William Mordue

Structure of locust adipokinetic hormone, a neurohormone that regulates lipid utilisation during flight

Adipokinetic hormone, isolated from locust corpora cardiaca, has been identified as a blocked pep-tide : PCA–Leu–Asn–Phe–Thr–Pro–Asn–Trp–Gly–Thr–NH2. The detailed structure is based on mass spectrometric data, substantiated in part by dansylEdman and carboxypeptidase data on thermolytic fragments. This is the first peptide hormone from an insect neuroendocrine organ to be fully characterised.

Actions of azadirachtin, a plant allelochemical, against insects

Investigations of the antifeedant mode of action of azadirachtin and four synthetic analogues, 22,23-dihydroazadirachtin, 3-tigloylazadirachtol, 11-methoxydihydroazadirachtin and 22,23-bromoethoxydihydroazadirachtin have revealed that both polyphagous and oligophagous insects are behaviourally responsive to azadirachtin, with the most responsive species being able to differentiate extremely small changes in the parent molecule. In Lepidoptera the antifeedant response is correlated also with increased neural activity of the chemoreceptors. When locusts are treated on crop plants, the antifeedant and physiological actions of azadirachtin and analogues work in concert and result in feeding deterrence, growth and moulting aberrations and mortality with the same order of potency as for antifeedancy. Specific binding studies using [3H]dihydroazadirachtin carried out on locust testes and Spodoptera Sf9 cells have shown that the competitive binding of the different analogues of azadirachtin to these binding sites occurs in a similar order of potency to that found with antifeedant and IGR bioassays. This suggests a causal link between specific binding to membrane proteins and the ability of the molecule to exert biological effects.

Identification of bloodmeals of the Scottish biting midge, Culicoides impunctatus, by indirect enzyme-linked immunosorbent assay (ELISA).

Abstract. An indirect enzyme‐linked immunosorbent assay (ELISA) has been developed to identify the bloodmeal sources of Culicoides impunctatus Goetghebuer. Nightly light trapping in Scotland, between April and September 1992, caught a total of 344 blood‐engorged female C. impunctatus, all in May, June and July. The insects were stored in 70% ethanol and tested 5–7 months later. Bloodmeal sources were positively identified for 246 (71.5%). Bovine hosts were most common (38.4%), followed by deer (23.0%) and sheep (9.9%). Only one midge had fed on human blood. Relatively freshly fed (<24h), fully engorged females gave the clearest results. The wider applications of the technique to the study of Culicoides host preferences is discussed.

5-hydroxytryptamine stimulates fluid secretion in locust malpighian tubules independently of cAMP

5-Hydroxytryptamine (5-HT) stimulates fluid secretion by semi-isolated Malpighian tubules of Locusta in a dose-dependent manner. The threshold of stimulation is between 10(-8) and 10(-7) M 5-HT; maximal activation occurs at doses greater than 10(-6) M. Relative to the activation induced by diuretic hormone (storage lobe extracts), 5-HT increases the rate of fluid secretion by only 65%. Phentolamine, the alpha-adrenergic blocker, failed to inhibit either DH or 5-HT stimulated secretion. Diuretic hormone raises the levels of intracellular of cAMP, and activates adenylate cyclase in plasma membrane preparations of Locusta Malpighian tubules. 5-HT (10(-4) M) has no effect in either assay system. Thus 5-HT can stimulate fluid secretion independently of cAMP. A hypothetical model for hormone stimulated fluid secretion by Locusta Malpighian tubules, involving dual-receptor activation, is proposed. Other biogenic amines, including octopamine, adrenalin, dopamine, synephrine and the formamidine chlordimeform were tested for their ability to stimulate fluid secretion. Only dopamine showed a weakly stimulatory effect.

