14 research outputs found
The Downregulation of Short Neuropeptide Receptor (SNPFR) in the Red Imported Fire Ant Solenopsis Invicta (Hymenoptera: Formicidae) and the Tawny Crazy Ant Nylanderia Fulva (Hymenoptera: Formicidae) using RNA Interference, and the Use of an Anthranilic Diamide as a Novel Management Technique
The red imported fire ant, Solenopsis invicta Buren (Hymenoptera: Formicidae), is an invasive species endemic to South America that was inadvertently introduced into the United States. This invasive species costs over six billion dollars (USD) annually and causes substantial ecological and agricultural damage. Anthranilic diamides are novel chemical insecticides that selectively target the insect ryanodine receptor in the endoplasmic reticulum, causing calcium release and insect mortality. In this study, cyclaniliprole, an anthranilic diamide, was used to cause significant mortality in S. invicta workers. RNA interference (RNAi) is a known regulator of biological systems in insects and was also used in this study to downregulate the short Neuropeptide F Receptor (sNPFR) in S. invicta workers and larvae. Gene expression was quantified for sNPFR in different S. invicta caste members, and dsRNA was produced for sNPFR and was administered to S. invicta brood and workers. Primers for the sNPFR sequence were also produced and tested on Tawny crazy ant workers, Nylanderia fulva (Hymenoptera: Formicidae), a natural enemy of S. invicta and invasive pest. By doing this, a single dsRNA construct could potentially be used to manage both S. invicta and N. fulva
Short Neuropeptide F Receptor in the Worker Brain of the Red Imported Fire Ant (Solenopsis invicta Buren) and Methodology for RNA Interference
The red imported fire ant (Solenopsis invicta Buren) is one of the worst invasive species in the United States. Investigating their physiology to understand its molecular basis could lead to the discovery of new gene targets for fire ant control. Neuropeptides are involved in the regulation of important physiological processes, and in insects the short neuropeptide F (sNPF) plays an important role as regulator of feeding, and involved in mechanisms of nutrient sensing, growth and reproduction. This study is focused into unveiling the physiological role of the sNPF and its receptor (sNPFR) in fire ants. In workers, we found a total of nine clusters of immunoreactive-sNPFR cells located near important neuropiles in the brain. These sNPFR-expressing cells are sensitive to the presence/absent of brood, perhaps in correlation with changes in the nutritional status of the colony. Also, these cell clusters are differentially expressed among worker subcastes, suggesting the sNPF/sNPFR pathway could be associated with mechanisms of division of labor. To discover where sNPF is synthesized, we attempted to localize the sNPF-expressing cells in the brain of queens and workers through in situ hybridization, unfortunately without success; thus, the site of synthesis of sNPF in the brain still remains unknown. Finally, we tried to elucidate the role of the sNPFR in social context by silencing its gene expression trough feeding of dsRNA using small laboratory colonies. We found differential effects when delivering the sNPFR-dsRNA through heat-killed dsRNA-producing bacteria or by delivering dsRNA purified from these bacterial cultures. Also, the type of food used as carrier and the concentration of dsRNA were crucial for gene silencing success. Our results showed that feeding a large concentration of dsRNA in liquid and solid foods is required to induce RNAi in the queen midgut; and that the silencing of the sNPFR in this organ did not induce mortality in these queens, but instead it appears to increase larval mortality. In summary, our results are consistent with the sNPF/sNPFR signaling pathway in fire ants being involved in the regulation of division of labor and in sensing the nutritional status, and suggest its function is fundamental for larval development
