A New Basal Subfamily of mariner Elements in Ceratitis rosa and Other Tephritid Flies

Several copies of highly related transposable elements, Crmar2, Almar1, and Asmar1, are described from the genomes of Ceratitis rosa, Anastrepha ludens, and A. suspensa, respectively. One copy from C. rosa, Crmar2.5, contains a full-length, uninterrupted ORF. All the other copies, from the three species contain a long deletion within the putative ORF. The consensus Crmar2 element has features typical of the mariner/Tc1 superfamily of transposable elements. In particular, the Crmar2 consensus encodes a D,D41D motif, a variant of the D,D34D catalytic domain of mariner elements. Phylogenetic analysis of the relationships of these three elements and other members of the mariner/Tc1 superfamily, based on their encoded amino acid sequences, suggests that they form a new basal subfamily of mariner elements, the rosa subfamily. BLAST analyses identified sequences from other diptera, including Drosophila melanogaster, which appear to be members of the rosa subfamily of mariner elements. Analyses of their molecular evolution suggests that Crmar2 entered the genome of C. rosa in the recent past, a consequence of horizontal transfer.     

Parasitoids of medfly, Ceratitis capitata, and related tephritids in Kenyan coffee: a predominantly koinobiont assemblage

Arabica coffee was sampled from two sites in the central highlands of Kenya (Rurima, Ruiru) and one site on the western side of the Rift Valley (Koru). Three species of ceratitidine Tephritidae, Ceratitis capitata (Wiedemann), C. rosa Karsch and Trirhithrum coffeae Bezzi, were reared from sites in the central highlands, and an additional species, C. anonae Graham, was recovered from the western-most site. Ten species of parasitic Hymenoptera were reared from these tephritids. The parasitoid assemblage was dominated by koinobionts. Eight of the species are koinobiont endoparasitoids, but only one idiobiont larval ectoparasitoid was reared, and only one idiobiont pupal endoparasitoid. The effects of sampling bias on determination of parasitoid assemblage size associated with concealed hosts are discussed. The potential for use of these parasitoids in biological control is also discussed. Most of the parasitoid species recovered during this study are capable of developing on C. capitata, while several also attack C. rosa. Both flies are notorious pests of tropical and subtropical fruits.     

Comparison of the mitotic karyotypes of Ceratitis capitata, Ceratitis rosa, and Trirhithrum coffeae (Diptera: Tephritidae) by C-banding and FISH.

The sex chromosomes of the tephritid fruit fly Ceratitis capitata (Wiedemann) are heteromorphic. The male-determining region was located on the Y chromosome by deletion mapping using unbalanced offspring from several translocation strains. In addition, we showed that only 15% of the Y chromosome is required for male determination and male fertility. Based on this result, we expected to find Y-chromosomal length polymorphism in natural populations. Using fluorescence in situ hybridization with two repetitive DNA probes that label the Y chromosome, no obvious size differences were detected in seven wild-type strains and three mutant strains. As the medfly is probably of East African origin, we also analyzed two wild-type strains established recently from pupae sampled in Kenya. The Y chromosomes show a polymorphism in the hybridization pattern of a repetitive Y-specific medfly clone. However, the overall size of the Y chromosome is similar to that of the other strains. Besides C. capitata, the tephritid fruit flies Ceratitis (Pterandrus) rosa Karsch and Trirhithrum coffeae Bezzi also emerged from pupae sampled in Kenya. Their karyotype was analyzed by C-banding. Furthermore, the ribosomal genes were mapped to the sex chromosomes in these two species. Key words : Ceratitis capitata, Tephritidae, C-Banding, FISH, rDNA.     

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.     

