Stable fly, Stomoxys calcitrans, mouthpart removal influences stress and anticipatory responses in mice

Abstract Biting fly attack induces a variety of stress and anxiety related changes in the physiology and behaviour of the target animals. Significant reductions in pain, or more appropriately, nociceptive sensitivity (latency of a foot-lifting response to an aversive thermal stimulus), are evident in laboratory mice after a 1 h exposure to stable flies, Stomoxys calcitrans. The role of the various components of biting fly attack in the development of this stress-induced reduction in pain sensitivity (analgesia) is, however, unclear. This study demonstrates that fly-naive mice do not exhibit a stress-induced analgesia when exposed to stable flies whose biting mouthparts have been removed. In contrast, mice that have been previously exposed to intact stable flies exhibit significant analgesia when exposed to flies that are incapable of biting. However, the level of analgesia induced is lower than that elicited by exposure to intact stable flies. Exposure to non-biting house flies, Musca domestica , has no effect on nociceptive sensitivity. It appears that the actual bite of the stable fly is necessary for the induction of analgesia and probably other stress and anxiety associated responses in fly naive mice. However, mice rapidly learn to recognize biting flies and exhibit significant, possibly anticipatory analgesic responses to the mere presence of biting flies.     

Exposure to stable flies reduces spatial learning in mice: involvement of endogenous opioid systems

Abstract. Biting flies influence both the physiology and behaviour of domestic and wild animals. This study demonstrates that relatively brief (60min) exposure to stable flies, Stomoxys calcitrans (L.), affects the spatial abilities of male mice. Stable fly exposure resulted in poorer subsequent performance in a water maze task in which individual mice had to learn the spatial location of a submerged hidden platform using extramaze visual cues. Determinations of spatial acquisition and retention were made with mice that had been previously exposed for 60min to either stable flies or house flies, Musca domestica (L.). Mice exposed to stable flies displayed over one day of testing (six blocks or sets of four trials) significantly poorer acquisition and retention of the water maze task than either mice that had been exposed to house flies or fly-naive mice. This attenuation of spatial learning occurred in the absence of any evident sensorimotor or motivational impairments. The reduction in spatial abilites involved endogenous opioid systems, as the decreased performance resulting from stable fly exposure was blocked by pre-treatment with the prototypic opiate antagonist, naltrexone. These results indicate that relatively brief exposure to biting flies can lead to a decrease in spatial abilities which is associated with enhanced endogenous opioid activity. These results support the involvement of endogenous opioid systems in the mediation of the behavioural and physiological effects of biting fly exposure. They further suggest that decreases in spatial abilities and performance may be part of the behavioural consequences of biting fly exposure in domestic and wild animals.     

Tsetse fly saliva biases the immune response to Th2 and induces anti-vector antibodies that are a useful tool for exposure assessment

Tsetse flies (Glossina sp.) are blood-feeding dipteran insects that transmit African trypanosomes, parasites that are responsible for human sleeping sickness and veterinary infections. Increasing attention is being paid to the effects of tsetse fly saliva deposited at the feeding site, which enables the blood-feeding process and putatively promotes parasite transmission. Here we demonstrate that saliva induces strong humoral responses against the major 43-45 kDa protein fraction (tsetse salivary gland proteins 1 and 2 - Tsal1 and Tsal2) in mice and humans and suppresses murine T and B cell responses to heterologous antigen. The saliva-induced immune response is associated with a Th2-biased cytokine profile and the production of mainly IgG1 and IgE antibody isotypes. Functionally, the antibodies raised in mice exposed to tsetse fly bites or induced after experimental saliva immunisation do not affect the fly's blood-feeding efficiency nor its survival. We propose that anti-saliva as well as anti-Tsal1/2 antibody responses can be used in epidemiological studies as a tool to analyze human exposure to tsetse flies.     

Evidence for activation of endogenous opioid systems in mice following short exposure to stable flies

Biting flies influence both physiology and behaviour of domestic and wild animals. This study demonstrates that brief (30 min) exposure of male and female mice to stable flies leads to significant increases in nociceptive responses, indicative of the induction of analgesia. The biting fly-induced analgesia was mediated by endogenous opioid systems as it was blocked by the prototypic opiate antagonist naloxone. Exposure for 30 min to the bedding of biting fly-exposed mice also induced significant opioid mediated analgesic responses in mice. Exposure to either house flies or the bedding of house fly-exposed mice had no significant effects on nociception. These results indicate that brief exposure to either stable flies, or to olfactory cues associated with mice exposed to stable flies, activates endogenous opioid systems leading to the induction of analgesia and likely other opioid mediated behavioural and physiological stress responses. These results suggest the involvement of endogenous opioid systems in the mediation of the behavioural and physiological consequences of biting fly exposure in domestic and wild animals.     

