The responsiveness of Bactrocera jarvisi (Diptera: Tephritidae) to two naturally occurring phenylbutaonids, zingerone and raspberry ketone

Journal of Insect Physiology 109 (2018) 41–46

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The responsiveness of Bactrocera jarvisi (Diptera: Tephritidae) to two naturally occurring phenylbutaonids, zingerone and raspberry ketone Suk-Ling Weea, a b

⁎,1

T

, Thelma Peekb, Anthony R. Clarkea

School of Earth, Environmental and Biological Sciences, Queensland University of Technology (QUT), GPO Box 2434, Brisbane, Queensland 4001, Australia Department of Agriculture and Fisheries, Ecosciences Precinct, Dutton Park, Queensland, Australia

A R T I C LE I N FO

A B S T R A C T

Keywords: Jarvis’s fruit fly Phytochemical Phenylbutanoid Sexual maturation Lure response

The males of different species of Bactrocera and Zeugodacus fruit flies are commonly attracted to plant-derived phenylpropanoids (e.g. methyl eugenol (ME)) or phenylbutanoids (e.g. raspberry ketone (RK)) but almost never to both. However, one particular plant-derived phenylbutanoid, zingerone (ZN), which possesses an intermediate chemical structure between ME and RK, weakly attracts both ME- and RK-responding fruit fly species. Bactrocera jarvisi, an Australian fruit fly species, is weakly attracted to cue lure (an analogue of RK) but strongly attracted to ZN. Here, we investigated the minimum olfactory threshold and optimum sensitivity of B. jarvisi males to ZN and RK as a function of dose, time and sexual maturation. Our results show that B. jarvisi males had a marked preferential response to ZN, with a much lower olfactory threshold and faster response time to ZN than RK. Probit analysis demonstrated that ZN was at least > 1600× more potent than RK as a male attractant to B. jarvisi. Although fruit fly male attraction to the phytochemicals is generally associated with sexual maturity, in B. jarvisi immature males were also attracted to ZN. Our results suggest that B. jarvisi males have a fine-tuned olfactory response to ZN, which appears to play a central role in the chemical ecology of this species.

1. Introduction The males of Dacini fruit flies (Diptera: Tephritidae, members of the genera Bactrocera Macquart and Zeugodacus Hendel predominantly) exhibit strong, positive chemotaxis to a small group of plant-derived secondary metabolites (referred to in the wider fruit fly literature, and hereafter, as ‘male lures’ or just ‘lures’, because of their history in applied entomology) (Raghu, 2004; Tan and Nishida, 2012). The response of flies to these phytochemicals is so strong that when mixed with a toxicant in a lure-and-kill pest management approach, and used in combination other control tactics such as protein bait spray, they can drive local populations to extinction (Cunningham and Steiner, 1972; Cantrell et al., 2002). As late as the 1980s why the flies responded to the lures was still unknown (Cunningham, 1989), but it is now widely recognised that the lures are associated with the mating systems of the flies (see reviews by Shelly, 2010; Tan and Nishida, 2012). Lure-fed males gain a competitive mating advantage over unfed males through incorporation of phytochemical-derived compounds into their pheromones which are subsequently more attractive to females (Shelly and Dewire, 1994; Tan

and Nishida, 1998; Wee et al., 2007, 2018a). Males may also show increased activity after lure feeding (Kumaran et al., 2014a); while mating with a lure-fed male (in some cases) may also have direct benefits (higher fecundity) (Kumaran et al., 2013) and cause complex indirect effects to females (Kumaran et al., in press). However, as research continues, it is also becoming clear that the lure effect varies between fruit fly species (see e.g. Kumaran et al., 2014a; Shelly, 2017) and within species based on lure (Kumaran et al., 2014b). Only a few years ago the association between lures and fly response was considered straightforward. Males of a species were thought to only ever respond to one of two lure types, those lures being methyl eugenol (ME) and cue lure (CL) (or raspberry ketone [RK], the hydrolysed form of CL) (Fig. 1), or the species was ‘non-lure responsive’ (Drew, 1974; Drew and Romig, 2013). Species may have been ‘weakly’ or ‘strongly’ attracted to a given lure (Drew, 1989), but nevertheless it was still one or the other. However, increasingly, it is clear that this simple dichotomy is inadequate to capture the complexity of the Dacini lure response, with new attractive chemicals being found (Royer, 2015; Siderhurst et al., 2016; Wee et al., 2018b), and previously ‘non-lure responsive flies’ being attracted to novel chemicals (Royer et al., 2018).

