CRISPR/Cas9-mediated knockout of a male accessory glands-specific gene takeout1 decreases the fecundity of Zeugodacus cucurbitae female.

BACKGROUND: The melon fly, Zeugodacus cucurbitae (Coquillett), is an invasive Tephritidae pest with robust fertility. The male accessory glands (MAGs) form a vital organ that ensures insect reproductive efficiency. Most of the secreted proteins by MAGs exhibit a male bias expression. Takeout, one of these proteins, is abundantly present in the MAGs of many insects. RESULTS: In this study, we identified 32 takeout genes in Z. cucurbitae. The phylogenetic analysis and multiple sequence alignment results showed that Zctakeout1 is the most related homolog to the MAGs-specific takeout in Tephritidae. The real-time quantitative PCR results showed that Zctakeout1 was exclusively expressed in the male adult stage, and its expression level gradually increased with the increase in age and then remained stable at the sexually matured stage. The distribution among tissues demonstrated the specific expression of Zctakeout1 in the MAGs, and fluorescence immunohistochemical results confirmed the presence of Zctakeout1 in close proximity to binuclear cells of the mesoderm epidermal MAGs. In continuation, CRISPR/Cas9-mediated genome editing was employed, resulting in successfully generating a homozygous strain with an +8 bp insertion. The mating experiments with the Zctakeout1-/- males resulted in significant reductions in both the mating rate and egg production of females. CONCLUSION: These findings prove that the MAGs-specific Zctakeout1 is essential in regulating fecundity in female Z. cucurbitae fruit flies. Our data suggests its utilization in future essential insect-specific gene-directed sterility insect technique (SIT) by the genetic manipulation to keep these important Tephritidae populations under control. © 2024 Society of Chemical Industry.

Two heat shock cognate 70 genes involved in spermatogenesis regulate the male fertility of Zeugodacus cucurbitae, as potential targets for pest control.

The melon fly Zeugodacus cucurbitae Coquillett (Diptera: Tephritidae) is an agricultural quarantine pest threatening fruit and vegetable production. Heat shock cognate 70 (Hsc70), which is a homolog of the heat shock protein 70 (Hsp70), was first discovered in mice testes and plays an important role in spermatogenesis. In this study, we identified and cloned five Hsc70 genes from melon fly, namely ZcHsc70_1/2/3/4/5. Phylogenetic analysis showed that these proteins are closely related to Hsc70s from other Diptera insects. Spatiotemporal expression analysis showed that ZcHsc70_1 and ZcHsc70_2 are highly expressed in Z. cucurbitae testes. Fluorescence in situ hybridization further demonstrated that ZcHsc70_1 and ZcHsc70_2 are expressed in the transformation and maturation regions of testes, respectively. Moreover, RNA interference-based suppression of ZcHsc70_1 or ZcHsc70_2 resulted in a significant decrease of 74.61% and 63.28% in egg hatchability, respectively. Suppression of ZcHsc70_1 expression delayed the transformation of sperm cells to mature sperms. Meanwhile, suppression of ZcHsc70_2 expression decreased both sperm cells and mature sperms by inhibiting the meiosis of spermatocytes. Our findings show that ZcHsc70_1/2 regulates spermatogenesis and further affects the male fertility in the melon fly, showing potential as targets for pest control in sterile insect technique by genetic manipulation of males.

Overexpression of miR-927-5p suppresses stalky expression and negatively reduces the spermatid production in Zeugodacus cucurbitae.