Variability in empathic fear response among 11 inbred strains of mice

Empathy is an important emotional process that involves the ability to recognize and share emotions with others. We have previously developed an observational fear learning (OFL) behavioral assay to measure empathic fear in mice. In the OFL task, a mouse is conditioned for context‐dependent fear when it observes a conspecific demonstrator receiving aversive stimuli. In the present study, by comparing 11 different inbred mouse strains that are commonly used in the laboratory, we found that empathic fear response was highly variable between different strains. Five strains – C57BL/6J, C57BL/6NTac, 129S1/SvImJ, 129S4/SvJae and BTBR T⁺ Itpr3^tf/J – showed observational fear (OF) responses, whereas AKR/J, BALB/cByJ, C3H/HeJ, DBA/2J, FVB/NJ and NOD/ShiLtJ mice exhibited low empathic fear response. Importantly, day 2 OF memory was significantly correlated with contextual memory in the classical fear conditioning among the 11 strains. Innate differences in anxiety, locomotor activity, sociability and preference for social novelty were not significantly correlated with OFL. Interestingly, early adolescent C57BL/6J mice exhibited an increase in acquisition of OF. The level of OFL in C57BL/6J strain was not affected by sex or strains of the demonstrator. Taken together, these data strongly suggest that there are naturally occurring OFL‐specific genetic variations modulating empathic fear behaviors in mice. The identification of causal genes may uncover novel genetic pathways and underlying neural mechanisms that modulate empathic fear and, ultimately, provide new targets for therapeutic intervention in human mental disorders associated with impaired empathy.     

Social learning of fear and safety is determined by the demonstrator's racial group

Social learning offers an efficient route through which humans and other animals learn about potential dangers in the environment. Such learning inherently relies on the transmission of social information and should imply selectivity in what to learn from whom. Here, we conducted two observational learning experiments to assess how humans learn about danger and safety from members (‘demonstrators'') of an other social group than their own. We show that both fear and safety learning from a racial in-group demonstrator was more potent than learning from a racial out-group demonstrator.     

Synergism Between Stress Responses Induced by Biting Flies and Predator Odours

We have previously shown that endogenous opioid systems (i.e. endorphins, enkephalins) are involved in the mediation of the behavioural and physiological effects of biting-fly exposure. Opioid mediated reductions in pain sensitivity, or, more appropriately, nociceptive sensitivity (latency of a foot-lifting response to a 50°C thermal surface), are evident in laboratory mice, Mus musculus domesticus, exposed to biting flies. Similar opioid-mediated reductions in pain sensitivity (opioid analgesia) are also seen after exposure to a variety of other natural stressors such as the threat of predation. Here, we demonstrate that brief (30-min) exposure of male mice to stable flies, Stomoxys calcitrans, significantly and synergistically augments either the concurrent or subsequent (60 min after fly exposure) analgesic effects induced by exposure to a predator (cat odour). These results demonstrate that the analgesic, and probably other opioid mediated behavioural and physiological stress responses induced by exposure to a relatively low number of biting flies, are markedly increased by the presence of another natural aversive stimulus. In addition, they show that biting-fly exposure significantly exacerbates the effects of a subsequent stressful stimulus. These findings raise a possible mechanism whereby exposure to a low number of biting flies, which by themselves may not appear to have a great impact, can have marked behavioural and physiological consequences.     

Big Baby,Little Mother: Tsetse Flies Are Exceptions to the Juvenile Small Size Principle

While across the animal kingdom offspring are born smaller than their parents, notable exceptions exist. Several dipteran species belonging to the Hippoboscoidea superfamily can produce offspring larger than themselves. In this essay, the blood-feeding tsetse is focused on. It is suggested that the extreme reproductive strategy of this fly is enabled by feeding solely on highly nutritious blood, and producing larval offspring that are soft and malleable. This immense reproductive expenditure may have evolved to avoid competition with other biting flies. Tsetse also transmit blood-borne parasites that cause the fatal diseases called African trypanosomiases. It is discussed how tsetse life history and reproductive strategy profoundly influence the type of vector control interventions used to reduce fly populations. In closing, it is argued that the unusual life history of tsetse warrants their preservation in the areas where human and animal health is not threatened.     

