Polytene chromosome maps of the melon fly Bactrocera cucurbitae (Diptera: Tephritidae).

Standard photographic maps of the polytene chromosomes are presented for the melon fly Bactrocera cucurbitae, a serious pest of fleshy fruits and vegetables. Five larval salivary gland polytene chromosomes (10 polytene arms) were isolated, and their characteristic features and landmarks have been recognized. Banding patterns of each of the polytene arms are presented, where variation in band intensity and puffs appear to reflect fundamental differences in chromosomes. The whole polytene genome has been typically mapped by dividing it into 100 sections and the subsections were lettered. The mitotic chromosomes of larval brain ganglia are also examined, five pairs of autosomes and an XX/XY sex chromosome pair. In addition, a heterochromatic mass corresponding to the sex chromosomes are observed in the polytene nuclei of salivary gland tissue. This investigation showed that B. cucurbitae has excellent cytological material for polytene chromosome analysis and proved to be very useful for obtaining more detailed genetic information on the pest's natural populations.     

Genetic relationship of the melon fly,Bactrocera cucurbitae (Diptera: Tephritidae) inferred from mitochondrial DNA

Abstract The melon fruit fly, Bactrocera cucurbitae (Coquillett) (Diptera: Tephritidae), has been the subject of worldwide quarantine and management efforts due to its widespread agricultural impact and potential for rapid range expansion. From its presumed native distribution in India, this species has spread throughout the hot‐humid regions of the world. We provide information that reveals population structure, invasion history and population connectivity from 23 locations covering nine countries based on DNA sequences of the mitochondrial cytochrome oxidase I (COI) gene. Forty‐two polymorphic sites were described among 38 haplotypes. The most common haplotype, H1, was observed in 73% of the samples distributed among all populations. Highest genetic diversity was seen within populations, and no isolation‐by‐distance was detected. The western regions (Nepal, Bangladesh, Thailand, Burma and China‐west) showed higher haplotype diversity than eastern regions (China‐east). China‐Yunnan showed highest levels of genetic diversity in China. Haplotype diversity decreased with longitude from west to east. Together, these analyses suggest that B. cucurbitae has expanded from west to east within a limited geographic scale and recently invaded China through Yunnan Province.     

瓜实蝇卵巢的发育阶段及分级

瓜实蝇是热带和亚热带地区最重要的经济害虫之一。本研究拟对卵巢发育进行分级,以判断田间捕获的瓜实蝇是否达到性成熟。室内饲养瓜实蝇成虫,对开始羽化至羽化后80 d的不同日龄成虫卵巢进行解剖,并记录了卵巢长度、卵巢宽度、卵巢指数（卵巢长度＆#215;卵巢宽度）和所含卵粒数。将瓜实蝇卵巢发育过程分为4期6个级别,分别为卵黄发生前期（Ⅰ级和Ⅱ级）、卵黄发生期（Ⅲ级和Ⅳ级）、抱卵期（Ⅴ级）、经产期（Ⅵ级）。随着卵巢的发育,Ⅰ～Ⅴ级卵巢长度和宽度迅速增长,卵巢发育成熟开始产卵以后,Ⅵ级卵巢长度和宽度逐渐减小。Ⅰ级和Ⅱ级卵巢长度与宽度大致相等,以后各期卵巢长度均大于宽度。Ⅲ级和Ⅳ级可见清晰的卵室,卵母细胞迅速增长,卵黄逐渐沉积。Ⅴ级可见清晰的卵粒,整个卵巢呈圆柱状。Ⅵ级开始排出卵子,所含卵粒数显著少于Ⅴ级;虽然此级卵巢长度大于Ⅳ级,但卵巢宽度、卵巢指数与Ⅳ级基本一致;Ⅵ级卵巢可见黄体,可结合黄体判定卵巢所处发育级别。本研究对评估粘虫板捕获的瓜实蝇雌虫生理日龄结构有潜在应用价值。     

Toxicity of gallic acid to melon fruit fly,Bactrocera cucurbitae (Coquillett) (Diptera: Tephritidae)

Melon fruit fly, Bactrocera cucurbitae (Coquillett) is an important pest of cucurbits and other vegetable crops. It is not only a serious pest of cucurbit crops but sometimes also attacks non-host plants. In an endeavour to explore secondary metabolites as important and safe means of pest management, we investigated the effects of gallic acid, a phenolic compound, on the growth and development of melon fruit fly, B. cucurbitae. Larval survival and emergence were severely affected by gallic acid treatment. Both decreased in a concentration dependent manner with increase in concentration. Gallic acid-treated larvae took longer duration to pupate and reach the adult stage as compared to control larvae. Inhibitory effects of gallic acid were also observed on larval weight, pupal weight, mean relative growth rate and food assimilated which decreased with treatment. The ability of gallic acid to disrupt the development of B. cucurbitae suggests that the phenolic compound might have caused oxidative stress in the body of the insect.     

