Mitotic and polytene chromosome analysis in Dacus oleae (Diptera: Tephritidae).

The present study constitutes the first attempt to construct a photographic map of the polytene chromosomes of Dacus oleae, a pest of the olive tree that causes serious financial damage in all olive oil producing countries. The map was constructed by using the larval fat body cells, the chromosomes of which are representative of the polytene chromosomes of other polytene tissues. In addition, the mitotic chromosomes of brain ganglia were examined, permitting tentative correlations between mitotic and polytene elements. This investigation shows that D. oleae is suitable for cytogenetic analysis in both mitotic and polytene chromosomes, a fact that may prove very useful for obtaining more detailed genetic information on the pest's natural populations.     

Malpighian tubule polytene chromosomes of Culex quinquefasciatus (Diptera,Culicinae)

Dipteran polytene chromosomes provide an excellent model for understanding in species complexes, as well as for structural and functional cytogenetics. The status of species in the Culex pipiens complex is controversial and the use of polytene chromosomes for cytogenetic analysis in the subfamily Culicinae has been difficult because of methodological problems. In this study, Malpighian tubule polytene chromosomes were obtained from young (0 to 12 h, 20 C) and old (20 to 42 h, 28 C) laboratory-bred C. pipiens quinquefasciatus pupae. The chromosome maps for this species were constructed and compared with published data for C. pipiens pipiens and C. p. quinquefasciatus. Although the banding patterns were conserved between subspecies, analysis of the structural variations in the bands and interbands revealed differences apparently related to the physiological stage and ecogeographical strain. The organization of the centromeric regions in larval and pupal chromosomes showed greater similarity to each other than did those of pupal and adult chromosomes. The use of pupal polytene chromosomes for in situ hybridization with vector competence probes is discussed.     

Sex chromosomes and associated rDNA form a heterochromatic network in the polytene nuclei of Bactrocera oleae (Diptera: Tephritidae)

The olive fruit fly, Bactrocera oleae, has a diploid set of 2n?=?12 chromosomes including a pair of sex chromosomes, XX in females and XY in males, but polytene nuclei show only five polytene chromosomes, obviously formed by five autosome pairs. Here we examined the fate of the sex chromosomes in the polytene complements of this species using fluorescence in situ hybridization (FISH) with the X and Y chromosome-derived probes, prepared by laser microdissection of the respective chromosomes from mitotic metaphases. Specificity of the probes was verified by FISH in preparations of mitotic chromosomes. In polytene nuclei, both probes hybridized strongly to a granular heterochromatic network, indicating thus underreplication of the sex chromosomes. The X chromosome probe (in both female and male nuclei) highlighted most of the granular mass, whereas the Y chromosome probe (in male nuclei) identified a small compact body of this heterochromatic network. Additional hybridization signals of the X probe were observed in the centromeric region of polytene chromosome II and in the telomeres of six polytene arms. We also examined distribution of the major ribosomal DNA (rDNA) using FISH with an 18S rDNA probe in both mitotic and polytene chromosome complements of B. oleae. In mitotic metaphases, the probe hybridized exclusively to the sex chromosomes. The probe signals localized a discrete rDNA site at the end of the short arm of the X chromosome, whereas they appeared dispersed over the entire dot-like Y chromosome. In polytene nuclei, the rDNA was found associated with the heterochromatic network representing the sex chromosomes. Only in nuclei with preserved nucleolar structure, the probe signals were scattered in the restricted area of the nucleolus. Thus, our study clearly shows that the granular heterochromatic network of polytene nuclei in B. oleae is formed by the underreplicated sex chromosomes and associated rDNA.     

Developmental puffing activity in the salivary gland and Malpighian tubule chromosomes ofAcricotopus lucidus (Diptera,Chironomidae)

A detailed map of the salivary gland chromosomes ofAcricotopus lucidus is presented. Differences in puffing and developmental Puffing sequences of the three salivary gland lobes were investigated from mid fourth larval instar to pupation and compared with the puffing pattern of the Malpighian tubules. The intraglandular differentiation is quite extensive; the differences in the pattern of gene activity between the anterior lobe and the main and side lobes are as great as between the salivary gland and the Malpighian tubules. In the main and side lobes all developmental puffing changes proceed synchronously whereas in the anterior lobe both asynchronous and synchronous changes occur. In the anterior lobe the asynchronous regression of BR 3 and BR 4 is followed by a characteristic sequence of activation and inactivation of puffs.     