Survival and development of different life stages of three Ceratitis spp. (Diptera: Tephritidae) reared at five constant temperatures

Fruit flies (Diptera: Tephritidae) are the most damaging pests on fruit crops on Réunion Island, near Madagascar. Survival and development of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann), the Natal fruit fly, C. rosa Karsch and the Mascarenes fruit fly, C. catoirii Guérin-Mèneville were compared at five constant temperatures spanning 15 to 35 degrees C. Durations of the immature stages of C. capitata, C. rosa and C. catoirii ranged from 14.5-63.8, 18.8-65.7 and 16.8-65.8 days, respectively, at 30-15 degrees C. The lower developmental threshold and thermal constant were calculated using the temperature summation model. The thermal constant for total development of the immature stages of C. capitata, C. rosa and C. catoirii were 260, 405 and 356 DD, respectively. Species differed mainly during the larval stages and ovarian maturation period, with smaller differences in the egg stage. Ceratitis rosa appeared to be better adapted to low temperatures than the two other species as it showed a lower larval developmental threshold of 3.1 degrees C compared to 10.2 degrees C for C. capitata and 8.9 degrees C for C. catoirii. Overall, C. catoirii had a low survival rate within the range of temperatures studied. The different responses of the three Ceratitis species to various temperatures explain to some extent their distribution on the island. The results obtained will be used for optimizing laboratory rearing procedures and for constructing computer simulation models to predict fruit fly population dynamics.     

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.     

Sequence Analysis of Active Mariner Elements in Natural Populations of Drosophila Simulans

Active and inactive mariner elements from natural and laboratory populations of Drosophila simulans were isolated and sequenced in order to assess their nucleotide variability and to compare them with previously isolated mariner elements from the sibling species Drosophila mauritiana and Drosophila sechellia. The active elements of D. simulans are very similar among themselves (average 99.7% nucleotide identity), suggesting that the level of mariner expression in different natural populations is largely determined by position effects, dosage effects and perhaps other factors. Furthermore, the D. simulans elements exhibit nucleotide identities of 98% or greater when compared with mariner elements from the sibling species. Parsimony analysis of mariner elements places active elements from the three species into separate groups and suggests that D. simulans is the species from which mariner elements in D. mauritiana and D. sechellia are most likely derived. This result strongly suggests that the ancestral form of mariner among these species was an active element. The two inactive mariner elements sequenced from D. simulans are very similar to the inactive peach element from D. mauritiana. The similarity may result from introgression between D. simulans and D. mauritiana or from selective constraints imposed by regulatory effects of inactive elements.     

Developmental rates at constant temperatures of three economically important Ceratitis spp. (Diptera: Tephritidae) from southern Africa

Three species of Ceratitis MacLeay are of economic importance in southern Africa. To learn more about the influence of temperature on the development of these species, the developmental rates of South African populations of Ceratitis (Ceratitis) capitata (Wiedemann), C. (Pterandrus) rosa Karsch, and C. (Ceratalaspis) cosyra (Walker) were compared at constant temperatures of 14, 18, 22, 26, and 30 degrees C. The duration of each life stage and the percentage survival of the immature life stages of each species were determined. One linear and three nonlinear developmental rate models (Briére, Lactin, and Logan-6) were found to fit the data well and were used to generate the minimum, optimum, and maximum developmental thresholds, in addition to the life cycle thermal constants for the three species. These parameter values were 9.6, 28.5, 33.0, and 338 for C. capitata, 9.7, 28.8, 33.2, and 376 for C. cosyra, and 8.6, 27.7, 33.0, and 429 for C. rosa, respectively. The parameters for C. capitata are similar to those found by other researchers for this species in Reunion but the parameters for C. rosa differ substantially from published values for a Reunion population of this species, suggesting that these are different biotypes. The similarities between the developmental parameters for C. capitata and C. cosyra do not support known differences in the distribution of these species so other limiting factors such as relative humidity and the availability of host species may be important. This finding therefore cautions against basing predictions of potential global distributions of species solely on life table or climatic parameter values.     

Pathogenicity of Metarhizium anisopliae (Metsch.) Sorokin and Beauveria bassiana (Balsamo) Vuillemin,to three adult fruit fly species: Ceratitis capitata (Weidemann), C. rosa var. fasciventris Karsch and C. cosyra (Walker) (Diptera :Tephritidae)

