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.     

Learning to fear and cope with a natural stressor: individually and socially acquired corticosterone and avoidance responses to biting flies

Animals learn to recognize and respond to a variety of dangerous factors, with biting and blood-feeding flies being among the most prevalent of natural stressors. Here we describe the behavioral avoidance and hormonal (corticosterone) stress responses to biting fly exposure and the roles of individual and social learning in the acquisition of these fear-associated responses. Male mice exposed to a single 30-min session of attack by intact biting flies (stable fly, Stomoxys calcitrans L.) exhibited increased plasma corticosterone levels and active self-burying responses to avoid the flies. When exposed 24 h later to altered flies whose biting mouth parts were removed and were incapable of biting, the mice displayed conditioned increases in corticosterone and avoidance responses. This conditioned increase in corticosterone and self-burying was also acquired through social learning without direct individual experience with the intact biting flies. Fly naive "observer" mice that witnessed other "demonstrator" mice being attacked by biting flies, but were not exposed to intact flies themselves, displayed increases in corticosterone levels and self-burying to avoid flies when exposed 24 h later to altered flies. The social learning was not due to social facilitation or sensitization. Observers had to witness the self-burying avoidance responses of the demonstrator to the biting flies in order to subsequently recognize a potential threat to themselves and display the appropriate responses. These individually and socially acquired conditioned fear responses are likely part of the mechanisms that allow animals to defend themselves from biting and blood-feeding arthropods.     

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.     

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.     

Neurotensin and pain modulation

Neurotensin (NT) can produce a profound analgesia or enhance pain responses, depending on the circumstances. Recent evidence suggests that this may be due to a dose-dependent recruitment of distinct populations of pain modulatory neurons. NT knockout mice display defects in both basal nociceptive responses and stress-induced analgesia. Stress-induced antinociception is absent in these mice and instead stress induces a hyperalgesic response, suggesting that NT plays a key role in the stress-induced suppression of pain. Cold water swim stress results in increased NT mRNA expression in hypothalamic regions known to project to periaqueductal gray, a key region involved in pain modulation. Thus, stress-induced increases in NT signaling in pain modulatory regions may be responsible for the transition from pain facilitation to analgesia. This review focuses on recent advances that have provided insights into the role of NT in pain modulation.     

Exposure to a hypogeomagnetic field or to oscillating magnetic fields similarly reduce stress-induced analgesia in C57 male mice

Previous studies have shown that exposure to altered magnetic fields alters analgesic responses in a variety of species, including humans. Here we examined whether deprivation of the normally occurring geomagnetic field also affects stress-induced analgesia, by measuring the nociceptive responses of C57 male mice that were restraint-stressed in a hypogeomagnetic environment (inside a mu-metal box). Stress-induced analgesia was significantly suppressed in a manner comparable to that observed in mice that were either exposed to altered oscillating magnetic fields or treated with the prototypic opiate antagonist naloxone. These results represent the first piece of evidence that a period in a hypogeomagnetic environment inhibits stress-induced analgesia.     

A change in the alarm level entails a change in behavioural strategy of mice in stress and a change in analgesia induced by it

The effects of anxiogenic (pentylentetrazole) and anxiolytic (diazepam) agents on and cold swim stress-induced analgesia were investigated in SHR and NMRI male mice. It was shown that behavioral response to acute stress was associated with a change in the pain tolerance threshold. Diazepam increased immobility time and attenuated stress-induced analgesia (SIA). NMRI mice were more responsive to anxiolytic than the SHR mice, but the lattes manifested more dramatic changes when anxiety was pharmacologically enhanced (immobility time was significantly reduced and the SIA exaggerated). Our findings suggest that the main parameters change in reciprocal manner following a pharmacologically altered anxiety, and reveal that differences between two strains of mice are determined by differences in their sensitivity to stress.     

Behavioural responses of dairy cattle to the stable fly, Stomoxys calcitrans, in an open field environment

Individual cows (25 in each of four herds) were monitored 8-10 times weekly for 12 weeks (stable fly season) on a southern California dairy, with 100 observations per cow. The numbers of biting stable flies, Stomoxys calcitrans (L.) (Diptera: Muscidae) on the front legs and the frequencies of four fly-repelling behaviours per 2-min observation period [head throws, front leg stamps, skin twitches (panniculus reflex) and tail flicks] were recorded. Fly numbers varied, peaking at 3.0-3.5 flies per leg in week 9 (late May). Weekly herd mean frequencies of fly-repelling behaviours were highly dependent on fly numbers, with a linear regression r(2) > 0.8. Head throws and stamps were less frequent than skin twitches and tail flicks. Individual cows differed in numbers of stable flies and behaviours. Behaviours were correlated with flies for individual cows, but at a lower level than were herd means (r = 0.3-0.7). Cows that stamped more within a herd tended to have lower fly counts; other fly-repelling behaviours were less effective. Cows maintained ranks within a herd with regard to fly numbers (r = 0.47), head throws (0.48), leg stamps (0.64), skin twitches (0.69) and tail flicks (0.64). Older cows tended to harbour higher fly numbers and to stamp less relative to younger adult cows. Ratios of leg stamps and head throws to fly numbers dropped significantly through time, suggesting habituation to pain associated with fly biting. Tail flicks were not effective for repelling Stomoxys, but were easiest to quantify and may help in monitoring pest intensity. At this low-moderate fly pressure, no consistent impacts on milk yield were detected, but methods incorporating cow behaviour are recommended for future studies of economic impact.     

