Establishment and characterization of the Bactrocera dorsalis (Diptera: Tephritidae) embryonic cell line QAU-Bd-E-2.

In this study, we successfully established a Bactrocera dorsalis (Diptera: Tephritidae) embryonic cell line, i.e., QAU-Bd-E-2, from the insect eggs. The cells have been stably passaged for more than 60 times in TNM-FH medium with 10% fetal bovine serum (FBS). QAU-Bd-E-2 cells are adherent cells. Most of the cells were round, spindle-shaped, and rod-shaped. Round cells accounted for 82.3%, with a diameter of 13.9 ± 2.6 µm; spindle-shaped cells accounted for 9.8%, with the size of 51.2 ± 11.2 µm × 10.3 ± 3.1 µm; the rod-shaped cells accounted for 7.9%, with the size of 35.2 ± 9.4 µm × 12.0 ± 2.5 µm. The mitochondrial cytochrome oxidase I subunit (CoI) gene from QAU-Bd-E-2 cells was amplified, and the 657 bp fragment had a 100% similarity with the CoI gene of B. dorsalis, suggesting that the cell line was derived from B. dorsalis. The chromosome number of QAU-Bd-E-2 cells was mostly 12, which is the same as the B. dorsalis chromosome number. The cell density of QAU-Bd-E-2 cells reached the maximum (3.4 × 106 cells/mL) at 192 h, and the population doubling time was 31.9 h. Bactrocera dorsalis cripavirus (BdCV) could replicate in QAU-Bd-E-2 cells, suggesting that this cell line could be used for in-depth study of the relationship between virus and host.

Giant polyploid epidermal cells and male pheromone production in the tephritid fruit fly Eurosta solidaginis (Diptera: Tephritidae).

Eurosta solidaginis males produce large amounts of putative sex pheromone compared to other insect species; however, neither the site of pheromone production nor the release mechanism has been characterized. We compared E. solidaginis males and females, focusing on sexually dimorphic structures that are known to be involved in pheromone production in other tephritid species. Morphological and chemical analyses indicated that the rectum and pleural epidermis are involved in male E. solidaginis pheromone production, storage, or emission. We detected large quantities of pheromone in the enlarged rectum, suggesting that it stores pheromone for subsequent release through the anus. However, pheromone might also discharge through the pleural cuticle with the involvement of unusual pleural attachments of the tergosternal muscles, which, when contracted in males, realign specialized cuticular surface elements and expose less-sclerotized areas of cuticle. In males, pheromone components were also detected in epidermal cells of the pleuron. These cells were 60-100 times larger in mature males than in females and, to our knowledge, are the largest animal epithelial cells ever recorded. Furthermore, because these large cells in males are multinucleated, we presume that they develop through somatic polyploidization by endomitosis. Consequently, the pheromone-associated multinuclear pleural epidermal cells of Eurosta solidaginis may provide an interesting new system for understanding polyploidization.

Functional characterization of olfactory receptors in the Oriental fruit fly Bactrocera dorsalis that respond to plant volatiles.

The Oriental fruit fly, Bactrocera dorsalis, is a highly destructive pest of various fruits. The reproductive and host-finding behaviors of this species are affected by several plant semiochemicals that are perceived through chemosensory receptors. However, the chemosensory mechanisms by which this perception occurs have not been fully elucidated. We conducted RNA sequencing analysis of the chemosensory organs of B. dorsalis to identify the genes coding for chemosensory receptors. We identified 60 olfactory receptors (ORs), 17 gustatory receptors and 23 ionotropic receptors-including their homologs and variants-from the transcriptome of male antennae and proboscises. We functionally analyzed ten ORs co-expressed with the obligatory co-receptor ORCO in Xenopus oocytes to identify their ligands. We tested 24 compounds including attractants for several Bactrocera species and volatiles from the host fruits of B. dorsalis. We found that BdorOR13a co-expressed with ORCO responded robustly to 1-octen-3-ol. BdorOR82a co-expressed with ORCO responded significantly to geranyl acetate, but responded weakly to farnesenes (a mixture of isomers) and linalyl acetate. These four compounds were subsequently subjected to behavioral bioassays. When each of the aforementioned compound was presented in combination with a sphere model as a visual cue to adult flies, 1-octen-3-ol, geranyl acetate, and farnesenes significantly enhanced landing behavior in mated females, but not in unmated females or males. These results suggest that the ORs characterized in the present study are involved in the perception of plant volatiles that affect host-finding behavior in B. dorsalis.