The Red Imported Fire Ant (Solenopsis invicta Buren) Kept Y not F: Predicted sNPY Endogenous Ligands Deorphanize the Short NPF (sNPF) Receptor
Short Neuropeptide F Receptor in the Worker Brain of the Red Imported Fire Ant (Solenopsis invicta Buren) and Methodology for RNA Interference
The red imported fire ant (Solenopsis invicta Buren) is one of the worst invasive species in the United States. Investigating their physiology to understand its molecular basis could lead to the discovery of new gene targets for fire ant control. Neuropeptides are involved in the regulation of important physiological processes, and in insects the short neuropeptide F (sNPF) plays an important role as regulator of feeding, and involved in mechanisms of nutrient sensing, growth and reproduction. This study is focused into unveiling the physiological role of the sNPF and its receptor (sNPFR) in fire ants. In workers, we found a total of nine clusters of immunoreactive-sNPFR cells located near important neuropiles in the brain. These sNPFR-expressing cells are sensitive to the presence/absent of brood, perhaps in correlation with changes in the nutritional status of the colony. Also, these cell clusters are differentially expressed among worker subcastes, suggesting the sNPF/sNPFR pathway could be associated with mechanisms of division of labor. To discover where sNPF is synthesized, we attempted to localize the sNPF-expressing cells in the brain of queens and workers through in situ hybridization, unfortunately without success; thus, the site of synthesis of sNPF in the brain still remains unknown. Finally, we tried to elucidate the role of the sNPFR in social context by silencing its gene expression trough feeding of dsRNA using small laboratory colonies. We found differential effects when delivering the sNPFR-dsRNA through heat-killed dsRNA-producing bacteria or by delivering dsRNA purified from these bacterial cultures. Also, the type of food used as carrier and the concentration of dsRNA were crucial for gene silencing success. Our results showed that feeding a large concentration of dsRNA in liquid and solid foods is required to induce RNAi in the queen midgut; and that the silencing of the sNPFR in this organ did not induce mortality in these queens, but instead it appears to increase larval mortality. In summary, our results are consistent with the sNPF/sNPFR signaling pathway in fire ants being involved in the regulation of division of labor and in sensing the nutritional status, and suggest its function is fundamental for larval development
Differences in sNPF Receptor-Expressing Neurons in Brains of Fire Ant (Solenopsis invicta Buren) Worker Subcastes: Indicators for Division of Labor and Nutritional Status?
Molecular characterization of a short neuropeptide F signaling system in the swimming crab, Portunus trituberculatus, and its role in ovarian development
Short neuropeptide F (sNPF) is a neuropeptide that widely distributed among arthropods. This neuropeptide has been proposed to play pleiotropic roles in insects, but its physiological functions in crustaceans are poorly understood. Here, we cloned the cDNA sequences of sNPF and its putative receptor (sNPFR) from the swimming crab, Portunus trituberculatus, and determined their possible roles during ovarian development. PtsNPF encodes three sNPF mature peptides with a conserved C-terminal āRLRFGā motif. All three PtsNPF peptides concentration-dependently activated PtsNPFR expressed in HEK293T cells, with EC50 values in the nanomolar range. PtsNPF and PtsNPFR transcripts showed a broad distribution among neural and non-neural tissues. During the ovarian development, expression of PtsNPF and PtsNPFR in hepatopancreas and ovary both increased to the highest levels at the late-vitellogenic stage, a period for rapid vitellogenesis. The in vitro experiments further showed that, among the three sNPF peptides, sNPF3 treatments can induce the vitellogenin (Vg) gene and protein levels in the hepatopancreas, as well as the Vg receptor (VgR) gene and protein levels and vitellogenin (Vn) deposition in the ovary. Opposing effects were observed for the dsPtsNPFR treatments, suggesting that PtsNPFR plays a role in the PtsNPF-mediated vitellogenesis. Additionally, it was found that the PtsNPF/PtsNPFR system can activate different second messengers species in hepatopancreas and ovary, suggesting it may act via different G proteins
