The role of cAMP-dependent protein kinase A in the formation of long-term memory in Bactrocera dorsalis

The cAMP-dependent protein kinase A(PKA)signaling pathway has long been considered critical for long-term memory(LTM)formation.Previous studies have mostly focused on the role of PKA signaling in LTM induction by multiple spaced conditioning with less attention to LTM induction by a single conditioning.Here,we conducted behavioral-pharmacology,enzyme immunoassay and RNA interference experiments to study the role of the PKA signaling pathway in LTM formation in the agricultural pest Bactrocera dorsalis,which has a strong memory capacity allowing it to form a two-day memory even from a single conditioning trial.We found that either blocking or activating PKA prior to conditioning pretreatment affected multiple spaced LTM,and conversely,they did not affect LTM formed by single conditioning.This was further confirmed by enzyme-linked immunosorbent assay(ELISA)and silencing of the protein kinase regulatory subunit 2 and catalytic subunit 1.Taken together,these results suggest that activating PKA during memory acquisition helps to induce the LTM formed by multiple spaced conditioning but not by a single conditioning.Our findings challenge the conserved role of PKA signaling in LTM,which provides a basis for the greater diversity of molecular mechanisms underlying LTM formation across species,as well as possible functional and evolutionary implications.

Characterization of Domeless receptors and the role of Bd Domeless3 in anti-symbiont-like virus defense in Bactrocera dorsalis

The Janus kinase/signal transducers and activators of transcription(JAK/STAT)signaling pathway play a pivotal role in innate immunity.Among invertebrates,Domeless receptors serve as the key upstream regulators of this pathway.In our study on Bactrocera dorsalis,we identified three cytokine receptors:BdDomeless1,BdDomeless2,and BdDomeless3.Each receptor encompasses five fibronectin-type-III-like(FN III)extracellular domains and a transmembrane domain.Furthermore,these receptors exhibit the increased responsiveness to diverse pathogenic challenges.Notably,only BdDomeless3 is upregulated during symbiont-like viral infections.Moreover,silencing BdDomeless3 enhanced the infectivity of Bactrocera dorsalis cripavirus(BdCV)and B.dorsalis picorna-like virus(BdPLV),underscoring BdDomeless3’s crucial role in antiviral defense of B.dorsalis.Following the suppression of Domeless3 expression,six antimicrobial peptide genes displayed decreased expression,potentially correlating with the rise in viral infectivity.To our knowledge,this is the first study identifying cytokine receptors associated with the JAK/STAT pathway in tephritid flies,shedding light on the immune mechanisms of B.dorsalis.

The Essential Oil of Ocimum americanum from Senegal and Gambia as a Source of Methyleugenol for the Control of Bactrocera dorsalis, Fruit Fly

The fruit fly, Bactrocera dorsalis (Diptera: Tephritidae) is one of the most important pests in all mango-producing areas, particularly in West Africa. In Senegal, O. americanum leaves have been used for several years to control this fly. However, to our knowledge, no chemical studies have been carried out. Thus, the aim of this study is to determine the chemical composition of the essential oil of O. americanum leaves collected in Senegal and Gambia. The essential oil obtained by hydrodistillation of these leaves is analyzed by GC/FID and GC/MS. Yields of essential oils from O. americanum leaves are 3.84% and 2.13%, respectively. Analysis of these essential oils by GC/FID and GC/MS allowed the identification of 23 compounds representing almost 100% of the total compositions. These essential oils are mainly dominated by methyleugenol (72.0% and 75.8%, respectively). Other components in significant percent are trans-β-caryophyllene (13.9% and 13.0%, respectively), germacrene D (4.1% and 3.7%, respectively), β-elemene (3.3% and 0.9%, respectively). Due to the high methyleugenol content, this study explains the attractive potential of O. americanum towards B. dorsalis. In perspective, we plan to evaluate the attractive effect of the essential oil and leaf powder of O. americanum against B. dorsalis, a real pest of mango orchards in Senegal.