Expression of stress-evoked analgesia, connected with activity of animals in a forced swimming test]

The influence of forced swimming on the development of stress-induced analgesia was studied in 35 SHR mice, 65 NMRI mice, and 23 white outbred male rats. Mice were subjected to swimming conditions (at a temperature of 11 degrees C) for a period of 4 minutes and rats for 6 minutes. Pain thresholds were measured by a footshock. It was shown that behavioral response to acute stress is associated with a change in the pain tolerance threshold: activity of an animal under test conditions positively correlated with stress-induced analgesia. The response to stress and parameters of stress-induced analgesia depend on the genetic factor and age, however, the correlation between the activity during exposure to stress and the extent of stress-induced analgesia conserves in all cases.     

Sex differences in the effects of neuropeptide FF and IgG from neuropeptide FF on morphine- and stress-induced analgesia.

There is evidence suggesting that the endogenous mammalian octapeptide FLFQPQRFamide (F8Fa or neuropeptide FF, NPFF) has modulatory effects on opioid-mediated analgesia in rodents. There is also substantial evidence for sex differences in opioid analgesia, whereby male rats and mice display greater levels of opioid-mediated analgesia than females. In the present study, determinations were made of the effects of NPFF and IgG from antiserum against NPFF on morphine- and restraint stress-induced opioid analgesia in male and female deer mice. Intracerebroventricular (ICV) administrations of NPFF (0.10-10 micrograms) reduced in a dose-dependent manner morphine- and stress-induced analgesia in both male and female mice, with NPFF having markedly greater antagonistic effects in the male than female mice. Additionally, ICV administrations of NPFF-IgG increased the levels of morphine- and stress-induced analgesia and significantly reduced basal nociceptive sensitivity in male mice, whereas, in female mice, NPFF-IgG had no significant effects on either opioid-mediated analgesia or nociceptive sensitivity. These results indicate that there are sex differences in the modulatory effects of NPFF on opioid-mediated analgesia.     

Mouse strain differences in autonomic responses to stress

In humans, anxiety disorders are often accompanied by an overactive autonomic nervous system, reflected in increased body temperature (BT) and heart rate (HR). In rodents, comparable effects are found after exposure to stress. These autonomic parameters can give important information on stress and anxiety responses in mice. In the present experiments, stress reactivity of three frequently used mouse strains [129 Sv/Ev, Swiss Webster (SW) and C57 BL/6] was assessed using their autonomic stress responses. BT, HR and activity were telemetrically measured. Undisturbed circadian rhythms already showed clear differences between the mouse strains. Hereafter, autonomic responses to stressors with increasing intensity were measured. Strain differences were found in magnitude and duration of the stress responses, especially after high-intensity stressors. Generally, C57BL/6 mice showed the largest autonomic response, SW the lowest and the 129Sv/Ev the intermediate response. Interestingly, the observed ranking in autonomic stress response does not match the behavioral stress responsivity of these strains. Finally, sensitivity to the anxiolytic diazepam (0, 1, 2, 4 and 8 mg/kg) was tested using the stress-induced hyperthermia paradigm. Pharmacological sensitivity to diazepam differed between the strains with the 129Sv/Ev being most sensitive. These studies show that simultaneous measurement of behavioral and autonomic parameters under stressful conditions contributes considerably to a better interpretation of anxiety and stress levels in mice.     

Improved capture of stable flies (Diptera: Muscidae) by placement of knight stick sticky fly traps protected by electric fence inside animal exhibit yards at the Smithsonian's National Zoological Park

Stable flies are noxious blood‐feeding pests of exotic animals at zoological parks, inflicting painful bites, and causing discomfort to animals. Stable fly management is difficult because of the flies’ tendency to remain on the host animals only when feeding. Non‐toxic traps can be efficient but traps placed around exhibit perimeters captured fewer‐than‐expected numbers of flies. By surrounding traps with square electric fence enclosures, traps could be placed in the exhibits with the host animals and compared with an equal number of traps placed along perimeter fences. During a 21‐week study, traps inside exhibits captured 5× more stable flies than traps placed along exhibit perimeters. Traps inside exhibits tended to show more fluctuations in fly populations than traps along perimeters. The increased numbers of flies captured using this technique should provide relief from this pestiferous fly and greatly improve animal health and welfare. We believe this to be the first study where traps were used to capture stable flies in exhibit yards at a zoological park.     