The pathogenicity of two isolates of Beauveria bassiana and 12 of Metarhizium anisopliae towards adult fruit flies, Ceratitis capitata and Ceratitis rosa var. fasciventris was tested in the laboratory. Fruit flies were exposed to dry conidia evenly spread on velvet material covering the inner side of a cylindrical plastic tube. All isolates tested were pathogenic to both species of fruit flies. Mortality ranged from 7 to 100% in C. capitata and from 11.4 to 100% in C. rosa var. fasciventris at 4 days post-inoculation. Six isolates, M. anisopliae ICIPE 18, 20, 32, 40, 41 and 62, were highly pathogenic to both C. capitata and C. rosa var. fasciventris. The LT90 values of the most pathogenic isolates ranged between 3-4 days in both insects. Because of the difficulties in rearing C. cosyra, only the isolates that were highly pathogenic to both C. rosa var. fasciventris and C. capitata were tested against adult C. cosyra. They caused mortality of between 72-78% at 4 days post-inoculation. The LT90 values in all the isolates did not exceed 4 days. One of the most pathogenic isolates, M. anisopliae ICIPE 20, was evaluated against C. capitata and C. rosa var. fasciventris in cage experiments using three autoinoculators (maize cob, cheesecloth and Petri dish) in an autoinoculative device consisting of plastic mineral bottle. Mortality of between 70-93% was observed in flies of both species that were captured from the cages and held under laboratory conditions. These results indicate the possibility of fruit fly suppression with entomopathogenic fungi using an autoinoculative device.     

Recent horizontal transfer of mellifera subfamily mariner transposons into insect lineages representing four different orders shows that selection acts only during horizontal transfer

We report the isolation and sequencing of genomic copies of mariner transposons involved in recent horizontal transfers into the genomes of the European earwig, Forficula auricularia; the European honey bee, Apis mellifera; the Mediterranean fruit fly, Ceratitis capitata; and a blister beetle, Epicauta funebris, insects from four different orders. These elements are in the mellifera subfamily and are the second documented example of full-length mariner elements involved in this kind of phenomenon. We applied maximum likelihood methods to the coding sequences and determined that the copies in each genome were evolving neutrally, whereas reconstructed ancestral coding sequences appeared to be under selection, which strengthens our previous hypothesis that the primary selective constraint on mariner sequence evolution is the act of horizontal transfer between genomes.     

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.     

The chorion genes of the medfly, Ceratitis capitata. II. Characterization of three novel cDNA clones obtained by differential screening of an ovarian library

We have isolated three new chorion cDNA clones from a Ceratitis capitata ovarian library. Their isolation was accomplished by differential screening of the library using as probes 32P-labeled poly(A)+ mRNAs obtained from hand-staged medfly choriogenic versus prechoriogenic follicles. RNA blot hybridization analysis revealed that the genes corresponding to these clones have unique temporal profiles of mRNA accumulation, restricted to specific choriogenic stages. In addition, in vitro translation products encoded by these cDNAs approximately comigrated with polypeptides synthesized de novo in culture by choriogenic follicles. All three genes are located in regions of the medfly genome that are specifically amplified in female ovaries. DNA sequence analysis has revealed that one of these clones is derived from a homolog of the Drosophila melanogaster s38 chorion gene. It appears that, although D. melanogaster and C. capitata are separated by at least 120 million years of evolution, the mechanisms by which chorion genes are expressed and regulated during development have been well maintained. We suggest that the regulatory elements controlling the expression of sex-specific (e.g., chorion) genes may be isolated and used to construct transgenic medfly strains from which females could be eliminated by negative selection; such strains could be used as part of an effort to control this agricultural pest.     

The Hobo Transposable Element Excises and Has Related Elements in Tephritid Species

Function of the Drosophila melanogaster hobo transposon in tephritid species was tested in transient embryonic excision assays. Wild-type and mutant strains of Anastrepha suspensa, Bactrocera dorsalis, B. cucurbitae, Ceratitis capitata, and Toxotrypana curvicauda all supported hobo excision or deletion both in the presence and absence of co-injected hobo transposase, indicating a permissive state for hobo mobility and the existence of endogenous systems capable of mobilizing hobo. In several strains hobo helper reduced excision. Excision depended on hobo sequences in the indicator plasmid, though almost all excisions were imprecise and the mobilizing systems appear mechanistically different from hobo. hobo-related sequences were identified in all species except T. curvicauda. Parsimony analysis yielded a subgroup including the B. cucurbitae and C. capitata sequences along with hobo and Hermes, and a separate, more divergent subgroup including the A. suspensa and B. dorsalis sequences. All of the sequences exist as multiple genomic elements, and a deleted form of the B. cucurbitae element exists in B. dorsalis. The hobo-related sequences are probably members of the hAT transposon family with some evolving from distant ancestor elements, while others may have originated from more recent horizontal transfers.     

