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Oxidative stress is commonly observed in both idiopathic and genetic cases of Parkinson's disease (PD). It plays an important role in the degeneration of dopaminergic neurons, and it has been associated with altered telomere length (TL). There is currently no cure for PD, and extracts of antioxidative plant, such as Mucuna pruriens and Withania somnifera, are commonly used in Ayurveda to treat patients with PD. In this study, we evaluated 2 enzymatic markers of oxidative stress, glutathione (GSH) system and superoxide dismutase (SOD), and TL in a Drosophila melanogaster model for PD [phosphatase and tensin homolog-induced putative kinase 1 (PINK1)B9]. This evaluation was also performed after treatment with the phytoextracts. PINK1B9 mutants showed a decrease in GSH amount and SOD activity and unexpected longer telomeres compared with wild-type flies. M. pruriens treatment seemed to have a beneficial effect on the oxidative stress conditions. On the other hand, W. somnifera treatment did not show any improvements in the studied oxidative stress mechanisms and even seemed to favor the selection of flies with longer telomeres. In summary, our study suggests the importance of testing antioxidant phytoextracts in a PINK1B9 model to identify beneficial effects for PD.-Baroli, B., Loi, E., Solari, P., Kasture, A., Moi, L., Muroni, P., Kasture, S., Setzu, M. D., Liscia, A., Zavattari, P. Evaluation of oxidative stress mechanisms and the effects of phytotherapic extracts on Parkinson's disease Drosophila PINK1B9 model.

Despite its great potentiality, little attention has been paid to modelling gastrointestinal symptoms of Parkinson's disease (PD) in Drosophila melanogaster (Dm). Our previous studies on standardized Mucuna pruriens extract (Mpe) have shown usefulness in the Drosophila model of PD. In this communication, we provide new information on the effect of Mpe on basal and serotonin treated contractions in the crop (i.e., an important and essential part of the gut) in Drosophila PD mutant for PTEN-induced putative kinase 1 (PINK1B9) gene. The effect of Mpe on PINK1B9 supplied with standard diet to larvae and/or adults, were assayed on 10–15 days old flies. Conversely from what we observed in the wild type flies, recordings demonstrated that exogenous applications of serotonin on crop muscles of untreated PINK1B9 affect neither the frequency nor the amplitude of the crop contraction, while the same muscle parameters are enhanced following brain injections of serotonin, thus suggesting that PINK1B9 mutants may likely have an impairment in the serotonergic pathways. Also, the mitochondrial morphology in the crop muscles is strongly compromised, as demonstrated by the transmission electron microscopy analysis. The Mpe treatment rescued the crop muscle parameters and also the mitochondrial morphology when supplied to both larvae and adults. Overall, this study strengthens the relevance of using PINK1B9 Dm as a translational model to study the gastrointestinal symptoms in PD and also confirms the useful employment of M. pruriens for PD treatment.

A morphofunctional investigation of the different neuronal subpopulations projecting through each of the nerves IV-VI emerging bilaterally from the terminal abdominal ganglion (TAG) was correlated with the octopaminergic activity in the ganglion that controls the ovipositor movements associated with calling behavior in the female gypsy moth Lymantria dispar. Tetramethylrodamine-dextran backfills from nerve stumps resulted in a relatively low number of TAG projections, ranging from 12 to 13 for nerve pair IV, 12 to 14 for nerve pair V, and 8 to 9 for nerve pair VI. Furthermore, as assessed by electrophysiological recordings, a number of fibers within each of these nerves displays spontaneous tonic activity, also when the ganglion is fully disconnected from the ventral nerve cord (VNC). Octopamine (OA) applications to the TAG strongly enhanced the activity of these nerves, either by increasing the firing rate of a number of spontaneously firing units or by recruiting new ones. This octopaminergic activity affected calling behavior, and specifically the muscle activity leading to cycling extensions of the intersegmental membrane (IM) between segments VIII and IX (ovipositor). Our results indicate that in the female gypsy moth the octopaminergic neural activity of the TAG is coupled with extensions and retractions of IM for the purpose of releasing pheromone, where motor units innervated by nerve pair IV appear antagonistic with respect to those innervated by nerve pair V.

The role of specific sex-related patterns in olfactory dysfunctions of Parkinson’s disease (PD) patients is unclear. The aim of this study was to assess the presence of specific sex-related patterns in olfactory dysfunctions excluding the possibility of confounding effects in patients with Parkinson’s disease. One hundred and sixty-eight participants (99 PD patients and 69 controls) were enrolled and evaluated using Sniffin’ Sticks Extended test (SSET). There was no significant sex difference in the control group for the SSET parameters. By contrast, in the PD group male patients scored significantly lower on odor discrimination (OD), identification (OI), and Threshold-Discrimination-Identification (TDI) score than females. On multivariable linear regression analysis, the only significant predictors of TDI score were sex and apathy. Among PD patients, men showed a significantly greater impairment compared to women in OI, OD and TDI score, but not in odor threshold (OT). These findings highlighted the possible role of sex differences in the development of associated PD non-motor symptoms.

Parkinson’s disease (PD) is one of the most common neurodegenerative diseases characterized by the clinical triad: tremor, akinesia, and rigidity. Several studies have suggested that PD patients show disturbances in olfaction as one of the earliest, nonspecific nonmotor symptoms of disease onset. We sought to use the fruit flyDrosophila melanogasteras a model organism to explore olfactory function in LRRKloss-of-functionmutants, which was previously demonstrated to be a useful model for PD. Surprisingly, our results showed that the LRRK mutant, compared to the wild flies, presents a dramatic increase in the amplitude of the electroantennogram responses and this is coupled with a higher number of olfactory sensilla. In spite of the above reported results, the behavioural response to olfactory stimuli in mutant flies is impaired compared to that obtained in wild type flies. Thus, behaviour modifications and morphofunctional changes in the olfaction of LRRKloss-of-functionmutants might be used as an index to explore the progression of parkinsonism in this specific model, also with the aim of studying and developing new treatments.

