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Isolation and identification of pheromones and their mode of action in African locusts

Objective

- To perform chemical analysis of pheromones in African locusts and comparable grasshoppers;
- To identify the mainly volatile and air borne compounds and prepare them for behavioural studies;
- To analyse the less volatile substances on the cuticle of the locust after solution in pentane;
- To find out the mode of action of the identified compounds in relation to their pheromone effect.
- Identification of headspace compounds
Headspace analysis of Schistocerca gregaria and Locusta migratoria were carried out extensively, 27 compounds were identified having mostly aromatic structure. Some of them are sexually specific. Benzylcyanide was found only in adult yellow males and for the first time in insects.
At the beginning of maturation gregarious males of S. gregaria produce 1, 2, 3-trimethoxybenzene, (Z)-5-octenoid acid, trans-3-ethyl-1,4-dimethyl-pyrrolidin-2,5-dione (I) and veratrole; the compound (I) could be synthesised and its configuration was identified by NOE-experiment. Adult gregarious females emit the same compounds as the nymphs that mainly produce phenol and guaiacol in both sexes. In S. gregaria and Aiolopus thalassinus (F.) males' methyl benzoate was released. In extracts of faeces obtained by steam distillation 4-vinylguaiacol was found in high concentrations that may be an artefact, because the compound was not detectable in headspace extract of faeces, in which phenol, guaiacol and benzylcyanide were found.

- Analysis of less volatile cuticular surface compounds
Only a weak dimorphism was found in the composition of hydrocarbons, 128 various alkanes were identified in S. gregaria, L. migratoria and A. thalassinus. It seems not likely that there is an alkane functioned as pheromone in locusts. Additionally, in S. gregaria 20 different long-chained branched and straight-lined ketones were identified, from which 18 compounds are new as natural products. The 2-alkanones 3,7-dimethylpentacosan-2-one, 3,7-dimethylheptacosan-2-one and 3,7,1,5-trimethylheptacosan-2-one are sexual and stage specific. In contrast to that 6-alkanones were found in both sexes of adults. In nymphs, mainly 5-alkanones were identified. Besides the ketones, in S. gregaria various 2,5-disubstituted tetrahydrofurans were identified in the polar fraction of the cuticle. Also long-chained bishomolog nitrogen compounds of unknown structure were detected.

- Molecular basis of pheromone reception
Due to the important functional role of c-AMP activated potassium channels in olfactory neurones of locusts, attempts were made to identify the channel protein using molecular cloning approaches. Screening a c-DNA library from locust antennae with appropriate molecular probes led to the isolation of highly reactive clones. It contains several sequence motifs that are characteristic for cyclic nucleotide-binding domain within partially hydrophobic region near the C-terminus, a pore-forming region and a residual voltage-sensor motif.

- Olfactometric responses to volatile compounds
Strong positive responses were observed for sexually mature males of L. migratoria, when offered either extracts concentrated over Poropak or air impregnated with volatiles emitted by females. The reverse was also observed for sexually immature females responded to mature males. The identified compound ethyl-acetophenone indicated a strong and prolonged arrestant effect in sexually mature males. In S. gregaria mature females responded significantly to guaiacol. Veratrole showed an inhibition effect. For the responses of mature males' guaiacol has to be combined with veratrole or benzylcyanide. For immature males' guaiacol, benzaldehyde and benzylcyanide in binary or tertiary combination gave high responses. Immature females responded only to the combination of four compounds. According to these observations, the aggregation pheromone of S. gregaria will be a blend of headspace compounds.

- Elektrophysiological reactions of the antennae to volatiles
Electroantennographically, all components of headspace tested evoked a significant response depending on their concentrations, showing that there are receptors on the antennae which percept the chemicals. A strong reaction to the mixture was observed in immature females and males, thus there may be an additive or synergistic effect of the compounds. From the singly tested compounds' benzylcyanide had the strongest antennographical reaction in adult locusts, especially in immature males.