⁎ Corresponding author at: School of Environmental and Natural Resource Sciences, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia. E-mail addresses: [email protected] (S.-L. Wee), [email protected] (T. Peek), [email protected] (A.R. Clarke). 1 Present address: School of Environmental and Natural Resource Sciences, Centre for Insect Systematics, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia.

https://doi.org/10.1016/j.jinsphys.2018.06.004 Received 30 April 2018; Received in revised form 29 May 2018; Accepted 8 June 2018 Available online 08 June 2018 0022-1910/ © 2018 Elsevier Ltd. All rights reserved.

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lighting between 07:00 and 16:00 hr daily in addition to natural sunlight. Except for flies used for the sexual maturity experiment, which were sex-segregated within a few hours of emergence, all flies were sexed within two days of emergence and maintained separately in screen cages (40 × 40 cm) at 27 °C and 70% RH. Sexually mature virgin flies (14–21 day-old) were used for all studies unless otherwise stated. 2.2. Chemicals Based on the results of a preliminary test, zingerone (ZN; 4-(4-hydroxy-3-methoxyphenyl)-2-butanone; CAS 122-48-5) and raspberry ketone (RK; 4-(4-hydroxyphenyl)butan-2-one; CAS 5471-51-2) (both > 96% purity, Sigma-Aldrich) were diluted serially in absolute ethanol (≥99.8% purity; Sigma-Aldrich) into the desired concentrations (ZN: 10, 50, 100, 400, 800 ng/10 µl; RK: 25, 50, 100, 300, 500, 1000 µg/10 µl) for Probit bioassays. For age-dependent lure response bioassays, 500 µg/10 µl of RK or ZN was used. Fig. 1. Chemical structures of methyl eugenol (ME; 3,4-dimethoxy-allylbenzene), cue lure (CL; 4-(p-acetoxyphenyl)-2-butanone), raspberry ketone (RK; 4(4-hydroxyphenyl)butan-2-one) and zingerone (ZN; 4-(4-hydroxy-3-methoxyphenyl)-2-butanone).

2.3. Sexual maturation One-hundred each of first-day emerged B. jarvisi males and females, after full-body colouration had developed, were placed in a medium size screen cage (40 × 40 × 40 cm) with food and water ad libitum. Daily observations for mating pairs were conducted after scotophase (between 20:30–21:30 hr) under dimmed red light conditions until the experimental flies were 50 days old. As soon as a pair settled in mating, the pair in copula was carefully coaxed into a specimen vial and removed from the cage. The experiment was replicated four times. At the end of the 50-day observation period, a graph of cumulative mating percent was plotted as a function of age after adult emergence. An explicit assumption was made that mating is directly correlated with sexual maturation attainment and that the sexual maturation development rate was the same in both sexes, as has been done elsewhere (Ooi and Wee, 2016; Wee et al., 2018a,b). This approach may slightly overestimate the time required for flies to reach sexual maturity (i.e. if flies are sexually mature for one or more days before mating), but it cannot underestimate the time taken to reach sexual maturity as sexually immature Bactrocera do not mate.

