Assessment of non-methyl eugenol-responding lines of Bactrocera dorsalis (Diptera: Tephritidae) males on lure response and mating.

Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), is a major global pest of fruits. Currently, the sequential male annihilation technique, followed by the sterile insect technique has been used to significantly reduce the population of feral males in this species. However, issues with sterile males being killed by going to male annihilation traps have reduced the efficacy of this approach. The availability of males that are non-methyl eugenol-responding would minimize this issue and increase the efficacy of both approaches. For this, we recently established two separate lines of non-methyl eugenol-responding males. These lines were reared for 10 generations and in this paper, we report on the assessment of males from these lines in terms of methyl eugenol response and mating ability. We saw a gradual decrease in non-responders from ca. 35 to 10% after the 7th generation. Despite that, there were still significant differences until the 10th generation in numbers of non-responders over controls using laboratory strain males. We did not attain pure isolines of non-methyl eugenol-responding males, so we used non-responders from the 10th generation of those lines as sires to initiate two reduced-responder lines. Using these reduced responder flies, we found that there was no significant difference in mating competitiveness when compared with control males. Overall, we suggest that it may be possible to establish lines of low or reduced responder males to be used for sterile release programs, that could be applied until the 10th generation of rearing. Our information will contribute to the further development of an increasingly successful management technique incorporating the use of SIT alongside MAT to contain wild populations of B. dorsalis.

Effects of low-concentration spinetoram wax-based bait stations on Bactrocera dorsalis (Diptera: Tephritidae).

Spinetoram wax-based bait station (SWBB) is a maintenance-free, long-lasting, and eco-friendly management measure for Bactrocera dorsalis. However, the impacts of low-concentration spinetoram on B. dorsalis have not yet been determined. Therefore, our study aimed to determine the impacts of low-concentration SWBBs on the biology, demographics, detoxifying enzymes, and gut microorganisms of B. dorsalis. Our results showed that low-concentration SWBBs posed dose-dependent effects on the lifespan and fecundity of B. dorsalis adults. Both the LC10 and LC30 treatments significantly reduced the fecundity, while only the latter led to significant deleterious effects on the longevity of adults. Transgenerational bioassays revealed that exposure to LC30 significantly affected the development period of larvae and pupae as well as the livability of pre-adult stage of the progeny. However, except for the ovipositional period, no significant effects on the biological traits of F1 adults were observed. In terms of the F1 demographic parameters, dose-dependent effects were observed. Moreover, both the LC10 and LC30 treatments significantly extended the mean generation time, while the latter remarkably decreased the finite and intrinsic rates. Additionally, the significant induction of CarE activity by the LC10 and LC30 treatment was maintained until 24 and 48 h respectively. The CYP450 O-deethylation activity in the LC30 treatment was significantly enhanced at 24 and 48 h intervals when compared to the control. Regarding the intestinal bacterial community, after B. dorsalis adults were exposed to low-concentration SWBBs, the relative abundances of Providencia and Vagococcus were significantly increased, whereas those of Lactococcus and Brachyspira experienced a significant decrease. The obtained results are expected to serve as a foundation for the application of spinetoram in "lure-and-kill" strategies against B. dorsalis.

Gallic acid induces constitutive resistance against Bactrocera dorsalis infestation in mango fruit by its dual action.

The Oriental fruit fly, Bactrocera dorsalis (Hendel) is a major insect pest of mango fruit worldwide resulting in huge loss of fruit quality and productivity. However, there exist a few mango varieties resistant to B. dorsalis infestation. The objective of the present study was, therefore to identify the major fruit component imparting resistance to B. dorsalis. Principal Component Analysis of phenolic acids in pulp and peel tissues of two resistant varieties, viz., Langra and EC 95862, revealed that among the phenolic acids present in the fruit, gallic acid was the most abundant component in both fruit peel and pulp while laboratory studies revealed that gallic acid was acutely toxic to B. dorsalis with its dual action as antioxidant in the host and a prooxidant in the insect. Field study with the preharvest application of gallic acid on young developing fruits of B. dorsalis susceptible Alphonso mango showed that it could protect the fruit against insect damage confirming that gallic acid is essentially responsible for providing constitutive resistance against B. dorsalis in Langra and EC 95862. Thus, preharvest application of gallic acid to developing fruits could be used as part of an Integrated Pest Management strategy to control infestation by B. dorsalis. Future work on breeding / development of transgenes of susceptible mango varieties with high levels of gallic acid in fruit peel is likely to provide the simplest means of inducing constitutive resistance against B. dorsalis infestation.

