Impact of Larval Food Source on the Stability of the Bactrocera dorsalis Microbiome.

Bacterial symbionts are crucial to the biology of Bactrocera dorsalis. With larval diet (fruit host) being a key factor that determines microbiome composition and with B. dorsalis using more than 400 fruits as hosts, it is unclear if certain bacterial symbionts are preserved and are passed on to B. dorsalis progenies despite changes in larval diet. Here, we conducted a fly rearing experiment to characterize diet-induced changes in the microbiome of female B. dorsalis. In order to explicitly investigate the impacts of larval diet on the microbiome, including potential stable bacterial constituents of B. dorsalis, we performed 16S rRNA sequencing on the gut tissues of teneral female flies reared from four different host fruits (guava, mango, papaya, and rose apple) infested using a single cohort of wild B. dorsalis that emerged from tropical almond (mother flies). Although B. dorsalis-associated microbiota were predominantly shaped by the larval diet, some major bacterial species from the mother flies were retained in progenies raised on different larval diets. With some variation, Klebsiella (ASV 1 and 2), Morganella (ASV 3), and Providencia (ASV 6) were the major bacterial symbionts that were stable and made up 0.1-80% of the gut and ovipositor microbiome of female teneral flies reared on different host fruits. Our results suggest that certain groups of bacteria are stably associated with female B. dorsalis across larval diets. These findings provide a basis for unexplored research on symbiotic bacterial function in B. dorsalis and may aid in the development of novel management techniques against this devastating pest of horticultural importance.

Estragole, a potential attractant of the winged melon aphid Aphis gossypii.

BACKGROUND: Aphids are sap-sucking insect pests of economic importance. They exhibit polyphenism, producing two kinds of morphotypes; winged (alate) and wingless (aptera) morphs. While wingless morphs can be controlled by insecticides, winged morphs are a challenge for targeted control measures as they can fly. Although colored sticky traps are used to control and monitor winged aphids, only a small population is trapped, making sticky traps less effective in controlling aphids. Studies have shown that fragrant oils applied to sticky traps increased attraction of sap-sucking insects like whiteflies and thrips. Here, we tested selected essential oils for their attractiveness to winged Aphis gossypii in potato fields. RESULTS: In field assays, selected essential oils with yellow or colorless sticky traps attracted more winged A. gossypii than controls. The combination of yellow traps baited with essential oils attracted ~2-3-fold more winged A. gossypii than did colorless traps baited with essential oils. In a multi-cycle 2 year study, yellow sticky traps baited with basil oil consistently attracted more winged A. gossypii than yellow sticky traps baited with lavender, geranium or tea tree oils. In electrophysiological studies, winged A. gossypii's antennae responded consistently to estragole in basil oil. In olfactometer assays with estragole, winged A. gossypii spent significantly more time in the treatment arm of the olfactometer than in the control arm, validating estragole's attractiveness. Furthermore, yellow sticky traps baited with pure estragole, in potato fields, attracted similar number of winged A. gossypii as yellow sticky traps baited with basil oil. CONCLUSION: Our findings demonstrate the potential of using basil oil as a potential attractant to improve the efficacy of sticky traps in the monitoring and control of winged aphids. © 2023 Society of Chemical Industry.

The greater wax moth, Galleria mellonella (L.) uses two different sensory modalities to evaluate the suitability of potential oviposition sites.

An ovipositing insect evaluates the benefits and risks associated with the selection of an oviposition site for optimizing the fitness and survival of its offspring. The greater wax moth, Galleria mellonella (L.), uses beehives as an oviposition site. During egg-laying, the gravid wax moth confronts two kinds of risks, namely, bees and conspecific larvae. While bees are known to attack the moth's offspring and remove them from the hive, the conspecific larvae compete for resources with the new offspring. To date, little is known about the mechanisms involved in the assessment of oviposition site by the greater wax moth, G. mellonella (L.). Here, we demonstrate that the wax moth uses two different sensory modalities to detect risks to its offspring in the hives of Apis cerena. Bees appear to be detected by the contact-chemoreception system of the gravid wax moth, while detection of conspecifics relies on the olfactory system. Hence, our findings suggest that two different sensory modalities are used to detect two different risks to the offspring and that the selection of oviposition sites by G. mellonella (L.) relies on the integration of inputs from both the olfactory and contact-chemoreception systems.

1-Octanol emitted by Oecophylla smaragdina weaver ants repels and deters oviposition in Queensland fruit fly.

