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Biological Control xxx (2017) xxx-xxx

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Biological Control

UN CO PR RR EP R EC IN TE T D PR OO F

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Response of Bactrocera oleae to different photoperiods and temperatures using a novel method for continuous laboratory rearing Valentina Baratellaa⁠ ,⁠ ⁎⁠ , Claudio Puccib⁠ , Bruno Paparattib⁠ , Stefano Speranzab⁠ a b

Council for Agricultural Research and Economics CREA – Research Centre for Agriculture and Environment, via della Navicella 2/4, 00184 Rome, Italy Department of Agriculture and Forestry Science DAFNE, Tuscia University, via San Camillo de Lellis, 01100 Viterbo, Italy

ARTICLE INFO

ABSTRACT

Keywords: Olive fruit fly Rearing Photoperiod Ecophysiology Artificial diet

Background: The development of a continuous rearing protocol for the olive fly Bactrocera oleae (Rossi) (Diptera: Tephritidae) remains a fundamental goal of biocontrol programs. We report a new method for continuous small-scale laboratory rearing on olive fruits. The effect of different photoperiod (long and short-day), in combination with two levels of temperature (16 °C and 27 °C), was verified. Results: Laboratory colonies were successfully maintained on olive fruits for more than 24 months, until project termination. Data showed a short-day photo-phase response: all tested parameters (pupation, emergence, life span, sex ratio) substantially increased on the short-day, except for the emergency rate. Also, changes in the population size were observed in response to photoperiodic condition. There was a significant interaction effect between temperature and photoperiod for pupal stage duration, adult emergence and population size. Conclusion: In earlier and recent literature, most of the rearing procedures adopted long-day photophase and high temperature, but scarce investigation was performed on the effects of the photoperiod on the olive fly. In the present work, we developed a novel method for continuous laboratory rearing of the olive fly on its natural host, which allowed to demonstrate a clear effect of the photoperiod on the effectiveness of the rearing procedures. Laboratory colonies with access to olive fruits showed a short-day photoperiodic response: the optimal combination of climatic parameters was photoperiod 8:16 LD and temperature 16 °C.

1. Introduction

The olive fruit fly (OLF) Bactrocera oleae (Rossi) (Diptera: Tephritidae) is the primary economic pest in all olive growing regions of Europe, North Africa, the Middle East and, recently, California and north-western Mexico (Rice et al., 2003; Zygouridis et al., 2009; Daane et al., 2011; Papadopoulos, 2014). The development of long-term management practices for the fly, focused on classical biological control and IPM strategies, has been investigated for over 80 years in the Mediterranean basin and has become the prime target of current control programs in California and Israel (Sime et al., 2007; Argov et al., 2012; Yokoyama et al., 2011, 2012; Speranza et al., 2003; Pucci et al., 2013). Despite the amount of research done in this sense, the release of parasitoids of the fly has not been particularly successful (Raspi and Loni, 1994; Miranda et al., 2008; Wang et al., 2013). European biological control programs for the OLF have relied almost exclusively on Psyttalia concolor (Szépigeti) (Hymenoptera: Braconidae), also thanks to the

⁎

Corresponding author. Email address: [email protected] (V. Baratella)

http://dx.doi.org/10.1016/j.biocontrol.2017.04.010 Received 9 August 2016; Received in revised form 18 April 2017; Accepted 22 April 2017 Available online xxx 1049-9644/ © 2016 Published by Elsevier Ltd.

ease of mass-rearing on the Mediterranean fruit fly (Medfly) Ceratitis capitata Wied. (Diptera: Tephritidae) on artificial diet. The use of the Medfly as a factitious host in artificial media has contributed to the poor performance of parasitoids both in the field and in laboratory rearing (Kimani-Njogu et al., 2001; Sime et al., 2007; Wang et al., 2011, 2013). In addition, the use of the Medfly can be difficult in places where this pest is not established, because of quarantine implications. This is the case of California, where OLF-specialized parasitoids, rather than Medfly-reared generalists, are at various stages of laboratory assessment to determine their suitability as biological control agents. Another issue of concern is the use of artificial diets for parasitoids rearing, which showed to affect their genetic make-up and behaviour, e.g. host finding and recognition. All the above underscores the need for improving rearing procedures on OLF. Since the developing of a rearing protocol for the olive fruit fly is a fundamental goal which still needs to be addressed, the success of control programs will depend on the ability to establish effective rearing methods in the laboratory, which can then be implemented for large-scale applications, i.e.