BACKGROUND: The melon fly, Zeugodacus cucurbitae Coquillett, is one of the major pests attacking Cucurbitaceae crops. Identifying critical genes or proteins regulating fertility is essential for sustainable pest control and a research hotspot in insect physiology. MicroRNAs (miRNAs) are short RNAs that do not directly participate in protein translation, but instead function in post-transcriptional regulation of gene expression involved in male fertility. RESULTS: We found that miR-927-5p is highly expressed in the testes and investigated its function in spermatogenesis in Z. cucurbitae. Fluorescence in situ hybridization (FISH) showed miR-927-5p in the transformation and maturation region of the testis, and overexpression of miR-927-5p reduced the number of sperms by 53%. In continuation, we predicted 12 target genes of miR-927-5p using bioinformatics combined with transcriptome sequencing data, and found that miR-927-5p targets the new gene Stalky in insects, which was validated by quantitative real-time PCR, RNA pull-down and dual luciferase reporter assays. FISH also confirmed the co-localization of miR-927-5p and the transcript Stalky_1 in the testis. Moreover, silencing of Stalky_1 by RNA interference reduced the number of sperms by 32% and reduced sperm viability by 39% in physiologically mature male adults. Meanwhile, the silencing of Stalky_1 also resulted in low hatchability. CONCLUSION: Our work not only presents a new, so far unreported mechanism regulating spermatogenesis by miR-927-5p targeting a new unknown target, Stalky, which is providing new knowledge on the regulatory network of insect spermatogenesis, but also lays a foundation for the development of SIT against important tephritid fly pests. © 2024 Society of Chemical Industry.

Suppressing the expression of glutathione S-transferase gene GSTd10 increases the sensitivity of Zeugodacus cucurbitae against ß-cypermethrin.

Zeugodacus cucurbitae Coquillett (Diptera: Tephritidae) is an agriculturally and economically important pest worldwide that has developed resistance to ß-cypermethrin. Glutathione S-transferases (GSTs) have been reported to be involved in the detoxification of insecticides in insects. We have found that both ZcGSTd6 and ZcGSTd10 were up-regulated by ß-cypermethrin induction in our previous study, so we aimed to explore their potential relationship with ß-cypermethrin tolerance in this study. The heterologous expression of ZcGSTd6 and ZcGSTd10 in Escherichia coli showed significantly high activities against 1-chloro-2,4-dinitrobenzene (CDNB). The kinetic parameters of ZcGSTd6 and ZcGSTd10 were determined by Lineweaver-Burk. The Vmax and Km of ZcGSTd6 were 0.50 µmol/min·mg and 0.3 mM, respectively. The Vmax and Km of ZcGSTd10 were 1.82 µmol/min·mg and 0.53 mM. The 3D modelling and molecular docking results revealed that ß-cypermethrin exhibited a stronger bounding to the active site SER-9 of ZcGSTd10. The sensitivity to ß-cypermethrin was significantly increased by 18.73% and 27.21%, respectively, after the knockdown of ZcGSTd6 and ZcGSTd10 by using RNA interference. In addition, the inhibition of CDNB at 50% (IC50) and the inhibition constants (Ki) of ß-cypermethrin against ZcGSTd10 were determined as 0.41 and 0.33 mM, respectively. The Ki and IC50 of ß-cypermethrin against ZcSGTd6 were not analysed. These results suggested that ZcGSTd10 could be an essential regulator involved in the tolerance of Z. cucurbitae to ß-cypermethrin.

Insecticidal Properties of Erythritol on Four Tropical Tephritid Fruit Flies, Zeugodacus cucurbitae, Ceratitis capitata, Bactrocera dorsalis, and B. latifrons (Diptera: Tephritidae).

Tephritid fruit flies are among the most destructive agricultural pests of fruits and vegetables worldwide and can impose trade barriers against the movement of fresh tropical commodities. Primary pre-harvest control methods for these flies rely on the spraying of conventional chemical insecticides or bait sprays. However, resistance to these control methods has been reported in fruit flies. Erythritol is a non-nutritive sugar alternative for human consumption, which has been tested and confirmed for its insecticidal properties against various insect pest species. In this study, using laboratory bioassays, we evaluated the insecticidal effect of erythritol alone or various erythritol formulations containing sucrose and/or protein on four tropical fruit fly species established in Hawaii (e.g., melon fly, Mediterranean fruit fly, oriental fruit fly, and Malaysian fruit fly). In addition, the effects of other non-nutritive hexose and pentose sugar alcohols, such as sorbitol, mannitol, and xylitol, were tested. Among the different standalone and combinatory treatments tested, 1M erythritol and a combinatory formulation of 2M erythritol + 0.5M sucrose appeared to be the most detrimental to the survival of all four species of tested flies, suggesting the potential of using erythritol as a non-toxic management tool for the control of tropical tephritid fruit flies.

