Purinoceptors in blood feeding behaviour in the stable fly, Stomoxys calcitrans

Abstract Both sexes of the stable fly, Stomoxys calcitrans L. (Diptera: Muscidae), have receptors on their mouthparts that mediate blood feeding. The potency ranking of the adenine nucleotides (ATP > ADP > AMP > adenosine) in eliciting feeding and suppressing the NaCl-sensitive cell may indicate the involvement of a P₂-type receptor. This is supported by the lack of effect on feeding by methyl xanthines. Feeding-related behavioural and electrophysiological results demonstrate that the potency of CH₃-S-ATP is not greater than that of ATP. These ATP-mediated responses are antagonized by ANAPP₃. Results support the conclusion that the putative 'ATP receptor' involved in stable fly phagostimulation resembles the P_2x purinoceptor of vertebrates.     

Circadian activity pattern in the stable fly, Stomoxys calcitrans

Abstract. Activity rhythms of Stomoxys calcitrans (L.) were assessed under constant conditions using actographs.In LD 12:12 h, activity was almost completely diurnal, had a 24 h period, and peaked 4 h after lights-on.In constant darkness, activity peaked in the early afternoon and the period of the rhythm was c. 26 h, indicating a circadian pacemaker.A delay in feeding shortened the free-running period in constant dark to c. 24 h.In constant light, flies were arrhythmic.Starvation also affected activity, with 24- and 48-h starved flies being as much as 20 times more active than recently fed flies.     

Electroantennogram responses of the stable fly, Stomoxys calcitrans, to components of host odour

Abstract. Electroantennograms (EAGs) were recorded from laboratory-reared male and female Stomoxys calcitrans (L.) in response to a range of synthetic chemicals known to be electrophysiologically-active for other biting flies. Of the eight compounds initially tested, only two - 1-octen-3-ol and 3-methylphenol - consistently elicited larger electroantennograms (EAGs) than did control treatments; 1-octen-3-ol was the most potent. EAG recovery time was inversely correlated with EAG amplitude. EAGs recorded with primary C₂-C₁₂ carbon chain-length primary aliphatic alcohols peaked at octan-1–ol with pentan-1-ol, hexan-1-ol and heptan-1-ol also eliciting EAG responses significantly larger than the controls. When different C₈ carbon chain compounds and nonane were tested: 1-octen-3-ol elicited the largest EAGs followed by, in decreasing activity, octan-1-ol, 1-bromooctane, octan-3-ol, octanal, 2-octanone, octanoic acid and nonane. The EAG response of 1-octen-3-ol increased sigmoidally with dose, with the threshold at between 2 and 20 ng, and the peak response at 200 μg on filter paper. EAGs larger than control were also elicited by entrained ox odour and ox breath. The behavioural implications are discussed.     

Characterization of mannosyl transferases during the pupal instar of Stomoxys calcitrans (L)

Particulate fractions (10,000g) from pupae of Stomoxys calcitrans transfer [¹⁴C]-mannose from GDP-[¹⁴C]-mannose to dolichol monophosphate and proteins. Production of the mannosyl lipid was inhibited by Mn²⁺, UDP, GMP, GDP, and EDTA. The insect growth regulator diflubenzuron had no effect on mannosyl transferase activity. Dolichol monophosphate and Mg²⁺ stimulated mannosyl transferase activity. The mannosyl lipid product was identified as mannosyl-phosphoryl-dolichol (Man-P-Dol). The apparent K_m and V_max values for the formation of Man-P-Dol using GDP-[¹⁴C]-Man while holding dolichol phosphate constant were 2.4 ± 0.9 μM and 9.4 ± 2.3 pmol Man-P-Dol·min^?1·mg^?1 protein, respectively. The apparent K_m and V_max values using dólichol phosphate while holding GDP-Man constant were 2.2 ± 1.2 μM and 18.5 ± 1.7 pmol Man-P-Dol·min^?1·mg^?1 protein.     

Sensory and behavioural responses of the stable fly Stomoxys calcitrans to rumen volatiles

Analysis of volatiles from rumen digesta by gas chromatography linked antennogram recordings from Stomoxys calcitrans (L) (Diptera: Muscidae) antennal receptor cells revealed about 30 electrophysiologically active constituents, the most important of which is dimethyl trisulphide with a sensory threshold in the femtogram range. The behavioural responses of S. calcitrans to five chemostimulants (dimethyl trisulphide, butanoic acid, p-cresol, oct-1-en-3-ol and skatole) were tested in a wind tunnel where activation and attraction of hungry flies to rumen volatiles were recorded. Dimethyl trisulphide, butanoic acid and p-cresol were found to attract S. calcitrans. This sensitivity to rumen volatile constituents, that also occur in animal wastes used for oviposition by Stomoxys spp., as well as in flowers used by stable flies as sources of nectar is discussed in the context of the behavioural ecology of these flies.     

