Regulation of mating behavior by nutrition and the corpus allatum in both male and female Phormia regina (Meigen)

Both dietary protein and the corpus allatum (CA) were required for normal mating behavior in both male and female black blow fly, Phormia regina (Meigen). Nutrition (protein diet) activated the CA in both sexes. More than 10 mg of dietary liver was required for each male to result in 80% insemination of females, while 20 mg of liver was required for each female to allow 78% of females to become inseminated. Between 10 to 15 mg of protein meal (i.e., liver) was required to activate sexual receptivity in 71% of the females, while between 15 to 20 mg of liver was needed to support full o?cyte development in 70% of the females (Yin, C.-M., Zou, B.-X., Li, M.-F., Stoffolano, J.G., Jr. 1994. Discovery of a midgut peptide hormone which activates the endocrine cascade leading to oogenesis in Phormia regina Meigen. Journal of Insect Physiology, 40, 283-292). Allatectomy suppressed mating behavior more than 2-fold in both sexes. Topical application of 10 &mgr;g of S-methoprene (a juvenile hormone analogue) at 12 h after the onset of liver feeding restored sexual activity of both allatectomized males and females. Incidence of successful insemination increased as the o?cyte development progressed. Ovariectomy suppressed sexual receptivity more than 3-fold in liver-fed females. Thus, in addition to nutrition and the CA, ovaries and their developmentaal status can also affect the sexual receptivity in female P. regina.     

Screening method for insecticidal activity using first instars of black blow fly (Diptera: Calliphoridae)

A bioassay method suitable for rapid mass screening of fermentation and synthetic organic compounds for insecticidal activity is described. The test, which uses first instars of susceptible black blow fly, Phormia regina (Meigen), in a bovine serum medium, detects insecticidal activity with reproducible results. It is capable of selecting the most active compound in structure-activity relationships by minimum effective dose concentration studies. The bioassay system is easy to operate and requires only a minute quantity of chemical compound.     

Amphetamine and Reserpine Deplete Brain Biogenic Amines and Alter Blow Fly Feeding Behavior

HPLC with electrochemical detection was used to determine the levels of p-hydroxyphenylethanolamine (octopamine), 3,4-dihydroxyphenylethylamine (dopamine), and 5-hydroxytryptamine (5-HT) in the brains of control, reserpine, and d-amphetamine-treated blow flies, Phormia regina Meigen. Parallel studies were carried out to assess the effects of the two drugs on fly feeding behavior, measured as mean acceptance threshold: the minimum sucrose concentration to which the average fly in a population will respond by proboscis extension when its tarsi contact the solution. In saline-injected control flies, all three amines were found at levels of approximately 2 pmol/brain. Thirty minutes after injection with d-amphetamine (12 micrograms/fly), brain octopamine was depleted by 85%, whereas dopamine and 5-HT were depleted by 70%. Reserpine (5 micrograms/fly) caused 70% depletion of dopamine and greater than 90% depletion of both octopamine and 5-HT 24 h after injection. However, the effect of reserpine was much slower in onset (hours versus minutes) and more persistent (days versus hours) than was the effect of d-amphetamine. With either drug, the time course of amine depletion closely matched the time course of the increase in feeding threshold observed in drug-treated flies. These results suggest that CNS pools of the biogenic amines, octopamine, dopamine, and 5-HT are important in governing blow fly responsiveness to food stimuli.     

Insect satiety: sulfakinin localization and the effect of drosulfakinin on protein and carbohydrate ingestion in the blow fly, Phormia regina (Diptera: Calliphoridae)

Sulfakinins, which are satiety factors in invertebrates, have previously been shown to inhibit feeding in the German cockroach and desert locust. This study examines the occurrence of sulfakinin immunoreactivity and the role of sulfakinin as a feeding satiety factor in the black blow fly, Phormia regina. Specifically, this study examines the effect of sulfakinin on two of the blow fly's nutrient requirements (i.e., carbohydrates and proteins). We observed sulfakinin immunoreactive cells in the brains of both male and female flies. We found that drosulfakinin I (DrmSKI, FDDY[SO(3)H]GHMRFa) significantly inhibited carbohydrate feeding by 44% at the most effective dose (10 nmol) in female flies. Statistically, there was no significant effect on males; however, injections of 10 nmol DrmSKI reduced carbohydrate feeding by 34% compared to the sham. Drosulfakinin had no effect on protein feeding and no significant inhibition was detected in females or males. The results of this study lend further support to the idea that carbohydrate and protein feeding are regulated by separate control mechanisms, especially in Calliphoridae.     

