Molecular sexing in the Mediterranean fruit fly, Ceratitis capitata

Molecular methods have been devised for sexing Mediterranean fruit fly (medfly) individuals using minimal amounts of material from any stage of the life cycle. Molecular sexing methods are particularly valuable when material is obtained from pre-adult stages and sex identification based on morphological characters is not possible. These methods may also be useful for adult stage material in situations where only limited amounts or poorly preserved specimens are available. The sexing methods described here use the polymerase chain reaction (PCR) to amplify sequences known to originate from the sex chromosomes of this species. One method co-amplifies homologous regions of the ITS1 ribosomal DNA from both the X and Y chromosomes. Males and females are distinguished based on the restriction fragment pattern produced after digestion of the PCR products with the restriction enzyme ApoI. A second method identifies males based on the positive amplification of a repetitive DNA sequence originating from the Y chromosome. Both methods are shown to be capable of establishing the sex identity of individuals using only minimal amounts of material from any stage of the life cycle.     

Host-specific demographic studies of the Mediterranean fruit fly Ceratitis capitata

Abstract. 1. This study was conducted in Greece using a wild strain of medflies ( Ceratitis capitata [Weidemann]) to assess age-specific vital rates in adults, and development and survival in pre-adults when reared on different host fruit. The results were used to construct life tables.
2. The demographic analyses suggested that there are basically four aspects of the medfly's life history which are of major importance: (i) multiple, highlyoverlapping generations; (ii) high net reproduction while young; (iii) high larval fitness in certain hosts; (iv) lack of diapause.
3. Reasons why these characteristics are felt to be important are briefly discussed.     

Identification and characterization of male specific serum proteins in the Mediterranean fruit fly Ceratitis capitata

Two major and three minor male specific serum proteins (MSSPs) have been identified in the Mediterranean fruit fly Ceratitis capitata. All five MSSPs accumulate in the haemolymph during the first 3 days of the adult development and represent more than 50% of the total haemolymph proteins in the adult males. All MSSPs are dimeric proteins consisting of two subunits with molecular weights between 13.5 and 14.5 kDa. MSSP-1, 3, 4 and 5 are homodimers while MSSP-2 is a heterodimer. The two major MSSPs (MSSP-1 and 5) have been isolated. Antisera against these two MSSPs cross-react partially with each other's antigens but did not give any reaction with haemolymph from adult females and third instar larvae.     

Isolation and characterization of the Xanthine dehydrogenase gene of the Mediterranean fruit fly, Ceratitis capitata

Xanthine dehydrogenase (XDH) is a member of the molybdenum hydroxylase family of enzymes catalyzing the oxidation of hypoxanthine and xanthine to uric acid. The enzyme is also required for the production of one of the major Drosophila eye pigments, drosopterin. The XDH gene has been isolated in many species representing a broad cross section of the major groups of living organisms, including the cDNA encoding XDH from the Mediterranean fruit fly Ceratitis capitata (CcXDH) described here. CcXDH is closely related to other insect XDHs and is able to rescue the phenotype of the Drosophila melanogaster XDH mutant, rosy, in germline transformation experiments. A previously identified medfly mutant, termed rosy, whose phenotype is suggestive of a disruption in XDH function, has been examined for possible mutations in the XDH gene. However, we find no direct evidence that a mutation in the CcXDH gene or that a reduction in the CcXDH enzyme activity is present in rosy medflies. Conclusive studies of the nature of the medfly rosy mutant will require rescue by germline transformation of mutant medflies.     

