Drosophila Oviposition Research Articles

Evaluating Learning and Memory in Drosophila melanogaster to Study the Neurodevelopmental Impacts of Toxicants.

Neurodevelopmental disorders are a heterogeneous group of behaviorally defined disorders with both genetic and environmental risk factors. Given that many neurodevelopmental disorders are characterized by impaired learning and/or intellectual abilities, behavioral paradigms that assess cognition in animal models have been effective tools in delineating underlying genetic variants that impact disease pathophysiology. For example, learning and memory paradigms in the common fruit fly Drosophila melanogaster have been successfully used to study risk genes and biological pathways associated with several neurodevelopmental disorders, including fragile X syndrome, autism spectrum disorder, and CHARGE syndrome. While these established Drosophila behavioral paradigms have historically been used to investigate genetic risk factors, they also have many other applications, including use in developmental neurotoxicology studies to determine environmental risk factors for neurodevelopmental disorders. There is, however, a deficit of step-by-step protocols that describe how to apply learning and memory assays in fruit flies to developmental neurotoxicology studies. Here, we describe two quantitative behavioral paradigms for Drosophila-predator-induced oviposition and courtship conditioning-that can be used to measure different forms of learning and memory in the context of a developmental neurotoxicology study. Non-associative learning and memory are measured here by examining female Drosophila oviposition behavior in response to endoparasitoid wasps, while associative learning and memory are measured in males using courtship conditioning. Our protocols outline procedures for oral toxicant exposure of developing fruit flies, culturing of endoparasitoid wasps, measuring Drosophila oviposition activity, and assessing conditioned courtship in order to identify the impact of toxicants on learning and memory in both females and males. As an example, we present the protocols using bisphenol A, a chemical utilized in the synthesis of polycarbonate plastics, to determine its impacts on learning and memory. These protocols are inexpensive and relatively simple to perform, and can be used by labs that are new to Drosophila behavioral research to evaluate how toxicant exposure influences learning and memory in male and female flies. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Preparation of toxicant-containing food and developmental exposure Basic Protocol 2: Predator-induced oviposition assay Support Protocol: Culture of Leptopilina heterotoma Basic Protocol 3: Conditioned courtship assay.

Search performance and octopamine neuronal signaling mediate parasitoid induced changes in Drosophila oviposition behavior

Making the appropriate responses to predation risk is essential for the survival of an organism; however, the underlying mechanisms are still largely unknown. Here, we find that Drosophila has evolved an adaptive strategy to manage the threat from its parasitoid wasp by manipulating the oviposition behavior. Through perception of the differences in host search performance of wasps, Drosophila is able to recognize younger wasps as a higher level of threat and consequently depress the oviposition. We further show that this antiparasitoid behavior is mediated by the regulation of the expression of Tdc2 and Tβh in the ventral nerve cord via LC4 visual projection neurons, which in turn leads to the dramatic reduction in octopamine and the resulting dysfunction of mature follicle trimming and rupture. Our study uncovers a detailed mechanism underlying the defensive behavior in insects that may advance our understanding of predator avoidance in animals.

Neem-Based Insecticides Deter Oviposition Activity in Spotted Wing Drosophila (Diptera: Drosophilidae) in Sweet Cherries.

Spotted wing drosophila, Drosophila suzukii (Matsumura), is an important invasive insect pest in the United States. Spotted wing drosophila females have serrated ovipositor that enables them to deposit eggs inside healthy, ripening softskinned fruits. In many berry and fruit crops, spotted wing drosophila is primarily controlled using synthetic insecticides in the organophosphate, pyrethroid, and spinosyn groups. However, alternative, reduced-risk insecticides with short residuals are necessary to minimize the possibilities of insecticide resistance and address other negative effects of synthetic insecticides. Neem-based products as alternative plant-based insecticides have antifeedant, repellent, antiovipository, and molting interference effects on insects and have been shown to affect the oviposition activities of some tephritid flies. We conducted laboratory studies to evaluate the oviposition deterrent activities of selected neem-based insecticides that contain either azadirachtin, or CHENO, or a combination of both, on spotted wing drosophila using sweet cherry in California. The results of the study showed that some of the neem oil products could reduce the degree of spotted wing drosophila oviposition by half compared with the control.