Aromatic plants in blue tit Cyanistes caeruleus nests: no negative effect on blood-sucking Protocalliphora blow fly larvae

Nesting birds use several behavioural or physiological defence mechanisms against parasites. On Corsica, female blue tits Cyanistes caeruleus incorporate fresh fragments of a limited number of aromatic plants in the nest cup, from the end of nest construction until fledging. Some of these plants negatively affect bacterial growth and host location by blood-sucking mosquitoes in laboratory conditions. In natural populations, Corsican blue tit chicks are exposed to the highest levels of blood-sucking ectoparasitic blow flies Protocalliphora spp. reported in Europe. These ectoparasites can have severe negative effects on chick development and survival probabilities, especially when food constraints are elevated. Here we investigated in several natural Corsican blue tit populations the hypothesis that aromatic plants brought to the nest have anti-blow fly effects during the chick-rearing stage. We predicted that: 1) the amount of aromatic plants should be negatively related to blow fly infestation intensity across nests, 2) experimental addition of aromatic plants in nests should reduce blow fly infestation intensity, and 3) nestlings should be in better physical condition in nests where aromatic plants were experimentally added. No significant relation was found between amount of aromatic plants in nests and blow fly infestation intensity. Experimental addition of aromatic plants did not reduce blow fly infestation intensity and did not affect the chick phenotypic parameters we measured. We conclude that aromatic plants in blue tit nests are not used as a defence against ectoparasitic Protocalliphora blow flies in our study population.     

Cuticular hydrocarbons and their role in copulatory behavior in Phormia regina (Meigen)

The cuticular hydrocarbons from adult Phormia regina (Meigen) were characterized by gas chromatography-mass spectrometry. Both sexes had similar components in nearly identical quantities, consisting of complex mixtures of saturated n-, monomethyl- and dimethylalkanes from 23 to 33 total carbons. Although no diet-, age-, or sex-specific differences were observed, cuticular hydrocarbons were shown to be involved in copulatory behavior. Hydrocarbon profiles of wild, compared to laboratory reared flies, showed no major differences. Behaviorally, males responded the same to dead decoys of either sex. Removal of the hydrocarbons, using hexane, from either male or female decoys, did not affect the number of mating strikes, but markedly reduced the number of copulatory attempts and the amount of time males spent mounted on either decoy. House fly, Musca domestica L., males when paired with a female M. domestica decoy produced copulatory attempts: whereas, when P. regina males were placed with M. domestica female decoys, there were no copulatory attempts. It is concluded that the cuticular hydrocarbons of P. regina function as species-specific but not sex-specific mating cues and elicit species-specific copulatory behavior in males.     

Immunolocalization and possible effect of a moth allatotropin-like substance in a fly, Phormia regina (Diptera: Calliphoridae)

Insect allatotropin upregulates the biosynthesis of juvenile hormones by the corpus allatum. We raised two rabbit antisera against the allatotropin of Manduca sexta (Mas AT) using a synthetic, multiple-antigenic-peptide that contains a branching heptalysine core and eight Mas AT molecules. Both antisera recognized specifically the same neurons in the larval brain, frontal ganglion and terminal abdominal ganglion of M. sexta as previously reported by others. Immunoassay showed reactivity specific to the Mas AT. Very low or nearly no cross-reactivity was found for two Mas AT-like peptides, a myotropin from Locusta migratoria and a Mas AT-like peptide deduced from the DNA sequence of Aedes aegypti, respectively. Immunopositive neurons also were identified in adult Phormia regina, Dacus dorsalis, Oncopeltus fasciatus, and Mythimna loreyi, and in larval M. loreyi, Bombyx mori, and Andraca bipunctata. At 20 pmol per 25 μl incubation medium (i.e. 8x10(-7) M), synthetic Mas AT significantly stimulated in vitro juvenile hormone biosynthesis by the corpus allatum of adult, sugar-fed females of P. regina to 2.64-fold that of controls. Thus, this study provides the first demonstration that at the higher end of the physiological concentration range, the Mas AT has allatotropic effect in vitro to CA of non-lepidopterans. However, in vivo functions of Mas AT and/or Mas AT-like peptide in P. regina remain to be defined.     

