Bulbophyllum apertum flower (Orchidaceae) releases raspberry ketone (RK) in its fragrance, which attracts males of several fruit fly species belonging to the genus Bactrocera. Besides RK as a major component, the flower contains smaller amounts of 4-(4-hydroxylphenyl)-2-butanol, plus two minor volatile components, veratryl alcohol and vanillyl alcohol. Within the flower, the lip (labellum) had the highest concentration of RK with much smaller quantities present in petals; other flower parts had no detectable RK. Male fruit flies attracted to the flower belong to RK-sensitive species--such as Bactrocera albistragata, B. caudatus, B. cucurbitae (melon fly), and B. tau. Removal and attachment of the pollinarium to a fly's thoracic dorsum occurred when a male of B. albistragata was toppled into the floral column cavity, due to an imbalance caused by it shifting its body weight while feeding on the see-saw lip, and then freeing itself after being momentarily trapped between the lip and column. During this process, the stiff hamulus (the pollinia stalk protruding prominently towards the lip) acted as a crowbar when it was brushed downwards by the toppled fly and lifted the pollinia out of the anther. If the fly was big or long for the small triangular lip, it would not be toppled into the column cavity and would just walk across the column, during which time the pollinarium could be accidentally removed by the fly's leg, resulting in a failed transport of the pollinarium. This suggests an unstable situation, where the orchid relies only on a particular pollinator species in the complex ecosystem where many RK-sensitive species inhabit. Wild males of B. caudatus (most common visitors) captured on Bulbophyllum apertum flowers were found to sequester RK in their bodies as a potential pheromonal and allomonal ingredient. Thus, RK can act either as a floral synomone (pollinarium transported) or kairomone (accidental removal of pollinarium leading to total pollen wastage), depending on the body size of the male fruit flies visiting the flowers.
Bactrocera carambolae and B. papayae are major fruit fly pests and sympatric sibling species of the B. dorsalis complex. They possess distinct differences in male pheromonal components. In the 1990's, wild Bactrocera fruit flies with morphological traits intermediate between those of B. carambolae and B. papayae were often captured in traps baited with methyl eugenol (ME). Chemical analyses of rectal glands of ME-fed males revealed that the laboratory Fl, F2, and backcross hybrids possessed ME-derived sex pheromonal components ranging from that typical of B. papayae to that of B. carambolae without any specific trend, which included a combination of pheromonal components from both parental species within an individual hybrid. ME-fed hybrids without any ME-derived pheromonal components were also detected. Further chemical analysis of rectal glands from wild Bactrocera males, after ME feeding in the laboratory, showed a combination of pheromonal components similar to that found in the ME-fed, laboratory-bred hybrids. These findings present circumstantial evidence for the occurrence of a natural hybrid of the two Bactrocera species.
Sexually mature males of Bactrocera papayae are strongly attracted to and consume methyl eugenol (ME). Upon consumption, ME is biotransformed to two phenylpropanoids, 2-allyl-4,5-dimethoxyphenol (DMP) and (E)-coniferyl alcohol (CF), that are transported in the hemolymph, sequestered and stored in the rectal glands, and subsequently released as sex and aggregation pheromones during courtship. To date, very little work on the ultrastructure and anatomy of the rectal gland has been done, and the accumulation of phenylpropanoids in the rectal glands of males has not been observed visually. Our objectives are to describe the anatomy and fine structures of the rectal glands of males and females and to observe the accumulation of autofluorescent compounds in the rectal glands of males. The rectal glands of males and females have four rectal papillae with each papilla attached to a rectal pad. The rectal pads protrude from the rectal gland as the only surfaces of the gland that are not surrounded by muscles. The rectal papillae of ME-fed males had oil droplets and autofluorescent compounds that were absent from those of ME-deprived males. The autofluorescent compounds accumulated in the rectal sac, which is an evagination that is not found in rectal glands of females. The accumulation of these compounds increased with time and reached maximum at a day post-ME feeding and decreased thereafter. This trend is similar to the accumulation pattern of phenylpropanoids, CF and DMP in the rectal gland.