Characterization of olfactory sensilla of Stomoxys calcitrans and electrophysiological responses to odorant compounds associated with hosts and oviposition media

Stable flies, Stomoxys calcitrans L. (Diptera: Muscidae), are economically important biting flies that have caused billions of dollars in losses in the livestock industry. Field monitoring studies have indicated that olfaction plays an important role in host location. To further our understanding of stable fly olfaction, we examined the antennal morphology of adults using scanning electron microscopy techniques. Four major types of sensillum were found and classified as: (a) basiconic sensilla; (b) trichoid sensilla with three subtypes; (c) clavate sensilla, and (d) coeloconic sensilla. No significant differences between male and female flies in abundances (total numbers) of these sensillum types were observed, except for medium-sized trichoid sensilla. The distinctive pore structures found on the surface of basiconic and clavate sensilla suggest their olfactory functions. No wall pores were found in trichoid and coeloconic sensilla, which suggests that these two types of sensillum may function as mechano-receptors. Details of the distributions of different sensillum types located on the funicle of the fly antenna were also recorded. Electroantennogram results indicated significant antennal responses to host-associated compounds. The importance of stable fly olfaction relative to host and host environment seeking is discussed. This research provides valuable new information that will enhance future developments in integrated stable fly management.     

Human head exposure to a 37 Hz electromagnetic field: effects on blood pressure, somatosensory perception, and related parameters

Previous studies have shown that exposure to an electromagnetic field (EMF) of 37 Hz at a flux density of 80 microT peak enhances nociceptive sensitivity in mice. Here we examined the effects on pain sensitivity and some indexes of cardiovascular regulation mechanisms in humans by measuring electrical cutaneous thresholds, arterial blood pressure, heart rate and its variability, and stress hormones. Pain and tolerance thresholds remained unchanged after sham exposure but significantly decreased after electromagnetic exposure. Systolic blood pressure was significantly higher during electromagnetic exposure and heart rate significantly decreased, both during sham and electromagnetic exposure, while the high frequency (150-400 mHz) component of heart rate variability, which is an index of parasympathetic activity, increased as expected during sham exposure but remained unchanged during electromagnetic exposure. Cortisol significantly decreased during sham exposure only. These results show that exposure to an EMF of 37 Hz also alters pain sensitivity in humans and suggest that these effects may be associated with abnormalities in cardiovascular regulation.     

Effects of stable flies (Diptera: Muscidae) and heat stress on weight gain and feed efficiency of feeder cattle.

Cattle respond to the feeding of stable flies, Stomoxys calcitrans (L.), by bunching to protect their front legs. This bunching can increase heat stress which indirectly accounts for much of the reduction in cattle weight gains. We used fly-screened, self-contained feedlot pens which allowed regulation of fly populations feeding on cattle. The indirect fly effects (bunching and heat stress) accounted for 71.5% of the reduced weight gain. The direct effect of the biting flies and energy loss involved in fighting flies accounted for 28.5% of the reduced weight gain.     

Visual targets for capture and management of house flies, Musca domestica L.

House flies and stable flies were collected on a Florida dairy farm using a commercial Alsynite sticky cylinder trap that was either used alone or covered with white, blue, or black outdoor awning fabric. Collections of both species of flies were highest on exposed Alsynite (house flies, 506.2 flies/day; stable flies, 19.1) followed by blue fabric (house flies, 308.1 flies/day; stable flies 12.5). Responses of both species to white and black fabric were 70% lower than to either of the former materials. When blue fabric was used to cover 50% of the surface area of Alsynite cylinders, house fly responses were significantly higher (290.2 flies/day) than to blue fabric alone (165.2); stable fly responses to the bi-colored target were significantly higher (152.6) than to Alsynite alone (93.8). Comparison of fly counts in the blue-covered versus uncovered Alsynite with traps of a single material indicated that house fly attraction to blue fabric was enhanced by the presence of clear Alsynite, whereas stable fly attraction to Alsynite was enhanced by the presence of blue fabric. The presence of blue+Alsynite visual targets increased collections of house flies in pans of dry fly bait but not in baited jug traps. Visual targets treated with 1.2% bifenthrin controlled >50% and 90% of house flies in large cages by days two and four after placement, respectively.     