Leishmania parasites (Kinetoplastida: Trypanosomatidae) reversibly inhibit visceral muscle contractions in hemimetabolous and holometabolous insects

Female sand flies can acquire protozoan parasites in the genus Leishmania when feeding on an infected vertebrate host. The parasites complete a complex growth cycle in the sand fly gut until they are transmitted by bite to another host. Recently, a myoinhibitory peptide was isolated from Leishmania major promastigotes. This peptide caused significant gut distension and reversible, dose-dependent inhibition of spontaneous hindgut contractions in the enzootic sand fly vector, Phlebotomus papatasi. The current study further characterizes myoinhibitory activity in L. major and other kinetoplastid parasites, using the P. papatasi hindgut and other insect organ preparations. Myoinhibitory activity was greatest in cultured promastigotes and in culture medium in late log-phase and early stationary-phase, coinciding with development of infective Leishmania morphotypes in the sand fly midgut. L. major promastigote lysates inhibited spontaneous contractions of visceral muscle preparations from hemimetabolous (Blattaria and Hemiptera) and holometabolous (Diptera) insects. Inhibition of visceral muscle contractions in three insect orders indicates a conserved mode of action. Myoinhibitory activity was detected also in Leishmania braziliensis braziliensis, a Sudanese strain of Leishmania donovani, and the kinetoplastid parasite Leptomonas seymouri. Protozoan-induced myoinhibition mimics the effect of insect myotropins. Inhibiting host gut contractions protects Leishmania parasites from being excreted after blood meal and peritrophic matrix digestion, allowing development and transmission of infective forms.     

Effects of age and blood sugar levels on the proboscis extension of the blow fly Phormia regina

In some insects the proboscis is extended to imbibe a sugar solution if the concentration of sugar applied to the chemosensilla exceeds the behavioural threshold value. Recently, I found a reversal of the threshold values of this "proboscis extension reflex" (PER) in the blow fly (Phormia regina M.) for glucose and fructose. It depended on maturation and physiological conditions, both of which are explicable in terms of changing concentration of haemolymph trehalose. The direct injection of trehalose into the fly haemocoele brought about a dramatic shift of the threshold values of PER measured on tarsi or labellar sensilla, suggesting a strong dependence of PER on the blood sugar level. Using the tip-recording method, the dose-response (impulse frequency) curves for glucose and fructose were obtained on individual largest labellar chemosensilla. The curves for glucose and fructose crossed at one point because the former had a steeper gradient and higher maximum response than the latter. Injection experiments with trehalose were also carried out to test for changes in gustatory response. The shifting of the behavioural dose-response curves for glucose and fructose two hours after injection of 1 M trehalose (2 μl) into the haemocoele of the fly was associated with significant reduction in responsiveness of labellar chemosensilla to glucose, but less so to fructose. No change in responsiveness was found following injection of mannose. A hypothesis to explain the reversal relation of the PER thresholds, based on a shift in the firing rate in gustatory sensilla and possibly also interneurons, is discussed.     

Effect of neurotensin on the smooth muscle of the chicken crop

1. The effect of neurotensin was studied in the isolated smooth muscle of the chicken crop. 2. This peptide induced concentration-dependent (10(-10)-10(-8) mol/l) contractions of muscle strips. 3. The response to neurotensin was not modified after incubation of the preparation with atropine, indomethacin, naloxone and tetrodotoxin. The neurotensin-exerted contractions are therefore probably myogenic. 4. These properties of neurotensin indicate a possible role for this peptide in the control of crop motility.     