Sex pheromonal components of the tephritid fruit fly Bactrocera dorsalis (Hendel), 2-allyl-4,5-dimethoxyphenol and (E)-coniferyl alcohol, are biosynthesized from a highly potent male attractant, methyl eugenol, then sequestered and stored in the rectal gland prior to their release during courtship at dusk. These sex pheromonal components have been detected in the haemolymph and crop organ. Hence, attempts were made to separate and identify the haemolymph fractions which contained the sex pheromonal components. Identification of these bioactive fractions in methyl eugenol-fed male flies using gel filtration column chromatography and biodetection using live male flies showed two fractions as highly attractive to conspecific males. These fractions show a significant increase in protein absorbance in the elution profile of haemolymph from methyl eugenol-fed males compared with that from methyl eugenol-deprived males. The molecular mass of these bioactive fractions as determined by using gel filtration was in the peptide range of 3.3 to 5.5 kDa. Subsequent gas chromatography-mass spectrometry analyses further confirmed the presence of the pheromonal components in the bioactive fractions. The presence of these methyl eugenol-derived sex pheromonal components in specific haemolymph fractions suggests the involvement of a sex pheromone binding complex.
Males of Bactrocera dorsalis (Diptera: Tephritidae) are attracted strongly to and feed compulsively on methyl eugenol (1,2-dimethoxy- 4 -(2-propenyl)benzene), a highly potent male attractant. Pharmacophagy of methyl eugenol results in the production of phenylpropanoids 2-allyl-4,5-dimethoxyphenol and (E)-coniferyl alcohol that are sequestered and stored in the rectal gland prior to release as sex pheromonal components during mating at dusk. While these pheromonal components have also been detected in the hemolymph and crop of methyl eugenol-fed males, there is currently little information on the transport of these compounds from the crop to rectal gland in male B. dorsalis. Therefore, using physiological techniques such as parabiosis, rectal gland transplantation and hemolymph transfusion coupled with gas chromatography-mass spectrometry (GC-MS) analyses, we were able to ascertain and confirm the role of the hemolymph in the transport of these sex pheromonal components from the crop to the rectal gland. Further, the temporal profile of these methyl eugenol-derived bioactive compounds in the hemolymph also shows an increase with time post-methyl eugenol-feeding, i.e., 2-allyl-4,5-dimethoxyphenol attaining maximum amounts 15 min after ME consumption and decreasing thereafter, while for (E)-coniferyl alcohol-the increase and decrease are more gradual. These results further demonstrate the ability of insect hemolymph to transport many diverse forms of bioactive molecules including attractant-derived sex pheromonal components.
After pharmacophagy of methyl eugenol (ME), males of Bactrocera carambolae (Diptera: Tephritidae) produced (E)-coniferyl alcohol (CF) along with its endogenously synthesized pheromonal compounds. CF was shown to be released into the air by the ME-fed males only during the courtship period at dusk and attracted significantly more males and females than the ME-deprived males in wind tunnel assays. However, earlier onset of sexual attraction and a higher mating success were observed only in the wind tunnel and field cage assays on the third day posttreatment of ME. Field cage observations on the male-to-male interaction indicated that the ME-deprived males did not exhibit aggregation behavior, but that ME feeding promoted aggregation behavior in B. carambolae. Field cage observations revealed that the ME-deprived males were not only attracted to the ME-fed males, but also appeared to feed on their anal secretions. The secretions were subsequently confirmed to contain CF along with endogenously produced pheromonal compounds. Results obtained for B. carambolae were compared to those previously obtained from its sibling species, Bactrocera dorsalis, and are discussed in light of species advancement in fruit fly-plant relationships.
