- Email: [email protected]
Effects of advanced age on olfactory response of male and female Queensland fruit fly, Bactrocera tryoni (Froggatt) (Diptera: Tephritidae)
Journal of Insect Physiology 122 (2020) 104024
Contents lists available at ScienceDirect
Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys
Effects of advanced age on olfactory response of male and female Queensland fruit fly, Bactrocera tryoni (Froggatt) (Diptera: Tephritidae)
T
⁎
Mst Shahrima Tasnin , Katharina Merkel, Anthony R. Clarke School of Biology and Environmental Science, Queensland University of Technology (QUT), Brisbane, QLD 4000, Australia
A R T I C LE I N FO
A B S T R A C T
Keywords: Olfactory response Ageing Exploratory activity Cue-lure Guava juice Bactrocera tryoni Dacinae
Olfaction is an essential sensory modality of insects which is known to vary with age. In short-lived insects odour response generally declines rapidly with increasing age, but how increasing age affects the olfactory response of long-lived insects is less known and there may be different life-time patterns of olfactory response. Here, we examine the effect of age on olfactory response and exploratory activity of a long-lived tephritid fruit fly, Bactrocera tryoni from sexual maturity (3 weeks) to advanced age (15 weeks). Males were tested against a malespecific attractant, cue-lure, which is associated with courtship and sexual selection in this species; while females were tested against guava-juice, a highly attractive oviposition host fruit odour. Trials were done in the laboratory using a Y-tube olfactometer at three weekly intervals. The probability of olfactory response of both males and females to tested odours declined with age. Males retained a constant attraction to cue-lure until 12 weeks of age, but then showed a significant drop in olfactory response at 15 weeks. However, females showed the highest attraction to guava-juice odour until six weeks of age and declined gradually thereafter. The change on odour response over time can be associated with an age-related change in initial locomotor activity for females as there was no change, over the life of the experiment, in selective female orientation to the odour source once flies started exploring within the olfactometer. However, for 15 week-old males, there was a simultaneous drop in both locomotor activity and selective olfactory orientation. The consistent attraction of male to cue-lure might be related to life-long reproductive activities of males, as males are thought to mate continuously during life. On the other hand, females’ highest attraction to guava-juice odour in early life followed by a gradual decline might be linked with their oviposition rate which peaks in early life.
1. Introduction For insects, olfaction is an essential sensory modality for the perception of stimuli from the environment (Krieger and Breer, 1999; Touhara and Vosshall, 2009). Olfaction plays a key role in behaviours such as the location of food, mates and oviposition substrates, as well as enemy avoidance (Lebreton et al., 2017; Wyatt, 2014). An insect’s response to odours may vary depending on physiological factors such as feeding and nutritional status, mating status and sexual maturation (Gadenne et al., 2016; Jin et al., 2017; Lemmen-Lechelt et al., 2018). For example, insects that are deprived of food generally have a higher attraction to food odours (Wäckers, 1994); while virgin insects are more responsive to odours from mating sites and partners than are mated insects (Jin et al., 2017; Sollai et al., 2018). An individual’s age is another factor that strongly affects the odour response of an insect (Gadenne et al., 2016; Reyes et al., 2017). In insects, age-related changes in olfactory responses are linked to
⁎
sexual maturation, as well as ageing (Gadenne et al., 2016; Martel et al., 2009). For example, in early life, insects are more attracted to food odours while, after sexual maturation, they may be more attracted to cues from mating partners (Klowden, 1990). Additionally, an insect’s response to the same odour may change with age, for example, females of the blowfly, Phormia regina Meigen, show increasing olfactory sensitivity to swormlure-4 and 1-hexanol during the first week of adult life (Crnjar et al., 1990). In contrast to increasing olfactory sensitivity, an insect’s response to odours can be negatively affected by ageing once past some optimal age (Gadenne et al., 2016; Iliadi and Boulianne, 2010). For instance, the antennal olfactory sensitivity of tsetse flies, Glossina morsitans morsitans Westwood, to various tested volatiles started declining after only five days of age (Otter et al., 1991), while the perception of aversive odours diminished in eight week old Drosophila melanogaster Meigen (Cook-Wiens and Grotewiel, 2002). For D. melanogaster, Cook-Wiens and Grotewiel (2002) attributed the reduction of olfactory response with increasing age to both the development
Corresponding author. E-mail addresses: [email protected] (M.S. Tasnin), [email protected] (K. Merkel), [email protected] (A.R. Clarke).
https://doi.org/10.1016/j.jinsphys.2020.104024 Received 24 October 2019; Received in revised form 7 February 2020; Accepted 10 February 2020 Available online 12 February 2020 0022-1910/ © 2020 Elsevier Ltd. All rights reserved.
Journal of Insect Physiology 122 (2020) 104024
M.S. Tasnin, et al.
this study evaluates age-dependant olfactory responses and exploratory activities of the fly from sexual maturity to advanced age (15 weeks). Specifically, we observed the effect of age on olfactory response and exploratory activity of male and female flies in the presence of cue-lure and guava-juice odour, respectively. We hypothesized that: (i) exploratory activities will be higher in younger flies compared to older flies; (ii) the flies’ response to odours will be higher at a younger age compared to old age; and (iii) younger flies will take less time to choose the odour source compared to old flies.
of age-related defects in the olfactory system and associated parts of the brain, as well as a decline in locomotor activity. Tephritid fruit flies (Diptera: Tephritidae) are globally significant insect pests of agriculture, affecting most horticultural crops (Norrbom et al., 1999). Female fruit flies oviposit into host fruits, where resultant larval feeding causes fruit damage (Fletcher, 1989; May, 1958; Sutherst et al., 2000). The behaviour and ecology of tephritid fruit flies are strongly tied to olfaction, including the pheromonal attraction of conspecifics during mating (Landolt et al., 1985; López-Guillén et al., 2011), location of host fruit by females for oviposition (Eisemann, 1980; Jang et al., 1999; Liu and Zhou, 2016), and for male tephritids of the tribe Dacini for the location of plant secondary chemicals which are linked to male sexual selection (Kumaran et al., 2013; Raghu, 2007; Shelly, 2010). The management of pest tephritids relies heavily on the manipulation of this chemical ecology, using olfactory attractants for surveillance and lure-and-kill controls (Aluja, 1996; Koyama et al., 2004). Despite the implications for management, and simply for better understanding tephritid biology, studies of ageing and changing olfactory responses in tephritids are limited. Multiple studies have investigated the effect of ageing from adult emergence to sexual maturity [which occurs on average from 10 to 20 days after emergence, (Clarke, 2019)] on the response of male Dacini to male-specific lures, such as methyl eugenol (ME) and cue-lure (Kamiji et al., 2018; Metcalf and Metcalf, 1992; Weldon et al., 2008; Wong et al., 1991). Most of these studies have found that lure attraction is dependent on the male becoming sexually mature, although in some species males may begin to respond prior to maturation (Wee et al., 2018). Fitt (1981), in the only study of its type, recorded the response of Bactrocera opiliae (Drew and Hardy) to ME significantly past sexual maturation and found no change in responsiveness of males up to 12 weeks of age. Outside of the Dacini male lures, age-related olfaction studies in the Tephritidae are rare. The response of Ceratitis capitata (Wiedemann) to synthetic food attractants was investigated until 40 days of age, where it was shown that sugarfed flies showed higher response at middle age (10–25 days age) while protein-fed flies showed higher response at a young age (1–5 days age) with a declining trend afterwards (Kouloussis et al., 2009). For Anastrepha obliqua (Macquart), antennal responses to male lure and host fruit volatiles were observed until 20 days of age and it was found that older flies exhibited lower antennal response compared to younger flies (Reyes et al., 2017). While such studies are valuable, and with the exception of Fitt (1981) for males, there are no studies of which we are aware that evaluate olfactory responses of both male and female tephritid fruit flies to olfactory cues until an advanced age, as has been done for Drosophila (Cook-Wiens and Grotewiel, 2002) and other insects (Gadenne et al., 2016). This is despite the fact that some tephritids can be very long-lived (Carey et al., 2008), with Dacini tephritids, the focus of this paper, surviving 11 weeks on average in the laboratory (Clarke, 2019) and over 28 weeks as over-wintering adults (Clarke et al., 2019). Bactrocera tryoni (Froggatt) (Diptera: Tephritidae) is an Australian endemic insect which is a key pest of the Australian horticultural industry (Clarke et al., 2011; Sutherst et al., 2000). Sexually mature Bactrocera tryoni males are attracted to cue-lure [4-(p-acetoxyphenyl)-2butanone] (Meats and Hartland, 1999), while mature females are attracted to volatiles released from ripening host fruits (Cunningham et al., 2016; Eisemann, 1980). Cue-lure is widely used for quarantine surveillance, monitoring and field control of B. tryoni (Dominiak et al., 2003; Lloyd et al., 2010; Reynolds et al., 2016; Stringer et al., 2017), while fruit-based odours are being used as the basis for female traps (Cunningham et al., 2016). Bactrocera tryoni can be long-lived, with some individuals surviving in excess of 20 weeks in controlled laboratory studies (Balagawi, 2006), but there is no knowledge of how olfactory responses in the fly change once they become sexually mature, which occurs at approximately 21 days for wild flies (Dalby-Ball and Meats, 2000). Given the importance of olfaction in the management of B. tryoni,
2. Materials and method 2.1. Study insect Bactrocera tryoni used for the experiment were collected from the field, bred from naturally infested fruits. Two cohorts of wild flies were obtained from South-east Queensland (approx. 27°S, 152°E): the first from a collection of 200 mango fruits made on the 5th Feb 2018; the second from a collection of 180 carambola fruits made on the 18th Mar 2018. Infested fruits were placed in plastic containers on a layer of vermiculite and kept in an incubator at 25 °C and 65% relative humidity. After two weeks the vermiculite was sieved for pupae. Pupae were placed in 32 × 32 × 32 cm white mesh cages supplied with water. Each evening, flies emerging during the preceding 24 h period were transferred to new holding cages to keep flies separate according to emergence date. The holding cages were supplied with standard diets (water, sugar cubes and yeast hydrolysate ad libitum) throughout the rearing period. Each cage contained 200 flies, 100 of each sex to allow for mating. Flies originating from mango and carambola were treated as two separate cohorts for experimental purposes. 2.2. Olfactometer bioassay To observe age-related changes in exploratory activities and olfactory responses, five age groups (3, 6, 9, 12 and 15 week old) of male and female B. tryoni were tested. Based on the authors’ unpublished data, and in agreement with Dalby-Ball and Meats (2000), the three weeks old flies can be regarded as newly sexually mature and mated. Male age-related olfaction was tested against 0.1 ml undiluted cue-lure, while females were tested against the odour of 1 ml of a commercial guava juice (water + 30% guava [Psidium guava] puree, Golden Circle Ltd, QLD 4013, Australia) exposed on a petri dish. Guava is a major host of B. tryoni (Hancock et al., 2000) and mature females are highly attracted to ripe guava odours (Cunningham et al., 2016). Dosages of both attractants were based upon preliminary concentration trials which sought to maximise positive fly orientation. Experiments were conducted using a glass Y-tube olfactometer (Model: CADS-2P, Sigma Scientific Ltd) with a single odour source (cuelure for males, and guava juice for females) and a no-odour (blank) control. The olfactometer consisted of a 15 cm long stem and two 10 cm long side-arms (75° angle and 5 cm internal diameter). Side-arms were attached to two sealed glass jars, each 20 cm in depth and 14 cm diameter. An adjustable air compressor pumped airstream at a rate of 2.5 L/min through a clean air delivery system which was connected separately to both glass jars. A table lamp with white light was placed just above the junction of Y-tube arms to minimise differences in light intensity in the experimental arena. The odour sources were placed in an open petri dish, which was sat within the glass jar of the treatment arm: odour sources were replaced every two hours. The arm of the odour-containing glass jar was changed after testing 17 flies (i.e. half of a cohort treatment), at which time the olfactometer was also cleaned with water and 70% ethanol. A total of 35 flies was tested from each cohort (i.e. mango and carambola) for each age group, resulting in 70 flies tested for each age group. In the case of three and six week agegroups, 70 males originating from the carambola cohort only were tested against cue-lure due to a logistic difficulty during the experiment 2
Journal of Insect Physiology 122 (2020) 104024
M.S. Tasnin, et al.
with the mango cohort flies. All tests were conducted in the same, small laboratory room with controlled temperature (26 ± 1 °C) and constant lighting.
