SRI LANKAN WEEVIL COLLECTION AND MAINTENANCE

Adult weevils were collected from a peach (P. persica) orchard in Vero Beach, Indian River County, Florida, USA. Weevils were collected by hand from peach trees into ventilated jars. The weevil adults were kept in BugDorm^™cages (BD-6630, Bioquip Inc., Rancho Dominguez, California, USA) provisioned with cocoplum leaves (Chrysobalanus icaco L.; Chrysobalanaceae) in a climate-controlled greenhouse at a mean temperature of 25 °C and a 12:12 h (L:D) photoperiod.

SEXUAL DIMORPHISM OF THE WEEVIL

Identifying the sexually dimorphic characteristics is very important in separating the male and female weevils for behavioral and electrophysiological studies. Length and breadth measurements of adult weevils were done with CellSense Dimension software (Olympus Corporation, Tokyo, Japan). Adult male and female weevils were measured from the center of the ecdysial line on the head to the tip of the abdomen. Separate measurements were done for length of the head, thorax, abdomen, and the length and width of the last 4 abdominal sternites (George et al. 2015).

OVIPOSITION PREFERENCE AND LARVAL DEVELOPMENT OF SRI LANKAN WEEVIL ON DIFFERENT HOST PLANTS

Choice assays were performed to study oviposition preference and larval development of Sri Lankan weevil on host plants. Three host plants including peach (P. persica; Rosaceae), Bahia grass (Paspalum notatum Flüggé; Poaceae), and cupid's shaving brush (Emilia sonchifolia (L.) DC; Asteraceae) were studied based on their abundance and distribution in peach orchards. Plants were maintained in 10 L (3 gallon) pots. Choice assays were performed inside a BugDorm^™cage (BD-6630, Bioquip Inc., Rancho Dominguez, California, USA) (60 × 60 × 180 cm) with the 3 different host plants. Plants were in non-flowering phase, except cupid's shaving brush. Fifty pairs of Sri Lankan weevils were introduced into the cage (50 males + 50 females). Weevils were allowed to feed and oviposit on the plants for 8 wk. Adult weevils were removed, and the larval distribution in the root zone was measured. The soil in each treatment pot was separated into 2 equal halves (top and bottom 5 inches). Sri Lankan weevil adult oviposition choice, oviposition depth, and larval development in the soil were measured.

ELECTROANTENNOGRAM RECORDINGS WITH PEACH VOLATILES

Volatile collections were performed on young and mature peach leaves with solid-phase microextraction fibers (polydimethylsiloxane/divinylbenzene) and analyzed by gas chromotogaphy-mass spectrometer analysis. Compounds were identified by comparing spectral data with those from commercially available standards and spectra from mass spectrometer libraries, and confirmed by retention times of authentic standards. Identified odorants were used in electroantennogram recordings (n = 6) as described by Lapointe et al. (2012). Headspace volatiles of synthetic odorant were puffed into a filtered and humidified airflow upstream to the male and female antenna. Benzaldehyde was puffed at the end of each run as a positive control. Responses were adjusted by subtracting responses to clean air during the corresponding run from odorant responses. Statistical analysis was performed with JMP ver. 10 (SAS Institute 2019). One-way ANOVA was performed to analyze electroantennogram responses (α = 0.05) followed by Tukey's HSD for comparison of means.

SEXUAL DIMORPHISM OF THE WEEVIL

Adult female weevils were significantly longer than males (F _{1, 19} = 38.5; P < 0.0001) (Fig. 1A). The mean weight of females (35.5 mg) was significantly greater than that of males (18.2 mg) (F _{1, 19} = 73.46; P < 0.0001). The lengths of the head and abdomen of female weevils were greater than those of males, but there was no difference in the length of the thorax (P = 0.008, < 0.0001, and 0.14, respectively) (George et al. 2015). Metathoracic inter-coxal distance was greater for females (1.5 mm) compared with males (1.3 mm) (F _{1, 19} = 22.16; P < 0.0001) (George et al. 2015). Female weevils have more pronounced black-gray markingd (Fig. 1B) compared to males (Fig. 1C). The distance between the metathoracic coxa was significantly greater for females, and the black-gray marking extended from the sternum of the mesothoracic segment to the second abdominal segment. The males had a small black-gray marking restricted to the anterior mesosternal area (Fig. 1C). Males had more white accumulation of (presumably) cuticular hydrocarbons present on their body surface compared with females. The presence of these hydrocarbons gives the males a more whitish appearance compared with females. The dark markings of females were characterized by a dense investiture of plumose and elongate scales and no oval scales. Scanning electron microscopic images revealed that females had fewer ovate-to-obovate scales in this region of characteristic black-gray marking, and more plumose scales compared with males (George et al. 2015). These black-gray markings on female abdominal sternum are visible to the unaided eye, and can be used as a sexually dimorphic character to distinguish males from females.