Prolonged mating in the melon fly,Dacus cucurbitae (Diptera: Tephritidae): Competition for fertilization by sperm-loading

Summary Males of the melon flyDacus cucurbitae mate with females for 10 hours or more, usually starting at dawn and terminating at dusk. We tested the sperm-loading hypothesis (Dickinson, 1986) that males remaining with females for long periods of time benefit by numerically overwhelming the sperm of their competitors. The amount of sperm transferred to a female increased with time after mounting. The number of feamles which laid eggs at least once during experimental periods was positively correlated with mating duration. Oviposition rate was positively correlated with mating duration, as well. Egg hatchability was not influenced by mating duration. Mating duration was a major determinant of paternity when females were doubly mated with one male for 6 hours and another male for 2 hours. Females whose first matings were longer showed first male sperm predominance, while females whose second matings were longer showed last male sperm predominance. The adaptive significance of prolonged mating by male melon flies is discussed.     

Circadian rhythm and time of mating in Bactrocera cucurbitae (Diptera: Tephritidae) selected for age at reproduction

Chrono-biological traits were changed by selecting for life-history traits via a genetic linkage controlling both time-related behavioural and life-history traits. Behavioural traits were compared between lines selected for young (Y-lines) and old (O-lines) age at reproduction in the melon fly, Bactrocera cucurbitae (Coquillett). Adults from O-lines, which survive longer than flies from Y-lines, mated later in the day and had a longer period of circadian rhythm in the locomotor activity than those from Y-lines. Flies from F(1) reciprocal crosses had an intermediate time of mating and periods of circadian rhythm between that of the parents, indicating a genetic basis to these traits. The presence of these behavioural differences across the selection lines indicates that chrono-biological traits exhibit correlated responses to selection on age at reproduction. The correlated responses in the behavioural traits to selection for life-history traits are discussed from two points of view: pleiotropy and inadvertent selection.     

Sperm competition in the melon fly,Bactrocera cucurbitae (Diptera: Tephritidae): Effects of sperm “longevity” on sperm precedence

Sperm competition inBactrocera cucurbitae was studied by double matings of one female with normal and sterile males, with different intervals between the first and the second matings and with or without allowing oviposition after the first or the second mating. When the interval was less than 4 days, the last-male sperm precedence,P ₂ , was not different from 0.5, but as the interval was prolonged,P ₂ was higher than 0.5. There was no significant difference between treatments in which females were allowed to oviposit after the first mating and only after the second mating. The reason for the higherP ₂ when the interval was long was therefore attributed not to sperm usage for egg fertilization during the two matings but, possibly, to sperm mortality. ThatP ₂ was 0.5 for shorter intervals suggests that particular sperm replacement mechanisms such as removal and inactivation are absent in B. cucurbitae. Our study is the first to demonstrate a significant effect of short sperm longevity on the last-male sperm precedence.     

Remating inhibition in the melon fly,Bactrocera (=Dacus) cucurbitae (Diptera: Tephritidae): Copulation with spermless males inhibits female remating

Eight hour copulation of the melon fly,Bactrocera cucurbitae, which usually mates at dusk and finishes copulation at dawn, inhibited female remating, while 3 h copulation did not. Copulation of females with either normal or virgin sterile males inhibited female remating. Sperm-depleted sterile males inhibited female remating at the same rate as normal males when the copulation duration was 8 h, indicating that existence or amount of sperm in females' spermathecae is not important in remating inhibition. Females of a wild strain remated later than females of a mass-reared strain, irrespective of strains of 1st and 2nd males. This suggests that the females may control their own remating, or that there is a difference between wild and mass-reared strains in their sensitivity to a male substance that inhibits females' receptivity.     