2.4. Data analysis All data analysis and visualizations were performed with the R statistical program (Team, 2018) and SigmaPlot (Systat Software, San Jose, CA). Male and female data were analysed separately as the sexes were examined against different odours as well as having differences in longevity. In the experiment, age-group and cohort were explanatory variables, while exploratory activity, olfactory response and time-toresponse were response variables. As exploratory activity and olfactory response were recorded as “1” or “0” for an individual fly, the resulting data were binary, while the time-to-response (measured in seconds) was continuous and followed a gamma distribution. Within each gender, generalized linear models (GLMs) were fitted to examine if the olfactory response, exploratory activity and time-to-response of individual flies were affected by the factors age-group and cohort. Agegroups and cohorts were initially fitted to test their interactive and additive effect on the response variables. A stepwise backward elimination approach was applied to determine the minimum adequate model. When a significant effect was detected by the model, a post hoc test was conducted for pairwise comparison of means using the package emmeans (Russell, 2018). The comparison tests were performed on the log odds ratio scale for binary data. Additionally, chi-square tests were conducted with the raw data which was the frequency of flies choosing the odour or control. This test examined if the number of flies choosing the odour source or the control differed from a 1:1 ratio depending on the age of flies. A deviation from a 1:1 ratio within an age group indicates directed movement, as opposed to a random walk into either arm.
2.3. Experimental design All experiments were carried out using individual flies and, for all experiments, the same procedure was followed. One hour before the experiment started, 35 flies were transferred from their maintenance cage in the insectary and brought to the experimental lab for acclimation. Flies were individually released at the entrance of the olfactometer stem. The fly’s behaviour in the olfactometer was observed for up to 10 min with the variables below recorded. If a fly showed no positive olfactory decision within 10 min the trial was terminated. i. Exploratory activity: If a fly moved upwind and crossed the marked halfway point of either side-arm of the olfactometer it was defined as an ‘explorer’ (coded as ‘1’); a fly which did not upwind forage in this way for the 10 min of observation was referred as a “non-explorer” (coded as ‘0’). ii. Olfactory response (explorer): When a fly responded positively to the odour containing arm (crossed the midpoint of the treatment side-arm) this was recorded as selective orientation (coded as ‘1’); when a fly went past the midpoint of the control arm this was defined as non-selective orientation (coded as ‘0’). iii. Time-to-response: The time taken after release for flies showing selective orientation to reach Y-tube arm with odour source (measured in seconds). These measured variables are illustrated in Fig. 1.
3. Results Male experiments were conducted between 08:00 to 13:00 h, because male B. tryoni are most attracted to cue-lure during morning hours (Weldon et al., 2008), while female experiments were conducted from 10:00 to 15:00 h which is considered the peak window for female oviposition in this species (Ero et al., 2011).
3.1. Effect of age on the male response to cue-lure GLMs were run with age-groups (excluding weeks 3 and 6) and cohorts to determine their interactive and additive effect on the response variables. In both models, there was no significant difference between the cohorts in how the olfactory response in males changed with age. Following this, both models were run with all the age-groups and cohorts which also produced non-significant effects of the cohort for all the response variables. Thus, the cohort was subsequently removed from the models and the following results only focus on the significant explanatory variable; age-group. The GLMs run with the response variable “olfactory response” showed that age had a significant effect on male response to cue-lure. Subsequently, GLMs were run with “exploratory activity” and “time-toresponse” to determine whether the reduction in olfactory response was related to changes in these parameters. Additionally, chi-square tests were conducted to examine whether reduced olfactory response of males was due to a decline in choosiness for the odour or not. The results of GLMs and chi-square tests are presented below. 3.1.1. Olfactory response of male to cue-lure GLM revealed that, for all flies tested (i.e. explorers + non-explorers), there was a significant difference in the probability of flies positively orientating to cue-lure among the five age groups [GLM (selective-orientation ~ age-groups, family = binomial), Chi2 = 19.431, Df = 4, P < 0.001]. The subsequent pairwise comparisons revealed that there was no significant difference in selective orientation rate from 3 to 12 week old flies (Wk. 3 vs. 6, Z = 0.511, P = 0.986; Wk. 3 vs. 9, Z = 0.680, P = 0.961; Wk. 3 vs. 12, Z = 0.171, P = 0.910; Wk. 6 vs. 9, Z = 0.169, P = 0.910; Wk. 9 vs. 12, Z = 0.509, P = 0.987), but 15 week old flies had a significantly lower positive orientation rate compared to all other younger age-groups (Z = 3.683, P = 0.002; Z = 3.218, P = 0.011; Z = 3.061, P = 0.019; and Z = 3.529, P = 0.004 in 15 vs. 3, 6, 9 and 12 week pairwise comparisons respectively) (Fig. 2 A). For explorer flies only, the
Fig. 1. Diagrammatic representation of response variables measured concerning Bactrocera tryoni foraging in a Y-tube olfactometer. Non-explorer, fly did not cross the mid-point of either olfactometer arm in 10 min (dark grey area); Explorer, fly did cross the mid-point of either olfactometer arm within 10 min (light grey area); Time-to-response, the time taken by an explorer fly to cross the arm mid-point; Selective and non-selective orientation, the response of an explorer fly to the odour arm or blank control arm, respectively. 3
Journal of Insect Physiology 122 (2020) 104024
M.S. Tasnin, et al.
Fig. 2. The mean ( ± 1SE) probability of male Bactrocera tryoni selective orientation to cue-lure at five ages for (A) all tested flies (explorer + non-explorer) and (B) explorer flies only. Different letters above the bars represent significant differences at P < 0.05. There is no significant age effect in Fig. 2B.
lure for male flies [GLM (time duration ~ age groups, family = gamma), F = 2.650, Df = 4, P = 0.035]. The subsequent post hoc test demonstrated a significant difference in time-to-response with 3 week old males taking significantly less time than 15 week old males (Z = 3.581, P = 0.003), but both age groups were not significantly different from the response times of 6, 9, and 12 week old males (Fig. 4).
probability of flies selectively orientating to cue-lure did not significantly differ depending on age-group [GLM (selective orientation ~ age-groups, family = binomial) (Chi2 = 9.188, Df = 4, P = 0.057, mean probability = 0.79 ± 0.06)] (Fig. 2 B). 3.1.2. Exploratory activity The probability of males exploring either arm of the olfactometer was significantly affected by the age of the flies [GLM: (explorer ~ agegroups, family = binomial), Chi2 = 15.393, Df = 4, P = 0.004], with the subsequent post hoc test revealing that there was a significant drop in the exploration probability of flies at 15 weeks of age compared to 3 weeks (Z = 3.711, P = 0.002, 3 vs. 15 week pairwise comparison), while the 6 to 12 week responses were intermediate to, and not significantly different from, the two extremes (Fig. 3).
3.1.4. Choice of male to cue-lure vs. control Explorer male flies showed a significant preference to cue-lure over the control until an age of 12 weeks (Week [Wk.] 3, Chi2 = 15.868, P < 0.001; Wk. 6, Chi2 = 23.273, P < 0.001; Wk. 9, Chi2 = 20.455, P < 0.001; Wk. 12, Chi2 = 25.130, P < 0.001), however the response of males to cue-lure or control did not significantly differ from a 1:1 ratio at week 15 (Chi2 = 1.581, P = 0.209) (Fig. 5).
3.1.3. Time-to-response There was a significant effect of age on the time-to-response to cue-
Fig. 3. The mean ( ± 1SE) probability of five age-groups of male Bactrocera tryoni exploring either arm of a Y-tube olfactometer in the presence of a cue-lure source in one arm and a blank control in the other. Different letters on the bars represent significant difference at P < 0.05.
Fig. 4. The mean ( ± 1SE) time taken (seconds) by five age-groups of male Bactrocera tryoni to locate a cue-lure source. Different letters on the bars represent significant differences at P < 0.05. 4
Journal of Insect Physiology 122 (2020) 104024
M.S. Tasnin, et al.
P < 0.001). However, there were no significant differences in the exploration probability between 3 and 6 week old females, as well as among 9 to 15 weeks old flies (Fig. 7). 3.2.3. Time-to-response There was no significant effect of age on the time-to-response to guava-juice odour for female flies [GLM (time duration ~ age-groups, family = gamma), F = 2.321, Df = 4, P = 0.059, mean time = 219.15 ± 28.60 s] (Fig. 8). 3.2.4. Choice of female to guava-juice vs. control Explorer female flies showed a significant preference to guava-juice over the control for all age groups tested (Wk. 3, Chi2 = 9.290, P = 0.002; Wk. 6, Chi2 = 8.643, P = 0.003; Wk. 9, Chi2 = 5.818, P = 0.016; Wk. 12, Chi2 = 5.232, P = 0.022; Wk. 15, Chi2 = 5.121, P = 0.023) (Fig. 9). 4. Discussion
Fig. 5. Age-related olfactory response of male Bactrocera tryoni to cue-lure. Results of chi-square analyses demonstrate significant deviation from 1:1 response ratio to cue-lure and blank-odour control in all age groups except 15 weeks (P < 0.001 = ***). Seventy flies were tested per age-group, not all responded. The number of males that went to cue-lure and blank control arm are presented in black and grey bars respectively.
4.1. Reduction in olfactory response with ageing Similar to other insects (Cook-Wiens and Grotewiel, 2002; Gadenne et al., 2016; Iliadi and Boulianne, 2010), advancing age was found to negatively affect the olfactory response of B. tryoni with respect to the tested odours. We found decreased orientation to odour sources in both male and female B. tryoni, with the probability of selective orientation of males and females dropping significantly at 15 weeks compared to three weeks. In D. melanogaster, a similar reduction of olfactory response was attributed to developing defects in the olfactory system as well as decreasing locomotor activity with ageing (Cook-Wiens and Grotewiel, 2002). The reduction in the selective orientation of female B. tryoni to odour sources can be more directly attributed to reduced exploratory activity (i.e. locomotion) with age because the selective orientation probability of females was constant at all ages among explorer flies. However, for males, the situation is more complex with, at 15 weeks, both a significant drop in the number of explorer flies and the failure of explorers to discriminate between lure and blank arms of the olfactometer.
3.2. Effect of age on the female response to guava-juice odour GLMs were run with age-groups and cohorts to determine their interactive and additive effect on female olfactory responses. The cohorts did not have significant effects on any of the response variables in both models. Subsequently, cohort was removed from the models and the following results only focus on the significant explanatory variable: agegroup. The GLMs run with the response variable “olfactory response” showed that age had a significant effect on female response to guavajuice odour. Subsequently, GLMs were run with exploratory activity and time-to-response to explore whether the reduction in olfactory response was related to changes in these parameters. Additionally, chisquare tests were conducted to examine whether reduced olfactory response of females was due to a decline in choosiness for the odour or not. The results of GLMs and chi-square tests are presented below.
4.2. Reduction in exploratory activity with ageing The current study revealed a decline in the exploratory activity of flies with ageing in the presence of the tested odours. As the flies aged, fewer crossed the mid-line of the olfactometer arms, with many simply sitting in the mouth of the Y-tube for the 10 min observation period. Additionally, at advanced age, male flies took a significantly longer time to choose the odour source compared to the youngest flies. However, female flies did not have a significant difference in time-toresponse at all tested ages. Reduction in exploratory activity and locomotion with increasing age has been recorded in other insects. In studies with D. melanogaster, 6 week old flies were 30-fold more likely to stay at the release point than 3 week old flies (Grotewiel et al., 2005), while 40–41 day old flies were less likely to cross a 27-cm-diameter arena from the release point than 6–7 day old flies (Iliadi and Boulianne, 2010). A similar trend was reported for C. capitata, where walking frequency declined after three weeks and was extremely low before death (Carey et al., 2006). Male and female B. tryoni, reared on a standard laboratory diet, showed different patterns in flight and walking frequency with age. Females increased mean locomotor activities from 4 to 10 to 30 days, while males showed increased locomotor activity from days 4 to 10, but had reduced activity at 30 days (Prenter et al., 2013). However, in contrast to a decline in locomotor activity at an early age (i.e. between 10 and 30 days), in the current study males and females were observed to retain their maximal exploratory activities for longer periods (6 weeks in females, 12 weeks in males). This may be because of the different experimental setup. All of
3.2.1. Olfactory response of female to guava-juice GLM revealed that, for all flies tested (i.e. explorers + non-explorers), there was a significant difference in the probability of flies selectively orientating to guava-juice among the five age-groups [GLM (selective orientation ~ age-groups, family = binomial), Chi2 = 15.121, Df = 4, P = 0.004]. The subsequent post hoc tests demonstrated that attraction to guava-juice odour was significantly higher for 3 week-old versus 15 week-old females (Z = 3.337, P = 0.008), with 6, 9 and 12 week old flies intermediate between these extremes (Fig. 6 A). However, the probability of selective orientation for explorer flies did not differ with age [GLM (selective orientation ~ age-group, family = binomial), Chi2 = 0.198, Df = 4, P = 0.995, mean probability = 0.69 ± 0.07] (Fig. 6B). 3.2.2. Exploratory activity The probability of females exploring either arm of the olfactometer was significantly affected by the age of the flies [GLM: (explorer ~ agegroups, family = binomial), Chi2 = 38.257, Df = 4, P < 0.001], with the subsequent post hoc tests revealing that there was a significant drop in the exploration probability of flies at 9, 12 and 15 weeks of age compared to 3 weeks (Z = 3.383, P = 0.006; Z = 3.526, P = 0.004 and Z = 4.979, P < 0.001 in 3 vs. 9, 12 and 15 week pairwise comparisons respectively). There was also a significant difference between the exploration probability of 6 week and 15 week old flies (Z = 4.066, 5
Journal of Insect Physiology 122 (2020) 104024
M.S. Tasnin, et al.