N-acetylglucosaminyl transferases from the pupal instar of the stable fly,Stomoxys calcitrans

N-acetylglucosaminyl transferases from pupae of Stomoxys calcitrans (L.) were studied in 10,000g pellet suspensions. Characterization of these enzymes was based on formation of glycolipids (ie, Dol·PP-GlcNAc and Dol·PP-(GlcNAc)₂), oligosaccharide lipids, and giycoproteins. Studies on transferase activity during the pupal instar showed that there were two peaks of activity; the first peak was on day 0 (prepupae) and the second at 3 days after pupation. Subcellular fractionation indicated that 10,000g and 100,000g pellets contained most of the transferase activities. The transferases required divalent cations (either Mn²⁺ or Mg²⁺). The pH optimum, which varied for each of the products formed, was 7.5 for glycolipids, 7.0 for oligosaccharide lipids, and 6.5 for glycoprotein. Inclusion of dolichol monophosphate doubled the amount of Dol·PP-GlcNAc and Dol·PP·(GlcNAc)₂ formed, but had little effect on oligosaccharide lipid and glycoprotein formation. Tunicamycin was a potent inhibitor of glycolipid formation with an I₅₀ of 1.8–4.8 nM. It was confirmed that tunicamycin acts by preventing the transfer of GlcNAc-1-P from UDP-GlcNAc to Dol·P. UMP reverses glycolipid formation, yielding UDP-GlcNAc. Some characterization of the products was performed. Glycolipids were shown to be Dol·PP-GlcNAc and Dol·PP-(GlcNAc)₂. Glycoprotein was rapidly solubilized by protease and detergent treatments, whereas oligosaccharide lipids appeared to be acid-labile, pyrophosphate-containing lipids. The apparent kinetic constants for the formation of glycolipids were as follows: UDP-GlcNAc K_m = 1.55 ± 0.47 μM, V_max = 0.66 ± 0.21 pmol·min^?1·mg^?1; Dol·P K_m = 2.08 ± 0.85 μM, V_max = 0.13 ± 0.06 pmol·min^?1·mg^?1 protein.     

Absence of female-specific protein in the hemolymph of stable fly Stomoxys calcitrans (L.) (Diptera: Muscidae)

Changes, during the reproductive cycle, in fat body, hemolymph, and ovarian proteins of the stable fly Stomoxys calcitrans were characterized quantitatively and qualitatively using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein content of all three tissues increased after blood feeding. Fat body protein increased first, followed by hemolymph and ovarian proteins. SDS-PAGE failed to identify vitellogenin in both female hemolymph and fat body samples. No single protein or group of proteins predominated at any stage of the reproductive cycle. Comparisons between male and female stable fly hemolymph and fat body proteins failed to detect female-specific proteins. Female-specific proteins, however, were detected in the hemolymph of four other species of Diptera.     

Retention and attempted mechanical transmission of Ehrlichia risticii by Stomoxys calcitrans

Abstract. The ability of adult Stomoxys calcitrans (L.) (Diptera: Muscidae) to retain viable Ehrlichia risticii (Rickettsiaceae), the aetiologic agent of Potomac horse fever (PHF), and mechanically transmit the pathogen from citrated bovine blood artificially infected with E.risticii to susceptible mice was studied. Viable E. risticii were found in the digestive tract of S.calcitrans 3h after the flies had engorged to repletion on infected blood; however, no E. risticii were detected in flies ≥2 days after feeding. Subsequently, groups of S.calcitrans were fed for 20 s on infected blood, then fed to repletion on mice 30, 60,130, 180 or 220 min after having feeding interrupted. Mice displayed no clinical signs of PHF and did not produce anti- E. risticii antibodies when assayed 30 days after S. calcitrans had fed. Although S.calcitrans were able to harbour viable E.risticii for at least 3h, transmission of the disease agent to susceptible mice during interrupted feeding was not demonstrated under these experimental conditions.     

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.     