This new line of research can be largely attributed to the discovery that certain orchid species, e.g. Bulbophyllum patens and Bu. baileyi, attract both ME and CL responsive species (Tan and Nishida, 2000) and that the attractive chemical is the phenylbutanoid zingerone (ZN) (the essence of ginger) (Fig. 1) (Tan and Nishida, 2007). ZN, as a phenylbutanoid, possesses an intermediate chemical structure between ME (a phenylpropanoid) and RK (a phenylbutanoid) explaining, from a chemical structure perspective, its attractiveness to both ME- and RK/ CL responsive groups of fruit flies (Tan and Nishida, 2000). Bactrocera jarvisi (Tryon) (Diptera: Tephritidae) (a.k.a. Jarvis’s fruit fly) is an endemic Australian fruit fly distributed across northern Australian and down the Australian east coast to the southern end of the subtropics (Drew, 1989). While recorded in the older literature as either non-lure responsive (Drew et al., 1978) or weakly responsive to CL (Drew, 1989), Fay (2012) reported the species was strongly attracted to ZN, a result confirmed by Royer (2015). As a species which sits between ME and CL/RK responsive species, understanding the biology and physiology of the species’ lure response is critical to helping develop a more informed knowledge of the larger lure-response pattern across the Dacini. Bactrocera jarvisi is only one of numerous Dacini fruit flies across Asia and the Pacific that are now known to respond to ZN (Tan and Nishida, 2000, 2007; Fay, 2012; Royer, 2015; Royer et al., 2018), so detailed research on a predominantly ZN responsive species fills a key gap in fruit fly male lure research. In this paper, we investigate the lure selectivity and sensitivity of B. jarvisi males towards the two naturally-occurring phenylbutanoids, RK and ZN, in association with male sexual maturation. Probit analysis (Finney, 1971) is used to determine the minimal response threshold and optimum sensitivity of the fly to each of the lures. In addition, we investigate the temporal aspect of phytochemical lure perception, i.e. total time taken to elicit a positive response to lure, to gain more insight into B. jarvisi’s fruit fly-lure interactions. This is the first paper of an intended series, which aims to build a comprehensive understanding the chemical ecology of this ZN responsive fruit fly.

2.4. Olfactory threshold determination via Probit analysis The response of male B. jarvisi to either ZN or RK was evaluated based on a precise evaluation method via the complete sequential total male lure reflex, which involves a sequential behavioural attraction, arrestment and feeding (hereafter referred to as complete sequential reflex) (Metcalf et al., 1979). A positive response was scored when an individual male, activated by the presence of the test chemical, approached by an oriented zig-zag flight, landed and fed on the lure source within a 10-minute bioassay duration. This zig-zag flight was readily observed in the observation arenas. Lure response bioassays were conducted in the morning between 09:00–11:30 hr in a laboratory which was illuminated with fluorescent lighting in addition to natural sunlight received through glass doors and windows. Room temperature and relative humidity were maintained between 22 and 25 °C and 65–70%, respectively. During each trial a group of 20 male flies, housed in a screen cage (20 × 20 × 30 cm), was assayed for their response to increasing quantities of either ZN or RK which was tested on different days. For each bioassay, 10 µl of lure solution was dispensed onto a 3.5 cm diam. filter paper that was placed in a 3.5 cm diam. disposable Petri dish using a pre-calibrated glass pipette (Drummond®, USA). A male fruit fly’s sensitivity to a chemical attractant can be measured both as a function of dose (dose-response via Probit analysis) as well as a function of time, i.e. time taken to complete the sequential response reflex (hereafter known as response time). We further differentiated the response time into two categories, i.e. the minimum time

2. Materials and methods 2.1. Insects Bactrocera jarvisi of 6–8th generation (reared from field-infested mangoes) were obtained as pupae from the [Queensland] Department of Agriculture and Fisheries, Brisbane, Queensland. Emerged flies were provided with protein hydrolysate and sugar mixture (ratio 3:1) and water ad libitum. The rearing room was illuminated with fluorescent 42

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for a positive response (i.e. a complete sequential reflex demonstrated by the first male fly) and the average response time of all positive responses. The bioassay began with the lowest concentration, and any fly respondents were then immediately removed from the cage with an aspirator without affecting the behaviour of the remaining flies. The bioassays were always run with fresh flies. For controls, 10 μl absolute ethanol was dispensed instead of a lure solution. Five to six replicates, with each replicate consisting of 20 males from multiple cohorts, were performed for each lure-dose tested. 2.5. Age-dependent response to lures For age-dependent lure response bioassays, similar procedures and conditions as for the dose-response bioassays were followed except that males of different ages were assayed separately to either ZN or RK. Bactrocera jarvisi males of 5, 7, 9, 11, 14, 21 and 28 days old were exposed to 500 µg of ZN or RK to determine age-dependent response. Five replications, each replicate consisting of 20 males, were tested without flies being re-used. In addition, the response time was also recorded for each lure and age tested.

Fig. 2. Mean ( ± 1 S.E.) cumulative mating percentages as a function of age of Bactrocera jarvisi over a 50-day observation period. Observations were made from a total of four replications with 100 pairs each in screen cage (40 × 40 × 40 cm).