Light intensity regulates the sexual behaviors of oriental fruit fly Bactrocera dorsalis under laboratory conditions

The oriental fruit fly,Bactrocera dorsalis(Hendel),is a devastating pest of citrus fruits.After successful mating,adult females insert their eggs into the ripened fruit,resulting in moldy and rotten fruit and causing great economic losses for the citrus industry.In the field,flies initiate copulatory behaviors as twilight approaches,and decreasing light intensity in this period is the normal stimulus for copulation.In this study,ten light intensities ranging from 0–30000 lux were set to identify the typical intensity that strongly regulates the copulation behavior of B.dorsalis.Three light intensities found to regulate the copulation behavior were then selected to verify their effects on adult male wing fanning and female chemotaxis towards 2,3,5-trimethylpyrazine(TMP).At last,strong light and complete darkness were artificially combined in the lab to verify whether they could prevent copulation to inform behavioral manipulation of oriental flies in the future.The results indicated that adult flies generally initiated copulatory behaviors at low light intensity(<1000 lux).Stronger light significantly prevented copulation in proportion to intensity,with nearly no copulation events initiated when light intensity was above 20000 lux.Both male wing fanning and female chemotaxis towards TMP were attenuated as light intensity became stronger.However,at 10000 lux,males still fanned their wings to a certain extent while TMP completely lost its attractiveness to females.In the darkness,adults did not initiate any sexual behaviors,e.g.,copulation,wing fanning,or chemotaxis to TMP.One hour of strong light(10000 lux)combined with continuous darkness completely prevented mating.These results show that light condition is an essential factor for copulatory behaviors in the oriental fruit fly.Researchers could thus manipulate light conditions artificially or disrupt the molecular target in flies’light transduction pathway to develop environmentally-friendly techniques to control this pest.

Research Progress on the Innate Immunity of Bactrocera dorsalis (Hendel) and the Regulation Mechanism of Parasitic Wasp Venom Protein

[Objectives]The use of natural enemies of living insects and their derivatives can effectively avoid the problems of pesticide residues,pest resistance,biodiversity decline,control effect weakening and so on.[Methods]Parasites inject various parasitic factors into hosts to inhibit the development of hosts,adjust the immunity of hosts,interfere with the growth and development of hosts,and reduce the nutrition metabolism of hosts,so as to ensure the growth and development of the offspring.Host pests can escape or conquer the parasitism of parasitic wasps through immune defense system in order to reproduce their own offspring.[Results]Under intense and strong selection pressure,in order to effectively ensure the success rate of parasitism,the adaptive diversity of parasitism strategies of parasitic wasps is finally caused.In the process of evolution and under the pressure of directional selection,the innate immunity and acquired immunity gradually evolve.[Conclusions]In-depth research on parasitic factors of parasitic wasps and their interaction with crop pests immunity and development can not only improve theoretical understanding of insect immunity and development biology,pest biological control and other disciplines,but also be expected to enable the application of some components of parasitic factors to agriculture,medicine and pharmacy.Bactrocera dorsalis is a destructive fruit and vegetable pest.This paper summarized the venom protein of B.dorsalis parasitoids and the immune interaction with hosts,in order to provide theoretical basis for biological control of plant pests by using parasitic natural enemies.

Relationship between Population Fluctuation of Oriental Fruit Fly Bactrocera dorsalis Hendel and Abiotic Factors in Yezin, Myanmar

Population fluctuation of oriental fruit fly, Bactrocera dorsalis （Hendel） （Diptera： Tephritidae）, was monitored by using methyl eugenol traps during May, 2011 to December, 2011 and May, 2016 to December, 2016 in mango orchard farms of Department of Agricultural Research in Yezin, Myanmar. In 2011, the highest mean number of male oriental fruit flies/trap/day （241.42 ± 11.79） was recorded in June and the lowest （2.21 ± 0.40） in December. The highest mean number of male oriental frui t flies/trap/day （388.95 ± 8.56） was recorded in the month of June and the lowest （2.33 ± 0.20） in December, 2016. Population data were correlated with meteorological data including temperature, duration of sunshine, and rainfall. Population fluctuation of male fruit flies was positively correlated with temperature and rainfall, and negatively correlated with the duration of sunshine.