Electroantennogram responses of the stable fly, Stomoxys calcitrans, to carbon dioxide and other odours

ABSTRACT. Electroantennograms (EAGs) were recorded from Stomoxys calcitrans (L.) in response to increasing concentrations of carbon dioxide in an airstream. The magnitude of the EAG increases logarithmically from +0.023% carbon dioxide up to approximately +2.0% where a maximum is reached. Flies deprived of food for 48–50 h are more responsive to small increases in the carbon dioxide concentration than those deprived of food for only 20–23 h. It is concluded that the sensitivity of carbon dioxide receptors on the antennae of S. calcitrans increases as hunger develops. EAGs were also recorded in response to cattle odour, odour from fresh cattle faeces, expired human breath, acetone, and l-octen-3-ol. Acetic acid vapour causes a reversal of the usual EAG response indicating inhibition.     

Larval experience of stable fly,Stomoxys calcitrans Linneaus, 1758 (Diptera: Muscidae) does not influence oviposition preference in gravid females

1. Hopkins' host selection principle (HHSP) proposes that, in holometabolous insects, gravid females prefer to oviposit on their developmental substrate. This hypothesis is widely explored in phytophagous insects, but few studies have considered blood‐feeding insects. In this study, the HHSP was tested using the haematophagous stable fly, Stomoxys calcitrans (Diptera: Muscidae). Using no‐choice and two‐choice tests, the study first sought to demonstrate whether S. calcitrans larvae recognise their rearing substrate. 2. It was found that when a rearing substrate is offered to S. calcitrans larvae singly (no‐choice) or associated with a non‐rearing substrate (two‐choice), they were able to recognise the substrate in which they developed. This ability disappeared when larvae were transferred to another substrate for 5 h. 3. Next, using oviposition bioassays, it was investigated whether information gathered by pre‐imaginal stages during their development can persist into the adult stages after metamorphosis. It was hypothesised that gravid females emerging from clean and uncleaned pupae reared on sheep and camel dung would still prefer to lay on their rearing substrate. The results revealed that gravid females did not prefer to oviposit on their developmental substrate. In addition, failure to recognise and prefer their developmental substrate was found in gravid females that had emerged from puparia that were cleaned as well as those contaminated with sheep dung. 4. It is concluded that S. calcitrans adult preference is not influenced by their offspring's experience. Overall, the HHSP was not supported by our study, suggesting that this principle may not apply to generalist insects characterised by high plasticity in oviposition substrate acceptance.     

Effect of entomopathogenic fungi upon adults of Stomoxys calcitrans and Musca domestica (Diptera: Muscidae)

The pathogenicity and virulence of 10 isolates of entomopathogenic fungi from the soil of lodging pens of dairy production units in Aguascalientes, Mexico, on adults of Stomoxys calcitrans and Musca domestica were determined. All isolates were pathogenic when exposed by aspersion to a concentration of 1×10⁸ conidia/ml, causing between 20.3 and 91.7% mortality in S. calcitrans and between 31 and 91.7% in M. domestica at 7 days post-exposure; in S. calcitrans isolates of Beauveria bassiana (Bb114) and Metarhizium anisopliae (Ma135), sensu lato, were the most noteworthy as mortality reached above 90% with an LC₅₀ of 3.5×10⁵ conidia/ml for Bb114, while for Ma135 reached 1.6×10⁴ conidia/ml. In M. domestica Ma134 and Ma135 showed mortality above 90% with an LC₅₀ of 4.3×10⁴ and 1.4×10⁵ conidia/ml, respectively.     

Microsatellite DNA markers for the Stable Fly,Stomoxys calcitrans (Diptera: Muscidae)

Eight polymorphic microsatellite markers have been isolated from a microsatellite‐enriched DNA library from the Stable Fly, Stomoxys calcitrans. These loci exhibited four to 15 alleles per locus and an expected heterozygosity ranging from 0.19 to 0.84 in the three populations studied from La Réunion island (Indian Ocean). They should therefore be valuable for studying genetic diversity and population structure. Cross‐species amplification of these eight loci in the closely allied species Stomoxys niger niger was successful for four of these loci, one of which was monomorphic and one of which strongly departed from Hardy–Weinberg expectations.     

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.     

