Deterioration of digestive physiology of Bactrocera cucurbitae larvae by trypsin inhibitor purified from seeds of Mucuna pruriens.

Peptidase inhibitors (PIs) are plant proteins that are found to be effective against various digestive peptidases of insects. The present study isolated and characterized a trypsin inhibitor from mature dry seeds of Mucuna pruriens and investigated its effect against Bactrocera cucurbitae larvae, a major pest of cucurbitaceae crops, for its inhibitory activity. The purified trypsin inhibitor from M. pruriens seeds gave a molecular weight of ~11 kDa on SDS-PAGE. M. pruriens trypsin inhibitor (MPTI) exhibited inhibitory effect on growth of melon fruit fly larvae (64-72 h old) as it resulted in prolongation of larval, pupal and total development period. There was a significant increase in larval mortality with increase in concentration of MPTI. Nutritional indices decreased significantly at all the concentrations of MPTI. Quantitative RT- PCR revealed that the mRNA expression level of trypsin and chymotrypsin genes was reduced while that of GST, esterases, AP, SOD and catalase were enhanced. It can therefore be inferred that MPTI can serve as a promising agent for biocontrol that can reduce the problems caused by fruit fly and other similar catastrophic pests. This study provides the fundamental information for future successful strategies for pest management.

Purified winged bean protease inhibitor affects the growth of Bactrocera cucurbitae.

The melon fruit fly, Bactrocera cucurbitae (Coquillett), is a serious agricultural pest which has defied the various control measures employed against it. Protease inhibitors present in plants which have the potential to inhibit the growth and development of associated insect pests can be a possible alternative which can be manipulated for developing resistance in plants to the pest. In the present study, winged bean (Psophocarpus tetragonolobus) protease inhibitor isolated through affinity chromatography was explored for its potential to disrupt the development of melon fruit fly, B. cucurbitae. Different concentrations (12.5, 25, 50, 100, 200, and 400 µg ml-1) of the winged bean protease inhibitor (WBPI) were incorporated into the artificial diet of the second instar (64-72 h old) larvae of B. cucurbitae. The WBPI significantly delayed the larval, pupal, and total development period. The percentage pupation and adult emergence of the treated larvae was reduced as compared with control. The activities of major digestive enzymes (trypsin, chymotrypsin, leucine aminopeptidase, and elastase) decreased significantly in the larvae treated with different concentrations (50, 100, 200, and 400 µg ml-1) of WBPI. The findings reveal that the inhibitor holds considerable promise for the management of the melon fruit fly.

Host plant accessions determine bottom-up effect of snapmelon (Cucumis melo var. momordica) against melon fly (Bactrocera cucurbitae (Coquillett)).

The melon fly, Bactrocera cucurbitae (Tephritidae: Diptera) is an important pest of snapmelon (Cucumis melo var. momordica), leading to significant losses in yield in the hot arid agro-climate of India. The accessions IC- 430190 (11.21%), DKS-AHS 2011/4 (14.97%) and DKS-AHS 2011/3 (18.57%) were found to be novel resistant accessions against melon fly, B. cucurbitae infestation. Free amino acid and total soluble solid (TSS) were in positive correlation with percent fruit infestation whereas phenols, tannin, total alkaloids and flavonoid contents had significant negative correlation with percent fruit infestation. The percent fruit infestation had significant positive correlation with fruit length, fruit diameter and flesh thickness and negative correlation with length of ovary pubescence, rind hardness at immature stage, rind hardness at mature stage and pericarp thickness. Based on Kaiser Normalization method, two principal components (PCs) were extracted explaining cumulative variation of 82.80% in melon fly infestation. PC1 explained 53.41% of the variation while PC2 explained 29.39% of variation. The flavonoid, total alkaloid, tannins, phenols content, length of ovary pubescence and rind hardness were the novel antibiosis and antixenotic characters found in snapmelon resistant melon fly, B. cucurbitae and therefore, could be used as marker traits in plant breeding programs to select resistant accessions.

Insecticidal Activity of the Leaf Essential Oil of Peperomia borbonensis Miq. (Piperaceae) and Its Major Components against the Melon Fly Bactrocera cucurbitae (Diptera: Tephritidae).