Influence of Host Shift on Genetic Differentiation of the Oriental Fruit Fly,Bactrocera dorsalis

Invasion of the oriental fruit lfy, Bactrocera dorsalis, into new niches containing different food sources （a process referred to as host shift）, may cause population genetic differentiation and sympatric speciation. To attempt to infer that experimentally, test populations were established by transferring a subset of the original populations, which had been grown on banana for many generations, onto navel orange, and then subculturing the navel orange population and banana population for at least 20 generations. Four pairs of SSR primers with high polymorphism on laboratory strains were used to detect population genetic differentiation. All six tested populations （the 5th, 10th and 15th generations of B. dorsalis fed on banana and navel orange, respectively） were found to have low genetic diversity. Furthermore, the genetic diversity of the navel orange populations was found to decline after being crossed for several generations. Populations initially were deviated from Hardy-Weinberg equilibrium, however, equilibrium was achieved with increasing numbers of generations in both of the host populations. Limited gene lfows were found among the six populations. The Nei’s standard genetic distances between the two host populations of the same generation were initially low, but increased with generation number. Genetic distances between banana and navel orange populations of the same generation were lower than genetic distances between different generations grown on the same host plant. Analysis of molecular distance （AMOVA） results based on generation groups and host groups demonstrated that genetic variation among generations was greater than that between the two host populations. The results indicated that population genetic differentiation occurred after the host shift, albeit at low level. Biogeography and taxonomy of the B. dorsalis complex revealed that speciation of B. dorsalis might be tightly associated with host shift or host specialization of B. dorsalis following dispersal.

Trapping Effects of Different Treatments on Bactrocera dorsalis and Bactrocera cucurbitae

Five different trapping treatments, spraying attractant on bottle surface and spraying attractant inside the bottles with 0, 50,100 and 200 mL water, were set to trap Bactrocera dorsalis and B. cucurbitae in guava （Psidium guajava） park. The results showed that when the usage of attractant was 1 g, both Haonian and Wende had trapping effect on B. dorsalis and B. cucurbitae. The trapping effect of Haonian on B. dorsalis was better than that of Wende, while their trapping effects on B. cucurbitae was just the opposite. The trapping effects of different treatments had great difference. The trapping effect of Haonian on two species of fruit flies enhanced with the increasing volume of water, and reached the ma^mum value as the water volume was 200 mL. With the increasing volume of water, the trap- ping effect of Wende on two species of fruit flies first increased, and then decreased, which reached the maximum value as the water volume was 50 mL. Different treatments with attractants spraying inside bottles had better trapping effects on two species of fruit flies than that spraying on bottle surface.

Advances in Detection Methods for Invasive Pest Bactrocera dorsalis

Bactrocera dorsalis Hendel （Diptera： Tephritidae） is an invasive pest around the world. The paper summarizes biological and ecological characteristics of B, dorsalis, and reviews its detection methods from the aspects of morphological identification, acoustic detection and molecular detection, in order to provide a reference for further research and development of new detection methods. The hot issues in the study of B. dorsalis, such as ecological adaptation pattern, diffusion pathways and mechanisms, sustainable control measures, are also put forward in the paper.

Monitoring and Control Technology of Bactrocera dorsalis

After systematic monitoring and field survey in orchard for three years from 2009 to 2011, it was found that Bactrocera dorsalis occurred for more than three generations each year on the north shore of the middle and lower reaches of Yangtze River, with overlapping generations; the activity peak of adults was from mid August to late September; the occurrence in citrus orchard in hilly and mountainous area was severe; the average insect-fruit rate in October reached 0.20% - 15.88% ; B. dorsalis had sporadic occurrence in other orchards. The integrated control measures of plowing in winter to kill pupae, removing insect-fruits, boycott- ing on the ground, trapping by sex attraetants and spraying pesticides could basically control the damage of B. dorsalis.

