Introduction

Wolbachia pipientis, an obligatory intracellular maternally transmitted alpha proteobacterium, was first identified in Culex pipiens in 1936 [1]. Recent studies have estimated that more than 40% of the terrestrial arthropod species have evolved symbiotic relationships with Wolbachia [2]. This extensively wide host spectrum stimulated abundant research aiming to establish factors explaining the evolutionary success of Wolbachia, which could be highly attributed to its ability to manipulate biological functions of its hosts in a way that assures both the bacterium and the host continuity through generations.

The broad array of reproductive manipulations used by Wolbachia include the induction of parthenogenesis, feminization, male killing, and Cytoplasmic Incompatibility (CI). CI is the most common Wolbachia-induced, reproductive phenomenon that results in embryonic lethality when a Wolbachia infected male mates with an uninfected female or a female that carries a different Wolbachia strain [3,4,5]. Considering the sterility induction to uninfected populations, the idea of exploiting the Wolbachia infection towards implementing insect pest control has been proposed since early ‘70s as an environmental friendly pest control method (Incompatible Insect Technique-IIT method) [6, 7]. “Dictation” of reproduction (sterility induction in this case) is not a stand-alone phenomenon and the presence of Wolbachia is often accompanied with a broad spectrum of responses in host organisms which have not been fully elucidated. Such effects may favor or limit the potential for IIT implementation in case of positive and negative effects respectively.

Unravelling the effects of Wolbachia infection on insects’ biology has become an intriguing experimental field over the last few decades. The Wolbachia-insect endosymbiotic relationship has been correlated with a wide range of effects (negative, neutral or positive) on major biological parameters, such as fecundity, fertility, mating behaviour and adult lifespan [8,9,10,11,12,13,14,15,16,17,18,19,20]. There are often contradictory experimental results regarding effects of Wolbachia on insect host life history and behavior, even among different populations of the same species, that are attributed to i) the “dynamic” nature of the Wolbachia-host symbiotic relationship, and ii) the determinant role of both insect and Wolbachia genomic backgrounds on the expression of a given biological modification. For example, Weeks et al. (2007) pointed out that the fecundity disadvantage imposed by wRi to D. simulans evolved into a fecundity benefit in less than 20 years, whereas among the mosquitoes’ genus Aedes, Wolbachia infection has been associated either with negative or neutral effects on adults’ longevity [12, 17, 18]. Additionally, studies on insect behavior conducted mainly on Drosophila spp. demonstrated that Wolbachia could contribute to reproductive isolation between infected and uninfected populations [19, 21,22,23,24].

The Mediterranean fruit fly (medfly), Ceratitis capitata (Wiedeman) (Diptera: Tephritidae), is one of the most damaging pests to fruit production worldwide. The broad range of host plants, the wide geographical distribution, combined with multivoltinism makes the control of this pest challenging and large scale Area-Wide Integrated Pest Management (AW-IPM) projects the most appropriate strategy to achieve sound control [25, 26]. Often the implementation of the Sterile Insect Technique (SIT), successfully implemented in several parts of the globe over the last three decades, consists the main element of many AW-IPM projects. In contrast to medfly, the implementation of SIT for other target species is facing quite challenging barriers. Some of the major obstacles are (a) the poor productivity of large scale rearing, and (b) the inadequate performance of the mass-reared insects under field conditions [27, 28]. Both aforementioned limiting factors are probably stemmed from the absence of suitable insect strains that are fully adapted to mass-rearing conditions and the currently used rearing protocols. Wolbachia infection could potentially abate some of the aforementioned productivity and biological quality concerns through the modifications imposed on hosts’ biology. In addition, CI expression in a novel host could set the scene for a combined application of irradiation and symbiont-based, sterility induced, pest control methods (combined SIT and IIT approach) [29,30,31].

