Taste detection of phytoecdysteroids in larvae of Bombyx mori, Spodoptera littoralis and Ostrinia nubilalis.

A number of plants produce significant amounts of phytoecdysteroids that can disrupt the hormonal levels of insects feeding upon them. Insects equipped with taste receptors sensitive to phytoecdysteroids are able to avoid such plants. How common is this strategy? By recording from the lateral and medial sensilla styloconica in two polyphagous species (Ostrinia nubilalis and Spodoptera littoralis) and in a monophagous species (Bombyx mori), we tested whether the receptors could detect three commonly occurring phytoecdysteroids 20-hydroxyecdysone (20E), ponasterone A (ponA) and ecdysone (E). In B. mori, 20E and ponA elicited dose-dependent responses with a threshold of 1 &mgr;M only in the medial sensilla. In O. nubilalis, 20E, E and ponA elicited responses at threshold of 1 &mgr;M in both sensilla. In S. littoralis, 20E elicited responses with a threshold of 10 &mgr;M in both sensilla. By means of behavioural choice tests, we show that 20E is an effective feeding deterrent for O. nubilalis and S. littoralis first instar larvae. This suggests that the perception of phytoecdysteroids is more common among phytophagous lepidoptera than previously thought, although their toxicity or antifeedancy varies between species.

[Accumulation and biomagnification of organochlorine insecticides in molluscs and fish of the Moulay Bouselham lagoon, Morocco]. / Accumulation et biomagnification des insecticides organochlorés dans les mollusques et les poissons de la lagune de Moulay Bouselham, au Maroc.

We studied the contamination by organochlorine insecticides of the clams and eels in Moulay Bousselham lagoon, on the west coast of Morocco. DDT and its metabolites, DDE and DDD, were found to have accumulated in the lipids of all specimens. Organochlorine concentration in clams was highest for samples taken from the mouth of channels draining agricultural land. Organochlorine content was more than 100 ng/g in some cases in this area, whereas it was less than 40 ng/g in the rest of the lagoon. Organochlorine content peaked in May and then decreased after egg laying. Marked concentration of these pesticides was observed in eels, which are mobile and carnivorous. Organochlorine content peaked in November, mainly among the large specimens preparing to migrate to the ocean. DDT was stored in lipids, at concentrations up to 2,000 ng/g. However, even this high concentration is below the upper limit for human consumption. The higher proportion of DDE than of DDT shows that most of the contamination is not recent. BHC and cyclodienes were also detected, at low concentrations. The contamination of eels and clams in the Moulay Bouselham does not currently pose a threat to public health.

Electrophysiological responses of gustatory sensilla of Mamestra brassicae (Lepidoptera, Noctuidae) larvae to three ecdysteroids: ecdysone, 20-hydroxyecdysone and ponasterone A.

Specialised phytophagous Lepidoptera such as Bombyx mori and Pieris brassicae have contact chemoreceptors that perceive ecdysteroids at very low concentrations. This sensory perception allows them to feed on substrates with a high content of phytoecdysteroids. We have evaluated if a polyphagous insect like Mamestra brassicae does possess contact chemoreceptor cells that are sensitive to these molecules. Electrophysiological recordings were performed from contact chemoreceptors located on the maxilla. These receptors were stimulated with some sugars, amino acids and salts and with three ecdysteroids. Our results demonstrate that a specific cell within the lateral sensilla responds to 20-hydroxyecdysone and ponasterone A but not to ecdysone.

cDNA cloning and sequence determination of the pheromone biosynthesis activating neuropeptide of Mamestra brassicae: a new member of the PBAN family.

Sex pheromone biosynthesis in a number of moth species is induced by a conserved 33-amino acid amidated neuropeptide PBAN (pheromone biosynthesis activating neuropeptide). Here, using immunoblotting and bioassay, we present evidence for the presence of a very similar peptide, called Mab-PBAN, in the brain-subesophageal ganglion complex of Mamestra brassicae females. A partial Mab-PBAN encoding cDNA was isolated using 3'RACE. The deduced amino acid sequence for Mab-PBAN is: LADDMPATPADQEMYRPDPEQIDSRTKYFSPRL with a presumed amidated C-terminus. Mab-PBAN has high homology to the other members of the PBAN peptide family: 94% with Hez-PBAN, 87.9% with Lyd-PBAN and 78.8% with Bom-PBAN. The Mab-PBAN gene encodes, beside Mab-PBAN, at least three putative amidated peptides in the same reading frame, all of them having a common C-terminal pentapeptide motif F(T/S)P(R/K)L-NH2.