Identification and functional verification of Y-chromosome-specific gene typo-gyf in Bactrocera dorsalis

Genes on the Y chromosome play important roles in male sex determination and development.The identification of Y-chromosome-specific genes not only provides a theoretical basis for the study of male reproductive development,but also offers genetic control targets for agricultural pests.However,Y-chromosome genes are rarely characterized due to their high repeatability and high heterochromatinization,especially in the oriental fruit fly.In this study,1011 Y-chromosome-specific candidate sequences were screened from 2 to 4 h Bactrocera dorsalis embryo datasets with the chromosome quotient method,6 of which were identified as Y-chromosome-specific sequences by polymerase chain reaction,including typo-gyf,a 19126-bp DNA sequence containing a 575-amino acid open reading frame.Testicular deformation and a significant reduction in sperm number were observed after typo-gyf knockdown with RNA interference in embryos.After typo-gyf knockout with clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated protein 9 in the embryonic stage,the sex ratio of the emergent adults was unbalanced,with far more females than males.A genotype analysis of these females with the Y-chromosome gene MoY revealed no sex reversal.Typo-gyf knockout led to the death of XY individuals in the embryonic stage.We conclude that typo-gyf is an essential gene for male survival,and is also involved in testicular development and spermatogenesis.The identification of typo-gyf and its functional verification provide insight into the roles of Y-chromosome genes in male development.

Formation characteristics of long-term memory in Bactrocera dorsalis

Studies on insects have contributed significantly to a better understanding of learning and memory,which is a necessary cognitive capability for all animals.Although the formation of memory has been studied in some model insects,more evidence is required to clarify the characteristics of memory formation,especially long-term memory(LTM),which is important for reliably storing information.Here,we explored this question by examining Bactrocera dorsalis,an agricultural pest with excellent learning abilities.Using the classical conditioning paradigm of the olfactory proboscis extension reflex(PER),we found that paired conditioning with multiple trials(>3)spaced with an intertrial interval(≥10 min)resulted in stable memory that lasted for at least 3 d.Furthermore,even a single conditioning trial was sufficient for the formation of a 2-d memory.With the injection of protein inhibitors,protein-synthesis-dependent memory was confirmed to start 4 h after training,and its dependence on translation and transcription differed.Moreover,the results revealed that the dependence of memory on protein translation exhibited a time-window effect(4-6 h).Our findings provide an integrated view of LTM in insects,suggesting common mechanisms in LTM formation that play a key role in the biological basis of memory.

Integrated analysis of miRNA and mRNA expression profiles in response to gut microbiota depletion in the abdomens of female Bactrocera dorsalis

Insect gut microbiota has been reported to participate in regulating host multiple biological processes including metabolism and reproduction.However,the corresponding molecular mechanisms remain largely unknown.Recent studies suggest that microRNAs(miRNAs)are involved in complex interactions between the gut microbiota and the host.Here,we used next-generation sequencing technology to characterize miRNA and mRNA expression profiles and construct the miRNA–gene regulatory network in response to gut microbiota depletion in the abdomens of female Bactrocera dorsalis.A total of 3016 differentially expressed genes(DEGs)and 18 differentially expressed miRNAs(DEMs)were identified.Based on the integrated analysis of miRNA and mRNA sequencing data,229 negatively correlated miRNA-gene pairs were identified from the miRNA-mRNA network.Gene ontology enrichment analysis indicated that DEMs could target several genes involved in the metabolic process,oxidation-reduction process,oogenesis,and insulin signaling pathway.Finally,real-time quantitative polymerase chain reaction further verified the accuracy of RNA sequencing results.In conclusion,our study provides the profiles of miRNA and mRNA expressions under antibiotics treatment and provides an insight into the roles of miRNAs and their target genes in the interaction between the gut microbiota and its host.