Fig. 6. . The mean ( ± 1SE) probability of female Bactrocera tryoni selectively orientating to guava-odour at five age-groups for (A) all tested flies (explorer + nonexplorer) and (B) explorer flies only. Different letters above the bars represent significant differences at P < 0.05. There is no significant age effect in Fig. 6B.
Fig. 8. The mean ( ± 1SE) time taken (seconds) by five age-groups of female Bactrocera tryoni to locate a guava-juice source did not show significant difference, Df = 4, P = 0.059.
Fig. 7. The mean ( ± 1SE) probability of five age-groups of female Bactrocera tryoni exploring either arm of a Y-tube olfactometer in the presence of a guavajuice source and a blank control. Different letters on the bars represent significant differences at P < 0.05.
orientation preference to guava-juice odour. Male and female olfactory responses are discussed separately in the following sections
the previous ageing research on locomotor and exploratory activities were conducted without an odour sources, while the current experiments were conducted in the presence of cue-lure and guava juice which have been reported to increase activity and flight of mature males and females in wind tunnel experiments (Dalby-Ball and Meats, 2000; Meats and Hartland, 1999). Additionally, the current research was conducted with wild flies, while the work of Prenter et al. (2013) used laboratory-reared flies, which may also influence results.
4.3.1. The constant attraction of males to cue-lure until an advanced age Males of B. tryoni retained positive olfactory responses to cue-lure for most of their life. A similar result was observed in B. opiliae, where males response to methyl eugenol remained unchanged until 12 weeks of age (Fitt, 1981). The Bactrocera male response to a small group of plant-derived secondary chemicals is considered a critical element of their biology and ecology (Clarke, 2019), as exposure to these chemicals enhances individual male fitness through increased sexual competitiveness (Kumaran et al., 2013; Kumaran et al., 2014; Metcalf and Metcalf, 1992; Weldon et al., 2008). Bactrocera tryoni males mate throughout their lives, even though older males have reduced mating success when in competition with young males (Ekanayake et al., 2017). There is thus an evolutionary driver for male flies to retain a strong lure response for as long in life as possible, and this is reflected in our results (to week 12) and those of Fitt (1981). In this light, we interpret the dramatic decrease of male olfactory response at 15 weeks, caused by simultaneous drops in locomotor activity and selective
4.3. Patterns in reduced olfactory response in males and females The olfactory responses and exploratory activities of both males and females declined at advanced age. The male olfactory response to cuelure was constant until 12 weeks of age, after which their olfactory response dropped dramatically at 15 weeks when there were few explorers, and those that did move orientated indiscriminately to control and treatment arms. However, female exploratory response decreased gradually over time and reached the lowest level at the oldest age group, although the oldest age group still showed a significant 6
Journal of Insect Physiology 122 (2020) 104024
M.S. Tasnin, et al.
the population (authors’ unpublished data), following the over-wintering quiescent period (Clarke et al., 2019; Fletcher, 1979; Muthuthantri et al., 2010; O'loughlin et al., 1984). It is possible that such populations may be undetected, or at least underestimated, if relying solely on cue-lure trapping. Also positively for pest management, the results suggest that new generation female traps based on host fruit odours (Gregg et al., 2018), can be expected to be most effective against female B. tryoni when they are at their reproductive peak, but also less effective against the very old spring populations. CRediT authorship contribution statement Mst Shahrima Tasnin: Conceptualization, Methodology, Data curation, Visualization. Katharina Merkel: Conceptualization, Methodology, Supervision. Anthony R. Clarke: Conceptualization, Methodology, Supervision. Acknowledgements Fig. 9. Age-related olfactory responses of female Bactrocera tryoni to guavajuice odour versus a blank control. Results of the chi-square test demonstrates significant deviation from 1:1 response ratio to guava-odour and blank-odour control in all age groups (P < 0.05 = *; P < 0.01 = **). Seventy flies were tested per age-group, not all responded.
We are thankful to the owner and staff, especially Bob Brinsmead and Mick O’Reilly, of Tropical Fruit World, Tweed Valley, NSW for providing information about infested fruits in the field and allowing us onto their property for collecting infested carambola. Similarly, we thank Brendan Missenden for helping us to collect the infested mangoes from his property. Thanks to QUT colleagues Karina Pyle, Rehan Silva, Francesca Strutt and Kiran Mahat for providing suggestions during experiments. A.R.C. and the QUT fruit fly lab is funded by the Australian Research Council through its Discovery Project (DP180101915) and Industrial Transformation Training Centre (IC150100026) schemes. The Australian Research Council had no role in the design, analysis or interpretation of this research.
olfactory orientation, as likely symptomatic of wider failures of several biological systems associated with advanced age, rather than a loss of “interest” in cue-lure per se. 4.3.2. Higher attraction to host fruit odour in younger females The females’ exploratory activities to guava-juice odour showed a gradual reduction with ageing, but with no corresponding decline for positive selection to the odour in explorer flies. A similar response was found in A. obliqua, where a decline in the olfactory response to volatiles of host fruits was found in 20 day old females compared to 1, 5 or 10 day old females (Reyes et al., 2017). In B. tryoni, at a given point in time, the olfactory response of gravid females to host fruit odour is linked with oviposition (Eisemann and Rice, 1992). We believe our results support this observation in a longitudinal temporal context, and this explains the female decline in response with ageing. In our experiment, we found young females (3–6 weeks) demonstrated the highest response to guava juice odour, with a continuous decline thereafter. This correlates closely with the lifetime fecundity patterns of female B. tryoni, which sees a peak in egg production at four weeks, with a continuous decline thereafter (Fitt, 1990; Kumaran et al., 2013). While the current study identified aging effects on olfaction of B. tryoni, a limitation of this study is that the male and female olfactory responses were each tested against a single odour type, i.e. cue-lure for males and guava juice for females. Thus, the study cannot determine whether the changes reported here are general patterns of age related olfactory change for males and females of this species, or if the patterns observed relate to the specific odours tested. Further studies are therefore required to confirm or deny the generality of our results.
Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jinsphys.2020.104024. References Aluja, M., 1996. Future trends in fruit fly management. In: Steck, M.A.G.J. (Ed.), Fruit Fly Pests: A World Assessment of Their Biology and Management. St. Lucie Press, Delray Beach, FL, pp. 309–320. Balagawi, S., 2006. Comparative ecology of Bactrocera Cucumis (French) and Bactrocera Tryoni (Froggatt) (Diptera: Tephritidae)-Understanding the Life History Consequences of Host Selection and Oviposition Behaviour. Griffith University. Carey, J.R., Papadopoulos, N., Kouloussis, N., Katsoyannos, B., Muller, H.G., Wang, J.L., Tseng, Y.K., 2006. Age-specific and lifetime behavior patterns in Drosophila melanogaster and the Mediterranean fruit fly, Ceratitis capitata. Experimenta Gerontol. 41, 93–97. https://doi.org/10.1016/j.exger.2005.09.014. Carey, J.R., Papadopoulos, N.T., Müller, H.G., Katsoyannos, B.I., Kouloussis, N.A., Wang, J.L., Wachter, K., Yu, W., Liedo, P., 2008. Age structure changes and extraordinary lifespan in wild medfly populations. Aging Cell 7, 426–437. https://doi.org/10.1111/ j.1474-9726.2008.00390.x. Clarke, A.R., 2019. Biology and Management of Bactrocera and Related Fruit Flies. CAB Internatinal, Wallingford, UK. Clarke, A.R., Merkel, K., Hulthen, A.D., Schwarzmueller, F., 2019. Bactrocera tryoni (Froggatt) (Diptera: Tephritidae) overwintering: an overview. Austral Entomol. 58, 3–8. https://doi.org/10.1111/aen.12369. Clarke, A.R., Powell, K.S., Weldon, C.W., Taylor, P.W., 2011. The ecology of Bactrocera tryoni (Diptera: Tephritidae): what do we know to assist pest management? Ann. Appl. Biol. 158, 26–54. https://doi.org/10.1111/j.1744-7348.2010.00448.x. Cook-Wiens, E., Grotewiel, M.S., 2002. Dissociation between functional senescence and oxidative stress resistance in Drosophila. Exp. Gerontol. 37, 1347–1357. https://doi. org/10.1016/S0531-5565(02)00096-7. Crnjar, R., Yin, C.-M., Stoffolano Jr, J., Barbarossa, I.T., Liscia, A., Angioy, A., 1990. Influence of age on the electroantennogram response of the female blowfly (Phormia regina) (Diptera: Calliphoridae). J. Insect Physiol. 36, 917–921. https://doi.org/10. 1016/0022-1910(90)90079-U. Cunningham, J.P., Carlsson, M.A., Villa, T.F., Dekker, T., Clarke, A.R., 2016. Do fruit ripening volatiles enable resource specialism in polyphagous fruit flies? J. Chem. Ecol. 42, 931–940. https://doi.org/10.1007/s10886-016-0752-5. Dalby-Ball, G., Meats, A., 2000. Influence of the odour of fruit, yeast and cue-lure on the flight activity of the Queensland fruit fly, Bactrocera tryoni (Froggatt) (Diptera: Tephritidae). Austr. J. Entomol. 39, 195–200. https://doi.org/10.1046/j.1440-6055.
4.4. Implications for management As the olfactory response of an insect may vary with age, the trapping effectiveness of pest insects for surveillance and management may also vary (Kouloussis et al., 2009). Cue-lure is an effective monitoring tool for male B. tryoni and is used to control field populations of this insect by mass-trapping and male-annihilation (Dominiak et al., 2003; Lloyd et al., 2010; Reynolds et al., 2016; Stringer et al., 2017). That male flies retain their attraction to cue-lure consistently until an advanced age is a positive finding for pest management. However, loss of attraction to cue-lure may be a problem for sampling B. tryoni males at the very end of winter, when there is a majority of very old males in 7
Journal of Insect Physiology 122 (2020) 104024
M.S. Tasnin, et al.