Cloning,recombinant expression and inhibitor profiles of dihydrofolate reductase from the Australian sheep blow fly,Lucilia cuprina

While dihydrofolate reductase (DHFR) is an important drug target in mammals, bacteria and protozoa, no inhibitors of this enzyme have been developed as commercial insecticides. We therefore examined the potential of this enzyme as a drug target in an important ectoparasite of livestock, the Australian sheep blow fly, Lucilia cuprina (Diptera: Calliphoridae) (Wiedemann). The non‐specific DHFR inhibitors aminopterin and methotrexate significantly inhibited the growth of L. cuprina larvae, with IC₅₀ values at µg levels. Trimethoprim and pyrimethamine were 5–30‐fold less active. Relative IC₅₀ values for the inhibition of recombinant L. cuprina DHFR by various inhibitors were in accordance with their relative effects on larval growth. The active‐site amino acid residues of L. cuprina DHFR differed by between 34% and 50% when compared with two mammalian species, as well as two bacteria and two protozoa. There were significant charge and size differences in specific residues between the blow fly and human DHFR enzymes, notably the L. cuprina Asn21, Lys31 and Lys63 residues. This study provides bioassay evidence to highlight the potential of blow fly DHFR as an insecticide target, and describes differences in active site residues between blow flies and other organisms which could be exploited in the design of blow fly control chemicals.     

Transduction pathways mediated by second messengers including cAMP in the sugar receptor cell of the blow fly: study by the whole cell clamp method

The whole cell clamp method was directly applied to the sensory receptor neurons isolated from the adult labellar hair of the blow fly Phormia regina to locate the signal transduction pathways mediated by second messengers. First, the cAMP-mediated transduction pathway was examined to specify its location in the sugar receptor cell. Sugar receptor cell was identified by recording inward current flow under the voltage clamp applying sucrose solution to the surface of the taste neurons. When cyclic nucleotides, such as cGMP and cAMP, were introduced into the sugar receptor cell, inward current was observed (cGMP, 70pA; cAMP, 300pA at 350microM). Inhibitors and activators for the second messengers (GDPbetaS and forskolin) and non-cyclic nucleotides were also examined. Second, non-nucleotide second messengers (IP3 and Ca2+) were examined. The sugar receptor cell was activated when it was injected with IP3 or Ca2+. All the obtained results suggest that the cAMP-mediated signal transduction pathway plays a major role in the sugar receptor cell. The possibility of other transduction pathways mediated by IP3 or Ca2+ was not excluded.     

Opposite effects of ecdysone and 20-hydroxyecdysone on in vitro uterus motility of a tsetse fly

In order to examine the possible effects of ecdysteroids on parturition, we studied in vitro the influence of ecdysone and 20-hydroxyecdysone on the motility of isolated uterus from virgin and pregnant female tsetse fly (Glossina fuscipes). Ecdysone initiates phasic uterine contractions or enhances the frequency of preexisting contractile activity. In contrast, uterine contractions are decreased or abolished by 20-hydroxyecdysone. Pharmacological data indicate that tsetse fly uterus exhibits myogenic and nerve-evoked contractions. Ecdysteroids mainly act on nervous structures that control muscle contractions. Our results provide evidence for a specific action of ecdysteroids on a nerve-muscle target involved in female reproduction.     

Clonidine, octopaminergic receptor agonist, reduces protein feeding in the blow fly, Phormia regina (Meigen)

Results in this study are consistent with those of Murdock and his colleagues who clearly demonstrated that clonidine, an agonist of octopaminergic receptors in some insects, significantly increases sucrose feeding. Their studies, however, did not examine the effect of clonidine on protein feeding. Injection of a 20 microg/microl/fly dose of clonidine significantly reduces protein feeding in both sexes of Phormia regina, instead of stimulating feeding as is observed with carbohydrate feeding. The manner in which the flies are fed prior to starvation and the method of testing influences the amounts of diet consumed. It is proposed that the biogenic amines influence the state of hunger (i.e., protein versus carbohydrates) while other chemicals and neural mechanisms (i.e., such as sulfakinins and stretch receptors, respectively) affect satiety.     