2.6. Data analyses Olfactory response data obtained were pooled and analyzed using PoloPlus 2.0 (LeOra Software, 2002) based on Finney (1971). For each lure, the best fitted line of Probit percent of B. jarvisi male response was plotted against logarithm of dosage. ED50 (effective median dose), the minimum dose required to elicit 50% response in the population tested, was obtained for RK and ZN. A total of 100–120 males per dose was tested in order to obtain a reliable estimate of ED50. The data were also subjected to a parallelism test. For age-dependent response to ZN or RK, proportionate data (number of responding flies over the total number tested, n = 20) was transformed to a modified arcsine square root (Anscombe, 1948) and subjected to Normality Test (Shapiro-Wilk) before further statistical analyses. Two-way analysis of variance (ANOVA) was used to determine the effects of male age, lure type and their interaction on male response. Tukey’s Test was used for pairwise multiple means comparison. Due to highly unequal replications resulted from the differences in lure-responding flies, one-way ANOVA was used to analyse response time of flies according lure type. When parametric assumptions were not met, Kruskal-Wallis on ranks was used, and means were separated by Dunn’s Method. For comparison at a particular age, Student’s t-test or Mann-Whitney U test, depending on whether the data were normal and had equal variances, was used to compare males’ responses between ZN and RK. Statistical analysis was performed using Sigma Plot 12.0, and α was set at P = 0.05 for all comparisons. 3. Results 3.1. Sexual maturation The first mating of B. jarvisi occurred at day seven (0.5% or 2 out of the 400 pairs tested), and the cumulative mating percentage gradually increased with age such that 50% of the test population had mated by days 16–17 (Fig. 2). By the end of the 50-day experimental period, 84.5% of the test population had mated. 3.2. Olfactory threshold determination

Fig. 3. Dose-sensitivity response curves of Bactrocera jarvisi to zingerone (ZN) and raspberry ketone (RK) at different doses.

The manner of B. jarvisi males’ response to both lures was identical, i.e. upon olfactory detection males would undertake directional flight towards the lures; upon landing, feeding on the lure source ensued with males extending their proboscis and imbibing the lures, leaving visible feeding marks on the filter paper. In the Probit bioassays, a dose as low as 10 ng ZN attracted

approximately 13% of B. jarvisi males (Fig. 3), with the ED50 response to ZN being 178.97 ng (Table 1). For RK, 25 µg was needed to elicit a similar percentage response (14.2%) in B. jarvisi males (Fig. 3) and the ED50 response was 292.70 µg (Table 1). The heterogeneity factors for both ED50 values were < 1 (ZN: 0.163; RK: 0.489), indicating the data 43

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Table 1 Probit analysis of male response to lures, zingerone (ZN) and raspberry ketone (RK), for Bactrocera jarvisi. Lure

na

ZN

100

RK

100

Regression equation

y = 0.899x – 2.026 y = 0.877x – 4.791

χ2

df

ED50b (µg)

95% fiducial limits (µg) Upper

Lower

Relative potency

0.490

3

0.179

0.133

0.249

1

1.956

4

292.70

222.9

401.00

1635

Total flies tested per dose. Effective median dose is the dose required to elicit a positive response in 50% of the insect population tested. a

b

fits well with the model of standardized residuals when plotted against the predicted/expected values. The parallelism test revealed that the slopes of regression lines for ZN and RK were not significantly different (χ2 = 0.03, df = 1; P > 0.05), which allowed valid relative potency comparison between the two lures. Based on the ED50 values obtained, ZN was approximately 1,635 times more potent than RK as a short distance male attractant to B. jarvisi males. When sexually mature B. jarvisi males were exposed to ZN of different dosages, there was a significant difference in the minimum response time (H = 14.738, df = 4; P = 0.005) (Fig. 4). The minimum time taken for a positive response to ZN was significantly shorter for 0.4 µg (17.4 ± 4.8 sec) than other doses tested while there was no significant difference in the average response time. For RK, however, there was no trend in either minimum or average response time across the doses tested (H = 2.815, df = 5; P = 0.728).