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larval intraspecific scramble competition, rather than contest competition, and field observations of multiple infestations support this assertion (Burrack et al., 2009). Several overlapping generations may develop per year, with four to five or even more generations occurring in highly favorable areas (Rice et al., 2003). OLF females may enter in a state of reproductive diapause (“white period”), induced when fruits are in short supply and during periods of high summer temperatures (Gutierrez et al., 2009 and references therein). Photoperiod and temperature commonly influence both reproductive diapause and termination of diapause in insects restricted to specific hosts, but for the OLF, the role of photoperiod still has to be fully clarified (Papaj, 2000; Raspi et al., 2005). The majority of insects, being summer active, show a long-day photoperiodic response, while the short-day response characterizes a small number of insect species that are spring-autumn or winter-active, passing the summer in diapause (Danilevskii, 1965; Saunders, 1982). In many different geographic areas, OLF females manifest ovarian immaturity and males do not respond to sex pheromone traps during late spring-early summer, suggesting a short-day photoperiodic response (Economopoulos et al., 1977; Neuenschwander and Michelakis, 1979; Fletcher and Kapatos, 1983; Ballatori et al., 1983). Nonetheless, Tzanakakis and Koveos (1986) reported that high percentages of OLF females did not mature their oocytes when preimaginal stages were reared in a short-day photoperiod, 18–20 °C of temperature. More recently, the research done by Raspi et al. (2005), in an attempt to verify the effect of different photoperiods on OLF egg maturation, reported a lower percentage of females with eggs, but at the same time a higher number of eggs per female, during the short-day photoperiod (LD 9:15) compared to the long-day photo-phase (LD 16:8). Overall, the few reports on the effects of photoperiodic conditions on the fly are contradictory, and the identification of the optimal constant photoperiod is a focus point of great importance that still remains to be investigated. In the present work, we propose a new technique for a low-input, continuous rearing of the olive fly on its natural host, under laboratory conditions. The OLF responses to different constant photoperiods (long-day and short-day), in combination with two levels of temperature (16 °C and 27 °C), were verified in relation to ecophysiological parameters of concern to facilitate OLF insectary production. The principal aim of this study is to provide a substantial contribution to the development of rearing facilities for the olive fruit fly and, as a result, for all those parasitoids, natural enemies of the fly, presently being investigated in Europe and California as means of biocontrol.


mass rearing (Genç and Nation, 2008; Genc, 2008; Estes et al., 2011; Wang et al., 2013). In this regard, several attempts have been made to rear OLF ex-situ, with the aim of studying different bio-ecological and behavioral aspects. Most of the studies referred to only one generation (Economopoulos et al., 1977; Remund et al., 1977; Pucci and Forcina, 1982; Tzanakakis and Koveos, 1986; Koveos and Tzanakakis, 1990; Raspi et al., 1997; Genç and Nation, 2008). Other investigations on multiple generations have relied on artificial diets (Tsitsipis, 1977a, b; De Magalhaes Silva, 1970; Genc, 2008). Nonetheless, when OLF is reared on artificial diets or factitious hosts, adverse effects occur on its fitness and performance compared to wild individuals (Tsakas and Zouros, 1980; Loukas et al., 1985; Kostantopoulou et al., 1996). The flies were found to differ in certain biological traits such as longevity, reproductive pattern and capacity, male competitiveness, flight ability, field dispersal, eye colour and vision and pheromone production (Prokopy and Economopoulos, 1975; Calkins, 2002; Calkins and Parker, 2005). Artificial-diet reared flies usually display a lower presence of transient and endosymbiotic bacteria compared to wild populations, because of laboratory antibiotics and preservatives used in artificial diets (Estes et al., 2011). Many laboratories renewed the stock flies to resolve issues of colony quality, but the genetic trade-off occurred in few generations, making the renewal of limited value. Researchers are now oriented at maintaining OLF laboratory colonies on their natural host (Sime et al., 2007; Genç and Nation, 2008; Spanedda and Baratella, 2010). The main issue related to this methodology is that OLF oviposits at the pit hardening stage of olive fruits (de Alfonso et al., 2014; Malheiro et al., 2015), and drupes picked at this phenological stage cannot be stored long enough without affecting their suitability for larval feeding. As a result, it is not possible to provide quality fruits for more than a few months a year, not even by collecting them in geographic progression of ripening (Sime et al., 2007). To continue the rearing over years, the protocols developed along this line, based on the procedures of Tzanakakis (Tzanakakis, 1989), have relied on the periodic collection of wild adults from the field. However, this approach is impractical and significantly affects the OLF response to specific, complex environmental conditions (Sime et al., 2007; Wang et al., 2009, 2013; Estes et al., 2011). Many authors have investigated the effect of temperature on development rates of tephritid fruit flies (Tsitsipis, 1977a, b; Tsitsipis, 1980; Moore, 1960; Tzanakakis et al., 1968; Girolami, 1979; Fletcher, 2002; Wang et al., 2009). Complete development of OLF larval and pupal stages occurs at temperatures between 12 °C and 35 °C. The OLF eggs don’t hatch at temperature threshold values of 7.5 °C and 35 °C (Tsitsipis, 1977a, b). Girolami (1979) tested the range of temperature for the complete development of immature stages of the fly on olive fruits: eggs complete their development at temperatures between 7.5 °C and 37.5 °C, larvae at temperatures 10 °C to 32.5 °C, pupae at temperatures 10 °C to 30 °C. On the contrary, little and limited research has been performed on the effects of photoperiod on OLF ecophysiological traits (Tzanakakis and Koveos, 1986; Koveos and Tzanakakis, 1990; Raspi et al., 1997). In the field, the life cycle of the fly is closely linked to the phenology of Olea spp. as well as to climatic factors, with strong agro-ecological effects (Petacchi et al., 2015). Pre-imaginal stages overwinter in the fruit or as pupae in the soil, adult flies overwinter in a facultative reproductive-dormancy, but, depending on climate, can remain active year-round in the trees canopy (Kapatos and Fletcher, 1984; Neuenschwander et al., 1981, Gutierrez et al., 2009; Petacchi et al., 2015). Dormancy breaks as fruits become available, and the oviposition starts when olive fruits reach the pit-hardening phenological stage. Prior of oviposition, a period from 6 to 10 days is required for ovarian maturation. Courtship and mating occur at dusk, at the end of daylight. Females prefer to lay one eggs per fruit, but there are evidences of a

2. Material and methods 2.1. Ethics statement

No specific authorization was required for insect field collection, nor for performing the experiments. All experiments were carried out at the Tuscia University facilities in Viterbo, Italy. Wild adults were collected from the field, no endangered or protected species were involved. 2.2. Experimental materials