Mitotic and polytene chromosomes analysis of the oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae)

The Oriental fruit fly, Batrocera dorsalis s.s. (Hendel) is one of the most destructive agricultural pests, belonging to a large group of difficult to distinguish morphologically species, referred as the B. dorsalis complex. We report here a cytogenetic analysis of two laboratory strains of the species and provide a photographic polytene chromosome map from larval salivary glands. The mitotic complement consists of six chromosome pairs including a heteromorphic sex (XX/XY) chromosome pair. Analysis of the polytene complement has shown a total of five polytene chromosomes (10 polytene arms) that correspond to the five autosomes. The most important landmarks of each polytene chromosome and characteristic asynapsis at a specific chromosomal region are presented and discussed. Chromosomal homology between B. dorsalis and Ceratitis capitata has been determined by comparing chromosome banding patterns. The detection of chromosome inversions in both B. dorsalis strains is shown and discussed. Our results show that the polytene maps presented here are suitable for cytogenetic analysis of this species and can be used for comparative studies among species of the Tephritidae family. They also provide a diagnostic tool that could accelerate species identification within the B. dorsalis complex and could shed light on the ongoing speciation in this complex. Polytene chromosome maps can facilitate the development of biological control methods and support the genome mapping project of the species that is currently in progress.     

Olive fruit fly [Bactrocera (Dacus) oleae (Rossi) (Diptera: Tephritidae)] adult rearing diet without antibiotic

The olive fruit fly [Bactrocera (Dacus) oleae (Rossi) (Diptera: Tephritidae)] adult diet since its development in the 1960’s regularly incorporates antibiotic. Considering recent findings on the importance and function of the indigenous microbial flora of insects, the effects of antibiotic removal were measured on the survival and egg laying of wild flies derived from McPhail trappings and from field infested olive fruits. In the first case wild flies fed no antibiotic laid significantly greater numbers of eggs for two generations (in 5 out of 10 and 2 out of 10 counting dates for G1 and G2 respectively), while there were no significant differences in survival in either test (P = 0.221 for P generation, P = 0.988 for G1 generation from McPhail traps, P = 0.056 for flies from infested fruits). Percent egg–pupa recovery and adult emergence were not significantly affected by lack of antibiotic. An antibiotic‐free strain has been maintained for eight generations, showing acceptable performance when compared to the long‐reared standard ‘Laboratory’ strain. Overall results suggest the feasibility of an adult diet free of antibiotic without negative effects on colony survival and performance.     

Genetic and cytogenetic analysis of the olive fruit fly Bactrocera oleae (Diptera: Tephritidae)

The genetic and cytogenetic characteristics of one of the major agricultural pests, the olive fruit fly Bactrocera oleae, are presented here. The mitotic metaphase complement of this insect consists of six pairs of chromosomes including one pair of heteromorphic sex chromosomes, with the male being the heterogametic sex. The analysis of the polytene complements of three larval tissues, the fat body, the salivary glands and the Malpighian tubules of this pest has shown (a) a total number of five long chromosomes (10 polytene arms) that correspond to the five autosomes of the mitotic nuclei and a heterochromatic mass corresponding to the sex chromosomes, (b) the constancy of the banding pattern of the three somatic tissues, (c) the absence of a typical chromocenter as an accumulation of heterochromatin, (d) the existence of reverse tandem duplications, and (e) the presence of toroid tips of the chromosome arms. The in situhybridization of genes or DNA sequences to the salivary gland polytene chromosomes of B. oleaeprovided molecular markers for all five autosomes and permitted the establishment of chromosomal homologies among B. olea, B. tryoniand Ceratitis capitata. The heat shock response of B. oleae, as revealed by heat-inducible puffing and protein pattern, shows a higher thermotolerance than Drosophila melanogaster.     