Aversive Responses of Female Mice to the Odors of Parasitized Males: Neuromodulatory Mechanisms and Implications for Mate Choice

The detection and avoidance of parasitized conspecifics is proposed to have important consequences for the behavior of animals, especially as related to mate choice. A reduction in pain sensitivity (i.e. analgesia) is a major correlate of exposure to real or potential danger and threatening stimuli, facilitating the expression of various active (e.g. fleeing) and passive (e.g. immobilization) defense responses. The present study examined pain sensitivity (latency of a foot-lifting response to 50 ° C thermal surface) of female mice, Mus musculus, that were exposed to the urine and other odor secretions of male mice subclinically infected with the naturally occurring, enteric, sporozoan parasite, Eimeria vermiformis. A 30-min exposure to the odors of a parasitized male induced an analgesia in the female mice that was found to be mediated by the increased activity of endogenous opioid peptide systems. A brief 1-min exposure to the male odors induced a shorter duration and lower amplitude analgesia of a non-opioid (serotonergic) nature. Maximum analgesic responses were induced by the odors of pre-infective [5 days post-infection (PI)] and infective (day 10 PI) males, with significantly lower responses elicited by the odors of post-infective (day 17 PI) male mice. Exposure to the odors of unparasitized males had no significant effects on the pain sensitivity of female mice. These results indicate that female mice can distinguish between the odors of parasitized and non-parasitized male mice, and find the odors of parasitized males threatening and/or stressful. These odor-induced analgesic responses and their neurohormonal correlates may be part of an adaptive preparatory defense mechanism that facilitates the detection and avoidance of parasitized males by female mice and contributes to female mate choice.     

Magnetic fields inhibit opioid-mediated ‘analgesic’ behaviours of the terrestrial snail,Cepaea nemoralis

1. The terrestrial snail, Cepaea nemoralis, when placed on a warmed surface (40 degrees C) displays a thermal avoidance behaviour that entails an elevation of the anterior portion of the fully extended foot. The latency of this nociceptive response was increased by the prototypical mu and specific kappa opiate agonists, morphine and U-50, 488H, respectively, in a manner indicative of anti-nociception and the induction of 'analgesia'. Pretreatment with the prototypical opiate antagonist, naloxone, blocked the morphine- and reduced the U-50, 488H-induced analgesia. Naloxone had no effects on the thermal response latencies of saline treated animals. 2. Exposure to either cold (7 degrees C) or warm (38 degrees C) temperature stress increased the nociceptive thresholds of Cepaea in a manner indicative of the induction of 'stress-induced analgesia'. The warm stress-induced analgesia was opioid mediated, being blocked by naloxone, whereas, the cold stress-induced analgesia was insensitive to naloxone. 3. Exposure for 15-30 min to 0.5 Hz weak rotating magnetic fields (1.5-8.0 G) significantly reduced the analgesic effects of the mu and kappa opiate agonists in a manner similar to that observed with naloxone. The magnetic stimuli also inhibited the endogenous opioid mediated warm stress-induced analgesia and significantly reduced the cold stress-induced analgesia. The magnetic stimuli had no evident effects on the nociceptive responses of saline-treated animals. The dihydropyridine (DHP) and non-DHP calcium channel antagonists diltiazem, verapamil. and nifedipine differentially and significantly reduced, while the DHP calcium channel agonist, BAY K8644, significantly enhanced the inhibitory effects of the magnetic fields on morphine-induced analgesia.     

Salivary gland hypertrophy virus of house flies in Denmark: prevalence, host range, and comparison with a Florida isolate

House flies (Musca domestica) infected with Musca domestica salivary gland hypertrophy virus (MdSGHV) were found in fly populations collected from 12 out of 18 Danish livestock farms that were surveyed in 2007 and 2008. Infection rates ranged from 0.5% to 5% and averaged 1.2%. None of the stable flies (Stomoxys calcitrans), rat-tail maggot flies (Eristalis tenax) or yellow dung flies (Scathophaga stercoraria) collected from MdSGHV-positive farms displayed characteristic salivary gland hypertrophy (SGH). In laboratory transmission tests, SGH symptoms were not observed in stable flies, flesh flies (Sarcophaga bullata), black dump flies (Hydrotaea aenescens), or face flies (Musca autumnalis) that were injected with MdSGHV from Danish house flies. However, in two species (stable fly and black dump fly), virus injection resulted in suppression of ovarian development similar to that observed in infected house flies, and injection of house flies with homogenates prepared from the salivary glands or ovaries of these species resulted in MdSGHV infection of the challenged house flies. Mortality of virus-injected stable flies was the highest among the five species tested. Virulence of Danish and Florida isolates of MdSGHV was similar with three virus delivery protocols, as a liquid food bait (in sucrose, milk, or blood), sprayed onto the flies in a Potter spray tower, or by immersiion in a crude homogenate of infected house flies. The most effective delivery system was immersion in a homogenate of ten infected flies/ml of water, resulting in 56.2% and 49.6% infection of the house flies challenged with the Danish and Florida strains, respectively.