Small-scale experimental rearing started in autumn 2008, when infested olive fruits were collected from the field, a typical organic olive grove of northern Lazio (40 years old, Canino cv), and moved to the laboratory. Emerging wild adults were divided in two parental adult populations in replicate, 30 adults each (sex ratio 1:1), which were placed in plastic cages. Rearing cages and oviposition chambers were plastic cylinders (diameter 30 cm, height 40 cm) with a removable bottom and a little side-window, the cage top was closed with 0.5 mm-mesh tulle. Feeders were cylindrical glass containers (diameter 3 cm

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further oviposition (n + 1 cycle). Theoretical premise is that breeding separately laboratory generations at different developmental stages allows collecting observational data on physiological parameters and stage transitions on a discrete-time basis. The activity of the adults was observed regularly from a minimum of 2 observations per day, 1 h each. When mature, the 3rd instar larvae exited the fruit and dropped to the bottom of the cages. To provide a comfortable pupation media, we successfully tested the use of moistened paper towels on the plastic countertop of the cages, nebulized daily with distilled water (Navrozidis and Tzanakakis, 2005; Genc, 2008). The collection of pupae was simple and there was no need to handle them manually: puparia were collected using a fine brush wet with distilled water, then inspected and transferred onto clean absorbent paper in transparent Petri dishes. The pupae were kept at the same environmental conditions as the adult population and monitored daily until the adult flies emerged. Unemerged puparia were removed and inspected under a stereomicroscope to check for pathogen contamination. An ordered sequence of operations was run daily in order to collect data: visual and microscope inspections, counting of live and dead flies separately per sex and generation cycle, counting and collecting of empty, live or dead puparia, counting of larvae. The mortality in each cage was recorded daily, the dead flies were inspected for eventual pathogen contamination and removed. Exhausted fruits were randomly selected and examined for the presence of larvae and puparia inside each fruit. In this study the short-day (8:16 LD) and the long-day (16:8 LD) photoperiods were evaluated with regard to their effects on the physiology of the fly and also regarding some management issues, in combination with two levels of temperature (16 °C and 27 °C). Cages were placed in a randomized design and periodically moved to compensate for differences in light intensity. Dawn and dusk were simulated by 50% reduced light for 1 h at the beginning and at the end of the photo-phase. Ecophysiological responses were evaluated by measuring the following biological traits: number of pupae (PN); duration of pupal stage (PD, days); adult emergence rate (ER); adult emergence duration (ED, days); total life span (from oviposition to adult death) (LS, days); sex ratio (females to males) (SR); population size (PS). The length of the pupal stage was calculated by recording the time interval from the emergence of the first pupa to the end of pupation. Similarly, the duration of the adult emergence was recorded starting from the first emerging adult until the end of eclosion. The population size (PS) was recorded counting the number of individuals at time intervals per each combination of temperature (T) and photoperiod (LD) (treatments) and expressed as mean number of adult flies per treatment. The population density (DE) was then calculated as number of adult flies within the given area of the cage. The adult emergence rate indicates the adult-pupa recovery expressed as number of eclosions to number of pupae, and provides an estimate of total mortality affecting laboratory populations at different environmental conditions. The separate breeding of laboratory generations at different developmental stages allowed collecting observational data on physiological parameters and stage transitions on a discrete-time basis. That means, all developmental parameters (PD, ED, LO, etc.) were collected from laboratory generations and expressed as mean value per adult cohort. The counting of live and dead flies separately per sex and generation cycle, the counting and collecting of empty, live or dead puparia, the counting of larvae, the mortality in each cage were recorded separately per each lab generation. This is quite a valuable difference compared with traditional rearing practices, and allowed us to obtain estimates that accounted also for stochastic variability in vital rates among individuals in the same stage (De Roos, 2008).


and height 10 cm) with a plastic screw top and a tray with a porous bottom to allow adults to feed comfortably (Spanedda and Baratella, 2010). Cages, feeders and all instruments were washed once a week using sodium hypochlorite then soaked in water for 3 h and washed again in abundant water. The OLF culture was placed permanently in a climatic chamber in the laboratory, where the parameters of temperature T, humidity RH and photoperiod LD were monitored daily using chamber detectors and digitally recorded with a data-logger. The chamber humidity was set to 75 ± 5% RH. To assess the physiological responses of the fly, the effect of two photoperiodic conditions, the short day (8:16 LD) and the long day (16:8 LD), were verified in combination with two levels of temperature. In biological systems as insect populations, there are usually different and important error sources, e.g. temporal/ spatial variation in driving variables, system parameters, demographic stochasticity etc. Consequently, to emphasize and make significant the fly’s response, we adopted two distant values of temperature of 16 °C and 27 °C, spanning the range of average monthly temperatures from July/August to September/October, in olive-growing regions of Viterbo province, Italy (aggregate data from the weather stations of Tuscia University and SIARL – Integrated Agro-Meteorological Service of Lazio). A constant supply of fresh olive drupes, suitable to oviposition, was ensured by applying a method for the in-situ permanent storage of olive fruits, recently developed by our research group (Spanedda and Baratella, 2010). In accordance with this methodology, coverings of straw and tulle were assembled on olive branches in the field, as soon as fruits were at the proper size but not yet at the pit-hardening stage, allowing for slower ripening and physical protection of fruits. From these coverings in the field, olive branches were picked regularly, microscopically inspected, defoliated to avoid rapid dehydration of fruits, put in a vase with water and then placed in the oviposition chambers in laboratory. This method provided not-punctured olives for more than 12 months (Spanedda and Baratella, 2010; Baratella, 2011). To allow the retention of endosymbiotic and transiently acquired bacteria, the OLF adults were supplied with non-sterile diets (Moore, 1959, 1962; Drew and Yuval, 2000; Sacchetti et al., 2008; Estes et al., 2011). The liquid diet was comprised of a solution of distilled water and sugar (1:8), while the proteic source was a non-sterile water soluble extract of autolyzed yeast cells, a mixture of amino acids, peptides, water soluble vitamins and carbohydrates (non-sterile Yeast Extract, Sigma-Aldrich). Water and nutrients were supplied regularly on a daily basis to avoid food competition that could have long-term effects on several adult fitness traits (i.e. longevity, fecundity) (Tsiropoulos and Manoukas, 1977; Tzanakakis, 1989). 2.3. Rearing protocol