Population dynamics of the oriental fruit fly, Bactrocera dorsalis （Diptera： Tephritidae） in the Kunming area,southwestern China

Population dynamics of Bactrocera dorsalis Hendel （Diptera： Tephritidae） were studied through pheromone trapping over 4 years （1997, 1999, 2000, 2003） in the Kunming region, a high plateau area in southwestern China. B. dorsalis immigrates from southern Yunnan to Kunming each year, and occurs during early May through November. Annual trap captures recorded an increase in the B. dorsalis populations from May to July, when they peaked in abundance, and a decline until November. No flies were detected from November to April. The fruit flies had two generations. There was considerable overlapping due to the continuous arrival of immigrating flies during the summer months. Annual capture rates were significantly related to numbers of flies caught in July when peak captures were recorded; whereas the peak captures, in turn, positively depended on numbers of flies recorded in May, the first month of fly appearance in the current year. It suggested that the annual population abundance was mainly dependent on the size of the initial emigrating population. A daily average temperature of 18℃ was probably the threshold temperature required for the flies to undertake long-range dispersal, which partially explained the start of the fly in May each year on this high plateau. Under field conditions, the fruit flies can withstand 13℃ as a daily average temperature. No flies were recorded in any of the study years at a daily temperature colder than 10 ℃.

Influence of various stressors on the expression of core genes of the small interfering RNA pathway in the oriental fruit fly, Bactrocera dorsalis

RNA interference （RNAi）-based technology has emerged as a potential tool for controlling insect pests, however, previous studies found that the efficiency of RNAi in Bactrocera dorsalis was variable. In nature, insects often meet various challenges, such as pathogen infections, extreme temperatures, lack of nutrition and heavy metals. To better understand the association of the stressors with efficiency of RNAi, in the current study we tested the expression of three core genes, dicer2 （Bddcr2）, r2d2 （Bdr2d2） and argonaute2 （Bdago2）, of the small interfering RNA （siRNA） pathway of B. dorsalis upon various stressors. Our results showed that all three genes were upregulated by the infection of invertebrate iridescent virus 6, which suggested a function of the siRNA pathway against viral infection. The loading of FeC13 could also increase the expression of Bddcr2. The treatments ofEscherichia coli, extremely high （40~C） and low （0~C） temperatures, as well as starvation, could negatively influence the expression of Bddcr2 and/or Bdago2. In total, our results showed that various stressors could influence the expression of core components ofB. dorsalis siRNA pathway. This highlights further speculation on the RNAi efficiency upon these stressors. Considering the complexity and variation of RNAi efficiency in different conditions, these results provide initial aspects in possible environmental stressors to influence the activity of the siRNA pathway, but the real impact of RNAi efficiency posed by these stressors requires further studies.

Octopaminergic neurons function in appetitive but not aversive olfactory learning and memory in Bactrocera dorsalis

The biogenic amine octopamine(OA,invertebrate counterpart of nora-drenaline)plays critical roles in the regulation of olfactory behavior.Historically,OA has been thought to mediate appetitive but not aversive learning in honeybees,fruit flies(Drosophila),and crickets.However,this viewpoint has recently been challenged because OA activity through aβ-adrenergic-like receptor drives both appetitive and aversive learn-ing.Here,we explored the roles of OA neurons in olfactory learning and memory retrieval in Bactrocera dorsalis.We trained flies to associate an orange odor with a sucrose reward or to associate methyl eugenol,a male lure,with N,N-diethyl-3-methyl benzoyl amide(DEET)punishment.We then treated flies with OA receptor antagonists before appetitive or aversive conditioning and a memory retention test.Injection of OA receptor antagonist mianserin or epinastine into the abdomen of flies led to impaired of appetitive learning and memory retention with a sucrose reward,while aversive learning and memory reten-tion with DEET punishment remained intact.Our results suggest that the OA signaling participates in appetitive but not aversive learning and memory retrieval in B.dorsalis through OA receptors.

Inducing and isolation of antibacterial peptides from oriental fruit fly, Bactrocera dorsalis Hendel

One antibacterial activity fraction from an immunized dipteran insect, Bactrocera dorsalis, was isolated and purified by prepurification, ion-exchange chromatography, gel filtration chromatography and reverse-phase high performance liquid chromatography （HPLC）. The final purified fraction was checked on the Smart system HPLC and was judged as a pure fraction. The results of physical and biological analysis revealed that this fraction is heat stable and showed strong activities against Gram-positive bacterial growth. It possesses antibicrobial peptide properties and is worth further investigation.