Current source density mapping of antennal sensory selectivity reveals conserved olfactory systems between tephritids and Drosophila.

Ecological specialization of insects involves the functional and morphological reshaping of olfactory systems. Little is known about the degree to which insect sensitivity to odorant compounds is conserved between genera, tribes, or families. Here we compared the olfactory systems of six tephritid fruit fly species spanning two tribes and the distantly related Drosophila melanogaster at molecular, functional, and morphological levels. Olfaction in these flies is mediated by a set of olfactory receptors (ORs) expressed in different functional classes of neurons located in distinct antennal regions. We performed a phylogenetic analysis that revealed both family-specific OR genes and putative orthologous OR genes between tephritids and Drosophila. With respect to function, we then used a current source density (CSD) analysis to map activity across antennae. Functional maps mirrored the intrinsic structure of antennae observed with scanning electron microscopy. Together, the results revealed partial conservation of the olfactory systems between tephritids and Drosophila. We also demonstrate that the mapping of olfactory responses is necessary to decipher antennal sensory selectivity to olfactory compounds. CSD analysis can be easily applied to map antennae of other species and therefore enables the rapid deriving of olfactory maps and the reconstructing of the target organisms' history of evolution.

The mitochondrial genome of the peach fruit fly, Bactrocera zonata (Saunders) (Diptera: Tephritidae): Complete DNA sequence, genome organization, and phylogenetic analysis with other tephritids using next generation DNA sequencing.

Mitochondrial genome can provide information for genomic structure as well as for phylogenetic analysis and evolutionary biology. The complete 15,935 bp mitochondrial genome of Bactrocera zonata (Diptera: Tephritidae), is assembled from Illumina MiSeq read data. The mitogenome information for B. zonata was compared to the homologous sequences of other tephritids. Annotation indicated that the structure and orientation of 13 protein coding genes (PCGs), 22 tRNA and 2 rRNA sequences were typical of, and similar to, the ten closely related tephritid species. The nucleotide composition shows heavily biased toward As and Ts accounting 73.34% and exhibits a slightly positive AT skew, which is similar to other known tephritid species. All PCGs are initiated by ATN codons, except for cox1 with TCG and atp8 with GTG. Nine PCGs use a common stop codon of TAA or TAG, whereas the remaining four use an incomplete termination codon T or TA likely to be completed by adenylation. All tRNAs have the typical clover-leaf structure, with an exception for trnS((AGN)). Four short intergenic spacers showed high degree of conservation among B. zonata and other ten tephritids. A poly(T) stretch at the 5' end followed by [TA(A)]n-like stretch and a tandem repeats of 39 bp has been observed in CR. The analysis of gene evolutionary rate revealed that the cox1 and atp6 exhibits lowest and highest gene substitution rates, respectively than other genes. The phylogenetic relationships based on Maximum Likelihood method using all protein-coding genes and two ribosomal RNA genes confirmed that B. zonata is closely related to Bactrocera correcta, Bactrocera carambolae, Bactrocera papayae, and Bactrocera philippinensis and Bactrocera dorsalis belonging to B. dorsalis species complex forms a monophyletic clade, which is in accordance with the traditional morphological classification and recent molecular works.

Genetic and cytogenetic characterization of genetic sexing strains of Bactrocera dorsalis and Bactrocera cucurbitae (Diptera: Tephritidae).

In the current study, we performed genetic and cytogenetic analyses of two genetic sexing strains (GSSs), one for Bactrocera dorsalis s.s. and one for melon fly, Bactrocera cucurbitae Coquillett, the first such strains ever constructed in these species. In both strains, the genetic sexing mechanism is based on a pupal color dimorphism (white or brown) and is the result of a reciprocal translocation between the Y chromosome and the autosome bearing the white pupae (wp) locus. Based on genetic analysis and cytological data on mitotic metaphases and larval salivary gland polytene chromosomes, we succeeded in mapping the autosome breakpoints in the two Y-autosome translocations even though the Y chromosome is not visible in polytene nuclei. We show that polytene chromosomes can be used in cytogenetic analyses toward the development of genetic control methods in these pest species. The results of the genetic analysis are in full agreement with the cytological description of the strains.