Gracillariidae). J. Chem. Ecol. 44, 276–287. https://doi.org/10.1007/s10886-0180927-3. Liu, L., Zhou, Q., 2016. Olfactory response of female Bactrocera minax to chemical components of the preference host citrus volatile oils. J. Asia-Pac. Entomol. 19, 637–642. https://doi.org/10.1016/j.aspen.2016.05.008. Lloyd, A.C., Hamacek, E.L., Kopittke, R.A., Peek, T., Wyatt, P.M., Neale, C.J., Eelkema, M., Gu, H., 2010. Area-wide management of fruit flies (Diptera: Tephritidae) in the Central Burnett district of Queensland, Australia. Crop Prot. 29, 462–469. https:// doi.org/10.1016/j.cropro.2009.11.003. López-Guillén, G., López, L.C., Malo, E.A., Rojas, J.C., 2011. Olfactory responses of Anastrepha obliqua (Diptera: Tephritidae) to volatiles emitted by calling males. Florida Entomol. 874–881. Martel, V., Anderson, P., Hansson, B.S., Schlyter, F., 2009. Peripheral modulation of olfaction by physiological state in the Egyptian leaf worm Spodoptera littoralis (Lepidoptera: Noctuidae). J. Insect Physiol. 55, 793–797. https://doi.org/10.1016/j. jinsphys.2009.04.012. May, A., 1958. Fruit fly problem in southern and central Queensland. Queensland Agric. J. 84, 153–159. Meats, A., Hartland, C., 1999. Upwind anemotaxis in response to cue-lure by the Queensland fruit fly, Bactrocera tryoni. Physiol. Entomol. 24, 90–97. https://doi.org/ 10.1046/j.1365-3032.1999.00118.x. Metcalf, R.L., Metcalf, E.R., 1992. Plant Kairomones in Insect Ecology and Control. Chapman and Hall Ltd, Springer, US. Muthuthantri, S., Maelzer, D., Zalucki, M.P., Clarke, A.R., 2010. The seasonal phenology of Bactrocera tryoni (Froggatt) (Diptera: Tephritidae) in Queensland. Austr. J. Entomol. 49, 221–233. https://doi.org/10.1111/j.1440-6055.2010.00759.x. Norrbom, A.L., Carroll, L.E., Thompson, F.C., White, I., Freidberg, A., 1999. Systematic Database of Names. Myia. O'loughlin, G., East, R., Meats, A., 1984. Survival, development rates and generation times of the Queensland fruit fly, Dacus Tryoni, in a marginally favourable climate: experiments in Victoria. Aust. J. Zool. 32, 353–361. https://doi.org/10.1071/ ZO9840353. Otter, C.D., Tchicaya, T., Schutte, A., 1991. Effects of age, sex and hunger on the antennal olfactory sensitivity of tsetse flies. Physiol. Entomol. 16, 173–182. https://doi.org/ 10.1111/j.1365-3032.1991.tb00554.x. Prenter, J., Weldon, C.W., Taylor, P.W., 2013. Age-related activity patterns are moderated by diet in Queensland fruit flies Bactrocera tryoni. Physiol. Entomol. 38, 260–267. https://doi.org/10.1111/phen.12023. Raghu, S., 2007. Functional significance of phytochemical lures to dacine fruit flies (Diptera: Tephritidae): an ecological and evolutionary synthesis. Bull. Entomol. Res. 94, 385–399. https://doi.org/10.1079/BER2004313. Reyes, H., Malo, E.A., Toledo, J., Cruz-Esteban, S., Rojas, J.C., 2017. Physiological state influences the antennal response of Anastrepha obliquato male and host volatiles. Physiol. Entomol. 42, 17–25. https://doi.org/10.1111/phen.12157. Reynolds, O.L., Osborne, T., Crisp, P., Barchia, I.M., 2016. Specialized pheromone and lure application technology as an alternative male annihilation technique to manage Bactrocera tryoni (Diptera: Tephritidae). J. Econ. Entomol. 109, 1254–1260. https:// doi.org/10.1093/jee/tow023. Russell, L., 2018. Emmeans: Estimated Marginal Means, aka Least-Squares Means. R package version 1. Shelly, T., 2010. Effects of methyl eugenol and raspberry ketone/cue lure on the sexual behavior of Bactrocera species (Diptera: Tephritidae). Appl. Entomol. Zool. 45, 349–361. https://doi.org/10.1303/aez.2010.349. Sollai, G., Solari, P., Crnjar, R., 2018. Olfactory sensitivity to major, intermediate and trace components of sex pheromone in Ceratitis capitata is related to mating and circadian rhythm. J. Insect Physiol. 110, 23–33. https://doi.org/10.1016/j.jinsphys. 2018.08.007. Stringer, L.D., Kean, J.M., Beggs, J.R., Suckling, D.M., 2017. Management and eradication options for Queensland fruit fly. Popul. Ecol. 59, 259–273. https://doi.org/10.1007/ s10144-017-0593-2. Sutherst, R.W., Collyer, B.S., Yonow, T., 2000. Fruit fly problem in southern and Central Queensland. Aust. J. Agric. Res. 51, 467–480. Team, R.C., 2018. R: a language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing; 2018. https://www.R‐project.org/. Touhara, K., Vosshall, L.B., 2009. Sensing odorants and pheromones with chemosensory receptors. Annu. Rev. Physiol. 71, 307–332. https://www.annualreviews.org/doi/ full/10.1146/annurev.physiol.010908.163209. Wäckers, F., 1994. The effect of food deprivation on the innate visual and olfactory preferences in the parasitoid Cotesia rubecula. J. Insect Physiol. 40, 641–649. https:// doi.org/10.1016/0022-1910(94)90091-4. Wee, S.-L., Peek, T., Clarke, A.R., 2018. The responsiveness of Bactrocera jarvisi (Diptera: Tephritidae) to two naturally occurring phenylbutaonids, zingerone and raspberry ketone. J. Insect Physiol. 109, 41–46. https://doi.org/10.1016/j.jinsphys.2018.06. 004. Weldon, C.W., Perez-Staples, D., Taylor, P.W., 2008. Feeding on yeast hydrolysate enhances attraction to cue-lure in Queensland fruit flies, Bactrocera tryoni. Entomol. Exp. Appl. 129, 200–209. https://doi.org/10.1111/j.1570-7458.2008.00768.x. Wong, T.T., McInnis, D., Ramadan, M.M., Nishimoto, J.I., 1991. Age-related response of male melon flies Dacus cucurbitae (Diptera: Tephritidae) to cue-lure. J. Chem. Ecol. 17, 2481–2487. Wyatt, T.D., 2014. Pheromones and Animal Behavior: Chemical Signals and Signatures. Cambridge University Press.
2000.00168.x. Dominiak, B., Westcott, A., Barchia, I., 2003. Release of sterile Queensland fruit fly, Bactrocera tryoni (Froggatt) (Diptera: Tephritidae), at Sydney, Australia. Aust. J. Exp. Agric. 43, 519–528. https://doi.org/10.1071/EA01146. Eisemann, C., 1980. An Investigation of Some Stimuli Influencing Host Finding and Oviposition Behaviours of the Queensland Fruit Fly, Dacus (Bactrocera) tyroni (Frogg.). University of Queensland. Eisemann, C., Rice, M., 1992. Attractants for the gravid Queensland fruit fly Dacus tryoni. Entomol. Exp. Appl. 62, 125–130. Ekanayake, E., Clarke, A.R., Schutze, M.K., 2017. Effect of body size, age, and premating experience on male mating success in Bactrocera tryoni (Diptera: Tephritidae). J. Econ. Entomol. 110, 2278–2281. https://doi.org/10.1093/jee/tox186. Ero, M.M., Hamacek, E., Clarke, A.R., 2011. Foraging behaviours of Diachasmimorpha kraussii (Fullaway) (Hymenoptera: Braconidae) and its host Bactrocera tryoni (Froggatt) (Diptera: Tephritidae) in a nectarine (Prunus persica (L.) Batsch var. nectarina (Aiton) Maxim) orchard. Austr. J. Entomol. 50, 234–240. https://doi.org/10. 1111/j.1440-6055.2011.00821.x. Fitt, G.P., 1981. The influence of age, nutrition and time of day on the responsiveness of male Dacus opiliae to the synthetic lure, methyl eugenol. Entomol. Exp. Appl. 30, 83–90. https://doi.org/10.1111/j.1570-7458.1981.tb03588.x. Fitt, G.P., 1990. Comparative fecundity, clutch size, ovariole number and egg size of Dacus tryoni and D. jarvisi, and their relationship to body size. Entomol. Exp. Appl. 55, 11–21. https://doi.org/10.1111/j.1570-7458.1990.tb01343.x. Fletcher, B., 1989. Life history strategies of tephritid fruit flies. In: Robison, A.S.H.G. (Ed.), Fruit Flies, Their Biology, Natural Enemies And Control. Elsevier Science, Amsterdam, The Netherlands, pp. 195–208. Fletcher, B.S., 1979. The overwintering survival of adults of the Queensland fruit fly, Dacus Tryoni, under natural conditions. Aust. J. Zool. 27, 403–411. https://doi.org/ 10.1071/ZO9790403. Gadenne, C., Barrozo, R.B., Anton, S., 2016. Plasticity in insect olfaction: to smell or not to smell? Annu. Rev. Entomol. 61, 317–333. https://doi.org/10.1146/annurev-ento010715-023523. Gregg, P.C., Del Socorro, A.P., Landolt, P.J., 2018. Advances in attract-and-kill for agricultural pests: beyond pheromones. Annu. Rev. Entomol. 63, 453–470. https://www. annualreviews.org/doi/abs/10.1146/annurev-ento-031616-035040. Grotewiel, M.S., Martin, I., Bhandari, P., Cook-Wiens, E., 2005. Functional senescence in Drosophila melanogaster. Ageing Res. Rev. 4, 372–397. https://doi.org/10.1016/j.arr. 2005.04.001. Hancock, D., Hamacek, E., Lloyd, A., Elson-Harris, M., 2000. The Distribution and Host Plants Of Fruit Flies (Diptera: Tephritidae) in Australia. Queensland Department of Primary Industries. Iliadi, K.G., Boulianne, G.L., 2010. Age-related behavioral changes in Drosophila. Ann. N. Y. Acad. Sci. 1197, 9–18. https://doi.org/10.1111/j.1749-6632.2009.05372.x. Jang, E.B., McInnis, D.O., Kurashima, R., Carvalho, L.A., 1999. Behavioural switch of female Mediterranean fruit fly, Ceratitis capitata: mating and oviposition activity in outdoor field cages in Hawaii. Agric. For. Entomol. 1, 179–184. https://doi.org/10. 1046/j.1461-9563.1999.00025.x. Jin, S., Zhou, X., Gu, F., Zhong, G., Yi, X., 2017. Olfactory plasticity: variation in the expression of chemosensory receptors in Bactrocera dorsalis in different physiological states. Front. Physiol. 8, 672. https://doi.org/10.3389/fphys.2017.00672. Kamiji, T., Kaneda, M., Sasaki, M., Ohto, K., 2018. Sexual maturation of male Bactrocera correcta (Diptera: Tephritidae) and age-related responses to β-caryophyllene and methyl eugenol. Appl. Entomol. Zool. 53, 41–46. Klowden, M., 1990. The endogenous regulation of mosquito reproductive behavior. Experientia 46, 660–670. https://doi.org/10.1007/bf01939928. Kouloussis, N.A., Papadopoulos, N.T., Muller, H.G., Wang, J.L., Mao, M., Katsoyannos, B.I., Duyck, P.F., Carey, J.R., 2009. Life table assay of field-caught Mediterranean fruit flies, Ceratitis capitata, reveals age bias. Entomol. Exp. Appl. 132, 172–181. https://doi.org/10.1111/j.1570-7458.2009.00879.x. Koyama, J., Kakinohana, H., Miyatake, T., 2004. Eradication of the melon fly, Bactrocera cucurbitae, in Japan: importance of behavior, ecology, genetics, and evolution. Annu. Rev. Entomol. 49, 331–349. https://www.annualreviews.org/doi/abs/10.1146/ annurev.ento.49.061802.123224. Krieger, J., Breer, H., 1999. Olfactory reception in invertebrates. Science 286, 720–723. https://doi.org/10.1126/science.286.5440.720. Kumaran, N., Balagawi, S., Schutze, M.K., Clarke, A.R., 2013. Evolution of lure response in tephritid fruit flies: phytochemicals as drivers of sexual selection. Anim. Behav. 85, 781–789. https://doi.org/10.1016/j.anbehav.2013.01.024. Kumaran, N., Hayes, R.A., Clarke, A.R., 2014. Cuelure but not zingerone make the sex pheromone of male Bactrocera tryoni (Tephritidae: Diptera) more attractive to females. J. Insect Physiol. 68, 36–43. https://doi.org/10.1016/j.jinsphys.2014.06.015. Landolt, P., Heath, R., King, J., 1985. Behavioral responses of female papaya fruit flies, Toxotrypana curvicauda (Diptera: Tephritidae), to male-produced sex pheromone. Ann. Entomol. Soc. Am. 78, 751–755. https://doi.org/10.1093/aesa/78.6.751. Lebreton, S., Borrero-Echeverry, F., Gonzalez, F., Solum, M., Wallin, E.A., Hedenstrom, E., Hansson, B.S., Gustavsson, A.L., Bengtsson, M., Birgersson, G., Walker 3rd, W.B., Dweck, H.K.M., Becher, P.G., Witzgall, P., 2017. A Drosophila female pheromone elicits species-specific long-range attraction via an olfactory channel with dual specificity for sex and food. BMC Biol. 15, 88. https://doi.org/10.1186/s12915-0170427-x. Lemmen-Lechelt, J.K., Wist, T.J., Evenden, M.L., 2018. State-dependent plasticity in response to host-plant volatiles in a long-lived moth, Caloptilia fraxinella (Lepidoptera:
8
Contents lists available at ScienceDirect
Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys
Effects of advanced age on olfactory response of male and female Queensland fruit fly, Bactrocera tryoni (Froggatt) (Diptera: Tephritidae)
T
⁎
Mst Shahrima Tasnin , Katharina Merkel, Anthony R. Clarke School of Biology and Environmental Science, Queensland University of Technology (QUT), Brisbane, QLD 4000, Australia
A R T I C LE I N FO
A B S T R A C T
Keywords: Olfactory response Ageing Exploratory activity Cue-lure Guava juice Bactrocera tryoni Dacinae
Olfaction is an essential sensory modality of insects which is known to vary with age. In short-lived insects odour response generally declines rapidly with increasing age, but how increasing age affects the olfactory response of long-lived insects is less known and there may be different life-time patterns of olfactory response. Here, we examine the effect of age on olfactory response and exploratory activity of a long-lived tephritid fruit fly, Bactrocera tryoni from sexual maturity (3 weeks) to advanced age (15 weeks). Males were tested against a malespecific attractant, cue-lure, which is associated with courtship and sexual selection in this species; while females were tested against guava-juice, a highly attractive oviposition host fruit odour. Trials were done in the laboratory using a Y-tube olfactometer at three weekly intervals. The probability of olfactory response of both males and females to tested odours declined with age. Males retained a constant attraction to cue-lure until 12 weeks of age, but then showed a significant drop in olfactory response at 15 weeks. However, females showed the highest attraction to guava-juice odour until six weeks of age and declined gradually thereafter. The change on odour response over time can be associated with an age-related change in initial locomotor activity for females as there was no change, over the life of the experiment, in selective female orientation to the odour source once flies started exploring within the olfactometer. However, for 15 week-old males, there was a simultaneous drop in both locomotor activity and selective olfactory orientation. The consistent attraction of male to cue-lure might be related to life-long reproductive activities of males, as males are thought to mate continuously during life. On the other hand, females’ highest attraction to guava-juice odour in early life followed by a gradual decline might be linked with their oviposition rate which peaks in early life.