From the parental adult populations, 2 adult cohorts in replicate were obtained, whose immature stages developed at same photophase and temperature of the adults (instar acclimation). Since the scalarity of oviposition and the longevity of adult flies determine the overlapping of vital stages succession in Bactrocera oleae (succeeding generations) with scramble competition among juvenile stages (Neuenschwander and Michelakis, 1979; Burrack et al., 2009), we discretized the cohort continuous reproductive cycle into segregated, overlapping laboratory generations. That is, each adult cohort started a series of laboratory reproductive cycles, obtained by segregating punctured fruits into separate cages every 10 days and providing 100 ± 2 fresh fruits to the adult cohort for further oviposition. After a constant oviposition time of 10 ± 1 days, punctured fruits were moved into separate cages where the development cycle from egg to adult proceeded independently (n cycle). At the same time, freshly picked olive branches laden with fresh fruits were microscopically inspected for the absence of punctures on fruits and provided to the adult population for

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the mean, 25th and 75th percentile and 95% of confidence interval of the parameters. The increase of ER with the short day was not significant, whereas its variation caused by temperature (T) was highly significant (Fig. 1). The interaction effect between T and photoperiod (LD) was not significant (Table 1), suggesting that the variation in the rate of emergence was the effect of T only. The emergence of new adults directly from fruits was observed at high values of temperature and long-day photoperiod, although some scattered occurrences were noticed at short-day photoperiod. LD affected the sex ratio, which decreased from 1.42 on the long-day to 1.01 on the short-day (Table 1, Fig.1). There was a significant interaction effect between T and LD for the duration of pupal stage (PD) and the adult emergence duration (ED) (Table 1): in fact, although the length of these stages decreased from the short day to the long-day at both temperatures, this trend was more pronounced when the fly developed at a lower temperature (Fig. 2). The analysis of simple effects indicates that for both PD and ED, the combination 16 °C, 8:16 LD showed highly significant differences (p < 0.001, HSD-Tukey) compared to the other means (Table 1). The mean differences relative to 27 °C, 8:16 and 16:8 LD are significant at p = 0.029 for PD and not significant for ED (p > 0.05) (Fig. 1). Results indicated that the interaction between photoperiod and temperature for PD and ED was due primarily to their longer duration at the short-day and lower temperature, than at the other three experimental conditions. The main effect of T on the mean number of adult flies in the colony, in other words the increase of the population size with increasing temperature, was not significant (Table 2). Instead, LD showed a highly significant effect, which resulted in a higher population size at the short-day photoperiod (Table 2). The interaction effect between T and LD was significant, and results from post hoc comparisons clearly showed that, during the long-day photophase, increasing temperature caused a reduction in the population size. Inversely, during the short-day the number of adult flies increased with temperature (Fig. 3). To better highlight the impact of temperature and photoperiod on the performance of the fly, we further analyzed our factorial data by computing the percentage variation (increase or decrease) of OLF ecophysiological parameters, in relation to the two independent variables of the rearing (T and LD treatments). The estimated marginal means for the independent variables, e.g. the effect of T and of LD by two-way factorial ANOVA, are reported in Tables 1 and 2. Relative positive or negative changes of the measured parameters were compared after normalizing each value to its alternative treatment, and plotted on integrated radar charts, which allowed for the simultaneous comparison of the strength of the difference between groups (Fig. 4). The radar charts give an overview on which ecophysiological parameters are more likely to be affected by changes in T and/or LD rearing levels, displaying at the same time the relative magnitude of this influence (Fig. 4). In Fig. 4A, each line represent a level of temperature, the greater the distance between the lines, the greater the impact of temperature changes on the parameters of the fly. The lower temperature during the rearing (dark line) had a generic, robust incremental effect on ecophysiological parameters of the fly, with the exception of population size (PS) and sex ratio (SR), whose relative variations were small (Fig. 4A). In fact, the main effect of T on PS and SR gave no statistical significance (Table 1). In Fig. 4B, the main effect of the photoperiod is more pronounced for PS and PN (number of puparia), but it is clearly noticeable for all the parameters with the only exception of ER (not significant differences). The short-day increased all the parameters significantly, even if with different extent; the greater relative variations were for PS and PN, while temperature had a stronger influence on PN, PD and ED. In fact, PD and ED showed also a significant interaction effect between photoperiod and temperature (Table 1), which, as analysis of simple ef


