Salicylic Acid Induces Changes in Mango Fruit that Affect Oviposition Behavior and Development of the Oriental Fruit Fly,Bactrocera dorsalis

The Oriental fruit fly, Bactrocera dorsalis (Hendel) is an important quarantine pest around the globe. Although measures for its control are implemented worldwide through IPM and male annihilation, there is little effect on their population. Hence, there is a need for new strategies to control this minacious pest. A strategy that has received negligible attention is the induction of ‘natural plant defenses’ by phytohormones. In this study, we investigated the effect of salicylic acid (SA) treatment of mango fruit (cv. Totapuri) on oviposition and larval development of B. dorsalis. In oviposition choice assays, gravid females laid significantly less eggs in SA treated compared to untreated fruit. Headspace volatiles collected from SA treated fruit were less attractive to gravid females compared to volatiles from untreated fruit. GC-MS analysis of the headspace volatiles from SA treated and untreated fruit showed noticeable changes in their chemical compositions. Cis-ocimene and 3-carene (attractants to B. dorsalis) were reduced in the headspace volatiles of treated fruit. Further, reduced pupae formation and adult emergence was observed in treated fruit compared to control. Increased phenol and flavonoid content was recorded in treated fruit. We also observed differential expression of anti-oxidative enzymes namely catalase (CAT), polyphenoloxidase (PPO) and peroxidase (POD). In summary, the results indicate that SA treatment reduced oviposition, larval development and adult emergence of B. dorsalis and suggest a role of SA in enhancing mango tolerance to B. dorsalis.     

植物源农药对橘小实蝇的毒杀及引诱作用研究

在室内条件下采用药膜法,分别测定了3种植物源生物杀虫剂对橘小实蝇成虫的毒力。其中GF-120（0.02%多杀霉素RB）对成虫的毒性最强,LC50值为0.23mg·kg^-1,其次为0.3%印楝素EC,毒性最小的为0.36%苦参碱EC。采用非选择性试验测定了橘小实蝇成虫对8种植物精油的趋性,结果表明,天竺葵和薄荷对橘小实蝇成虫的诱集效果最好,引诱率可达36%以上,与甲基丁香酚处理比较无显著差异,并且对雌雄虫都有效,其次为洋甘菊、鼠尾草和丁香,甜橙、罗勒和柠檬的诱集效果较差。     

BdorCSP2 Is Important for Antifeed and Oviposition-Deterring Activities Induced by Rhodojaponin-III against Bactrocera dorsalis

Rhodojaponin-III is a nonvolatile botanical grayanoid diterpene compound, which has antifeedant and oviposition deterrence effects against many kinds of insects. However, the molecular mechanism of the chemoreception process remains unknown. In this study, the important role of BdorCSP2 in the recognition of Rhodojaponin-III was identified. The full length cDNA encoding BdorCSP2 was cloned from legs of Bactrocera dorsalis. The results of expression pattern revealed that BdorCSP2 was abundantly expressed in the legs of adult B. dorsalis. Moreover, the expression of BdorCSP2 could be up-regulated by Rhodojaponin-III. In order to gain comprehensive understanding of the recognition process, the binding affinity between BdorCSP2 and Rhodojaponin-III was measured by fluorescence binding assay. Silencing the expression of BdorCSP2 through the ingestion of dsRNA could weaken the effect of oviposition deterrence and antifeedant of Rhodojaponin-III. These results suggested that BdorCSP2 of B. dorsalis could be involved in chemoreception of Rhodojaponin-III and played a critical role in antifeedant and oviposition behaviors induced by Rhodojaponin-III.     