Effet du neuropeptide sNPF sur le comportement de l'abeille domestique (Apis mellifera)
Chez les vertĆ©brĆ©s, le neuropeptide Y (NPY) joue un rĆ“le crucial dans la survie individuelle en modulant Ć la fois les comportements liĆ©s Ć la nourriture et au stress. Des niveaux Ć©levĆ©s de NPY corrĆØlent avec une augmentation de la faim provoquant une ingestion plus importante et rĆ©duisent la sensibilitĆ© aux stimuli stressants. Chez les invertĆ©brĆ©s, deux homologues indĆ©pendants de NPY ont Ć©tĆ© identifiĆ©s : le neuropeptide F (NPF) et le neuropeptide F court (sNPF). Chez l'abeille domestique (Apis mellifera), npf et snpf ainsi que leur peptides respectifs NPF et sNPF ont Ć©tĆ© identifiĆ©s or seul sNPF possĆØde un rĆ©cepteur, suggĆ©rant un rĆ“le fonctionnel de ce neuropeptide chez cet insecte. Nous avons Ć©tudiĆ© l'impact de sNPF sur une multitude de comportements comprenant l'ingestion de nourriture de bonne et mauvaise qualitĆ©, les rĆ©ponses appĆ©titives et aversives, les apprentissages et la mĆ©moire appĆ©titifs et aversifs. Nos rĆ©sultats rĆ©vĆØlent qu'une Ć©lĆ©vation artificielle des niveaux de sNPF via une application topique chez les butineuses augmente la prise alimentaire de nourriture bonne et mauvaise qualitĆ©. De plus, en utilisant une variĆ©tĆ© de tests pour Ć©tudier les rĆ©ponses sensorielles, nous avons montrĆ© que sNPF a un rĆ“le clĆ© dans la modulation des rĆ©ponses appĆ©titives, mais cet effet est absent pour les rĆ©ponses aversives. Les abeilles nourries et traitĆ©es avec du sNPF augmentent leur rĆ©ponse au saccharose et aux stimuli olfactifs appĆ©titifs, de faƧon similaire aux abeilles affamĆ©es. En adĆ©quation avec les derniers rĆ©sultats, des enregistrements in vivo multi photoniques de l'activitĆ© neuronale du lobe antennaire, le premier centre olfactif dans le cerveau de l'abeille, montrent une baisse des rĆ©ponses aux odeurs appĆ©titives chez les abeilles nourries qui est rĆ©tablie par le traitement avec le sNPF au mĆŖme niveau que les abeilles affamĆ©es. Par ailleurs, l'effet modulatoire du sNPF Ć©tait totalement absent sur les rĆ©ponses aversives contrairement Ć ce qui a Ć©tĆ© observĆ© chez la drosophile et les vertĆ©brĆ©s, indiquant que chez les abeilles, sNPF n'augmente pas la tolĆ©rance aux stimuli stressants. Etant donnĆ© l'amplification causĆ©e par le traitement sNPF sur la rĆ©ponse au saccharose, nous avons Ć©tudiĆ© si cet effet se retrouvait dans des protocoles d'apprentissage pour lesquels les abeilles Ć©taient entraĆ®nĆ©es Ć discriminer un stimulus rĆ©compensĆ© par du saccharose d'un autre qui ne l'est pas. Nous avons Ć©tudiĆ© l'effet du sNPF sur les apprentissages et mĆ©moires appĆ©titifs visuels et olfactifs. Dans le premier cas, des abeilles en semi libre vol ont Ć©tĆ© entraĆ®nĆ©es Ć discriminer deux couleurs dans un labyrinthe en Y aprĆØs une application topique de sNPF. Dans le second cas, des abeilles en contention ont Ć©tĆ© entraĆ®nĆ©es Ć discriminer deux odeurs aprĆØs une application topique de sNPF via le conditionnement du rĆ©flexe d'extension du proboscis. En parallĆØle, nous avons Ć©tudiĆ© les effets du sNPF sur l'apprentissage aversif gustatif pour lequel les abeilles en contention apprennent l'association entre une stimulation gustative de l'antenne avec un choc Ć©lectrique aprĆØs une application topique de sNPF. Nos rĆ©sultats montrent une nette amĆ©lioration de l'apprentissage et mĆ©moire appĆ©titifs visuels et des tendances allant dans le mĆŖme sens dans le cas de l'apprentissage appĆ©titif olfactifs. A l'inverse, aucun effet n'a Ć©tĆ© observĆ© quant Ć l'apprentissage et la mĆ©moire aversifs gustatifs, ce qui est cohĆ©rent avec l'absence d'effet de sNPF sur les rĆ©ponses sensorielles aversives.