Assessment of 2-allyl-4,5-dimethoxyphenol safety and attractiveness to mature males of Bactrocera dorsalis (Hendel).

Males of the Oriental fruit fly Bactrocera dorsalis (Hendel) are highly attracted to, and compulsively feed, on methyl eugenol (ME). ME is converted into 2-allyl-4,5-dimethoxyphenol (DMP) and (E)-coniferyl alcohol (E-CF), which are temporarily sequestered in the fly's rectal gland prior to being released at dusk. Previous research initially confirmed that DMP is a relatively strong lure to B. dorsalis males. However, the characteristics of males' response to DMP and toxicology of DMP remains largely unclear. In our study, we demonstrated that DMP was more attractive to sexually mature males than E-CF tested in laboratory bioassays. Interestingly, the responsiveness of mature males to DMP was not uniform throughout the day, eliciting the highest response during the day and dropping to a low level at night. Furthermore, there were no significant differences between the olfactory responses of virgin and mated mature males to DMP. No obvious signs of toxic symptom and deaths were observed in mice during a 14-day acute oral toxicity testing. Further, toxicologically significant changes were not observed in body weight, water intake, food consumption, and absolute and relative organ weights between control and treated groups, implying DMP could be regarded as nontoxic. Lastly, the cytotoxicity data of DMP on cells showed that it exhibited no significant cytotoxicity to normal human and mouse cells. Taken together, results from both the acute and cellular toxicity experiments demonstrated the nontoxic nature of DMP. In conclusion, DMP shows promise as an effective and eco-friendly lure for B. dorsalis males, and may contribute to controlling B. dorsalis in the flied.

The adipokinetic hormone signaling system regulates the sensitivity of Bactrocera dorsalis to malathion.

The neuropeptide adipokinetic hormone (AKH) binds to the AKH receptor (AKHR) to regulate carbohydrate and lipid metabolism. It also participates in the insect anti-stress response. We used RT-qPCR to detect the expression levels of 39 neuropeptides in malathion-susceptible (MS) and malathion-resistant (MR) strains of Bactrocera dorsalis. AKH and AKHR were highly expressed in the MR strain. Using a malathion bioassay and RNA interference (RNAi), we demonstrated that AKHR is involved in the susceptibility of B. dorsalis to malathion. We found significantly reduced expression of two detoxification enzyme genes (glutathione-S-transferase, GST and α-esterase, CarE) after AKHR RNAi. Based on our previous data, GSTd10 and CarE6 participate the direct metabolism of malathion in this fly, which is also verified by a malathion metabolism assay by HPLC using the crude enzymes in the current study. These results suggest that AKHR plays an important role in affecting malathion susceptibility via detoxification enzyme genes.

Genetic stability, genetic variation, and fitness performance of the genetic sexing Salaya1 strain for Bactrocera dorsalis, under long-term mass rearing conditions.

BACKGROUND: A genetic sexing strain (GSS) is an essential component for pest control using the sterile insect technique (SIT). A GSS is developed using a combination of Y-autosome translocation and a selectable marker such as pupal color, resulting in heterozygous males and homozygous females that possess wild-type brown pupae (wp+) and mutant white pupae (wp) alleles, respectively. The genetic sexing Salaya1 strain developed for Bactrocera dorsalis was evaluated using a clean stream and scaled-up for subsequent production lines (e.g., initiation, injection, and release). Colony management under small- and large-scale conditions for long-term rearing may affect the sexing system, genetic background, and fitness performance of the strain. Routine monitoring was applied to study genetic stability, genetic variation, and male mating competitiveness. RESULTS: The percentage of recombinants was significantly different between males (wp) and females (wp+), ranging between 0.21-0.43% and 0.01-0.04%, respectively. Using 106 bands from six ISSR markers, the genetic backgrounds of two generations (F40 and F108) of the clean stream were found to be almost identical (0.960), and between those two generations and the wild population, the similarities were 0.840 and 0.800, respectively. In addition, the sterile males performed well in competitive mating with fertile females (Relative Sterility Index = 0.67 ± 0.13). The rates of fliers calculated from both clean and release streams were higher than 0.95. Regarding the fitness of the Salaya1 strain, the fertility and pupal recovery were similar in all production lines. The sex ratio (Male/Female) distortion was also recorded. CONCLUSIONS: The Salaya1 strain reared at the mass-rearing facility retained its genetic stability, genetic variation, behavior (e.g., competitive mating and flight ability), and traits related to fitness for at least 10 consecutive generations. The filter rearing system is effective at minimising the selection pressure while maintaining the genetic background and fitness performances of the clean stream. These characteristics were stable throughout the production lines. In addition, the production efficiency is comparable among the different production lines and other similar types of GSSs.