Findings from studies using animal models expressing amyotrophic lateral sclerosis (ALS) mutations in RNA-binding proteins, such as Transactive Response DNA-binding protein-43 (TDP-43), indicate that this protein, which is involved in multiple functions, including transcriptional regulation and pre-mRNA splicing, represents a key candidate in ALS development. This study focuses on characterizing, in a Drosophila genetic model of ALS (TDP-43), the effects of Mucuna pruriens (Mpe) and Withania somnifera (Wse). Electrophysiological and behavioural data in TDP-43 mutant flies revealed anomalous locomotion (i.e. impaired climbing with unexpected hyperactivity) and sleep dysregulation. These features, in agreement with previous findings with a different ALS model, were at least partially, rescued by treatment with Mpe and Wse. In addition, electrophysiological recordings from dorsal longitudinal muscle fibers and behavioral observations of TDP-43 flies exposed to the volatile anaesthetics, diethyl ether or chloroform, showed paradoxical responses, which were normalized upon Mpe or Wse treatment. Hence, given the involvement of some potassium channels in the effects of anaesthetics, our results also hint toward a possible dysregulation of some potassium channels in the ALS-TDP-43 Drosophila model, that might shed new light on future therapeutic strategies pertaining to ALS.

Oogenesis in most adult insects is a nutrient-dependent process involving ingestion of both proteins and carbohydrates that ultimately depends on peripheral input from chemoreceptors. The main goal of this study was to characterize, in the female blowfly Phormia regina, the responsive changes of the labellar chemoreceptors to carbohydrates and proteins in relation to four different stages along the ovarian cycle: (1) immature ovaries, (2) mid-mature ovaries, (3) mature ovaries and ready for egg-laying and (4) post egg-laying ovaries. Then, the possible effects exerted by exogenous serotonin on the chemoreceptor sensitivity profiles were investigated. Our results show that ovary length, width and contraction rate progressively increase from stage 1 to 3, when all these parameters reach their maximum values, before declining in the next stage 4. The sensitivity of the labellar "sugar" chemoreceptors to both sucrose and proteins varies during the ovarian maturation stages, reaching a minimum for sucrose in stage 3, while that to proteins begins. Exogenous 5-HT supply specifically increases the chemoreceptor sensitivity to sugar at the stages 3 and 4, while it does not affect that to proteins. In conclusion, our results provide evidence that in female blowflies the cyclic variations in the sensitivity of the labellar chemosensilla to sugars and proteins are time-related to ovarian development and that during the stages 3 and 4 the responsiveness of the sugar cell to sucrose is under serotonergic control.

Phote-HrTH (Phormia terraenovae hypertrehalosemic hormone) has been demonstrated in the Diptera to be involved in flight metabolism, reproduction, and diapause. Each of these events needs the hormone's action and requirement for carbohydrates is the common denominator. In Diptera, carbohydrates are taken up during feeding by action of the cibarial pump and are then stored in the crop. Using adult Phormia regina, both a bioassay and electrophysiological recordings show that Phote-HrTH slows down or inhibits the crop lobe muscles (P5) and, at the same time, stimulates the muscles of the pump 4 (P4) involved in pushing fluids out of the crop and up into the midgut for digestion. The EC50 for P4 was in the nanomolar range while the IC50 for P5 was 1.4-75.1 pM. The effect of Phote-HrTH on P4/5 suggests that the peptide is important in coordinating the two pumps, which are involved in moving carbohydrates up into the midgut for digestion. The adult crop organ is an essential storage organ for carbohydrates and now should be considered an important structure capable of delivering nutrients to the midgut for digestion.

This study showed that in adult Drosophila melanogaster, the type of sugar-either present within the crop lumen or in the bathing solution of the crop-had no effect on crop muscle contraction. What is important, however, is the volume within the crop lumen. Electrophysiological recordings demonstrated that exogenous applications of serotonin on crop muscles increases both the amplitude and the frequency of crop contraction rate, while adipokinetic hormone mainly enhances the crop contraction frequency. Conversely, octopamine virtually silenced the overall crop activity. The present study reports for the first time an analysis of serotonin effects along the gut-brain axis in adult D. melanogaster. Injection of serotonin into the brain between the interocellar area shows that brain applications of serotonin decrease the frequency of crop activity. Based on our results, we propose that there are two different, opposite pathways for crop motility control governed by serotonin: excitatory when added in the abdomen (i.e., directly bathing the crop) and inhibitory when supplied within the brain (i.e., by injection). Finally, our results point to a double brain-gut serotonergic circuitry suggesting that not only the brain can affect gut functions, but the gut can also affect the central nervous system. On the basis of our results, and data in the literature, a possible mechanism for these two discrete serotonergic functions is suggested.

Bioassays and electrophysiological recordings were conducted in the adult blowfly Phormia regina to provide new insights into the regulatory mechanisms governing the crop filling and emptying processes of the supercontractile crop muscles. The cibarial pump drives ingestion. Simultaneous multisite extracellular recordings show that crop lobe (P5) distension during ingestion of a 4.7 μl sugar meal does not require muscle activity by any of the other pumps of the system. Conversely, pumping of fluids toward the anterior of the crop system during crop emptying is brought about by active muscle contraction, in the form of a highly coordinated peristaltic wave starting from P5 and progressively propagating to P6, P4 and P3 pumps, with P5 contracting with a frequency about 3.4 times higher than the other pumps. The crop contraction rate is also modulated by hemolymph-borne factors such as sugars, through ligand recognition at a presumptive receptor site rather than by an osmotic effect, as assessed by both behavioural and electrophysiological experiments. In this respect, sugars of equal osmolarity produce different effects, glucose being inhibitory and mannose ineffective for crop muscles, while trehalose enhances crop activity. Finally, voltage and current clamp experiments show that the muscle action potentials (mAPs) at the P4 pump are sustained by a serotonin-sensitive calcium conductance. Serotonin enhances calcium entry into the muscle cells and this could lead, as an indirect modulatory effect, to activation of a Ca2+-activated K+ conductance (IK(Ca)), which sustains the following mAP repolarization phase in such a way that further mAPs can be generated early and the frequency consequently increased.