Humans have used weaver ants, Oecophylla smaragdina, as biological control agents to control insect pests in orchards for many centuries. Over recent decades, the effectiveness of weaver ants as biological control agents has been attributed in part to deterrent and oviposition inhibiting effects of kairomones produced by the ants, but the chemical identity of these kairomones has remained unknown. We have identified the kairomone responsible for deterrence and oviposition inhibition by O. smaragdina, providing a significant advance in understanding the chemical basis of their predator/prey interactions. Olfactometer assays with extracts from weaver ants demonstrated headspace volatiles to be highly repellent to Queensland fruit fly, Bactrocera tryoni. Using electrophysiology and bioassays, we demonstrate that this repellence is induced by a single compound, 1-octanol. Of 16 compounds identified in O. smaragdina headspace, only 1-octanol evoked an electrophysiological response from B. tryoni antennae. Flies had greatly reduced oviposition and spent significantly less time in an olfactometer arm in the presence of 1-octanol or a synthetic blend of headspace volatiles containing 1-octanol than in the presence of a synthetic blend of headspace volatiles without 1-octanol, or clean air. Taken together, our results demonstrate that 1-octanol is the functional kairomone component of O. smaragdina headspace that explains repellence and oviposition deterrence, and is hence an important contributor to the effectiveness of these ants as biological control agents.

Endosymbiotic Bacteria Aid to Overcome Temperature Induced Stress in the Oriental Fruit Fly, Bactrocera dorsalis.

Endosymbiotic microbiota are known to have an enormous impact on their host, influencing its physiology, behavior, fitness, and various other aspects. The present study hypothesizes that certain bacterial symbionts aid the Oriental fruit fly, Bactrocera dorsalis in its adaptation to survive thermal stress encountered in the environment. Investigative studies on the change in gut and reproductive tract microbiota diversity of male and female B. dorsalis revealed that certain genera of Acinetobacter, Brevibacillus, Bacillus, Enterobacter, Enterococcus, Pseudomonas, and Staphylococcus were involved in the adaptation of B. dorsalis to temperature stresses. The intestinal and reproductive tract bacterial community of B. dorsalis varied depending on the temperature the insects were reared at. We hypothesized that the microbiota present in B. dorsalis' gut helped it endure temperature stresses over prolonged periods. Out of 54 bacterial isolates, 25, 15, and 14 isolates were obtained from flies reared at 27 °C, 18 °C, and 35 °C, respectively. A 16S rDNA analysis revealed that the bacterial isolates (reared at different temperatures) belonged to different genera. The flies were supplemented with antibiotics to suppress the existing gut microbiota and subsequently fed with bacterial isolates from flies reared at 18 °C, 27 °C (control) or 35 °C separately. When these flies were placed in incubators pre-set at the above temperatures, the survival rate exhibited by the flies differed significantly. The flies fed with bacterial isolates from 18 °C could survive only in incubators pre-set at 18 °C, while flies fed with bacterial isolates from 35 °C could survive only at 35 °C and not vice versa. The microbiota supplementation assay established that the presence of specific bacterial isolates aided the flies' survival under varied thermal stresses.

Overlooked Scents: Chemical Profile of Soma, Volatile Emissions and Trails of the Green Tree Ant, Oecophylla smaragdina.

The green tree ant, Oecophylla smaragdina, is one of only two recognized species of weaver ants. While the identity and functions of chemicals produced and emitted by its congener O. longinoda have been studied quite extensively and serve as a valuable model in chemical ecology research, little comparable information is available about O. smaragdina. Although some analyses of chemicals produced and emitted by O. smaragdina have been reported, the literature is fragmentary and incomplete for this species. To address this knowledge gap, and to enable comparisons in the chemical ecology of the two weaver ant species, we here describe diverse chemicals from the cuticle, Dufour's glands, poison glands, head, headspace volatiles, and trails of O. smaragdina.

Forewarned is forearmed: Queensland fruit flies detect olfactory cues from predators and respond with predator-specific behaviour.

Animals can gain significant advantages from abilities to detect cues from predators, assess risks, and respond adaptively to reduce the likelihood of injurious interactions. In contrast, predator cue-induced changes in behaviour may interfere with fitness-associated activities such as exploration, foraging and reproduction. Despite the ecological importance of predator-prey interactions in insects, remarkably little is known about the abilities of insects to detect and respond to olfactory cues from predators, or the potential costs of such responses. We here demonstrate that a tephritid fruit fly, the Queensland fruit fly Bactrocera tryoni, is able to detect and respond differentially to volatile olfactory cues from four potential predators (three spiders and an ant) that vary in prevalence and diurnal activity. Male and female flies increased or decreased motility (velocity, active time, distance moved), or exhibited no change in motility, depending on which predator volatiles they encountered. Further, flies significantly reduced foraging, oviposition and mating propensity in the presence of volatiles from any of the predators. This study is the first report of predator-specific responses to olfactory cues in a tephritid fruit fly, and highlights that such anti-predator responses can impose costs on general activity and reproductive behaviour.