Regulation of <i>Drosophila</i> Visceral Muscle Contractions by Multiple, Indirect Octopaminergic and Glutamatergic Signaling Pathways

Mammalian visceral muscles are indirectly regulated by receptors expressed in peripheral neurons and other non-contractile cells. The complex interactions between these pathways remain poorly understood. It also remains unknown whether invertebrates use pathways similar to mammals to indirectly regulate visceral contractility. To elucidate the principles of organization for the signaling pathways responsible for visceral motility and the more general logic of aminergic neuromodulation, we are using the Drosophila oviposition circuit. We find that the receptors for the adrenergic neurotransmitter octopamine as well as the metabotropic glutamate receptor are not expressed in muscle cells, but rather, in epithelial cells that line the oviduct lumen in a regionally specific pattern and in multiple subtypes of peripheral neurons, some of which co-express mechanosensory markers. We have used live imaging to demonstrate that most of the functional effects of octopamine and glutamate are mediated by indirect pathways and that contractions of the lateral and common oviducts are regulated independently. Neurons intrinsic to the lateral oviduct indirectly activate muscle cells, and mutational and optogenetic analyses indicate that Octβ2 is required for these effects. An additional indirect pathway for lateral oviduct contractions is controlled by glutamate receptors expressed on a distinct subset of neuronal processes outside of the lateral oviduct. In addition to contractions of the lateral oviduct, octopamine regulates dilation, but this occurs in both the lateral and common oviducts and is blocked by mutation of OAMB but not Octβ2. Additional, unsuspected interactions between octopamine and glutamate include contractions of the common oviduct that requires an interaction between octopamine and with glutamate, blockade of lateral oviduct contractions by preincubation with glutamate and opposing effects of octopamine and glutamate in epithelial cells. These data indicate that multiple indirect pathways regulate the oviposition circuit similar to the mechanisms described for mammalian visceral muscles. They also uncover a complex network of neuromodulatory effects that provide insight into the regulation of behavioral circuits in other systems.

Susceptibility of Bunch Grape and Muscadine Cultivars to Berry Splitting and Spotted-Wing Drosophila Oviposition

Fruit splitting is a physiological disorder that produces surface cracks that promote disease and insect damage. The spotted-wing drosophila (SWD), <i>Drosophila suzukii</i> (Matsumura), is a species of vinegar fly that attacks berry crops worldwide, including grapes, and reduces fruit quality and yield. In this study, we tested the susceptibility of various hybrid bunch grapes and muscadines to berry splitting and SWD attack. Ten grape cultivars were harvested in 2016 and 2017, and Thompson Seedless (<i>Vitis vinifera</i>) was purchased locally. Fruit quality traits examined included Brix (soluble solids), total acidity (TA), pH, fruit firmness, and skin break force. Villard blanc and OK392 were the least susceptible to berry splitting (0%). Although muscadines showed the highest fruit firmness, they were also susceptible to splitting, especially Fry Seedless (88% splitting incidence). There were no significant correlations among soluble solids, firmness, and splitting; however, there was a negative correlation between splitting and TA. Pierce’s disease (PD)-resistant and PD-tolerant grapes also appear to be mostly resistant to or highly tolerant of SWD infestation. SWD females do not appear to be able to oviposit in fruit hosts that have a fruit firmness exceeding 300 g/mm. Wounds or splits in the grape epidermis increased reproductive success of SWD by 400%, suggesting that a well-managed vineyard will not host large SWD populations. Vineyards suffering from severe berry damage might provide SWD with a summer fruit host. Overall, PD-resistant hybrids and muscadines are highly adaptive to subtropical climates and are tolerant of this new invasive vinegar fly.

Agar-polydimethylsiloxane devices for quantitative investigation of oviposition behaviour of adult Drosophila melanogaster.

