E:\ 82(1) 19136-A T Rani 2020

Indian Journal of Entomology, 82(1): 000-000 (2020) DoI No.:

ELECTROPHYSIOLOGY AND BEHAVIOUR IN BANANA PSEUDOSTEM WEEVIL ODOIPORUS LONGICOLLIS OLIVIER TO CONSPECIFIC CHEMICAL CUES

A T Rani*, K R M Bhanu**, Vasudev Kammar*** and A K Chakravarthy****

*Division of Vegetable Protection, ICAR-IIVR, Varanasi 221305, Uttar Pradesh **Biocontrol Research Laboratory, PCI, Bengaluru 562163 ***Department of Food and Public Distribution, Ministry of Consumer affairs and Food and Public Distribution, Govt. of India, Krishi Bhawan, New Delhi 110001 ****Division of Entomology and Nematology, Indian Institute of Horticulture Research, Hessaraghatta 560089 *Email: [email protected] (corresponding author)

ABSTRACT

An understanding of the role of semiochemicals in biology can be used for developing IPM tools. The olfactory and behavioural responses of banana pseudostem weevils (BPW) Odoiporus longicollis Olivier to semiochemicals isolated from conspecific were investigated by electroantennography (EAG) and Y-tube olfactometer bioassays. Volatiles were collected from male and female weevils, separately and together from both sexes by air entrainment. Dichloromethane extracts of male and female weevils were used as stimuli in electrophysiological study. The observations revealed that the antennae of both male and female produced significant EAG responses (1.378±0.350 mV and 1.704±0.246, respectively) to male volatile compared to other stimuli tested. In two-choice experiments, more males (p=0.001, χ2=11.84) and females (p= 0.033, χ2=4.57) were attracted to male volatiles. Significant responses of both sexes to headspace volatiles from male extracts during EAG and olfactometer experiments revealed that specific volatile compounds emitted by male played a significant role in aggregation. These results might enable identification of male specific pheromone components, which can enhance effectiveness of pheromone trapping for O. longicollis.

Key words: Air entrainment, dichloromethane, semiochemicals, Odoiporus longicollis, electroantennography, Y-tube olfactometer, male and female volatiles, differences Banana Musa spp. is cultivated about 0.858 million that BPW incurs 10-90% yield oss (Padmanaban and ha with the production of 29.16 million MT and the Sathiamoorthy, 2001). productivity of 34 MT/ ha in India (Horticultural Statistics, 2017). The incidence of insect pests is a major The biology, ecology, seasonal incidence and constraint for banana cultivation. About 19 pests are chemical control of BPW had been previously found associated with banana in India (Padmanaban studied (Azam et al., 2010; Priyadarshini et al., 2014; et al., 2001). Of these, the banana pseudostem weevil Khairmode et al., 2015). Because of the long life (BPW) or banana pseudostem borer Odoiporus span of adults and endophytic behavior of the larvae, longicollis Olivier (Coleoptera: ) is conventional methods of control, especially insecticides gaining importance as a serious pest and its occurrence were found less effective (Prasuna et al., 2008). Also, had been reported in nontraditional areas too (Justin et insecticides can be harmful to non-target species and al., 2008). The weevil is becoming a serious threat to leave residues. Hence, alternative control methods that banana crop in South India. BPW is a monophagous are safe and efficient are warranted. Semiochemicals pest and the adult femalesPreview lay eggs in the outermost like pheromones provide effective control (Welter leaf sheath of banana (Visalakshi et al., 1989). Larvae et al., 2005). Successful management of weevils in hatched from the eggs bore into the living tissue, cotton, coconut and sweet potato using aggregation producing frass- filled tunnels and permit the invasion pheromones had been reported (Dickerson et al., 1987; of fungal and bacterial pathogens. Mature larvae pupate Chinchilla et al., 1996; Downham et al., 2001). Sordidin, in cocoons made from plant fibres close to the exit holes. the aggregation pheromone of Cosmopolites sordidus The severity of the loss is greater when infestation Germar, another important pest of banana, had been occurs at the early vegetative stage. It is estimated successfully used (Jayaraman et al., 1997; Tinzaara et al., 2000). 2 Indian Journal of Entomology, 82(1), 2020

It had been reported that male BPW produced a pheromone, 2-methyl-4-heptanol which attracted both male and female weevils (Gunawardena and Dissanayake, 2000a). Ravi and Palaniswami (2002) reported the presence of its female sex pheromone. The aggregation pheromones of several weevil species consist of multiple behaviorally active components. The advantage of multicomponent pheromones is that they convey more information than a component (Silverstein, and Young, 1976). The combination of one or more pheromone components increases the attractiveness of insects to pheromone baited traps. Gunawardena and Dissanayake, (2000) reported that 2-methyl-4-heptanol works only with food bait under field conditions. The single component aggregation pheromone ineffective in trapping more weevils. Therefore, the present study to determine the presence of other pheromone components to improve the existing pheromone trapping method by increasing the attractiveness to the sexes and also to develop multicomponent pheromone- based control method for BPW.