Molecular Characteristics,mRNA Expression,and Alternative Splicing of a Ryanodine Receptor Gene in the Oriental Fruit Fly,Bactrocera dorsalis (Hendel)

Ryanodine receptors (RyRs) are a distinct class of ligand-gated channels controlling the release of calcium from intracellular stores. The emergence of diamide insecticides, which selectively target insect RyRs, has promoted the study of insect RyRs. In the present study, the full-length RyR cDNA (BdRyR) was cloned and characterized from the oriental fruit fly, Bactrocera dorsalis (Hendel), a serious pest of fruits and vegetables throughout East Asia and the Pacific Rim. The cDNA of BdRyR contains a 15,420-bp open reading frame encoding 5,140 amino acids with a predicted molecular weight of 582.4 kDa and an isoelectric point of 5.38. BdRyR shows a high level of amino acid sequence identity (78 to 97%) to other insect RyR isoforms. All common structural features of the RyRs are present in the BdRyR, including a well-conserved C-terminal domain containing consensus calcium-binding EF-hands and six transmembrane domains, and a large N-terminal domain. Quantitative real-time PCR analyses revealed that BdRyR was expressed at the lowest and highest levels in egg and adult, respectively, and that the BdRyR expression levels in the third instar larva, pupa and adult were 166.99-, 157.56- and 808.56-fold higher, respectively, than that in the egg. Among different adult body parts, the highest expression level was observed in the thorax compared with the head and abdomen. In addition, four alternative splice sites were identified in the BdRyR gene, with the first, ASI, being located in the central part of the predicted second spore lysis A/RyR domain. Diagnostic PCR analyses revealed that alternative splice variants were generated not only in a tissue-specific manner but also in a developmentally regulated manner. These results lay the foundation for further understanding the structural and functional properties of BdRyR, and the molecular mechanisms for target site resistance in B. dorsalis.     

The Role of Dopamine in Drosophila Larval Classical Olfactory Conditioning

Learning and memory is not an attribute of higher animals. Even Drosophila larvae are able to form and recall an association of a given odor with an aversive or appetitive gustatory reinforcer. As the Drosophila larva has turned into a particularly simple model for studying odor processing, a detailed neuronal and functional map of the olfactory pathway is available up to the third order neurons in the mushroom bodies. At this point, a convergence of olfactory processing and gustatory reinforcement is suggested to underlie associative memory formation. The dopaminergic system was shown to be involved in mammalian and insect olfactory conditioning. To analyze the anatomy and function of the larval dopaminergic system, we first characterize dopaminergic neurons immunohistochemically up to the single cell level and subsequent test for the effects of distortions in the dopamine system upon aversive (odor-salt) as well as appetitive (odor-sugar) associative learning. Single cell analysis suggests that dopaminergic neurons do not directly connect gustatory input in the larval suboesophageal ganglion to olfactory information in the mushroom bodies. However, a number of dopaminergic neurons innervate different regions of the brain, including protocerebra, mushroom bodies and suboesophageal ganglion. We found that dopamine receptors are highly enriched in the mushroom bodies and that aversive and appetitive olfactory learning is strongly impaired in dopamine receptor mutants. Genetically interfering with dopaminergic signaling supports this finding, although our data do not exclude on naïve odor and sugar preferences of the larvae. Our data suggest that dopaminergic neurons provide input to different brain regions including protocerebra, suboesophageal ganglion and mushroom bodies by more than one route. We therefore propose that different types of dopaminergic neurons might be involved in different types of signaling necessary for aversive and appetitive olfactory memory formation respectively, or for the retrieval of these memory traces. Future studies of the dopaminergic system need to take into account such cellular dissociations in function in order to be meaningful.     