Potential of a fly gut microbiota incorporated gel-based larval diet for rearing Bactrocera dorsalis (Hendel).

BACKGROUND: The Oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), is an important polyphagous pest of horticultural produce. The sterile insect technique (SIT) is a proven control method against many insect pests, including fruit flies, under area-wide pest management programs. High quality mass-rearing process and the cost-effective production of sterile target species are important for SIT. Irradiation is reported to cause severe damage to the symbiotic community structure in the mid gut of fruit fly species, impairing SIT success. However, studies have found that target-specific manipulation of insect gut bacteria can positively impact the overall fitness of SIT-specific insects. RESULTS: Twelve bacterial genera were isolated and identified from B. dorsalis eggs, third instars larval gut and adults gut. The bacterial genera were Acinetobacter, Alcaligenes, Citrobacter, Pseudomonas, Proteus, and Stenotrophomonas, belonging to the Enterobacteriaceae family. Larval diet enrichment with the selected bacterial isolate, Proteus sp. was found to improve adult emergence, percentage of male, and survival under stress. However, no significant changes were recorded in B. dorsalis egg hatching, pupal yield, pupal weight, duration of the larval stage, or flight ability. CONCLUSIONS: These findings support the hypothesis that gut bacterial isolates can be used in conjunction with SIT. The newly developed gel-based larval diet incorporated with Proteus sp. isolates can be used for large-scale mass rearing of B. dorsalis in the SIT program.

Tudor knockdown disrupts ovary development in Bactrocera dorsalis.

One of the main functions of the piwi-interacting RNA pathway is the post-transcriptional silencing of transposable elements in the germline of many species. In insects, proteins belonging to the Tudor superfamily proteins belonging to the Tudor superfamily play an important role in to play an important role in this mechanism. In this study, we identified the tudor gene in the oriental fruit fly, Bactrocera dorsalis, investigated the spatiotemporal expressional profile of the gene, and performed a functional analysis using RNA interference. We identified one transcript for a tudor homologue in the B. dorsalis transcriptome, which encodes a protein containing the typical 10 Tudor domains and an Adenosine triphosphate (ATP) synthase delta subunit signature. Phylogenetic analysis confirmed the identity of this transcript as a tudor homologue in this species. The expression profile indicated a much higher expression in the adult and pupal stages compared to the larval stages (up to a 60-fold increase), and that the gene was mostly expressed in the ovaries, Malpighian tubules and fat body. Finally, gene knockdown of tudor in B. dorsalis led to clearly underdeveloped ovaries in the female adult and reductions in copulation rate and amount of oviposition, indicating its important role in reproduction. The results of this study shed more light on the role of tudor in ovary development and reproduction.

Incongruence between molecules and morphology: A seven-gene phylogeny of Dacini fruit flies paves the way for reclassification (Diptera: Tephritidae).

Molecular and morphological research often suggest conflicting results. Selective pressure on certain morphologies can confound understanding of evolutionary relationships. Dacini is one of the most diverse tribes of tephritid flies and contains many economically important pest species. Their black and yellow patterned body markings are presumed to act as wasp mimicry, and the characters separating species and groups are limited and in some cases phenotypically plastic. The traditional taxonomy of the tribe is controversial because groupings are based on unique combinations of morphological characters without the use of cladistic methods, though recent phylogenetic and taxonomic analyses have resulted in significant changes to their taxonomy. The monophyly of the three largest genera in the tribe has been tested with only small numbers of representatives per genus and a limited number of genes. To further understand the taxonomy and evolution of Dacini we sequenced seven genes from 167 Dacini species and five dipteran outgroups to construct a robust phylogeny and test phylogenetic relationships between genera, subgenera, and species complexes. Our phylogeny confirms the monophyly of Dacus, Bactrocera, and Zeugodacus. However, most groups below the genus level are not monophyletic, and only through further revision will we be able to understand their evolution and clarify the taxonomy within this tribe.