Male lures and male mating competitiveness in a genetic sexing strain of the melon fly (Diptera: Tephritidae)

The Sterile Insect Technique (SIT) has been used to suppress or eradicate populations of the melon fly, Zeugodacus cucurbitae (Coquillett), a pest of various fruits and vegetables throughout Asia, Africa, and South Pacific Islands. The success of the SIT depends largely on the ability of released, sterile males to compete successfully with wild males for copulations with wild females. Unfortunately, both the sterilization process (via gamma irradiation) and artificial selection under mass-rearing conditions generally reduce the biological competence, including the mating competitiveness, of the released males. However, previous data on Z. cucurbitae show that irradiation has minimal adverse effects and prolonged mass-rearing may or may not result in decreased mating ability. The present study first confirms that neither irradiation nor mass-rearing had deleterious effects on male mating ability of males from a pupal-color, genetic sexing strain (termed T1). Then, the influence of male lures (cue-lure and its natural analogue raspberry ketone) on the mating success of sterile T1 males was measured as a potential pre-release procedure to improve the effectiveness of SIT. Feeding on cue-lure enhanced male mating success, but the effect was short-lived (1 d but not 3 d post-feeding). Feeding on raspberry ketone boosted mating success over a longer time, i.e., at least 5 d but not 10 d post-feeding. However, mixing raspberry ketone into the adult diet had no detectable effect on male mating performance. The implications of these results for SIT against Z. cucurbitae are discussed.     

Population genetic structure of the melon fly, Bactrocera cucurbitae (Coquillett) (Diptera: Tephritidae) based on mitochondrial cytochrome oxidase (COI) gene sequences

Population genetic structure of melon fly analysed with mitochondrial cytochrome oxidase I gene suggested that melon fly populations across the globe is homogeneous with non-significant variation of 0.000-0.003 base substitutions per site. Test isolates representing various geographic situations across the world were placed in 26 mitochondrial haplotypes based on variations associated with a maximum of three mutational steps and the predominant haplotype i.e. H1 was present in all melon fly populations except Hawaiian population. Evolution of mtCOI gene suggested that the fly could have originated some 0.4 million years ago. The present study also indicated that the B. cucurbitae population expansion is an event of post Pleistocene warm climatic conditions with small number of founder population. The invasion of B. cucurbitae in Hawaii was associated with the large population size and the global presence of the fly is associated with human mediated dispersal. The very low genetic variation suggested that the fly management might be possible by large scale sterile insect techniques programme.     

Acoustic studies on the mechanism of sound production in the mating songs of the melon fly,Dacus cucurbitae Coquillett (Diptera: Tephritidae)

The sound production mechanism in the male mating songs for the melon flyDacus cucurbitae Coquillett was acoustically investigated to determine whether sounds resulted from free wing-fanning or stridulation produced by contact between wing and abdominal pecten. Waveforms in the songs of normal (pecten-present) males showed more complex vibrations than those of pecten-removed males. The total harmonic distortion in normal songs was greater than that of the pecten-removed songs. Female (pecten-absent) songs showed different sound signatures on the oscillogram and frequency spectrum from normal male songs. The female songs had more harmonic components than the normal male songs. These results suggest that wing/pecten stridulation of normal male songs produces complicated wing oscillations with a small number of harmonics of near-periodic waveforms and a high harmonic distortion. They also suggest that the free wing-fanning observed during female and pecten-removed male songs produces simple and uniform wing oscillations with many harmonic contents of complex-periodic waveforms and low harmonic distortion. Despite the occurrence of some acoustic differences, pecten-removal did not positively influence the rate of copulation.     

The production of pheromone clouds by spraying in the melon fly,Dacus cucurbitae coquillett (Diptera: Tephritidae)

Pheromone clouds sprayed by melon fly males were visually detected by focusing a beam of light at them during dusk when the males were vibrating their wings. The clouds were sprayed to the front, rear and upper sides of the male. We found that special morphological structures are used for spraying the pheromone clouds. When a male melon fly engages in calling behavior, sex pheromone droplets are excreted from his anus. This excretion is wiped off with the tarsus of his hind leg, and then it is deposited on the sexually dimorphic cubital cell hairs on the wing. During wing vibration, the targal bristles on the 3rd abdominal segment, which are peculiar to males, are rubbed against the specialized hairs of the cubital cell. Calling males sprayed clouds of pheromone with these actions. This paper was presented at the 32nd Annual Meeting of the Japanese Society of Applied Entomology and Zoology (Kochi, April, 1988).     