To assess the mean number of adults in the laboratory colony during each treatment, the population size (PS), generally modeled as continuous in number and discrete in time (Shaffer, 1984; Valpine et al., 2014), was calculated by counting the adult flies of the n lab generations across the 2 cohorts at discrete time intervals (every 10 days). More specifically, given n laboratory generations at time t, the population size distribution was calculated as an aggregate function:

where A is the number of adult flies, E is the number of newly emerged adult flies and M is the number of dead flies of the ith laboratory generation. To cope with the different developmental times of the fly at different environmental conditions, we obtained discrete and comparable snapshots of the population consistency for a given treatment by considering the second and third order quartiles of the PS distribution (2 treatment sequences × 2 quartile measures = 4 PS measures per treatment). In our opinion, compared to the simple counting of the total flies, this mathematical structure is more effective in visualize and evaluate the population consistency of the fly across different treatments, taking into account both temporal variation in driving variables and demographic stochasticity. The laboratory colony was maintained continuously for over 24 months, and during this time was subjected to different photoperiodxtemperature treatments. The TxLB treatments were applied in 2 different sequences (16 °C × 8:16, 27 °C × 8:16, 16 °C × 16:8, 27 °C × 16:8, and then 27 °C × 8:16, 27 °C × 16:8, 16 °C × 8:16, 16 °C × 16:8), in order to avoid eventual side-effects related to environmental-driven variables, e.g. the ripening stage of fruits coming from the field. The colony passed from one treatment to another through transitional periods of 2–3 months at standard laboratory conditions (12:12 photoperiod and 22 °C of temperature) during which no data were collected. The transitional periods were used for the colony acclimation, which consisted in obtaining (at least) one generation of adult flies at standard environmental conditions, before applying any treatment. On this basis, we assumed the absence of trans-generational effects. For the purposes of this project, 28 laboratory generations per cohort were considered (56 in total). We obtained 14 ± 2 laboratory generations per treatment, on time periods that lasted consistently to the developing time of the flies at the different TxLB conditions (see Table 1). Each treatment were ended as soon as a proper number of lab generations were obtained. Then, all the remaining flies of the last generations (which were obviously not taken into account for the treatment effect) entered the transitional period. 2.4. Statistical analysis

All data were statistically analysed by two-way factorial analysis of variance with Tukey’s HSD post hoc pair wise comparisons and follow-up tests in case of significant result for interaction effect (SPSS, IBM Corp., Armonk, NY). 3. Results

As expected, T affected all ecophysiological parameters, which decreased significantly passing from 16 °C to 27 °C, with the exception of the sex ratio, where the trend was not significant (Table 1). Results showed also a highly significant effect of the photoperiod: the short-day photophase substantially increased the value of all tested parameters, except for the emergence rate ER (Table 1). In Fig. 1, boxplots reports the main effects of temperature and photoperiod on OLF life span, number of pupae, emergence rate and sex ratio, displaying

4

PN (nr.)

ED (days)

ER (%)

D E T C E R R O C N U ± 6,32 ± 6,32

32,55 16,41

± 0,84 ± 0,84

74,50 35,06

± 5,85 ± 6,76

28,75 20,21

± 0,78 ± 0,90

95,63 42,54 53,38 27,58

± 8,28 ± 9,56 ± 8,28 ± 9,56

38,69 a 26,42 b 18,81 c 14,00 d

** *** NS

*** *** **

± 1,10 ± 1,27 ± 1,10 ± 1,27

LS (days)

SR

31,10 17,51

± 0,94 ± 0,94

87 67

±4 ±4

95,85 70,35

± 1,58 ± 1,58

1,28 1,15

± 0,09 ± 0,09

28,42 20,19

± 0,87 ± 1,01

82 73

±3 ±4

91,58 74,63

± 1,47 ± 1,69

1,01 1,42

± 0,08 ± 0,09

± 1,24 ± 1,43 ± 1,24 ± 1,43

93 81 70 65

±5 ±5 ±5 ±5

103,53 88,17 79,63 61,08

± 2,07 ± 2,39 ± 2,07 ± 2,39

1,01 1,55 1,00 1,29

± 0,11 ± 0,13 ± 0,11 ± 0,13

EP

69,08 40,48

PR

Treatmenta⁠ T 16 °C 27 °C LD 8:16 16:8 T*LDb⁠ 16 °C * 8:16 16 °C * 16:8 27 °C * 8:16 27 °C * 16:8 Significancec⁠ T LD T*LD

PD (days)

RI NT

Table 1 Main effects of temperature (T) and photoperiod (LD) combinations on ecophysiological parameters of Bactrocera oleae.

F O O R P

37,03 a 25,17 b 19,81 c 15,21 c *** *** **

*** NS NS

*** *** NS

NS ** NS

a PN number of puparia; PD duration of pupal stage; ED adult emergence duration; ER adult emergence rate (number of eclosions on number of pupae); LS Life Span (including immature development); SR sex ratio (females/males). Data reported as mean ± standard error, interaction and main effect by two-way factorial ANOVA. b Data followed by different letters within a T*LD column are significantly different (p < 0.05, Tukey HSD Post Hoc Test for Simple Effects). c *⁠ ** = p < 0.001, *⁠ * = p < 0.01, *⁠ = p < 0.05, NS = not significant.



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Fig. 1. Main effect of temperature (A) and photoperiod (B) on OLF life span (LS, days), number of pupae (PN), emergence rate (ER) and sex ratio (SR). Boxplots showing mean (bold line), the 25th and 75th percentile (box), 95% of confidence interval (whiskers), main effect by two-way factorial ANOVA: *⁠ ** = p < 0.001, *⁠ * = p < 0.01.