Cytoplasmic glutamine synthetase gene expression regulates larval development in Bactrocera dorsalis (Hendel).

In insects, glutamine synthetase (GS), a key enzyme in the synthesis of glutamine, has been reported to be associated with embryonic development, heat shock response, and fecundity regulation. However, little is known about the influence of GS on postembryonic development. In this study, we demonstrate that blocking the activity of GS in the oriental fruit fly (Bactrocera dorsalis) with use of a GS-specific inhibitor (L-methionine S-sulfoximine), led to a significant delay in larval development, pupal weight loss, and inhibition of pupation. We further identify cloned and characterized two GS genes (BdGS-c and BdGS-m) from B. dorsalis. The two GS genes identified in B. dorsalis were predicted to be located in the cytosol (BdGS-c) and mitochondria (BdGS-m), and homology analysis indicated that both genes were similar to homologs from other Dipterans, such as Drosophila melanogaster and Aedes aegypti. BdGS-c was highly expressed in the larval stages, suggesting that cytosolic GS plays a predominant role in larval development. Furthermore, RNA interference experiments against BdGS-c, to specifically decrease the expression of cytosolic GS, resulted in delay in larval development as well as pupal weight loss. This study presents the prominent role played by BdGS-c in regulating larval development and suggests that the observed effect could have been modulated through ecdysteroid synthesis, agreeing with the reduced expression of the halloween gene spook. Also, the direct effects of BdGS-c silencing on B. dorsalis, such as larval lethality, delayed pupation, and late emergence, can be further exploited as novel insecticide target in the context of pest management.

Molecular characterizations of natalisin and its roles in modulating mating in the oriental fruit fly, Bactrocera dorsalis (Hendel).

Initially, natalisin (NTL) was identified from three holometabolous insect species, Drosophila melanogaster, Tribolium castaneum and Bombyx mori, and was documented to regulate reproductive behaviours in D. melanogaster and T. castaneum. In this study, we report the sequences of the NTL precursor and its receptor (NTLR) from an important agricultural pest, Bactrocera dorsalis (Hendel). NTLR is a typical G-protein coupled receptor and phylogenetic analysis showed that B. dorsalis NTLR was closely related to insect natalisin receptors from other species. A functional assay of NTLR transiently expressed in Chinese hamster ovary cells showed that it was activated by putative natalisin mature peptides in a concentration-dependent manner, with 50% effective concentrations (EC50 ) at nanomolar or micromolar levels. As indicated by quantitative real-time PCR, both NTL and NTLR had the highest expression in the central nervous system of B. dorsalis compared with the other tested tissues. Three pairs of adult brain neurones of B. dorsalis were identified with immunohistochemical antibody staining against D. melanogaster NTL4, and in situ hybridization with specific DNA probes. Moreover, RNA interference mediated by double-stranded RNA injection in adults provided evidence for the important roles of NTL in regulating both male and female mating frequencies in this fly.

Functional analysis of five trypsin-like protease genes in the oriental fruit fly, Bactrocera dorsalis (Diptera: Tephritidae).

Insect midgut proteases catalyze the release of free amino acids from dietary proteins and are essential for insect normal development. To date, digestive proteases as potential candidates have made great progress in pest control. To clarify the function of trypsin-like protease genes in the digestive system of Bactrocera dorsalis, a serious pest of a wide range of tropical and subtropical fruit and vegetable crops, five trypsin genes (BdTry1, BdTry2, BdTry3, BdTry4 and BdTry5) were identified from transcriptome dataset, and the effects of feeding condition on their expression levels were examined subsequently. RNA interference (RNAi) was applied to further explore their function on the growth of B. dorsalis. The results showed that all the BdTrys in starving midgut expressed at a minimal level but up-regulated upon feeding (except BdTry3). Besides, RNAi by feeding dsRNAs to larvae proved to be an effective method to cause gene silencing and the mixed dsRNAs of the five BdTrys slowed larvae growth of B. dorsalis. The current data suggest that trypsin genes are actively involved in digestion process of B. dorsalis larvae and thereafter play crucial roles in their development.