1. Introduction For insects, olfaction is an essential sensory modality for the perception of stimuli from the environment (Krieger and Breer, 1999; Touhara and Vosshall, 2009). Olfaction plays a key role in behaviours such as the location of food, mates and oviposition substrates, as well as enemy avoidance (Lebreton et al., 2017; Wyatt, 2014). An insect’s response to odours may vary depending on physiological factors such as feeding and nutritional status, mating status and sexual maturation (Gadenne et al., 2016; Jin et al., 2017; Lemmen-Lechelt et al., 2018). For example, insects that are deprived of food generally have a higher attraction to food odours (Wäckers, 1994); while virgin insects are more responsive to odours from mating sites and partners than are mated insects (Jin et al., 2017; Sollai et al., 2018). An individual’s age is another factor that strongly affects the odour response of an insect (Gadenne et al., 2016; Reyes et al., 2017). In insects, age-related changes in olfactory responses are linked to
⁎
sexual maturation, as well as ageing (Gadenne et al., 2016; Martel et al., 2009). For example, in early life, insects are more attracted to food odours while, after sexual maturation, they may be more attracted to cues from mating partners (Klowden, 1990). Additionally, an insect’s response to the same odour may change with age, for example, females of the blowfly, Phormia regina Meigen, show increasing olfactory sensitivity to swormlure-4 and 1-hexanol during the first week of adult life (Crnjar et al., 1990). In contrast to increasing olfactory sensitivity, an insect’s response to odours can be negatively affected by ageing once past some optimal age (Gadenne et al., 2016; Iliadi and Boulianne, 2010). For instance, the antennal olfactory sensitivity of tsetse flies, Glossina morsitans morsitans Westwood, to various tested volatiles started declining after only five days of age (Otter et al., 1991), while the perception of aversive odours diminished in eight week old Drosophila melanogaster Meigen (Cook-Wiens and Grotewiel, 2002). For D. melanogaster, Cook-Wiens and Grotewiel (2002) attributed the reduction of olfactory response with increasing age to both the development
Corresponding author. E-mail addresses: [email protected] (M.S. Tasnin), [email protected] (K. Merkel), [email protected] (A.R. Clarke).
https://doi.org/10.1016/j.jinsphys.2020.104024 Received 24 October 2019; Received in revised form 7 February 2020; Accepted 10 February 2020 Available online 12 February 2020 0022-1910/ © 2020 Elsevier Ltd. All rights reserved.
Journal of Insect Physiology 122 (2020) 104024
M.S. Tasnin, et al.
this study evaluates age-dependant olfactory responses and exploratory activities of the fly from sexual maturity to advanced age (15 weeks). Specifically, we observed the effect of age on olfactory response and exploratory activity of male and female flies in the presence of cue-lure and guava-juice odour, respectively. We hypothesized that: (i) exploratory activities will be higher in younger flies compared to older flies; (ii) the flies’ response to odours will be higher at a younger age compared to old age; and (iii) younger flies will take less time to choose the odour source compared to old flies.
of age-related defects in the olfactory system and associated parts of the brain, as well as a decline in locomotor activity. Tephritid fruit flies (Diptera: Tephritidae) are globally significant insect pests of agriculture, affecting most horticultural crops (Norrbom et al., 1999). Female fruit flies oviposit into host fruits, where resultant larval feeding causes fruit damage (Fletcher, 1989; May, 1958; Sutherst et al., 2000). The behaviour and ecology of tephritid fruit flies are strongly tied to olfaction, including the pheromonal attraction of conspecifics during mating (Landolt et al., 1985; López-Guillén et al., 2011), location of host fruit by females for oviposition (Eisemann, 1980; Jang et al., 1999; Liu and Zhou, 2016), and for male tephritids of the tribe Dacini for the location of plant secondary chemicals which are linked to male sexual selection (Kumaran et al., 2013; Raghu, 2007; Shelly, 2010). The management of pest tephritids relies heavily on the manipulation of this chemical ecology, using olfactory attractants for surveillance and lure-and-kill controls (Aluja, 1996; Koyama et al., 2004). Despite the implications for management, and simply for better understanding tephritid biology, studies of ageing and changing olfactory responses in tephritids are limited. Multiple studies have investigated the effect of ageing from adult emergence to sexual maturity [which occurs on average from 10 to 20 days after emergence, (Clarke, 2019)] on the response of male Dacini to male-specific lures, such as methyl eugenol (ME) and cue-lure (Kamiji et al., 2018; Metcalf and Metcalf, 1992; Weldon et al., 2008; Wong et al., 1991). Most of these studies have found that lure attraction is dependent on the male becoming sexually mature, although in some species males may begin to respond prior to maturation (Wee et al., 2018). Fitt (1981), in the only study of its type, recorded the response of Bactrocera opiliae (Drew and Hardy) to ME significantly past sexual maturation and found no change in responsiveness of males up to 12 weeks of age. Outside of the Dacini male lures, age-related olfaction studies in the Tephritidae are rare. The response of Ceratitis capitata (Wiedemann) to synthetic food attractants was investigated until 40 days of age, where it was shown that sugarfed flies showed higher response at middle age (10–25 days age) while protein-fed flies showed higher response at a young age (1–5 days age) with a declining trend afterwards (Kouloussis et al., 2009). For Anastrepha obliqua (Macquart), antennal responses to male lure and host fruit volatiles were observed until 20 days of age and it was found that older flies exhibited lower antennal response compared to younger flies (Reyes et al., 2017). While such studies are valuable, and with the exception of Fitt (1981) for males, there are no studies of which we are aware that evaluate olfactory responses of both male and female tephritid fruit flies to olfactory cues until an advanced age, as has been done for Drosophila (Cook-Wiens and Grotewiel, 2002) and other insects (Gadenne et al., 2016). This is despite the fact that some tephritids can be very long-lived (Carey et al., 2008), with Dacini tephritids, the focus of this paper, surviving 11 weeks on average in the laboratory (Clarke, 2019) and over 28 weeks as over-wintering adults (Clarke et al., 2019). Bactrocera tryoni (Froggatt) (Diptera: Tephritidae) is an Australian endemic insect which is a key pest of the Australian horticultural industry (Clarke et al., 2011; Sutherst et al., 2000). Sexually mature Bactrocera tryoni males are attracted to cue-lure [4-(p-acetoxyphenyl)-2butanone] (Meats and Hartland, 1999), while mature females are attracted to volatiles released from ripening host fruits (Cunningham et al., 2016; Eisemann, 1980). Cue-lure is widely used for quarantine surveillance, monitoring and field control of B. tryoni (Dominiak et al., 2003; Lloyd et al., 2010; Reynolds et al., 2016; Stringer et al., 2017), while fruit-based odours are being used as the basis for female traps (Cunningham et al., 2016). Bactrocera tryoni can be long-lived, with some individuals surviving in excess of 20 weeks in controlled laboratory studies (Balagawi, 2006), but there is no knowledge of how olfactory responses in the fly change once they become sexually mature, which occurs at approximately 21 days for wild flies (Dalby-Ball and Meats, 2000). Given the importance of olfaction in the management of B. tryoni,
2. Materials and method 2.1. Study insect Bactrocera tryoni used for the experiment were collected from the field, bred from naturally infested fruits. Two cohorts of wild flies were obtained from South-east Queensland (approx. 27°S, 152°E): the first from a collection of 200 mango fruits made on the 5th Feb 2018; the second from a collection of 180 carambola fruits made on the 18th Mar 2018. Infested fruits were placed in plastic containers on a layer of vermiculite and kept in an incubator at 25 °C and 65% relative humidity. After two weeks the vermiculite was sieved for pupae. Pupae were placed in 32 × 32 × 32 cm white mesh cages supplied with water. Each evening, flies emerging during the preceding 24 h period were transferred to new holding cages to keep flies separate according to emergence date. The holding cages were supplied with standard diets (water, sugar cubes and yeast hydrolysate ad libitum) throughout the rearing period. Each cage contained 200 flies, 100 of each sex to allow for mating. Flies originating from mango and carambola were treated as two separate cohorts for experimental purposes. 2.2. Olfactometer bioassay To observe age-related changes in exploratory activities and olfactory responses, five age groups (3, 6, 9, 12 and 15 week old) of male and female B. tryoni were tested. Based on the authors’ unpublished data, and in agreement with Dalby-Ball and Meats (2000), the three weeks old flies can be regarded as newly sexually mature and mated. Male age-related olfaction was tested against 0.1 ml undiluted cue-lure, while females were tested against the odour of 1 ml of a commercial guava juice (water + 30% guava [Psidium guava] puree, Golden Circle Ltd, QLD 4013, Australia) exposed on a petri dish. Guava is a major host of B. tryoni (Hancock et al., 2000) and mature females are highly attracted to ripe guava odours (Cunningham et al., 2016). Dosages of both attractants were based upon preliminary concentration trials which sought to maximise positive fly orientation. Experiments were conducted using a glass Y-tube olfactometer (Model: CADS-2P, Sigma Scientific Ltd) with a single odour source (cuelure for males, and guava juice for females) and a no-odour (blank) control. The olfactometer consisted of a 15 cm long stem and two 10 cm long side-arms (75° angle and 5 cm internal diameter). Side-arms were attached to two sealed glass jars, each 20 cm in depth and 14 cm diameter. An adjustable air compressor pumped airstream at a rate of 2.5 L/min through a clean air delivery system which was connected separately to both glass jars. A table lamp with white light was placed just above the junction of Y-tube arms to minimise differences in light intensity in the experimental arena. The odour sources were placed in an open petri dish, which was sat within the glass jar of the treatment arm: odour sources were replaced every two hours. The arm of the odour-containing glass jar was changed after testing 17 flies (i.e. half of a cohort treatment), at which time the olfactometer was also cleaned with water and 70% ethanol. A total of 35 flies was tested from each cohort (i.e. mango and carambola) for each age group, resulting in 70 flies tested for each age group. In the case of three and six week agegroups, 70 males originating from the carambola cohort only were tested against cue-lure due to a logistic difficulty during the experiment 2
Journal of Insect Physiology 122 (2020) 104024
M.S. Tasnin, et al.
with the mango cohort flies. All tests were conducted in the same, small laboratory room with controlled temperature (26 ± 1 °C) and constant lighting.