Identification,mRNA Expression,and Functional Analysis of Chitin Synthase 1 Gene and Its Two Alternative Splicing Variants in Oriental Fruit Fly,Bactrocera dorsalis

Two alternative splicing variants of chitin synthase 1 gene (BdCHS1) were cloned and characterized from the oriental fruit fly, Bactrocera dorsalis (Hendel). The cDNA of both variants (BdCHS1a and BdCHS1b) consisted of 5,552 nucleotides (nt), with an open reading frame (ORF) of 4,776 nt, encoding a protein of 1,592 amino acid residues, plus 685- and 88-nt of 5′- and 3′-noncoding regions, respectively. The alternative splicing site was located between positions 3,784-3,960 and formed a pair of mutually exclusive exons (a/b) that were same in size (177 nt), but showed only 65% identity at the nucleotide level. During B. dorsalis growth and development, BdCHS1 and BdCHS1a were both mainly expressed during the larval-pupal and pupal-adult transitions, while BdCHS1b was mainly expressed during pupal-adult metamorphosis and in the middle of the pupal stage. BdCHS1a was predominately expressed in the integument whereas BdCHS1b was mainly expressed in the trachea. The 20-hydroxyecdysone (20E) induced the expression of BdCHS1 and its variants. Injection of dsRNA of BdCHS1, BdCHS1a, and BdCHS1b into third-instar larvae significantly reduced the expression levels of the corresponding variants, generated phenotypic defects, and killed most of the treated larvae. Furthermore, silencing of BdCHS1 and BdCHS1a had a similar result in that the larva was trapped in old cuticle and died without tanning completely, while silencing of BdCHS1b has no effect on insect morphology. These results demonstrated that BdCHS1 plays an important role in the larval-pupal transition and the expression of BdCHS1 in B. dorsalis is regulated by 20E.     

福建地区橘小实蝇田间种群动态监测研究

利用Steiner诱捕器内置诱捕剂Me或Cue与0.2%马拉硫磷混合物制成棉球诱芯,在福建省各个地区共设置50个监测点,诱捕监测橘小实蝇（Bactrocera dorsalis Hendel）种群动态。从2002～2005年,连续4年的诱捕结果表明,橘小实蝇在福建省的发生呈单峰特点,7～9月为发生盛期,高峰出现在8月份。瓜菜园生境、石榴芒果园生境和龙眼香蕉园生境中,石榴芒果园生境橘小实蝇种群密度最高。运用地理信息系统（MapInfo）对该虫监测数据进行地图分析表明,橘小实蝇种群密度的地域特征明显,橘小实蝇有不断向外扩散的趋势。     

Selection of Pupation Habitats by Oriental Fruit Fly Larvae in the Laboratory

We performed a series of laboratory experiments to determine the effects of shade, soil moisture, and soil compaction on the selection of pupation habitats by wandering late-instar Oriental fruit flies, Bactrocera dorsalis (Hendel). Larvae showed a strong preference toward pupating in shaded rather than brightly lit areas, in moist rather than dry soil, and in soil with larger particle sizes. These behavioral preferences are likely to lead to clumped distribution of Oriental fruit fly pupae in natural habitats. The implications of this for management of localized populations by chemical and biological methods are discussed.     

Local Enhancement of Alighting in the Melon Fly, Bactrocera cucurbitae: Effect of Olfactory, Visual, and Acoustical Stimuli

In studies conducted in Hawaii under both greenhouse and field conditions, we evaluated the propensity of melon fly females, Bactrocera cucurbitae (Coquillett) (Diptera: Tephritidae), to alight on either fruit mimics (agar spheres) or host fruit that were or were not occupied by conspecific resident females. We also examined the extent to which occurrence of local enhancement of alighting found in B. cucurbitae females was affected by a variety of factors such as the presence or the absence of host fruit odor (zucchini or ivy gourd), the number of conspecifics present on a host, the degree of isolation of assayed females from other females prior to testing, and the kinds of stimuli (acoustical, visual, olfactory) emanating from conspecifics present on a host mimic. In addition, we asked whether local enhancement might be operative in the food-foraging behavior of melon flies. We found that in a variety of situations, melon fly females alighted in significantly greater numbers at resources (food, fruit mimics, or host fruit) occupied by conspecific females than at unoccupied resources. Such positive influence of resident conspecific females was more pronounced in greenhouse cage assays when one or two rather than four residents were present on a host mimic (but was more pronounced when four rather than one or two residents were present on a host fruit in a field test), and was more evident when test females were grouped with conspecific females than when test females were isolated from conspecific females for 5 days before testing. Rather than acoustical or olfactory stimuli associated with resident conspecific females, the mere physical presence (visual stimulus) of a motionless dead resident melon fly female provided sufficient stimulation for test females to alight in significantly greater numbers at resources occupied by conspecific females than at unoccupied resources. We consider our findings as good evidence of local enhancement in the melon fly and discuss our results in relation to monitoring tactics for adult melon flies.     

