COMPOUNDS FROM PEPPER FLOWERS AND FRUITS AS POTENTIAL ATTRACTANTS FOR THE CAPTURE OF PEPPER WEEVIL

Two field experiments were conducted to evaluate synthetic attractants derived from pepper flowers, flower buds and fruits, alone or in combination with the aggregation pheromone. The evaluation was carried out with the release and recapture of Anthonomus eugenii adults at different distances from the four cardinal points in separate trials. The volatility of the synthetic mixture and aggregation pheromone was determined by gas chromatographic analysis of the volatiles captured by dynamic headspace. The traps with synthetic mixture and essential oil captured insects at 10 m, while the aggregation pheromone trapped up to 60 m. The combination of synthetic mixture or essential oil with the aggregation pheromone did not increase the number of recaptures compared to the single pheromone. The synthetic mixture together with geranic acid recaptured adults up to 15 m, although they were not significantly different from the control. The exclusion of geranic acid from the aggregation pheromone significantly reduced the number of recaptured insects (P<0.05), while geranic acid alone failed to capture weevils. The results could be improved by increasing the concentrations of the compounds or by adding other compounds released during the reproductive stages of pepper. These results could guide future efforts for the development of tools based on synthetic plant volatiles for the monitoring of this pest.


ISSN: 2320-5407
Int. J. Adv. Res. 9(06), 357-364 358 abscission and reduction in the field ranging from 30 to 90% of production if timely action measures are not implemented (Campbell, 1924;Fernández et al., 2020). Traditional methods are difficult because part of its development takes place inside the fruit, which allows the insect to evade any chemical application. In Mexico, in open fields, up to 15 insecticide applications are performed per season (Avendaño-Meza, 2017), which generates negative effects for beneficial organisms (Rodríguez-Leyva et al., 2007;Labbé et al., 2020).
Since the discovery of the aggregation pheromone, it has been used effectively to monitor the insect in the field (Eller et al., 1994;Eller and Palmquis 2014), but this strategy is only efficient before flowering and at the end of the harvest, because the effect of the pheromone is diluted by the large number of volatiles released by flowers. Recent studies indicated that the pepper weevil responds to odors released by host plants (Addesso and McAuslane, 2009 Although the compounds released by the reproductive structures of peppers have been identified, no studies have been conducted to determine their effectiveness for monitoring insects in the field. Therefore, this work aimed to evaluate the attraction of synthetic compounds derived from flowers, flower buds, fruits and the aggregation pheromone for the capture of pepper weevil adults under field conditions.

Materials And Methods:-Insects
Insects of unknown age and mating status were collected in Ejido Vallejo (23.118245 º N, -100.545644 º W), municipality of Villa de Guadalupe, San Luis Potosí, Mexico, on serrano pepper cultivars during August 2018. The colony was established at the Colegio de Postgraduados, under controlled conditions of temperature (26±2 º C) and photoperiod (13:11 h light: dark). Every third day, emerged adults were removed and transferred to 3 L capacity containers, where they remained until use, continuously fed with developing jalapeño pepper fruits (≤ 30 mm in length). For field experiments, weevils more than 10 days old were used, separated by sex according to the characteristics described by Eller et al., (1995). The insects were left without food and water for 12 hours prior to field trials.

Attractants
Compounds (Z)-β-ocimene, 2-Isobutyl-3-methoxypyrazine, (Z)-3-hexenyl acetate, terpinolene, geraniol and geranic acid were purchased from Sigma Aldrich®, while (E)-β-ocimene was purchased from Chemos®. The components of the aggregation pheromone of the pepper weevil Z Glandlure II, E Glandlure II and Glandlure III & IV mixture (1:1) were purchased from Bedoukian Research® and were formulated according to the concentrations reported by Eller et al., (1994). The essential oil was extracted by steam entrainment with 100 g of poblano pepper cv. flower buds using the methodology described by Zheljazkov et al., (2013), with some modifications. For the synthetic mixture and essential oil, microcentrifuge tubes were used as releasers, which were loaded with 500 mg (mixture or essential oil) diluted in mineral oil (Herschi Trading®) with a final volume of 1 mL for field evaluation. The treatments used during the 2018 and 2019 experiments are shown in Table 1. 359 a Treatment 2 corresponds to the essential oil extracted from the pepper flower buds; b 1:1 Mixture; c Combination between treatments 1 and 3; d Combination between treatments 2 and 3; e The geranic acid was formulated separately and mixed with an equal amount of mineral oil, using microcentrifuge tubes as dispersers; f Same formulation as treatment 3 of the 2018 experiment. * Name of each treatment.

