Do age and mating status affect olfactory response of the parasitoid, Microplitis croceipes (Hymenoptera: Braconidae) to host-related plant odors?

Insects

Cocoons of Microplitis croceipes were provided by the USDA-ARS, Insect Biology and Population Management Research Laboratory (Tifton, Georgia, USA) and reared in our laboratory (Auburn University, AL, USA) on 2^nd – 3^rd instar larvae of H. virescens. Upon emergence, adult wasps were transferred to aerated plastic BugDorm® cages (Megaview Science Co. Taichung, Taiwan) and supplied with 10% sucrose/water solution (w/v). Naive (untrained) parasitoids were used in both EAG and Y-tube olfactometer bioassays to test innate responses of parasitoids to host-related plant odors. Eggs of H. virescens were initially purchased from Benzon Research Inc. (Carlislie, PA, USA) and reared in our laboratory at Auburn University. Larvae of H. virescens were reared on pinto bean artificial diet according to Shorey and Hale³². In total, about 640 female parasitoids were used for bioassays. The general conditions for insect rearing and bioassays were 25 ± 1°C, 75 ± 5 % RH and L14:D10 h photoperiod.

Plants

Cotton (Gossypium hirsutum, var. max 9, All-Tex Seed Inc., Levelland, TX, USA) plants were grown in individual pots (9 cm high, 11 cm diameter) in a growth chamber at 26.6°C day, 25.6°C night, 60% RH, L16:D8 h (L:D) photoperiod. Seeds were planted in a top soil/vermiculate mixture. Plants used for headspace volatile collections were 4–6 weeks-old.

Age and mating status treatments

Upon emergence, parasitoids were separated based on their sex. Subsequently, an equal number of females were randomly designated to ‘mated’ or ‘unmated’ cages. Male parasitoids were put inside cages (19 × 13 × 10 cm) designated to mated females at a 2:1 (male: female) ratio while cages designated to unmated females contained females only. Female parasitoids were allowed to mate for at least 24 h before use in bioassays. Both mated and unmated females were further designated to separate cages based on their age. Age groups 1–3, 4–6 and 7–9 d-old were used in Y-tube olfactometer bioassays while age groups 1–3, 4–6, 7–9, and 10–12 d-old were used in electroantennogram (EAG) recording. The 10-12 d-old age group was not included in behavioral bioassays due to low numbers of insects surviving beyond 10 days in the laboratory, resulting in a low number of replicates. It should be noted that Y-tube olfactometer bioassays required more replicates than EAG recording.

Headspace volatile collection

Headspace volatiles were collected from H. virescens-infested cotton plants using the protocol described by Ngumbi et al.³¹. A plant pot with soil was wrapped with aluminum foil to minimize contamination. The plant was then placed in a volatile collection chamber (Analytical Research Systems, Inc., Gainesville, FL, USA) consisting of a 5-L air-tight glass jar. A purified air stream of 500 ml/min was passed through the jar at room temperature using Teflon tubing connected to an air delivery system. To induce volatiles from plants, 30 2^nd –3^rd instar larvae of H. virescens were allowed to feed on a cotton plant for 24 h during volatile collection from 1100 h one day to 1100 h of the following day. Headspace volatiles were collected with a trap containing 50 mg of Super-Q (CAT#: 2735, Alltech Associates, Deerfield, IL, USA) and eluted with 300 µl of methylene chloride. The resulting extract was stored in a freezer (-20°C) until use.

Y-tube olfactometer bioassays

A Y-tube olfactometer (Analytical Research Systems, Inc., Gainesville, FL, USA) was used to test attraction of female M. croceipes to four odor stimuli of varying complexity. The setup and procedure was similar to that reported by Morawo and Fadamiro³³. Parasitoids were introduced individually into the olfactometer and allowed to make a choice between test stimulus and control. Insects were tested once and discarded. Parasitoids that made no choice within 5 min were removed and excluded from the analyses. The number of non-responding parasitoids in the 24 sets of bioassays ranged from 0 to 5 with a mean of 1.2 insects per test.

cis-3-Hexenol (CAT#: W256307) and α-pinene (CAT#: 147524) (purity 95-99%; Sigma-Aldrich®, St. Louis, MO, USA) were individually formulated in hexane (HPLC-grade) at 1 µg/µl concentration. A 50/50 v/v binary mixture (cis-3-Hexenol + α-pinene) of the two compounds was also prepared. A central dose of 10 µg (10 µl sample) was previously determined to be optimal in a related study³⁰. In separate bioassays, each compound or binary mixture was delivered as a 10 µl sample on filter paper strips (40 × 7 mm, CAT#: 1001090, Whatman® No. 1) in the treatment arm while the control arm contained the same volume of hexane (solvent control). Humidified and purified (charcoal filtered) air was pushed into each arm at the rate of 250 ml/min and removed by suction from the central arm of the olfactometer at the rate of 500 ml/min to avoid odor mix-up.

To test parasitoid response to H. virescens-infested cotton, one arm of the olfactometer was connected to an air-tight glass jar (5-L) containing an infested plant (with host larvae). The other arm was connected to a similar glass jar containing a pot of soil covered with aluminum foil, which served as control. A new plant was used on different days during which bioassays were conducted (5–7 plant replicates). Inlet air was pushed into the olfactometer through each jar at the rate of 300 ml/min and sucked out at the rate of 600 ml/min. Experiments were performed in a randomized complete block design with equal number of insect replicates from each age and mating status groups tested per day (n = 20 per test). All olfactometer bioassays were conducted between 1100 h and 1700 h on different days.