MATERIALS AND METHODS

Odoiporus longicollis adults of mixed age and sex and of unknown mating status were collected from damaged banana plants around Tubgere hobli, Fig. 1. Banana pseudostem weevil (A) male (B) female Doddaballapur Taluk (1306’, 77036’, 930 masl) and Hesaraghatta hobli, Bengaluru (1305’,77035’, 930 m every 5 min for a 12 hr period both during scotophase AMSL). Males and females were separated based (6 pm to 6 am) and photophase (6 am to 6 pm). During on the rostrum characteristics (Fig. 1). Weevils were the scotophase, observations were facilitated using a maintained in separate plastic containers (29×17×33 red bulb (5 W). Observations on number of copulation cm) in laboratory under a photoperiod of 12L: 12D events/ pair and mean number of mating pairs were at 25± 20 C, 70± 10% RH at the Biocontrol Research recorded and the observations repeated twice. laboratories (BCRL), Pest Control (India) Pvt. Ltd., Prior to insect volatile collection, preliminary trials Sriramanahalli, Bengaluru. Weevils were provided with were conducted to determine the peak time of mating freshly cut pseudostem pieces as food. Unfed pieces activity of male and female BPWs during scotophase contaminated with fecal matter were replaced weekly. and photophase under caged conditions. The volatile A laboratory study was conducted to determine the collection was carried out between 1100 to 1400 hr of peak time of mating activity and timing of pheromone photophase by air entrainment method. Groups of 50 release. Experiments were conducted during scotophase males and 50 females were maintained separately in and photophase of weevils in the Biocontrol Research different glass aeration chambers (6 x 19 cm). Thirty Laboratory, Bengaluru to understand the time of activity. males and 30 females were held separately in wire mesh In this study, number of mating pairs and frequency of and wooden cages (0.3 m3) and both cages were kept Previewin a single plastic acrylic container (6.5×12×17 cm) mating in each hour were recorded. Five pairs of male and female in three replicates (n= 15 pairs) were placed for volatile collection from male and female BPWs. in separate transparent acrylic boxes (19×15×9 cm) A charcoal filtered humidified airstream was pushed covered by a muslin cloth tied with an elastic ribbon. through the aeration system at 1.8 L min-1 (Fig. 3). Weevils were provided with fresh pieces of banana Emitted volatiles were collected daily in glass column pseudostem (3×2 cm). The study was conducted at trap filled with a 10 mg of Porapak-Q adsorbent. The 25± 20 C, 70± 10% RH. Weevil pairs were analyzed weevil volatiles were collected from the Porapak-Q with Electrophysiology and behaviour in banana pseudostem weevil odoiporus longicollis olivier to conspecific chemical cues 3 A T Rani et al

Fig. 2. Frequency of matings (n=15 pairs); estimated hourly in 1st to 12th hr

Fig. 3. Insect volatile collection (air entrainment method)