Genetic changes of life history and behavioral traits during mass-rearing in the melon Fly,Bactrocera cucurbitae (Diptera: Tephritidae)

Quantitative genetic studies for life history and behavioral traits are important in quality control for insect mass-rearing programs. Firstly, a brief history of quality control in mass-reared insects is described. Next, the differentiation of many traits of wild and mass-reared melon flies,Bactrocera cucurbitae, in Okinawa is reviewed, and the factors which have caused variation in these traits are considered. As artificial selection pressures are thought to be more important than inbreeding depression and genetic drift in the mass-reared strain of the Okinawan melon fly, two artificial selection experiments were conducted to evaluate genetic variations and genetic correlations among life history and behavioral traits. These are divergent selections for age at reproduction and for developmental period. The genetic relationship among 5 traits, i.e. longevity, age at reproduction, developmental period, circadian period, and time of mating was clarified and discussed in relation to genetic changes of traits during the mass-rearing. The results suggest that the genetic trade-off relationships between traits should be taken into account in mass-rearing programs.     

Development of immature stages and comparative demography of two cucurbit-attacking fruit flies in Reunion Island: Bactrocera cucurbitae and Dacus ciliatus (Diptera Tephritidae)

On Reunion Island, two species of Dacini, Bactrocera cucurbitae (Coquillett) and Dacus ciliatus Loew, infest 16 host plant species belonging to the family Cucurbitaceae from sea level to 1,600 m. These two species represent two primary pests of this plant family on the island. Melon fly, Bactrocera cucurbitae, and Ethiopian fruit fly, D. ciliatus, larval development was studied at four different constant temperatures (15, 20, 25, and 30 degrees C) with three host plants (cucumber, pumpkin, and squash). Adult life histories of these two species were studied at 25 degrees C with the three host plants. The results led to the conclusion that B. cucurbitae had a faster egg incubation time. Its preimaginal instars developed significantly faster than those of D. ciliatus independent of temperature. B. cucurbitae and D. ciliatus had similar mean preoviposition duration and egg hatching success. Fecundity was significantly higher for the melon fly on cucumber and pumpkin and lower on squash. Two distinctly different life- history patterns were evident: (1) later onset of reproduction, longer oviposition time, longer life span, and higher fecundity (B. cucurbitae) and (2) early reproduction, lower oviposition time, shorter life span, and lower fecundity (D. ciliatus). These results are useful for improving laboratory-rearing methods and for building simulation models to predict Dacini population dynamics.     

Genetic structure and demographic history of the melon fly Zeugodacus cucurbitae (Coquillet) (Diptera: Tephritidae) in Thailand

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Comparison of the efficacy of different food attractants and their concentration for melon fly (Diptera: Tephritidae)

The relative attractiveness of six commercially available protein hydrolysates and the influence of their concentration were evaluated in field cages by a release-capture method of lab-reared melon fly adults, Bactrocera cucurbitae (Coquillett). Buminal, Corn Steepwater, Hym-Lure, Pinnacle, Nulure, and SolBait were tested for both sexes of the melon fly. The tested products exhibited clear differences in attractiveness. SolBait was the most effective protein hydrolysate. Pinnacle and Corn Steepwater also gave promising results. A general tendency for an increase in effectiveness with increasing concentration within the range 0.5 to 10% was shown. This study will allow pest control practitioners to choose more effective attractants for use in bait sprays to control the melon fly thus reducing the intensive use of insecticides currently practiced in Reunion island and enabling the development of Integrated pest management (IPM) methods for cucurbit crops.     