Fig. 2. Estimated marginal means for temperature (T) and photoperiod (LD) on OLF adult emergence and pupal stage. (A) Interaction effect T*LD on adult emergence duration, p = 0.009. (B) Interaction effect T*LD on the duration of the pupal stage, p = 0.003. Significant interaction effect by two-way factorial ANOVA.

fects indicated, was due primarily to their longer duration at the short-day and lower temperature. SR showed a peculiar, opposite trend, with a decline of female flyes at the short-day, and a rise at the long-day (Fig. 4B).

than comparable groups provided with sugar, water and protein; sugar, water and olives; or sugar and water only. Wang et al. (2009) observed that, when deprived of host fruit, females appeared to virtually terminate egg production after 10 days, but when provided with olive fruit, females continued to produce mature eggs. The presence and maintenance of natural microbiota occurring on non-sterile olive drupes has been hypothesized as one of significant factors affecting egg maturation (Moore, 1959, 1962; Papaj, 2000). Ghiardi (2009) hypothesized that the olive juice and the proliferation of bacteria harboured in the fly’s gut could release different metabolites, such as essential amino acids, required for ovarian development and egg maturation. Following these authors’ recommendations, in our experimental conditions OLF adults were provided with non-sterile olive fruits together with sugar, distilled water and non-sterile water soluble extract of autolyzed yeast cells. In our work, consistent to what was found by Dimou et al. (2009), the presence of non-sterile olive fruits and the ab

4. Discussion

4.1. Laboratory rearing

4.1.1. Access to (non-sterile) olive fruits The access to olive fruits has been widely confirmed in literature as fundamental for several physiological and behavioral traits of the fly. Fletcher et al., 1978 first speculated that the lack of olive fruits might cause OLF reproductive dormancy. Fletcher and Kapatos (1983) reported that OLF females provided with olive fruits together with sugar, water and protein hydrolysate, matured faster at any time of the year

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eggs was easily overcome by the natural protection of the oily flesh of the olives. In nature, the OLF females prefer to oviposit only one egg in a host. After oviposition, OLF ingests olive juice from the oviposition site and regurgitates onto the drupe, deterring further ovipositions by conspecifics (Cirio, 1971). Under rearing conditions or in case of high infestation rates, the average number of larvae feeding in a fruit may be greater than one, revealing a larval intraspecific scramble competition, rather than contest competition (Burrack et al., 2009). Nonetheless, chemical mechanisms that ensure adequate spacing of progeny on limited resources continue to operate (Koveos and Tzanakakis, 1990; Renwick, 1989; Aluja and Mangan, 2007). In the present research, the periodic replacement of punctured drupes with freshly harvested fruits allowed to elude repelling mechanisms and to better express the reproductive potential of the OLF population. In addition, since the scalarity of ovipositions determines the overlapping of succeeding generations, discretizing the reproductive cycle into segregated laboratory generations on a discrete-time basis allowed to synchronize the development time of instars. Then, handling procedures and data gathering on physiological parameters and stage transitions became more manageable, and the fly's response to environmental variables was more precisely discerned. One shortcoming of this technique could be the amount of work required in the field, with a consequent need for the prototyping and automation of field operations for the scaling up of the method. Two further aspects of laboratory rearing practices on fresh fruits must be considered, when dealing with OLF population reared on artificial diets: (i) damage caused to pupae and larvae by prolonged crawling over diet and (ii) in-diet pupation (Estes et al., 2011). Rearing larvae in freshly harvested olives, we facilitate the pupation and an easy collection of pupae simply by providing moistened paper towels as a media to pupate outside the fruits (Navrozidis and Tzanakakis, 2005; Sime et al., 2007; Genç and Nation, 2008). The larvae crawled out of the drupes, dropped on the pupation medium and then pupated between the layers of the moistened paper towels. The collection of pupae from the paper towel layers was quite simple and with no need of manual handling: we did not record incomplete adult eclosion nor adult wing damage caused by pupae damages. We noticed that the combined influence of temperature and photoperiod also seems to determine the site of pupation, inside or outside the fruits: part of the new adults emerged directly from fruits at high temperatures and long-day photoperiod, although some scattered occurrences were observed during the short-day photoperiod. It’s not clear, however, if the preference for the pupation site directly undergoes the effect of temperature and/or photoperiod, or if there are indirect influences, for instance of the length of time during which fruits remain suitable to safely host pupae. The optimal density of OLF adults inside the rearing cages still needs to be empirically determined. It is possible, however, to refer to the average density of the Democritus laboratory colony held in Crete for mass-rearing purposes: 120 dm3⁠ cages housed 1800–2400 adults (Tsitsipis, 1977a, b; Tsitsipis, 1975). Factors such as access to food, water, oviposition sites, interaction between flies (fighting and mating), and resting space have a high impact on both acquisition and consumption of food (Estes et al., 2011). Reduced egg production, infertility, and early adult mortality result from overcrowding (Tzanakakis, 1989). By discretizing OLF overlapping generations into segregated laboratory generations, the maximum OLF density recorded in our cages (28,3 dm3⁠ ) barely reached 300 adults/cage, in other words the available volume was never less than 94cm3⁠ /adult, a value much greater than the maximum spacing of the Democritus colony (66,7cm3⁠ /adult). The average number of larvae per fruit was always lower than the threshold values above which the deleterious effects of competition appear. After dissection of drupes under optical microscope examination, we very rarely found dead larvae and/or aborted eggs. Brawls sporadically ob

Table 2 Main effects of temperature (T) and photoperiod (LD) on size and density of OLF adult population and analysis of simple effects.