Comparative multi-tissue analyses identify testis-specific serine/threonine protein kinase (TSSK) genes involved in male fertility in the melon fly Zeugodacus cucurbitae.

BACKGROUND: Zeugodacus cucurbitae is an agricultural pest species with robust reproductive capabilities capable of causing extensive damage. The advent of novel male fertility-related pest control strategies has been an area of active entomological research focused on the sterile insect technique (SIT) strategy. RESULTS: RNA-sequencing analyses were conducted using 16 tissue samples from adult male Z. cucurbitae, leading to the identification of 5338 genes that were differentially expressed between the testes and three other analyzed tissue types. Of these genes, 808 exhibited high levels of testis expression. A quantitative polymerase chain reaction (qPCR) approach was used to validate the expression of ten of these genes selected at random, including ZcTSSK1 and ZcTSSK3, which are testis-specific serine/threonine protein kinase (TSSK) genes. Evaluation via a loss-of-function-based knockdown assay showed that the down-regulation of either of these two genes in males was associated with significantly decreased egg hatching rates. In situ hybridization analyses revealed the expression of both of these transcripts in the transformation zone, and significant decreases in Z. cucurbitae sperm numbers were observed following double-stranded RNA treatment. Together, these results suggested that inhibiting ZcTSSK1 and ZcTSSK3 expression was sufficient to alter male fertility in Z. cucurbitae. CONCLUSION: These transcriptional sequencing results provide a foundation for further efforts to clarify the regulators of Z. cucurbitae male fertility. These preliminary analyses of the functions of ZcTSSK family genes as regulators of spermatogenesis underscore their importance in the processes integral to male fecundity and their potential as targets for pest control efforts centered on the genetic manipulation of males. © 2023 Society of Chemical Industry.

Genome-wide screening and expression of glutathione S-transferase genes reveal that GSTe4 contributes to sensitivity against ß-cypermethrin in Zeugodacus cucurbitae.

Glutathione S-transferases (GSTs) are an essential multifunctional protein family with common detoxifying enzymes. In this study, 34 GST genes were identified from the melon fly, Zeugodacus cucurbitae, one of the most destructive pests worldwide. These GSTs include 32 cytosolic genes and two microsomal genes. Furthermore, these cytosolic GSTs were classified into six classes: 11 delta, 13 epsilon, three theta, one sigma, two zeta, and two omega. Most of these showed dynamic expression during the developmental stage, some of which showed stage-specific expression. The expression in various adult tissues showed that most of them were expressed in anti-stress-related tissues. The transcriptional response of the delta and epsilon families was determined when Z. cucurbitae was exposed to three insecticides, abamectin, dinotefuran, and ß-cypermethrin. Seven genes were significantly up-regulated by abamectin exposure. Moreover, five and four genes were significantly up-regulated with dinotefuran and ß-cypermethrin exposure, respectively, demonstrating their involvement in the detoxification of these such toxic substances in Z. cucurbitae. One example of these genes, ZcGSTe4, was randomly selected to explore its function in response to ß-cypermethrin exposure. Over-expressed ZcGSTe4 in E. coli showed significant tolerance to ß-cypermethrin, and RNAi-mediated suppression of ZcGSTe4 also increased the sensitivity of melon fly to this agent. This study provides a foundation for further studies on the mechanism of detoxification metabolism in the melon fly.

Preference and performance of peach fruit fly (Bactrocera Zonata) and Melon fruit fly (Bactrocera Cucurbitae) under laboratory conditions.