The essential oil from the leaves of Peperomia borbonensis from Réunion Island was obtained by hydrodistillation and characterized using GC-FID, GC/MS and NMR. The main components were myristicin (39.5%) and elemicin (26.6%). The essential oil (EO) of Peperomia borbonensis and its major compounds (myristicin and elemicin), pure or in a mixture, were evaluated for their insecticidal activity against Bactrocera cucurbitae (Diptera: Tephritidae) using a filter paper impregnated bioassay. The concentrations necessary to kill 50% (LC50 ) and 90% (LC90 ) of the flies in three hours were determined. The LC50 value was 0.23 ± 0.009 mg/cm2 and the LC90 value was 0.34 ± 0.015 mg/cm2 for the EO. The median lethal time (LT50 ) was determined to compare the toxicity of EO and the major constituents. The EO was the most potent insecticide (LT50  = 98 ± 2 min), followed by the mixture of myristicin and elemicin (1.4:1) (LT50  = 127 ± 2 min) indicating that the efficiency of the EO is potentiated by minor compounds and emphasizing one of the major assets of EOs against pure molecules.

Isolation and Molecular Characterization of the Transformer Gene From Bactrocera cucurbitae (Diptera: Tephritidae).

transformer (tra) is a switch gene of sex determination in many insects, particularly in Dipterans. However, the sex determination pathway in Bactrocera cucurbitae (Coquillett), a very destructive pest on earth, remains largely uncharacterized. In this study, we have isolated and characterized one female-specific and two male-specific transcripts of the tra gene (Bcutra) of B. cucurbitae. The genomic structure of Bcutra has been determined and the presence of multiple conserved Transformer (TRA)/TRA-2 binding sites in Bcutra has been found. BcuTRA is highly conservative with its homologues in other tephritid fruit flies. Gene expression analysis of Bcutra at different developmental stages demonstrates that the female transcript of Bcutra appears earlier than the male counterparts, indicating that the maternal TRA is inherited in eggs and might play a role in the regulation of TRA expression. The conservation of protein sequence and sex-specific splicing of Bcutra and its expression patterns during development suggest that Bcutra is probably the master gene of sex determination of B. cucurbitae. Isolation of Bcutra will facilitate the development of a genetic sexing strain for its biological control.

Identification of candidate chemosensory genes in Bactrocera cucurbitae based on antennal transcriptome analysis.

The melon fly, Bactrocera cucurbitae (Coquillett) (Tephritidae: Diptera), is an invasive pest that poses a significant threat to agriculture in Africa and other regions. Flies are known to use their olfactory systems to recognise environmental chemical cues. However, the molecular components of the chemosensory system of B. cucurbitae are poorly characterised. To address this knowledge gap, we have used next-generation sequencing to analyse the antenna transcriptomes of sexually immature B. cucurbitae adults. The results have identified 160 potential chemosensory genes, including 35 odourant-binding proteins (OBPs), one chemosensory protein (CSP), three sensory neuron membrane proteins (SNMPs), 70 odourant receptors (ORs), 30 ionotropic receptors (IRs), and 21 gustatory receptors (GRs). Quantitative real-time polymerase chain reaction quantitative polymerase chain reaction was used to validate the results by assessing the expression profiles of 25 ORs and 15 OBPs. Notably, high expression levels for BcucOBP5/9/10/18/21/23/26 were observed in both the female and male antennae. Furthermore, BcucOROrco/6/7/9/13/15/25/27/28/42/62 exhibited biased expression in the male antennae, whereas BcucOR55 showed biased expression in the female antennae. This comprehensive investigation provides valuable insights into insect olfaction at the molecular level and will, thus, help to facilitate the development of enhanced pest management strategies in the future.

Effect of host shift on the gut microbes of Bactrocera cucurbitae (Coquillett) (Diptera: Tephritidae).