Biochemical and immunocytochemical analysis of vitellogenesis in the olive fruit fly Dacus (bactrocera) oleae (Diptera: Tephritidae)

Synthesis and selective accumulation of the major yolk proteins in the developing oocytes of the species Dacus oleae (Diptera: Tephritidae) was studied biochemically and by immunoelectron microscopy. In the hemolymph of adult females, two yolk proteins precursors (or vitellogenins) have been detected. They each exhibit a similar molecular weight and isoelectric point to their respective mature yolk proteins (or vitellins), while electrophoretic analysis of their synthetic profile shows that their levels in the hemolymph increase rapidly during development. Immunogold electron microscopy of ovarian sections, revealed that the hemolymph vitellogenins reach the oocyte through enlarged inter-follicular spaces and demonstrated vitellogenin synthesis by the follicle cells of the vitellogenic follicles. The newly synthesized vitellogenins follow a distinct secretory pathway into these cells as compared to other components being synthesized at the same time (e.g. the vitelline envelope proteins), since they were found in secretory vesicles that appeared to be differentiated from those destined to participate in the vitelline envelope. The vitellogenin-containing vesicles exocytose their contents directionally into the follicle cell/vitelline envelope boundary, and subsequently the vitellogenins diffuse among the gaps of the forming vitelline envelope and reach the oocyte plasma membrane. Their internalization by the oocyte includes the formation of an endocytic complex consisting of coated pits, coated vesicles, endosomes, transitional yolk bodies, and finally mature yolk bodies, in which the storage of the vitellins and other yolk proteins occur. These results are discussed in relation to data obtained from other Dipteran species.     

Microflora species and their volatile compounds affecting development of an alcohol dehydrogenase homozygous strain (Adh-I) of Bactrocera (Dacus) oleae (Diptera: Tephritidae)

Microflora species and volatiles emitted from artificial diets were examined from the larvae of three homozygous alcohol dehydrogenase (Adh) strains of the olive fruit fly, Bactrocera (Dacus) oleae (Gmelin), reared under identical conditions. Differences in volatile composition were detected when Adh-I homozygous larvae developed in a diet lacking the preservative p-hydroxybenzoic acid methyl ester (nipagin). Larval development of the Adh-I strain in the preservative-free diet was reduced by 50%, whereas pupal emergence was completely inhibited. The larval development and pupal emergence of Adh-F and Adh-S strains were not affected. Unique microorganisms with characteristic volatile profiles were isolated from the preservative-free diet of the Adh-I strain that were different from those, isolated from Adh-S, Adh-F, laboratory colony, and wild insect populations. Our results indicated that the variations in volatile composition of the artificial diets, and the inhibition of larval development and pupal emergence in Adh-I strain were related to changes in the microflora that developed in the diets of the Adh-I strain.     

Digestive proteolytic activity in larvae and adults of Bactrocera oleae Gmelin (Diptera: Tephritidae)

Digestive proteolytic activity in larvae and adults of Bactrocera oleae was studied using specific substrates and inhibitors. The optimal pH for general proteolytic activity was 4 and 10 for soluble and membrane-bound fractions of larvae, and 9 for the soluble fraction of adults. The highest activities of general proteases were revealed at temperatures of 25 °C and 45 °C for both the soluble and membrane-bound fractions of larvae as well as the soluble fraction of adults. Determination of the specific protease activities demonstrated the presence of serine and cysteine proteases in addition to two exopeptidases in the larvae and adults. However, trypsin-like protease, chymotrypsin-like protease, and two exopeptidases of larvae, and chymotrypsin-like protease as well as cathepsin L of adults had no activity in the soluble fraction. The presence of specific proteases was verified by using specific inhibitors such as PMSF, TLCK, TPCK, E-64, EDTA, phenanthroline, and DTT. Finally, feeding of B. oleae larvae on different olive varieties revealed the highest trypsin-like protease, chymotrypsin-like protease, elastase, cathepsin B, cathepsin L, and cathepsin D on Amigdalifolia, Coratina, Baladi, Mari, Conservalia, Baladi, and Arbequina, respectively. These results showed digestive proteolytic activities in B. oleae for the first time, and could be the basic knowledge required for finding a control procedure to decrease the damage of this destructive pest around the world.     