A novel point mutation of acetylcholinesterase in a trichlorfon-resistant strain of the oriental fruit fly Bactrocera dorsalis (Diptera: Tephritidae)

Acetylcholinesterase (AChE) is the target enzyme of organophosphorus and carbamate insecticides. We applied trichlorfon to select resistant strains of Bactrocera dorsalis Hendel in the laboratory. Two trichlorfon-resistant strains, the Tri-R₁ strain with 18.23-fold resistance and the Tri-R₂ strain with 69.5-fold resistance, were obtained. Three known mutations, I159V, G433S and Q588R were identified in AChE of two resistant strains, and a novel mutation, G365A, was identified in the more resistant Tri-R₂ strain. The modeled 3-D-structure of AChE showed that G365A and G433S are closely adjacent in the gorge above the catalytic site S235. Mutations of G365A and G433S resulted in a steric hindrance by stronger Van der Waals force between two sites. Such a minor structural change might block insecticides from squeezing through the gorge to reach the active site, but not the natural substrate. Compared with the susceptible strain, the AChE activity of the Tri-R₁ strain and the Tri-R₂ strain was 0.87- and 0.67-fold, the K _m value of the Tri-R₁ strain and the Tri-R₂ strain was 0.11- and 0.10-fold, the V _max value of two resistant strains was 0.26- and 0.15-fold, whereas, the I ₅₀ to trichlorfon significantly increased by 9.07- and 13.19-fold. These results suggested that the novel point mutation G365A of AChE might be involved in increasing resistance to trichlorfon in the resistant strain of oriental fruit fly.     

基于线粒体COI基因的桔小实蝇种群遗传分化研究

【目的】推测桔小实蝇Bactrocera dorsalis在中国的扩散路径和新发生地区的入侵来源。【方法】本研究测序获得来自中国、泰国、日本、老挝、孟加拉国和美国等地31个种群的192头桔小实蝇个体的COI序列（1 496 bp,占COI基因全长97.3%）,并以软件DnaSP 5.0, MEGA 6.0和Arlequin 3.5等完成各种群的遗传多样性、种群间的遗传分化以及单倍型分析。【结果】所测31个桔小实蝇种群总体表现出较高水平的核苷酸多样性（0.00663）和高水平的单倍型多样性（0.98069）。以F-统计法度量种群间遗传分化程度, 结果显示中国桔小实蝇地理种群间遗传分化较弱, 中国种群与泰国、日本、老挝、孟加拉国、美国种群间的遗传分化程度不同, 中国种群与美国种群及日本种群的遗传分化最大。而Mantel检验发现,中国、泰国、日本、老挝、孟加拉国和美国种群间的遗传分化与空间距离有关（R=0.670, P<0.0001）, 中国种群间的遗传分化不是地理隔离所造成的（R=0.038, P=0.534）。中国种群与泰国、日本、老挝、孟加拉国和美国种群间不存在共享单倍型。根据种群系统发育树,可将中国原发生地区种群划分为西南、东南两大分支。中性检验和错配分析结果均表明桔小实蝇曾存在大规模的扩张。【结论】桔小实蝇以东南地区和西南地区为源头向中国内陆扩散,其中广东、福建、广西和贵州种群为中国内陆种群较为有影响力的源头。根据遗传多样性、遗传分化、单倍型分析,推测新发生地区桔小实蝇的来源, 例如安徽合肥桔小实蝇种群主要来源于福建长乐、广东珠海和上海。     

Effects of the juvenile hormone analogue methoprene and dietary protein on male melon fly Bactrocera cucurbitae (Diptera: Tephritidae) mating success

The effect of access to dietary protein (P) and the topical application of a juvenile hormone analogue (methoprene (M)) on mating behaviour of male melon fly Bactrocera cucurbitae was assessed in the laboratory and in field cages. Age, dietary protein and methoprene application increased the mating success and influenced the mating behaviour. Treatment with methoprene (M+) to protein-deprived (P−) males had only a modest effect on the acceleration of sexual maturity, but application of methoprene (M+) to protein-fed (P+) males greatly accelerated sexual maturity. Protein diet (P+) increased mating success of males in comparison to protein-deprived (P−) males. Protein and methoprene have a synergistic effect on mating behaviour, since M + P+ treated males exhibit reduced mating latency and achieved higher mating in younger ages than methoprene and/or protein-deprived males. Copulation duration was correlated with nutritional status and M + P+ males copulated longer at the age of advanced sexual maturity than M − P+ males. Our results suggest that in this species with a lek mating system, females discriminate between the males based on their sexual signals, which were influenced by protein in the adult diet, methoprene application and age. The results are discussed in the light of mating competitiveness of precocious treated young males and their relevance to Sterile Insect Technique application against this pest species.