Background: Gut microbes play an important role in the adaptation of insects. Polyphagous insects usually undergo changes in gut microbiota after host shift. The Bactrocera cucurbitae have a wide range of hosts, but the dynamic of gut microorganisms during host shift have not been thoroughly investigated. To understand the role of gut microbes in insect adaptation, it is necessary to study the dynamics of insect gut microorganisms during host transfer. Methods: Using Bactrocera cucurbitae (Coquillett) (Diptera: Tephritidae) and its four hosts as study subjects, we investigated the dynamics of gut microbes during host transfer and the effects of different hosts on the gut microbial composition of B. cucurbitae. Results: The results showed that the Chao1 index of B. cucurbitae decreased significantly during host transfer, and the intestinal microorganisms were significantly affected by the original host, host, and generations. Furthermore, predicated changes in the abundance of secondary metabolite pathways after host transfer suggested that microorganisms may play an important role in the degradation of secondary metabolites, among which Providencia and Morganella have important functions in the gut of B. cucurbitae. Conclusion: This implied that microorganisms play a function in the host transfer process of B. cucurbitae and may be an important cofactor in the adaptation of B. cucurbitae to different hosts and environments, providing new research ideas for the future control of B. cucurbitae.

Effects of Sublethal Concentrations of Insecticides on Survival and Reproduction of Two Bactrocera Species (Diptera: Tephritidae).

The melon fruit fly, Bactrocera cucurbitae (Coquillett) and the pumpkin fruit fly, Bactrocera tau (Walker) (Diptera: Tephritidae), are important invasive pests on Cucurbitaceous hosts. The acute toxicity of five insecticides to females of these two fruit fly species was bio-assayed by membrane method, and effects of their sublethal concentration on the survival, reproduction, and ovary development of females were evaluated in the laboratory. Results showed that based on the LC50 values, both B. cucurbitae and B. tau were the most susceptible to trichlorfon. After treatment with sublethal concentration (LC30) of trichlorfon, abamectin+ß-cypermethrin, spinetoram, and lambda-cyhalothrin, the female survival days of the two Bactrocera species were significantly shortened compared with the control. Moreover, the fecundity of two Bactrocera species was remarkably decreased, after exposure to abamectin+ß-cypermethrin and trichlorfon LC30. However, the sublethal concentration (LC30) of insecticides had no significant influence on the egg hatchability of the fruit flies. Furthermore, after treatment with abamectin+ß-cypermethrin LC30, the ovary length, width, and egg load of B. cucurbitae were significantly lower than that of the control; however, only the ovarian length and egg load of B. tau were significantly decreased on the 16th and 20th day. In conclusion, abamectin+ß-cypermethrin has an excellent insecticidal activity against B. cucurbitae and B. tau.

Exploration of insecticidal potential of Cry protein purified from Bacillus thuringiensis VIID1.

Insect pests are a threat to agriculture as they cause a loss of 15-22% to economically important crops every year. Bacillus thuringiensis produces parasporal crystal inclusions that have insecticidal 'Cry' proteins which are toxic to insect larvae of the order Lepidoptera, Coleoptera and Diptera, etc. In the present study, 40 different soil samples from Amritsar and its surrounding areas were selected for isolation of B. thuringiensis. The rod shaped, gram-positive bacterial isolates were further analyzed for characteristic crystal formation using phase contrast and scanning electron microscopy. 6 Bacillus samples containing cry genes were identified using the universal primers for cry genes, of which one isolate exhibited a protein band of ~95 kDa. This protein was purified using a Sephadex G-75 column. The insecticidal assays conducted with purified Cry protein on insect larvae of lepidopteran and dipteran orders viz. Spodoptera litura, Galleria malonella, Bactrocera cucurbitae and Culex pipens revealed considerable detrimental effects. A significant increase in larval mortality was observed for the larvae of all insects in a concentration dependent manner when treated with Cry protein purified from B. thuringenisis VIID1. The purified Cry protein did not have any significant effect on honey bee larvae.

Effect of Short-Term Low Temperature on the Growth, Development, and Reproduction of Bactrocera tau (Diptera: Tephritidae) and Bactrocera cucurbitae.