In Pakistan and all over the world, the Peach Fruit Fly (PFF), Bactrocera zonata (Saunders.) and the Melon Fruit Fly (MFF), Bactrocera cucurbitae (Coquillett.) are considered severe and polyphagous insect pests for various fruits and vegetables. The current study was conducted to check the Laboratory preference and performance of B. cucurbitae and B. zonata on selected Fruits Citrus (Citrus sinensis), Apple (Malus domestica), Banana (Musa acuminate), and vegetable, Sponge gourd (luffa aegyptiaca), Bitter gourd (Momordica charantia) Pumpkin (Cucurbita moschata) under laboratory conditions. The study showed that Sponge Gourd was the preferable host with the mean pupae resurgence of (242.33), followed by Bitter Gourd (78.333) among selected vegetables. At the same time, among fruits, a banana was the preferable host with mean pupae resurgence (204.33), followed by orange (158.33). The pumpkin and apple was the least preferable host for both B. cucurbitae and B. zonata, with mean pupae resurgence (35.667) and (79.000), respectively. Furthermore, the study showed that Banana was the preferable host for B. Zonata among intact and infested fruits, whereas B. cucurbitaee showed the most preference to Bitter gourd among intact and infested vegetables showing significantly different results among intact and infested fruits and vegetables. Maximum number of eggs, pupa, female flies, male flies, adult emergence from pupa (flies) and period of pupa of B. zonata and B. cucurbitae on banana and bitter gourd. While, other fruits and vegetables showed the minimum number of eggs, pupa, female flies, male flies, adult emergence from pupa (flies) and period of the pupa. The current study concluded there is a need to evaluate other host plants against these fruit fly species for effective control.

Expression and Role of Vitellogenin Genes in Ovarian Development of Zeugodacus cucurbitae.

Vitellogenin (Vg) genes encode the major egg yolk protein precursor in arthropods. In this study, four Vgs were identified in Zeugodacus cucurbitae (Coquillett). Sequence analysis showed that four ZcVgs had the conserved Vg domain. Phylogenetic analysis indicated that four ZcVgs were homologous to the Vgs of Tephritidae insects. The temporal and spatial expression patterns of ZcVgs were analyzed by quantitative real-time polymerase chain reaction (RT-qPCR), and the four ZcVgs showed high expression levels in female adults, especially in the fat body. The expression of ZcVg1 and ZcVg3 was down-regulated by a low dosage (0.5 µg) of 20-hydroxyecdysone (20E), and ZcVg2, ZcVg3, and ZcVg4 were up-regulated by a high dosage (1.0 and 2.0 µg) of 20E. The expression of ZcVg1 and ZcVg2 was up-regulated by 5 µg of juvenile hormone (JH), while all of the ZcVgs were down-regulated by a low and high dosage of JH. Expression of ZcVgs was down-regulated after 24 h of starvation and recovered to normal after nutritional supplementation. After micro-injection of the gene-specific double-stranded RNA, the ZcVgs' expression was significantly suppressed, and ovarian development was delayed in Z. cucurbitae females. The results indicate that RNA interference of reproduction-related genes is a potential pest control method that works by manipulating female fertility.

Poor Host Status of Australian Finger Lime, Citrus australasica, to Ceratitis capitata, Zeugodacus cucurbitae, and Bactrocera dorsalis (Diptera: Tephritidae) in Hawai'i.

We examined the host status of the Australian finger lime, Citrus australiasica F. Muell. (Rutaceae), to Hawai'i's tephritid fruit fly pests using laboratory and field studies. In high-density (500 flies, 1:1 males and females) no-choice cage exposures (25 × 25 × 25-cm cage size), both undamaged and punctured finger limes were infested by Ceratitis capitata and Zeugodacuscucurbitae at a low rate compared to papaya controls, whereas Bactrocera dorsalis did not infest undamaged fruit, suggesting finger lime is a nonhost. In low-density (50 females) no-choice cage exposures, C. capitata and Z. cucurbitae readily oviposited in undamaged fruit but individuals rarely developed to the pupal or adult stage. For C. capitata, 274 finger limes exposed to 2000 gravid females, which laid an estimated 14,384 eggs, produced two pupae and no adults. For Z. cucurbitae, 299 fruit exposed to 2000 gravid females, which laid an estimated 4484 eggs, produced four pupae and one adult. Field sampling of undamaged fruit from the tree and off the ground from commercial farms produced five C. capitata pupa and one adult from 1119 fruit, for an infestation rate of 0.05 flies per kilogram of fruit; field collections found no natural Z. cucurbitae or B. dorsalis infestation, but the number of fruit available was too low to demonstrate nonhost status with a high degree of confidence.