BACKGROUND The gypsy moth Lymantria dispar L. is a widespread pest that causes economic damage to cork oak forests. Females produce the sex pheromone (+)-(7R,8S)-epoxy-2-methyloctadecane, known as (+)-disparlure [(+)D], for long-distance attraction of conspecific males. A (+)D analogue, 2-decyl-1-oxaspiro[2.2]pentane (OXP-01), neither stimulating nor attractive by itself, causes short-time inhibition of male response in a 1:1 blend with (+)D. The authors investigated whether and how the biological activity of the natural pheromone is affected by OXP-01 on a long-time basis (up to 16 days), also by looking at possible physicochemical reciprocal interactions. RESULTS Blending of (+)D with OXP-01 decreased, under low evaporation rate, the pheromone effectiveness, as assessed by electroantennogram recordings. In male trappings, within the first 24 h, OXP-01 decreased and later enhanced the blend attractiveness, but only under high evaporation rate. Gas chromatography–mass spectroscopy indicates that quantitative retrieval of (+)D from blend cartridges is higher than for pure pheromone, and nuclear magnetic resonance measurements show that OXP-01 produces, possibly by Van der Waals interactions, a bimolecular entity with pheromone causing retention and lengthening of its attractiveness over time. CONCLUSION The biological and physicochemical interactions between (+)D and OXP-01 may provide valuable information for the optimisation of pheromone-based control strategies for gypsy moths. © 2013 Society of Chemical Industry

The responses of olive fly (Bactrocera oleae) antennal and palpal olfactory receptors to odors emitted by Pseudomonas putida bacterial filtrate and to volatiles from a host plant were evaluated using electrophysiological and behavioral bioassays. Morphological identification of olfactory receptors was also performed. The third antennal segment (flagellum) bears four types of multiporous sensilla: trichoid, short basiconica, clavate and grooved. Maxillary palps have mechanosensory bristles and multiporous basiconica sensilla. In wind-tunnel bioassays, olive fly responses to volatiles emitted by bacterial filtrate were higher than those to culture medium. Bacterial filtrate was more attractive than ammonium carbonate or a mixture of ethyl acetate and acetic acid in ethanol. GC-MS of bacterial filtrate identified some of the chemicals produced by bacterial activity, including methyl thiolacetate, ammonia, 2-pentanone, 2-heptanone, ethyl tiglate and methyl thiocyanate. Electrophysiological investigations proved that antennal sensilla are responsive to bacterial filtrate odor, methyl thiolacetate, olive leaves and olives, as well as to α-pinene, while acetic acid elicited an inhibitory response. Electropalpgrams recorded a specific response to bacterial filtrate by mated males and females, as well as a dose-dependent response relationship to methyl thiolacetate by mated females. The identification of new active volatile compounds in the semiochemical system of the olive fly is promising for the development of innovative control strategies in area-wide management.

The present study was aimed at characterizing the effects of Withania somnifera (Wse) and Mucuna pruriens (Mpe) on a Drosophila melanogaster model for Amyotrophic Lateral Sclerosis (ALS). In particular, the effects of Wse and Mpe were assessed following feeding the flies selectively overexpressing the wild human copper, zinc-superoxide dismutase (hSOD1-gain-of-function) in Drosophila motoneurons. Although ALS-hSOD1 mutants showed no impairment in life span, with respect to GAL4 controls, the results revealed impairment of climbing behaviour, muscle electrophysiological parameters (latency and amplitude of ePSPs) as well as thoracic ganglia mitochondrial functions. Interestingly, Wse treatment significantly increased lifespan of hSDO1 while Mpe had not effect. Conversely, both Wse and Mpe significantly rescued climbing impairment, and also latency and amplitude of ePSPs as well as failure responses to high frequency DLM stimulation. Finally, mitochondrial alterations were any more present in Wse- but not in Mpe-treated hSOD1 mutants. Hence, given the role of inflammation in the development of ALS, the high translational impact of the model, the known anti-inflammatory properties of these extracts, and the viability of their clinical use, these results suggest that the application of Wse and Mpe might represent a valuable pharmacological strategy to counteract the progression of ALS and related symptoms.

Bioassays and electrophysiological recordings were conducted to determine the role of serotonin and calcium on the supercontractile pump muscles of the diverticulated crop of adult blowflies. Using in situ crop preparations, serotonin was found to significantly increase the rates of contractions of a specific pump in the crop wall, pump P4. The addition of the serotonin antagonist, mianserin, or calcium free saline, both significantly reduced the contraction rates of this pump. Recordings, using suction electrodes from pump P4, confirm the in situ bioassay data and show that serotonin promotes muscle activity in empty crops in which no pump activity is normally observed. Moreover, our data indicate the crucial role of extracellular calcium ions in crop pump contractile activity. These results provide new information on how the crop of adult dipterans is modulated and suggest that serotonin, possibly supplied by neurons in the thoracico-abdominal neural plexus, may be involved in modulating the pumping of crop contents into the midgut for digestion or triggering antiperistalsis from the foregut in the process known as regurgitation or 'bubbling'.

The aim of the present study was to examine the presence and the possible role of abdominal olfactory sensilla in Culicoides imicola mediating the search for potential hosts and oviposition sites, by means of a morphological, electrophysiological and behavioural approach. The results reported here show that in the midge C. imicola the whole abdomen, comprising the ovipositor, are endowed with three morphotypes of multiporous sensilla that display olfactory sensitivity towards kairomones related to the host-animal skin such as L: -(+)-lactic acid and 1-octen-3-ol, to the host-animal urine such as 3-ethylphenol and 4-propylphenol, and to the potent attractant sesame seed oil. Electrophysiological and behavioural data for the first time suggest in the midge the involvement of abdominal olfactory structures in the choice of the oviposition sites and allow in discussing their possible role in the host-animal localisation. Field experiments showed that light traps baited with the aforementioned compounds elicited a stronger degree of attractiveness on midges with respect to the unbaited traps (control), although to a different extent. Our results, while implying a number of considerations concerning the role of molecules tested as kairomones, also suggest their use in the control of the midge C. imicola population.