The fruit flies Bactrocera tau (Walker) and B. cucurbitae (Coquillett) are economically important invasive pests on numerous vegetable and fruit species in China. Due to the instability of the early spring climate, temperatures often deviate far below the normal temperature for short periods of time. Such a sudden short-term low temperature may impact the reproduction and development of the two fruit fly species. In this study, the effects of low temperatures (8, 6, 4, 2, 0, -2, and -4°Ð¡) on the development and reproduction of these two closely related fruit fly species were studied under laboratory condition. The results showed that their survival rates decreased gradually with corresponding decreases in the tested temperatures. On the other hand, their pre-oviposition periods and their adult female to male sex ratios increased, while the average number of eggs per female of each species and longevity of male adult of B. cucurbitae initially increased and later decreased after exposure to the low-temperature treatments. Overall, low temperatures promoted reproduction in B. cucurbitae and B. tau at temperatures ranging from 24°Ð¡ to as low as 8°Ð¡. Development and reproduction of the two species were negatively affected when temperatures were between 8 to -4°Ð¡. The cold resistance of each developmental stage was higher in B. tau than in corresponding stages of B. cucurbitae.

Purification of a trypsin inhibitor from Psoralea corylifolia seeds and its influence on developmental physiology of Bactrocera cucurbitae.

A trypsin inhibitor was purified from the seeds of Psoralea corylifolia by ion-exchange and affinity chromatography. The purified fractions were subjected to RP- HPLC which resolved into a single peak. SDS-PAGE analysis gave an apparent molecular weight of 18 kDa. P. corylifolia trypsin inhibitor (PCTI) was found to be a competitive inhibitor and was active over a broad temperature (10-100 °C) and pH (6-11) range. It was shown to have a deleterious effect on growth and development of larvae of the melon fruit fly, Bactrocera cucurbitae, when incorporated in artificial diet using various concentrations. The larval, pupal, total development period and larval mortality significantly increased during the treatment. Inhibitory effects were also observed on percentage emergence which was significantly reduced. Nutritional indices namely food assimilated (FA) and mean relative growth rate (MRGR) also decreased significantly with increase in concentration of PCTI. qRT-PCR results indicated that the expression of trypsin and chymotrypsin genes were down-regulated while elastase, catalase, GST, SOD and AP were up-regulated. PCTI was also effective against certain bacterial strains. These results indicated that the peptidase inhibitor from P. corylifolia may be a potential bio-control agent which can decrease the damage caused by B. cucurbitae and other related destructive pests.

Response Mechanism of Oviposition and Relevant Protein Expression of Bactrocera cucurbitae (Coquillet) to Short-Term High-Temperature Conditions.

Bactrocera cucurbitae (Coquillett) (Diptera: Tephritidae) is an important pest of vegetables in Asia, the Middle East, Africa, and Hawaii. High temperature can significantly influence B. cucurbitae reproduction. The effect of short-term high-temperature exposure on proteins that affect oviposition was analyzed by proteomics. Among six key target genes for oviposition, the expression of Vitellogenin-1, Vitellogenin-2, and Vitellogenin receptor was similar in B. cucurbitae exposed to higher temperature compared to controls. However, levels of Vitellogenin-3 were reduced. Juvenile hormone (Jh)-inducible protein was downregulated and then upregulated, while the expression of Jh-epoxide hydrolase-2 showed the opposite Jh-inducible protein trend. Therefore, short-term high-temperature stress can cause differential expression of proteins related to oviposition in B. cucurbitae, which in turn further triggers the hormesis of oviposition. High-temperature conditions have become more frequent because of climate warming and are predicted to continue. The data indicate that climate effects on insect reproduction pose a significant threat to agriculture in a world of increasing population.

Diversity of bacterial communities in the midgut of Bactrocera cucurbitae (Diptera: Tephritidae) populations and their potential use as attractants.