Knockdown of specific cuticular proteins analogous to peritrophin 3 genes disrupt larval and ovarian development in Bactrocera dorsalis(Diptera:Tephritidae)

Cuticular proteins(CPs)are critical components of the insect cuticle and play important roles in maintaining normal insect development and defense against various environmental stresses.The oriental fruit fly(Bactrocera dorsalis)is one of the most destructive pests worldwide,and its eight CPs analogous to peritrophin 3(BdCPAP3)family genes have been identified in our prev ious study.In the present study,we further explored the possible roles of CPAP3 genes in B.dorsalis development.Each sequence of BdCPAP3 genes contained three conserved ChtBD2(chitin-binding)domains.Spatial and temporal expression patterns revealed that the four BdCPAP3 genes(BdCPAP3-AI,B,E,and E2)might play important roles in larval pupariation of B.dorsalis.Moreover,treatment with a juvenile hormone analog(methoprene)significantly restricted expression of these four CPAP3 genes,whereas treatment with 20-hydroxy-ecdysone induced expression.The RNA interference(RNAi)results revealed that down-regulated CPAP3 genes led to signif-icant delay of pupariation,and injection of dsBdCPAP3-E into 5-d-old B.dorsalis larvae caused approximately 40%mortality.Interestingly,we also confirmed that BdCPAP3-D2 was involved in B.dorsalis ovarian development.This study showed that some specific CP4P3 genes had crucial roles in B.dorsalis development,and these CP genes could be used as potential targets to control this pest via RNAi.

Neuropeptide signaling systems are involved in regulating thermal tolerance in the oriental fruit fly

Neuropeptides and their receptors are involved in the regulation and coordination of various physiological processes in insects.Although various neuropeptides have been identified previously,the corresponding receptors remain unknown in the oriental fruit fly,Bactrocera dorsalis,an important agricultural insect pest.It is also unclear whether neuropeptide signaling systems are involved in regulating the thermal tolerance of this notorious pest.Here,we systematically identified 44 putative neuropeptide receptor genes which encode 66 protein sequences,and analyzed the spatio-temporal expression patterns of the neuropeptide ligands and their receptor genes in B.dorsalis.We also analyzed changes in their transcript accumulation in two thermo-tolerant populations(heat and cold)of B.dorsalis.The results showed that numerous neuropeptides and receptors participate in responding to thermal stresses during acclimation.In particular,the expression of short neuropeptide F(sNPF)was up-regulated in the heat-tolerant population of B.dorsalis.Moreover,proteomic data showed that sNPF was up-regulated in both thermo-tolerant populations of B.dorsalis.The functional verification based on CRISPR/Cas9 demonstrated that sNPF is involved in regulating the tolerance to thermal stresses.The results of this study enrich our knowledge on the function of neuropeptide sNPF in insects.Moreover,this study demonstrated the role of neuropeptide signaling systems in thermal adaptation,contributing to a better understanding of the rapid invasiveness of B.dorsalis around the world.

玉溪市新平县桔小实蝇成虫种群消长特征及其与气象因子的关系

为了揭示玉溪市新平县桔小实蝇[Bactrocera dorsalis(Hendel)]成虫种群消长特征及其与气象因子的关系,于2020—2022年采用甲基丁香酚为诱芯的诱捕器,监测新平县桔小实蝇雄成虫的消长动态,并根据当地气象资料分析气象因子与桔小实蝇雄成虫数量的关系。结果表明,桔小实蝇在新平县全年皆发生,冬季成虫数量较少,夏季为桔小实蝇成虫暴发期,5月以后种群数量开始增长,种群数量暴发期为6—10月。气象因子显著影响了桔小实蝇雄虫的数量,其中主要受月平均气温和月平均最低气温的影响。由此可知,影响新平县桔小实蝇种群数量变动的主要因素是气温、降雨、寄主种类和海拔等,这些因素对桔小实蝇的作用方式、强度和时间各不相同,在成虫种群消长的不同阶段发挥不同作用,但通过特定时空下的相互组合,综合决定着新平县桔小实蝇种群消长的基本特征。