Electroantennogram recording (EAG)

EAG response of female M. croceipes was recorded to measure odor perception in mated parasitoids. The EAG protocol used was previously described by Ngumbi et al.³¹. Glass capillaries (1.1 mm I.D.) filled with Ringer solution served as reference and recording electrodes. The reference electrode was connected to the back of the head of a female M. croceipes while the recording electrode was connected to the cut tip of the terminal segment of the antenna. The analog signal was detected through a probe (INR-II, Syntech, the Netherlands), and was captured and processed with a data acquisition controller (IDAC-4, Syntech, the Netherlands). EAG 2000 software v2.7 (Syntech, the Netherlands) was used to analyze digital signal readouts. Test stimuli were delivered as 10-µl samples (10 µg dose) on filter paper strips (7 × 40 mm) placed inside 14 cm Pasteur pipettes (Fisher Scientific, Pittsburgh, PA, USA).

Four treatment stimuli and two control stimuli were individually delivered as 0.2-s puffs of air, with 2 min interval between puffs. For each antennal preparation, the following stimuli were presented: hexane (control), methylene chloride (control), cis-3-hexenol, α-pinene, binary mixture, headspace volatile extract, hexane and methylene chloride. Thus, hexane and methylene chloride (solvent controls) were applied at the beginning and end of each recording series while the position of other test stimuli was randomized across replicates (see Morawo et al.³⁴). Recordings were performed in a randomized complete block design with equal number of insect replicates (n = 10) from each age group tested per day.

Data analyses

Attraction of parasitoids to each of four test stimuli in Y-tube olfactometer was modeled as a binary response (stimulus = 1, control = 0) using logistic regression to analyze possible interactions between age and mating status factors. The model adequacy for each set of experiment was confirmed with a likelihood ratio test³⁵. When no significant interaction was recorded, each factor was analyzed separately. For olfactometer data, deviation of parasitoid responses from a 50:50 (stimulus: control) distribution was analyzed using a Chi-square goodness-of-fit test. Absolute EAG responses (EAG response to solvent control deducted from EAG response to test stimuli) of mated parasitoids across age groups were compared using Kruskal-Wallis test. All analyses were performed in SAS v9.2 (SAS Institute Inc., Cary, NC, USA) with P = 0.05 level of significance.

Effect of age and mating on attraction of parasitoids in Y-tube olfactometer

Overall, there was no significant interaction between age and mating status factors for any of the four odor stimuli tested (cis-3-hexenol: P = 0.7295, Figure 1A; α-pinene: P = 0.7352, Figure 1B; binary mixture: P = 0.1136, Figure 1C; host-infested cotton: P = 0.7044, Figure 1D; Logistic Regression). However, mated parasitoids, 4-6 d-old were significantly (80/20%, χ²= 7.20, df = 1, P =0.0073) more attracted to the binary mixture (cis-3-hexenol + α-pinene) compared to hexane control (Figure 1C). Similarly, 1-3 d-old mated parasitoids were significantly (75/25%, χ²= 5.00, df = 1, P =0.0253) more attracted to H. virescens-infested cotton compared to control (Figure 1D). Parasitoids did not show significant attraction to test stimuli in other Y-tube olfactometer bioassays.

Figure 1. Effect of age and mating status on the attraction of female Microplitis croceipes to four host-related plant odors in Y-tube olfactometer bioassays.

Bars represent percentage of mated and unmated parasitoids of ages 1–3, 4–6, and 7–9 d-old when given a choice between hexane (solvent control) and synthetic compounds cis-3-hexenol (A), α-pinene (B), a 50/50 v/v binary mixture of cis-3-hexenol and α-pinene (C), and a choice between control jar (with no plant) and Heliothis virescens-infested cotton (D). Synthetic compounds were formulated in hexane at 1 µg/µl and presented as 10 µl samples (10 µg dose). Thirty 2^nd–3^rd instar larvae of H. virescens were allowed to infest cotton plants for 24 h before bioassays. N = 20 responding parasitoids per choice test. Numbers in the bars indicate actual number of responding individuals that chose each arm of the olfactometer. Asterisk (*) indicates significant deviation of parasitoid responses from a 50:50 (stimulus: control) distribution (χ² goodness of fit test, P < 0.05).

Effect of age on EAG response of parasitoids

In general, the age of mated female M. croceipes did not have a significant effect on their EAG response to test odor stimuli (Figure 2). Both relatively young and older parasitoids showed similar levels of innate antennal sensitivity to single components, binary mixture and headspace volatile extract of host-infested cotton. These results are mostly in agreement with those recorded in Y-tube olfactometer bioassays.

Figure 2. Effect of age on the electroantennogram (EAG) response of mated female Microplitis croceipes to four host-related plant odors.

Bars represent mean absolute EAG responses (mV± SE, N = 10) of mated parasitoids age 1–3, 4–6, 7–9 and 10–12 d-old to cis-3-hexenol (A), α-pinene (B), a 50/50 v/v binary mixture of cis-3-hexenol and α-pinene (C), and Heliothis virescens-infested cotton headspace volatile extract (D). Absolute EAG for each stimulus is the actual EAG value minus EAG value of solvent control. Synthetic compounds were formulated in hexane at 1 µg/µl and presented as 10 µl samples (10 µg dose). NS indicates no significant difference.