Drosophila melanogaster (fruit fly) is a model organism and its behaviours including oviposition (egg-laying) on agar substrates have been widely used for assessment of a variety of biological processes in flies. Physical and chemical properties of the substrate are the dominant factors affecting Drosophila's oviposition, but they have not been investigated precisely and parametrically with the existing manual approaches. As a result, many behavioral questions about Drosophila oviposition, such as the combined effects of the aforementioned substrate properties (e.g., exposure area, sugar content, and stiffness) on oviposition and viability, and their threshold values, are yet to be answered. In this paper, we have devised a simple, easily implementable, and novel methodology that allows for modification of physical and chemical composition of agar substrates in order to quantitatively study survival and oviposition of adult fruit flies in an accurate and repeatable manner. Agar substrates have been modified by surface patterning using single and hexagonally arrayed through-hole polydimethylsiloxane (PDMS) membranes with various diameters and interspacing, as well as by substrate stiffness and sugar content modification via alteration of chemical components. While pure PDMS substrates showed a significant lethal effect on flies, a 0.5 mm diameter through-hole access to agar was found to abruptly increase the survival of adult flies to more than 93%. Flies avoided ovipositing on pure PDMS and on top of substrates with 0.5 mm diameter agar exposure areas. At a hole diameter of 2 mm (i.e., 0.25% exposure area) or larger, eggs were observed to be laid predominately inside the through-holes and along the edges of the PDMS-agar interface, showing a trending increase in site selection with 4 mm (i.e., 1% exposure area threshold) demonstrating natural oviposition rates similar to pure agar. The surface-modified agar-PDMS hybrid devices and the threshold values reported for the substrate physical and chemical conditions affecting oviposition are novel; therefore, we advocate their use for future in-depth studies of oviposition behaviour in Drosophila melanogaster with accuracy and repeatability. The technique is also useful for development of novel assays for learning and decision-making studies as well as miniaturized devices for self-assembly of eggs and embryonic developmental investigations.

Use of Early Ripening Cultivars to Avoid Infestation and Mass Trapping to Manage Drosophila suzukii (Diptera: Drosophilidae) in Vaccinium corymbosum (Ericales: Ericaceae).

Use of early ripening highbush blueberry cultivars to avoid infestation and mass trapping were evaluated for managing spotted wing drosophila, Drosophila suzukii (Matsumura). Fourteen highbush blueberry cultivars were sampled for spotted wing drosophila infestation. Most 'Earliblue', 'Bluetta', and 'Collins' fruit were harvested before spotted wing drosophila oviposition commenced, and so escaped injury. Most fruit from 'Bluejay', 'Blueray', and 'Bluehaven' were also harvested before the first week of August, after which spotted wing drosophila activity led to high levels of blueberry infestation. In a separate experiment, damage to cultivars was related to the week in which fruit were harvested, with greater damage to fruit observed as the season progressed. Attractant traps placed within blueberry bushes increased nearby berry infestation by 5%, irrespective of cultivar and harvest date. The significant linear reduction in infestation with increasing distance from the attractant trap suggests that traps are influencing fly behavior to at least 5.5 m. Insecticides applied to the exterior of traps, compared with untreated traps, revealed that only 10-30% of flies visiting traps enter the traps and drown. Low trap efficiency may jeopardize surrounding fruits by increasing local spotted wing drosophila activity. To protect crops, traps for mass trapping should be placed in a perimeter outside fruit fields and insecticides need to be applied to the surface of traps or on nearby fruit to function as an attract-and-kill strategy.

Choice alters Drosophila oviposition site preference on menthol.

SummaryFood choice and preference relies on multiple sensory systems that are under the control of genes and sensory experience. Exposure to specific nutrients and nutrient-related molecules can change food preference in vertebrates and invertebrates. For example, larval exposure of several holometabolous insects to menthol can change their adult response to this molecule. However, studies involving Drosophila melanogaster exposure to menthol produced controversial results due maybe to methodological differences. Here, we compared the oviposition-site preference of wild-type D. melanogaster lines freely or forcibly exposed to menthol-rich food. After 12 generations, oviposition-site preference diverged between the two lines. Counterintuitively, menthol ‘forced’ lines showed a persistent aversion to menthol whereas ‘free choice’ lines exhibited a decreased aversion to menthol-rich food. This effect was specific to menthol since the ‘free choice’ lines showed unaltered responses to caffeine and sucrose. This suggests that the genetic factors underlying Drosophila oviposition site preference are more rapidly influenced when flies have a choice between alternative sources compared to flies permanently exposed to the same aversive substance.