Fig. 5. Age-related attraction of male Bactrocera jarvisi to zingerone (ZN; n = 5 replicates of 20 males per replicate for each age group) and raspberry ketone (RK; n = 4), assayed in screen cages (20 × 20 × 40 cm). Letter designations shown indicate statistically significant differences across days within a lure type (lower case for ZN, upper case for RK) at P = 0.05 (Tukey’s test). Comparison of mean attraction between lures at a particular age was done by pairwise multiple comparison procedures (Tukey’s test); P < 0.01 (**), P < 0.005 (***) and P < 0.001 (****).

3.3. Age-dependent response to lures Two-way ANOVA analysis showed that there were significant lure and age effects on B. jarvisi male response, but no interaction effect (lure: F = 133.599, df = 1, 56, P < 0.001; age: F = 66.175, df = 7,56, P < 0.001). At three days old, approximately 10% of B. jarvisi males showed positive responses to ZN (Fig. 5). Male response to ZN increased significantly to 36% at five days old and by 7 days old had surpassed 50% population response. The maximum response of B. jarvisi males to ZN was at nine days old (76%) and older males did not cause a significant increase in ZN response (Fig. 5). The first response of B. jarvisi males to RK was at day five, when 8.8% of males positively responded (Fig. 5). The profile of age-related lure attraction was different from that of ZN. An initial increase in response occurred with age but not did not peak (at 60–70%) until day 11. Thereafter, the response level decreased slightly from 14 to 28 days old, although this decline was not statistically significant. Males of B. jarvisi showed significantly greater attraction to ZN than RK at all ages tested (0.01 < P < 0.001; Tukey’s test) (Fig. 5). When the minimum response time to lures was compared across all ages, there was a significant difference in the temporal response of B. jarvisi males (ZN: F = 18.084, df = 7, 32, P < 0.001; RK: F = 3.975, df = 6, 22, P = 0.008) (Fig. 6A). When exposed to ZN, the minimum response time of a 3 days old B. jarvisi male was significantly longer (average 260.8 ± 47.17 sec; range: 180–418 sec) than older males. The minimum response time of older males to ZN, including both sexually immature (≤7 days old; range: 48.8–63.6 sec) and sexually mature (≥9 days old; range: 13.6–36.2 sec) was significantly shorter (P < 0.05; Holm-Sidak method) (Fig. 6A). When exposed to RK, the minimum response time of a five days old B. jarvisi male was significantly longer than older males (P < 0.05; Holm-Sidak method). When compared between lures, the minimum response time differed significantly at five days old (t = 8.383, df = 6; P < 0.001), nine, 11 and 28 days old (Student’s t-test; P < 0.05) (Fig. 6A). For the average response time, a significant difference was evident when males were exposed to ZN (H = 42.359, df = 7; P < 0.001), but not to RK (Fig. 6B). Eleven day old males responded more quickly to ZN compared to all other ages (P < 0.05; Dunn's Method). Over all ages,

Fig. 4. Minimum (when the first fly respondent demonstrated a complete sequential reflex) and average response time (means of summation responses from all respondent flies) of Bactrocera jarvisi males when assayed to different dosage of zingerone (ZN) and raspberry ketone (RK) in screen cages. Letter designations indicate statistically significant differences at P = 0.05 (Tukey’s test). 44