Defensive Mechanisms in Cucurbits against Melon Fly (Bactrocera cucurbitae) Infestation through Excessive Production of Defensive Enzymes and Antioxidants.

Melon fly (Bactrocera cucurbitae) is the most common pest of cucurbits, and it directly causes damage to cucurbit fruits in the early developmental stage. The infection of fruit tissues induces oxidative damage through increased generation of cellular reactive oxygen species. The effects of melon fly infestation on the production of defensive enzymes and antioxidant capabilities in five cucurbit species, namely, bottle gourd, chayote, cucumber, snake gourd, and bitter gourd, were investigated in this study. The total phenolic and flavonoid content was considerably higher in melon fly infestation tissues compared to healthy and apparently healthy tissues. The chayote and bottle gourd tissues expressed almost 1.5- to 2-fold higher phenolic and flavonoid contents compared to the tissues of bitter gourd, snake gourd, and cucumber upon infestation. Defensive enzymes, such as peroxidase (POD), superoxide dismutase (SOD), polyphenol oxidase (PPO), and catalase (CAT), were high in healthy and infected tissues of chayote and bottle gourd compared to bitter gourd, snake gourd, and cucumber. The activity of POD (60-80%), SOD (30-35%), PPO (70-75%), and CAT (40-50%) were high in infected chayote and bottle gourd tissue, representing resistance against infestation, while bitter gourd, snake gourd, and cucumber exhibited comparatively lower activity suggesting susceptibility to melon fly infection. The antioxidant properties were also high in the resistant cucurbits compared to the susceptible cucurbits. The current research has enlightened the importance of redox-regulatory pathways involving ROS neutralization through infection-induced antioxidative enzymes in host cucurbit resistance. The melon fly infestation depicts the possible induction of pathways that upregulate the production of defensive enzymes and antioxidants as a defensive strategy against melon fly infestation in resistant cucurbits.

60Co-γ Radiation Alters Developmental Stages of Zeugodacus cucurbitae (Diptera: Tephritidae) Through Apoptosis Pathways Gene Expression.

Radiation is considered as a promising insect pest control strategy for minimizing postharvest yield losses. Among various techniques, irradiation is a method of choice as it induces lethal biochemical or molecular changes that cause a downstream cascade of abrupt physiological abnormalities at the cellular level. In this study, we evaluated the effect of 60Co-γ radiation on various developmental stages of Zeugodacus cucurbitae Coquillett and subsequent carry-over effects on the progeny. For this purpose, we treated eggs with 30- and 50-Gy radiation doses of 60Co-γ. We found that radiation significantly affected cellular antioxidants, insect morphology, and gene expression profiles. Our results indicate that in response to various doses of irradiation reactive oxygen species, catalase, peroxidase, and superoxide dismutase activities were increased along with a significant increase in the malondialdehyde (MDA) content. We observed higher mortality rates during the pupal stage of the insects that hatched from irradiated eggs (50 Gy). Furthermore, the life span of the adults was reduced in response to 50 Gy radiation. The negative effects carried over to the next generation were marked by significantly lower fecundity in the F1 generation of the irradiation groups as compared to control. The radiation induced morphological abnormalities at the pupal, as well as the adult, stages. Furthermore, variations in the gene expression following irradiation are discussed. Taken together, our results signify the utility of 60Co-γ radiation for fruit fly postharvest management.

lnc94638 is a testis-specific long non-coding RNA involved in spermatozoa formation in Zeugodacus cucurbitae (Coquillett).