Fig. 1.

(A) Lateral view showing the difference in size of female and male Sri Lankan weevils. Dimorphism appears as black-gray markings on the ventral mesosternum of female (B) and male weevils (C).

OVIPOSITION PREFERENCE AND LARVAL DEVELOPMENT OF SRI LANKAN WEEVIL ON DIFFERENT HOST PLANTS

Oviposition preference and larval development of Sri Lankan weevil were observed only in pots containing a peach plant (47.5 ± 8.2 larvae). No Sri Lankan weevil larvae were found in pots of Bahia grass and Aster weed (Asteraceae). No significant differences were observed in the distribution of first (P = 0.74), second (P = 0.80), third (P = 0.51), fourth (P = 0.12), and fifth (P = 0.35) instars in the top and bottom halves of the peach soil (Fig. 2). Egg to adult development of Sri Lankan weevils took an average of 105 d. In this experiment, we showed that Sri Lankan weevil larvae can be reared successfully in association with peach roots under greenhouse conditions.

Fig 2.

Sri Lankan weevil larval distribution in top (black columns) and bottom (gray columns) 5 inches of soil in pots containing peach seedlings. No significant differences were observed in the distribution of larval stages.

ELECTROANTENNOGRAM RECORDINGS WITH PEACH VOLATILES

Peach volatiles identified by solid-phase microextraction and gas chromatography-mass spectrometry included alcohols, aldehydes, terpenes, esters, and other compounds. Benzaldehyde and Z-3-hexenyl acetate were the major odorants in peach flush, whereas mature peach leaves produced very low amounts of benzaldehyde (Fig. 3). Some of these odorants, but not benzaldehyde, also were present in the headspace of Valencia citrus (Rutaceae), also a host plant for Sri Lankan weevils.

Electroantennogram recordings were performed with excised Sri Lankan weevil antennae (Fig. 4A). The club of the antenna (Fig. 4B) was placed on a gold wire connected to the recording electrode (Fig. 4C). Gold wire for the electrode significantly improved the signal-to-noise ratio of the recordings. Many of the peach volatiles identified by solid-phase microextraction and gas chromatography-mass spectrometry elicited Sri Lankan weevil antennal responses. Benzaldehyde produced consistent antennal responses from male and female weevils, and was used as a positive control to compare electroantennogram responses from other peach odorants. Benzaldehyde has been reported as an attractant for plum curculio weevil Conotrachelus nenuphar Herbst (Coleoptera: Curculionidae) (Akotsen-Mensah & Fadamiro 2015). Z-2-hexenol produced the highest antennal response from male weevils (F _10,55 = 15.8, P < 0.0001), followed by Z-3-hexenol. Z-3-hexenol has been reported as a component of an attractant blend for Otiorhynchus sulcatus (F.) (Coleoptera: Curculionidae) (Van Tol et al. 2012). Hexanal also produced higher electroantennogram responses from male Sri Lankan weevil antennae compared to other odorants tested (Table 1). Female weevil antennal responses were highest for Z-2-hexenol (F _10,55 = 9.15, P < 0.0001), followed by Z-3-hexenol and hexanal (Table 1). Male and female Sri Lankan weevil antennae responded to peach odorants such as Z-2-hexenol, Z-3-hexenol, and hexanal and benzaldehyde. These antennaly active odorants will be tested further in olfactometer assays. Olfactometer assays will help to study the behavioral activity of these odorants, and to identify the principal odorants and blends that could be developed as attractants for monitoring and trapping of these weevils in the field.

Fig. 3.

Chromatographic detection of volatiles present in headspace of peach flush, mature peach leaves, and Valencia (sweet orange) leaves.

Identification of semiochemicals/odorant blends will contribute to development of monitoring tools to detect, monitor, and manage populations in peaches and other specialty crops. Analyzing the behavior and chemical ecology of the weevil is expected to result in identification of aggregation and sex pheromones for monitoring traps and mating disruption techniques. Our findings will help reduce the use of pesticides in groves, reduce production costs, and reduce pesticide residue load on marketed fruit. In the longer term, knowledge of weevil biology and feeding behavior, and host plant resistance will help develop resistant cultivars as conventional breeding programs progress.

Fig. 4.

(A) Antennae of Sri Lankan weevil; (B) scanning electron microscopy of olfactory and mechanoreceptor hairs on the club of Sri Lankan weevil antennae; (C) arrangement of antennal preparation for electroantennogram recordings.

Table 1.

Electroantennogram responses from male and female Myllocerus weevils to odorants present in headspace volatile collection from peach leaf flushes (Mean ± SEM (n = 6). Mean electroantennogram responses of male and female Sri Lankan weevils expressed as percent of response to benzaldehyde.