The transfer of Wolbachia to medfly, a non-host species (but see [32]) that constitutes the most optimized pest in terms of the Area-Wide SIT control, constitutes an interesting biological “framework” to clarify pending symbiotic-related inquires at both basic and applied level. Zabalou et al. (2004, 2009) [33, 34] set the stage for such an experimental approach by using embryonic cytoplasmic injections and managed to establish three Wolbachia-infected medfly lines carrying two different, Rhagoletis cerasi derived, Wolbachia strains: the “S10.3” carrying the wCer4 and the “88.6” and “56S2 Genetic Sexing Strain (GSS)” carrying the wCer2 bacterium strain. A series of laboratory experiments conducted to evaluate the artificially infected lines, showed that all three Wolbachia-infected medfly lines exhibit considerable stability, inducing 100% of cytoplasmic incompatibility in the novel host [33, 34]. Additional laboratory studies revealed that Wolbachia infection seems to reduce medfly fertility, fecundity and lifespan whereas also shorten the developmental duration [35]. However, it is not known whether the Wolbachia impact on C. capitata biological traits has “evolved” over time or conferred additional alterations. Moreover, none of the previous studies evaluated Wolbachia effects on C. capitata behaviour nor under mass rearing conditions.

Given the impact that Wolbachia infections may have on the life history traits of a host species, and particularly on its rearing efficiency and male mating competitiveness, in the present study we used the medfly-Wolbachia symbiotic associations, as a model system, to study the effect of two different Wolbachia strains, on the life history traits of two medfly lines with different genomic background. Exploiting this system under small scale mass-rearing conditions is expected to pave the ground for utilizing Wolbachia symbiosis as a tool to enhance Sterile Insect Technique approaches. Our findings are discussed in the context of the evolution of symbiotic association, the effects of Wolbachia on novel hosts, as well as from an applied perspective since Wolbachia is part of the tool kit towards the development of environmental friendly methods for population control of insect pest species of agricultural, veterinary and human health importance.

Materials and methods

Flies used

Laboratory lines: We used five Mediterranean fruit fly laboratory lines (see Additional file 1). The Wolbachia uninfected lines: (a) “Benakeio”, a laboratory line that has been maintained under laboratory conditions for more that 30 years, and (b) the Vienna 8 medfly genetic sexing strain (“Vienna 8 GSS” hereafter) reconstructed in 2012, carrying the D53 inversion and two mutations that allow male-only releases (the temperature sensitive lethal mutation (tsl) eliminates females after egg exposure to specific temperatures and the white pupae colour mutation (wp) assigns different colour to male and female pupae) [36]. And, the Wolbachia infected lines: (a) ‘88.6’, a transinfected Benakeio line carrying the wCer2 Wolbachia strain, (b) ‘S10.3’, a transinfected Benakeio line carrying the wCer4 Wolbachia strain, and (c) ‘56S2 GSS’, a transinfected Vienna 8 GSS line carrying the wCer2 Wolbachia strain. Both Wolbachia strains (wCer2 and wCer4) are naturally found in field populations of Rhagoletis cerasi, which was the donor species for the establishment of the Wolbachia-infected medfly lines [33, 34], (from now on any reference to medfly genetic background will be noted by capital letters, VIENNA 8 GSS or BENAKEIO, whereas any reference to each one of the five medfly lines will be noted by small letters, Vienna 8 GSS, Benakeio, 56S2 GSS, 88.6, S10.3).

Wildish flies: Male mating competitiveness of the five medfly laboratory lines were tested against the F1 generation of a medfly population collected from Volos (Central Greece), from field infested bitter oranges (pupae were sent to the FAO/IAEA Insect Pest Control Laboratory, that reared for one generation).

Rearing conditions

Experiments were conducted at the FAO/IAEA Insect Pest Control Laboratory, Seibersdorf Austria, from June 2013 to February 2014. Adults from the laboratory strains were reared in fine mesh covered, rectagular cages (200x180x20 cm, ≈200,000 flies in each) provided with water and adult diet consisting of yeast hydrolysate (MP Biochemicals) and sugar at a 1:3 ratio, respectively [37]. Females oviposited through the fine mesh, and eggs dropped (and are collected) in trays containing water (placed below the mesh). Eggs were placed on carrot diet where the larval development took place [38]. The wild adult females recovered from Greece were allowed to oviposit on bananas (the banana peel was pierced with a needle in order to facilitate oviposition), where larvae developed. All medfly colonies were kept at 22 °C and 65 ± 2% RH and a photoperiod of 14 L:10D with the light phase starting at 07:30.