Long non-coding RNAs (lncRNAs) generally display tissue-specific distributions, and testis-specific lncRNAs form the highest proportion of lncRNAs in many species. Here, we presented a detailed analysis of testis-specific lncRNAs in the melon fly, Zeugodacus cucurbitae, a highly destructive insect pest of cucurbitaceous and other related crops. Most testis-specific lncRNAs were found to be long intergenic non-coding RNAs (lincRNA). The size distribution of these lncRNAs ranged between 600 and 1000 nucleotides. Testis-specific lncRNAs that harboured one isoform number and two exons were the most abundant. Compared to other male tissues, the testis had more highly expressed lncRNAs. The quantitative real-time polymerase chain reaction results of 10 randomly selected testis-specific lncRNAs showed expression patterns consistent with RNA-seq data. Further analysis of the most highly expressed testis-specific lncRNA, lnc94638, was undertaken. Fluorescent in situ hybridization assays localized lnc94638 to the apical region of the testis that contains mature spermatozoa. RNA interference-mediated knockdown of lnc94638 expression reduced spermatozoa numbers and impaired the fertility of Z. cucurbitae male. This study provides a catalogue of testis-specific lncRNAs, shows that the testis-specific lnc94638 is involved in spermatogenesis and has the potential to be used for treating male sterility.

New Insights on the Zeugodacus cucurbitae (Coquillett) Bacteriome.

Various factors, including the insect host, diet, and surrounding ecosystem can shape the structure of the bacterial communities of insects. We have employed next generation, high-throughput sequencing of the 16S rRNA to characterize the bacteriome of wild Zeugodacus (Bactrocera) cucurbitae (Coquillett) flies from three regions of Bangladesh. The tested populations developed distinct bacterial communities with differences in bacterial composition, suggesting that geography has an impact on the fly bacteriome. The dominant bacteria belonged to the families Enterobacteriaceae, Dysgomonadaceae and Orbaceae, with the genera Dysgonomonas, Orbus and Citrobacter showing the highest relative abundance across populations. Network analysis indicated variable interactions between operational taxonomic units (OTUs), with cases of mutual exclusion and copresence. Certain bacterial genera with high relative abundance were also characterized by a high degree of interactions. Interestingly, genera with a low relative abundance like Shimwellia, Gilliamella, and Chishuiella were among those that showed abundant interactions, suggesting that they are also important components of the bacterial community. Such knowledge could help us identify ideal wild populations for domestication in the context of the sterile insect technique or similar biotechnological methods. Further characterization of this bacterial diversity with transcriptomic and metabolic approaches, could also reveal their specific role in Z. cucurbitae physiology.

Real Power Loss Reduction by Melon Fly Optimization and Spontaneous Process Algorithm's

Abstract In this work Melon Fly Optimization (MFO) Algorithm and Spontaneous Process Algorithm (SPA) is designed to reduce the Real power loss, voltage stability enhancement and reducing the Voltage deviation. In this work real power loss measured and how much loss has been reduced is also identified by suitable comparison with standard algorithms. In this society from common consumer to industry needs better quality of power continuously and constantly without much variation. One way to improve the quality of the power is to reduce the power loss. Also reduction of power loss will improve the economic conditions of the nation indirectly and it improves the productivity of the nation with any hurdles. Around the world all nations sequentially identifying the method to reduce the power loss in the transmission and subsequently it improve the quality of power. MFO algorithm has been formed based on the innate events of Melon fly. Due their very excellent eyesight and mutual supportive behaviour Melon fly will find the food without difficulty. By smell and vision the Melon fly will move to the best location form the current location. In the preliminary level Melon flies will search the food in multiple directions and they may be far away from the food source, it like scattering in the plane. Then Spontaneous Process Algorithm (SPA) is designed to solve the optimal reactive power problem Formulation of the projected algorithm is done by imitating the process done during nuclear fission and fusion. Every item of a nucleus attribute symbolizes each solution variable. Sequence of operators directs the nucleus and in order to avoid the local optimum it will imitate the dissimilar condition of reaction. In the exploration space nucleus symbolizes the variables and potential solution. Levy flight has been intermingled in the procedure to enhance the diversification and intensification in the search. Evaluation of validity of the Melon Fly Optimization (MFO) Algorithm and Spontaneous Process Algorithm (SPA) is done in IEEE 30-bus system by considering voltage stability (L-index) and also devoid of L-index criterion. Minimization of voltage deviation, voltage stability enhancement and power loss minimization has been achieved.