Taxonomic Identity of the Invasive Fruit Fly Pest,Bactrocera invadens: Concordance in Morphometry and DNA Barcoding

In 2003, a new fruit fly pest species was recorded for the first time in Kenya and has subsequently been found in 28 countries across tropical Africa. The insect was described as Bactrocera invadens, due to its rapid invasion of the African continent. In this study, the morphometry and DNA Barcoding of different populations of B. invadens distributed across the species range of tropical Africa and a sample from the pest''s putative aboriginal home of Sri Lanka was investigated. Morphometry using wing veins and tibia length was used to separate B. invadens populations from other closely related Bactrocera species. The Principal component analysis yielded 15 components which correspond to the 15 morphometric measurements. The first two principal axes contributed to 90.7% of the total variance and showed partial separation of these populations. Canonical discriminant analysis indicated that only the first five canonical variates were statistically significant. The first two canonical variates contributed a total of 80.9% of the total variance clustering B. invadens with other members of the B. dorsalis complex while distinctly separating B. correcta, B. cucurbitae, B. oleae and B. zonata. The largest Mahalanobis squared distance (D² = 122.9) was found to be between B. cucurbitae and B. zonata, while the lowest was observed between B. invadens populations against B. kandiensis (8.1) and against B. dorsalis s.s (11.4). Evolutionary history inferred by the Neighbor-Joining method clustered the Bactrocera species populations into four clusters. First cluster consisted of the B. dorsalis complex (B. invadens, B. kandiensis and B. dorsalis s. s.), branching from the same node while the second group was paraphyletic clades of B. correcta and B. zonata. The last two are monophyletic clades, consisting of B. cucurbitae and B. oleae, respectively. Principal component analysis using the genetic distances confirmed the clustering inferred by the NJ tree.     

Mexican Fruit Fly Populations in the Semi-Arid Highlands of the Sierra Madre Oriental in Northeastern Mexico

The Mexican fruit fly, Anastrepha ludens Loew (Diptera: Tephritidae), is one of the most important pests of citrus in Mexico. We report the results of an analysis of A. ludens populations that inhabit the semi-arid highlands of the Sierra Madre Oriental in northeastern Mexico. This study aimed to provide information on population fluctuation of A. ludens and how it relates to climate variables, as well as insights into habitat and native parasitoids. Population peaked in the period July–November when ripe fruits of the wild host, Casimiroa pubescens Ramírez, were available. No adults were captured the rest of the year, suggesting that high populations depend on the availability of wild host fruit. No significant relationships between population fluctuation and climatic variables were observed, except for minimum temperature. Fruit samples of citron (Citrus medica L.), pomegranate (Punica granatum L.), and C. pubescens were collected to determine degree of infestation. Infestation levels (pupae/g) ranged between 0.0006 for citron, 0.0047 for pomegranate, and 0.0240 for C. pubescens. A native parasitoid of Tephritidae, Doryctobracon crawfordii (Viereck) (Braconidae), was identified. Parasitism percentage was calculated at 12.5% on C. pubescens fruits. No parasitoids were observed on citron or pomegranate fruit samples. These results contribute to knowledge on behavior of A. ludens native to temperate environments where no commercial hosts are available. Further research on host expansion of this pest in light of scenarios of global climate change is suggested.     