The role of 1‐octen‐3‐ol as a host‐odour attractant for the biting midge, Culicoides impunctatus Goetghebuer, and interactions of 1‐octen‐3‐ol with a volatile pheromone produced by parous female midges

Abstract. 1‐octen‐3‐ol, a component of the body odour of ruminants, is demonstrated to be an attractant for host‐seeking, parous female Culicoides impunctatus Goetghebuer (Diptera, Ceratopogonidae).This compound was electrophysiologically and behaviourally active and, in the field, released at 0.11 ± 0.01 mg/day, increased female C.impunctatus catches over a seven‐day period compared with control catches.When 1‐octen‐3‐ol was combined either with live female midges or an extract of female midges in an electroantennogram assay, additive effects were recorded.Behaviourally, the same combination resulted in both enhanced attraction and repellency, depending on concentration.Attraction was recorded mainly when sub‐threshold concentrations of the separate components were combined.The potential of such a combination as an effective field bait is discussed.

Field studies on efficacy of host odour baits for the biting midge Culicoides impunctatus in Scotland

The efficacy of some putative attractants for the biting midge Culicoides impunctatus (Goetghebuer) (Diptera: Ceratopogonidae) was assessed using odour‐baited ‘delta traps’ and suction traps. 1‐octen‐3‐ol was confirmed as a potent olfactory attractant for C. impunctatus when released at 0.06 mg/h. Acetone (23 mg/h) and a mix of six phenolic compounds (phenol, 3‐ethylphenol, 4‐ethylphenol, 3‐methylphenol, 4‐methylphenol and 4‐propylphenol), at undetermined release rate, also significantly increased delta trap catches compared to unbaited controls. When tested in combination, there was evidence of synergism between CO2 (0.2 L/min) and acetone, 1‐octen‐3‐ol or cow urine, trap catches being, respectively, 4.7, 6.2 and 9.3‐fold greater than for CO2 alone. Highest catches were obtained with triple bait combinations comprising cow urine + acetone + CO2 or cow urine + 1‐octen‐3‐ol + CO2, which increased trap catches by × 22 and × 24, respectively, compared to CO2 alone. Culicoides impunctatus was found to be extremely sensitive to CO2 and responses, gauged over two field seasons, showed a significant dose‐dependent increase in catch across the entire range of release rates (0.2–2.5 L/min). Responses to these release rates, ranging from small to large mammal equivalents, emphasized the important role of CO2 in host location by C. impunctatus. Uses of olfactory attractants for monitoring and control of Culicoides are reviewed on the basis of these results.

Stimulated fluid secretion is sodium dependent in the Malpighian tubules of Locusta migratoria

The ionic dependencies of stimulated and unstimulated Locusta tubules have been studied. K+, Na+, Cl− are essential to both basal and stimulated secretion. K+ is secreted against a concentration gradient in unstimulated tubules. In response to diuretic hormone or cAMP application, there is a dramatic influx of K+ into the lumen. A high level of Na+ and Cl− in the bathing medium is required to allow maximal fluid secretion. The tubules show an apparent impermeability to Na+; its concentration in the secreted fluid is always much less than in the bathing medium. If Na+ is omitted from the medium and excess K+ added (80 mM K), then although basal secretion occurs (2.5 nl/min), the tubules fail to respond to stimulation. Clearly Na+ has an important indirect role to play in stimulated fluid secretion.

Quantification of adipokinetic hormones I and II in the corpora cardiaca of Schistocerca gregaria and Locusta migratoria

Abstract 1. 1. A method is described to quantify the amounts of adipokinetic hormones I and II (AKHI and AKHII) in the corpora cardiaca (CC) of Schistocerca gregaria and Locusta migratoria . These two hormones can be separated in a single reversed-phase high-performance liquid chromatography step with 0.1% trifluoroacetic acid and acetonitrile as the solvent system. The peaks were identified by their retention times with reference to synthetic and/or natural peptides. The peaks were detected at 206 nm, their area integrated and compared with a series of areas obtained for known amounts of synthetic AKHI chromatographed under the same conditions. 2. 2. The first quantification data for AKHI and AKHII are presented for 5th instar larvae and adult S. gregaria and L. migratoria 3. 3. At any given time during the period studied it was found that the CC always contained more AKHI than AKHII; the molar ratio AKHI: AKHII ranged from 2:1 to 6:1. 4. 4. S. gregaria contained more of the two peptides than L. migratoria . 5. 5. In most of the developmental stages studied more hormone was found in females than in males.

