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Selection of favorable oviposition site is a complex process vital in progeny development and fitness of insects1. Owing to strong selection pressure, the female has to evaluate and direct her eggs quickly into suitable oviposition sites for her generation to proceed2, 17. Site specific chemical cues aid in the evaluation and egg-laying process, but, this process is time consuming and costly for the insects in terms of fitness3-4. Therefore insects, through long term association or co-evolution, construct recognition templates for a particular chemical cue that aid in easier and faster processing of information in their brain5-7. Although acquired recognition templates are common, congenital recognition templates (CRT) are quite challenging to detect and is not exhibited by all insects7. But, we observed the presence of a CRT is the egg-laying behavior of the mango fruit fly, Bactrocera dorsalis. Gravid females of B. dorsalis readily lay eggs in ripe mangoes but shows explorative behavior on un-ripe fruits with scanty egg laying8. Here we show that the "ready oviposition" behavior in B. dorsalis is because of a conserved CRT to a lactone (Gamma-Octalactone) present in ripe mangoes (cv. Alphonso). The compound activates the specific CRT inducing oviposition in B. dorsalis without the need of oviposition site or evaluation process. This behavior can be attributed to a long association or co-evolution between mango and the mango fruit fly, B. dorsalis.
We asked what the evolutionary reason might be for the strong oviposition response to Gamma-Octalactone. Gamma-lactones are strong anti-fungal agents and inhibit growth of molds at even small concentrations9. Apart, it is a cue that is emitted from ripe mangoes and its presence means less terpene compounds10 that are detrimental to fruit flies. The heavy competition for suitable oviposition sites within and among species poses a threat. Thus, a conserved oviposition stimulant detection template makes it easier for flies to identify suitable oviposition sites quickly. The CRT, we think, does not alert the flies to only the presence of appropriate oviposition site but to a clean, healthy nursery devoid of competition from fungal saprophytes and insecticidal terpenes that can harm the development of its larvae. Thus, a fly should have the essential ability to expedite detection of a suitable oviposition site.
To test the presence of CRT and its conserved nature, we reared 52 generation of B. dorsalis from a parental line where only the first generation was exposed to mangoes. Thereafter, the succeeding generations was reared on banana or guava and was not exposed to mango or mango cues until the experiment. Since we were interested in the oviposition behavior, we used fresh gravid females for all our experiments. Cues from ripe mango (cv. Alphonso), collected by head-space air-entrainment technique with Porapak-Q were analyzed by GC-EAD and GC-MS. Using a simple bioassay technique, synthetic compounds that were EAD active (10 ÂµL/disc) were presented to gravid females on filter paper discs (50 mm diameter) to study their behavior to the cue. It was observed that some cues were highly attractive (Fig. 1a) but a specific cue, Gamma-Octalactone, triggered oviposition behavior where the flies extended and probed the filter paper using the ovipositor. Although filter papers are not appropriate oviposition sites, the females showed probing action seen only during egg-laying (Fig. 1b). This observation indicated the involvement of a CRT in oviposition site selection and suggested that this particular CRT is conserved in mango fruit flies and passed on through succeeding generations even after being reared on non-specific fruits like banana and guava that is devoid of the cue11, 12. Such behavior in dipterans is thought to be directed through a conserved olfactory circuit that overrides other exploratory pathways such as aversion, chemo-taxis and feeding7.
Substantial studies have focused on recognition templates and chemical ecology of oviposition behavior13, yet little is known about cues involved in activating such templates. Our behavioral studies indicate that B. dorsalis use Gamma-Octalactone to recognize oviposition sites readily. As Gamma-Octalactone is a volatile compound, the antenna plays a crucial role in its recognition. Therefore, we recorded their electrophysiological responses to the volatile cue. The recordings were carried out using the antenna (Fig. 1c) of fresh gravid females that were reared on banana or guava. Recording was done by pulsing air from filter paper (control) or filter paper smeared with different concentrations of the cue, directly over the antenna. The responses were recorded in an EAG and were compared with that of the control. From electrophysiological studies it was found that the fly's antenna responded equally to all concentration ranges (0.05 - 1 ppm) of the chemical cue (Fig. 1d). One-way ANOVA of the mean response among the concentration ranges showed no significant difference (n = 6 per group, F = 1.322, P = 0.2950). The concentration of the cue did not influence the choice of oviposition site. However, presence of the cue was crucial for inducing oviposition response. These results suggest that Gamma-Octalactone activates the CRT of B. dorsalis and helps it choose the right oviposition site.
To test this possibility, we investigated the ovipositional response of B. dorsalis to ripe mango pulp with or without Gamma-Octalactone. If the ripeness was the sole reason for oviposition, the flies must lay eggs in both Gamma-Octalactone treated and un-treated pulp (control). The results of the two-choice assay reveled that B. dorsalis directed significantly large number of eggs (95.85 Â± 8.7% SEM) into treated pulp than the control (paired t-test, n = 20, t = 11.270, df = 19, P < 0.0001) (Fig. 2a). Next, we tested if the concentration of the cue influences the oviposition site preference. The results indicated that the number of eggs laid into pulp containing different concentration of the cue was not significantly different (One-way ANOVA, n = 6 replicates per concentration, F = 0.267, P = 0.848) (Fig. 2b). Gravid flies sharply differentiate between treated and untreated pulp indicating the existence of a dedicated CRT that recognize Gamma-Octalactone alone and if activated, instigate flies to lay eggs into the pulp containing the cue.