The late-aged egg and third-instar life stages of laboratory-reared Malaysian fruit fly, Bactrocera latifrons (Hendel); Mediterranean fruit fly, Ceratitis capitata (Wiedemann); melon fly, B. cucurbitae Coquillett; and oriental fruit fly, B. dorsalis (Hendel), (Diptera: Tephritidae); and the third instars of wild Mediterranean fruit fly were exposed to thermal treatments. A heating block system was used to determine the thermal death kinetics of the four fruit fly species. Treatments consisted of heating the fruit fly life stages to 44, 46, 48, and 50 degrees C and holding for different times ranging from 0 to 120 min depending on the thermal mortality response and time required to obtain 100% mortality for each species and life stage. The 0.5-order kinetic model had the best fit to the survival ratio for all the treatment temperatures and was used to predict lethal times. The thermal death time (TDT) curves showed a tolerance order of Mediterranean fruit fly eggs < or = third instars at 44, 46, and 50 degrees C, third instars < or = eggs at 48 degrees C, and wild third instars < the laboratory-reared third instars. Comparison between Mediterranean fruit fly third instar thermotolerance from Hawaii and Israel showed that Israel Mediterranean fruit fly was more thermotolerant. A comparison of minimum treatment times at a given temperature required to obtain 100% mortality of laboratory-reared Malaysian, Mediterranean (Hawaii and Israel strains), melon, Mexican, and oriental fruit fly eggs or third instars and wild Mediterranean fruit fly (Hawaii strain) eggs or third instars showed that oriental fruit fly was the most thermotolerant among the third instars, and the difference in heat tolerance between third instars and eggs was negligible at 50 degrees C.
Identification of Bactrocera carambolae Drew and Hancock, B. papayae Drew and Hancock, B. tau Walker, B. latifrons Hendel, B. cucurbitae Coquillett, B. umbrosa Fabricius and B. caudata Fabricius would pose a problem if only a body part or an immature stage were available. Analysis of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of cytochrome oxidase I (COI) gene using primers COIR, COIF, UEA7 and UEA10 and restriction enzymes (MseI, RsaI and Alu1) was carried out. The banding profiles in the electrophoresis gel were analysed.
Differentiation of Bactrocera papayae Drew & Hancock and Bactrocera carambolae Drew & Hancock (Diptera: Tephritidae) based on morphological characters has often been problematical. We describe here a single-nucleotide polymorphism (SNP)-based polymerase chain reaction (PCR) assay to differentiate between these two species. For detection of SNPs, fragments derived from each species were amplified using two primer pairs, COIF/COIR and UEA7/UEA10, sequenced, and aligned to obtain a contiguous 1,517-bp segment. Two new sets of primers were designed based on the 11 SNPs identified in the region. Results of the SNP-PCR test using any one of these species-specific primer sets indicate that these two species could be differentiated on basis of presence or absence of a band in the gel profile. We also tested the SNP-PCR primers on Bactrocera umbrosa F., Bactrocera cucurbitae Coquillett, Bactrocera latifrons Hendel, and Bactrocera tau (Walker) but did not detect any band in the gel, indicating the likelihood of a false positive for B. papayae is nil. This SNP-PCR method is efficient and useful, especially for immature life stages or when only adult body parts of the two species are available for identification, as encountered often in quarantine work.
Four morphologically cryptic species of the Bactrocera dorsalis fruit fly complex (B. dorsalis s.s., B. papayae, B. carambolae and B. philippinensis) are serious agricultural pests. As they are difficult to diagnose using traditional taxonomic techniques, we examined the potential for geometric morphometric analysis of wing size and shape to discriminate between them. Fifteen wing landmarks generated size and shape data for 245 specimens for subsequent comparisons among three geographically distinct samples of each species. Intraspecific wing size was significantly different within samples of B. carambolae and B. dorsalis s.s. but not within samples of B. papayae or B. philippinensis. Although B. papayae had the smallest wings (average centroid size=6.002 mm±0.061 SE) and B. dorsalis s.s. the largest (6.349 mm±0.066 SE), interspecific wing size comparisons were generally non-informative and incapable of discriminating species. Contrary to the wing size data, canonical variate analysis based on wing shape data discriminated all species with a relatively high degree of accuracy; individuals were correctly reassigned to their respective species on average 93.27% of the time. A single sample group of B. carambolae from locality 'TN Malaysia' was the only sample to be considerably different from its conspecific groups with regards to both wing size and wing shape. This sample was subsequently deemed to have been originally misidentified and likely represents an undescribed species. We demonstrate that geometric morphometric techniques analysing wing shape represent a promising approach for discriminating between morphologically cryptic taxa of the B. dorsalis species complex.
Bactrocera dorsalis s.s. is a pestiferous tephritid fruit fly distributed from Pakistan to the Pacific, with the Thai/Malay peninsula its southern limit. Sister pest taxa, B. papayae and B. philippinensis, occur in the southeast Asian archipelago and the Philippines, respectively. The relationship among these species is unclear due to their high molecular and morphological similarity. This study analysed population structure of these three species within a southeast Asian biogeographical context to assess potential dispersal patterns and the validity of their current taxonomic status.