Conditioning odor-shock associations in the black blowfly,Phormia regina

Discriminative classical conditioning of an olfactory avoidance response was demonstrated in the blowfly, Phormia regina.Learning indices were calculated as the fraction of flies avoiding the CS+ (conditioned stimulus paired with electric shock reinforcement) minus the fraction of flies avoiding the CS-(conditioned stimulus not paired with electric shock), averaged over two different groups of flies, in which reciprocal odors were used as the CS+. Avoidance responses to both odors presented simultaneously at a T-maze choice point yielded mean learning indices of zero for naive flies or pseudoconditioned (shock alone) or sensitized (odors alone) controls. In contrast, pairing an odor with electric shock produced a mean learning index significantly greater than zero. These results are similar to those reported for Drosophila melanogaster.Blow flies, however, showed much lower levels of associative learning than fruit flies.     

The reactivity of the hypophyseal-adrenal system in rats with different forms of adaptive behavioral pathology]

The dexamethasone suppression test (DST) was applied to male Wistar rats with different models of depression: group with the learned helplessness, group with informational neurosis provided by time-deficit conditioned avoidance training, as well as groups of rats of two strains selected for low (KLA)--and high (KHA) avoidance learning. The pre-dexamethasone basal and stress-induced corticosterone levels were similar in intact rats and those exposed to inescapable shock. The dexamethasone administration (5 mkg/kg) failed to decrease the serum corticosterone level in rats with learned helplessness. The informational neurosis increased significantly the basal corticosterone level and decreased the stress response. Serum corticosterone levels were similar in KLA and KHA rats. These results give evidence that two stress-induced rat models of depression with similar behavioural disturbances (reduction of escape/avoidance reactions) exhibit marked differences in the activity of hypothalamo-pituitary-adrenal (HPA) axis.     

A new paradigm for operant conditioning of Drosophila melanogaster

A freely walking single fly (Drosophila melanogaster) can be conditioned to avoid one side of a small test chamber if the chamber is heated whenever the fly enters this side. In a subsequent memory test without heat it keeps avoiding the heat-associated side. The memory mutants dunce ¹ and rutabaga ¹ successfully avoid the heated side but show no avoidance in the memory test. Wildtype flies can be trained to successively avoid alternating sides in a reversal conditioning experiment. Every single fly shows strong avoidance and a positive memory score. The new conditioning apparatus has several advantages: (1) Statistically significant learning scores can be obtained for individual flies. (2) Learning scores are obtained fully automatically without interference of the experimenter. (3) The procedure is fast, robust and requires little handling. Therefore the apparatus is suitable for largescale mutant screening. (4) Animals are not attached to a hook and thus can easily be used for breeding.Abbreviations dnc dunce gene - PI performance Index - rut rutabaga gene - S.E.M. standard error of mean     

Leishmania chitinase facilitates colonization of sand fly vectors and enhances transmission to mice

Chitinases of trypanosomatid parasites have been proposed to fulfil various roles in their blood-feeding arthropod vectors but so far none have been directly tested using a molecular approach. We characterized the ability of Leishmania mexicana episomally transfected with LmexCht1 (the L. mexicana chitinase gene) to survive and grow within the permissive sand fly vector, Lutzomyia longipalpis. Compared with control plasmid transfectants, the overexpression of chitinase was found to increase the average number of parasites per sand fly and accelerate the escape of parasites from the peritrophic matrix-enclosed blood meal as revealed by earlier arrival at the stomodeal valve. Such flies also exhibited increased damage to the structure of the stomodeal valve, which may facilitate transmission by regurgitation. When exposed individually to BALB/c mice, those flies with chitinase-overexpressing parasites spent on average 2.4-2.5 times longer in contact with their host during feeding, compared with flies with control infections. Furthermore, the lesions that resulted from these single fly bite infections were both significantly larger and with higher final parasite burdens than controls. These data show that chitinase is a multifunctional virulence factor for L. mexicana which assists its survival in Lu. longipalpis. Specifically, this enzyme enables the parasites to colonize the anterior midgut of the sand fly more quickly, modify the sand fly stomodeal valve and affect its blood feeding, all of which combine to enhance transmission.     