Genetic differences among natural and laboratory-reared populations of the olive-fruit fly Dacus oleae (Diptera: Tephritidae)

Twelve wild populations of the olive-fruit fly Dacus oleae from around the Mediterranean basin and two laboratory populations were examined for their electrophoretic profiles at two esterase loci. The genetic distances among wild and laboratory populations were consistently greater than the distances among wild populations. There was a pattern in this genetic differentiation, in that the most common allele decreased in frequency during culture in the laboratory, thus causing an increase of heterozygosity in laboratory populations. The findings are discussed in the light of assessing the genetic quality of mass-laboratory (or factory) cultures of economically important insects.

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Zusammenfassung Die elektrophoretischen Profile zweier Esterase-Loci von zwölf natürlichen Populationen der Olivenfliege Dacus oleae aus Ländern des Mittelmeerbeckens und von zwei Laboratorium-Populationen wurden untersucht. Die genetischen Abstände zwischen natürlichen und Laboratorium-Populationen waren konsistent grösser als die Abstände zwischen natürlichen Populationen. Bei dieser genetischen Differenzierung zeigte sich eine Regelmässigkeit. Die häufigsten Allele nahmen während der Zucht im Laboratorium in ihrer Häufigkeit ab, was zu einer Zunahme der Heterozygosis in Laboratorium-Populationen führte.Diese Resultate werden diskutiert im Zusammenhang mit der genetischen Qualität von Massenzuchten wirtschaftlich wichtiger Insekten im Laboratorium und in der Industrie.

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On leave of absence from Department of Biology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4J1.

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This is paper No. VII of the series The Genetics of Dacus oleae     

Suppression of Bactrocera oleae (Diptera: Tephritidae) pupae by soil arthropods in the olive grove

Soil arthropods can provide ecosystem services, such as biological control of crop pests that spend part of their life cycle in the soil. This is the case of Bactrocera oleae (Rossi) (Diptera: Tephritidae), one of the most important pests of olives. The impact of edaphic arthropods on the abundance of B. oleae pupae was evaluated and their contribution for biological control of the pest was quantified. Exclusion and exposed boxes with B. oleae pupae were installed in olive groves in parallel with pitfall traps used for sampling arthropods and the percentage of pupae suppression was evaluated from January to May 2014. Forficulidae dominated the community during the winter period while Formicidae dominated in spring. Pupae suppression reached the maximum value in the beginning of spring and these results indicate that soil arthropods have strong impact in the decline of B. oleae pupae in olive groves.     

Studies on mitotic and salivary chromosomes of Dacus cucurbitae Coquillett (Diptera: Trypetidae)

Dacus cucurbitae is a serious pest of various types of fleshy fruits and vegetables. The mitotic and salivary chromosomes were reinvestigated using the air-drying and different (C-and H-) banding techniques with a view to rectify the existing controversy regarding the Indian populations of this species. A standard map of its salivary chromosomes was constructed and some important identifiable landmarks were recognized in each arm.     

Population structure and colonization history of the olive fly, Bactrocera oleae (Diptera, Tephritidae)

The olive fly, Bactrocera oleae, is the major pest of olives in most commercial olive-growing regions worldwide. The species is abundant in the Mediterranean basin and has been introduced recently into California and Mexico, creating problems for quarantine protection and international trade. Here, we use nuclear microsatellite markers and mitochondrial sequences to examine the history of olive fly range expansion and colonization. Sampled populations span the current distribution of the olive fly worldwide, including South and Central Africa, Pakistan, Mediterranean Europe and Middle East, California, and Mexico. The Pakistani populations appear to be genetically well differentiated from the remaining populations, though rooting the origins of the species is problematic. Genetic similarity and assignment tests cluster the remaining populations into two genetic groups--Africa and a group including the Mediterranean basin and the American region. That Africa, and not the Mediterranean, is the origin of flies infesting cultivated olive is supported by the significantly greater genetic diversity at microsatellite loci in Africa relative to the Mediterranean area. The results also indicate that the recent invasion of olive flies in the American region most likely originated from the Mediterranean area.     

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.     