Medfly and Wolbachia infection status

Samples collection and DNA extraction: Prior to experiments, 20 adults (10 males and 10 females) were collected upon emergence and immediately placed at -20 °C. DNA was isolated using the Qiagen DNeasy kit (Qiagen, Valencia, CA), following the manufacturer’s instructions.

PCR based Wolbachia screening: the Wolbachia presence was tested for all individuals by amplifying a Wolbachia-specific 16S rRNA gene fragment of about 438 bp using the Wolbachia specific primers wspecF and wspecR [39].

PCR based screening for “wCer” strains: All individuals that were found Wolbachia-positive were screened for the presence of different Wolbachia strains (wCer1 to wCer5) using the previously reported wsp gene-based PCR [40].

Three individuals from each of the infected strains were sequenced for the five Multilocus sequencing typing (MLST) genes (gatB, coxA, hcpA, fbpA and ftsZ), to verify beyond doubt the presence of the expected wCer strain (wCer2 or wCer4). Amplicons were amplified using the primers and PCR conditions described in the Wolbachia MLST database (https://pubmlst.org/wolbachia/info/protocols.shtml).

Effect of Wolbachia on demographic traits

We used the rearing-cages described above (see 2.) to collect the biological material for the demographic experiments. Eggs laid within a period of 24 h were placed on strips of black filter paper on a wet sponge infused with 3o/oo, Propionic acid to prevent fungal growth. Twenty-four hours after the egg collection, 1000 eggs were transferred into a Petri dish (radius x height: 70 × 15 mm), containing 150 g of a carrot larval diet [38]. Petri dishes were placed over sawdust, the larvae popped out of the diet to pupate and the pupae were collected by sieving the sawdust. We performed at least three replicates with 980–1000 eggs each, for each one of the medfly lines tested. Egg hatch, pupation, and adult emergence were recorded once a day at 11:00. Immature development took place under controlled temperature, humidity and illumination (22 °C, 65 ± 2% RH, 14 L:10D). In order to determine effects on adult lifespan and fecundity, upon emergence one female and two males were placed in 40 cm3 rectangular cages, having ample access to adult diet and water. At least 10 cages were tested for each of the five medfly lines. One side of the cages was covered with fine mesh, which was used by females to lay eggs through the fine mesh on a piece of moist black filter paper placed below the mesh. The eggs were counted under a stereoscope and the cages were inspected for dead flies at 12:00 daily throughout their lifespan. The cages were kept under constant environmental conditions (22 °C, 65 ± 2% RH, 14 L:10D) until the end of the experiment.

Assessing Wolbachia effect on males’ mating competitiveness

Male mating competitiveness of the five medfly lines (Vienna 8 GSS, 56S2 GSS, Benakeio, 88.6 and S10.3) was assessed against wild males for wild females (F1 progenies of a field collected population). The experiment was conducted in standard field cages (2.0 × 1.6 × 1.9 m) (placed in a glasshouse) housing one potted Citrus sinensis Osbeck (Rutaceae) tree, under controlled temperature and humidity (26 ± 1 °C, 45–55% RH respectively). Soon after emergence (within 24 h) flies were sorted by sex and kept in cylindrical Plexiglas cages (≈100 flies in a volume of 6.5 L). Water and a standard adult diet were supplied ad libitum. Male mating competitiveness was tested at the age of 5–7 days, against 11–13 days old wild males, for wild females of same age. The day before conducting the mating tests, adult males were marked on the thorax with a non-toxic dye (red or yellow colour) in order to distinguish the type of the male that achieves copulation. The colour used for the wild and treated males was alternated between treatments during different replications to exclude any possible effect on females’ mate choice. On the day of the test, males were released into a field cage at 07:30 and were allowed to occupy positions on foliage and perform the typical sexual performance before the release of females that took place at 09:00. At least two observations per hour were made until the termination of the mating test at 15:00. Mating couples were removed and placed into transparent plastic vials where they were kept until the end of copulation. Twenty five females and 50 males (25 wild and 25 of each individual population tested) were released in each field cage. The field cages were randomly allocated to treatments. We performed at least five replicates (field cages) for each medfly line.