BACKGROUND: The microbiota plays an important role in insect development and fitness. Understanding the gut microbiota composition is essential for the development of pest management strategies. Midgut bacteria were isolated from nine wild B. cucurbitae populations collected from different agroecological zones of India. These isolates were further studied for attractant potential of fruit fly adults, and the chemical constituents in the supernatants of gut bacteria were analysed. RESULTS: Twenty-six bacterial isolates belonging to the families Enterobacteriaceae, Bacillaceae, Micrococcaceae and Staphylococcaceae were isolated and identified on the basis of 16S rRNA gene sequence analysis. The dominant species in the midgut of melon fly were from the genera Enterobacter (34.6%), Klebsiella (19.2%), Citrobacter (7.7%), Bacillus (15.4%) and Providencia (7.7%), and 3.8% each of Micrococcus, Staphylococcus, Leclercia and Exiguobacterium. Bactrocera cucurbitae and B. dorsalis adults were significantly attracted to bacterial whole cell cultures and their supernatants in the fruit fly attraction bioassays. Bacillus cereus, Enterobacter, Klebsiella, Citrobacter and Providencia species attracted both male and females of Bactrocera species. The supernatants of Klebsiella, Citrobacter and Providencia species attracted a significantly greater number of females than males. The most abundant chemical constituents in supernatants of K. oxytoca and C. freundii were 3-methyl-1-butanol, 2-phenylethanol, butyl isocyanatoacetate, 2-methyl-1-propanol and 3-hydroxy-2-butanone, as identified by gas chromatography-mass spectrometry. CONCLUSIONS: The bacterial endosymbionts associated with melon fly exhibited attractant potential which could facilitate eco-friendly insect control strategies. © 2015 Society of Chemical Industry.

Assessment of Navel Oranges, Clementine Tangerines, and Rutaceous Fruits as Hosts of Bactrocera cucurbitae and Bactrocera latifrons (Diptera: Tephritidae).

Export of Citrus spp. fruits may require risk mitigation measures if grown in areas with established tephritid fruit fly (Diptera: Tephritidae) populations capable of infesting the fruits. The host status of Citrus spp. fruits is unclear for two tephritid fruit fly species whose geographic ranges have expanded in recent years: melon fly, Bactrocera cucurbitae (Cocquillett), and Bactrocera latifrons (Hendel). In no choice cage infestation studies, B. latifrons oviposited into intact and punctured Washington navel oranges (Citrus sinensis [L.] Osbeck) and Clementine tangerines (C. reticulata L. var. Clementine), but eggs rarely developed to the adult stage. B. cucurbitae readily infested intact and punctured tangerines, and to a lesser extent punctured oranges, but did not infest intact oranges. Limited cage infestation and only a single literature report of field Citrus spp. infestation suggest that risk mitigation of Citrus spp. for B. latifrons is not needed. Risk mitigation options of Citrus spp. for B. cucurbitae, including heat and cold treatments and systems approaches, are discussed.

Melon Fly, Bactrocera cucurbitae (Diptera: Tephritidae), Infestation in Host Fruits in the Southwestern Islands of Japan Before the Initiation of Island-wide Population Suppression, as Recorded in Publications of Japanese Public Institutions.

Bactrocera cucurbitae (Coquillett) is a tephritid fruit fly native to the Indo-Malayan region. Its distribution, though, has extended to include Africa, temperate Asia, and a number of Pacific islands. It became established in Japan in 1919 in the Yaeyama Islands and spread north in the Southwestern Islands of Japan. It was subsequently eradicated from these islands by an eradication program that extended from 1972 to 1993. As part of an effort to develop a worldwide database on the status of fruits as hosts of melon fly, the infestation data gathered from host fruits collected in this eradication program, before the initiation of suppression activities, are summarized here. Bactrocera cucurbitae infestation was documented in 24 plant taxa of four plant families (Caricaceae, Cucurbitaceae, Moraceae, and Solanaceae), with the following four new hosts identified: Ficus erecta Thunb., F. pumila L. (Moraceae), Solanum erianthum D. Don (Solanaceae), and Zehneria liukiuensis Jeffrey ex Walker (Cucurbitaceae).

An Overview of Pest Species of Bactrocera Fruit Flies (Diptera: Tephritidae) and the Integration of Biopesticides with Other Biological Approaches for Their Management with a Focus on the Pacific Region.

Fruit flies (Diptera: Tephritidae) are among the most economically important pest species in the world, attacking a wide range of fruits and fleshy vegetables throughout tropical and sub-tropical areas. These species are such devastating crop pests that major control and eradication programs have been developed in various parts of the world to combat them. The array of control methods includes insecticide sprays to foliage and soil, bait-sprays, male annihilation techniques, releases of sterilized flies and parasitoids, and cultural controls. During the twenty first century there has been a trend to move away from control with organophosphate insecticides (e.g., malathion, diazinon, and naled) and towards reduced risk insecticide treatments. In this article we present an overview of 73 pest species in the genus Bactrocera, examine recent developments of reduced risk technologies for their control and explore Integrated Pest Management (IPM) Programs that integrate multiple components to manage these pests in tropical and sub-tropical areas.