The fruit fly Bactrocera caudata is a pest species of economic importance in Asia. Its larvae feed on the flowers of Cucurbitaceae such as Cucurbita moschata. To-date it is distinguished from related species based on morphological characters. Specimens of B. caudata from Peninsular Malaysia and Indonesia (Bali and Lombok) were analysed using the partial DNA sequences of cytochrome c oxidase subunit I (COI) and 16S rRNA genes. Both gene sequences revealed that B. caudata from Peninsular Malaysia was distinctly different from B. caudata of Bali and Lombok, without common haplotype between them. Phylogenetic analysis revealed two distinct clades, indicating distinct genetic lineage. The uncorrected 'p' distance for COI sequences between B. caudata of Malaysia-Thailand-China and B. caudata of Bali-Lombok was 5.65%, for 16S sequences from 2.76 to 2.99%, and for combined COI and 16S sequences 4.45 to 4.46%. The 'p' values are distinctly different from intraspecific 'p' distance (0-0.23%). Both the B. caudata lineages are distinctly separated from related species in the subgenus Zeugodacus - B. ascita, B. scutellata, B. ishigakiensis, B. diaphora, B. tau, B. cucurbitae, and B. depressa. Molecular phylogenetic analysis indicates that the B. caudata lineages are closely related to B. ascita sp. B, and form a clade with B. scutellata, B. ishigakiensis, B. diaphora and B. ascita sp. A. This study provides additional baseline for the phylogenetic relationships of Bactrocera fruit flies of the subgenus Zeugodacus. Both the COI and 16S genes could be useful markers for the molecular differentiation and phylogenetic analysis of tephritid fruit flies.
The Malaysian fruit fly, Bactrocera latifrons (Hendel), is a pest of peppers (Capsicum spp.) in Thailand. A field trial was undertaken to determine whether five commonly used cultivars of C. annuum, with marked differences in morphology and pungency, varied in their susceptibility to infestation by B. latifrons. Experiments carried out in both the dry and rainy seasons showed temporal differences in the number of fruits per cultivar, but there was no effect of variety or season on the proportion of fruits attacked or the number of pupae obtained per infested fruit However, the number of dead larvae per infested fruit was significantly higher, and the percent of pupae giving rise to adults was lower for the larger sweet pepper than other cultivars tested.
Sweet cherries, Prunus avium (L.) L., grown in the western United States are exported to many countries around the world. Some of these countries have enforced strict quarantine rules and trade restrictions owing to concerns about the potential establishment and subsequent spread of western cherry fruit fly, Rhagoletis indifferens Curran (Diptera: Tephritidae), a major quarantine pest of sweet cherry. We used 1) niche models (CLIMEX and MaxEnt) to map the climatic suitability, 2) North Carolina State University-Animal and Plant Health Inspection Service Plant Pest Forecasting System to examine chilling requirement, and 3) host distribution and availability to assess the potential for establishment of R. indifferens in areas of western North America where it currently does not exist and eight current or potential fresh sweet cherry markets: Colombia, India, Indonesia, Malaysia, Taiwan, Thailand, Venezuela, and Vietnam. Results from niche models conformed well to the current distribution of R. indifferens in western North America. MaxEnt and CLIMEX models had high performance and predicted climatic suitability in some of the countries (e.g., Andean range in Colombia and Venezuela, northern and northeastern India, central Taiwan, and parts of Vietnam). However, our results showed no potential for establishment of R. indifferens in Colombia, Indonesia, Malaysia, Taiwan, Thailand, Venezuela, and Vietnam when the optimal chilling requirement to break diapause (minimum temperature < or = 3 degree C for at least 15 wk) was used as the criterion for whether establishment can occur. Furthermore, these countries have no host plant species available for R. indifferens. Our results can be used to make scientifically informed international trade decisions and negotiations by policy makers.