K+ channels of expiratory neurones in the brain stem preparation of the newborn rat.

Single channel activity of expiratory neurones was studied in outside-out recordings. Expiratory neurones were identified in the ventrolateral region of the in vitro isolated brain stem-spinal cord preparation of newborn rats in cell-attached and whole-cell configurations by their pattern of firing related to phrenic motor output. Three potassium (K+) channels of 10, 30 and 70 pS exhibited steady-state activity during long voltage commands (up to 5 min) and could be found associated together in the same patches. The 30pS channel showed voltage dependency, being most active at small depolarizations. The 70 pS channel showed little activity with < 1% of openings per sample time and 1 mM tetraethylammonium (TEA) sensitivity. At similar concentrations, the discharge of the phrenic nerve was also altered, as shown by the increase of the respiratory frequency and a tonic discharge. The association of these K+ channel types on the same patches may be specific of respiratory neurones and could contribute to their bursting activity.

Evidence for the involvement of 3-oxo-delta 4 intermediates in ecdysteroid biosynthesis.

Although the involvement of 3-oxo-delta 4 compounds as intermediates in arthropod ecdysteroid biosynthesis has been postulated for a long time, it has not yet been directly demonstrated. In the present study, 3-oxo-delta 4-steroids have been synthesized and incubated in vitro with dissociated moulting gland cells from the crab Carcinus maenas. The tritiated compounds were converted into 3-dehydroecdysone, ecdysone and/or 25-deoxyecdysone, i.e. final ecdysteroids. This means that the 3-oxo-delta 4 compounds had undergone a 5 beta-reduction, to give the 5 beta-conformation of ecdysteroids. Our results suggest that the 3-oxo-delta 4-steroid 4,7-cholestadien-14 alpha-ol-3,6-dione may be an intermediate in the biosynthetic pathway. The 5 beta-reduction reaction involves a cytosolic enzyme which requires NADPH as electron donor and seems specific for 3-oxo-delta 4 substrates. This reaction was the most active in crab Y-organs, as compared with other tissues. The characteristics of the 5 beta-reductase (subcellular localization, substrate and cofactor requirements) appear similar to those of the vertebrate 3-oxo-delta 4-steroid 5 beta-reductase involved in steroid hormone catabolism and bile acid biosynthesis.

[Ethyl 4-methyloctanoate, major component of male pherome in Oryctes rhinoceros (L.) (Coleoptera, Dynastidae)]. / Le 4-méthyloctanoate d'éthyle, composant principal de la phéromone mâle de Oryctes rhinoceros (L.) (Coleoptera, Dynastidae).

Ethyl 4-methyloctanoate, which has already been described in Oryctes monoceros, has been identified, using extracts of effluvia collected from males, as being a major component of the male pheromone of O. rhinoceros. Field trials have been carried out in North Sumatra, Indonesia. Ethyl 4-methyloctanoate synthesized in the laboratory and released at 10 mg/d resulted in the capture of 6.8 insects per week per trap, whereas ethyl chrysanthemate (40 mg/d), an allelochemical compound once used as an attractant, only led to the capture of 0.3 insects, and the control none at all. The insects captured with the pheromone were 81% females, the majority being sexually mature. Discovery of this compound opens up new prospects for O. rhinoceros control.

Identification of PBAN-like peptides in the brain-subesophageal ganglion complex of lepidoptera using Western-blotting.

An immunoblotting technique used to visualize pheromone-biosynthesis-activating-neuropeptide (PBAN)-like peptides in insect tissues is described. This technique involves a tricine-SDS-PAGE system and a chemiluminescent revelation of the antigens. Using this technique, PBAN-like immunoreactive peptides were found in the brain-subesophageal ganglion complex of various lepidopteran species, including moths: Heliothis zea, Mamestra brassicae, Spodoptera littoralis, S. latifascia and S. descoinsi (Noctuidae), Eldana saccharina (Pyralidae), and a butterfly: Pieris brassicae (Pieridae). PBAN-like peptides were detected in both sexes of the species studied, and even in a butterfly species that does not use pheromone to mate. This suggests that those peptides are widely distributed among Lepidoptera and confirms that they could be involved in functions other than regulation of sex pheromone production.

Purification and characterization of multiple forms of odorant/pheromone binding proteins in the antennae of Mamestra brassicae (Noctuidae).