Aplysia brasiliana neurons R3-R14: primary structure of the myoactive histidine-rich basic peptide and its prohormone

Neurons R3-R14 of the marine mollusc Aplysia are model neuroendocrine cells thought to regulate cardiovascular activity in vivo. The cells express a gene encoding three peptides--peptides I, II and the histidine-rich basic peptide (HRBP)--each of which has been chemically characterized in Aplysia californica. In the studies presented here, HRBP and its prohormone (proHRBP) were purified from A. brasiliana abdominal ganglion extracts by reversed-phase high-performance liquid chromatography and characterized by amino acid compositional and sequence analyses. ProHRBP was an 85-residue peptide whose sequence was: NH2-Glu-Glu-Val-Phe-Asp-Asp-Thr-Asp-Val-Gly-Asp-Glu-Leu-Thr-Asn-Ala-Leu- Glu-Ser - Val-Leu-Thr-Asp-Leu-Lys-Asp-Lys-Arg-Asp-Ala-Glu-Glu-Pro-Ser-Ala-Phe-Met- Thr-Arg - Leu-Arg-Arg-Gln-Val-Ala-Gln-Met-His-Ile-Trp-Arg-Ala-Asn-His-Asp-Arg-His- His-Ser - Thr-Gly-Ser-Gly-Arg-His-Ser-Arg-Phe-Leu-Thr-Arg-Asn-Arg-Tyr-Gly-Gly-Gly- His-Leu - Ser-Asp-Ala-COOG. It differed from A. californica pro-HRBP at seven of the 85 positions. Compositional and sequence analyses demonstrated that A. brasiliana HRBP was a 43-residue peptide corresponding to residues 43 through 85 of proHRBP, and that a significant proportion of the isolated peptide possessed a blocked NH2 terminus. Although this sequence differed from that of A. californica HRBP at five of 43 residues, the two peptides were approximately equipotent in inducing contractions of A. californica crop muscle in vitro, suggesting that the substituted residues may not be critical for biological activity.     

Control of pupal fat body disappearance in the female black blow fly,Phormia regina (Meigen) by the brain and the corpus allatum

Neck-ligation, brain implantation, allatectomy, methoprene treatment, and ovariectomy indicated that the disappearance of pupal fat body cells in newly emerged adult female blow fly, Phormia regina, is controlled by the brain and the corpus allatum (CA). Absence of brain or CA greatly lowered the rate of fat body cell disappearance (i.e. death). Dependency on the CA decreased from 0 to 36h post-emregence, indicating that the CA was active during the earlier part of this timespan. Methoprene treatment enhanced pupal fat body cell disappearance in allatectomized females. Brain implantation restored the rate of pupal fat body cell disappearance in neck-ligated flies. Brains from day 1 sugar-fed flies proved to be more effective than those from day 2 sugar-fed flies, indicating that there may be a window after adult emergence that allows the brain to act directly or indirectly on the death of pupal fat body cells. Ovariectomy did not alter the rate of pupal fat body cell death in test animals. Dying pupal fat body cells were smaller in size, less dense (i.e. did not sink in saline like normal pupal fat body cells), and stickier (i.e. attached to other tissues tighter) than the healthy cells. A possible role played by ecdysteroids is also discussed.     

Characterization of NADPH-cytochrome P-450 reductase from house flies (Musca domestica,L.) susceptible and resistant to insecticides,and the blow fly (Phormia regina,meigen)

NADPH-cytochrome $\text{c}$ reductase was purified by affinity chromatographic techniques from microsomes prepared from the abdomens of insecticide-resistant (R) and -susceptible (S) house flies Musca domestica and of the black blow fly Phormia regina. Data are presented which describe (1) the ability of the purified enzymes to support an in vitro reconstitution of mono-oxygenase activity, (2) the changes in activity of these preparations observed in buffers of varying ionic strength, (3) comparative kinetic behaviour between microsomal and purified forms of the enzymes, (4) the immunochemical characteristics of these preparations, and (5) their amino acid composition. The reductases from the three sources were found to be very similar in all of these tests. Substrate binding constants were 5 μM for NADPH, 12 μM for cytochrome $\text{c}$, and the catalytic mechanism was interpreted as ordered Bi Bi. The inhibitory constant of the reductase from the resistant fly for 2′-AMP, an analogue of NADP⁺, was 187 μM; whereas no assciation of the inhibitor was observed below concentrations of 400 μM for the enzyme of either the susceptible house fly or the blow fly. However, the data are insufficient to suggest that the reductase is a significant factor in insecticide resistance. Compared to the same enzymes from rat and rabbit liver, the insect reductases show a different ionic strength optimum (0.14), have distinct antigenic determinants, and have different levels of acidic and basic amino acids in the membrane-binding peptide.     