Bactrocera dorsalis s.s. (Hendel) and B. papayae Drew & Hancock, are invasive pests belonging to the B. dorsalis complex. Their species status, based on morphology, is sometimes arguable. Consequently, the existence of cryptic species and/or population isolation may decrease the effectiveness of the sterile insect technique (SIT) due to an unknown degree of sexual isolation between released sterile flies and wild counterparts. To evaluate the genetic relationship and current demography in wild populations for guiding the application of area-wide integrated pest management using SIT, seven microsatellite-derived markers from B. dorsalis s.s. and another five from B. papayae were used for surveying intra- and inter-specific variation, population structure, and recent migration among sympatric and allopatric populations of the two morphological forms across Southern Thailand and West Malaysia.
The whole mitochondrial genome of the pest fruit fly Bactrocera arecae was obtained from next-generation sequencing of genomic DNA. It had a total length of 15,900 bp, consisting of 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and a non-coding region (A + T-rich control region). The control region (952 bp) was flanked by rrnS and trnI genes. The start codons included 6 ATG, 3 ATT and 1 each of ATA, ATC, GTG and TCG. Eight TAA, two TAG, one incomplete TA and two incomplete T stop codons were represented in the protein-coding genes. The cloverleaf structure for trnS1 lacked the D-loop, and that of trnN and trnF lacked the TΨC-loop. Molecular phylogeny based on 13 protein-coding genes was concordant with 37 mitochondrial genes, with B. arecae having closest genetic affinity to B. tryoni. The subgenus Bactrocera of Dacini tribe and the Dacinae subfamily (Dacini and Ceratitidini tribes) were monophyletic. The whole mitogenome of B. arecae will serve as a useful dataset for studying the genetics, systematics and phylogenetic relationships of the many species of Bactrocera genus in particular, and tephritid fruit flies in general.
Males of certain species belonging to the Bactrocera dorsalis complex are strongly attracted to, and readily feed on methyl eugenol (ME), a plant secondary compound that is found in over 480 plant species worldwide. Amongst those species is one of the world's most severe fruit pests the Oriental fruit fly, Bactrocera dorsalis s.s., and the former taxonomic species Bactrocera invadens, Bactrocera papayae and Bactrocera philippinensis. The latter species have been recently synonymised with Bactrocera dorsalis based on their very similar morphology, mating compatibility, molecular genetics and identical sex pheromones following consumption of ME. Previous studies have shown that male fruit fly responsiveness to lures is a unique phenomenon that is dose species-specific, besides showing a close correlation to sexual maturity attainment. This led us to use ME sensitivity as a behavioural parameter to test if Bactrocera dorsalis and the three former taxonomic species had similar sensitivity towards odours of ME. Using Probit analysis, we estimated the median dose of ME required to elicit species' positive response in 50% of each population tested (ED50). ED50 values were compared between Bactrocera dorsalis and the former species. Our results showed no significant differences between Bactrocera dorsalis s.s., and the former Bactrocera invadens, Bactrocera papayae and Bactrocera philippinensis in their response to ME. We consider that the Bactrocera males' sensitivity to ME may be a useful behavioural parameter for species delimitation and, in addition to other integrative taxonomic tools used, provides further supportive evidence that the four taxa belong to one and the same biological species, Bactrocera dorsalis.