Comparative Proteomic Profiling Reveals Molecular Characteristics Associated with Oogenesis and Oocyte Maturation during Ovarian Development of Bactrocera dorsalis (Hendel).

Time-dependent expression of proteins in ovary is important to understand oogenesis in insects. Here, we profiled the proteomes of developing ovaries from Bactrocera dorsalis (Hendel) to obtain information about ovarian development with particular emphasis on differentially expressed proteins (DEPs) involved in oogenesis. A total of 4838 proteins were identified with an average peptide number of 8.15 and sequence coverage of 20.79%. Quantitative proteomic analysis showed that a total of 612 and 196 proteins were differentially expressed in developing and mature ovaries, respectively. Furthermore, 153, 196 and 59 potential target proteins were highly expressed in early, vitellogenic and mature ovaries and most tested DEPs had the similar trends consistent with the respective transcriptional profiles. These proteins were abundantly expressed in pre-vitellogenic and vitellogenic stages, including tropomyosin, vitellogenin, eukaryotic translation initiation factor, heat shock protein, importin protein, vitelline membrane protein, and chorion protein. Several hormone and signal pathway related proteins were also identified during ovarian development including piRNA, notch, insulin, juvenile, and ecdysone hormone signal pathways. This is the first report of a global ovary proteome of a tephritid fruit fly, and may contribute to understanding the complicate processes of ovarian development and exploring the potentially novel pest control targets.

LARVAL X-RAY IRRADIATION INFLUENCES PROTEIN EXPRESSION IN PUPAE OF THE ORIENTAL FRUIT FLY, BACTROCERA DORSALIS.

The sterile insect technique (SIT) was developed to eradicate the new world screwworm from the southern United States and Mexico, and became a component of many area-wide integrated pest management programs, particularly useful in managing tephritid fruit flies. SIT is based on the idea of rearing and sterilizing male pests, originally by ionizing radiation, and then releasing into field, where they compete for and mate with wild females. Mating with sterile males leads to reduced fecundity to lower pest populations. There are concerns with the use and distribution of radioisotopes for SIT programs, which have led to developing X-ray irradiation protocols to sterilize insects. We considered the possibility that X-ray irradiation exerts sublethal impacts aside form sterilizing insects. Such effects may not be directly observable, which led us to the hypothesis that X-ray irradiation in one life stage creates alterations in biological fitness and protein expression in the subsequent stage. We tested our hypothesis by irradiating larvae of Bactrocera dorsalis. There are two major points. One, exposing larvae to X-ray treatments led to reduced adult emergence, fecundity, fertility, and flight capacity from the corresponding pupae and emerged adults. Two, the X-ray treatments led to substantial expression changes in 27 pupal proteins. We assorted the 67 spots representing these proteins into three groups, metabolism, development, and structure. Our interpretation is these X-ray induced changes in biological performance and protein expression indicate their adult counterparts may be disabled in their abilities to successfully compete for and mate wild females in native habitats.

High genetic diversity in the offshore island populations of the tephritid fruit fly Bactrocera dorsalis.

BACKGROUND: Geographic isolation is an important factor that limit species dispersal and thereby affects genetic diversity. Because islands are often small and surrounded by a natural water barrier to dispersal, they generally form discrete isolated habitats. Therefore, islands may play a key role in the distribution of the genetic diversity of insects, including flies. RESULTS: To characterize the genetic structure of island populations of Bactrocera dorsalis, we analyzed a dataset containing both microsatellite and mtDNA loci of B. dorsalis samples collected from six offshore islands in Southern China. The microsatellite data revealed a high level of genetic diversity among these six island populations based on observed heterozygosity (Ho), expected heterozygosity (HE), Nei's standard genetic distance (D), genetic identity (I) and the percentage of polymorphic loci (PIC). These island populations had low F ST values (F ST = 0.04161), and only 4.16 % of the total genetic variation in the species was found on these islands, as determined by an analysis of molecular variance. Based on the mtDNA COI data, high nucleotide diversity (0.9655) and haplotype diversity (0.00680) were observed in all six island populations. F-statistics showed that the six island populations exhibited low or medium levels of genetic differentiation among some island populations. To investigate the population differentiation between the sampled locations, a factorial correspondence analysis and both the unweighted pair-group method with arithmetic mean and Bayesian clustering methods were used to analyze the microsatellite data. The results showed that Hebao Island, Weizhou Island and Dong'ao Island were grouped together in one clade. Another clade consisted of Shangchuan Island and Naozhou Island, and a final, separate clade contained only the Wailingding Island population. Phylogenetic analysis of the mtDNA COI sequences revealed that the populations on each of these six islands were closely related to different populations on mainland China. CONCLUSIONS: Our study suggests that these island populations have high genetic diversity, experience frequent gene flow and exhibit low or medium levels of genetic differentiation among some island populations. Therefore, the geographic isolation of the six islands does not appear to be a major dispersal barrier to B. dorsalis. Such knowledge is helpful for a better understanding of evolutionary processes of the species of island populations.