Proteins extracted form the antennae of Mamestra brassicae (L.) (Lepidoptera: Noctuidae) adults were biochemically characterized as pheromone-binding proteins (PBP) and general odorant-binding proteins (GOBP). PBP and GOBP were purified by two successive and different HPLC (high performance liquid chromatography) systems and native polyacrylamide gel electrophoresis (native-PAGE). Their N-terminal sequence was determined by Edman microsequencing. The combined results showed evidence for three different PBPs in males, and two different PBPs in females. In addition, one GOBP was characterized in both males than in females antennae. In the males, two isoforms of PBP have the same N-terminal sequence, but different apparent mobilities and hydrophobicities: they could be separated by electrophoresis and reverse phase-HPLC (RP-HPLC). The other PBP sequence (SQEIM) showed particularly high homology (88%) with the PBP of Heliothis virescens, another noctuid moth. The existence of several forms of PBP in the same animal strongly supports the hypothesis of the specificity of binding between the proteins and their odorant ligands, the pheromonal compounds. The observed microdiversity at the soluble proteins level could provide a good model for studying their involvement in the initial stages of odor discrimination.

Electrophysiological and field activity of halogenated analogs of (E,E)-8,10-dodecadien-1-ol, the main pheromone component, in codling moth (Cydia pomonella L.).

Pest monitoring and control of the codling moth,Cydia pomonella L., have been developed using the main pheromone component of this species, (E,E)-8,10-dodecadienol (codlemone). However, the activity of codlemone is not satisfactory for pest control by mating disruption. Thus, we have synthesized halogenated analogs of codlemone to see if they could be used as new agents for pest control of the codling moth. Their biological activity was measured by electrophysiological techniques. In EAG screening, codlemone was the most active compound. F(10,11)-codlemone [(E,E)-10,11-difluoro-8,10-dodecadienol] and Cl-codlemone [(E,E)-11-chloro-8,10-undecadienol] elicited significant EAG responses, F(10,11)-codlemone triggering responses not significantly different from responses to codlemone. EAG cross-adaptation experiments and single sensillum recordings revealed that these compounds were detected by the same receptor neuron type as codlemone. No competitive inhibition with codlemone was observed from nonactive compounds. In field trapping, F(10,11)-codlemone and Cl-codlemone were more attractive to male codling moths than codlemone itself. Possible explanations of this activity are discussed.

Dorsal abdominal glands in nymphs of southern green stink bug,Nezara viridula (L.) (heteroptera: Pentatomidae): Chemistry of secretions of five instars and role of (E)-4-oxo-2-decenal, compound specific to first instars.

We investigated the exocrine secretions from the five nymphal instars in the southern green stink bug,Nezara viridula, by analyzing separately the contents of the three dorsal abdominal reservoirs. All DAGs 1 produced a mixture of five alkanes with 12, 13, 14, 15, and 16 carbons. No differences were found between DAGs 2 and DAGs 3, for the five instars: the glands of first instars produce the same alkanes as DAGs 1,n-tridecane, traces of (E)-2-decenal, and a specific compound: (E)-4-oxo-2-decenal. In the other instars (second to fifth), (E)-4-oxo-2-decenal is absent from the secretion but another compound is present: (E)-4-oxo-2-hexenal. The kinetics of production of the different compounds were studied, the maximum amounts produced occurring 36 hr after hatching. The biological function of (E)-4-oxo-2-decenal was investigated. Using olfactometry, we showed that this compound acts as an attractant and an arrestant on second instars, at physiological doses. Moreover, this semiochemical was shown to be repellent to the fire-antSolenopsis geminata, a potential predator ofN. viridula and we established the dose-response curve for the repellent activity.

Defensive secretion of first-instar larvae of rootstalk borer weevil,Diaprepes abbreviatus L. (Coleoptera: Curculionidae), to the fire-antSolenopsis geminata (F.) (Hymenoptera: Formicidae).

Since several species of predatory ants show some kind of repulsion towards the first-instar larvae (FIL) ofDiaprepes abbreviatus L., the predatory behavior ofSolenopsis geminata (F.), a common ant in the citrus groves in Guadeloupe, was studied. Different extracts of larvae were disposed on egg masses ofD. abbreviatus and presented as prey to the ants, both in the field and in the laboratory. The ants are repelled by the FIL extracts. The allelochemicals involved are produced in large amounts, from 5 to 20 ng per larva. Physiochemical analyses have led to the identification of two sesquiterpenes of molecular weight 218 and 234, secreted in the respective proportions of 65 and 35%.