A number of oxaspiropentane derivatives (OXPs) were tested as potential (+)-disparlure analogues, with the aim of identifying any possible interaction of these compounds, be it additive, synergetic, or inhibitory, with the pheromone response in the male gypsy moth Lymantria dispar. As assessed by male electroantennograms, 2 OXPs, 2-decyl-1-oxaspiro[2.2]pentane (OXP-01) and 4-(1-oxaspiro[2.2]pent-2-yl)butan-1-ol (OXP-04), were found to be effective. OXP-01 had no stimulatory effect but strongly decreased the response to (+)-disparlure in a blend in a 1:1 ratio. By contrast, OXP-04 proved to be more stimulating than (+)-disparlure and also had an additive effect in the blend. Single-cell recordings from the sensilla trichoidea showed the activity of 2 cells, one of which responded to (+)-disparlure. OXP-01 reduced the stimulating effectiveness of pheromone by silencing the pheromone-responding unit when the 2 compounds were presented in blend, whereas OXP-04 mimicked the pheromone response, evidenced by exciting the pheromone-responding neuron when tested alone. Behavioral observations are in agreement with electrophysiological results.

The aim of this study was to examine the role of the olfactory system of the midge Culicoides imicola Kieffer as the major system mediating repellency to antihelminthic avermec- tins. Incidental observations indicate that treatment with Dectomax or Ivomec (commercial formula of the avermectins doramectin and ivermectin, respectively) protects sheep from in- fection by bluetongue (BT) viruses. Our electrophysiological data from midge antennae showed that the stimulating effectiveness of L-()-lactic acid, butanone, and sheep seece odor decreased after addition of avermectins. The results show that these antihelminthics affect the olfactory sensitivity of the insect toward the animal host by reducing the response to those compounds that attract the insect, consequently reducing the possibility of biting the sheep and thereby trans- ferring the BT virus.

The common fruit fly Drosophila melanogaster (Dm) is a simple animal species that contributed significantly to the development of neurobiology whose leucine-rich repeat kinase 2 mutants (LRRK2) loss-of-function in the WD40 domain represent a very interesting tool to look into physiopathology of Parkinson’s disease (PD). Accordingly, LRRK2 Dm have also the potential to contribute to reveal innovative therapeutic approaches to its treatment. Withania somnifera Dunal, a plant that grows spontaneously also in Mediterranean regions, is known in folk medicine for its anti-inflammatory and protective properties against neurodegeneration. The aim of this study was to evaluate the neuroprotective effects of its standardized root methanolic extract (Wse) on the LRRK2 loss-of-function Dm model of PD. To this end mutant and wild type (WT) flies were administered Wse, through diet, at different concentrations as larvae and adults (L+/A+) or as adults (L-/A+) only. LRRK2 mutants have a significantly reduced lifespan and compromised motor function and mitochondrial morphology compared to WT flies 1% Wse-enriched diet, administered to Dm LRRK2 as L-/A+and improved a) locomotor activity b) muscle electrophysiological response to stimuli and also c) protected against mitochondria degeneration. In contrast, the administration of Wse to Dm LRRK2 as L+/A+, no matter at which concentration, worsened lifespan and determined the appearance of increased endosomal activity in the thoracic ganglia. These results, while confirming that the LRRK2 loss-of-function in the WD40 domain represents a valid model of PD, reveal that under appropriate concentrations Wse can be usefully employed to counteract some deficits associated with the disease. However, a careful assessment of the risks, likely related to the impaired endosomal activity, is required.

Taste sensitivity in response to diet components was studied in larvae of the gypsy moth, Lymantria dispar, by means of electro‐physiological and behavioural approaches. Dose‐response rela tionships were studied for NaCl, sucrose, and nicotine solutions with the aim of better characterizing chemoreceptor cells involved in gustation. Results showed that medial sensilla styloconica in the galea are specifically sensible to deterrent substances like salt and nicotine; instead, lateral sensilla showed a marked sensitivity to sugars. Morphological and electrophysiological data on the possi ble role of the sensilla basiconica of the maxillary palps are also reported. On the basis of the results obtained, it is proposed that taste chemoreceptor cells in the gypsy moth should be catego rized as phagostimulatory or deterrent on the basis of their behav ioural effects, rather than of their responding to specific stimuli.

Blowflies respond to sugars, salts, and water through the activation of specific chemoreceptor neurons in the labellar taste chemosensilla. These insects also detect deterrent stimuli, but identification of a specific “deterrent” chemoreceptor within their sensilla has been elusive. Here electrophysiological evidence is provided that the so-called “fifth” cell in taste chemosensilla of blowflies responds to deterrent compounds, such as quinine, amiloride, nicotine, and caffeine, which are also known to be bitter tasting for vertebrates. Therefore, comparison of behavioral and electrophysiological data, including crossadaptation analysis, suggests that the blowfly can detect “bitter” stimuli by activation of the “fifth” cell. A possible chemoreception mechanism is discussed.

The feeding of Aedes aegypti (L.) on blood is induced by the presence of phagostimulants: adenine nucleotides. Three chemoreceptive cells in the labral apical sensilla can distinguish the presence of adenine nucleotides depending on the other stimulus components. This work aims at correlating the sensory information arising from the labral apical sensilla with the feeding behavior in response to the same stimuli. The saline stimulating solution, containing adenine nucleotides, is modulated by changing one of the following components: salt concentration, buffer or pH. Cell 3 that responds to NaCl in a dose dependent manner seems to have another unique modality. The response of this cell is unaffected by ATP when the stimulating solution is NaCl buffered by NaHCO3. It responds at a higher spike frequency to the presence of ATP in a NaCl solution without NaHCO3. Thus in the presence of ATP Cell 3 detects whether the NaCl solution is buffered by NaHCO3. Both the blood feeding response and the sensory information from Cell 2 (which responds at high spike frequencies to the presence of ATP) are modulated by pH in a similar way. Both responses present a bi-modal response, with a major peak at pH 4.0 and a moderate peak at the most alkaline pH value tested.