Characterization and Evolution of mariner Elements from Closely Related Species of Fruit Flies (Diptera: Tephritidae)

Mariner elements were amplified using the polymerase chain reaction from two species of tephritid flies, Ceratitis rosa and Trirhithrum coffeae. The sequences were ∼1.3 kb in length. None of these elements appeared to be functional, as in every case the open reading frame (ORF) was disrupted by the presence of frameshifts or stop codons. These elements, Crmar1 and Tcmar1, are very similar to the Ccmar1 element previously amplified from the closely related tephritid species C. capitata and are members of the mellifera subfamily of mariner elements. The phylogeny and pattern of divergence of these elements were examined in relation to the phylogeny of the host species. It is highly probable that the elements were present in the ancestral lineage prior to the divergence of the three species. The copy numbers of the elements within each species are very different, ranging from about 10 in T. coffeae to 5,000 in C. rosa. The possible mechanisms which determine the copy number of an element in the host genome are discussed. Received: 25 April 1997 / Accepted: 31 July 1997     

Climatic niche partitioning following successive invasions by fruit flies in La Réunion

1. Biological invasions have profound effects on community structure. The community composition following invasions can be influenced by the habitat diversity and the species' responses to abiotic factors. 2. We evaluated the tolerance to climatic factors and analysed the field distribution of four polyphagous fruit flies (Diptera: Tephritidae) of La Réunion Island (three exotic species that successively invaded the island and the endemic species Ceratitis catoirii) in order to evaluate the opportunities of coexistence by niche differentiation. 3. Atmospheric humidity and immersion in water in the laboratory greatly influence the survival of fruit fly pupae. While C. catoirii and C. rosa are very sensitive to desiccation, C. capitata and especially Bactrocera zonata are relatively tolerant. B. zonata also tolerated immersion in water much longer than did C. rosa and C. catoirii, that in turn were more resistant than C. capitata. Overall, field distributions agree with the predictions based on this study of humidity combined with previous data on the effects of temperature. 4. Climatic niche partitioning promotes coexistence between some but not all pairs of invasive species. Thus, C. rosa can coexist with both C. capitata and B. zonata at the regional scale, while climatic niches are not different enough to promote coexistence of the latter two species. The endemic species has no private climatic niche either and this now very rare species could be in the process of extinction. 5. By promoting coexistence or not, climatic diversity in invaded areas can directly affect the community composition following invasions.     

Introduction of the Transposable Element Mariner into the Germline of Drosophila Melanogaster

A chimeric white gene (wpch) and other constructs containing the transposable element mariner from Drosophila mauritiana were introduced into the germline of Drosophila melanogaster using transformation mediated by the P element. In the absence of other mariner elements, the wpch allele is genetically stable in both germ cells and somatic cells, indicating that the peach element (i.e., the particular copy of mariner inserted in the wpch allele) is inactive. However, in the presence of the active element Mos1, the wpch allele reverts, owing to excision of the peach element, yielding eye-color mosaics and a high rate of germline reversion. In strains containing Mos1 virtually every fly is an eye-color mosaic, and the rate of wpch germline reversion ranges from 10 to 25%, depending on temperature. The overall rates of mariner excision and transposition are approximately sixfold greater than the rates in comparable strains of Drosophila simulans. The activity of the Mos1 element is markedly affected by position effects at the site of Mos1 insertion. In low level mosiac lines, dosage effects of Mos1 are apparent in the heavier level of eye-color mosaicism in Mos1 homozygotes than in heterozygotes. However, saturation occurs in high level mosaic lines, and then dosage effects are not observed. A pBluescribe M13+ plasmid containing Mos1 was injected into the pole plasm of D. melanogaster embryos, and the Mos1 element spontaneously integrated into the germline at high efficiency. These transformed strains of D. melanogaster presently contain numerous copies of mariner and may be useful in transposon tagging and other applications.     