Ce travail de thĆØse a montrĆ© que le sNPF affecte plusieurs modalitĆ©s de comportements (ingestion, gustation, olfaction, vision, apprentissage, mĆ©moire) et les processus neuronaux (lobe antennaire) liĆ©s aux comportements appĆ©titifs, mais non aversifs, chez l'abeille. Par consĆ©quent, ce travail fournit de nouvelles perspectives pour Ć©tudier les processus d'ingestion et le comportement alimentaire des abeilles.Neuropeptide Y (NPY) signalling plays a crucial role for individual survival in vertebrates as it mediates both food- and stress-related behaviours. High NPY level correlates with increased hunger and leads to a larger food intake while it also reduces sensitivity to stressful stimuli. In invertebrates, two independent homologs of NPY have been identified: the neuropeptide F (NPF) and the short neuropeptide F (sNPF). In honey bees (Apis mellifera), both NPF and sNPF have been reported but only sNPF was found to have a dedicated receptor sNPFR, thus indicating that sNPF/sNPFR provides a functional signalling pathway in this insect. We thus studied the impact of sNPF on multiple behavioural components, including food-related behaviours such as ingestion of palatable and unpalatable food, appetitive and aversive responsiveness, and appetitive and aversive associative learning and memory retention. Our results show that increasing artificially sNPF levels in honey bee foragers via topical exposure, increases significantly their consumption of both palatable and unpalatable food. In addition, using various responsiveness tests, we showed that sNPF is a key player in the modulation of appetitive but not aversive responsiveness. Fed foragers treated with sNPF exhibited a significant increase in their responsiveness to sucrose solutions and to appetitive olfactory stimuli, matching the levels of starved bees. In agreement with this last finding, in vivo multiphoton recordings of neural activity in the antennal lobe, the primary olfactory centre of the bee brain, showed a decreased responsiveness to appetitive odours in fed bees, which was rescued by treatment with sNPF to the level exhibited by starved bees. Interestingly, the modulatory effect of sNPF was totally absent in responsiveness to aversive stimuli contrarily to what has been observed in vertebrates and flies, thus indicating that in bees, sNPF dos not increase tolerance to stressors. Given the enhancing effect of sNPF on appetitive responsiveness, we next studied if this effect translates to different appetitive learning protocols in which bees are trained to discriminate a stimulus that is rewarded with a sucrose solution from another that is not. We studied the effect of sNPF on both appetitive visual and olfactory learning and memory retention. In the first case, free-flying bees were trained to discriminate two colors in a Y-maze following topical increase of sNPF. In the second case, harnessed bees were trained to discriminate two odorants following topical application of sNPF, using the conditioning of the proboscis extension reflex. In parallel, we studied the effect of sNPF for aversive gustatory learning in which harnessed bees learning the association of antennal taste with electric shock, following topical application of sNPF. Our results revealed a clear improvement of appetitive color learning and retention and a mitigated tendency in the same direction in the case of appetitive olfactory learning. On the contrary, no effect was observed in the case of the aversive gustatory learning and retention, consistently with the lack of effect of sNPF on aversive responsiveness. To sum up, this work showed that sNPF affects multiple appetitive behavioural modules (ingestion, gustation, olfaction, vision, learning, memory) and central processing (antennal lobe activity) in the honey bee while being dispensable for aversive ones. It provides therefore a rich and multifaceted view of the effects of this neuropeptide on the behaviour of a social insect and opens new research perspective to study ingestion processes and appetitive behaviour in bees
Nutrition, Hormones, Transcriptional Regulatory Networks and Division of Labor in Honey Bee Colonies
Phenotypic plasticity ā one genotype producing alternative phenotypes ā is increasingly understood to be an important force in phenotypic evolution, but its mechanistic basis remains poorly understood. This thesis describes research into the molecular mechanisms underlying age-related behavioral and physiological plasticity in worker honey bees.