Field experiments
They were conducted in an area of the Colegio de Postgraduados, municipality of Texcoco, State of Mexico, Mexico (19.468861 º N, -98.898833 º W), in a field with a flat topography, without the presence of pepper cultivation to avoid interference from weevils coming from the field. Yellow traps (30.5 cm x 15 cm) impregnated with glue (Adhequim®) were used, where the treatments were placed. The traps were placed one day before starting the experiment at a height of 0.30 m in the direction of the prevailing winds. They were distributed in a completely randomized block design, with three replicates per treatment with a separation of 100 m between each replicate and treatment.
In the first experiment (August-December 2018) treatments were evaluated at six distances in separate trials, each lasting 15 days. A. eugenii adults were released at the four cardinal points of each treatment from each distance used. For evaluation 1, 720 adults were used, 5 females and 5 males were released from 5 m. For evaluation 2, the same number of insects was released from a distance of 10 m. In evaluation 3, 864 weevils were used, 6 females and 6 males were released from 15 m. In evaluation 4, 1008 weevils were used, 7 females and 7 males were released from a distance of 30 m. In evaluation 5, 1296 adults were used, 9 females and 9 males were released from 60 m. The 360 second experiment was conducted during August-October 2019, where the treatments were evaluated at 5, 10 and 15 m. At each distance, 600 weevils were used, of which 10 adults (5 females and 5 males) were released at the four cardinal points of each treatment. The releases were carried out during 13:00 to 17: 00 hours in the afternoon, according to the period of greatest activity of females and males (Muñiz-Merino et al., 2014). The number of insects used was subject to their availability in the established colony. The traps were checked once a week, during the time each evaluation lasted (distance evaluated), while the volume level of each dispenser was checked every third day.

Collection of volatiles in dispersers
The compounds of the synthetic mixture and aggregation pheromone were captured by dynamic headspace on days 1, 7, 14, 21 and 28. The releasers were placed in a cylindrical glass flask with a 29/42 ground-glass neck, 21 cm high, 6 cm internal diameter and 500 mL capacity (Pyrex®). The flask had a ground-glass stopper (2942), with two glass tubes to which a Nalgene hose (3/16 ID) was attached, through which air was passed with an Elite 802 pump with a flow rate of 60 mL/min, regulated with a flow meter (Gilmont®).Three flasks were placed at the same time for each releaser, a 150 mm pasteur pipette (Brand®) was placed at each air inlet point, packed with 50 mg of Tenax TA 60/80 adsorbent (Sigma Aldrich®), which served as a filter. Another cartridge of the same type was placed in line at the outlet of each flask to collect the compounds from the disperser, with a capture time of 3 hours. The volatiles captured in each cartridge were eluted with 4 mL of HPLC grade hexane and brought to a concentration of 100 µL by a gentle stream of nitrogen. The resulting solution was placed in 3 mL amber vials (Agilent-Technologies®) and stored at -4 °C until analysis by gas chromatography.
Chromatographic analysis of the samples of the releasers 1 μL of each concentrated sample was injected into a Hewlett Packard gas chromatograph (5890) with a flame ionization detector (GC-FID). Gas Chromatography conditions were: nitrogen as carrier gas, with a flow rate of 1 mL min -1 , the detector and injector temperature was 250 ° C. The run conditions were an initial temperature of 40 °C stable for 5 min, then increased by 5 °C min -1 until reaching 100 °C, then increased by 10 ° C min -1 until reaching 210 °C, maintained for 5 min, with a total run time of 33 min. Identification and confirmation of the compounds were obtained by comparing retention times with commercial standards.

Statistical analysis
Data from the 2018 and 2019 field experiments were analyzed using linear mixed models, treatments were used as a fixed factor and replicates nested in weeks as a random factor. A post hoc analysis with Bonferroni correction with a probability of 0.05 was performed to test for significant differences within each group of means. All analyses were performed using SPSS v. 25.0 for Windows (IBM Corp. 2019).