Treatmenta⁠ T 16 °C 27 °C LD 8:16 16:8 T*LDb⁠ 16 °C * 8:16 16 °C * 16:8 27 °C * 8:16 27 °C * 16:8 Significancec⁠ T LD T*LD

DE (adult/dm3⁠ )


PS (nr)

135,50 156,00

±10,22 ±10,22

4,79 5,51

±0,36 ±0,36

219,38 72,13

±10,22 ±10,22

7,75 2,55

±0,36 ±0,36

191,25 a 79,75 b 247,50 a 64,50 b

±14,45 ±14,45 ±14,45 ±14,45

6,76 a 2,82 b 8,75 a 2,28 b

±0,51 ±0,51 ±0,51 ±0,51

NS *** *

NS *** *

⁠ ). Data a PS, population size (mean number of adults); DE, density (DE, adults/dm3 reported as mean ± standard error, interaction and main effect by two-way factorial ANOVA. b Data followed by different letters within a T*P column are significantly different (p < 0.05, Tukey HSD Post Hoc Test for Simple Effects). c *⁠ ** = p < 0.001, *⁠ * = p < 0.01, *⁠ = p < 0.05, NS = not significant.

Fig. 3. Estimated marginal means for temperature (T) and photoperiod (LD) on the size of OLF adult population. Interaction effect T*LD on population size, p = 0.029. Significant interaction effect by two-way factorial ANOVA.

sence of antibiotics in adults’ diets did not cause any apparent pathogen-induced mortality, nor negative effects on colony survival and growth.

4.1.2. Rearing practices With respect to previous laboratory rearings on a natural host, the procedures applied in our work deal with some critical issues related to the biology of the fly (Sime et al., 2007; Genç and Nation, 2008; Wang et al., 2009; Spanedda and Baratella, 2010). The OLF females are hesitant to oviposit in artificial devices in the laboratory (Hagen et al., 1963), and the various egging devices developed so far (e.g. wax-coated oviposition substrates like domes and cones) have proven to be difficult, time-consuming and costly (Tsitsipis, 1977a, b; Genc, 2008; Estes et al., 2011; Hagen et al., 1963). In our rearing protocol, by using olive drupes as oviposition substrate, the complicated procedures of egg collection has been avoided and the low desiccation tolerance of

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Fig. 4. Impact of temperature (A) and photoperiod (B) treatments on OLF ecophysiological parameters. Main effects by two-way factorial ANOVA expressed as percentage variations normalized to the alternative treatment, superscripts represent no significant differences between treatments. PN number of puparia; PD duration of pupal stage; ED adult emergence duration; ER adult emergence rate; LS Life Span; SR sex ratio.

served at the higher densities, particularly between females, should therefore be attributed to the natural aggressive behaviour of the fly (Benelli, 2014).

(Navrozidis and Tzanakakis, 2005; Sime et al., 2007; Genç and Nation, 2008; Yokoyama et al., 2011; Wang et al., 2013) adopted long-day photophases and high temperatures. Regarding the effect of temperature, in the present work, as expected, all ecophysiological parameters decreased significantly passing from 16 °C to 27 °C, with the exception of the sex ratio, where the trend was not significant (Fig. 1). At higher temperature, our data reported the shortening of the pupal stage, which must be linked to a more rapid development of larvae (Fig. 1). According to Neuenschwander and Michelakis (1979), the effect of temperature on the difference in development times may have been enhanced by the quicker ripening of fruits at high temperatures, with faster larval developments attributed to increasing nutritional quality as olive fruits ripened. Nonetheless, these different speeds in the ripening of fruits, and their subsequent quicker senescence, could probably also explain the variation in vitality of the OLF pupae: in fact, the emergence rate was the only parameter dependent only on the temperature, showing a significant decrease at the higher temperature (Fig. 2 and 4). Regarding the fly's performances in relation to photoperiodic conditions, the main novel finding of the present work was the short-day photophase response of the olive fruit fly: in agreement with Tzanakakis (2003), the combination of short-day and low temperature showed a higher number of pupae and a higher adult emergence rate and longevity (including immature development) (Table 1). There was a significant interaction effect between temperature and photoperiod for the duration of pupal stage and adult emergence, indicating that the effect of temperature on these parameters is dependent on the photoperiodic conditions (Fig. 2). In fact, at higher latitudes the prevalence of the photoperiod as internal signal related to reproductive physiology and behaviours, widespread among insects, should have strong selective advantages, being more reliable and noise-free than temperature and thermoperiod. The interactions between photoperiod and temperature may also affect the seasonal changes in insects at a number of levels (Saunders, 2014). The mean number of OLF adults in the laboratory colonies was primarily adjusted in response to the photoperiodic conditions: the population size reached greater values during shorter days (8:16 LD) (Table 2). In our experimental data, the main effect of T on the population size gave no statistical significance, whilst the main effect of LD was highly significant (Table 2). Again, the interaction effect between tem

4.2. Ecophysiological responses

In different geographic areas the OLF exhibits two annual reproductive peaks in March-April and September-October, with a lack of mature eggs in the ovaries during late spring and early summer which suggests a reproductive dormancy presumably caused by high temperatures, lack of olive fruit and low humidity (Papaj, 2000; Raspi et al., 2005). Tzanakakis speculated that, from the ecophysiological point of view, the OLF resembles a short-day species, having adapted to develop best in autumn, when olive fruits are suitable for larval growth (Tzanakakis, 2003). The lack of ovarian maturation during late spring-early summer and the laboratory induction of reproductive diapause under similar environmental conditions supported this hypothesis (Tzanakakis and Koveos, 1986). The photoperiodic regulation, i.e. the measurement of successive photoperiods and their accumulation, are thought to be active for the majority of insects, and for a number of species in Diptera (Saunders, 2014), being the most predictive environmental cue for the seasonal timing of physiology and behaviour. Circadian locomotor rhythms of the adult flies may reflect endogenous oscillations with a temperature compensated period, with rhythms synchronised to LD cycles that become arrhythmic in constant light above a certain intensity. The photoperiodic effect is also based on the circadian system but seems to involve a separate mechanism at both the molecular and neuronal levels (Saunders, 1997). The internal coincidence model (Saunders, 2014) suggests that the photoperiodic mechanism is based on (at least) two circadian oscillators: seasonal changes are sensed by changing “internal” phase relationships between them as days (or nights) lengthen or shorten. Among the higher Diptera, an array of daily behaviours such as general locomotor activity, flight, mating, oviposition, egg hatch, pupation and pupal eclosion are governed by circadian oscillators, whereas various seasonal phenomena such as the onset of diapause, or larval growth rates, are governed by photoperiodic clocks (Saunders, 1997). Nevertheless, the existence of reproductive diapause in the OLF is still under question and most of the rearing procedures in earlier works (De Magalhaes Silva, 1970; Tzanakakis, 1989; Koveos and Tzanakakis, 1990) and in recent literature