Discovery of genes related to formothion resistance in oriental fruit fly (Bactrocera dorsalis) by a constrained functional genomics analysis.

Artificial selection can provide insights into how insecticide resistance mechanisms evolve in populations. The underlying basis of such phenomena can involve complex interactions of multiple genes, and the resolution of this complexity first necessitates confirmation that specific genes are involved in resistance mechanisms. Here, we used a novel approach invoking a constrained RNA sequencing analysis to refine the discovery of specific genes involved in insecticide resistance. Specifically, for gene discovery, an additional constraint was added to the traditional comparisons of susceptible vs. resistant flies by the incorporation of a line in which insecticide susceptibility was 'recovered' within a resistant line by the removal of insecticide stress. In our analysis, the criterion for the classification of any gene as related to insecticide resistance was based on evidence for differential expression in the resistant line as compared with both the susceptible and recovered lines. The incorporation of this additional constraint reduced the number of differentially expressed genes putatively involved in resistance to 464, compared with more than 1000 that had been identified previously using this same species. In addition, our analysis identified several key genes involved in metabolic detoxification processes that showed up-regulated expression. Furthermore, the involvement of acetylcholinesterase, a known target for modification in insecticide resistance, was associated with three key nonsynonymous amino acid substitutions within our data. In conclusion, the incorporation of an additional constraint using a 'recovered' line for gene discovery provides a higher degree of confidence in genes identified to be involved in insecticide resistance phenomena.

Weathering and Chemical Degradation of Methyl Eugenol and Raspberry Ketone Solid Dispensers for Detection, Monitoring, and Male Annihilation of Bactrocera dorsalis and Bactrocera cucurbitae (Diptera: Tephritidae) in Hawaii.

Solid male lure dispensers containing methyl eugenol (ME) and raspberry ketone (RK), or mixtures of the lures (ME + RK), and dimethyl dichloro-vinyl phosphate (DDVP) were evaluated in area-wide pest management bucket or Jackson traps in commercial papaya (Carica papaya L.) orchards where both oriental fruit fly, Bactrocera dorsalis (Hendel), and melon fly, Bactrocera cucurbitae (Coquillett), are pests. Captures of B. dorsalis with fresh wafers in Jackson and bucket traps were significantly higher on the basis of ME concentration (Mallet ME [56%] > Mallet MR [31.2%] > Mallet MC [23.1%]). Captures of B. cucurbitae with fresh wafers in Jackson and bucket traps were not different regardless of concentration of RK (Mallet BR [20.1%] = Mallet MR [18.3%] = Mallet MC [15.9%]). Captures of B. dorsalis with fresh wafers, compared with weathered wafers, were significantly different after week 12; captures of B. cucurbitae were not significantly different after 16 wk. Chemical analyses revealed presence of RK in dispensers in constant amounts throughout the 16-wk trial. Degradation of both ME and DDVP over time was predicted with a high level of confidence by nonlinear asymptotic exponential decay curves. Results provide supportive data to deploy solid ME and RK wafers (with DDVP) in fruit fly traps for detection programs, as is the current practice with solid TML dispensers placed in Jackson traps. Wafers with ME and RK might be used in place of two separate traps for detection of both ME and RK responding fruit flies and could potentially reduce cost of materials and labor by 50%.