Effect of Wolbachia on flight ability

The procedure described in detail in [41] was followed to assess effects of Wolbachia infection on adult flight ability. Two days before emergence, 100 pupae were placed within a ring of paper, which was centered in the bottom of a Petri dish (100 × 15 mm). One black Plexiglass tube (89 mm diameter, 10 mm high) was placed over a Petri dish. The inside of the tube was lightly coated with unscented talcum powder to prevent the flies from walking out. We recorded the number of individuals that could fly out of the tube. Five replicates (100 pupae each) were set up for each medfly line tested. All tests were conducted in a controlled environment (26 °C and 65% RH, 14 L:10D and 1500 lx light intensity over the tubes).

Effect of Wolbachia on adult longevity under food and water deprivation

Within 4 h of adult emergence (07:30–11:30 am), 30 males and 30 females were placed in a large Petri dish (150 × 15 mm) with a mesh-covered window in the lid and a hole of approximately 15 mm in the center of the lid. All dishes were kept in the dark at 26 °C and 65% RH, until the death of the last fly. Dead flies were sorted by sex, counted and removed from the Petri dishes twice a day (every 12 h; at 19:30 pm and 07:30 am). We performed five replications (Petri dishes) for each medfly line tested.

Statistical analysis

Data analyses were performed using the SPSS v20.0 (SPSS Inc., Chicago, IL, U.S.A.). The effect of medfly genetic background (VIENNA 8 GSS and BENAKEIO) and Wolbachia infection on all biological parameters studied in this paper was determined by analyzing the data of the two uninfected medfly lines (Vienna 8 GSS and Benakeio) and the two infected with wCer2 medfly lines (88.6, Vienna 8 GSS). The effect of the Wolbachia strain (wCer2 and wCer4) on the same biological parameters was determined by analyzing the three laboratory populations (Benakeio, 88.6, S10.3).

Binary logistic regression analysis was used to infer the effects of medfly genetic background and Wolbachia infection on egg hatch and the survival rates during larval and pupal stages. Chi-square tests, followed by the Bonferroni correction for pairwise comparisons, were used to infer the effects of Wolbachia strain on egg hatch, larval and pupal survival rates.

Cox regression analysis was used to determine the effects of medfly genetic background and Wolbachia infection on the developmental duration of the immature stages (pre-pupa duration) [42]. The effect of Wolbachia strain on the developmental duration of the immature stages was exlored by Kaplan-Meier estimators followed by pair-wise comparisons using the log-rank test (Mantel-Cox).

The effect of medfly genetic background and Wolbachia infection on adult sex ratio, fecundity, males’ mating competitiveness and flight ability was determined by two-way ANOVA. The effect of the Wolbachia strain on the aforementioned parameters was estimated by one way ANOVA.

The effect of medfly genetic background, Wolbachia infection, Wolbachia strain and adult sex on longevity under water and food deprivation were determined by Cox regression analysis [42].

In all tests, the level of significance was set at α = 0.05.

Results

Wolbachia status of the five laboratory strains

All flies of the three Wolbachia infected medfly lines (60 out of 60 individuals) produced the expected 16S rRNA gene amplicon, while all screened flies of the two uninfected medfly lines (40 out of 40) were found negative (data not shown). The wsp based PCR screening that discriminates among the different wCer Wolbachia strains was performed on the same flies of the three Wolbachia-infected medfly lines and 56S2 plus 88.6 produced only the expected wCer2-specific wsp amplicon (40 out of the 40 flies), while all flies from the S10.3 line produced only the wCer4-specific wsp amplicon (data not shown). Three individuals (out of the 20) were selected per Wolbachia-infected medfly line and the MLST profile was analyzed through sequencing of the MLST genes. Again, all flies presented the expected MLST profile, as described in the Wolbachia MLST database.