1.5 ml of HPLC grade Dichloromethane (DCM) for × 0.25 μm film (J and W Scientific, Folsom, CA, USA). 15 min. The extracts were concentrated under nitrogen The GC was run at a flow rate of 5 ml min-1 with a stream and reduced to 1 ml so that the volatiles are in oven temperature from 60 to 220 °C with a hold of 2 diluted form for direct Previewuse for different studies. Extracts min at 60°C. The chromatograms were analysed for were stored at -200C in a refrigerator when not in use. the presence of common and sex-specific pheromone components. Gas chromatographic (GC) analyses of the contents of volatiles collected from both sexes of O. longicollis The electroantennographic system used for the study separately were performed on Agilent 7890A GC system was Syntech, Hilversum, The Netherlands. Antenna equipped with flame ionization detector. The GC was of the weevils was excised using a micro scissor in a fitted with a DB-5 capillary column (30 m × 0.25 mm way that all the antennal segments and the basal nerve 4 Indian Journal of Entomology, 82(1), 2020 remained intact. An excised antenna was mounted female volatile in DCM (100 μl) vs DCM (100 μl) and on to an antenna holder for the EAG probe in such a (4) only DCM (100 μl) (Control). The bioassays were way that the tip of the antenna was in contact with -ve carried out between 1100 to 1400 h during photophase. electrode and base i.e. scape touched +ve electrode. This was accomplished using electroconductivity gel Electroantennogram responses were compared (Sigma-gel, Parker Laboratory Fories. Inc. USA). using a one-way ANOVA followed by Honestly The different stimuli tested were (1) male volatile, (2) Significant Different (HSD) Tukey test (SPSS 16.0 female volatile, (3) male + female volatile and (4) DCM version software). Results of all olfactometer bioassays 2 solvent (control). Each sample was subjected to EAG were analyzed with a binomial test (χ test; SPSS 16.0 applying 20 μl on Whatman No.1 filter paper strips (6 version software) and means compared by Tukey HSD cm length and 5 mm width) and placed into the Pasteur test (p<0.05). pipette (5.75 mm dia; 145 mm length; tip 47 mm). The RESULTS AND DISCUSSION stimulus puffs were given at 30 sec intervals and the control stimulus was given at the beginning and end of The results of trails conducted to determine the each EAG run. Each sample had 5 replicates/ sex, with timing of pheromone release by BPWs are presented each replicate being an antenna. in Fig. 2. Observations on the time of day for mating observed revealed that weevils mated during both day The behavioral responses of males and females to and night. The pairs started mating in the 1st hour of volatile extracts were evaluated in a dual choice Y-tube scotophase and photophase. During scotophase, the olfactometer. The olfactometer consisted of Y-shaped maximum mating activity was observed in the 1st hour acrylic tube of 6 cm dia. The main tube (stem) of the (3.67 ± 1.33) itself, later the number of copulations olfactometer and the two arms were each 30 cm in 0 decreased as the scotophase progressed. The peak length at 90 . The air-delivery unit was connected to mating period occurred during 6th h (5.33 ± 1.45) and the two arms of the Y-tube to draw purified air to pass 9th h (5.67 ± 2.40) of photophase when 100 % of the through the odor sources in the Y-tube. Air flow through pairs mated. During scotophase (p=0.255, F=1.358) each of the olfactometer arms was maintained at 0.5 L -1 and photophase (p=0.227, F=1.420) there were min . Fixed number of insect (50 males or females/30 no significant differences between individual time pairs) volatile extracts collected above in DCM (100 periods. When photoperiods were compared, they were μl ) were loaded on Whatman filter paper strips of 1 × significantly different (t=2.524, p=0.028) and distinct 3 cm and were placed in one of the Y-tube chambers pattern was observed with maximum number of mating after evaporating solvent and the other chamber served during day time. Since, mating activity was observed as control (equal volume of HPLC-grade DCM was constantly from 4th to 11th hr of photophase with two placed after solvent evaporation). New filter papers peak periods at 6th and 9th h, the volatile collection from prepared with the volatile extracts and DCM were used male and female weevils and laboratory bioassay were for each trial (3 insects). The position of treatments was carried out during this period (1100 to 1400 hr). alternated after each trial, to avoid directional bias. The number of males and females evaluated varied from In coleopterans, the mating occurs at any time 15 to 21. A group of 3 BPWs were introduced into the of the day (Silva et