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response by the species to utilise ZN’s known biological activity as a metabolism enhancer in fruit flies (Kumaran et al., 2014a), as it is for a variety of other organisms (Venkatramalingam et al., 2007; Park, 2010; Chang et al., 2012). Forced early feeding of RK to sexually immature adult B. tryoni (Froggatt) (this species does not naturally respond to lures as immature flies) increased their sexual maturation rate over unfed flies (Akter et al., 2017). Given the variety of behavioural and physiological interactions being discovered between dacine fruit flies and the male lures, natural selection acting on a species to feed on a lure to increase their maturation is perhaps not unexpected. Strong attraction to lures before commencement of mating activity should facilitate fruit fly population suppression or eradication using the male annihilation technique (MAT). The success of such programs relies on the attraction of the males of fruit fly pest species to the poisoned lure (Steiner et al., 1965, 1970; Vargas et al., 2014). Early male response would deprive receptive females of suitable males, as males would have been attracted-and-killed at a younger age. In theory, this should make MAT against B. jarvisi highly effective. Lure efficacy is affected by the release rate of a compound, which is associated with its vapour pressure (Park et al., 2016). Based on calculated values, the vapour pressure of ZN (0.011 Pa at 25.00 °C; Hanssen, 2015) is six times higher than RK (0.0018 Pa at 25.00 °C; Park et al., 2016) but ca. 327 times lower than ME (3.6 Pa at 25.00 °C; Park S.J., pers. comm.). However, ZN had been shown earlier a weaker lure than ME and CL/RK in attracting other Bactrocera species (Tan and Nishida, 2007). In the present study, ZN is significantly more attractive to B. jarvisi males than RK. This result provides further behavioural evidence to corroborate previous findings that chemical molecular conformation is a more important factor of consideration than vapour pressure in interpreting lure attractiveness, at least in the case of tephritid fruit flies (Metcalf et al., 1979, 1983; Park et al., 2016). For phenylbutanoids, it has been suggested that the 2-butanone side chain is a primary constituent for fruit fly attractiveness (Metcalf and Metcalf, 1992). Both ZN and RK possess the 2-butanone side chain, but ZN has an additional methoxy-group side chain. This additional structural moiety may be appropriately aligned and triggered to create a conformational change in the receptor protein of specific olfactory sensillae in B. jarvisi, thus eliciting the typical lure response reflex. Although nothing about the olfactory circuit of this fruit fly species is yet known, based on the work done on B. dorsalis and methyl eugenol (Zheng et al., 2012; Liu et al., 2017), our results suggest that B. jarvisi males have evolved olfactory receptor(s) finely tuned to ZN.

Fig. 6. Mean ( ± 1 SEM) (A) minimum and (B) average response times for Bactrocera jarvisi males of different ages exposed to zingerone (ZN) and raspberry ketone (RK) in screen cages. Letter designations shown indicate statistically significant differences at P = 0.05 (Tukey’s test) across days for the same lure type (lower case for ZN, upper case for RK). Asterisks (*) indicate the level of statistical significance between ZN and RK treatment by Mann-Whitney UTest at a particular time step (* = P < 0.05, **** = P < 0.001).

B. jarvisi males showed a significantly shorter mean response time to ZN than RK, except for seven day old flies (Fig. 6B). 4. Discussion Evaluation of RK and ZN for attractiveness showed that B. jarvisi males had a marked and consistent preferential response for ZN over RK. Response to ZN began at a younger age (and remained higher at all subsequent ages), required a much lower olfactory threshold, and elicited a faster response time. The olfactory threshold of B. jarvisi to ZN is at the nanogram level, with an ED50 of 179 ng. This is the first report of a dacine fruit fly species that demonstrates a level of sensitivity to a male lure that is comparable to that of male B. dorsalis to ME (ED50: 222–268 ng) (Wee et al., 2002; Hee et al., 2015). Attraction of B. jarvisi to both lures is generally associated with age and sexual development, which corresponds with previous studies on other Bactrocera and Zeugodacus species (Wong et al., 1989, 1991; Wee and Tan, 2000; Ooi and Wee, 2016; Wee et al., 2018a,b). However, the attraction of B. jarvisi males to ZN began even before the commencement of any mating activity, i.e. as early as three to five days of age. By day seven, when < 10% of the test population had mated, the male attraction to ZN was over 50%. The strong ZN response by B. jarvisi males so soon after emergence is highly unusual in comparison to other fruit fly species. To the best of our knowledge, this is the first report of a fruit fly species belonging to the CL/RK-sensitive group that shows this early and strong response to a phytochemical lure (Wong et al., 1991, Ooi and Wee, 2016), although it is known in some ME responsive flies (e.g. B. umbrosa, Wee et al. 2018a). This strong attraction at the juvenile stage might be an evolved

5. Funding source Supported by the Endeavour Research Fellowship 2016 (55962016) awarded to S.L.W. from the Australian Government. 6. Declaration of interest We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. We confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. We further confirm that the order of authors listed in the manuscript has been approved by all of us. We confirm that we have given due consideration to the protection of intellectual property associated with this work and that there are no impediments to publication, including the timing of publication, with respect to intellectual property. In so doing we confirm that we have followed the regulations of our institutions concerning intellectual property. We understand that the Corresponding Author is the sole contact for the Editorial process (including Editorial Manager and direct communications with the office). He/she is responsible for communicating 45

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