The phagostimulants from the cellular fraction of blood induce gorging of Aedes aegypti (L.), and this process is enhanced by some plasma components. This project examines the responses of the labral apical chemoreceptors to plasma components enhancing phagostimulation. From the electrophysiological responses of the labral apical chemoreceptors four cells were identified by the waveform of their action potentials. Three of the cells (Cell 2, Cell 3 and Cell 4) responded in a dose dependent manner to NaCl. The responses of Cell 2 and Cell 3 to NaCl concentrations from 1 to 500 mmol/l can be described by a logarithmic equation. The response of Cell 2 to 150 mmol/l NaCl is modulated when a buffer is added. The magnitude of the modulation of the response is determined by the nature of the buffer: NaHCO3 inhibits while Na2HPO4 enhances the response. High osmotic pressure inhibits the response of Cell 4, regardless of how it is achieved. Cell 4 responds with a high frequency to the presence of l -alanine, the C-terminal amino acid of albumin, but shows a reduced response to the same concentration of albumin. From these results it can be concluded that labral apical chemoreceptors of A. aegypti are capable of detecting the plasma components involved in blood recognition.

The gypsy moth Lymantria dispar, like many other forest defoliator species, exhibits periodic population outbreak dynamics, whose variability in different years and in different places can be attributed to different microclimatic parameters. This study was focused on the correlation between climatic variations in two different localities of Sardinia over a three‐year period and on changes in sensitivity of the male antennal olfactory receptors. Our electrophysiological results show that olfactory EAG responses to the sex pheromone of the antennae are inversely correlated with rainfall during gypsy moth larval development. On the other hand, male antennal sensitivity and ambient temperature were not correlated, possibly due to the fact that no significant climatic variations were detected in the period examined. Our findings, especially for rainfall, are in good agreement with data in the literature concerning the effects of weather on gypsy moth population dynamics.

The role of amiloride in the labellar responses to various taste stimuli in the blowfly Protophormia terraenovae was studied with the aim of ascertaining whether amiloride-sensitive cation conductances are present in the chemosensory systems of insects. Results include that: 1) amiloride has no effect on the "salt" cell response to any stimulus; 2) amiloride decreases the "sugar" cell response to fructose, but does not affect that to sucrose; 3) the effects of amiloride on the responses of the "water" cell and the "fifth" cell are less clearly definable, due to the probable superimposition of osmotic mechanisms in the former and the poorly known response modalities of the water. In conclusion, amiloride-sensitive receptor sites seem to exist also in insects. However, unlike most vertebrates investigated, they are principally located in the sugar receptor cell, not on the salt cell.

Electrophysiological responses to NaCl in the concentration range 1–1000 mM were obtained from each of the 11 intermediate and 13 large labellar chemosensilla in the blowfly Protophormia terraenovae . Up to three of the four chemoreceptor cells associated with each chemosensillum responded to NaCl in both sensillum types; the spike discharges of two of these cells were analysed and cross-compared. Differences in chemoreceptor sensitivity and response pattern resulted between intermediate and large sensilla. Sensitivity differences were found, by means of the cluster analysis, within each sensillum type in relation to sensillar topology and are discussed in the light of a possible spatial contribution to the sensory coding process.

Electrophysiological recordings show that the hair-like sensilla on the tip of the labrum in Culex pipiens L. have neurones that respond to adenine nucleotides. Adenine nucleotides show the following descending order of stimulating effectiveness: ADP > ATP=AMP-PNP=AMP. Sensillar sensitivity to these adenine nucleotides were tested at the concentration range of 10 −5 –10 −3 M. These electrophysiological data are in good accordance with data reported from behavioural studies of this species.

The results of the present study demonstrate that series of gustatory stimulations can induce shortening of the cardiac cycle in the blowfly, Protopbormia terraenovae. This effect is due to a decrease in duration of one of the two phases which compose the cardiac cycle, that is the Fast‐phase. No changes in action potential frequency of either phase were evoked by stimulation. The data are discussed in terms of sensory‐induced changes in hemolymph circulation, which may function to optimize the insect's performance with respect to a state of alertness.

The study on transduction mechanisms underlying bitter stimuli is a particularly intriguing challenge for taste researchers. The present study investigates, in the labellar chemosensilla of the blowfly Protophormia terraenovae, the transduction mechanism by which saccharin evokes the response of the "deterrent" cell, with particular attention to the contribution of K(+) and Ca(2+) current and the role of cyclic nucleotides, since second messengers modulate Ca(2+), Cl(-) and K(+) currents to different extents. As assessed by extracellular single-sensillum recordings, our results show that the addition of a Ca(2+) chelator such as EGTA or the Ca(2+) current blockers SKF-96365, Mibefradil, Nifedipine and W-7 decrease the response of the "deterrent" cell to saccharin. A similar decreasing effect was also obtained following the addition of 4-aminopyridine, a K(+) current blocker. On the contrary, the membrane-permeable cyclic nucleotide 8-bromoguanosine 3',5'-cyclic monophosphate (8Br-cGMP) activates this cell and shows an additive effect when presented mixed with saccharin. Our results are consistent with the hypothesis that in the labellar chemosensilla of the blowfly both Ca(2+) and K(+) ions are involved in the transduction mechanism of the "deterrent" cell in response to saccharin. Our results also suggest a possible pathway common to saccharin and 8Br-cGMP.

An electrogenic K(+) transport in the tormogen cell of insect chemosensilla is involved in the generation and maintenance of the transepithelial potential (TEP). To gain more information about the K(+) transport system underlying the TEP generation and the location of its components in the plasma membrane of the tormogen cell, we studied the effects of inhibitors of K(+)/H(+) P-ATPase (bafilomycin A1, omeprazole and Na-orthovanadate), of K(+)/Cl(-) co-transport (bumetanide), of Cl(-) channels (NPPB) and of a K(+) channel blocker (BaCl(2)). The relationship between TEP amplitude and spike firing activity was also studied. Experiments were performed on the labellar chemosensilla of the blowfly Protophormia terraenovae using a modified tip-recording technique. Results show that: (a) K(+)/H(+) P-ATPase inhibitors significantly decrease the TEP, when properly applied to the labellum for 20 min, so as to reach the basolateral side of the plasma membrane, while no effect was detected when applied to the apical side, (b) bumetanide, NPPB and BaCl(2) decrease the TEP value only when administered to the apical side, (c) spike activity is positively correlated with the TEP. A model is proposed of the active and passive K(+) transports sustaining the TEP associated with the blowfly chemosensilla.