Reconstructing a comprehensive transcriptome assembly of a white-pupal translocated strain of the pest fruit fly Bactrocera cucurbitae.

BACKGROUND: Bactrocera cucurbitae is a serious global agricultural pest. Basic genomic information is lacking for this species, and this would be useful to inform methods of control, damage mitigation, and eradication efforts. Here, we have sequenced, assembled, and annotated a comprehensive transcriptome for a mass-rearing sexing strain of this species. This forms a foundational genomic and transcriptomic resource that can be used to better understand the physiology and biochemistry of this insect as well as being a useful tool for population genetics. FINDINGS: A transcriptome assembly was constructed containing 17,654 transcript isoforms derived from 10,425 unigenes. This transcriptome size is similar to reports from other Tephritid species and probably includes about 70-80% of the protein-coding genes in the genome. The dataset is publicly available in NCBI and GigaDB as a resource for researchers. CONCLUSIONS: Foundational knowledge on the protein-coding genes in B. cucurbitae will lead to improved resources for this species. Through comparison with a model system such as Drosophila as well as a growing number of related Tephritid transcriptomes, improved strategies can be developed to control this pest.

Methoprene application and diet protein supplementation to male melon fly, Bactrocera cucurbitae, modifies female remating behavior.

Methoprene (an analogue of juvenile hormone) application and feeding on a protein diet is known to enhance male melon fly, Bactrocera cucurbitae Coquillett (Diptera: Tephritidae), mating success. In this study, we investigated the effect of these treatments on male B. cucurbitae's ability to inhibit female remating. While 14-d-old females were fed on protein diet, 6-d-old males were exposed to one of the following treatments: (i) topical application of methoprene and fed on a protein diet; (ii) no methoprene but fed on a protein diet; (iii) methoprene and sugar-fed only; and (iv) sugar-fed, 14-d-old males acted as controls. Treatments had no effect on a male's ability to depress the female remating receptivity in comparison to the control. Females mated with protein-deprived males showed higher remating receptivity than females first mated with protein-fed males. Methoprene and protein diet interaction had a positive effect on male mating success during the first and second mating of females. Significantly more females first mated with sugar-fed males remated with protein-fed males and females first mated with methoprene treated and protein-fed males were more likely to remate with similarly treated males. Females mating latency (time to start mating) was significantly shorter with protein-fed males, and mating duration was significantly longer with protein-fed males compared with protein-deprived males. These results are discussed in the context of methoprene and/or dietary protein as prerelease treatment of sterile males in area-wide control of melon fly integrating the sterile insect technique (SIT).

GENETIC CORRELATIONS BETWEEN LIFE-HISTORY AND BEHAVIORAL TRAITS CAN CAUSE REPRODUCTIVE ISOLATION.

Reproductive isolation may often evolve as an indirect (pleiotropic) consequence of populations adapting to different environments or habitats. For example, niches that are temporally or seasonally offset can select for organisms with different developmental characteristics. These developmental differences can inadvertently cause reproductive isolation by a variety of means including shifts in mating activity patterns. Here, we show a genetic correlation between a life-history trait (developmental period) and a behavioral trait (time of mating) that causes significant premating isolation in the melon fly, Bactrocera cucurbitae (Diptera: Tephritidae). Fly lines selected for short and long developmental periods differ in their preferred times of mating during the evening. This difference translates into significant prezygotic isolation, as measured by mate choice tests. If the time of mating between two populations differed more than one hour, the isolation index was significantly higher than zero. These indicate that premating isolation can be established if the developmental period is divergently selected for. If such genetic correlations are ubiquitous in many organisms, multifarious divergent selection for life-history traits would often accelerate the evolution of reproductive isolation. We speculate that reproductive isolation may have been evolved via genetic correlations among time-related traits, for example, developmental period and time of mating, as in other organisms.