- Benzylcyanide and colour change in S. gregaria males
Two different populations were tested for colour change in males and the influence of benzylcyanide on copulation and egg-laying were studied. Benzylcyanide was produced only from yellow males. For copulation and egg deposition a colour change was not necessary. The colour change did not depend on the maturation process. Benzylcyanide was unable to induce yellow coloration in immature and mature males. But the maturation process in males and females increased rapidly if a yellow male was induced. For the induction of yellow coloration, i.e. production of benzylcyanide, a body contact with a yellow male was necessary for rapid coloration.


- Pheromones and reproduction in S. gregaria
In unmated females egg-pod production increased in the presence of males by means of a male volatile chemical factor. Mislaid egg-pods were highest in females without antennae and in virgin females near to males. A high number of eggs per pod were correlated to a low production rate of egg-pods. The egg-laying interval was shorter than in virgin females; in the latter it was shortened in the presence of males producing volatile pheromones.

- Effect of Melia extract on larval development, maturation and egg production in S. gregaria
Oral applications of Melia extract prolonged the nymphal development, delayed the maturation of the adults, thus no oviposition took place. Males did not become yellow and their fat body was reduced. In oocytes no formation of chorion was observed.

- Population differences in S. gregaria
In S. gregaria normally 4 to 5 generations can be obtained in gregarious populations if continuous breeding takes place through one year. A population found in Tenerife reproduced only twice per year with a higher number of eggs per egg-pod, but with low hatching rate. The lowest number of eggs/pod was correlated with the fasted reproduction. No difference was found in nymphal development. The period of preoviposition was much longer in the population from Tenerife than from other places. Yellow coloration of mature males occurred only under crowded conditions.

Follow-up

- What is the real blend of volatiles acting as pheromone ?
- Are there different blends of volatiles having various pheromone effects ?
- How induce the body contact with yellow males the production of benzylcyanide in conspecific mature males and what is the meaning of the yellow coloration ?
- Do the less volatile compounds on the cuticle, i.e. long-chain ketones, tetrahydrofurans, di-n-alkylethers and long-chained bishomolog nitrogen compounds have any pheromone character ?
- Heterologous expression of the c-AMP activated channel type from locusts may be allowed to fully characterise for the first time a cyclic nucleotide gated potassium channel to understand the structure/function relationship between cyclic nucleotide gated channel types;
- What are the real pheromones influencing reproduction and gregarisation ?
- Besides behavioural mechanisms, there may be a genetic factor influencing aggregation and reproduction differing in various populations.
- Identification of the headspace volatiles from adults, hoppers and faeces by using CLSA stripping method and GC + MS;
- Applying an olfactometer to various adult and hopper stages to find out, whether the moving air-column contains stage specific volatiles, and to identify them by means of the analysed compounds;
- Performing research on the mode of action of the analysed volatiles by special rearing experiments and antennograms;
- Using special less volatile identified compounds as cuticular layer to find out whether there is a substance functioning as a contact pheromone;
- Treatment of immature adult females of locusts with the main headspace compounds of mature males demonstrating their influence on reproduction process;
- Investigations of the olfactory signalling by patch-champ techniques;
- Generation of monoclonal antibodies which interact with distinct subpopulations of cells in locust antenna;
- Carry out special experiments showing the influence of locust pheromones on the endocrine system;
- Evaluation of botanicals interfering the pheromone action chain to reduce reproduction and on this way population density.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

Universität Hannover
Address
Herrenhäuser Straße 2
30419 Hannover
Germany

Participants (5)

UNIVERSIDADE NOVA DE LISBOA
Portugal
Address
Quinta Da Torre
2825 Monte Da Caparica
UNIVERSITE CADI AYYAD
Morocco
Address

40000 Marrakech
UNIVERSITY OF HAMBURG
Germany
Address
Martin-luther-king-platz 6
20146 Hamburg
University of Cairo
Egypt
Address
Cairo University Street
Cairo - Gizeh
Universität Hohenheim
Germany
Address
Garbenstraße 30
70599 Stuttgart