Reproductive behavior of fruit flies: courtship, mating, and oviposition.

Many species of the Tephritidae family are invasive and cause huge damage to agriculture and horticulture, owing to their reproductive characteristics. In this review, we have summarized the existing studies on the reproductive behavior of Tephritidae, particularly those regarding the genes and external factors that are associated with courtship, mating, and oviposition. Furthermore, we outline the issues that still need to be addressed in fruit fly reproduction research. The review highlights the implications for understanding the reproductive behavior of fruit flies and discusses methods for their integrated management and biological control. © 2023 Society of Chemical Industry.

Differences in rectal amino acid levels determine bacteria-originated sex pheromone specificity in two closely related flies.

Sex pheromones are widely used by insects as a reproductive isolating mechanism to attract conspecifics and repel heterospecifics. Although researchers have obtained extensive knowledge about sex pheromones, little is known about the differentiation mechanism of sex pheromones in closely related species. Using Bactrocera dorsalis and Bactrocera cucurbitae as the study model, we investigated how the male-borne sex pheromones are different. The results demonstrated that both 2,3,5-trimethylpyrazine (TMP) and 2,3,5,6-tetramethylpyrazine (TTMP) were sex pheromones produced by rectal Bacillus in the two flies. However, the TMP/TTMP ratios were reversed, indicating sex pheromone specificity in the two flies. Bacterial fermentation results showed that different threonine and glycine levels were responsible for the preference of rectal Bacillus to produce TMP or TTMP. Accordingly, threonine (glycine) levels and the expression of the threonine and glycine coding genes were significantly different between B. dorsalis and B. cucurbitae. In vivo assays confirmed that increased rectal glycine and threonine levels by amino acid feeding could significantly decrease the TMP/TTMP ratios and result in significantly decreased mating abilities in the studied flies. Meanwhile, decreased rectal glycine and threonine levels due to RNAi of the glycine and threonine coding genes was found to significantly increase the TMP/TTMP ratios and result in significantly decreased mating abilities. The study contributes to the new insight that insects and their symbionts can jointly regulate sex pheromone specificity in insects, and in turn, this helps us to better understand how the evolution of chemical communication affects speciation.

Lethal and sublethal effects of broflanilide on four tephritid pests (Diptera: Tephritidae).

BACKGROUND: Fruit flies are internationally important quarantine or invasive pests of many fruits and vegetables and can cause serious economic losses. Long-term reliance on insecticides for controlling these pests has led to increasing resistance to multiple insecticides; hence, a new agent is needed. In this study, the acute toxicity and sublethal effects of the novel insecticide broflanilide on four adult fruit fly species, Bactrocera dorsalis, Bactrocera cucurbitae, Bactrocera tau, and Bactrocera correcta, were evaluated. RESULTS: Broflanilide was effective against B. dorsalis and B. correcta, with lethal concentration values (amount required to kill 50% of animals; LC50 ) of 0.390 and 1.716 mg/L. However, for B. cucurbitae (19.673 mg/L) and B. tau (24.373 mg/L), the LC50 was 50-60 times higher than that of B. dorsalis. The survival rates of B. correcta and B. cucurbitae were significantly lower under LC50 treatment than those of the control (corrected for mortality rate). Sublethal concentrations of broflanilide stimulated fecundity in all species except B. tau. The hatching rate at LC50 was significantly lower for B. correcta and B. tau compared with the control and even more so for B. correcta, which was zero. CONCLUSION: Broflanilide is potentially an effective insecticide for controlling B. dorsalis and B. correcta. However, the variation in toxicity of broflanilide to the four fruit flies suggests that species variation needs to be carefully considered. Our results highlight the importance of clarifying the sublethal effects of insecticides on target insects to ensure the comprehensive evaluation and rational use of insecticides. © 2023 Society of Chemical Industry.

Protein feeding mediates sex pheromone biosynthesis in an insect.

Protein feeding is critical for male reproductive success in many insect species. However, how protein affects the reproduction remains largely unknown. Using Bactrocera dorsalis as the study model, we investigated how protein feeding regulated sex pheromone synthesis. We show that protein ingestion is essential for sex pheromone synthesis in male. While protein feeding or deprivation did not affect Bacillus abundance, transcriptome analysis revealed that sarcosine dehydrogenase (Sardh) in protein-fed males regulates the biosynthesis of sex pheromones by increasing glycine and threonine (sex pheromone precursors) contents. RNAi-mediated loss-of-function of Sardh decreases glycine, threonine, and sex pheromone contents and results in decreased mating ability in males. The study links male feeding behavior with discrete patterns of gene expression that plays role in sex pheromone synthesis, which in turn translates to successful copulatory behavior of the males.