Purification and characterization of two cysteine peptidases of the Mediterranean fruit fly Ceratitis capitata during metamorphosis

In holometabolous insects, there is a complete body remodeling from larva to adult. We determined in Ceratitis capitata that the transition from pre-pupa to pupa, 40 to 48 h after puparium formation (h APF), is a key moment of metamorphosis; when salivary glands, intestine, fat body, and muscles are in different stages of cell death. At 44-46 h APF, muscles from segments 1-3 (thoracic region) appeared fully disintegrated, whereas posterior muscles just started death processes. To understand some of the biochemical events eventually involved in histolytic processes during early metamorphosis, two cysteine peptidases coined "Metamorphosis Associated Cysteine Peptidase" (MACP-I and MACP-II) were purified to homogeneity from 40-46-h APF insects. Both enzymes were inhibited by Ep-475, a specific inhibitor of papain-like cysteine-peptidases. MACP-I is a single chain protein with an apparent molecular mass of 80 kDa and includes several isoforms with pI values of pH 6.25-6.35, 6.7, and 7.2. The enzyme has an optimum pH of 5.0 and its pH stability ranges from pH 4.0 to 6.0. The molecular weight and N-terminal sequence suggest that MACP-I might be a novel enzyme. MACP-II is an acidic single chain protein with a pI of pH 5.85 and an apparent molecular mass of 30 kDa. The enzyme is labile with a maximum stability in the pH range of 4.0 to 6.0 and an optimum pH among 5.0 to 6.0. MAPCP-II characteristics suggest it is a cathepsin B-like enzyme.     

Two-way selection for mating activity in the Mediterranean fruit fly, Ceratitis capitata

No significant variation in mating activity was observed among eight laboratory strains of the Mediterranean fruit fly (medfly), Ceratitis capitata (Wiedemann) (Diptera: Tephritidae). Two methods were used to select strains showing high and low mating activity: a single pair technique (SP) assessing time to mating and a mass technique (M) based on a mating index. Reciprocal pairings between fast and slow selected lines showed that the difference between the SP lines was female-determined whereas the difference between the M lines depended on the behaviour of both sexes. M selection, irrespective of its direction, affected two courtship parameters, vibration distance and vibration duration both of which tended to be shorter. M selection was also associated with a reduction in startle activity in females.

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Résumé Aucune variation significative n'a été observée dans les activités sexuelles de huit souches de laboratoire de la mouche méditerranéenne du fruit, Ceratitis capitata (Wiedemann) (Diptera Tephritidae).Deux méthodes ont été utilisées pour sélectionner des souches manifestant une activité sexuelle basse ou élevée: une technique utilisant un seul couple (SP), basée sur la durée de la période avant copulation, et une technique de masse (M), basée sur un index d'accouplement.Des appariements réciproques entre les lignées rapides et lentes ont montré que la différence entre les lignées SP était déterminée par les femelles, tandis que la différence entre les lignées M résultait du comportement des deux sexes. La sélection M, quelle que soit sa direction, affects deux paramètres, la distance entre partenaires lors de la vibration et la durée de la vibration qui tous deux tendent à se raccourcir. La sélection M est aussi associée à une réduction de la réaction des femelles à un brusque stimulus lumineux (Startle activity).

     

地中海实蝇地理种群遗传分化研究

以mtDNA的CO Ⅰ基因的部分序列作为分子标记,研究了来自安哥拉、黎巴嫩、刚果共和国、尼日利亚、贝宁、多哥、加纳、科特迪瓦、南非、埃及、法国、约旦、秘鲁、智利、美国佛罗里达和美国夏威夷等16个国家和地区共75个个体的地中海实蝇Ceratitis capitaza地理种群序列特征和单倍型特征,并分析了各种群间的遗传分化水平和进化关系,结果表明,在供试的样品中发现单倍型的数目是29,其中有5种为共享倍型.地中海实蝇各地理种群的遗传多态性水平差异较大,总体遗传距离D等于0.007.非洲南撒哈拉区(安哥拉、刚果、尼日利亚、贝宁、多哥、加纳、科特迪瓦)的种群的单倍型数、单倍型百分比和群内遗传距离等遗传多态性参数值都高于其它供试地区,多样性明显较其他地区丰富.     