Characterization of proteolytic enzymes of the midgut and excreta of the biting fly Stomoxys calcitrans

Proteolytic enzymes were characterized in the midgut and the excreta of the stable fly Stomoxys calcitrans (L) with proteins, synthetic substrates, and inhibitors. Inhibition studies suggested trypsinlike activity in sugar-fed fly midguts, whereas excreta and blood-fed fly guts exhibited other proteases. Trypsinlike activity in midguts removed 20 and 30 h after a blood meal increased from 20% to 50% of the total proteolytic enzymes present. Trypsinlike activity was inhibited with human sera, trypsin-specific inhibitors, and a protein isolated from the stable fly thorax. When human albumin and globulin fractions were incubated with trypsinlike enzymes isolated from the midgut and excreta, the albumin fraction was less inhibitory than the globulin fractions and was readily hydrolyzed by the proteolytic enzymes. These results may indicate that the proteolytic enzymes produce an abortive complex with the globulin fractions of the sera. Such a complex may explain the temporary inhibition of proteolysis by the blood meal. Soybean trypsin inhibitor fed to stable flies caused 50% inhibition in proteolytic activity in the midguts of sugar-fed stable flies and 25% inhibition in the midguts of blood-fed stable flies. Complete inhibition of proteolytic enzyme activity was achieved only in vitro. pH profiles of proteolytic enzyme activity isolated from the excreta of blood-fed stable flies indicated that several proteolytic enzymes were excreted.     

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.     

Effects of carbon dioxide, acetone and 1-octen-3-ol on the flight responses of the stable fly, Stomoxys calcitrans, in a wind tunnel

Abstract. The flight behaviour of Stomoxys calcitrans (L.) in odour plumes containing carbon dioxide, acetone or l-octen-3-ol was assessed from video recordings. A downwind bias was evident in clean air, whereas all three test chemicals elicited upwind anemotaxis. Response thresholds were ∼0.006% for CO2, between 0.001 and 0.01 μg/l for acetone, and ∼0.0002 u.g/1 for l-octen-3-ol. Sinuosity (° cm^-1) and angular velocity (° s^-1) increased with C0₂ concentration, but velocity (cm s"¹) decreased. Similar, but less clear effects were observed for acetone and l-octen-3-ol.     

Isolation and in vitro synthesis of trypsin from the biting fly,Stomoxys calcitrans

The synthesis of [3H]trypsinlike enzyme by the fat body was followed in Stomoxys calcitransin vitro using a radioimmunoassay (RIA) developed against mammalian trypsin. Using high specific activity [3H]valine, trypsinlike activity was followed in midgut epithelial cells, thoracic muscle, and fat body removed from sugar-fed flies. Excreta protease of S. calcitrans was partially purified using charge and hydroxylapatite gel chromatography. Seventy-five percent of the enzyme eluted from these gels was inhibited by tosyl-L-lysine chloromethyl ketone HCI (TLCK) and was classified as trypsinlike. Electrophoresis of the trypsinlike enzyme indicated that it was only 50% pure. Trypsinlike activity from S. calcitrans bound to α₁-globulin IV-I and formed a complex that was dissociated on a P-100 Bio-Gel column. Binding between the protease and the α₁-gobulin IV-I caused a 1.4-fold increase in the apparent molecular weight of the protease on the P-100 Bio-Gel column. Trypsinlike activity was characterized in the midgut and excreta by affinity binding to covalently linked TLCK and tosyl-L-lysine chloromethyl ketone HCI (TAME)Sepharose 4B gels. Between 50% and 55% of the excreta protease and 5669% of the midgut protease bound to the affinity gels and was trypsinlike. Protease activity that did not bind to the gels was not inhibited by TLCK and did not have the esterolytic activity of trypsin.     

High relative abundance of the stable fly Stomoxys calcitrans is associated with lumpy skin disease outbreaks in Israeli dairy farms

The vector of lumpy skin disease (LSD), a viral disease affecting Bovidae, is currently unknown. To evaluate the possible vector of LSD virus (LSDV) under field conditions, a yearlong trapping of dipterans was conducted in dairy farms that had been affected by LSD, 1‐2 years previously. This was done in order to calculate monthly relative abundances of each dipteran in each farm throughout the year. The relative abundances of Stomoxys calcitrans (Diptera: Muscidae) in the months parallel to the outbreaks (December and April) were significantly higher than those of other dipterans. A stable fly population model based on weather parameters for the affected area was used to validate these findings. Its results were significantly correlated with S. calcitrans abundance. This model, based on weather parameters during the epidemic years showed that S. calcitrans populations peaked in the months of LSD onset in the studied farms. These observations and model predictions revealed a lower abundance of stable flies during October and November, when LSD affected adjacent grazing beef herds. These findings therefore suggest that S. calcitrans is a potential vector of LSD in dairy farms and that another vector is probably involved in LSDV transmission in grazing herds. These findings should be followed up with vector competence studies.