The electrophysiological response of labellar and tarsal chemosensilla in the blowfly Phormia regina was studied in response to a complex stimulus naturally encountered by flies such as sheep faeces, and to beef liver, a proteinaceous feeding source. Responses were investigated both before or after injection of clonidine, an octopamine agonist previously shown to enhance sucrose ingestion, while decreasing that of proteins. As assessed by single sensillum recordings, the four different chemosensory - "salt", "sugar", "deterrent" and "water" - cells were all activated by both stimuli, regardless of sex and sensillum type, the "sugar" one being in all cases the most sensitive to beef liver before clonidine injection. Clonidine treatment affected neither labellar nor tarsal sensitivity to sucrose. Conversely, clonidine-injected flies showed a significant increase in the activity of the "deterrent" cell to beef liver, thus accounting for a decrease in protein ingestion. This study for the first time provides evidence of a key role of a clonidine-sensitive peripheral taste sensitivity in down-regulation of protein ingestion in blowflies. Correlation between peripheral sensitivity and behavioural output is discussed.

By means of electroantennogram recording techniques, we have monitored the antennal olfactory sensitivity, from the time of eclosion until complete egg maturation, of female blowflies (Phormia regina) fed either a protein diet or a protein free one. The tested stimuli were swormlure-4 (SL-4) which is a potent lure for calliphorid flies, 1-hexanol as a reference stimulus and air as a control. After taking the electroantennograms, egg and ovarian development were evaluated. Stimulation with SL-4 and 1-hexanol evoked electroantennograms increasing in amplitude with age regardless of whether or not flies were fed protein during the first 5 days of adult life. In the protein-fed flies eggs were fully developed whereas those of the no-protein group remained undeveloped. The peak olfactory sensitivity occurs at a time when the female fly begins to search for an oviposition substrate.

A morpho-functional investigation of the sex pheromone-producing area was correlated with the pheromone release mechanism in the female gypsy moth Lymantria dispar. As assessed by male electroantennograms (EAG) and morphological observations, the pheromone gland consists of a single-layered epithelium both in the dorsal and ventral halves of the intersegmental membrane between the 8th and 9th abdominal segments. By using the male EAG as a biosensor of real-time release of sex pheromone from whole calling females, we found this process time coupled with extension movements of the ovipositor. Nevertheless, in females in which normal calling behavior was prevented, pheromone release was detected neither in absence nor in presence of electrical stimulation of the ventral nerve cord/terminal abdominal ganglion (TAG) complex. Tetramethylrhodamine-conjugated dextran amine stainings also confirm the lack of any innervation of the gland from nerves IV to VI emerging from the TAG. These findings indicate that the release of sex pheromone from the glands in female gypsy moths is independent of any neural control exerted by the TAG on the glands, at least by way of its three most caudally located pairs of nerves, and appears as a consequence of a squeezing mechanism in the pheromone-producing area.

The biogenic amine octopamine is known to be present in the abdominal ganglia of some insects, but the expression of functional octopamine receptors in these neuronal structures has not yet been characterized. In the present study, we describe the presence in the female gypsy moth terminal abdominal ganglion (TAG), a key structure in the control of the insect reproductive behavior, of an octopamine receptor coupled to stimulation of adenylyl cyclase through the GTP-binding protein G(s). The rank order of potency of different antagonists, which discriminate between the different classes of octopamine receptors, indicated the involvement of the neuronal type 3 receptor. The octopamine-stimulated adenylyl cyclase activity was inhibited by Ca(2+) in the low micromolar range and by activation of either protein kinase A or protein kinase C. In the isolated TAG, bath application of octopamine caused an increase of the spontaneous bursting activity of the emerging nerve of the 5th pair (V), whereas the antagonist mianserin reduced the nerve spiking activity and blocked the stimulatory effect of octopamine. These data demonstrate that the gypsy moth TAG expresses functional octopamine receptors, which may participate in the neuronal control of the insect reproductive behavior.

The present study investigates the effects of W-7 (a calmodulin antagonist involved in the Ca(++) cascade) on the response of the 'sugar' and 'water' cells of labellar chemosensilla in the blowfly Protophormia terraenovae to stimulation with sucrose or fructose. In order to ascertain whether Ca(++) conductance is involved, the effects of EGTA, one of the most used Ca(++) chelating agent, and of SK&F-96365, an inhibitor of receptor mediated calcium influx, were also studied. Our electrophysiological data indicate that W-7 addition strongly depresses the 'sugar' chemoreceptor response to both sugars and in the case of sucrose stimulation also influences adaptation rate. The Ca(++) chelator has no significant effects on the response of the 'sugar' cell following stimulation with sucrose, but lowers fructose stimulating effectiveness. In the presence of SK&F-96365 both sucrose and fructose responses are inhibited. A possible transduction mechanism for sugar reception is discussed.

The chemoreceptor spike activity in response to sucrose in the concentration range 1–500 mM was recorded from each of the 11 Intermediate and 13 Large labellar sensilla in the blowfly Protophormia terraenovae . The results showed that: (1) three of the four cells present in each sensillum are activated by sucrose stimulation; (2) differences between the Large and Intermediate types exist in the dose-response profiles of one of these cells (the `water' cell), possibly reflecting different sugar receptor site populations on the dendritic membranes of homologous cells in the two types; (3) sensilla of both types are differentially responsive to sucrose solutions according to their location on the labellum. These differences may provide elements for a spatial representation of the stimulus source within the sensory coding process.