Susceptibility of the Mediterranean fruit fly (Ceratitis capitata) and the Natal fruit fly (Ceratitis rosa) to entomopathogenic nematodes

The potential of entomopathogenic nematodes, Heterorhabditis bacteriophora, Heterorhabditis zealandica and Steinernema khoisanae, to infect pupariating larvae, pupae and adults of Ceratitis capitata and Ceratitis rosa was investigated in laboratory bioassays. Pupariating larvae and adult flies were susceptible to nematode infection, with no infection recorded for the pupae. Pupariating larvae of C. capitata were generally more susceptible to infection than those of C. rosa. Significantly more larvae of C. capitata were infected by H. bacteriophora. For C. rosa, highest infectivity of larvae was obtained with H. zealandica. In contrast, adults of both species were highly infected by S. khoisanae.     

Cytogenetic analysis of chromosome 5 from the Mediterranean fruit fly,Ceratitis capitata

A cytogenetic map of chromosome 5 from the Mediterranean fruit fly, Ceratitis capitata, was constructed. Six mutations were located by translocation, transposition or deletion mapping. This knowledge allowed alignment and orientation of the existing linkage map with the polytene chromosomes. In addition, mapping of mutations used as selectable markers in genetic sex-separation strains is an essential prerequisite for the improvement of genetic stability during mass-rearing.     

Ecological niches and potential geographical distributions of Mediterranean fruit fly (Ceratitis capitata) and Natal fruit fly (Ceratitis rosa)

Aim To predict and compare potential geographical distributions of the Mediterranean fruit fly (Ceratitis capitata) and Natal fruit fly (Ceratitis rosa). Location Africa, southern Europe, and worldwide. Methods Two correlative ecological niche modelling techniques, genetic algorithm for rule‐set prediction (GARP) and a technique based on principal components analysis (PCA), were used to predict distributions of the two fly species using distribution records and a set of environmental predictor variables. Results The two species appear to have broadly similar potential ranges in Africa and southern Europe, with much of sub‐Saharan Africa and Madagascar predicted as highly suitable. The drier regions of Africa (central and western regions of southern Africa and Sahelian zone) were identified as being less suitable for C. rosa than for C. capitata. Overall, the proportion of the region predicted to be highly suitable is larger for C. capitata than for C. rosa under both techniques, suggesting that C. capitata may be tolerant of a wider range of climatic conditions than C. rosa. Worldwide, tropical and subtropical regions are highlighted as highly suitable for both species. Differences in overlap of predictions from the two models for these species were observed. An evaluation using independent records from the adventive range for C. capitata and comparison with other predictions suggest that GARP models offer more accurate predictions than PCA models. Main conclusions This study suggests that these species have broadly similar potential distributions worldwide (based on climate), although the potential distribution appears to be broader for C. capitata than for C. rosa. Ceratitis capitata has become invasive throughout the world, whereas C. rosa has not, despite both species having broadly similar potential distributions. Further research into the biology of these species and their ability to overcome barriers is necessary to explain this difference, and to better understand invasion risk.     

Microsatellite analysis reveals remating by wild Mediterranean fruit fly females,Ceratitis capitata

Accurate estimates of remating in wild female insects are required for an understanding of the causes of variation in remating between individuals, populations and species. Such estimates are also of profound importance for major economic fruit pests such as the Mediterranean fruit fly (Ceratitis capitata). A major method for the suppression of this pest is the sterile insect technique (SIT), which relies on matings between mass-reared, sterilized males and wild females. Remating by wild females will thus impact negatively on the success of SIT. We used microsatellite markers to determine the level of remating in wild (field-collected) Mediterranean fruit fly females from the Greek Island of Chios. We compared the four locus microsatellite genotypes of these females and their offspring. Our data showed 7.1% of wild females remated. Skewed paternity among progeny arrays provided further evidence for double matings. Our lowest estimate of remating was 3.8% and the highest was 21%.     