Sex pheromone of the european sunflower moth,Homoeosoma nebulellum (Den. & Schiff.) (Lepidoptera: Pyralidae).

Four components, (Z)-9-tetradecenal (8.6%), (Z,E)-9,12-tetradecadienal (4.8%), (Z)-11-hexadecenal (49.5%), and (Z)-13-octadecenal (37.1%), were identified in extracts of female pheromone glands of the European sunflower moth,Homoeosoma nebulellum (Lepidoptera: Pyralidae) using GC and GC-MS analyses. EAG and single-cell recordings of male antennal receptors gave strong evidence for (Z,E)-9,12-tetradecadienal as the antennal key compound of sex pheromone detection in this species. This result was confirmed by field trapping; removal of (Z,E)-9,12-tetradecadienal from quaternary blends completely suppressed the male catches. The synthetic blends with this compound as a major component caught five times less males than the blends reproducing the ratio found in the female extracts [5% of (Z,E)-9,12-tetradecadienal only]. The occurrence of a minor component perceived as the most biologically relevant compound is discussed.

Male-produced aggregation pheromone of the american palm weevil,Rhynchophorus palmarum (L.) (Coleoptera, Curculionidae): Collection, identification, electrophysiogical activity, and laboratory bioassay.

Male American palm weevils (APWs),Rhynchophorus palmarum (L.) produced two sex-specific compounds, which were disclosed by volatile collections on Supelpak-2 and gas chromatography. One was a minor compound, not always detected. The major male-produced volatile was identified as (2E)-6-methyl-2-hepten-4-ol through coupled gas chromatography-mass spectrometry and gas chromatography-Fourier transform infrared spectrometry, proton nuclear magnetic resonance spectrometry, and rational synthesis. We propose the trivial name rhynchophorol for this new molecule, which proved to be the essential component of the APW aggregation pheromone by electroantennography, coupled gas chromatography-electroantennography and behavioral bioassays.

Sex pheromone ofPandemis heparana (Den. and Schiff.) (Lepidoptera:Tortricidae).

90% (Z)-11-tetradecen-1-yl acetate, 5% (Z)-9-tetradecen-1-yl acetate, 5% (Z)-11-tetradecen-1-ol, dodecyl acetate (<1%), and tetradecyl acetate (<1%) were identified from gland extracts ofPandemis heparana females by gas chromatography and gas chromatography-mass spectrometry analysis (including mass fragmentography studies), chemical characterization, total synthesis, laboratory and field bioassays. In the field, a mixture of (Z)-11-tetradecen-l-yl acetate and (Z)-9-tetradecen-l-yl acetate (95∶5) was found to be essential for attractiveness ofP. heparana males.

[Adsorption on "Porapak Q" of the effluvia emitted by virgin females of Choristoneura fumiferana (Clem.) (Lepidoptera, Tortricidae). Identification of tetradecene-11(E)al-1, principal sex pheromone of this species]. / Adsorption sur "Porapak Q" des effluves émis par la femelle vierge de Choristoneura fumiferana (Clem.) (Lépidoptère Tortricidae). Mise en évidence du tétradécène-11(E)al-1, principale phéromone sexuelle de cette espèce

Air which had been drawn over virgin female Choristoneura fumiferana was passed through "Porapak Q", in this way an extract containing all the volatile compounds emitted by the insect may be obtained. (E)-11-tetradecenal but not (E)-11-tetradecen-1-ol was identified among the components of the extract. The latter compound, an inhibitor of the aldehyde, has previously been shown to be present in the secretory gland of the insect.

[The sex-trapping of Sterrha biselata (Hufn.) (Lepidoptera Geometridae, Sterrhinae) by acetoxy-1 dodecadiens-7 E, 9Z, the sex pheromone of Lobesia botrana (Schiff.) (Lepidoptera Tortricidae, Olethreutinae)]. / Sur le piégeage sexuel de Sterrha biselata (Hufn.) (Lépidoptère Geometridae, Sterrhinae) par l'acétoxy-1 dodécadiène-7 E, 9 Z phéromone sexuelle de Lobesia botrana (Schiff.) (Lépidoptère Tortricidae, Olethreutinae)

7E, 9Z dodecadienyl acetate, a sex-heromone for Lobesia botrana, is selectively attractive, in vineyards, for males of this species. By testing this compound in woods and orchards, we have found that it was also selectively attractive for males of a geometrid moth; Sterrha biselata. This is one of the first examples where a definite chemical substance is active for a geometrid species.