A Novel Solid Artificial Diet for Zeugodacus cucurbitae (Diptera: Tephritidae) Larvae With Fitness Parameters Assessed by Two-Sex Life Table.

The melon fly, Zeugodacus cucurbitae (Coquillett), is a serious pest of many fruits and vegetables throughout the world. Here we have developed an easy and quick-to-prepare solid medium with multiple benefits including reductions in post-rearing waste, storage space, and labor for rearing Z. cucurbitae larvae. The development time from egg to pupa was 19.11 d when larvae were reared on the artificial diet, slightly longer than 17.73 d on pumpkin and 17.13 d on cucumber. Zeugodacus cucurbitae achieved higher values of pupal weight, length, and width on the artificial diet than two natural diet controls. The rates of pupation and adult emergence of Z. cucurbitae grown on the solid medium were comparable with those on pumpkin and cucumber. Furthermore, determined by age-specific two-sex life table method, the age-specific survival rate of Z. cucurbitae was higher on the artificial diet than cucumber but lower than pumpkin. The reproductive ability and population dynamics of Z. cucurbitae were not significantly affected on the solid medium compared with those on the two natural diets. The results suggest that our solid artificial diet is excellent for rearing Z. cucurbitae larvae in laboratory and may be used for its mass rearing, therefore facilitating its research and control.

The effect of diet and radiation on the bacterial symbiome of the melon fly, Zeugodacus cucurbitae (Coquillett).

BACKGROUND: Symbiotic bacteria contribute to a multitude of important biological functions such as nutrition and reproduction and affect multiple physiological factors like fitness and longevity in their insect hosts. The melon fly, Zeugodacus cucurbitae (Coquillett), is an important agricultural pest that affects a variety of cultivated plants belonging mostly to the Cucurbitaceae family. It is considered invasive and widespread in many parts of the world. Several approaches are currently being considered for the management of its populations including the environmentally friendly and effective sterile insect technique (SIT), as a component of an integrated pest management (IPM) strategy. In the present study, we examined the effect of diet and radiation on the bacterial symbiome of Z. cucurbitae flies with the use of Next Generation Sequencing technologies. RESULTS: Melon flies were reared on two diets at the larval stage, an artificial bran-based diet and on sweet gourd, which affected significantly the development of the bacterial profiles. Significant differentiation was also observed based on gender. The effect of radiation was mostly diet dependent, with irradiated melon flies reared on the bran diet exhibiting a significant reduction in species diversity and richness compared to their non-irradiated controls. Changes in the bacterial symbiome of the irradiated melon flies included a drastic reduction in the number of sequences affiliated with members of Citrobacter, Raoultella, and Enterobacteriaceae. At the same time, an increase was observed for members of Enterobacter, Providencia and Morganella. Interestingly, the irradiated male melon flies reared on sweet gourd showed a clear differentiation compared to their non-irradiated controls, namely a significant reduction in species richness and minor differences in the relative abundance for members of Enterobacter and Providencia. CONCLUSIONS: The two diets in conjunction with the irradiation affected significantly the formation of the bacterial symbiome. Melon flies reared on the bran-based artificial diet displayed significant changes in the bacterial symbiome upon irradiation, in all aspects, including species richness, diversity and composition. When reared on sweet gourd, significant changes occurred to male samples due to radiation, only in terms of species richness.