An EST database of the Caribbean fruit fly, Anastrepha suspensa (Diptera: Tephritidae).

Invasive tephritid fruit flies are a great threat to agriculture worldwide and warrant serious pest control measures. Molecular strategies that promote embryonic lethality in these agricultural pests are limited by the small amount of nucleotide sequence data available for tephritids. To increase the dataset for sequence mining, we generated an EST database by 454 sequencing of the caribfly, Anastrepha suspensa, a model tephritid pest. This database yielded 95,803 assembled sequences with 24% identified as independent transcripts. The percentage of caribfly sequences with hits to the closely related tephritid, Rhagoletis pomonella, transcriptome was higher (28%) than to Drosophila proteins/genes (18%) in NCBI. The database contained genes specifically expressed in embryos, genes involved in the cell death, sex-determination, and RNAi pathways, and transposable elements and microsatellites. This study significantly expands the nucleotide data available for caribflies and will be a valuable resource for gene isolation and genomic studies in tephritid insects.

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.

Cytological characterization of sex chromosomes and ribosomal DNA location in Anastrepha species (Diptera, Tephritidae).

This paper reports a comparative analysis of heterochromatin organization in the sex chromosomes of the fruit fly Anastrepha. Mitotic chromosomes of eight Anastrepha species from different taxonomic groups were stained with DAPI and chromomycin A3 fluorochromes followed by C-banding. A specific sex-chromosome banding pattern was obtained for each of the analyzed species. Fluorescence in situ hybridization (FISH) was performed to investigate the chromosomal location of rDNA loci. In all cases the rDNA sequences were found to localize exclusively to the sex chromosomes. The results further extend the chromosomal knowledge of Anastrepha and allow a precise species identification.

Programmed cell death of follicular epithelium during the late developmental stages of oogenesis in the fruit flies Bactrocera oleae and Ceratitis capitata (Diptera, Tephritidae) is mediated by autophagy.

In the present study, we describe the features of programmed cell death of ovarian follicle cells, occurring during the late developmental stages of oogenesis in the olive fruit fly, Bactrocera oleae and the medfly, Ceratitis capitata. During stage 14, the follicle cells contain autophagic vacuoles, and they do not exhibit caspase activity in all parts of the egg chamber. Their nuclei are characterized by condensed chromatin, accompanied with high- but not low-molecular weight DNA fragmentation events exclusively detected in distinct cells of the anterior pole. These data argue for the presence of an autophagy-mediated cell death program in the ovarian follicle cell layer in both species. The above results are likely associated with the abundant phagocytosis observed at the entry of the lateral oviducts, where numerous cell bodies are massively engulfed by epithelial cells. We strongly believe that during the termination of the above Dipteran oogenesis, an efficient mechanism of absorption of the degenerated follicle cells is selectively activated, in order to prevent the blockage of the ovarioles and thus robustly support the physiological completion of the ovulation process.

Hemocyte types and total and differential counts in unparasitized and parasitized Anastrepha obliqua (Diptera, Tephritidae) larvae

The hemocyte types, in addition to total and differential hemocyte counts were studied in parasitized and unparasitized Anastrepha obliqua larvae at the beginning and at the end of the third instar. In both developmental phases, in parasitized and unparasitized larvae, prohemocytes, plasmatocytes, granulocytes, adipohemocytes, spherulocytes and oenocytoids cells were observed. Mitotic figures indicate prohemocytes as stem cells. Prohemocytes, plasmatocytes and granulocytes are the most numerous cells in the hemolymph of A. obliqua. Difference in the total number of hemocytes was observed between unparasitized and parasitized larvae at the end of the third instar, but not at the beginning

Hemocyte types and total and differential counts in unparasitized and parasitized Anastrepha obliqua (Diptera, Tephritidae) larvae.

The hemocyte types, in addition to total and differential hemocyte counts were studied in parasitized and unparasitized Anastrepha obliqua larvae at the beginning and at the end of the third instar. In both developmental phases, in parasitized and unparasitized larvae, prohemocytes, plasmatocytes, granulocytes, adipohemocytes, spherulocytes and oenocytoids cells were observed. Mitotic figures indicate prohemocytes as stem cells. Prohemocytes, plasmatocytes and granulocytes are the most numerous cells in the hemolymph of A. obliqua. Difference in the total number of hemocytes was observed between unparasitized and parasitized larvae at the end of the third instar, but not at the beginning.