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Field experiment during August-October, 2019. The mean number of adults recaptured at 5 m distance showed a significant difference among the evaluated treatments (F=23.024; df= 5, 40; P < 0.05). The mean difference in AP (2.48±0.14) was statistically higher than the remaining treatments ( Figure 1; 5 m). SM: GA with GA (0.62±0.14), AP (-0.57±0.14) and CO (0.74±0.14); RS with GA (0.70±0.14), AP (-0.49±0.14) and CO (0.81±0.14); GA with SM: GA (-0.62±0.14), RS (-0.70±0.14) and AP (-1.19±0.14); AP with SM: GA (0. 57±0.14), RS (0.49±0.14), GA (1.19±0.14) and CO (1.31±0.14); as well as CO with SM: GA (-0.74±0.14), RS (-0.81±0.14) and AP (-1.31±0.14) treatments recorded a significant difference according to post hoc comparisons (P<0.05). At 10 m, a significant difference was again found in the mean number of recaptured adults among the evaluated treatments (F = 8.895; df = 5, 40; P < 0.05). Means between AP (1.61±0.12) and SM: GA (1.31±0.12) were higher compared to CO (0.86±0.12) used as a control (Figure 1; 10   The essential oil had a limited longevity in the field, since its volume decreased more than 50 % in five days. The short permanence of the extract in the field may have caused the low captures in the traps, since the compounds essential for attraction were not present. Perhaps, the higher release is due to the high vapor pressure of the compounds, linked to the ambient temperature recorded as suggested by Mette-Cecilie et al., (2019), who mention that a substance with a higher vapor pressure volatilizes more easily. The study of essential oils as attractants in the genus Anthonomus is scarce (McKibben et al., 1997), most have focused on repellent and insecticidal activity (Brito et al. 2021). Kendra et al., (2018) demonstrated the potential of essential oils for attracting X. glabratus, so their implementation in insect management is an option that would be worth exploring.
The combination of aggregation pheromone and synthetic mixture from the host plant did not increase the number of recaptures of A. eugenii compared to the single pheromone. Szendrei et al., (2011) in field experiments observed that the addition of Z-3-hexenyl acetate and hexyl acetate with pheromone components from A. musculus did not improve attraction. Possibly, the ratio of the release of the components in the mixture used was not sufficient to attract the weevil and cause synergism, as happened with A. rubi, where the addition of 1, 4-dimethoxybenzene, together with the aggregation pheromone caused higher captures than with the single pheromone (Wibe et al., 2014; Mozūraitis et al., 2020). Although this combination failed to improve attraction, this work provides the first evaluation of pepper volatiles together with the pheromone, which in future studies could be refined to increase attraction.
The synthetic mixture and geranic acid placed in the same trap captured insects at 15 m distance, although they were not significantly different from the control. Rodriguez-Saona et al., (2020) found that the response of A. musculus is not affected by the addition of geranic acid to the aggregation pheromone; furthermore, the addition of the sex pheromone of L. rugulipennis and 1, 4 dimethoxybenzene to the aggregation pheromone of A. rubi did not cause significant changes in the number of captures (Baroffio et al., 2018). In the case of A. eugenii, recaptures decreased significantly when adults were released at greater distances from the release point, similarly Dissanayaka et al., (2020) observed a decrease as the release distance of R. dominica increased. Perhaps this is because as distance increases, the concentration of the compound decreases, reducing insect attraction.
The aggregation pheromone (without geranic acid) captured fewer insects than the complete pheromone. In this respect, Eller et al., (1994) in field tests observed higher attraction of A. eugenii adults in traps baited with pheromone with the mixture of six compounds than with the mixture of five. Surely, the absence of geranic acid caused a reduction in the attraction of the insect, however, the study of the effect of different doses of geranic acid would help improve the attractiveness of the pheromone in the field. Geranic acid was ineffective in attracting A. eugenii adults, since traps with this compound showed lower captures compared to the control. Perhaps, as suggested by Eller et al., (1994), geranic acid is inactive when used individually and only works when the complete pheromone is present. In addition, the rapid volatility in the field may have caused a null response, since according to chromatographic analysis, this compound was only present for up to 14 days.

Conclusion:-
The traps with the mixture of (E)-β-ocimene, (Z)-β-ocimene, 2-Isobutyl-3-methoxypyrazine, (Z)-3-hexenyl acetate and terpinolene presented insects at short distance, similar to the essential oil. Improvement in detection systems for A. eugenii with the synthetic mixture should be possible by increasing the concentration of the components or by adding other compounds present in pepper. It is clear that the aggregation pheromone is an effective strategy for capturing A. eugenii adults, as long as it is placed before flowering and after harvest to capture as many adults as possible on alternate hosts. Further studies are required to develop a formulation that increases the efficacy of the synthetic mixture with the aggregation pheromone evaluated here.

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Local de Sanidad Vegetal del Altiplano Centro del estado de San Luis Potosí) for the support in the collection of samples of peppers infested with Anthonomus eugenii.