Exceptional longevity in the tephritid, Ceratitis rosa, a close relative of the Mediterranean fruit fly

This study shows that the fruit fly, Ceratitis rosa (Karsch), has a significantly longer life span than the medfly, C. capitata (Wiedemann); the species used as a model organism for the demographics of insect aging. This was somewhat surprising given that both have similar distributions and overlapping niches. We postulate that the greater longevity of C. rosa is related to the fact that it can occupy colder habitats where the availability of suitable host plants may be very unpredictable in both time and space.     

Molecular and Functional Analysis of the Mariner Mutator Element Mos1 in Drosophila

The white-peach allele in Drosophila results from insertion of the transposable element mariner. The particular copy that is inserted in white-peach is an inactive copy referred to as the peach element. The peach element is excised at a high rate in the presence of active copies of mariner located elsewhere in the genome, and the excision of peach in somatic cells is recognized phenotypically by the occurrence of eye-color mosaicism in white-peach flies. Active mariner elements identified by their ability to induce high levels of white-peach mosaicism are denoted Mos (Mosaic) factors. We have sequenced and functionally analyzed the factor Mos1 originally identified in Drosophila mauritiana. The Mos1 element is 1286 base pairs in length, the same length as the peach element. It differs from the peach element in 11 nucleotide positions distributed throughout its length, including four amino acid replacements in the long open reading frame. Analysis of chimeric constructs between Mos1 and peach implies that functionally important differences occur in both the 5' and 3' halves of Mos1. A mariner element identical in sequence to Mos1 yields lower levels of mosaicism in transformants, implying that adjacent flanking sequences have important effects on Mos1 activity. Another mariner element, designated Ma351, isolated from a nonmosaic strain of D. mauritiana, differs from Mos1 in just three nucleotide positions. When introduced into the germline, Ma351 yields various levels of white-peach mosaicism depending on insertion site. These results imply that the activity of mariner elements is determined jointly by their own nucleotide sequences, by the effects of adjacent flanking sequences, and by longer-range position effects.     

Identification of Nucleotide Substitutions Necessary for Trans-Activation of Mariner Transposable Elements in Drosophila: Analysis of Naturally Occurring Elements

Six copies of the mariner element from the genomes of Drosophila mauritiana and Drosophila simulans were chosen at random for DNA sequencing and functional analysis and compared with the highly active element Mos1 and the inactive element peach. All elements were 1286 base pairs in length, but among them there were 18 nucleotide differences. As assayed in Drosophila melanogaster, three of the elements were apparently nonfunctional, two were marginally functional, and one had moderate activity that could be greatly increased depending on the position of the element in the genome. Both molecular (site-directed mutagenesis) and evolutionary (cladistic analysis) techniques were used to analyze the functional effects of nucleotide substitutions. The nucleotide sequence of the element is the primary determinant of function, though the activity level of elements is profoundly influenced by position effects. Cladistic analysis of the sequences has identified a T----A transversion at position 1203 (resulting in a Phe----Leu amino acid replacement in the putative transposase) as being primarily responsible for the low activity of the barely functional elements. Use of the sequences from the more distantly related species, Drosophila yakuba and Drosophila teissieri, as outside reference species, indicates that functional mariner elements are ancestral and argues against their origination by a novel mutation or by recombination among nonfunctional elements.     

The clock gene period in the medfly Ceratitis capitata

We have isolated the clock gene period (per) from the medfly Ceratitis capitata, one of the most economically important insect pest species. The overall pattern of conserved, non-conserved and functional domains that are observed within dipteran and lepidopteran per orthologues is preserved within the coding sequence. Expression analysis from fly heads revealed a daily oscillation in per mRNA in both light : dark cycles and in constant darkness. However PER protein levels from head extracts did not show any significant evidence for cycling in either of these two conditions. When the Ceratitis per transgene under the control of the Drosophila per promoter and 3'UTR was introduced into Drosophila per -null mutant hosts, the transformants revealed a low level of rescue of behavioural rhythmicity. Nevertheless, the behaviour of the rhythmic transformants showed some similarities to that of ceratitis, suggesting that Ceratitis per carries species-specific information that can evidently affect the Drosophila host's downstream rhythmic behaviour.