Acetobacter tropicalis Is a Major Symbiont of the Olive Fruit Fly (Bactrocera oleae)

Following cultivation-dependent and -independent techniques, we investigated the microbiota associated with Bactrocera oleae, one of the major agricultural pests in olive-producing countries. Bacterial 16S rRNA gene libraries and ultrastructural analyses revealed the presence of several bacterial taxa associated with this insect, among which Acetobacter tropicalis was predominant. The recent increased detection of acetic acid bacteria as symbionts of other insect model organisms, such as Anopheles stephensi (G. Favia et al., Proc. Natl. Acad. Sci. USA 104:9047-9051, 2007) or Drosophila melanogaster (C. R. Cox and M. S. Gilmore, Infect. Immun. 75:1565-1576, 2007), prompted us to investigate the association established between A. tropicalis and B. oleae. Using an A. tropicalis-specific PCR assay, the symbiont was detected in all insects tested originating from laboratory stocks or field-collected from different locations in Greece. This acetic acid bacterium was successfully established in cell-free medium, and typing analyses, carried out on a collection of isolates, revealed that different A. tropicalis strains are present in fly populations. The capability to colonize and lodge in the digestive system of both larvae and adults and in Malpighian tubules of adults was demonstrated by using a strain labeled with a green fluorescent protein.Associations of insects with bacteria, protozoa, and fungi are complex and intimate, ranging from parasitism to mutualism, and may be extracellular or intracellular and may play a role in the nutrition, the physiology, or the reproduction of the insect host (10). Petri (1909 to 1910) described one of the first bacterial symbiotic associations in an insect species, the olive fly, Bactrocera (Dacus) oleae (31, 32).The olive fruit fly B. oleae is one of the major pests of the olive tree, strongly affecting olive production worldwide, especially in the Mediterranean area, where more than 90% of the world''s olive tree cultivation takes place (24, 27). Although there have been reports on the isolation of potentially effective Bacillus thuringiensis strains against B. oleae, olive fly control strategies remain almost exclusively based on insecticides, despite the awareness of a need for the use of more environmentally friendly control methods (29). Recently, new concepts are emerging, among which the symbiotic control approach is particularly noteworthy (4). This strategy includes the use of symbionts as vectors of antagonistic factors able to block the life cycle of the plant pathogen in the insect host or, alternatively, their use for the suppression of host natural populations (45). In any case, a prerequisite for developing a symbiotic control approach is the knowledge of the microbiota associated with the insect pest.The nature of the olive fruit fly-associated microbiota is controversial. The culturable bacterium Pseudomonas savastanoi has been suspected to be a mutualist of B. oleae for more than 50 years (6, 17, 22, 25, 32, 33). In addition, traditional microbiological approaches have identified other bacteria of the genera Bacillus, Erwinia, Lactobacillus, Micrococcus, Pseudomonas, Streptococcus, Citrobacter, Proteus, Providencia, Enterobacter, Hafnia, Klebsiella, Serratia, and Xanthomonas as associated with the olive fruit fly (3, 14, 19, 37). Recently, it was suggested that the bacterium housed within the esophageal bulb and the midgut of B. oleae is unculturable, and the novel name “Candidatus Erwinia dacicola” was proposed (7). The presence of “Ca. Erwinia dacicola” was confirmed in Italian natural populations (36).The contradictory results obtained in previous studies prompted us to investigate the microbiota associated with both laboratory and natural populations of the olive fruit fly by employing both cultivation-independent and -dependent methods.     

Polymorphic microsatellite markers in the Melon Fruit Fly, Bactrocera cucurbitae (Coquillett) (Diptera: Tephritidae)

Bactrocera cucurbitae (Coquillett) is an important insect pest of melon fruit causing extensive losses of production in Asia and Pacific areas. Twelve polymorphic microsatellite loci were isolated from an enriched DNA library of this species and their amplification characteristics are described. Genetic parameters were estimated on 80 individual flies from four natural populations. Allele numbers per locus ranged from three to 20. These microsatellite markers have potential utility in population structure and gene flow studies of B. cucurbitae.     