Many animals are able to alter their behavior and physiology in response to changes in the environment. At times, these changes in behavior and physiology are stable for long periods, a phenomenon known as phenotypic plasticity [1]. For instance, short periods of food deprivation stimulate feeding and the mobilization of stored nutrients to meet an individualās immediate energetic needs. But prolonged food deprivation can also lead to much longer-term effects, causing individuals to enter extended periods of inactivity, alter their reproductive strategy, or lose their position in a dominance hierarchy. In humans, chronic food deprivation early in life may lead to a propensity toward obesity and diabetes in later life (for an expanded and fully-referenced discussion of nutritionally-mediated phenotypic plasticity see Chapter 4). The mechanisms that enable and constrain plasticity in behavior and physiology are not well understood, but it is clear that they often involve coordinated and long-lasting changes in gene expression, brain circuitry, brain chemistry, and endocrine signaling [2]. My doctoral research has focused on understanding the molecular basis for nutritionally- and hormonally-mediated plasticity in the behavior and physiology of worker honey bees.
Honey bees are social insects, living together in colonies containing tens of thousands of individuals [3]. Colony life is organized by a complex and sophisticated division of labor. Each colony contains a single queen, who is specialized for reproduction and spends most of her time laying eggs. Males, called drones, are relatively rare, and their sole role is to mate. The vast majority of the individuals in the hive are sterile worker bees that are responsible for all of the other tasks performed by the colony. The tasks performed by worker bees are further divided up among individuals via a process of behavioral maturation that is the focus of this thesis. For the first 2-3 weeks of adult life, worker bees specialize on broodcare (ānursingā). They then switch for a few days to any of a number of more specialized tasks such as building honeycomb cells, storing food in honeycomb cells, or guarding the hive entrance against intruders. Finally, for the remaining 1-2 weeks of their life, worker bees forage outside the hive for nectar and pollen, the colonyās sole sources of food.
The work presented in this thesis builds on previous findings demonstrating links between worker honey bee division of labor and nutrition (reviewed in Chapter 4). Behavioral maturation in worker bees is coupled to changes in nutritional physiology, including a dramatic and stable loss of abdominal lipid that occurs prior to the onset of foraging. Moreover, previous studies had demonstrated that nutritional status can have causal influences on the timing of behavioral maturation and manipulations of a few feeding- or nutritionally-related genes accelerates or delays the age at onset of foraging.
In the work described here, I first test the hypothesis that worker bee behavioral maturation, a highly derived trait, is regulated, in part, by conserved nutritionally-related hormones (Chapter 1). I demonstrate that genes related to insulin signaling are differentially expressed in the brains and fat bodies of nurses and foragers. Furthermore, I show that manipulation of the insulin-related TOR pathway influences the age at which bees initiate foraging. These results suggest that the evolution of honey bee social behavior involved new roles for ancient nutritionally-related pathways. However, my subsequent work shows that not all nutritionally-related pathways have been coopted in the same way. I describe a more complex, and less resolved, relationship between behavioral state, nutrition and brain gene expression for a second nutritionally-related hormone, Neuropeptide Y (Chapter 2).
Next, using transcriptomic experiments, I demonstrate that maturation, as well as age-related stable lipid loss, involve massive changes in gene expression in the fat bodies (Chapter 3). I show that these changes in gene expression involve age-related changes in the responsiveness of hormonally and metabolically related pathways to nutrition, and roles for two evolutionarily novel, non-dietary factors: the storage protein vitellogenin and Queen Mandibular Pheromone, each of which influenced many maturationally-related genes in the fat bodies. These results also suggest the involvement in the responses to all these factors of a single nutritionally-related hormone, juvenile hormone (JH), which had previously been shown to pace behavioral maturation.
In Chapter 4, I review my findings from chapters 1-2 of this thesis, and previous studies, and propose a molecular systems biology approach to understanding division of labor. Specifically, I propose that phenotypic plasticity in worker honey bees involves nutritionally- and hormonally-driven changes in transcriptional regulatory networks in the fat bodies (as well in the brain), and I suggest methodologies for their elucidation.
Finally, in Chapter 5, I utilize the molecular systems biology approach outlined in Chapter 4 to show that a transcriptional regulatory network in the fat bodies underlies division of labor. I show that a juvenile hormone-related transcription factor, Ultraspiracle (USP), influences the age at onset of foraging. I then use a combination of chromatin immunoprecipitationāgenomic tiling microarrays, RNAi and deep mRNA sequencing to develop a model of the USP transcriptional regulatory network in fat cells. My results suggest that JH and USP function together to induce and maintain alternative states of a transcriptional regulatory network. These alternative states may well underlie the two basic phases of worker bee life, the in-hive and foraging phases.