The feeding of Aedes aegypti (L.) on blood and nectar is induced by phagostimulants: adenine nucleotides and sugars respectively. This work examines the responses of the four chemoreceptor cells in the labral apical sensilla to these phagostimulants. The apical chemoreceptors can detect the presence of adenine nucleotides. This part of the response is in good agreement with the gorging behavior. The output of the chemoreceptors cannot distinguish among different adenine nucleotides or among their concentrations (0.01–1 mmol/l), whereas gorging behavior is affected by the identity of adenine nucleotides and by their concentrations. Hence the gorging behavior cannot be driven by the output of these chemoreceptors alone. To the presence of adenine nucleotides Cell 2 was the only cell that responded with high frequencies, while the response of Cell 4 was almost abolished. The response of Cell 2 to ATP depended on the mosquito's physiological state. This dependence accorded well with the gorging behavior; Cell 2 responded with a higher frequency to ATP in the gorging state, than when not in a gorging state. The responses to sucrose and fructose constituted the only case recorded in which all these chemoreceptors failed to respond. This depression of response implies that other chemoreceptors must be present as sugar detectors.

In the attempt to gain more information on the mechanisms underlying bitter and/or sweet taste reception, we have investigated the responses of labellar chemosensilla in the blowfly Protophormia terraenovae to Na-saccharin, as compared to sweet stimuli (sucrose or fructose) and bitter stimuli (denatonium benzoate or amiloride). Electrophysiological and behavioral results indicate that the sweetener Na-saccharin inhibits the "sugar" cell in the labellar taste sensilla of the blowfly P. terraenovae. In multichoice preference tests, flies ingested more of the solutions containing sugar to those with sugar+Na-saccharin. This finding is in good agreement with the spike frequency reduction observed for the "sugar" cell activity. Analysis of the spike discharges also shows a positive dose-response for the "deterrent" cell following stimulation with Na-saccharin and denatonium benzoate. Flies drank any of the Na-saccharin solutions, regardless of their concentration, less than water, thus indicating a weak deterring effect on water drinking. The prevailing activation of the "deterrent" cell by stimulation with Na-saccharin is not directly coupled with a coherent behavioral output. Cross adaptation was found to occur between responses to Na-saccharin and denatonium benzoate or amiloride regardless of the order of adapting stimuli. In the case of sweet stimuli, cross adaptation occurred when the adapting stimulus was Na-saccharin, but it did not when the adapting stimuli were sucrose or fructose. Addition of Na-saccharin to both sugars significantly depressed the spike firing frequency, while an increase was observed with denatonium benzoate or amiloride.

The fruit fly Drosophila melanogaster (Dm) mutant for PTEN-induced putative kinase 1 (PINK1B9) gene is a powerful tool to investigate physiopathology of Parkinson's disease (PD). Using PINK1B9 mutant Dm we sought to explore the effects of Mucuna pruriens methanolic extract (Mpe), a L-Dopa-containing herbal remedy of PD. The effects of Mpe on PINK1B9 mutants, supplied with standard diet to larvae and adults, were assayed on 3-6 (I), 10-15 (II) and 20-25 (III) days old flies. Mpe 0.1% significantly extended lifespan of PINK1B9 and fully rescued olfactory response to 1-hexanol and improved climbing behavior of PINK1B9 of all ages; in contrast, L-Dopa (0.01%, percentage at which it is present in Mpe 0.1%) ameliorated climbing of only PINK1B9 flies of age step II. Transmission electron microscopy analysis of antennal lobes and thoracic ganglia of PINK1B9 revealed that Mpe restored to wild type (WT) levels both T-bars and damaged mitochondria. Western blot analysis of whole brain showed that Mpe, but not L-Dopa on its own, restored bruchpilot (BRP) and tyrosine hydroxylase (TH) expression to age-matched WT control levels. These results highlight multiple sites of action of Mpe, suggesting that its effects cannot only depend upon its L-Dopa content and support the clinical observation of Mpe as an effective medication with intrinsic ability of delaying the onset of chronic L-Dopa-induced long-term motor complications. Overall, this study strengthens the relevance of using PINK1B9 Dm as a translational model to study the properties of Mucuna pruriens for PD treatment.

Parkinson's disease (PD) is one of the most common neurodegenerative disease characterized by the clinical triad: tremor, akinesia and rigidity. Several studies have suggested that PD patients show disturbances in olfaction at the earliest onset of the disease. The fruit fly Drosophila melanogaster is becoming a powerful model organism to study neurodegenerative diseases. We sought to use this system to explore olfactory dysfunction, if any, in PINK1 mutants, which is a model for PD. PINK1 mutants display many important diagnostic symptoms of the disease such as akinetic motor behavior. In the present study, we describe for the first time, to the best of our knowledge, neurophysiological and neuroanatomical results concerning the olfactory function in PINK1 mutant flies. Electroantennograms were recorded in response to synthetic and natural volatiles (essential oils) from groups of PINK1 mutant adults at three different time points in their life cycle: one from 3-5 day-old flies, from 15-20 and from 27-30 days. The results obtained were compared with the same age-groups of wild type flies. We found that mutant adults showed a decrease in the olfactory response to 1-hexanol, α-pinene and essential oil volatiles. This olfactory response in mutant adults decreased even more as the flies aged. Immunohistological analysis of the antennal lobes in these mutants revealed structural abnormalities, especially in the expression of Bruchpilot protein, a marker for synaptic active zones. The combination of electrophysiological and morphological results suggests that the altered synaptic organization may be due to a neurodegenerative process. Our results indicate that this model can be used as a tool for understanding PD pathogensis and pathophysiology. These results help to explore the potential of using olfaction as a means of monitoring PD progression and developing new treatments.

1. 1. The addition of cAMP to stimulating solutions of NaCl, fructose (furanose sugar), sucrose, or glucose (pyranose sugars) decreases the responsiveness of labellar chemosensilla in Phormia . 2. 2. The addition of ATP, while decreasing the responsiveness to NaCl or fructose enhances the responsiveness to glucose and sucrose. 3. 3. The inhibiting effect of ATP on NaCl or fructose responses is suppressed by GDPsS, an inhibitor of adenylate cyclase (and thus of cAMP synthesis); moreover GDPsS further enhances the increase in response due to ATP when added to the sucrose or glucose solutions. 4. 4. Results suggest a possible involvement of cAMP and ATP in the taste reception mechanism in the blowfly.

