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Effects of Volatiles from Clavigralla tomentosicollis Stål. (Hemiptera: Coreidae) Adults on the Host Location Behavior of the Egg Parasitoid Gryon fulviventre (Crawford) (Hymenoptera: Scelionidae)

The egg parasitoid Gryon fulviventre is a potential biological control agent of Clavigralla tomentosicollis, a coreid pod-sucking pest of Vigna unguiculata. The host location behavior of naive parasitoid females was studied using a four-armed olfactometer. Two strains of G. fulviventre parasitoids from Burkina Faso and Benin were exposed to odors provided by healthy and infested pods as well as C. tomentosicollis females and males. The time spent in each odor zone was recorded to determine the preference of parasitoid females. Results show that odors from healthy pods, infested pods, and pest females did not attract the parasitoid. However, a significantly attractive response of both strains of G. fulviventre was recorded in the presence of volatiles from males of C. tomentosicollis. Moreover, experiments testing G. fulviventre females’ behavior when simultaneously exposed to volatiles from cowpea pods (healthy and infested) and increasing numbers of C. tomentosicollis males revealed a significantly higher attraction of parasitoid females of both strains by volatiles from ten males of C. tomentosicollis. The results suggest that the males of the insect pest emit a pheromone used as kairomone by parasitoids to locate their host. The conditions determining this attractiveness at field level and its impact on host-searching efficiency are discussed.


Introduction
Insect pests are an important constraint to cowpea (Vigna unguiculata (L.) Walp.) production in the tropics, including in Burkina Faso where the coreid pod-sucking bug, Clavigralla tomentosicollis Stål. (Hemiptera: Coreidae) is a key pest. 1 Clavigralla tomentosicollis nymphs and adults cause high economic losses on cowpea by feeding on the pods. 2 The damage is greater when the infestation occurs during flowering or pod-filling stage, and a treatment threshold is set at two to four larvae. 3 Several control strategies, including cultural control methods and host plant resistance, have been explored with limited success. 4 To date, the most effective control strategy relies on the spraying of synthetic insecticides. However, these pesticides are often not affordable to small-scale farmers and illiteracy can lead to their misuse which can generate many adverse effects. 1 To find healthy and ecologically sustainable control method, our research has focused on biological control. Biological control of C. tomentosicollis using parasitoids has been less explored. Natural parasitism by the egg parasitoid Gryon fulviventre (Hymenoptera: Scelionidae) has been recorded on over 52% of the target insects in Nigeria and Burkina Faso. 5,6 Natural parasitism by the egg parasitoid is low at the beginning of cowpea growing season but becomes high by the end of season leading to substantial reductions in the pest population. 5,6 Although this observed high parasitism suggests a potential use of G. fulviventre to control C. tomentosicollis, to date, little attention has been given to this alternative control measure. Moreover, there is very little information on G. fulviventre's bio-ecology and behavior.
The natural occurrence of parasitoids on a given pest does not necessarily make them effective biocontrol candidates. The selection of a parasitoid as a biological control agent relies on several requirements, including reproductive potential, effective mass rearing, and host-searching efficiency. Usually, parasitoid host searching is activated by a stimulus, such as complex volatiles from host insect and/or its host plant. 7,8 In several parasitoid species, effective detection of this stimulus depends on the parasitoid's ability to discriminate between a blend of volatiles produced both by herbivore-damaged plants and host insects. 9,10 High host-searching efficiency is also known to be a key factor determining the success of several parasitoid species used in large-scale biological control attempts. [11][12][13] Sources of volatile compounds that may influence the parasitoid's research behavior include the host plant (cowpea pods) and International Journal of Insect Science insect pest stages (particularly adults and eggs). Preliminary studies testing egg masses of C. tomentosicollis as volatiles source for females of G. fulviventre showed no significant host-searching behavior. Since then, in this study, we investigated the role of volatiles produced by cowpea pods and C. tomentosicollis adults on the host selection process of females from two strains of the parasitoid wasp G. fulviventre. The results could contribute to identify factors influencing host searching and to determine their importance in the implementation of a large-scale biological control strategy based on G. fulviventre.

Insect mass rearing
Strains of both C. tomentosicollis and G. fulviventre used in this study were derived from a laboratory mass rearing facility at the International Institute of Tropical Agriculture (IITA) in Cotonou, Benin. In the rearing room, the average temperature was 26.15°C ± 1.54°C and the mean relative humidity was 65.9% ± 7.6%. Clavigralla tomentosicollis nymphs and adults were supplied with fresh pods of a continuous crop of the sensitive Benin landrace cowpea variety, Kpodji-guê-guê, and were reared in cylindrical boxes (1900 mL each). Two strains (one from Burkina Faso and one from Benin) of the parasitoid G. fulviventre were reared on eggs of C. tomentosicollis. For this purpose, one mated G. fulviventre female was introduced in a cylindrical box (360 mL) for 48 hours with fifty fresh eggs of C. tomentosicollis. Emerging bug larvae hatching from nonparasitized eggs were removed from the box. The parasitized eggs were then monitored, and the subsequent generation of parasitoids emerged 16 days after parasitism occurred. Both strains of parasitoids were used in rearing and experiments as well.
For the olfactory tests, naive emerging G. fulviventre females were kept by pairs with males for mating prior to testing them. Burkina Faso and Benin are quite different in their climatic conditions which can influence insect ecology and behavior. That is the reason why both parasitoid strains were used in the hypothesis of a behavioral difference.