Together, the studies presented in this thesis provide insights into the relationship between nutrition, hormones, transcriptional regulation, and phenotypic plasticity.
References
1. West-Eberhard, MJ. Developmental Plasticity and Evolution. 2003. Oxford University Press, New York, NY. 794 pp.
2. Robinson, GE, Fernald, RD, Clayton, DF. Genes and social behavior. Science. 2008 Nov 7; 322(5903):896-900. doi:10.1126/science.1159277
3. Winston, ML. The Biology of the Honey Bee. 1987. Harvard University Press, Cambridge, MA. 294 pp
Feeding and the rhodopsin family G-Protein Coupled Receptors (GPCRs) in nematodes and arthropods
In vertebrates, receptors of the rhodopsin G-protein coupled superfamily (GPCRs) play an important role in the regulation of feeding and energy homeostasis and are activated by peptide hormones produced in the brain-gut axis. These peptides regulate appetite and energy expenditure by promoting or inhibiting food intake. Sequence and function homologues of human GPCRs involved in feeding exist in the nematode roundworm, Caenorhabditis elegans (C. elegans) and the arthropod fruit fly, Drosophila melanogaster (D. melanogaster), suggesting that the mechanisms that regulate food intake emerged early and have been conserved during metazoan radiation. Nematodes and arthropods are the most diverse and successful animal phyla on Earth. They can survive in a vast diversity of environments and have acquired distinct life styles and feeding strategies. The aim of the present review is to investigate if this diversity has affected the evolution of invertebrate GPCRs. Homologues of the C. elegans and D. melanogaster rhodopsin receptors were characterized in the genome of other nematodes and arthropods and receptor evolution compared. With the exception of bombesin receptors (BBR) that are absent from nematodes, a similar gene complement was found. In arthropods, rhodopsin GPCR evolution is characterized by species-specific gene duplications and deletions and in nematodes by gene expansions in species with a free-living stage and gene deletions in representatives of obligate parasitic taxa. Based upon variation in GPCR gene number and potentially divergent functions within phyla we hypothesize that life style and feeding diversity practiced by nematodes and arthropods was one factor that contributed to rhodopsin GPCR gene evolution. Understanding how the regulation of food intake has evolved in invertebrates will contribute to the development of novel drugs to control nematodes and arthropods and the pests and diseases that use them as vectors
Modulation of sleep and activity in Drosophila: a systems biology approach. Genetics, pharmacology and high-throughput analysis of behaviour
Drosophila melanogaster is a widely used model organism which for the past 20 years
has been employed in a variety of contexts to understand aspects of sleep, activity and
more complex forms of behaviour. A challenge within the field of behaviour is how
to accurately classify and quantify behaviours that arise from an organism when these
behaviours are observed in different contexts. Technological advances have increased
the availability of quantitative tools which can be used to examine activity and sleep
behaviour. With the use of these tools, we can now answer new questions about the
underlying mechanisms of behaviour in different conditions. In the thesis herein, I have
examined activity and sleep behaviour in two different contexts, utilising some of these
new technological tools, including a novel activity monitoring device and statistical
classification techniques. In the first part of this thesis, I use this activity monitoring
system to elucidate some of the mechanisms involved in homeostatic sleep behaviour.
Specifically, I examine the effect of two different sleep deprivation methods on mated
and virgin Drosophila females to examine their responses in terms of homeostatic sleep
regulation. Using the same methodology and protocol, I then extend this work to exam-
ine the role of the neuropeptide, Corazonin, and its receptor, the Corazonin receptor,
in these contexts. In the second part of this thesis, I use the same activity monitor-
ing system to record the behavioural responses of flies exposed to different insecticide
compounds. I then use both a statistical classification technique and behavioural anal-
ysis to attempt to classify these compounds based on their mode of action (MoA) and
symptomology. Finally, I apply this methodology to answer other biological questions
of interest, classifying both rebound sleep and flies with varying genotypes.Open Acces