transgenic markers will serve as an ideal initial condition to transfer insect transgenesis technology from the laboratory to field applications. Moreover, effective and easily recognizable sperm marking will make novel studies possible on medfly reproductive biology which will help to further improve SIT programs.     

Male position and calling effort together influence male attractiveness in leks of the medfly,Ceratitis capitata (Diptera: Tephritidae)

Despite the close association between lek mating systems and the study of female mate choice, male mating success in leks is often associated with other aspects of sexual selection as well as female choice of male display traits. Males of the medfly Ceratitis capitata form leks on the undersides of leaves of their host plants. By experimentally creating artificial leks, we show that male success at attracting females depends not only on male calling effort (pheromone production dispersed by wing movement), but also on the position of the male within a lek. Males in the highest position in the artificial lek (closest to the light) attracted more females, and received more visits from those females. In our experiment, we deliberately minimized the visual cues that females approaching a male could use and, under these conditions, found no associations between male attractiveness and male size, weight or fluctuating asymmetry, either of the wings or sex setae (a pair of bilateral supra‐fronto‐orbital bristles). The latter result contrasts with earlier studies showing a significant negative association between sex setae fluctuating asymmetry and mating success. Accordingly, we conclude that symmetry of the male sex setae has no role in nonvisual communication (e.g. through directing pheromone plumes). Mating patterns associated with this insect are therefore every bit as complex as those in vertebrate leks. © 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 95 , 479–487.     

Phloxine B effect on immature stages of the Mediterranean fruit fly,Ceratitis capitata (Diptera: Tephritidae) (Wiedemann)

A laboratory bioassay was developed to determine both the chemical toxicity and the phototoxicity of the xanthene dye, phloxine B (D&C Red No 28), to the immature stages of the Mediterranean fruit fly, Certitis capitata (Wiedemann). An additional goal was to find out which main tissues are affected first. A low, but significant, level of toxicity was observed when the insects were maintained in the dark: at the point of adult ecdysis, the LC50 was 11.03 mM. As expected, after 8-h exposure of late larva III to light, a high level of mortality was produced (LC50 at ecdysis: 0.45 mM) as a dose-dependent function of dye concentration. At sublethal concentrations of the dye, the surviving insects showed a number of physiological abnormalities. Phloxine B appeared to mainly affect the larval longitudinal muscles as well as the abdominal muscles of ecdysing adults, giving rise to abnormal puparia and failed adult ecdysis, respectively. Moreover, a significant phloxine B-dependent delay in the jumping of surviving larvae for dispersal was documented. This could be attributed to a delay in attaining a threshold weight for jumping and/or to abnormalities in neuromuscular coordination, thus reinforcing the idea of pleiotropic effects of the dye.     

Ultrastructure of the male reproductive accessory glands in the medfly Ceratitis capitata (Diptera: Tephritidae) and preliminary characterization of their secretions

The morphology and the ultrastructure of the male accessory glands and ejaculatory duct of Ceratitis capitata were investigated. There are two types of glands in the reproductive apparatus. The first is a pair of long, mesoderm-derived tubules with binucleate, microvillate secretory cells, which contain smooth endoplasmic reticulum and, in the sexually mature males, enlarged polymorphic mitochondria. The narrow lumen of the gland is filled with dense or sometimes granulated secretion, containing lipids. The second type consists of short ectoderm-derived glands, finger-like or claviform shaped. Despite the different shape of these glands, after a cycle of maturation, their epithelial cells share a large subcuticular cavity filled with electron-transparent secretion. The ejaculatory duct, lined by cuticle, has epithelial cells with a limited involvement in secretory activity. Electrophoretic analysis of accessory gland secretion reveals different protein profiles for long tubular and short glands with bands of 16 and 10 kDa in both types of glands. We demonstrate that a large amount of accessory gland secretion is depleted from the glands after 30 min of copulation.     

Structural organization of the copulation site in the medfly Ceratitis capitata (Diptera: Tephritidae) and observations on sperm transfer and storage

The copulation site of the medfly Ceratitis capitata was investigated at anatomical and ultrastructural levels. It consists of the anterior vagina, with a ventral fertilization chamber and a dorsal insemination pocket into which the two spermathecal ducts open. The fertilization chamber is an organ comprised of a number of alveoli that in virgin females are filled with a filamentous secretion, whereas in mated females contain sperm bundles. Through study of the internal morphology of the aedeagus, its position in the anterior vagina, and the direct observation of sperm transfer and storage, we confirmed that sperm are ejaculated through two gonopores at the top of the distiphallus and another at the base of the genital rod. The sperm flow dorsally into the insemination pocket and ventrally into the fertilization chamber. During copulation, the two spermathecae and the fertilization chamber are progressively filled with spermatozoa.     