Tissue-Specific Activation of a Single Gustatory Receptor Produces Opposing Behavioral Responses in Drosophila

Understanding sensory systems that perceive environmental inputs and neural circuits that select appropriate motor outputs is essential for studying how organisms modulate behavior and make decisions necessary for survival. Drosophila melanogaster oviposition is one such important behavior, in which females evaluate their environment and choose to lay eggs on substrates they may find aversive in other contexts. We employed neurogenetic techniques to characterize neurons that influence the choice between repulsive positional and attractive egg-laying responses toward the bitter-tasting compound lobeline. Surprisingly, we found that neurons expressing Gr66a, a gustatory receptor normally involved in avoidance behaviors, receive input for both attractive and aversive preferences. We hypothesized that these opposing responses may result from activation of distinct Gr66a-expressing neurons. Using tissue-specific rescue experiments, we found that Gr66a-expressing neurons on the legs mediate positional aversion. In contrast, pharyngeal taste cells mediate the egg-laying attraction to lobeline, as determined by analysis of mosaic flies in which subsets of Gr66a neurons were silenced. Finally, inactivating mushroom body neurons disrupted both aversive and attractive responses, suggesting that this brain structure is a candidate integration center for decision-making during Drosophila oviposition. We thus define sensory and central neurons critical to the process by which flies decide where to lay an egg. Furthermore, our findings provide insights into the complex nature of gustatory perception in Drosophila. We show that tissue-specific activation of bitter-sensing Gr66a neurons provides one mechanism by which the gustatory system differentially encodes aversive and attractive responses, allowing the female fly to modulate her behavior in a context-dependent manner.

Logjam encodes a predicted EMP24/GP25 protein that is required for Drosophila oviposition behavior.

A newly characterized Drosophila melanogaster gene, logjam (loj), functions in female reproduction by modulating oviposition behavior. The locus encodes at least six overlapping transcripts with unique 5' ends. P-element mutants that express very low levels of loj transcripts are unable to oviposit mature eggs. This phenotype can be rescued by the introduction of a transgene expressing the most abundant loj transcript. As for many genes that specify behavioral outputs, loj is present in the adult central nervous system (CNS). Interestingly, it is also observed in vitellogenic egg chambers, suggesting that there may be multiple functions for this gene in egg-laying behavior. loj encodes a predicted protein with homology to the EMP24/GP25 transmembrane components of cytoplasmic vesicles and likely functions in intracellular trafficking.

Drosophila Species, Breeding in the Stinkhorn (Phallus Impudicus Pers.) and Their Larval Parasitoids

A field survey was carried out in the seasons of 1985 and 1986 in a woodland area, situated in the central western part of The Netherlands, to study the community of Drosophila breeding in the stinkhorn, Phallus impudicus, and their larval parasitoids. The fluctuations of the fungi, Drosophila, and parasitoid populations are presented. During a large part of the season the system is relatively simple, comprising one Drosophila species, D. phalerata, and one parasitoid, Leptopilina clavipes. In autumn other parasitoids become common in Ph. impudicus. Most parasitoid species are polyphagous with respect to the host species they parasitize. As a consequence of high rates of parasitism, host populations regularly pass bottlenecks within one season, especially in July when parasitism can rise up to 100%. Mean Drosophila egg to adult development lasts 25-32 days in the field and species can realize at most 4-5 generations in one season. Parasitoid development takes 40-55 days and not more than 2 generations can be completed. Temperature explains 94% of the rate of development of the egg and first two larval stages of D. phalerata. Drosophila oviposition behaviour and larval development creates patches containing suitable hosts during several successive days. Parasitoids arrive in new patches from the first day of their appearance and, in general, patches are exploited by more than one parasitoid. Some implications of these features on the behavioural ecology of the parasitoids and population dynamics are discussed.

Drosophila Control in Tomato Fields1

Insecticidal sprays were tested for the field control of Drosophila oviposition—principally that of Drosophila melanogaster Meig—in tomato fruits during 1955 and 1957. Diazinon® ( O,O -diethyl O -(2-isopropyl-4-methyl-6-pyrimidinyl) phosphorothioate) applied at the rate of one-half pound per acre gave a satisfactory reduction of Drosophila activity. Wettable powder, emulsifiable concentrate and a granular formulation of Diazinon were compared and found to be equally effective at the rate of one-half pound per acre. The granular formulation exhibited long residual action and there appeared to be some cumulative effect from repeated applications. Malathion applied to small plots and in commercial plantings was only effective for 2 to 3 days and was followed by a very rapid fly buildup. Phosdrin® (1-methoxycarbonyl-1-propen-2-yl dimethyl phosphate) had somewhat the same weakness as malathion—short residual action. Ronnel and Dibrom®(1,2-dibromo-2,2-dichloroethyl dimethyl phosphate) were tested during 1957 only and compared favorably with Diazinon. Hydrolyzed yeast did not prove to be an added attractant to the flies when used alone.