al., 2012; Martins et al., 2013; base tube, and their behaviour was observed for 15 min. Khairmode et al., 2015 and Triana et al., 2014). BPW When a weevil crossed the choice line 10 cm after the mates throughout the year at day time and even at night division of the base tube and remained there for at least (Khairmode et al., 2015). Mating is observed at any 20 sec, it was recorded as a choice for the odour source time outside the pseudostem or in the space between in that arm. If the weevils stayed in the common tube the outermost and inner sheaths of the pseudostem or at the junction of the two arms and did not make a and frequent matings were observed (Dutt and Maiti, choice, were consideredPreview a non-responding and excluded 1972). In rice water weevil, Oryzophagus oryzae adults from the statistical analyses. Age and mating status of mated during day and night and the peak mating period the weevils were not controlled during the bioassays occurred during the 2nd h of photophase (Martins et al., because of the difficulty of rearing these insects in 2013). The banana weevil C. sordidus mates repeatedly laboratory. The different experiments carried out for in its lifetime. Mating occurred under both light and dark behavioural responses of male and female were (1) male conditions, but significantly more in the dark (Uzakah volatile in DCM (100 μl) vs DCM (100 μl), (2) female and Odebiyi, 2015). Sirot and Lapointe (2008) reported volatile in DCM (100 μl) vs DCM (100 μl), (3) male + that in tropical root weevil Diaprepes abbreviates (L.) Electrophysiology and behaviour in banana pseudostem weevil odoiporus longicollis olivier to conspecific chemical cues 5 A T Rani et al matings occurred throughout the day but the % of peaks in both the volatiles providing chemical support individuals mating tended to peak during the mid of to the bioassay data. the photophase. The antenna of male and female elicited significant In red palm weevil, Rhynchophorus ferrugineus EAG responses to all the tested stimuli compared to (Olivier), mating activity occurred during photophase control. Mean EAG responses of male antennae to at 02.00-06.00 pm (Kaakeh, 1998). Neochetina different insect volatiles revealed significant differences eichhorniae Waner mate repeatedly, both day and night, (F3,16= 4.288; p=0.021). The antennae of male produced in their life-time. However, mating frequency was significantly higher EAG response to male volatile 50 % greater at night than during the day (Stark and compared to other stimuli; maximum EAG response Goyer, 1983; Oke, 2011). Both sexes of Callisphyris to the male volatile was 1.378 ± 0.350 mV; and to apicicornis Fairmaire and Germain remain motionless female and mixed volatiles was statistically on par during the scotophase and during most of the day light (0.756±0.133 and 0.801±0.204 mV, respectively). The hours, except during a clear peak of activities in both EAG responses of female antennae to test volatile sexes between 09.00 am and 01.00 pm. Light acts as a samples were significantly different (F3,16 = 6.686; p= triggering factor for sexual behaviour in Callisphyris 0.004); female antenna gave significantly more EAG apicicornis and suggest that this time window would response to both male and female volatiles (1.704 ± be the optimal time for collecting the pheromone 0.246 and 1.368 ± 0.239, mV, respectively) which are from females and for testing the responses of males statistically on par; and gave a lesser response to mixed to pheromone components (Curkovic and Ferrera, volatile (1.250 ± 0.209 mV). Control evoked the least 2012). Light illumination is considered a necessary EAG response in both the sexes (Table 1). environmental condition during attempts to collect pheromones from calling females under laboratory Results of the present study reveal that male volatile conditions (Wang et al., 2002). elicited significantly more EAG responses in male and female antennae. There was a significant difference in The chromatographic profiles of volatiles from male EAG response of male and female antennae to different and female shown in Fig. 4 reveal a total of 25 peaks treatments. Antennae of female weevils revealed higher in male, among them the major peak was observed EAG amplitude than those of male. These results are at 6.465 rt with an area % of 30.313; in female 24 in agreement with those of Prasuna et al. (2008), who peaks were detected including six major peaks. The observed that whole body extract of males alone and chromatograms also indicate the presence of five male its combination with banana sheath extract elicited and eight female specific peaks with fourteen common significantly maximum EAG response in male and Preview