Gut bacteria induce oviposition preference through ovipositor recognition in fruit fly.

Gut bacteria play important roles in insect life cycle, and various routes can be used by insects to effectively transmit their gut bacteria. However, it is unclear if the gut bacteria can spread by actively attracting their insect hosts, and the recognition mechanisms of host insects are poorly understood. Here, we explore chemical interactions between Bactrocera dorsalis and its gut bacterium Citrobacter sp. (CF-BD). We found that CF-BD could affect the development of host ovaries and could be vertically transmitted via host oviposition. CF-BD could attract B. dorsalis to lay eggs by producing 3-hexenyl acetate (3-HA) in fruits that were hosts of B. dorsalis. Furthermore, we found that B. dorsalis could directly recognize CF-BD in fruits with their ovipositors in which olfactory genes were expressed to bind 3-HA. This work reports an important mechanism concerning the active spread of gut bacteria in their host insects.

Effects of four chemosterilants on Bactrocera tau.

Bactrocera tau (Walker) is a fly pest species mainly distributed in Southeast Asia and the South Pacific; it causes substantial ecological and economic issues because of its destructiveness and rapid reproduction. Chemical sterilization technology can reduce the use of insecticides and is widely applied for insect pest control. In this study, the sterilization efficacy of varying concentrations of four chemosterilants, namely, hexamethylphosphoramide (HMPA), CSII Aqua, 5-fluorouracil (5-FU), and colchicine, on adult pumpkin flies was investigated. The results indicated that a solution of 0.03% HMPA had the highest sterilization efficacy. When the number of sterile males was equal to or exceeded 20 times that of untreated males, the hatching rate of offspring eggs was less than 10%. Chemosterilant treatment significantly altered the levels of acid phosphatase (ACP), alkaline phosphatase (AKP), and B. tau vitellogenin (BtVg); these substances have an important impact on reproductive development. The treatment also decreased the size of the reproductive organs (i.e., testes and ovaries). Our results suggest that 0.03% HMPA has unique sterilization properties and may represent a new chemical agent for the control of B. tau populations in agricultural settings.

Rectal bacteria produce sex pheromones in the male oriental fruit fly.

Volatile molecules produced by the microbiota play a primary role in chemical communication between insects,1 and direct production of pheromone components by the microbiota is one of the most obvious mechanisms.2 Here, we investigated the production of male-borne sex pheromones of the oriental fruit fly, Bactrocera dorsalis. As observed in previous studies,3,4 2,3,5-trimethylpyrazine (TMP) and 2,3,5,6-tetramethylpyrazine (TTMP) are sex pheromones produced in the male rectum. Mature virgin females are strongly attracted to TMP and TTMP. 16S rRNA sequencing results show that the rectal bacteria are dominated by Bacilli that harbor the pathway to produce TMP and TTMP.5-8 The levels of Bacilli, TMP, and TTMP in the male rectum can be significantly decreased by feeding male flies with antibiotics. In vitro assays show that Bacillus species isolated from the male rectum can produce TMP and TTMP when provided with the substrates glucose and threonine, the levels of which are significantly higher in the rectum of mature males. These findings highlight the influence of microbial symbionts on insect pheromones and provide an example of direct bacterial production of pheromones in insects.

The nesting preference of an invasive ant is associated with the cues produced by actinobacteria in soil.

Soil-dwelling animals are at risk of pathogen infection in soils. When choosing nesting sites, animals could reduce this risk by avoiding contact with pathogens, yet there is currently little evidence. We tested this hypothesis using Solenopsis invicta as a model system. Newly mated queens of S. invicta were found to nest preferentially in soil containing more actinobacteria of Streptomyces and Nocardiopsis and to be attracted to two volatiles produced by these bacteria, geosmin and 2-methylisoborneol. Actinobacteria-rich soil was favored by S. invicta and this soil contained fewer putative entomopathogenic fungi than adjacent areas. Queens in such soil benefited from a higher survival rate. In culture, isolated actinobacteria inhibited entomopathogenic fungi, suggested that their presence may reduce the risk of fungal infection. These results indicated a soil-dwelling ant may choose nest sites presenting relatively low pathogen risk by detecting the odors produced by bacteria with anti-fungal properties.

