| Aprostocetus prolixus is an important egg parasitoid of A. germari, stem borer infesting many species of trees. This paper investigated the chemical mechanism in discrimination of previously prarasitized hosts by the parasitoid, which is very important to reveal the mechanism of parasitization by A. prolixus and use the parasitoid to control A. germari.When the healthy host and the host previously parasitized (conspecific-parasitized or self-parasitized) in the same pacth, the number of healthy host firstly contacted is significantly higher than the previously parasitized host (healthy host vs conspecific-parasitized, P < 0.01; healthy hosts vs self-parasitized, P < 0.001). The time for A. prolixus finding healthy host was much less than that for searching previously prarsitized host (healthy host vs conspecific-parasitized host, P < 0.001; healthy host vs self-parasitized host, P < 0.008). It was demonstrated that A. prolixus had marked the host in some stage of the process of parasitization.The markers can be perceived by the parasitoid in short distance and have repellent effect to herself or other individuals, which make the parasitoid prefer choosing the healthy host. The searching time, probing time and parasitization rate were significantly higher than previously parasitized hosts (searching time: healthy host vs conspecific-parasitized host, P = 0.002; healthy hosts vs self-parasitized host: P = 0.006. probing time: healthy host vs conspecific-parasitized host, P = 0.006; healthy host vs self-parasitized hosts, P = 0.008. parasitization rate: healthy host vs conspecific-parasitized host, P < 0.01; healthy host vs self-parasitized host, P < 0.001). It was indicated that parasitoid prefer parasitizing healthy host and avoiding superparasitism.When A. prolixus facing both conspecific-parasitized and self-parasitized host, the behavioral parameters of A. prolixus on the two hosts have no significant differences (the number of hosts firstly contacted, P > 0.05; time for finding each host, P = 0.499; searching time, P = 0.612; probing time, P = 0.310; parasitization rate, P > 0.05). It was showed that A. prolixus couldn't discriminate between consecific-parasitized and self-parasitized host.In non-choice tests, A. prolixus pefered parasitizing the healthy host (healthy host vs conspecific-parasitized host, P = 0.024; healthy host vs self-parasitized host, P = 0.010). It was also proved that the parasitoid marked the host in the process of prasitization. When there was only parasized host in the patch, the superparasitization rate of the previously parasitized host is 56%, but it is 14.3% when both healthy host and parasitized host were simultaneously provided to A. prolixus. It was indicated that A. prolixus could use parasitized hosts for parasitization when there is no healthy host available in the patch.In choice tests, there were no significant differences in the behavioral parameters of A. prolixus between on the healthy host and on host with self-footprints or conspecific-footprints. (healthy host vs host with conspecific-footpints: the number of host firstly contacted, P > 0.05; time for finding each host, P = 0.476; searching time, P = 0.931; probing time, P = 0.420; parasitization rate, P > 0.05; healthy host vs host with self-footprints: the number of host firstly contacted, P > 0.05; time for finding each host, P = 0.931; searching time, P = 0.678; probing time, P = 0.859; parasitization rate, P > 0.05). It was indicated that A. prolixus couldn't discriminate between healthy host and host searched by conspecifc or itself and the parasitoid didn't marke the host when searching on the host (searching with antenna and probing with ovipositor).When A. prolixus facing both healthy host and host previously parasitized but not smeared with ovipositor of the parsitoid , the behavioral parameters of A. prolixus on two kinds of hosts were not signicantly different(the number of each host firstly contacted, P > 0.05; time for contacting each host,P = 0.638; searching time,P = 0.538; probing time, P = 0.433; parasitization rate, P > 0.05). It was demonstrated that A. prolixus couldn't discriminate between healthy host and host parasized but not smeared with ovipositor of the parasitoid, and the parasioid didn't mark the host when searching, probing, detecting with ovipositor and ovipositing.From the results above, It can be easily found that only the hosts smeared with ovipositor of A. prolixus have ovipositing marker, which is not physicalA mark but chemical mark, namely marking pheromones.In the non-choice test, A. prolixus the parasitization rate on the healthy hosts are higher than hosts conspecific-parasitezed once or twice (healthy host vs host parasitezed once: P = 0.028; healthy host vs host parasitezed twice: P = 0.000). The parasitization rate on the conspecific parasitized once hosts is higher than hosts conspecific-parasitized twice (P = 0.013). It was indicated that more marking pheromones were accumulated on the host with times of parasitoid parasitizing the host increasing.In the choice tests, the numbers of firstly contacted hosts applied dichloromethane extracts of the abdomen are significantly less than the controls (host applied dichloromethane) (P < 0.05). The searching time on the hosts applied dichloromethane extracts of the abdomen is significant less than the controls (P < 0.05). However, there were no significant differences between hosts applied other solvent extracts and the controls, which indicates that the marking pheromone is from the abdomen of the parasitoid and dichloromethane is the optimal solvent.The results from GC-MS analysis showed that five groups of chemicals, including hydrocarbons, aldehydes, fatty acids, esters and terpenes, were found in the dichloromethane extracts of abdomen. The contents of hydrocarbons compounds consisting of 20 Akinds of chemicals is 40.55%, and aldehydes 3 kinds 0.32%, fatty acids 7 kinds 54.96%, esters 2 kinds 3.7% and terpenes 2 kinds 0.18%.The bioassays were conducted to measure the effects of dichloromethane of extrats of the Dufour's gland, posion sac (gland) and ovaries of A. prolixus on the behavior of the parasitoid using choice tests The time for the parasitoid firstly contacting the hosts applied Dufour's glands was significantly more than control hosts (applied only dichloromethane) (P < 0.048). The searching time of the parasitoid on the host treated with the extracts of Dufour's gland was significantly less than control host(P = 0.039). The parasitization rate of host applied extracts of Dufour's gland was also lower than control host(P < 0.001). However, there were no significant differences between the controls and hosts applied posion sac (gland) or ovaries in behavioral parameters. It shows that the extracts of Dufour's gland have repellent and ovipostion-deterrent effects to the A. prolixus. The behavioral evidence suggests that Dufour's gland is the host marking pheromone source.In the choice tests, it was found that the parasitization rate on the controls (host applied dichloromethane) was significantly higher than the hosts applied 10μg tetracosane (P < 0.05), and the numbers of the controls firstly contacted are higher than the hosts treated with 10μg palmitic acid (P < 0.05). However, 1μg palmitic acid, 1μg tetracosane, hexadecane (1μg and 10μg) and heptacosane (1μg and 10μg) have no effects on the host discrimination, which indicates palmitic acid and tetracosane are important components of marking pheromones.
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