Commensal Bacteria Aid Mate-selection in the Fruit Fly, Bactrocera dorsalis.

Commensal bacteria influence many aspects of an organism's behaviour. However, studies on the influence of commensal bacteria in insect mate-selection are scarce. Here, we present empirical evidence that commensal bacteria mediate mate-selection in the Oriental fruit fly, Bactrocera dorsalis. Male flies were attracted to female flies, but this attraction was abolished when female flies were fed with antibiotics, suggesting the role of the fly's microbiota in mediating mate-selection. We show that male flies were attracted to and ejaculated more sperm into females harbouring the microbiota. Using culturing and 16S rDNA sequencing, we isolated and identified different commensal bacteria, with Klebsiella oxytoca being the most abundant bacterial species. This preliminary study will enhance our understanding of the influence of commensal bacteria on mate-selection behaviour of B. dorsalis and may find use in devising control operations against this devastating pest.

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

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

Aspergillus flavus impairs antioxidative enzymes of Sternochetus mangiferae during mycosis.

Insects depend upon cuticular, humoral and cellular defenses to resist mycosis. However, entomopathogenic fungi through co-evolution have developed mechanisms to counter such defenses. Although a plethora of mechanisms of mycosis by entomopathogenic fungi are well-established, studies on the impairment of insects' antioxidative enzymes during mycosis remain elusive. Here, we used the interaction of Sternochetus mangiferae and its associated entomopathogenic fungus, Aspergillus flavus, as a model to validate our hypothesis. Uninfected insects were exposed to fungal spores for infection to occur. We observed symptoms of mycosis within 48 h of incubation period. Biochemical studies on antioxidative enzymes namely catalase, peroxidase and phenoloxidase, in infected and uninfected insects revealed decreased activity of these enzymes. It appears that A. flavus disables the host's antioxidative enzyme system that plays a crucial role in elimination of oxidative toxins produced during mycosis.

Centuries of domestication has not impaired oviposition site-selection function in the silkmoth, Bombyx mori.

Oviposition site-selection in insects is mediated through innate recognition templates (IRTs) tuned to specific chemical cues. These cues aid gravid insects in choosing suitable oviposition sites and may even enhance the fitness of their offspring by warding off predators and parasitoids. However, studies on the evolution of oviposition site-selection and cues instigating oviposition in domesticated insects remain elusive. Using the interaction between the silkmoth, Bombyx mori, and its host plant mulberry, Morus alba, as a model system, we demonstrate that centuries of domestication of silkmoth has not impaired its oviposition site-selection function. Silkmoths significantly preferred mulberry leaves to filter paper as oviposition sites. Oviposition assays with filter paper, filter paper treated with leaf volatiles and leaf alone proved that surface texture was not a significant criterion for oviposition site-selection, but volatile cues were. Oviposition assays with electrophysiologically active compounds from mulberry revealed that two of the volatiles, valencene and α-humulene, aided moths in choosing suitable oviposition sites and enhanced egg-laying significantly. Moreover, we show that generalist egg-parasitoids are strongly repelled by valencene and α-humulene. Our results demonstrate that IRTs tuned to cues that aid crucial functions like oviposition site-selection are less likely to be impaired even after centuries of domestication.

Computational reverse chemical ecology: virtual screening and predicting behaviorally active semiochemicals for Bactrocera dorsalis.

BACKGROUND: Semiochemical is a generic term used for a chemical substance that influences the behaviour of an organism. It is a common term used in the field of chemical ecology to encompass pheromones, allomones, kairomones, attractants and repellents. Insects have mastered the art of using semiochemicals as communication signals and rely on them to find mates, host or habitat. This dependency of insects on semiochemicals has allowed chemical ecologists to develop environment friendly pest management strategies. However, discovering semiochemicals is a laborious process that involves a plethora of behavioural and analytical techniques, making it expansively time consuming. Recently, reverse chemical ecology approach using odorant binding proteins (OBPs) as target for elucidating behaviourally active compounds is gaining eminence. In this scenario, we describe a "computational reverse chemical ecology" approach for rapid screening of potential semiochemicals. RESULTS: We illustrate the high prediction accuracy of our computational method. We screened 25 semiochemicals for their binding potential to a GOBP of B. dorsalis using molecular docking (in silico) and molecular dynamics. Parallely, compounds were subjected to fluorescent quenching assays (Experimental). The correlation between in silico and experimental data were significant (r2 = 0.9408; P < 0.0001). Further, predicted compounds were subjected to behavioral bioassays and were found to be highly attractive to insects. CONCLUSIONS: The present study provides a unique methodology for rapid screening and predicting behaviorally active semiochemicals. This methodology may be developed as a viable approach for prospecting active semiochemicals for pest control, which otherwise is a laborious process.