Egg-Surface Bacteria Are Indirectly Associated with Oviposition Aversion in Bactrocera dorsalis.

Finding a suitable oviposition site is a challenging task for a gravid female fly, because the hatched maggots have limited mobility, making it difficult to find an alternative host. The oriental fruit fly, Bactrocera dorsalis, oviposits on many types of fruits. Maggots hatching in a fruit that is already occupied by conspecific worms will face food competition. Here, we showed that maggot-occupied fruits deter B. dorsalis oviposition and that this deterrence is based on the increased ß-caryophyllene concentration in fruits. Using a combination of bacterial identification, volatile content quantification, and behavioral analyses, we demonstrated that the egg-surface bacteria of B. dorsalis, including Providencia sp. and Klebsiella sp., are responsible for this increase in the ß-caryophyllene contents of host fruits. Our research shows a type of tritrophic interaction between micro-organisms, insects, and insect hosts, which will provide considerable insight into the evolution of insect behavioral responses to volatile compounds.

Symbiotic microbiota may reflect host adaptation by resident to invasive ant species.

Exotic invasive species can influence the behavior and ecology of native and resident species, but these changes are often overlooked. Here we hypothesize that the ghost ant, Tapinoma melanocephalum, living in areas that have been invaded by the red imported fire ant, Solenopsis invicta, displays behavioral differences to interspecific competition that are reflected in both its trophic position and symbiotic microbiota. We demonstrate that T. melanocephalum workers from S. invicta invaded areas are less aggressive towards workers of S. invicta than those inhabiting non-invaded areas. Nitrogen isotope analyses reveal that colonies of T. melanocephalum have protein-rich diets in S. invicta invaded areas compared with the carbohydrate-rich diets of colonies living in non-invaded areas. Analysis of microbiota isolated from gut tissue shows that T. melanocephalum workers from S. invicta invaded areas also have different bacterial communities, including a higher abundance of Wolbachia that may play a role in vitamin B provisioning. In contrast, the microbiota of workers of T. melanocephalum from S. invicta-free areas are dominated by bacteria from the orders Bacillales, Lactobacillales and Enterobacteriales that may be involved in sugar metabolism. We further demonstrate experimentally that the composition and structure of the bacterial symbiont communities as well as the prevalence of vitamin B in T. melanocephalum workers from S. invicta invaded and non-invaded areas can be altered if T. melanocephalum workers are supplied with either protein-rich or carbohydrate-rich food. Our results support the hypothesis that bacterial symbiont communities can help hosts by buffering behavioral changes caused by interspecies competition as a consequence of biological invasions.

Microbial Communities in Different Developmental Stages of the Oriental Fruit Fly, Bactrocera dorsalis, Are Associated with Differentially Expressed Peptidoglycan Recognition Protein-Encoding Genes.

The insect microbiota can change dramatically to enable adaptation of the host in different developmental stages and environments; however, little is known about how the host maintains its microbiota to achieve such adaptations. In this study, 16S rRNA sequencing revealed that the microorganisms in larvae and adults of the Oriental fruit fly, Bactrocera dorsalis, are primarily Gram-negative bacteria but that the major components in pupae are Gram-positive bacteria. Using suppression subtractive hybridization (SSH) and transcriptome analysis, we screened two specifically expressed genes encoding peptidoglycan recognition proteins (PGRP-LB and PGRP-SB1) and analyzed their relationship to B. dorsalis microbial communities. Knockdown of the PGRP-LB gene in larvae and adults led to increased ratios of Gram-positive bacteria; knockdown of the PGRP-SB1 gene in pupae led to increased ratios of Gram-negative bacteria. Our results suggest that maintenance of the microbiota in different developmental stages of B. dorsalis may be associated with the PGRP-LB and PGRP-SB1 genes.IMPORTANCE Microorganisms are ubiquitous in insects and have widespread impacts on multiple aspects of insect biology. However, the microorganisms present in insects can change dramatically in different developmental stages, and it is critical to maintain the appropriate microorganisms in specific host developmental stages. Therefore, analysis of the factors associated with the microbiota in specific development stages of the host is needed. In this study, we applied suppression subtractive hybridization (SSH) combined with transcriptome analysis to investigate whether the microbiota in development stages of the Oriental fruit fly, Bactrocera dorsalis, is associated with expression of PGRP genes. We found that two different PGRP genes were specifically expressed during development and that these genes may be associated with changes in microbial communities in different developmental stages of B. dorsalis.