The Carambola fruit fly, Bactrocera carambolae, is an invasive pest in Southeast Asia. It has been introduced into areas in South America such as Suriname and Brazil. Bactrocera carambolae belongs to the Bactrocera dorsalis species complex, and seems to be separated from Bactrocera dorsalis based on morphological and multilocus phylogenetic studies. Even though the Carambola fruit fly is an important quarantine species and has an impact on international trade, knowledge of the molecular ecology of Bactrocera carambolae, concerning species status and pest management aspects, is lacking. Seven populations sampled from the known geographical areas of Bactrocera carambolae including Southeast Asia (i.e., Indonesia, Malaysia, Thailand) and South America (i.e., Suriname), were genotyped using eight microsatellite DNA markers. Genetic variation, genetic structure, and genetic network among populations illustrated that the Suriname samples were genetically differentiated from Southeast Asian populations. The genetic network revealed that samples from West Sumatra (Pekanbaru, PK) and Java (Jakarta, JK) were presumably the source populations of Bactrocera carambolae in Suriname, which was congruent with human migration records between the two continents. Additionally, three populations of Bactrocera dorsalis were included to better understand the species boundary. The genetic structure between the two species was significantly separated and approximately 11% of total individuals were detected as admixed (0.100 ≤ Q ≤ 0.900). The genetic network showed connections between Bactrocera carambolae and Bactrocera dorsalis groups throughout Depok (DP), JK, and Nakhon Sri Thammarat (NT) populations. These data supported the hypothesis that the reproductive isolation between the two species may be leaky. Although the morphology and monophyly of nuclear and mitochondrial DNA sequences in previous studies showed discrete entities, the hypothesis of semipermeable boundaries may not be rejected. Alleles at microsatellite loci could be introgressed rather than other nuclear and mitochondrial DNA. Bactrocera carambolae may be an incipient rather than a distinct species of Bactrocera dorsalis. Regarding the pest management aspect, the genetic sexing Salaya5 strain (SY5) was included for comparison with wild populations. The SY5 strain was genetically assigned to the Bactrocera carambolae cluster. Likewise, the genetic network showed that the strain shared greatest genetic similarity to JK, suggesting that SY5 did not divert away from its original genetic makeup. Under laboratory conditions, at least 12 generations apart, selection did not strongly affect genetic compatibility between the strain and wild populations. This knowledge further confirms the potential utilization of the Salaya5 strain in regional programs of area-wide integrated pest management using SIT.
An FAO/IAEA-sponsored coordinated research project on integrative taxonomy, involving close to 50 researchers from at least 20 countries, culminated in a significant breakthrough in the recognition that four major pest species, Bactrocera dorsalis, Bactrocera philippinensis, Bactrocera papayae and Bactrocera invadens, belong to the same biological species, Bactrocera dorsalis. The successful conclusion of this initiative is expected to significantly facilitate global agricultural trade, primarily through the lifting of quarantine restrictions that have long affected many countries, especially those in regions such as Asia and Africa that have large potential for fresh fruit and vegetable commodity exports. This work stems from two taxonomic studies: a revision in 1994 that significantly increased the number of described species in the Bactrocera dorsalis species complex; and the description in 2005 of Bactrocera invadens, then newly incursive in Africa. While taxonomically valid species, many biologists considered that these were different names for one biological species. Many disagreements confounded attempts to develop a solution for resolving this taxonomic issue, before the FAO/IAEA project commenced. Crucial to understanding the success of that initiative is an accounting of the historical events and perspectives leading up to the international, multidisciplinary collaborative efforts that successfully achieved the final synonymization. This review highlights the 21 year journey taken to achieve this outcome.
Bactrocera caudata is a pest of pumpkin flower. Specimens of B. caudata from the northern hemisphere (mainland Asia) and southern hemisphere (Indonesia) were analysed using the partial DNA sequences of the nuclear 28S rRNA and internal transcribed spacer region 2 (ITS-2) genes, and the mitochondrial cytochrome c oxidase subunit I (COI), cytochrome c oxidase subunit II (COII) and 16S rRNA genes. The COI, COII, 16S rDNA and concatenated COI+COII+16S and COI+COII+16S+28S+ITS-2 nucleotide sequences revealed that B. caudata from the northern hemisphere (Peninsular Malaysia, East Malaysia, Thailand) was distinctly different from the southern hemisphere (Indonesia: Java, Bali and Lombok), without common haplotype between them. Phylogenetic analysis revealed two distinct clades (northern and southern hemispheres), indicating distinct genetic lineage. The uncorrected 'p' distance for the concatenated COI+COII+16S nucleotide sequences between the taxa from the northern and southern hemispheres ('p' = 4.46-4.94%) was several folds higher than the 'p' distance for the taxa in the northern hemisphere ('p' = 0.00-0.77%) and the southern hemisphere ('p' = 0.00%). This distinct difference was also reflected by concatenated COI+COII+16S+28S+ITS-2 nucleotide sequences with an uncorrected 'p' distance of 2.34-2.69% between the taxa of northern and southern hemispheres. In accordance with the type locality the Indonesian taxa belong to the nominal species. Thus the taxa from the northern hemisphere, if they were to constitute a cryptic species of the B. caudata species complex based on molecular data, need to be formally described as a new species. The Thailand and Malaysian B. caudata populations in the northern hemisphere showed distinct genetic structure and phylogeographic pattern.