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the influence of environmental conditions on OLF physiology, the limited number of studies made so far on the effects of the photoperiod are, in any case, contradictory. Most of the rearing procedures attempted in earlier and recent literature adopted long-day photophase and high temperature, despite the fact that the OLF resembles a short-day species, with lack of ovarian maturation during late spring-early summer. In the present work, in agreement with Tzanakakis (2003), OLF laboratory colonies with access to olive fruits gave a short-day photophase response (8:16 LD). In our findings, all tested parameters (pupation, emergence, life span, sex ratio) substantially increased on the short-day, except for the emergency rate. The combination of short-day and low temperature showed a higher number of pupae and a higher adult emergence rate and life span. In partial agreement with Raspi et al. (2005), changes in the population size were observed in response to photoperiodic condition: the population size reached greater values on the shorter days (8:16 LD). There was also a significant interaction effect between temperature and photoperiod for the duration of pupal stage and adult emergence, indicating that the effect of temperature on these parameters is dependent on the photoperiodic conditions. Overall, our experimental data demonstrated that the OLF ecophysiological traits are affected by a combination of stimuli, among which the photoperiodic conditions play a key role. These innovative results form an excellent base of knowledge for future challenges to advance in the rearing of OLF parasitoids.


perature and photoperiod was also significant: during the long-day photophase, increasing temperature caused a reduction in the population size. Inversely, during the short-day the number of adult flies increased with temperature (Fig. 3). Our data partially confirm the findings of Raspi et al. (2005), which, at the short-day photoperiod, showed a higher number of eggs per female, but a lower percentage of females with eggs, compared to the long-day photophase (LD 16:8). It’s worth remembering that in the work of Raspi, OLF adult females were obtained from fully-grown larvae from field infested olives, collected in central Italy during October-November. Hence, Raspi’s larvae developed at the short-day photophase, whilst adults underwent photoperiodic treatments for a period of only 15 days after emergence. Similarly, Tzanakakis and Koveos (1986) in their experimental induction of ovarian immaturity, developed OLF adults at higher temperature and long day photophase compared to preimaginal stages. The induction of responses to a photophase (long-day) different from those of the larval stage, and the short duration of the photoperiodic treatments may have some way affected the authors first findings. Later studies supported the hypothesis that the ovarian development of the OLF is affected by a combination of host stimuli: the abiotic factors of photoperiod and temperature, but also the presence of olive fruits (Papaj, 2000; Wang et al., 2009). In this regards, our data indicated that, with access to olive fruits, the photoperiod of 8:16 LD and the temperature of 16 °C is the combination showing higher number of pupae and high population size (Tables 1 and 2). Our findings are supported by the works of Tzanakakis and Koveos (Tzanakakis and Koveos, 1986; Koveos and Tzanakakis, 1990) and Tzanakakis, 2003, in which short photoperiod, low temperature and access to olive fruits resulted in a much quicker female egg maturation compared to females deprived of olive fruits. These innovative results, which now seem to be supported by the work of Kokkari et al. (2017), seem to confirm that in the Mediterranean area the olive fruit fly have adapted to develop best in autumn, when olive fruits are suitable for larval growth.

Acknowledgments

The authors gratefully acknowledge Nikos T. Papadopoulos for his contribution. His suggestions and encouragements have been fundamental for the proper evaluation and presentation of our findings. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. References

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5. Conclusions

The olive fruit fly rearing technique is (usually) an issue when performing bioecological research on this pest and its parasitoids (Wang et al., 2013). With this paper, we aim to contribute useful information for improving rearing protocols for the olive fruit fly, the primary economic pest in all olive growing regions worldwide. Management practices for the fly have historically focused on classical biological control and have been investigated for over 80 years in the Mediterranean basin. Actually, it has become the prime target of classical biological control programs in California and Israel. As many authors agree, the success of biocontrol programs for the olive fly will depend on the developing of effective laboratory rearing methods, as well as on the ability to close the knowledge gap on the physiological role of the photoperiod. In the present work, we proposed a new technique for a continuous rearing on natural host, under laboratory conditions, and investigated the ecophysiological responses of the fly to different combinations of temperature and photoperiod. Since the studies of Moore (1959) and Fletcher et al. (1978), the access to olive fruits was widely confirmed in literature as fundamental for several physiological and behavioral traits of the OLF, but only few generations has been maintained on harvested olives under laboratory conditions. Compared to previous attempts, the rearing method developed in our work succeeded to obtain the continuous rearing of the fly on olive fruits in laboratory (Sime et al., 2007; Wang et al., 2009; Spanedda and Baratella, 2010). This novel, continuous rearing protocol allowed us to demonstrate for the first time a clear effect of the photoperiod on the effectiveness of the rearing procedures. Despite the effort involved in the study of

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