Obtaining the odors for olfactory tests
Several sources were used to obtain the tested odors. First of all, cowpea pods were collected from field at the IITA station. The harvested pods were previously washed with tap water to clean any other odor before use. The pods were placed under a stream of tap water for a few minutes without scrubbing to remove any insect odors that may be initially present. This does not change the structure of the pod and its odors and allows infestation with a controlled number of insects.
Infested pods were obtained by placing freshly harvested and cleaned pods with C. tomentosicollis adults and nymphs for 24 hours (24-hour infested pods) and 48 hours (48-hour infested pods). A total of five pods of each type were used for the experiment. Pods infested in this way for 48 hours are damaged enough to change the volatile compounds they emit. The healthy pods were not exposed to the insect prior to the trial. Three-day-old males and females of C. tomentosicollis collected from the rearing boxes were also used as source of odor. The insects used to produce odors were not provided with food over the course of the test.

Olfactometer setup
The response of G. fulviventre to volatiles produced by cowpea pods and C. tomentosicollis adults was investigated using a fourarmed olfactometer previously described. 14 This device was expected to determine the discriminatory ability of insects to more than two sources of odor at the same time. The device allows testing three different odors and one control at the same time as in a previous study. 15 The four-armed olfactometer is a complex device that includes a central unit called the orientation chamber in which the insect can move freely. The chamber has four openings 90° apart that allow the entry of air. A hole which ensures the exit of the air is in the middle of the room. The latter is finally covered with a transparent glass plate. From the end to the orientation chamber, the olfactometer consists of a glass tube containing charcoal for the purification of air, another container of distilled water for humidifying the air, and four flowmeters that calibrate the air inlet. Each flowmeter was connected to an odor source ( Figure 1). Clean airflow (wind speed: 300 mL/min) was divided into four subflows, and each subflow passed through one of the four odor sources connected to the four arms of the olfactometer. The device thus made it possible to deliver air loaded with each of the four test odors into the exposure chamber. The air exit hole in the middle of the orientation chamber prevents an accumulation of odors inside.

Experimental procedure
Mated, three-day-old, naive G. fulviventre females (without any prior contact with cowpea pods or C. tomentosicollis adults) were introduced individually into the exposure chamber and their behavior was observed for a maximum of 5 minutes. Six sets of four-odor combinations (three odors and one control) were tested ( Table 1). The choice of three,  3 five, ten, and fifteen individuals of male and female for pheromone experiment was based on preliminary tests. The increasing number of individuals was expected to yield increasing amount of pheromone which affects parasitoid attraction. 16 A test began once the female started moving, and the time spent in each odor field was recorded. Indirectly, the frequencies of entering in each odor field were recorded. Females remaining motionless for more than 2 minutes at the release point were discarded from the analysis. After testing five naive females, the positions of the odor sources were exchanged to correct for any potentially unforeseen asymmetry in the experimental setup. After testing ten naive female wasps, odor sources were renewed. In total, sixty naive female parasitoids from each strain were tested for each combination. Before each test, the arena was cleaned with laboratory alcohol. In the olfactometer room, the average temperature was 27.2°C and the mean relative humidity was 63.4%.

Data analysis
Data were analyzed to determine differences in the effect of the odor source on the behavior of female parasitoids. The Shapiro-Wilk and Levene tests in R (R Development Core Team 2014) were used to analyze the times spent by the parasitoids in each odor source to check for normality and homogeneity of variances, respectively. Several series of transformations (log ,log( ), , ( . ),ln ,ln( )) x x x x x x + + + 1 05 1 were also used without normalizing the data. So, the Kruskal-Wallis test, as an alternative to analysis of variance, was used with a = .05. Friedman tests 17,18 with a = .05 were used to analyze the frequency of choice of G. fulviventre females for each of the odors tested using XLSTAT752 software (Addinsoft; XLSTAT752).

Response of parasitoid females to odors from healthy and infested cowpea pods (Experiment 1)
The time spent by the parasitoids in the arms receiving odors from cowpea pods and the clean air ranged from 56 to 98 seconds for both strains of parasitoid (Burkina Faso and Benin). Females of both G. fulviventre strains were not significantly attracted by volatiles from either healthy or damaged cowpea pods (P = .495 and P = .558, respectively; Table 2). Similarly, mean frequencies of parasitoid choice among the 4 odors tested did not significantly differ for the Burkina Faso (P = .492) and Benin (P = .321) strains.