Next we set out to determine if the pulp (nutrition) was required for oviposition or is the cue enough to induce oviposition. Using a single-plate dual choice assay, we examined the above possibility. The result was surprising as the flies laid 98.13 Â± 5.6% of eggs (paired t-test, n = 9 replicates, t = 6.610, df = 8, P < 0.0001) into agarose discs (no nutrition) with Gamma-Octalactone (Fig. 2c). The outcome confirmed that Gamma-Octalactone was enough to induce oviposition. This response is channeled through a CRT that overrides inputs from gustatory and other olfactory pathways7.
Being a frugivorous insect, the larvae of B. dorsalis grow and complete their development on fruits. But, the placement of eggs by gravid females is a crucial component in the fitness of the larvae. Therefore, it is beneficial for the flies to develop a recognition template to specific cue that signals them of a site that provides optimal growth conditions for their larvae. Here we show three important findings. Firstly, the ripe mango cue, Gamma-Octalactone activates a dedicated CRT thus overriding exploratory behavior in B. dorsalis. Secondly, we discovered an oviposition stimulant for B. dorsalis. Thirdly, we proved that the concentration of the cue does not play a role in CRT related neural pathways. The identification of such cues that mediate astonishing behavior are important and may be a lead in understanding the olfactory neural pathways underlying such conserved recognition templates14, 15. The study may also open new avenues in pest management, agriculture, neurology and chemical ecology.
A wild type strain of B. dorsalis flies were established and maintained from 2010 at the Division of Entomology and Nematology, Indian Institute of Horticultural Research, Bangalore, India. The first generation was reared on mango while the subsequent generations were reared on guava or banana. Fruits were exposed to gravid females for oviposition. Oviposited fruits were placed on fine sand to aid larval development and pupation. Pupae were separated by sieving the sand and placed in screened cages (30 x 30 x 30 cm) for the emergence of adults. Adult flies that emerged were provided with yeast, sugar and honey solution moistened on cotton swabs ad libitum. Adults, 7-days old, were allowed to mate and gravid females (15 days old) were separated into another cage for behavioral assays. All colonies were maintained at optimum growth condition of 28 Â± 2Â°C, 75% RH and a photoperiod of 12-h light/12-h dark cycles. For all experiments progeny (newly gravid females, n = 30) of the final generation were used in all experiments.
Chemical extraction, GC/EAD/MS and identification
The method described by Jayanthi et. al was followed exactly as described16.
Concentration preference and electrophysiology studies
Electroantennogram (EAG) recordings were done with Ag-AgCl glass microelectrodes filled with saline solution. During recording, gravid female flies were anaesthetized by chilling and there antennae were removed with a pair of micro-scissors for EAG preparations. The antenna was placed between the electrodes to and the signals passed were detected by a high impedance amplifier and analyzed using customized software. The recordings were taken by pulsing air containing the identified oviposition stimulants at different concentration ranging from 0.05 to 1 ppm over the antenna. Six recording per EAG active peaks was done and the recordings in mA were noted. Oviposition substrate was prepared as described in two-choice assay method. The molten substrate containing appropriate concentrations of oviposition stimulants was poured into 6-well culture plates in a manner that each alternate well received a known test concentration. The plates were placed in cages with newly gravid females (n = 30) and allowed for 24 h for oviposition to occur.
Screening for oviposition stimulants
Authentic chemical standard EAD active fruit-cues were screened for their oviposition stimulation to fruit flies. Briefly, new gravid female flies (n = 30) were released into screened cages. They were allowed to acclimatize for 3-h prior to screening. Singly, each selected fruit-cue (10-uL) was applied on 50 mm filter paper discs and presented to the flies. Hallmark behaviors of oviposition stimulation like extending of ovipositor and puncturing action due to excitation was observed. Cue that elicited such behavior were selected for further studies. The screening for each cue was conducted in triplicates.
The two-choice assay was carried out using transparent 90-mm Petri dish containing an oviposition substrate. Briefly, oviposition substrate was made by mixing mango pulp (var.Alphonso) (1 % w/v) with molten agarose (0.8% w/v) and poured into Petri dish. Each dish received 10-ml of the media and was allowed to cool. Gamma-octalactone was added to the test plates and the control plates did not receive the cue. These plates were presented to the flies to sample for 24-h. The number of eggs laid was counted manually under a stereo-microscope.
Single-plate dual choice assay
The oviposition substrate was divided into exactly 2 halves by drawing a line with a permanent marker behind the Petri dish. One of the halves was swabbed with oviposition stimulants and the other half was used as control. For each test, 30 newly gravid females were released into screened cages and allowed to acclimatize for 3-h after which the Petri plates containing oviposition substrate was placed inside the cage to allow flies to sample for 24-h. To determine the oviposition preference, the number of eggs in each half of the dual-choice assay Petri dish was counted. Percent oviposition was calculated by converting the number of eggs laid in test and control to proportion of the total number of eggs laid.