2.4. Data analysis All data analysis and visualizations were performed with the R statistical program (Team, 2018) and SigmaPlot (Systat Software, San Jose, CA). Male and female data were analysed separately as the sexes were examined against different odours as well as having differences in longevity. In the experiment, age-group and cohort were explanatory variables, while exploratory activity, olfactory response and time-toresponse were response variables. As exploratory activity and olfactory response were recorded as “1” or “0” for an individual fly, the resulting data were binary, while the time-to-response (measured in seconds) was continuous and followed a gamma distribution. Within each gender, generalized linear models (GLMs) were fitted to examine if the olfactory response, exploratory activity and time-to-response of individual flies were affected by the factors age-group and cohort. Agegroups and cohorts were initially fitted to test their interactive and additive effect on the response variables. A stepwise backward elimination approach was applied to determine the minimum adequate model. When a significant effect was detected by the model, a post hoc test was conducted for pairwise comparison of means using the package emmeans (Russell, 2018). The comparison tests were performed on the log odds ratio scale for binary data. Additionally, chi-square tests were conducted with the raw data which was the frequency of flies choosing the odour or control. This test examined if the number of flies choosing the odour source or the control differed from a 1:1 ratio depending on the age of flies. A deviation from a 1:1 ratio within an age group indicates directed movement, as opposed to a random walk into either arm.
2.3. Experimental design All experiments were carried out using individual flies and, for all experiments, the same procedure was followed. One hour before the experiment started, 35 flies were transferred from their maintenance cage in the insectary and brought to the experimental lab for acclimation. Flies were individually released at the entrance of the olfactometer stem. The fly’s behaviour in the olfactometer was observed for up to 10 min with the variables below recorded. If a fly showed no positive olfactory decision within 10 min the trial was terminated. i. Exploratory activity: If a fly moved upwind and crossed the marked halfway point of either side-arm of the olfactometer it was defined as an ‘explorer’ (coded as ‘1’); a fly which did not upwind forage in this way for the 10 min of observation was referred as a “non-explorer” (coded as ‘0’). ii. Olfactory response (explorer): When a fly responded positively to the odour containing arm (crossed the midpoint of the treatment side-arm) this was recorded as selective orientation (coded as ‘1’); when a fly went past the midpoint of the control arm this was defined as non-selective orientation (coded as ‘0’). iii. Time-to-response: The time taken after release for flies showing selective orientation to reach Y-tube arm with odour source (measured in seconds). These measured variables are illustrated in Fig. 1.
3. Results Male experiments were conducted between 08:00 to 13:00 h, because male B. tryoni are most attracted to cue-lure during morning hours (Weldon et al., 2008), while female experiments were conducted from 10:00 to 15:00 h which is considered the peak window for female oviposition in this species (Ero et al., 2011).
3.1. Effect of age on the male response to cue-lure GLMs were run with age-groups (excluding weeks 3 and 6) and cohorts to determine their interactive and additive effect on the response variables. In both models, there was no significant difference between the cohorts in how the olfactory response in males changed with age. Following this, both models were run with all the age-groups and cohorts which also produced non-significant effects of the cohort for all the response variables. Thus, the cohort was subsequently removed from the models and the following results only focus on the significant explanatory variable; age-group. The GLMs run with the response variable “olfactory response” showed that age had a significant effect on male response to cue-lure. Subsequently, GLMs were run with “exploratory activity” and “time-toresponse” to determine whether the reduction in olfactory response was related to changes in these parameters. Additionally, chi-square tests were conducted to examine whether reduced olfactory response of males was due to a decline in choosiness for the odour or not. The results of GLMs and chi-square tests are presented below. 3.1.1. Olfactory response of male to cue-lure GLM revealed that, for all flies tested (i.e. explorers + non-explorers), there was a significant difference in the probability of flies positively orientating to cue-lure among the five age groups [GLM (selective-orientation ~ age-groups, family = binomial), Chi2 = 19.431, Df = 4, P < 0.001]. The subsequent pairwise comparisons revealed that there was no significant difference in selective orientation rate from 3 to 12 week old flies (Wk. 3 vs. 6, Z = 0.511, P = 0.986; Wk. 3 vs. 9, Z = 0.680, P = 0.961; Wk. 3 vs. 12, Z = 0.171, P = 0.910; Wk. 6 vs. 9, Z = 0.169, P = 0.910; Wk. 9 vs. 12, Z = 0.509, P = 0.987), but 15 week old flies had a significantly lower positive orientation rate compared to all other younger age-groups (Z = 3.683, P = 0.002; Z = 3.218, P = 0.011; Z = 3.061, P = 0.019; and Z = 3.529, P = 0.004 in 15 vs. 3, 6, 9 and 12 week pairwise comparisons respectively) (Fig. 2 A). For explorer flies only, the
Fig. 1. Diagrammatic representation of response variables measured concerning Bactrocera tryoni foraging in a Y-tube olfactometer. Non-explorer, fly did not cross the mid-point of either olfactometer arm in 10 min (dark grey area); Explorer, fly did cross the mid-point of either olfactometer arm within 10 min (light grey area); Time-to-response, the time taken by an explorer fly to cross the arm mid-point; Selective and non-selective orientation, the response of an explorer fly to the odour arm or blank control arm, respectively. 3
Journal of Insect Physiology 122 (2020) 104024
M.S. Tasnin, et al.
Fig. 2. The mean ( ± 1SE) probability of male Bactrocera tryoni selective orientation to cue-lure at five ages for (A) all tested flies (explorer + non-explorer) and (B) explorer flies only. Different letters above the bars represent significant differences at P < 0.05. There is no significant age effect in Fig. 2B.
lure for male flies [GLM (time duration ~ age groups, family = gamma), F = 2.650, Df = 4, P = 0.035]. The subsequent post hoc test demonstrated a significant difference in time-to-response with 3 week old males taking significantly less time than 15 week old males (Z = 3.581, P = 0.003), but both age groups were not significantly different from the response times of 6, 9, and 12 week old males (Fig. 4).
probability of flies selectively orientating to cue-lure did not significantly differ depending on age-group [GLM (selective orientation ~ age-groups, family = binomial) (Chi2 = 9.188, Df = 4, P = 0.057, mean probability = 0.79 ± 0.06)] (Fig. 2 B). 3.1.2. Exploratory activity The probability of males exploring either arm of the olfactometer was significantly affected by the age of the flies [GLM: (explorer ~ agegroups, family = binomial), Chi2 = 15.393, Df = 4, P = 0.004], with the subsequent post hoc test revealing that there was a significant drop in the exploration probability of flies at 15 weeks of age compared to 3 weeks (Z = 3.711, P = 0.002, 3 vs. 15 week pairwise comparison), while the 6 to 12 week responses were intermediate to, and not significantly different from, the two extremes (Fig. 3).
3.1.4. Choice of male to cue-lure vs. control Explorer male flies showed a significant preference to cue-lure over the control until an age of 12 weeks (Week [Wk.] 3, Chi2 = 15.868, P < 0.001; Wk. 6, Chi2 = 23.273, P < 0.001; Wk. 9, Chi2 = 20.455, P < 0.001; Wk. 12, Chi2 = 25.130, P < 0.001), however the response of males to cue-lure or control did not significantly differ from a 1:1 ratio at week 15 (Chi2 = 1.581, P = 0.209) (Fig. 5).
3.1.3. Time-to-response There was a significant effect of age on the time-to-response to cue-
Fig. 3. The mean ( ± 1SE) probability of five age-groups of male Bactrocera tryoni exploring either arm of a Y-tube olfactometer in the presence of a cue-lure source in one arm and a blank control in the other. Different letters on the bars represent significant difference at P < 0.05.
Fig. 4. The mean ( ± 1SE) time taken (seconds) by five age-groups of male Bactrocera tryoni to locate a cue-lure source. Different letters on the bars represent significant differences at P < 0.05. 4
Journal of Insect Physiology 122 (2020) 104024
M.S. Tasnin, et al.
P < 0.001). However, there were no significant differences in the exploration probability between 3 and 6 week old females, as well as among 9 to 15 weeks old flies (Fig. 7). 3.2.3. Time-to-response There was no significant effect of age on the time-to-response to guava-juice odour for female flies [GLM (time duration ~ age-groups, family = gamma), F = 2.321, Df = 4, P = 0.059, mean time = 219.15 ± 28.60 s] (Fig. 8). 3.2.4. Choice of female to guava-juice vs. control Explorer female flies showed a significant preference to guava-juice over the control for all age groups tested (Wk. 3, Chi2 = 9.290, P = 0.002; Wk. 6, Chi2 = 8.643, P = 0.003; Wk. 9, Chi2 = 5.818, P = 0.016; Wk. 12, Chi2 = 5.232, P = 0.022; Wk. 15, Chi2 = 5.121, P = 0.023) (Fig. 9). 4. Discussion
Fig. 5. Age-related olfactory response of male Bactrocera tryoni to cue-lure. Results of chi-square analyses demonstrate significant deviation from 1:1 response ratio to cue-lure and blank-odour control in all age groups except 15 weeks (P < 0.001 = ***). Seventy flies were tested per age-group, not all responded. The number of males that went to cue-lure and blank control arm are presented in black and grey bars respectively.
4.1. Reduction in olfactory response with ageing Similar to other insects (Cook-Wiens and Grotewiel, 2002; Gadenne et al., 2016; Iliadi and Boulianne, 2010), advancing age was found to negatively affect the olfactory response of B. tryoni with respect to the tested odours. We found decreased orientation to odour sources in both male and female B. tryoni, with the probability of selective orientation of males and females dropping significantly at 15 weeks compared to three weeks. In D. melanogaster, a similar reduction of olfactory response was attributed to developing defects in the olfactory system as well as decreasing locomotor activity with ageing (Cook-Wiens and Grotewiel, 2002). The reduction in the selective orientation of female B. tryoni to odour sources can be more directly attributed to reduced exploratory activity (i.e. locomotion) with age because the selective orientation probability of females was constant at all ages among explorer flies. However, for males, the situation is more complex with, at 15 weeks, both a significant drop in the number of explorer flies and the failure of explorers to discriminate between lure and blank arms of the olfactometer.
3.2. Effect of age on the female response to guava-juice odour GLMs were run with age-groups and cohorts to determine their interactive and additive effect on female olfactory responses. The cohorts did not have significant effects on any of the response variables in both models. Subsequently, cohort was removed from the models and the following results only focus on the significant explanatory variable: agegroup. The GLMs run with the response variable “olfactory response” showed that age had a significant effect on female response to guavajuice odour. Subsequently, GLMs were run with exploratory activity and time-to-response to explore whether the reduction in olfactory response was related to changes in these parameters. Additionally, chisquare tests were conducted to examine whether reduced olfactory response of females was due to a decline in choosiness for the odour or not. The results of GLMs and chi-square tests are presented below.
4.2. Reduction in exploratory activity with ageing The current study revealed a decline in the exploratory activity of flies with ageing in the presence of the tested odours. As the flies aged, fewer crossed the mid-line of the olfactometer arms, with many simply sitting in the mouth of the Y-tube for the 10 min observation period. Additionally, at advanced age, male flies took a significantly longer time to choose the odour source compared to the youngest flies. However, female flies did not have a significant difference in time-toresponse at all tested ages. Reduction in exploratory activity and locomotion with increasing age has been recorded in other insects. In studies with D. melanogaster, 6 week old flies were 30-fold more likely to stay at the release point than 3 week old flies (Grotewiel et al., 2005), while 40–41 day old flies were less likely to cross a 27-cm-diameter arena from the release point than 6–7 day old flies (Iliadi and Boulianne, 2010). A similar trend was reported for C. capitata, where walking frequency declined after three weeks and was extremely low before death (Carey et al., 2006). Male and female B. tryoni, reared on a standard laboratory diet, showed different patterns in flight and walking frequency with age. Females increased mean locomotor activities from 4 to 10 to 30 days, while males showed increased locomotor activity from days 4 to 10, but had reduced activity at 30 days (Prenter et al., 2013). However, in contrast to a decline in locomotor activity at an early age (i.e. between 10 and 30 days), in the current study males and females were observed to retain their maximal exploratory activities for longer periods (6 weeks in females, 12 weeks in males). This may be because of the different experimental setup. All of
3.2.1. Olfactory response of female to guava-juice GLM revealed that, for all flies tested (i.e. explorers + non-explorers), there was a significant difference in the probability of flies selectively orientating to guava-juice among the five age-groups [GLM (selective orientation ~ age-groups, family = binomial), Chi2 = 15.121, Df = 4, P = 0.004]. The subsequent post hoc tests demonstrated that attraction to guava-juice odour was significantly higher for 3 week-old versus 15 week-old females (Z = 3.337, P = 0.008), with 6, 9 and 12 week old flies intermediate between these extremes (Fig. 6 A). However, the probability of selective orientation for explorer flies did not differ with age [GLM (selective orientation ~ age-group, family = binomial), Chi2 = 0.198, Df = 4, P = 0.995, mean probability = 0.69 ± 0.07] (Fig. 6B). 3.2.2. Exploratory activity The probability of females exploring either arm of the olfactometer was significantly affected by the age of the flies [GLM: (explorer ~ agegroups, family = binomial), Chi2 = 38.257, Df = 4, P < 0.001], with the subsequent post hoc tests revealing that there was a significant drop in the exploration probability of flies at 9, 12 and 15 weeks of age compared to 3 weeks (Z = 3.383, P = 0.006; Z = 3.526, P = 0.004 and Z = 4.979, P < 0.001 in 3 vs. 9, 12 and 15 week pairwise comparisons respectively). There was also a significant difference between the exploration probability of 6 week and 15 week old flies (Z = 4.066, 5
Journal of Insect Physiology 122 (2020) 104024
M.S. Tasnin, et al.