Classical conditioning and retention in normal and mutantDrosophila melanogaster

Summary By changing the conditioned discrimination paradigm of Quinn et al. (1974) from an instrumental procedure to a classical (Pavlovian) one, we have demonstrated strong learning in type flies. About 150 flies were sequestered in a closed chamber and trained by explosing them sequentially to two odors in air currents. Flies received twelve electric shock pulses in the presence of the first odor (CS+) but not in the presence of the second odor (CS–). To test for conditioned avoidance responses, flies were transported to a Tmaze choice point, between converging currents of the two odors. Typically, 95% of trained flies avoided the shock-associated odor (CS+).Acquisition of learning was a function of the number of shock pulses received during CS+ presentation and was asymptotic within one training cycle. Conditioned avoidance increased with increasing shock intensity or odor concentration and was very resistant to extinction. Learning was best when CS+ presentations overlap shock (delay conditioning) and then decreased with increasing CS-US interstimulus intervals. Shocking flies immediately before CS+ presentation (backward conditioning) produced no learning. Nonassociative control procedures (CS Alone, US Alone and Explicitly Unpaired) produced slight decreases in avoidance responses, but these affected both odors equally and did not alter our associative learning index (A).Memory in wild-type flies decayed gradually over the first seven hours after training and still was present 24 h later. The mutantsamnesiac, rutabaga anddunce showed appreciable learning acquisition, but their memories decayed very rapidly during the first 30 min. After this, the rates of decay slowed sharply; conditioned avoidance still was measurable at least three hours after training.Abbreviations OCT 3-octanol - MCH 4-methylcyclohexanol - C-S Canton-Special - CS conditioned stimulus - US unconditioned stimulus     

Cooperative Blood-feeding and the Function and Implications of Feeding Aggregations in the Sand Fly,Lutzomyia longipalpis (Diptera: Psychodidae)

Given the importance that the evolution of cooperation bears in evolutionary biology and the social sciences, extensive theoretical work has focused on identifying conditions that promote cooperation among individuals. In insects, cooperative or altruistic interactions typically occur amongst social insects and are thus explained by kin selection. Here we provide evidence that in Lutzomia longipalpis, a small biting fly and an important vector of leishmaniasis in the New World, cooperative blood-feeding in groups of non-kin individuals results in a strong decrease in saliva expenditure. Feeding in groups also strongly affected the time taken to initiate a bloodmeal and its duration and ultimately resulted in greater fecundity. The benefits of feeding aggregations were particularly strong when flies fed on older hosts pre-exposed to sand fly bites, suggesting that flies feeding in groups may be better able to overcome their stronger immune response. These results demonstrate that, in L. longipalpis, feeding cooperatively maximizes the effects of salivary components injected into hosts to facilitate blood intake and to counteract the host immune defences. As a result, cooperating sand flies enjoy enormous fitness gains. This constitutes, to our knowledge, the first functional explanation for feeding aggregations in this species and potentially in other hematophagous insects and a rare example of cooperation amongst individuals of a non-social insects species. The evolution of cooperative group feeding in sand flies may have important implications for the epidemiology of leishmaniasis.     

Predictors of biting fly harassment and its impact on habitat use by feral horses (Equus caballus) on a barrier island

Feral horses on Assateague Island, Maryland, were observed in June and August 2000 to determine what behavioral and ecological factors affect the intensity of biting fly harassment and whether habitat use by horses was influenced by biting flies. Fly counts and frequencies of comfort movements (i.e., movements designed to dislodge insects) were recorded during focal animal samples, as well as data on sex, group size, habitat type, temperature, humidity, wind speed, and behavior. Seasonal habitat use patterns were assessed using 7 years of monthly census data on the horses. The number of biting flies on the horse was affected by horse sex, habitat, temperature, and group size. The number of comfort movements a horse showed was affected by habitat, temperature, wind speed, group size, and number of horses within one body length of the focal. The number of comfort movements made by a horse was found to be highly correlated with fly numbers. Though marshes were used most throughout the year, the pattern of use of dune, scrub, and human-altered habitats reflects a pattern of biting fly avoidance and refuge-seeking by the horses.     

The role of context, colour and location cues in socially learned novel food source preferences in starlings, Sternus vulgaris

Although the opportunity for errors in social learning is widely recognised, as yet little research has been directed towards understanding specific inaccuracies, biases and limitations in social learning and the mechanisms that give rise to them. In two experiments I ask how starlings, Sternus vulgaris, identify exemplars of novel feeders previously learned about socially. I find that starlings have a stronger response to feeders in the same context as that in which social learning took place, compared to identical and nonidentical feeders in a different context. Within a context that matches where social learning took place, starlings prefer feeders that show the same location and colour as the feeder demonstrated by the demonstrator starling, and show no preference when colour and location cues are dissociated. This suggests that starlings are relatively accurate social learners, since they show strong responses to novel foraging options only if they match the context, colour and location of options learned about socially, and they do so after very few trials. Furthermore, the responses of the subjects were compatible with conditioned learning-like mechanisms, which provide a useful basis for the further investigation of the origins and implications of errors in social learning.     