The construction of the first balancer chromosome for the Mediterranean fruit fly, Ceratitis capitata

The construction of the first balancer chromosome, FiM1, for the medfly Ceratitis capitata is described. This chromosome has three overlapping pericentric inversions and is marked with dominant and recessive mutations. The inversion breakpoints of FiM1 suppress recombination throughout the length of the fifth chromosome, allowing lethal mutations to be recovered and maintained. This chromosome will provide a powerful tool for the manipulation of laboratory stocks, in particular, the recovery of new mutant and transgenic strains. We demonstrate the use of FiM1 for the recovery and maintenance of chromosomes carrying lethal mutations.     

Sexual competitiveness of a transgenic sexing strain of the Mediterranean fruit fly, Ceratitis capitata

The sterile insect technique (SIT), when used for the control of the Mediterranean fruit fly (medfly), Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), generally relies on the release of sterile flies of only the male sex. Male selection is achieved through the use of a genetic sexing strain (GSS) in which females are killed by heat treatment in the generation prior to release. Transgenic sexing strains (TSS) have been developed that perform the same function of female-lethality, this time by withholding tetracycline (or related compounds) from the larval diet. The use of TSS may allow for certain problems associated with conventional GSS, such as strain instability and reduced productivity in mass-rearing, to be avoided. The performance, and principally the sexual competitiveness, of released male flies is important for the success of an SIT control programme. This study describes field cage experiments in which the competitiveness of males from a TSS (OX3376B) was compared with that of a conventional GSS (VIENNA-8) and two wild-type strains (TOLIMAN and ARG). When competing for female mates with wild-type males, OX3376B male performance was acceptable. When OX3376B males competed directly for mates with VIENNA-8 males, VIENNA-8 slightly outperformed the TSS males. Parallel tests, in which wild-type males competed with either OX3376B or VIENNA-8 males, showed that males from both sexing strains were highly competitive with wild-type males. These results suggest that OX3376B in particular, and TSS in general, show sufficiently good mating competitiveness to merit further research into their suitability for eventual use in SIT programmes.     

Performance of controlled-release formulations of trimedlure to attract the Mediterranean fruit fly, Ceratitis capitata

Mediterranean fruit flies (Ceratitis capitata Wied.) are detected by traps baited with the attractant, trimedlure (TML), deposited on the standard cotton dental roll dispenser. Since this dispenser is effective for only 2–4 weeks, controlled-release dispensers may extend the field life of TML-baited traps. A polymeric plug containing 70% by weight TML was highly effective for 8–12 weeks. Other formulations were less effective in the tests with released flies. A release rate of ca. 17 mg of standard TML/day or higher gave insect captures which were generally lower but statistically equivalent to those with the reference dispenser which released 61–63 mg/day. Normalized insect catches increased approximately as the square root of the release rate; the curvilinear regression equation suggested that TML release rates of ca. 52 and 15 mg/day give insect captures equal to ca. 100% and 50%, respectively, of the reference catches. The most attractive TML isomer (TML-C) was a highly effective attractant at a release rate of ca. 4 mg/day.

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Résumé C. capitata était attiré par des pièges alimentés avec de la trimedlure (TML), attractif déposé sur un distributeur commercial de coton dentaire en rouleau. Puisque ce distributeur ne fonctionne que 2 à 4 semaines, des distributeurs à libération contrôlée peuvent augmenter le temps de fonctionnement de pièges fournis en TML. Un bouchon en polymère contenant en poids 70% de TML a été très efficace pendant 8 à 12 semaines. D'autres formules ont été moins efficaces avec des mouches lâchées. Un taux de libération, de 17 mg ou plus de TML moyen par jour, capture moins, mais sans différence statistique significative, qu'un distributeur témoin qui libérait 61 à 63 mg/j. Les captures d'insectes ont augmenté à peu près comme le carré du taux de libération; l'équation de régression curviligne a conduit à penser que la libération de TML à des taux de 52 et 15 mg/j, provoque des captures égales à 100% et 50% des captures de référence. L'isomère le plus attractif (TML-C) a été un attractif très efficace au taux de libération de 4 mg/j.

     

Ecdysone-regulated chromosome puffing in the salivary glands of the Mediterranean fruit fly, Ceratitis capitata.