Fig. 4. GC analysis of volatile extracts from adults 6 Indian Journal of Entomology, 82(1), 2020

Table 1. EAG response of BPW male and female antennae to conspecific volatiles

Mean (± SE) EAG Response (mV) S. No. Treatments Male Female T1 Male volatile 1.378± 0.350a 1.704± 0.246a T2 Female volatile 0.756± 0.133ab 1.368± 0.239a T3 Male + Female volatile 0.801± 0.204ab 1.250± 0.209ab T4 Control (DCM) 0.287± 0.065b 0.452± 0.080b df 3, 16 3, 16 F test 4.288 6.686 P value 0.021* 0.004**

Values followed by same letters in each column not significantly different (Tukey HSD post-hoc test); *Significant <0.05, and ** at p<0.01; n=5. female antennae of BPWs. Fürstenau et al. (2012) Table 2. Response of male and female BPW reported maximum EAG deflections in males of black (in Y-tube olfactometer) banded oak borer, Coroebus florentinus than females to Odour sources Frequencies the identified compounds in the headspace of both sexes. Male Female However, the EAG responses do not indicate, Male volatile vs. Control whether the stimulus is an attractant or repellant. These Male volatile 17** 11* results suggest that male and female BPW antennae are Control 02 03 equally sensitive in perceiving the chemical signals Not decided 02 07 and also indicated the presence of specific chemical Total 21 21 information in the headspace volatile of both sexes. Female volatile vs. Control Female volatile 10* 12* Dual choice Y-tube olfactometer studies were Control 02 03 conducted to determine the behavioural responses of Not decided 09 06 BPW males and females to volatiles collected from Total 21 21 conspecific weevils. These indicated that significantly M+F volatile vs. Control more number of males were attracted to the male M+F volatile 12* 12 volatile (χ2=11.842, df=1, p=0.001, 17/2), male + Control 02 06 Not decided 07 03 female (χ2=7.143, df=1, p=0.008, 12/2) and female Total 21 21 volatile (χ2=5.333, df=1, p=0.021, 10/2) compared to Control vs. Control control. The responses of females to different volatiles Control 06 06 of BPW indicated that significantly more number of Control 07 08 females were attracted to the male (χ2=4.571, df=1, Not decided 08 07 p=0.033, 11/3) and female volatile (χ2=5.400, df=1, Total 21 21 p=0.020, 12/3). The female attraction to the male + Control: DCM, statistically significant (binomial test at female volatile was not significantly different (p<0.05) *p < 0.05; ** p< 0.01) from the control (Table 2). information for mating. Males were more attracted to The results of Y-tubePreview olfactometer study indicated the male produced aggregation pheromone compared that the attraction of both sexes to male volatile, and to female, suggesting that these compounds be used as male weevil’s attraction to male + female volatile, while chemical cues primarily by males to find an appropriate female weevil’s attraction to female volatile indicated site for mating, feeding and aggregation to get the presence of male specific chemicals responsible for protection against predation. The results of this study the aggregation and female specific sexual behaviour are in consistency with Prasuna et al. (2008). Ambrogi components. The attraction of male weevils to female and Zarbin (2008) reported the attraction of both sexes volatiles indicated the presence of female specific of Sternechus subsignatus weevils to male extracts 7 Indian Journal of Entomology, 82(1), 2020 but not to the female extracts. Similarly, males and Conotrechelus psidii Marshall were highly attracted females of guava weevil Conotrachelus psidii showed towards male extracts than to host plant extracts significant attraction towards natural headspace extract (Palacio-Cortes et al., 2015). of males containing host plant (Palacio-Cortes et al., 2015). Uzakah et al. (2015) reported that, the olfactory During EAG and olfactometer experiments, responses of C. sordidus to trapped adult volatiles significant responses of both male and female BPW revealed that males responded significantly to the towards male volatile showed that volatile compounds trapped volatiles of both males and females. However, emitted by males play a major role in aggregation. females responded significantly to only the male but Males of many coleopteran species were reported to not to the female volatiles. produce aggregation pheromones (Rao et al., 2003). Male specific pheromone components had been The % behavioral responses of both male and female identified in several curculionid species and become weevils to all the three natural volatiles from BPWs in useful for monitoring populations (Bartelt, 1999; Mayer comparison with control are depicted in Fig. 5. These and McLaughlin, 1991). In the subfamily , demonstrate that the communication among BPW Conotrachelus nenuphar (Herbst) (Leskey and Wright, conspecifics is mediated by pheromones. The male 2004), S. subsignatus (Ambrogi and Zarbin, 2008; volatile with soya bean stem was more attractive to Ambrogi et al., 2012) and weevils of the genus Pissodes male and female soya bean stalk weevils Sternechus (Booth et al.,1983; Philips et al., 1984) had also been subsignatus Boheman, than soya bean stem alone described using male specific pheromones as a part (Ambrogi and Zarbin, 2008). Males of guava weevil of their chemical communication system. These male

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Fig. 5. Behavioral response of (A) males and (B) females to volatiles (Y-tube olfactometer). Differences (χ2 test)- *p<0.05, **p<0.01, NS=non-significant. 8 Indian Journal of Entomology, 82(1), 2020

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(Manuscript Received: August, 2019; Revised: December, 2019; Accepted: January, 2020; Online Published: January, 2020)

Figure legends:

Figure 2 Frequency of copulations estimated for every 1 hour throughout the 1st-12th hour of photophase and scotophase

Figure 3 Insect volatile collection by air entrainment method.

Figure 4 Gas chromatographic analysis of volatile extracts obtained from male and female Odoiporous longicollis adults.

Figure 5 Behavioral responses (%) of Odoiporus longicollis (A) males and (B) females to volatiles from BPW in a Y-tube olfactometer. Differences between paired bars (χ2 test) indicated by *P<0.05, **P<0.01, NS=Non- significant. male volatile vs. control; female volatile vs. control; male + female volatile vs. control; control vs. control. Preview