An assessment of cold hardiness and biochemical adaptations for cold tolerance among different geographic populations of the Bactrocera dorsalis (Diptera: Tephritidae) in China.

The cold hardiness of larvae, pupae, and adults of the oriental fruit fly, Bactrocera Dorsalis (Hendel) (Diptera: Tephritidae) was characterized first, and then body water, total sugar and glycerol contents, and activities of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and alcohol dehydrogenase (ADH) of different geographical populations subjected to suitable rearing conditions and under sublethal low-temperature stress were compared. The cold hardiness of different populations was well correlated with the latitudes of distributions. The northern marginal population (31.6° N) had higher cold tolerance than southern populations (23.1° N and 24.3° N). Among different life stages, larvae had the least cold tolerance, whereas pupae had the most tolerance. Under suitable rearing conditions, the marginal population had lower activities of all four tested enzymes than that of the southern populations and also had lower body water and higher total sugar and glycerol contents. The low-temperature stress induced higher SOD, CAT, POD, and ADH activities of all tested life stages and of all tested populations with higher increase intensity in adults and pupae than in larvae. The increase intensity was higher in the marginal population than in the southern populations. Pupae in the marginal population and adults in the southern populations showed the largest activity enhancement, which agreed with the insect's overwinter stages in their respective locations. Lower temperature stress lowered body water and total sugar contents and increased glycerol contents. The results revealed a strong correlation between the cold hardiness of a population and the concentration or activity of various biochemicals and enzymes known to be involved in cold tolerance. The marginal population of B. dorsalis might have evolved a new biotype with better adaption to low temperature.

Towards a better future for DNA barcoding: Evaluating monophyly- and distance-based species identification using COI gene fragments of Dacini fruit flies.

The utility of a universal DNA 'barcode' fragment (658 base pairs of the Cytochrome C Oxidase I [COI] gene) has been established as a useful tool for species identification, and widely criticized as one for understanding the evolutionary history of a group. Large amounts of COI sequence data have been produced that hold promise for rapid species identification, for example, for biosecurity. The fruit fly tribe Dacini holds about a thousand species, of which 80 are pests of economic concern. We generated a COI reference library for 265 species of Dacini containing 5601 sequences that span most of the COI gene using circular consensus sequencing. We compared distance metrics versus monophyly assessments for species identification and although we found a 'soft' barcode gap around 2% pairwise distance, the exceptions to this rule dictate that a monophyly assessment is the only reliable method for species identification. We found that all fragments regularly used for Dacini fruit fly identification >450 base pairs long provide similar resolution. 11.3% of the species in our dataset were non-monophyletic in a COI tree, which is mostly due to species complexes. We conclude with recommendations for the future generation and use of COI libraries. We revise the generic assignment of Dacus transversus stat. rev. Hardy 1982, and Dacus perpusillus stat. rev. Drew 1971 and we establish Dacus maculipterus White 1998 syn. nov. as a junior synonym of Dacus satanas Liang et al. 1993.

Diagnostic Tool for the Identification of Bactrocera dorsalis (Hendel) (Diptera: Tephritidae) Using Real-Time PCR.

Accurate identification of Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), commonly known as the Oriental fruit fly, is a significant challenge due to the morphological convergence and taxonomic uncertainties of species belonging to the same genus. This highly polyphagous species poses a significant threat to fruit crops. With its potential establishment in Europe becoming a growing concern, there is an urgent need for rapid and efficient diagnostic methods. The study presented here introduces a diagnostic protocol based on real-time PCR using a TaqMan probe for the early and reproducible identification of B. dorsalis. Specimens representing the genetic diversity of the Italian population were collected and analyzed. Specific primers and probe were designed based on the conserved regions and an in silico analysis confirmed their specificity. The assay conditions were optimized, and analytical sensitivity, specificity, repeatability, and reproducibility were evaluated. The protocol showed high sensitivity and specificity, accurately detecting low DNA concentrations of B. dorsalis. This standardized method provides a reliable tool for routine diagnostics, enhancing the accuracy and efficiency of identifying the Oriental fruit fly at all stages of its development, thereby facilitating effective pest management measures. The development of this diagnostic protocol is crucial for monitoring and supporting efforts to prevent the passive spread of B. dorsalis in Europe, particularly in light of the recent active infestations detected in Italy.