Determination of host status of citrus fruits against the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae)

Abstract  The Mediterranean fruit fly (Medfly), Ceratitis capitata (Wiedemann) is a pest of citrus in parts of Western Australia. Three citrus cultivars: Valencia oranges, Eureka lemons and Imperial mandarins, as well as non-citrus control fruits, were examined for attractiveness and suitability to Medfly in the field and in the laboratory using choice and no-choice experiments. Oranges were more susceptible to Medfly than mandarins and lemons. Punctures in the skin had a significant impact on the degree of infestation in both citrus and non-citrus control fruit. Artificial infestation and larval survivorship were used to investigate the suitability of each cultivar to Medfly under laboratory conditions. Oranges and mandarins were suitable for the development of Medfly, but lemons were a poor host. When each cultivar was in season, field cage trials demonstrated that infestation occurred in oranges and mandarins but not in lemons.     

Structure and morphogenesis of the eggshell and micropylar apparatus in the olive fly, Dacus oleae (Diptera: Tephritidae)

The egg of the olive fly, Dacus oleae (Diptera, Tephritidae), is laid inside olives and the larva eventually destroys the fruit. The oocyte is surrounded by several distinct layers which are produced during choriogenesis. The chorion covering the main body of the egg outside of the vitelline membrane includes a "wax" layer, an innermost chorionic layer, an endochorion consisting of inner and outer layers separated by pillars and cavities similar to their counterparts in Drosophila melanogaster, as well as inner and outer exochorionic layers. The anterior pole is shaped like an inverted cup, which is chiefly hollow around its base and has very large openings communicating with the environment. Holes through the surface of the endochorion result from deposition of endochorionic substance around follicular cell microvilli. An opening at the apex of the cup provides an entrance for sperm entering the micropylar canal, which traverses the endochorion and continues into a "pocket" in a thickened vitelline protrusion. The micropylar canal is formed by deposition of endochorion and vitelline membrane around an elongated pair of follicular cell extensions. These extensions later degenerate and leave an empty canal about 5 microns in diameter and the narrower pocket about 1 micron in diameter. Respiration is thought to be facilitated by openings at the base of the anterior pole as well as by openings through the "plastron" around the main body of the shell.     

Hosts of Mediterranean fruit fly Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) from Broome and the Broome Peninsula, Western Australia

Abstract  In the Kimberley region of Western Australia, Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is only established at Broome. From 1998 to 2001 extensive fruit sampling was carried out at Broome during a trial investigating the eradication of medfly from the Kimberley using the sterile insect technique. Fruit was collected from 82 native and introduced plants growing in and surrounding the town of Broome and held for emergence of medfly. Eighteen plants, including the native tree Terminalia petiolaris , were found to be medfly hosts . On the basis of abundance, fruiting phenology and host suitability, eight species (kumquat Fortunella japonica , mango Magnifera indica , Barbados cherry Malpighia glabra , orange jessamine Murraya paniculata , guava Psidium guajava , Pacific almond Terminalia catapa , blackberry tree Terminalia petiolaris and yellow oleander Thevetia peruviana ) were the most important hosts ensuring medfly survival and population growth. Despite medfly being reared from T. petiolaris , it is likely that medfly can only maintain populations in areas close to human habitation, and eradication from these areas would lead to eradication from the whole Kimberley region.     

Inability of Ceratitis capitata (Diptera: Tephritidae) to overwinter in the Judean hills

The overwintering potential of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), in cold winter areas within its northern distribution is a key element in understanding its ecology. Recent studies have suggested that although originating in tropical Africa, the fly has become adapted to the cold weather that prevails within its northernmost areas of distribution. We address the question of whether the Mediterranean fruit fly has expanded its overwintering range to include the mountains of central Israel. Doing so would imply that the fly has developed either a behavioral or a physiological mechanism to cope with low temperature and/or damp conditions in combination with cold. We monitored adult populations year round, sampling fruit, calculating expected emergence days for overwintering flies, and studying adults captured within dense and sparse apple orchards. We also performed several manipulative experiments to study preimago ability to survive the winter under natural or seminatural conditions. The study was conducted in the central mountains of Israel at 700-m altitude from 1994 to 2003. Comparison experiments also were conducted at 400 m and at sea level. Our results show 1) no adults captured during the winter and spring, 2) an absence of new infestations during the winter and spring, and 3) inability of preimago stages to overwinter in the central mountains of Israel. Thus, we conclude that the fly does not overwinter in the central mountains of Israel. We discuss the ecological and applied significance of our findings.