Biochemical Differences Between Products of the ADH Locus in Olive Fruit Fly (Bactrocera oleae)

Purified alcohol dehydrogenases from olive fruitflies of genotypes SS, II, and SI were biochemicallycompared. The enzymes were found to differ in thespecific activity, in the influence of pH andtemperature on activity, and in the affinity with differentsubstrate-alcohols. The probable relationships of thesefindings with the dramatic changes in allele frequenciesobserved when natural populations are introduced in the laboratory are discussed.     

A Single Hot Event Stimulates Adult Performance but Reduces Egg Survival in the Oriental Fruit Moth,Grapholitha molesta

Climate warming is expected to increase the exposure of insects to hot events (involving a few hours at extreme high temperatures). These events are unlikely to cause widespread mortality but may modify population dynamics via impacting life history traits such as adult fecundity and egg hatching. These effects and their potential impact on population predictions are still largely unknown. In this study, we simulated a single hot event (maximum of 38°C lasting for 4 h) of a magnitude increasingly found under field conditions and examined its effect in the oriental fruit moth, Grapholitha molesta. This hot event had no impact on the survival of G. molesta adults, copulation periods or male longevity. However, the event increased female lifespan and the length of the oviposition period, leading to a potential increase in lifetime fecundity and suggesting hormesis. In contrast, exposure of males to this event markedly reduced the net reproductive value. Male heat treatment delayed the onset of oviposition in the females they mated with, as well as causing a decrease in the duration of oviposition period and lifetime fecundity. Both male and female stress also reduced egg hatch. Our findings of hormetic effects on female performance but concurrent detrimental effects on egg hatch suggest that hot events have unpredictable consequences on the population dynamics of this pest species with implications for likely effects associated with climate warming.     

Diet-Induced Over-Expression of Flightless-I Protein and Its Relation to Flightlessness in Mediterranean Fruit Fly,Ceratitis capitata

The Mediterranean fruit fly (medfly), Ceratitis capitata is among the most economically important pests worldwide. Understanding nutritional requirement helps rearing healthy medfly for biocontrol of its population in fields. Flight ability is a high priority criterion. Two groups of medfly larvae were reared with two identical component diets except one with fatty acids (diet A) and another without it (diet B). Adults from larvae reared on diet B demonstrated 20±8% of normal flight ability, whereas those from larvae reared on diet A displayed full flight ability of 97±1%. Proteomes were profiled to compare two groups of medfly pupae using shotgun proteomics to study dietary effects on flight ability. When proteins detected in pupae A were compared with those in pupae B, 233 and 239 proteins were, respectively, under- and over-expressed in pupae B, while 167 proteins were overlapped in both pupae A and B. Differential protein profiles indicate that nutritional deficiency induced over-expression of flightless-I protein (fli-I) in medfly. All proteins were subjected to Ingenuity Pathway Analysis (IPA) to create 13 biological networks and 17 pathways of interacting protein clusters in human ortholog. Fli-I, leucine-rich repeat (LRR)-containing G protein-coupled receptor 2, LRR protein soc-2 and protein wings apart-like were over-expressed in pupae B. Inositol-1,4,5-trisphosphate receptor, protocadherin-like wing polarity protein stan and several Wnt pathway proteins were under-expressed in pupae B. These results suggest down-regulation of the Wnt/wingless signaling pathway, which consequently may result in flightlessness in pupae B. The fli-I gene is known to be located within the Smith-Magenis syndrome (SMS) region on chromosome 17, and thus, we speculate that nutritional deficiency might induce over-expression of fli-I (or fli-I gene) and be associated with human SMS. However, more evidence would be needed to confirm our speculation.