EAGs were recorded from various locations on the flagellar surface of the antennae of the olive fly Dacus oleae stimulated with compounds of behavioural significance to this species. This information was compared with data on the typology and distribution patterns of the olfactory sensilla. Results showed that: a) 3 types of olfactory sensilla are present on the antennae: long basiconica, short grooved basiconica and trichodea; b) 1-hexanol, ethanol and volatiles belonging to the oily fraction of the olive pulp are the strongest stimuli, while those of the water fraction are little or not effective; c) EAG amplitude values vary as a function of the recording location on the flagellar surface; d) a significant correlation was found, for 2 stimuli of the oily fraction, between EAG amplitude and population density of long sensilla basiconica, thus suggesting this type as specifically responding to these substances. Resume Morphologie des antennes de Dacus oleae et localisation de leurs recepteurs olfactifs par electroantennogramme Des EAG ont ete obtenus en differents points de la surface de flagelles d'antennes de la mouche de l'olive, stimulees par des substances ayant une signification pour le comportement de cette espece. Ces informations ont ete comparees avec les donnees tirees de la typologie et de la distribution des sensilles olfactives. Les resultats mettent en evidence: a) 3 types de sensilles: longue basiconique, basiconique a petit sillon et trichodes; b) les stimuli les plus puissants de la pulpe sont l'1-hexanol, l'ethanol et les substances volatiles solubles dans l'huile, tandis que les substances hydrosolubles sont peu ou pas actives; c) une ampleur de l'EAG variant en fonction du lieu d'enregistrement sur la surface du flagelle; d) une correlation significative pour 2 stimuli liposolubles entre l'amplitude de l'EAG et la densite des longues sensilles basiconiques, ce qui suggere une reaction specifique de ce type de recepteur a ces substances.

Electrophysiologic techniques were used to study the bioelectri‐cal activity of the ovipositor chemosensilla of the apple maggot fly, Rhagoletis pomonella Walsh (Diptera, Tephritidae), in response to contact and volatile stimulation with various chemicals associated with fruits. The spike amplitude peaks evoked by contact stimuli showed that the chemosensilla are sensitive to glucose, malic acid, and quinic acid but not to fructose. There is evidence that ovipositor chemosensilla may provide information allowing glucose to be distinguished from the two acids. In addition, slow compound potentials were recorded in response to various volatiles associated with fruits. The contact stimulation experiments indicate that the ovipositor chemosensilla permit females to distinguish between a sugar and two acids that occur in apples in variable concentrations depending on the stage of ripeness, and that this difference may provide the fly with information about host suitability and/or quality.

In the present investigation it is shown that a reflex change in the heart activity of Protophormia flies is evoked by olfactory stimulation with several volatile substances, and particularly with those which are repellent for blowflies. Among these, i-pentanal and hexanal vapours evoked a fast, highly persistent cardiac response, whereas in the case of hexanol and ammonia vapours the response resulted slower and could be suppressed as a function of repeated stimulation.

1. 1. The stimulatory effect of a fructose (furanose sugar) solution on labellar taste chemosensilla in the blowfly Protophormia terraenovae is enhanced in the presence of GTP or cGMP. 2. 2. The increase in stimulating effectiveness due to cGMP is double with respect to that due to GTP. 3. 3. Chemosensillar responsiveness to the fructose solution is enhanced by the addition of an adenylate cyclase inhibitor, but is decreased by the addition of a phosphodiesterase inhibitor; these results are consistent with an inhibitory effect of cAMP.

1. 1. The addition of GTP and cGMP to an NaCl stimulating solution has an inhibitory effect on “cation” receptor cell responsiveness of taste chemosensilla in the blowfly, Protophormia terraenovae. 2. 2. The responsiveness of the same taste sensilla to an NaCl solution is enhanced by the addition of an adenylate cyclase inhibitor, but decreased by the addition of a phosphodiesterase inhibitor; this confirms the inhibitory effect of cAMP.

Using a modified “grease electrode”, we recorded extracellular spike activity from either member of each of the six pairs of peripheral nerves of the terminal abdominal ganglion in the female gypsy moth. Our results show that the three most caudally located nerve pairs (nerve pairs IV, V and VI) carry most of the spike activity related to the ovipositor movements associated with calling. The spike trains from nerve pair VI presented a temporal pattern which correlated most closely to the extension and retraction of the ovipositor during calling. Transection of the ventral nerve cord anterior to the terminal ganglion stopped neither the spike activity nor the calling behaviour, but some slight changes in both the spike frequency patterns and the duration of ovipositor extension and retraction were detected. We conclude that the terminal abdominal ganglion exhibits a considerable degree of autonomy in its control of the calling behaviour in the female gypsy moth.

Feeding enhances resitance of wing, tarsal and labellar hairs ofPhormia regina Meig. The observed increase in resistance might be related to variations in the secretory function of the accessory cells at the hair sockets. The importance of this fact in feeding control is discussed.

The effectiveness of labellar taste input in evoking the feeding response of labellar lobe spreading in Protophormia was greatly increased by injecting flies with clonidine or dl -octopamine. This effect was suppressed when either drug was administered together with yohimbine; when injected alone, the latter determined a strong decrease in the feeding responses. Drug treatment did not determine variations in labellar taste input or evoke spontaneous activity in the denervated muscle. These results suggest that the injected substances lower the feeding-related reflex threshold at the CNS level.

Stimulation with bovine serum albumin (BSA) evokes spikes discharges from three receptor cells of the labellar chemosensilla in Protophormia , the “sugar” cell being the most sensitive. Confrontation of the spike frequency profiles across all three chemoreceptor cells by the vector space analysis suggests that l -alanine (the C-terminal amino acid of the BSA molecule), but not l -aspartic acid (N-terminal amino acid), may account for the stimulator effectiveness of BSA.