The different effects of three Drosophila melanogaster dFMRFamide-containing peptides on crop contractions suggest these structurally related peptides do not play redundant functions in gut

A Drosophila melanogaster dFMRFamide gene product, TPAEDFMRFamide, decreased crop contractions. However, DPKQDFMRFamide and SDNFMRFamide, also encoded in dFMRFamide, did not affect crop motility, which suggests these peptides are not functionally redundant in the crop and their unique N-terminal structures are important for activity. TPAEDFMRFamide-specific antisera did not stain the crop, which suggests it acts as a hormone. TDVDHVFLRFamide (DMS), encoded in D. melanogaster myosuppressin, stops crop contractions. TPAEDFMRFamide and DMS each contains a RFamide C-terminus; however, their effects on crop contractions differ, which suggests that unique receptors or different ligand:receptor binding requirements exist for these structurally related peptides.     

Plant terpenoids: acute toxicities and effects on flight motor activity and wing beat frequency in the blow fly Phaenicia sericata

We evaluated the acute toxicities and the physiological effects of plant monoterpenoids (eugenol, pulegone, citronellal and alpha-terpineol) and neuroactive insecticides (malathion, dieldrin and RH3421) on flight muscle impulses (FMI) and wing beat signals (WBS) of the blow fly (Phaenicia sericata). Topically-applied eugenol, pulegone, citronellal, and alpha-terpineol produced neurotoxic symptoms, but were less toxic than malathion, dieldrin, or RH3421. Topical application of eugenol, pulegone, and citronellal reduced spike amplitude in one of the two banks of blow fly dorsolongitudinal flight muscles within 6-8 min, but with citronellal, the amplitude of FMIs reverted to a normal pattern within 1 hr. In contrast to pulegone and citronellal, where impulse frequency remained relatively constant, eugenol caused a gradual increase, then a decline in the frequency of spikes in each muscle bank. Wing beating was blocked permanently within 6-7 min of administering pulegone or citronellal and within 16 mins with eugenol. alpha-Terpineol-treated blow flies could not beat their wings despite normal FMI patterns. The actions of these monoterpenoids on blow fly flight motor patterns are discussed and compared with those of dieldrin, malathion, RH3421, and a variety of other neuroactive substances we have previously investigated in this system. Eugenol, pulegone and citronellal readily penetrate blow fly cuticle and interfere with flight muscle and/or central nervous function. Although there were differences in the effects of these compounds, they mainly depressed flight-associated responses, and acted similarly to compounds that block sodium channels and facilitate GABA action.     

Role of capsaicin-sensitive nerve fibers in uterine contractility in the rat

The possible participation of capsaicin-sensitive sensory nerves in the modulation of neurogenic contractions was studied in nonpregnant and term pregnant rat uteri. Neurogenic contractions were elicited by electric field stimulation (40 V, 1-70 Hz, 0.6 msec) in intact uteri and uteri that were previously exposed to capsaicin in vitro. In capsaicin pretreated preparations obtained both from nonpregnant and term pregnant rats, a dose-dependent increase in the amplitude of uterine contractions was detected. Prior systemic treatment of the rats with capsaicin (130 mg/kg, s.c.) abolished the effect of in vitro capsaicin administration on the amplitude of neurogenic contractions. Use of a specific antagonist of calcitonin gene-related peptide revealed that depletion of this peptide, which normally elicits uterine smooth muscle relaxation, may be responsible for the increased responsiveness of the uterus to low-frequency stimulation. Experiments on the localization of calcitonin gene-related peptide in uterine tissue specimens exposed to capsaicin revealed dose-dependent depletion of calcitonin-gene related peptide-immunoreactive nerves innervating blood vessels and the myometrium. The findings indicate that capsaicin-sensitive afferent nerves, by the release of sensory neuropeptides, significantly contribute to the modulation of uterine contractility both in nonpregnant and term pregnant rats. It is suggested that uterine sensory nerve activation may be part of a trigger mechanism leading to preterm contractions evoked by, for example, inflammation.