Effect of a cyclic analog of enkephalin on learning and memory in the mouse

In experiments on mice, the effect of cyclic analogue of enkephalin (CAE) on the processes of learning and memory was studied in control animals and in animals with changed functional state of monoaminergic brain systems. Administration of the peptide to intact animals significantly accelerated the acquisition of conditioned reflexes of two-way avoidance and did not significantly affect the retention of these reflexes and their subsequent reproduction. Retention and reproduction of conditioned reflexes elaborated in one combination, was disturbed. Administration of iprazid did not eliminate the "accelerating effect" of CAE on the formation of conditioned reflexes of the two-way avoidance but sharply disturbed their retention. In such conditions, the amnesing iprazid effect increased still more. Besides, under CAE effect, the activity of acetylcholinesterase in the motor and especially in the visual cortex of the mice increased. The obtained data testify to an important role of the monoaminergic and cholinergic brain mechanisms in realization of CAE effects on the processes of learning and memory.     

Impaired discrimination of and aversion to parasitized male odors by female oxytocin knockout mice

A major cost of social behavior is the increased risk of exposure to parasites, with animals utilizing social information to recognize and avoid infected conspecifics. In mice, females can discriminate between infected and uninfected males on the basis of social cues, displaying aversive responses to the odors of infected males. In the present study, using female mice whose gene for oxytocin (OT) has been selectively deleted (OT knockout mice (OTKO)), we show that at least one normal allele for OT is required for the mediation of the recognition and avoidance of parasitized males. Female wild type (OTWT) and heterozygous (OTHZ) mice distinguished between the odors of individual males infected with the louse, Polyplax serrata , and uninfected males while the KO mice did not. Exposure to the odors of infected males induced analgesia in OTWT and OTHZ females, with OTKO females displaying attenuated analgesia. OTWT and OTHZ females, but not the OTKO females, also distinguished between the odors of novel and familiar infected males and modulated their analgesic responses on the basis of prior familiarity. In an odor choice test, OTWT and OTHZ females displayed a marked initial choice for the odors of uninfected males, whereas the OTKO females showed no consistent choice. This impairment was specific to the odors of infected males. OTKO females displayed normal analgesic responses to another aversive social odor, that of a stressed male, and an aversive non-social odor, that of a cat. The OTKOs had normal non-social olfactory memory, but were impaired in their social odor memory. These findings indicate that a normal OT gene comprises an essential part of the central recognition mechanism whereby females can both reduce the transmission of parasites to themselves and select for parasite-free males.     

Increased corticosterone levels in mice subjected to the rat exposure test

In recent years, there has been a notable interest in studying prey-predator relationships to develop rodent-based models for the neurobehavioral aspects of stress and emotion. However, despite the growing use of transgenic mice and results showing important differences in the behavioral responses of rats and mice, little research has been conducted regarding the responses of mice to predators. The rat exposure test (RET), a recently developed and behaviorally validated prey-predator (mouse-rat)-based model, has proven to be a useful tool in evaluating the defensive responses of mice facing rats. To further validate the RET, we investigated the endocrine and behavioral responses of mice exposed to this apparatus. We first constructed a plasma corticosterone secretion curve in mice exposed to a rat or to an empty cage (control). Rat-exposed mice showed a pronounced rise in corticosterone levels that peaked 15 min from the beginning of the predator exposure. The corticosterone levels and behavioral responses of mice exposed to a rat or to a toy in the RET apparatus were then measured. We observed high plasma corticosterone levels along with clear avoidance behaviors represented by decreases in tunnel and surface area exploration and increases in risk assessment behaviors and freezing. This strongly suggests that the test elicits a repertoire of behavioral responses compatible with an aversion state and indicates that it is a promising model for the evaluation of prey-predator interactions. However, more physiological, neurochemical, and pharmacological studies are needed to further validate the test.     

Aggressive behaviours of female Drosophila melanogaster are influenced by their social experience and food resources

Abstract Aggressive behaviours of female Drosophila melanogaster (Meigen) (Diptera: Drosophilidae) were studied in the laboratory. These behaviours included 'approach', a seemingly intentional movement of a fly to another fly, 'lunge', a dash at another fly, and 'wings erect', a brief erection of wings toward another fly. These behaviours were influenced by social experience and food resources. First, event frequencies of aggressive behaviours were higher in flies that had been held in isolation than in those that had been held in crowded conditions. This difference was the result of the social experience of attacking flies and not that of attacked flies. Second, flies showed aggressive behaviours more frequently when they were on food containing yeast colonies than when they were on food without yeast colonies. Third, flies conditioned to a food solution containing live yeast prior to observation showed lower frequency of aggressive behaviours than flies conditioned to a boiled food solution. Development of aggressive behaviours became apparent one day after eclosion.