The effect of ecdysone on the puffing activity of the polytene chromosomes of Ceratitis capitata has been studied in organ cultures of late-larval salivary glands. Culture of glands from 120-h-old larvae (puff stage 1) in the presence of ecdysone resulted in the initiation of the late-larval puffing cycle that is normally observed in 145-h-old larvae (puff stage 4). During a 7-h period in the presence of ecdysone, the puffing patterns of most loci resembled the in vivo patterns observed in the period between puff stages 4 and 10, indicating that the first puffing cycle can be initiated by the hormone and proceed almost to completion, in vitro. Culture of salivary glands in the presence of ecdysone and a protein-synthesis inhibitor, as well as ecdysone withdrawal and readdition experiments, indicated that most of the ecdysone-regulated puffs could be categorized into three classes: (i) the puffs that were suppressed immediately by ecdysone, even in the absence of protein synthesis; (ii) the puffs that were induced directly by ecdysone; and (iii) the puffs that were induced indirectly by ecdysone, that is, they were induced after a lag period of a few hours and required protein synthesis for their induction.     

Improved stability of genetic sex-separation strains for the Mediterranean fruit fly, Ceratitis capitata.

In the existing genetic sexing strains for the medfly, Ceratitis capitata, male recombination leads to breakdown of the sexing mechanism under mass rearing conditions. The rate of breakdown depends on the recombination frequency and on the fitness of the recombinants. We have tested two different sexing genes, white pupa and a temperature sensitive lethal, in combination with the translocation T(Y;5)30C. Both sexing strains broke down, although at very different rates. In the case of the white pupa strain, 3.5% recombinants were observed after rearing the strain for 15 generations. The second strain, utilizing white pupa and the temperature sensitive lethal as selectable markers, already reached a comparable level after six generations and was broken down completely in the ninth generation. In these strains the frequency of recombination is high because the breakpoint of T(Y;5)30C and the sexing gene(s) are far apart. To remedy the situation, we have isolated four new translocations with breakpoints located closer to the sexing genes. Mass rearing was simulated for several generations with strains based on these translocations and no breakdown was observed under the conditions used.     

Field cage studies with a genetic sexing strain in the Mediterranean fruit fly, Ceratitis capitata

Using a genetic sexing line based on pupal colour dimorphism in Ceratitis capitata Wied., field cage experiments were carried out with a wild population in Italy. The experiments were designed to monitor the competitiveness of males of this line in comparison with wild males and to assess the effect of removal of females prior to release on the proportion of wild females mated by the released males.The results showed that the genetic sexing strain competed well with the wild population but there was a large degree of intra-strain mating. Removal of females prior to release radically changed the mating pattern from intra to inter-strain mating. This change greatly increases the efficiency of the sterile insect method of control.It was concluded that as well as the obvious economic advantages of genetic sexing, significant biological advantages would also be associated with the integration of such a strain in a sterile insect control programme.

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Résumé Des expériences en cage en champ ont été réalisées en Italie avec des populations naturelles de Ceratitis capitata en utilisant une lignée dont le sexage était basé sur le dimorphisme de la couleur des pupes. Les expériences ont été conçues pour contrôler la compétitivité des mâles de cette lignée face aux mâles sauvages, et d'évaluer l'effet du retrait des femelles avant le lâcher sur le pourcentage de femelles sauvages accouplées avec des mâles lâchés.Les expériences montrent que la compétition ne défavorise pas la lignée génétiquement sexable mais révèlent une fréquence élevée d'accouplement intralignée.Le retrait des femelles avant le lâcher modifie radicalement l'orientation des accouplements d'intra vers interlignées. Ce changement accroît fortement l'efficacité de la technique d'utilisation des mâles stériles dans la lutte biologique. On en a conclu que non seulement des intérêts économiques évidents mais aussi des avantages biologiques significatifs seraient retirés de l'intégration de telles lignées sexables génétiquement dans les programmes de lutte avec des mâles stériles.