Insecticidal activity of Citrus aurantium peel extract against Bactrocera oleae and Ceratitis capitata adults (Diptera: Tephritidae)

Citrus aurantium (L.) peel extracts in petroleum ether were evaluated for toxicity against olive fruit fly, Bactrocera oleae (Gmelin) and medfly, Ceratitis capitata (Wiedemann) adults. Bactrocera oleae flies were more susceptible to the extract than C. capitata in contact and residual bioassays. Fumigation bioassay had no effect. Both sexes of B. oleae were equally susceptible in both types of bioassays. However, males of C. capitata were more susceptible than the conspecific females. LD₅₀ values (concentration causing 50% mortality) after 96 h for the males and females of B. oleae were 44.8 and 40.1 μg/insect in contact bioassay through topical application. Whereas, LD₅₀ values for the males and females of C. capitata were 38.8 and 67.8 μg/insect respectively. LC₅₀ values after 96 h for the males and females of B. oleae were 18.8 and 17.8 μg/cm² in Petri dish residual bioassay. Whereas, LC₅₀ values for the males and females of C. capitata were 70.6 and 147.1 μg/cm² respectively.
Fractionation of the extract on a silica gel column with three different polarity solvents resulted in three fractions with only the intermediate polarity solvent fraction having substantial insecticidal activity. Toxicity of the mixtures of active and inactive fractions was equal to the original extract. Our results indicate that C. aurantium has potential for controlling insect pests.     

Cage study of spinosad-based bait efficacy on Bactrocera cucurbitae, Dacus ciliatus, and Dacus demmerezi (Diptera: Tephritidae) in Reunion Island

On Reunion Island, Bactrocera cucurbitae (Coquillett), Dacus ciliatus (Loew), and Dacus demmerezi (Bezzi) cause severe damage to Cucurbit crops. The aim of the study was to test in field cages the effectiveness of Synéis-appat (Dow AgroSciences), a spinosad-based bait (0.02% of spinosad) on both attraction and mortality of young adults (6-9 d old) of these three species. The effects of gender were also evaluated for all species whereas the effects of protein deprivation were tested with B. cucurbitae only. For the first 15 min after application, significantly more B. cucurbitae adults (21.7 +/- 1.8%) were attracted to the bait than D. demmerezi (7.6 +/- 2.4%) and D. ciliatus (2.7 +/- 1.4%); the subsequent response (30-75 min after bait application) of D. demmerezi was statistically similar to that recorded for B. cucurbitae; whereas the response ofD. ciliatus to the bait was consistently significantly lower. Adult mortality was significantly higher for B. cucurbitae (94.6 +/- 0.7%) than for D. demmerezi (85.7 +/- 2.1%), and was significantly higher for the latter than for D. ciliatus (60.4 +/- 4.4%). Sex had no significant effect on the mortality rate for each species. The efficiency of the bait was the same for B. cucurbitae adults regardless whether or not the diet included proteins. Overall, Synéis-appat appears to be more effective against B. cucurbitae and B. demmerezi than against D. ciliatus. In Reunion Island, this bait could constitute a useful component in the framework of Integrated Pest Management.     

The importance of dietary amino acids on the reproduction and longevity of adult Dacus oleae (Gmelin) (Diptera Tephritidae)

When the amino-acid mixture of an effective chemically defined diet was replaced by single amino acids, keeping the total nitrogen at the same level, the egg production of Dacus oleae was minimal with all the 19 amino acids tested. Male survival was adversely affected by the amino acids : alanine, aspartic acid, glutamic acid, glycine, hydroxyproline, lysine, serine and tyrosine, while female survival was shortened when the amino acids : glycine, hydroxyproline and lysine were added. The creation of amino-acid imbalances, by deleting the 19 amino acids individually, from the complete amino-acid mixture, showed that the amino acids : arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tryptophane and valine were indispensable to the adult Dacus oleae flies, as far as egg production is concerned. Survival of the male flies was significantly shortened when the amino acids : alanine, hydroxyproline and tryptophane were omitted. Significant differences in longevity between males and females were scored, when the amino acids : alanine, aspartic acid, cystine, glycine and tryptophane, were omitted.