Cyclic AMP and locust diuretic hormone action: Hormone induced changes in cAMP levels offers a novel method for detecting biological activity of uncharacterized peptide

Abstract The role of cAMP in diuretic hormone (DH) action on Locusta migratoria Malpighian tubules has been investigated. The rate of fluid secretion by semi-isolated tubules is elevated by the phosphodiesterase inhibitors theophylline (10 −3 M) and 3-isobutyl-1-methylxanthine (IBMX, 10 −3 M), although the latter is more effective. Removal of these inhibitors returns the rate of fluid secretion to basal levels. IBMX (10 −4 M) is shown to elevate the intracellular cAMP levels of tubules, but no significant elevation was observed in response to theophylline (10 −4 M). Stimulation of isolated tubules with DH (methanol extracted storage lobes) increases the intracellular cAMP levels of tubules by 7 to 8-fold, after 3 min stimulation at 30°C; the response to DH stimulation is lower after 10 min. Using plasma membrane preparations of Locusta migratoria Malpighian tubules, DH stimulates adenylate cyclase catalysed synthesis of cAMP from ATP, with optimum stimulation using 0.5–2 mg tubule membrane protein/ml. Dose-response data show that a saturated response occurs at doses of DH of greater than 0.2 storage lobe equivalents/75 μl of incubation medium. The proteolytic enzymes trypsin (0.01 mg/ml) and chymotrypsin (0.01 mg/ml) both destroy diuretic activity, tested using the semi-isolated tubule preparation. Trypsin 0.01 mg/ml also reduces the activity of the “DH” activating adenylate cyclase. Chromatography of methanol extracts of storage lobes on a TSK 2000 SW high performance size-exclusion chromatography column eluted in 0.1% TFA, separate the DH, activating adenylate cyclase, in fractions 7 and 8. Comparison of these active fractions with those tested using semi-isolated Malpighian tubule preparations suggests two distinct receptor sites on Locusta tubules.

Role of Semiochemicals in Mate Location by Parasitic Sea Louse, Lepeophtheirus salmonis

The role of olfaction and diffusible pheromones in mate location behavior of sea lice, Lepeophtheirus salmonis, was assessed with Y-tube behavioral bioassays. The pheromone “emitting” animals were located in a chamber in one arm of a Y-tube arena, with artificial seawater flowing through both arms. Adult male sea lice displayed both activation and directional responses to seawater conditioned with preadult II virgin females, but were only activated by mated adult female conditioned water. Further, when males were given the choice of preadult II virgin females or mated adult females, a significant number of males chose the arm with the preadult II virgin females. Adult males showed activation responses when presented with water conditioned with adult males but were not attracted to them. When presented with adult males, preadult II virgin females showed only directional responses, but not activation responses. Preadult II virgin female conditioned water was extracted using solid-phase extraction (SPE) protocols pioneered for semiochemical isolation. Adult male sea lice showed significant directional responses to the preadult II virgin female SPE extract. Distillation under vacuum was performed on the extract to give a distillate comprising components with a molecular weight range and physical properties comparable to those of compounds utilized as volatile semiochemicals by terrestrial organisms and a residue comprising components with higher molecular weight range comparable to those utilized as involatile semiochemicals. Adult males were found to be both significantly activated and attracted to the distillate, but not to the residue. This research provides evidence that small, lipophilic organic molecules are used by sea lice as sex pheromone signals to locate a member of the opposite sex.