Performance of Three Isolates of Metarhizium Anisopliae and Their Virulence against Zeugodacus Cucurbitae under Different Temperature Regimes, with Global Extrapolation of Their Efficiency.

The performance of entomopathogenic fungi in pest control is usually affected by both biotic and abiotic factors. This study aimed to determine the effects of various temperatures (15, 20, 25 and 30 °C) on conidial germination, mycelial growth and conidial density and virulence to the melon fly Zeugodacus cucurbitae of three selected isolates of Metarhizium anisopliae. The three isolates, ICIPE 18, ICIPE 30 and ICIPE 69, had previously been selected in laboratory bioassays. Percentage mortality by the three isolates ranged between 16.25% and 100.0% across the different temperatures. The isolates ICIPE 69 and ICIPE 18 recorded the highest percentage mortality of 96.25% and 100% and the shortest LT50 values of 2.61 and 2.63 days, respectively, at 30 °C. However, at 30 °C, ICIPE 69 produced the highest number of conidia of 90.5 × 107 /mL and was therefore selected for global mapping to predict its efficacy against Z. cucurbitae using the geospatial temperature data layer and the best fitted quadratic model. The map showed that the isolate would be more effective in the tropics than in temperate climates.

Cucumber and Tomato Volatiles: Influence on Attraction in the Melon Fly Zeugodacus cucurbitate (Diptera: Tephritidae).

The main hosts of the melon fly Zeugodacus cucurbitate are cultivated and wild cucurbitaceous plants. In eastern Africa, the melon fly is a major pest of the Solanaceae plant Solanum lycopersicum (tomato). We hypothesized that shared species-specific volatiles may play a role in host attraction. We tested this hypothesis by comparing the olfactory responses of the melon fly to Cucumis sativus (cucumber) (Cucurbitaceae) and tomato plant odors in behavioral and electrophysiological assays, followed by chemical analysis to identify the key compounds mediating the interactions. Our results identified 13 shared components between cucumber and tomato plant odors. A synthetic blend of seven of the shared components dominated by monoterpenes at concentrations mimicking the volatile bouquet of cucumber and tomato attracted both sexes of the melon fly. Our results suggest that the presence and quantity of specific compounds in host odors are the main predictors for host recognition in Z. cucurbitate.

Cucumber Lure Trapping of Zeugodacus cucurbitae (Diptera: Tephritidae) in Hawaii and Taiwan: Longevity and Nontargets Captures.

The melon fly, Zeugodacus cucurbitae (Coquillett), is a serious pest of tropical horticulture, causing damage to cucurbits, tree fruits, and fruiting vegetables. Melon flies are especially attractive to freshly sliced cucumber, and this has led to the identification of a nine-compound kairomone lure that can be used to trap both female and male flies. In this study, a seven-compound lure, containing (Z)-6-nonenal, (Z)-6-nonen-1-ol, 1-octen-3-ol, (E,Z)-2,6-nonadienal, (E)-2-nonenal, hexanal, and 1-hexanol, was formulated into PVC plugs (100 or 300 mg/plug) for field testing in wet traps. In Hawaii, 100 mg of the seven-compound cucumber lure, loaded in either plugs or glass capillaries, attracted more flies than traps containing Solulys protein over a 9-wk period. However, both cucumber lure formulations showed marked declines in the number of flies trapped after 3 wk. Similar results were obtained during a 6-wk field trial using 100 mg cucumber lure plugs in Taiwan. Increasing the cucumber lure loading rate to 300 mg/lure increased the effective trapping life of the attractant during a second 9-wk field trial conducted in Hawaii. The synthetic cucumber lure showed female-biased sex ratios in trap captures in the Taiwanese and second Hawaiian field trials. Protein lures captures were female-biased in all three field trials. Wet traps in Hawaii containing the cucumber lure were found to capture 25-30 nontarget insects/trap/week, less than half that captured with Solulys. Captured nontarget insects represented 37 families in 10 orders. The most common families caught were Ceratopogonidae (∼9 flies/trap) and Gryllidae (∼7 crickets/trap).