Fig. 6. . The mean ( ± 1SE) probability of female Bactrocera tryoni selectively orientating to guava-odour at five age-groups for (A) all tested flies (explorer + nonexplorer) and (B) explorer flies only. Different letters above the bars represent significant differences at P < 0.05. There is no significant age effect in Fig. 6B.
Fig. 8. The mean ( ± 1SE) time taken (seconds) by five age-groups of female Bactrocera tryoni to locate a guava-juice source did not show significant difference, Df = 4, P = 0.059.
Fig. 7. The mean ( ± 1SE) probability of five age-groups of female Bactrocera tryoni exploring either arm of a Y-tube olfactometer in the presence of a guavajuice source and a blank control. Different letters on the bars represent significant differences at P < 0.05.
orientation preference to guava-juice odour. Male and female olfactory responses are discussed separately in the following sections
the previous ageing research on locomotor and exploratory activities were conducted without an odour sources, while the current experiments were conducted in the presence of cue-lure and guava juice which have been reported to increase activity and flight of mature males and females in wind tunnel experiments (Dalby-Ball and Meats, 2000; Meats and Hartland, 1999). Additionally, the current research was conducted with wild flies, while the work of Prenter et al. (2013) used laboratory-reared flies, which may also influence results.
4.3.1. The constant attraction of males to cue-lure until an advanced age Males of B. tryoni retained positive olfactory responses to cue-lure for most of their life. A similar result was observed in B. opiliae, where males response to methyl eugenol remained unchanged until 12 weeks of age (Fitt, 1981). The Bactrocera male response to a small group of plant-derived secondary chemicals is considered a critical element of their biology and ecology (Clarke, 2019), as exposure to these chemicals enhances individual male fitness through increased sexual competitiveness (Kumaran et al., 2013; Kumaran et al., 2014; Metcalf and Metcalf, 1992; Weldon et al., 2008). Bactrocera tryoni males mate throughout their lives, even though older males have reduced mating success when in competition with young males (Ekanayake et al., 2017). There is thus an evolutionary driver for male flies to retain a strong lure response for as long in life as possible, and this is reflected in our results (to week 12) and those of Fitt (1981). In this light, we interpret the dramatic decrease of male olfactory response at 15 weeks, caused by simultaneous drops in locomotor activity and selective
4.3. Patterns in reduced olfactory response in males and females The olfactory responses and exploratory activities of both males and females declined at advanced age. The male olfactory response to cuelure was constant until 12 weeks of age, after which their olfactory response dropped dramatically at 15 weeks when there were few explorers, and those that did move orientated indiscriminately to control and treatment arms. However, female exploratory response decreased gradually over time and reached the lowest level at the oldest age group, although the oldest age group still showed a significant 6
Journal of Insect Physiology 122 (2020) 104024
M.S. Tasnin, et al.
the population (authors’ unpublished data), following the over-wintering quiescent period (Clarke et al., 2019; Fletcher, 1979; Muthuthantri et al., 2010; O'loughlin et al., 1984). It is possible that such populations may be undetected, or at least underestimated, if relying solely on cue-lure trapping. Also positively for pest management, the results suggest that new generation female traps based on host fruit odours (Gregg et al., 2018), can be expected to be most effective against female B. tryoni when they are at their reproductive peak, but also less effective against the very old spring populations. CRediT authorship contribution statement Mst Shahrima Tasnin: Conceptualization, Methodology, Data curation, Visualization. Katharina Merkel: Conceptualization, Methodology, Supervision. Anthony R. Clarke: Conceptualization, Methodology, Supervision. Acknowledgements Fig. 9. Age-related olfactory responses of female Bactrocera tryoni to guavajuice odour versus a blank control. Results of the chi-square test demonstrates significant deviation from 1:1 response ratio to guava-odour and blank-odour control in all age groups (P < 0.05 = *; P < 0.01 = **). Seventy flies were tested per age-group, not all responded.
We are thankful to the owner and staff, especially Bob Brinsmead and Mick O’Reilly, of Tropical Fruit World, Tweed Valley, NSW for providing information about infested fruits in the field and allowing us onto their property for collecting infested carambola. Similarly, we thank Brendan Missenden for helping us to collect the infested mangoes from his property. Thanks to QUT colleagues Karina Pyle, Rehan Silva, Francesca Strutt and Kiran Mahat for providing suggestions during experiments. A.R.C. and the QUT fruit fly lab is funded by the Australian Research Council through its Discovery Project (DP180101915) and Industrial Transformation Training Centre (IC150100026) schemes. The Australian Research Council had no role in the design, analysis or interpretation of this research.
olfactory orientation, as likely symptomatic of wider failures of several biological systems associated with advanced age, rather than a loss of “interest” in cue-lure per se. 4.3.2. Higher attraction to host fruit odour in younger females The females’ exploratory activities to guava-juice odour showed a gradual reduction with ageing, but with no corresponding decline for positive selection to the odour in explorer flies. A similar response was found in A. obliqua, where a decline in the olfactory response to volatiles of host fruits was found in 20 day old females compared to 1, 5 or 10 day old females (Reyes et al., 2017). In B. tryoni, at a given point in time, the olfactory response of gravid females to host fruit odour is linked with oviposition (Eisemann and Rice, 1992). We believe our results support this observation in a longitudinal temporal context, and this explains the female decline in response with ageing. In our experiment, we found young females (3–6 weeks) demonstrated the highest response to guava juice odour, with a continuous decline thereafter. This correlates closely with the lifetime fecundity patterns of female B. tryoni, which sees a peak in egg production at four weeks, with a continuous decline thereafter (Fitt, 1990; Kumaran et al., 2013). While the current study identified aging effects on olfaction of B. tryoni, a limitation of this study is that the male and female olfactory responses were each tested against a single odour type, i.e. cue-lure for males and guava juice for females. Thus, the study cannot determine whether the changes reported here are general patterns of age related olfactory change for males and females of this species, or if the patterns observed relate to the specific odours tested. Further studies are therefore required to confirm or deny the generality of our results.
Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jinsphys.2020.104024. References Aluja, M., 1996. Future trends in fruit fly management. In: Steck, M.A.G.J. (Ed.), Fruit Fly Pests: A World Assessment of Their Biology and Management. St. Lucie Press, Delray Beach, FL, pp. 309–320. Balagawi, S., 2006. Comparative ecology of Bactrocera Cucumis (French) and Bactrocera Tryoni (Froggatt) (Diptera: Tephritidae)-Understanding the Life History Consequences of Host Selection and Oviposition Behaviour. Griffith University. Carey, J.R., Papadopoulos, N., Kouloussis, N., Katsoyannos, B., Muller, H.G., Wang, J.L., Tseng, Y.K., 2006. Age-specific and lifetime behavior patterns in Drosophila melanogaster and the Mediterranean fruit fly, Ceratitis capitata. Experimenta Gerontol. 41, 93–97. https://doi.org/10.1016/j.exger.2005.09.014. Carey, J.R., Papadopoulos, N.T., Müller, H.G., Katsoyannos, B.I., Kouloussis, N.A., Wang, J.L., Wachter, K., Yu, W., Liedo, P., 2008. Age structure changes and extraordinary lifespan in wild medfly populations. Aging Cell 7, 426–437. https://doi.org/10.1111/ j.1474-9726.2008.00390.x. Clarke, A.R., 2019. Biology and Management of Bactrocera and Related Fruit Flies. CAB Internatinal, Wallingford, UK. Clarke, A.R., Merkel, K., Hulthen, A.D., Schwarzmueller, F., 2019. Bactrocera tryoni (Froggatt) (Diptera: Tephritidae) overwintering: an overview. Austral Entomol. 58, 3–8. https://doi.org/10.1111/aen.12369. Clarke, A.R., Powell, K.S., Weldon, C.W., Taylor, P.W., 2011. The ecology of Bactrocera tryoni (Diptera: Tephritidae): what do we know to assist pest management? Ann. Appl. Biol. 158, 26–54. https://doi.org/10.1111/j.1744-7348.2010.00448.x. Cook-Wiens, E., Grotewiel, M.S., 2002. Dissociation between functional senescence and oxidative stress resistance in Drosophila. Exp. Gerontol. 37, 1347–1357. https://doi. org/10.1016/S0531-5565(02)00096-7. Crnjar, R., Yin, C.-M., Stoffolano Jr, J., Barbarossa, I.T., Liscia, A., Angioy, A., 1990. Influence of age on the electroantennogram response of the female blowfly (Phormia regina) (Diptera: Calliphoridae). J. Insect Physiol. 36, 917–921. https://doi.org/10. 1016/0022-1910(90)90079-U. Cunningham, J.P., Carlsson, M.A., Villa, T.F., Dekker, T., Clarke, A.R., 2016. Do fruit ripening volatiles enable resource specialism in polyphagous fruit flies? J. Chem. Ecol. 42, 931–940. https://doi.org/10.1007/s10886-016-0752-5. Dalby-Ball, G., Meats, A., 2000. Influence of the odour of fruit, yeast and cue-lure on the flight activity of the Queensland fruit fly, Bactrocera tryoni (Froggatt) (Diptera: Tephritidae). Austr. J. Entomol. 39, 195–200. https://doi.org/10.1046/j.1440-6055.
4.4. Implications for management As the olfactory response of an insect may vary with age, the trapping effectiveness of pest insects for surveillance and management may also vary (Kouloussis et al., 2009). Cue-lure is an effective monitoring tool for male B. tryoni and is used to control field populations of this insect by mass-trapping and male-annihilation (Dominiak et al., 2003; Lloyd et al., 2010; Reynolds et al., 2016; Stringer et al., 2017). That male flies retain their attraction to cue-lure consistently until an advanced age is a positive finding for pest management. However, loss of attraction to cue-lure may be a problem for sampling B. tryoni males at the very end of winter, when there is a majority of very old males in 7
Journal of Insect Physiology 122 (2020) 104024
M.S. Tasnin, et al.