The Divergence in Bacterial Components Associated with Bactrocera dorsalis across Developmental Stages.

Eco-evolutionary dynamics of microbiotas at the macroscale level are largely driven by ecological variables. The diet and living environment of the oriental fruit fly, Bactrocera dorsalis, diversify during development, providing a natural system to explore convergence, divergence, and repeatability in patterns of microbiota dynamics as a function of the host diet, phylogeny, and environment. Here, we characterized the microbiotas of 47 B. dorsalis individuals from three distinct populations by 16S rRNA amplicon sequencing. A significant deviation was found within the larvae, pupae, and adults of each population. Pupae were characterized by an increased bacterial taxonomic and functional diversity. Principal components analysis showed that the microbiotas of larvae, pupae, and adults clearly separated into three clusters. Acetobacteraceae, Lactobacillaceae, and Enterobacteriaceae were the predominant families in larval and adult samples, and PICRUSt analysis indicated that phosphoglycerate mutases and transketolases were significantly enriched in larvae, while phosphoglycerate mutases, transketolases, and proteases were significantly enriched in adults, which may support the digestive function of the microbiotas in larvae and adults. The abundances of Intrasporangiaceae, Dermabacteraceae (mainly Brachybacterium) and Brevibacteriaceae (mainly Brevibacterium) were significantly higher in pupae, and the antibiotic transport system ATP-binding protein and antibiotic transport system permease protein pathways were significantly enriched there as well, indicating the defensive function of microbiotas in pupae. Overall, differences in the microbiotas of the larvae, pupae, and adults are likely to contribute to differences in nutrient assimilation and living environments.

The plant pathogen Gluconobacter cerinus strain CDF1 is beneficial to the fruit fly Bactrocera dorsalis.

Plant pathogens can build relationships with insect hosts to complete their life cycles, and they often modify the behavior and development of hosts to improve their own fitness. In order to unravel whether some bacteria that can make fruit rot could have developed symbiotic interactions with Bactrocera dorsalis, we studied the symbiont bacteria profiles of the fly. We identified the bacterium Gluconobacter cerinus strain CDF1 from the ovaries and eggs of the oriental fruit fly B. dorsalis and the amount of Gluconobacter cerinus strain CDF1 increased significantly as the ovaries developed and in fruits on which non-sterile eggs were laid. Gluconobacter cerinus strain CDF1 addition to bananas fastens the rotting process and its addition to the eggs fastens their development/hatching rate. All in all, our data suggest that Gluconobacter cerinus strain CDF1 is beneficial to the fruit fly.

A Chemosensory Protein Gene Si-CSP1 Associated With Necrophoric Behavior in Red Imported Fire Ants (Hymenoptera: Formicidae).

Necrophoric behavior is essential to colony health in social insects. Little is known about the genes that are responsible for necrophoric behavior. Here, we show that a chemosensory protein gene Si-CSP1 was expressed significantly higher in the antennae than in other tissues such as the legs and heads of Solenopsis invicta Buren workers. Furthermore, Si-CSP1-silenced workers moved significantly fewer corpses of their nestmates than normal workers. Finally, Si-CSP1-silenced workers exhibited weaker antennal responses to oleic acid and linoleic acid than controls. These results suggest that Si-CSP1 functions by sensing oleic acid and linoleic acid associated with dead colony members and regulating the necrophoric behavior of workers in S. invicta.

Gut symbiont enhances insecticide resistance in a significant pest, the oriental fruit fly Bactrocera dorsalis (Hendel).

BACKGROUND: Symbiotic bacteria affect insect physiology and ecology. They may also mediate insecticide resistance within their hosts and thereby impact pest and vector control practices. Here, we document a novel mechanism of insecticide resistance in which a gut symbiont of the tephritid pest fruit fly Bactrocera dorsalis enhances resistance to the organophosphate insecticide trichlorphon. RESULTS: We demonstrated that the gut symbiont Citrobacter sp. (CF-BD) plays a key role in the degradation of trichlorphon. Based on a comparative genomics analysis with other Citrobacter species, phosphatase hydrolase genes were identified in CF-BD. These CF-BD genes had higher expression when trichlorphon was present. Bactrocera dorsalis inoculated with isolated CF-BD obtained higher trichlorphon resistance, while antibiotic-treated flies were less resistant confirming the key role of CF-BD in insecticide resistance. CONCLUSIONS: Our findings suggest that symbiont-mediated insecticide resistance can readily develop in B. dorsalis and may represent a more widely relevant insecticide resistance mechanism than previously recognized.