Specific volatile compounds from mango elicit oviposition in gravid Bactrocera dorsalis females.

Selecting a suitable oviposition site is crucial to the fitness of female insects because it determines the successful development of their offspring. During the oviposition process, an insect must use cues from the external environment to make an appropriate choice of where to lay eggs. Generalist insects can detect and react to a plethora of cues, but are under selection pressure to adopt the most reliable ones to override noise and increase efficiency in finding hosts. The oriental fruit fly, Bactrocera dorsalis (Hendel), is a generalist that utilizes a multitude of fruits as oviposition sites. However, the identity and nature of oviposition stimulants for B. dorsalis is not well known. Recently, we identified a volatile compound γ-octalactone that elicits an innate oviposition response in B. dorsalis. We screened 21 EAD-active volatiles, identified from mango, for their oviposition stimulant activity. 1-Octen-3-ol, ethyl tiglate, and benzothiazole instigated oviposition in gravid B. dorsalis females. Flies deposited most of their eggs into pulp discs with oviposition-stimulants, and only a small fraction of eggs were laid into control discs. In a binary choice oviposition assay, 95.1, 93.7, and 65.6 % of eggs were laid in discs treated with 1-octen-3-ol, ethyl tiglate, and benzothiazole, respectively. Single plate two-choice assays proved that oviposition-stimulants were crucial in oviposition site selection by gravid female B. dorsalis. In simulated semi-natural assays, gravid B. dorsalis females accurately differentiated between fruits with and without 1-octen-3-ol, ethyl tiglate, and γ-octalactone by laying more eggs on the treated fruit. However, benzothiazole did not elicit an increase in oviposition when presented in this context. Our results suggest that the identified oviposition-stimulants are 'key' compounds, which the flies associate with suitable oviposition sites.

Oviposition site-selection by Bactrocera dorsalis is mediated through an innate recognition template tuned to γ-octalactone.

Innate recognition templates (IRTs) in insects are developed through many years of evolution. Here we investigated olfactory cues mediating oviposition behavior in the oriental fruit fly, Bactrocera dorsalis, and their role in triggering an IRT for oviposition site recognition. Behavioral assays with electrophysiologically active compounds from a preferred host, mango, revealed that one of the volatiles tested, γ-octalactone, had a powerful effect in eliciting oviposition by gravid B. dorsalis females. Electrophysiological responses were obtained and flies clearly differentiated between treated and untreated substrates over a wide range of concentrations of γ-octalactone. It triggered an innate response in flies, overriding inputs from other modalities required for oviposition site evaluation. A complex blend of mango volatiles not containing γ-octalactone elicited low levels of oviposition, whereas γ-octalactone alone elicited more oviposition response. Naïve flies with different rearing histories showed similar responses to γ-octalactone. Taken together, these results indicate that oviposition site selection in B. dorsalis is mediated through an IRT tuned to γ-octalactone. Our study provides empirical data on a cue underpinning innate behavior and may also find use in control operations against this invasive horticultural pest.

Does the growth temperature of a prokaryote influence the purine content of its mRNAs?

The formation and breaking of hydrogen bonds between nucleic acid bases are dependent on temperature. The high G+C content of organisms was surmised to be an adaptation for high temperature survival because of the thermal stability of G:C pairs. However, a survey of genomic GC% and optimum growth temperature (OGT) of several prokaryotes revoked any direct relation between them. Significantly high purine (R=A or G) content in mRNAs is also seen as a selective response for survival among thermophiles. Nevertheless, the biological relevance of thermophiles loading their unstable mRNAs with excess purines (purine-loading or R-loading) is not persuasive. Here, we analysed the mRNA sequences from the genomes of 168 prokaryotes (as obtained from NCBI Genome database) with their OGTs ranging from -5 °C to 100 °C to verify the relation between R-loading and OGT. Our analysis fails to demonstrate any correlation between R-loading of the mRNA pool and OGT of a prokaryote. The percentage of purine-loaded mRNAs in prokaryotes is found to be in a rough negative correlation with the genomic GC% (r(2)=0.655, slope=-1.478, P<000.1). We conclude that genomic GC% and bias against certain combinations of nucleotides drive the mRNA-synonymous (sense) strands of DNA towards variations in R-loading.