Response of parasitoid females to odors from Clavigralla tomentosicollis females (Experiment 2)
The parasitoid females were not significantly attracted by odors emitted by females of C. tomentosicollis (Table 3). Differences in time spent in the four arms of the olfactometer, as well as frequencies of choice between odors, did not significantly differ for both strains ( Table 3).

Response of parasitoid females to odors from C. tomentosicollis males (Experiment 3)
Gryon fulviventre females were attracted by odors from males of C. tomentosicollis depending on not only the strain tested but also concentration (Table 4). For the Burkina Faso strain, the time spent by the parasitoids in the arm diffusing volatiles from C. tomentosicollis males was significantly different (P = .037), whereas the frequency of choice among the four odor sources did not differ (P > .05). For the Benin strain, only the frequency of choice of G. fulviventre females for the odors from C. tomentosicollis males was significantly different (P = .001).

Discriminatory response of parasitoid females between combined odors from healthy pods, infested pods, and C. tomentosicollis males (Experiments 4-6)
As the compounds released by males of C. tomentosicollis attracted G. fulviventre females, we also tested their response to odors from an increasing number of males in comparison with volatiles from healthy and infested cowpea pods (Tables 5 to 7). Females of both strains showed no significant difference for the time spent or the frequency of choice for the odor sources tested with five males (Table 5). However, females were attracted by the odors released by ten males of C. tomentosicollis as evidenced by the higher frequencies of choice in Burkina Faso strain (P = .028). Females in Benin strain showed a discriminatory capacity of the different odors (P = .008), but they did not discriminate odors between infested pods and ten males ( Table 6). Again parasitoid females of both strains were not significantly attracted when the tested odors were provided by cowpea pods and 15 C. tomentosicollis males (Table 7).

Discussion
This study demonstrated that olfactory stimuli are used by G. fulviventre females to locate their host C. tomentosicollis.  However, the parasitoid females did not discriminate between volatiles produced by healthy and infested cowpea pods when they were exposed only to cowpea volatiles. For many parasitoid species, pest-damaged plants, including maize 19 and soybean, 20    International Journal of Insect Science hypothesize that G. fulviventre may be attracted either to a sexual pheromone used by C. tomentosicollis males to attract females or an aggregation pheromone released by the males. Such an assumption is based on previous observations indicating the possibility for parasitoids to use host-male insect's pheromones as kairomones to locate the hosts. [27][28][29][30][31] However, the results suggest that this odor attractiveness effect could depend on the number of C. tomentosicollis males used. At low and high densities (five and fifteen individuals, respectively), G. fulviventre females did not discriminate odors from the males. This finding suggests, on one hand, that a minimal amount of pheromone is first needed to trigger a response in parasitoids and that, on the other hand, the amount of pheromone released may in some way decrease once the pest densities have exceeded a certain threshold. Moreover, simply due to their regrouping in the olfactometer and regulating their density, the insects can inhibit the secretion of the congeners' attractive pheromone. [32][33][34] Such inhibition involves the secretion of antiaggregation substances, 33,35 which would not be perceived by the parasitoid or which would not play an attractive role for G. fulviventre female. Otherwise, pheromone is a mixture of several compounds and some of the compounds would act as a repellent for parasitoids. 36,37 An experimental olfactometer offers small and confined space distinct from real environmental conditions. Further experiments in actual environmental conditions could better establish what pest density, if any, triggers parasitoid infestation. Moreover, the precise identification of attractants for female parasitoids is an important step in considering the release of such substances to attract more parasitoids at the right time to prevent the growth of pest populations. In the wild, visual stimuli can also be involved, with or without olfactory signals, in triggering parasitoid activities. [38][39][40][41] Any role of this for host location behavior in G. fulviventre remains to be investigated. Provided that a precise attractive mechanism is characterized to ensure parasitism, then mass rearing and release of G. fulviventre at the beginning of the growing season when pest infestation initially occurs could potentially be used to control C. tomentosicollis populations.

Conclusions
The results obtained from this study are of great importance in the understanding of host-parasitoid interactions using a model that has been poorly studied, such as the egg parasitoid G. fulviventre and the major cowpea pod-sucking bug pest, C. tomentosicollis. The main findings indicated that the parasitoid females are attracted by olfactory cues provided by the males of their host species alone, cowpea pods (infested or not) being not attractive. Although precise determination of attractive volatile compounds emitted by C. tomentosicollis males remains for future research, the attractiveness of the involved compound depended on the density of males present and probably on the dose of volatiles produced. Subsequent studies could also characterize any role of other factors influencing the biological potential of this control agent, a necessary step for developing a best practice strategy for an effective protection of cowpea fields in West Africa.