Si-CSP9 regulates the integument and moulting process of larvae in the red imported fire ant, Solenopsis invicta.

Chemosensory proteins (CSPs) have been predicted to be involved in development; however, direct evidence for their involvement is lacking, and genetic basis is largely unknown. To determine the function of the chemosensory protein 9 (Si-CSP9) gene in Solenopsis invicta, we used RNA interference to silence Si-CSP9 in 3rd-instar larvae. The 3rd-instar larvae failed to shed their cuticle after being fed Si-CSP9-directed siRNA, and expression profiling of RNAi-treated and untreated control larvae showed that 375 genes were differentially expressed. Pathway enrichment analysis revealed that 4 pathways associated with larval development were significantly enriched. Blast analysis revealed that one fatty acid amide hydrolase (FAAH) gene was up-regulated and 4 fatty acid synthase (FAT) genes and one protein kinase DC2 gene (PKA) were down-regulated in the enriched pathways. Significantly higher expression of these genes was found in 4th-instar larvae, and Pearson correlation analysis of the expression patterns revealed significant relationships among Si-CSP9, PKA, FAAH, and FAT1-4. Moreover, we confirmed that expression levels of Si-CSP9, FAAH, and FAT1-4 were significantly reduced and that the development of 3rd-instar larvae was halted with PKA silencing. These results suggest that Si-CSP9 and PKA may be involved in the network that contributes to development of 3rd-instar larvae.

Association between changes in reproductive activity and D-glucose metabolism in the tephritid fruit fly, Bactrocera dorsalis (Hendel).

Reproduction is an important life process in insects; however, few studies have attempted to demonstrate the association between reproductive activity and energy metabolism. To address this problem, we focused on the reproductive changes in Bactrocera dorsalis males. We analyzed B. dorsalis male gene expression profiles during mating (DM), 3 h after mating (A3HM) and 12 h after mating (A12HM). Gene annotation and pathway analyses of differentially expressed genes show that galactose metabolism and the starch and sucrose metabolism pathway activities were significantly higher in A12HM group. Moreover, the maltase D gene was the most strongly up-regulated gene. The D-glucose levels were significantly higher in A12HM group. Maltase D expression level was significantly higher in males reared with sucrose. Body weights of the males reared with D-glucose and sucrose were significantly higher than those of the males reared with yeast extract. We observed more mated males from the groups fed sucrose and D-glucose than from those fed yeast extract. The D-glucose levels in individual males were highest at 18:00 h, when flies exhibit the most active mating behavior. This study shows that the maltase D gene and D-glucose are the critical gene and substrate, respectively, in male B. dorsalis mating process.

Validation of reference genes in Solenopsis invicta in different developmental stages, castes and tissues.

To accurately assess gene expression levels, it is essential to normalize real-time quantitative PCR (RT-qPCR) data with suitable internal reference genes. For the red imported fire ant, Solenopsis invicta, reliable reference genes to assess the transcript expression levels of the target genes have not been previously investigated. In this study, we examined the expression levels of five candidate reference genes (rpl18, ef1-beta, act, GAPDH, and tbp) in different developmental stages, castes and tissues of S. invicta. To evaluate the suitability of these genes as endogenous controls, three software-based approaches (geNorm, BestKeeper and NormFinder) and one web-based comprehensive tool (RefFinder) were used to analyze and rank the tested genes. Furthermore, the optimal number of reference gene(s) was determined by the pairwise variation value. Our data showed that two of the five candidate genes, rpl18 and ef1-beta, were the most suitable reference genes because they have the most stable expression among different developmental stages, castes and tissues in S. invicta. Although widely used as reference gene in other species, in S. invicta the act gene has high variation in expression and was consequently excluded as a reliable reference gene. The two validated reference genes, rpl18 and ef1-beta, can be widely used for quantification of target gene expression with RT-qPCR technology in S. invicta.