Gracillariidae). J. Chem. Ecol. 44, 276–287. https://doi.org/10.1007/s10886-0180927-3. Liu, L., Zhou, Q., 2016. Olfactory response of female Bactrocera minax to chemical components of the preference host citrus volatile oils. J. Asia-Pac. Entomol. 19, 637–642. https://doi.org/10.1016/j.aspen.2016.05.008. Lloyd, A.C., Hamacek, E.L., Kopittke, R.A., Peek, T., Wyatt, P.M., Neale, C.J., Eelkema, M., Gu, H., 2010. Area-wide management of fruit flies (Diptera: Tephritidae) in the Central Burnett district of Queensland, Australia. Crop Prot. 29, 462–469. https:// doi.org/10.1016/j.cropro.2009.11.003. López-Guillén, G., López, L.C., Malo, E.A., Rojas, J.C., 2011. Olfactory responses of Anastrepha obliqua (Diptera: Tephritidae) to volatiles emitted by calling males. Florida Entomol. 874–881. Martel, V., Anderson, P., Hansson, B.S., Schlyter, F., 2009. Peripheral modulation of olfaction by physiological state in the Egyptian leaf worm Spodoptera littoralis (Lepidoptera: Noctuidae). J. Insect Physiol. 55, 793–797. https://doi.org/10.1016/j. jinsphys.2009.04.012. May, A., 1958. Fruit fly problem in southern and central Queensland. Queensland Agric. J. 84, 153–159. Meats, A., Hartland, C., 1999. Upwind anemotaxis in response to cue-lure by the Queensland fruit fly, Bactrocera tryoni. Physiol. Entomol. 24, 90–97. https://doi.org/ 10.1046/j.1365-3032.1999.00118.x. Metcalf, R.L., Metcalf, E.R., 1992. Plant Kairomones in Insect Ecology and Control. Chapman and Hall Ltd, Springer, US. Muthuthantri, S., Maelzer, D., Zalucki, M.P., Clarke, A.R., 2010. The seasonal phenology of Bactrocera tryoni (Froggatt) (Diptera: Tephritidae) in Queensland. Austr. J. Entomol. 49, 221–233. https://doi.org/10.1111/j.1440-6055.2010.00759.x. Norrbom, A.L., Carroll, L.E., Thompson, F.C., White, I., Freidberg, A., 1999. Systematic Database of Names. Myia. O'loughlin, G., East, R., Meats, A., 1984. Survival, development rates and generation times of the Queensland fruit fly, Dacus Tryoni, in a marginally favourable climate: experiments in Victoria. Aust. J. Zool. 32, 353–361. https://doi.org/10.1071/ ZO9840353. Otter, C.D., Tchicaya, T., Schutte, A., 1991. Effects of age, sex and hunger on the antennal olfactory sensitivity of tsetse flies. Physiol. Entomol. 16, 173–182. https://doi.org/ 10.1111/j.1365-3032.1991.tb00554.x. Prenter, J., Weldon, C.W., Taylor, P.W., 2013. Age-related activity patterns are moderated by diet in Queensland fruit flies Bactrocera tryoni. Physiol. Entomol. 38, 260–267. https://doi.org/10.1111/phen.12023. Raghu, S., 2007. Functional significance of phytochemical lures to dacine fruit flies (Diptera: Tephritidae): an ecological and evolutionary synthesis. Bull. Entomol. Res. 94, 385–399. https://doi.org/10.1079/BER2004313. Reyes, H., Malo, E.A., Toledo, J., Cruz-Esteban, S., Rojas, J.C., 2017. Physiological state influences the antennal response of Anastrepha obliquato male and host volatiles. Physiol. Entomol. 42, 17–25. https://doi.org/10.1111/phen.12157. Reynolds, O.L., Osborne, T., Crisp, P., Barchia, I.M., 2016. Specialized pheromone and lure application technology as an alternative male annihilation technique to manage Bactrocera tryoni (Diptera: Tephritidae). J. Econ. Entomol. 109, 1254–1260. https:// doi.org/10.1093/jee/tow023. Russell, L., 2018. Emmeans: Estimated Marginal Means, aka Least-Squares Means. R package version 1. Shelly, T., 2010. Effects of methyl eugenol and raspberry ketone/cue lure on the sexual behavior of Bactrocera species (Diptera: Tephritidae). Appl. Entomol. Zool. 45, 349–361. https://doi.org/10.1303/aez.2010.349. Sollai, G., Solari, P., Crnjar, R., 2018. Olfactory sensitivity to major, intermediate and trace components of sex pheromone in Ceratitis capitata is related to mating and circadian rhythm. J. Insect Physiol. 110, 23–33. https://doi.org/10.1016/j.jinsphys. 2018.08.007. Stringer, L.D., Kean, J.M., Beggs, J.R., Suckling, D.M., 2017. Management and eradication options for Queensland fruit fly. Popul. Ecol. 59, 259–273. https://doi.org/10.1007/ s10144-017-0593-2. Sutherst, R.W., Collyer, B.S., Yonow, T., 2000. Fruit fly problem in southern and Central Queensland. Aust. J. Agric. Res. 51, 467–480. Team, R.C., 2018. R: a language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing; 2018. https://www.R‐project.org/. Touhara, K., Vosshall, L.B., 2009. Sensing odorants and pheromones with chemosensory receptors. Annu. Rev. Physiol. 71, 307–332. https://www.annualreviews.org/doi/ full/10.1146/annurev.physiol.010908.163209. Wäckers, F., 1994. The effect of food deprivation on the innate visual and olfactory preferences in the parasitoid Cotesia rubecula. J. Insect Physiol. 40, 641–649. https:// doi.org/10.1016/0022-1910(94)90091-4. Wee, S.-L., Peek, T., Clarke, A.R., 2018. The responsiveness of Bactrocera jarvisi (Diptera: Tephritidae) to two naturally occurring phenylbutaonids, zingerone and raspberry ketone. J. Insect Physiol. 109, 41–46. https://doi.org/10.1016/j.jinsphys.2018.06. 004. Weldon, C.W., Perez-Staples, D., Taylor, P.W., 2008. Feeding on yeast hydrolysate enhances attraction to cue-lure in Queensland fruit flies, Bactrocera tryoni. Entomol. Exp. Appl. 129, 200–209. https://doi.org/10.1111/j.1570-7458.2008.00768.x. Wong, T.T., McInnis, D., Ramadan, M.M., Nishimoto, J.I., 1991. Age-related response of male melon flies Dacus cucurbitae (Diptera: Tephritidae) to cue-lure. J. Chem. Ecol. 17, 2481–2487. Wyatt, T.D., 2014. Pheromones and Animal Behavior: Chemical Signals and Signatures. Cambridge University Press.
2000.00168.x. Dominiak, B., Westcott, A., Barchia, I., 2003. Release of sterile Queensland fruit fly, Bactrocera tryoni (Froggatt) (Diptera: Tephritidae), at Sydney, Australia. Aust. J. Exp. Agric. 43, 519–528. https://doi.org/10.1071/EA01146. Eisemann, C., 1980. An Investigation of Some Stimuli Influencing Host Finding and Oviposition Behaviours of the Queensland Fruit Fly, Dacus (Bactrocera) tyroni (Frogg.). University of Queensland. Eisemann, C., Rice, M., 1992. Attractants for the gravid Queensland fruit fly Dacus tryoni. Entomol. Exp. Appl. 62, 125–130. Ekanayake, E., Clarke, A.R., Schutze, M.K., 2017. Effect of body size, age, and premating experience on male mating success in Bactrocera tryoni (Diptera: Tephritidae). J. Econ. Entomol. 110, 2278–2281. https://doi.org/10.1093/jee/tox186. Ero, M.M., Hamacek, E., Clarke, A.R., 2011. Foraging behaviours of Diachasmimorpha kraussii (Fullaway) (Hymenoptera: Braconidae) and its host Bactrocera tryoni (Froggatt) (Diptera: Tephritidae) in a nectarine (Prunus persica (L.) Batsch var. nectarina (Aiton) Maxim) orchard. Austr. J. Entomol. 50, 234–240. https://doi.org/10. 1111/j.1440-6055.2011.00821.x. Fitt, G.P., 1981. The influence of age, nutrition and time of day on the responsiveness of male Dacus opiliae to the synthetic lure, methyl eugenol. Entomol. Exp. Appl. 30, 83–90. https://doi.org/10.1111/j.1570-7458.1981.tb03588.x. Fitt, G.P., 1990. Comparative fecundity, clutch size, ovariole number and egg size of Dacus tryoni and D. jarvisi, and their relationship to body size. Entomol. Exp. Appl. 55, 11–21. https://doi.org/10.1111/j.1570-7458.1990.tb01343.x. Fletcher, B., 1989. Life history strategies of tephritid fruit flies. In: Robison, A.S.H.G. (Ed.), Fruit Flies, Their Biology, Natural Enemies And Control. Elsevier Science, Amsterdam, The Netherlands, pp. 195–208. Fletcher, B.S., 1979. The overwintering survival of adults of the Queensland fruit fly, Dacus Tryoni, under natural conditions. Aust. J. Zool. 27, 403–411. https://doi.org/ 10.1071/ZO9790403. Gadenne, C., Barrozo, R.B., Anton, S., 2016. Plasticity in insect olfaction: to smell or not to smell? Annu. Rev. Entomol. 61, 317–333. https://doi.org/10.1146/annurev-ento010715-023523. Gregg, P.C., Del Socorro, A.P., Landolt, P.J., 2018. Advances in attract-and-kill for agricultural pests: beyond pheromones. Annu. Rev. Entomol. 63, 453–470. https://www. annualreviews.org/doi/abs/10.1146/annurev-ento-031616-035040. Grotewiel, M.S., Martin, I., Bhandari, P., Cook-Wiens, E., 2005. Functional senescence in Drosophila melanogaster. Ageing Res. Rev. 4, 372–397. https://doi.org/10.1016/j.arr. 2005.04.001. Hancock, D., Hamacek, E., Lloyd, A., Elson-Harris, M., 2000. The Distribution and Host Plants Of Fruit Flies (Diptera: Tephritidae) in Australia. Queensland Department of Primary Industries. Iliadi, K.G., Boulianne, G.L., 2010. Age-related behavioral changes in Drosophila. Ann. N. Y. Acad. Sci. 1197, 9–18. https://doi.org/10.1111/j.1749-6632.2009.05372.x. Jang, E.B., McInnis, D.O., Kurashima, R., Carvalho, L.A., 1999. Behavioural switch of female Mediterranean fruit fly, Ceratitis capitata: mating and oviposition activity in outdoor field cages in Hawaii. Agric. For. Entomol. 1, 179–184. https://doi.org/10. 1046/j.1461-9563.1999.00025.x. Jin, S., Zhou, X., Gu, F., Zhong, G., Yi, X., 2017. Olfactory plasticity: variation in the expression of chemosensory receptors in Bactrocera dorsalis in different physiological states. Front. Physiol. 8, 672. https://doi.org/10.3389/fphys.2017.00672. Kamiji, T., Kaneda, M., Sasaki, M., Ohto, K., 2018. Sexual maturation of male Bactrocera correcta (Diptera: Tephritidae) and age-related responses to β-caryophyllene and methyl eugenol. Appl. Entomol. Zool. 53, 41–46. Klowden, M., 1990. The endogenous regulation of mosquito reproductive behavior. Experientia 46, 660–670. https://doi.org/10.1007/bf01939928. Kouloussis, N.A., Papadopoulos, N.T., Muller, H.G., Wang, J.L., Mao, M., Katsoyannos, B.I., Duyck, P.F., Carey, J.R., 2009. Life table assay of field-caught Mediterranean fruit flies, Ceratitis capitata, reveals age bias. Entomol. Exp. Appl. 132, 172–181. https://doi.org/10.1111/j.1570-7458.2009.00879.x. Koyama, J., Kakinohana, H., Miyatake, T., 2004. Eradication of the melon fly, Bactrocera cucurbitae, in Japan: importance of behavior, ecology, genetics, and evolution. Annu. Rev. Entomol. 49, 331–349. https://www.annualreviews.org/doi/abs/10.1146/ annurev.ento.49.061802.123224. Krieger, J., Breer, H., 1999. Olfactory reception in invertebrates. Science 286, 720–723. https://doi.org/10.1126/science.286.5440.720. Kumaran, N., Balagawi, S., Schutze, M.K., Clarke, A.R., 2013. Evolution of lure response in tephritid fruit flies: phytochemicals as drivers of sexual selection. Anim. Behav. 85, 781–789. https://doi.org/10.1016/j.anbehav.2013.01.024. Kumaran, N., Hayes, R.A., Clarke, A.R., 2014. Cuelure but not zingerone make the sex pheromone of male Bactrocera tryoni (Tephritidae: Diptera) more attractive to females. J. Insect Physiol. 68, 36–43. https://doi.org/10.1016/j.jinsphys.2014.06.015. Landolt, P., Heath, R., King, J., 1985. Behavioral responses of female papaya fruit flies, Toxotrypana curvicauda (Diptera: Tephritidae), to male-produced sex pheromone. Ann. Entomol. Soc. Am. 78, 751–755. https://doi.org/10.1093/aesa/78.6.751. Lebreton, S., Borrero-Echeverry, F., Gonzalez, F., Solum, M., Wallin, E.A., Hedenstrom, E., Hansson, B.S., Gustavsson, A.L., Bengtsson, M., Birgersson, G., Walker 3rd, W.B., Dweck, H.K.M., Becher, P.G., Witzgall, P., 2017. A Drosophila female pheromone elicits species-specific long-range attraction via an olfactory channel with dual specificity for sex and food. BMC Biol. 15, 88. https://doi.org/10.1186/s12915-0170427-x. Lemmen-Lechelt, J.K., Wist, T.J., Evenden, M.L., 2018. State-dependent plasticity in response to host-plant volatiles in a long-lived moth, Caloptilia fraxinella (Lepidoptera:
8