Effects of Wolbachia infection on hatch rate

Logistic regression analysis revealed that both the genetic background of medfly and the Wolbachia infection, as well as their interaction were significant predictors of egg hatch (Wald’s t-test =55.68, df = 1, P < 0.0001; Wald’s t-test = 782.96, df = 1, P < 0.0001, and Wald’s t-test = 7.39, df = 1, P = 0.007, respectively). As shown in Fig. 1a, in both medfly genetic backgrounds, Wolbachia infection detrimentally reduced female fertility. Different Wolbachia strains exerted differential reduction in egg hatch rates on the same medfly genetic background (x2 = 1757.49, df = 2, P < 0.001). Chi-square test revealed significant differences between the infected 88.6 and S10.3 lines, and the uninfected Benakeio line (x2 = 1757.49, df = 2, P < 0.001). Both Wolbachia strains (wCer2 or wCer4) reduced hatch rates compared to the uninfected flies (x2 = 833.37 and 1666.67, df = 1, P < 0.001). Hatch rates were lower in wCer4 than in wCer2 infected lines (x2 = 174.72, df = 1, P < 0.0001) (Fig. 1b).

Fig. 1
figure 1

Egg hatch. Effect of (a) Wolbachia infection and medfly genotype, and (b) Wolbachia strain on the same medfly genotype on egg hatch rates. White columns represent average percent egg hatch of Wolbachia uninfected and grey columns that of Wolbachia infected lines. Columns headed with different letter are significantly different (P < 0.05) (sample sizes: Vienna 8: 4036, 56S2 eggs: 2984, Benakeio: 2886 eggs, 88.6: 2899 eggs, S10.3: 2934 eggs)

Full size image

Effects of Wolbachia infection on larval and pupal survival

Wolbachia infection was not a significant predictor of larval survival (Wald’s t-test t = 0.521, df = 1, P = 0.470) (Fig. 2a). However, it increased survival rates of pupae (Wald’s t-test t = 7.805, df = 1, P = 0.005) in both medfly genetic backgrounds (Fig. 2b). The effect of the different medfly genetic backgrounds was also significant predictor of both larval and pupal stage survival (Wald’s t-test t = 11.842, df = 1, P = 0.001 and Wald’s t-test t = 48.016, df = 1, P < 0.001, respectively). The interaction between Wolbachia infection and medfly genetic background was a significant predictor of pupal survival indicating a differential response of the two medfly genotypes (Wald’s t-test t = 17.386, df = 1, P < 0.001) (Fig. 2a). As far as effects of different Wolbachia strains on survival during larval and pupal stages are regarded, chi-square test revealed significant differences between the 88.6 and S10.3 lines, and the uninfected Benakeio line (x2 = 94.159 and 25.642, df = 2, P < 0.0001) (Figs. 2c, d). The Wolbachia strain wCer2 increased both larval and pupal survival compared to the uninfected flies (x2 = 5.525, df = 1, P = 0.019 and x2 = 7.948, df = 1, P = 0.005). Conversely, the Wolbachia strain wCer4 reduced the survival in the larval stage whereas it increased the survival in the pupal stage when compared to the uninfected lines (x2 = 66.693, df = 1, P < 0.001 and x2 = 25.304, df = 1, P = 0.001). Both survival during the larval and pupal stage were lower in the wCer4 infected flies compared to wCer2 infected ones (x2 = 81.615, df = 1, P < 0.001 and x2 = 5.274, df = 1, P = 0.021, respectively) (Figs. 2c, d).

Fig. 2
figure 2

Larval and pupal survival. Effect of Wolbachia infection and medfly genotype (a, b), and Wolbachia strain on the same medfly genotype (c, d), on percent survival of immature stages [larval (a, c) and pupal (b, d)]. White columns represent percent survival of Wolbachia uninfected immature medflies and grey columns that of Wolbachia infected lines. Columns headed with different letter are significantly different (P < 0.05) (sample sizes: Vienna 8: 2975 and 2314, 56S2: 913 and 683, Benakeio: 2231 and 1768, 88.6: 1157 and 956, S10.3: 698 and 447, larvae and pupae respectively)

Full size image

Effects of Wolbachia infection on immature development

The effect of Wolbachia infection on the embryonic and larval developmental duration of the five medfly laboratory lines is depicted in Fig. 3. Cox regression analysis revealed that both the genetic background of medfly and Wolbachia infection were significant predictors of egg to pupae developmental duration (Wald’s t-test = 290.51 and 30.12, df = 1, P < 0.0001, respectively), as well as their interaction (Wald’s t-test = 9.36, df = 1, P < 0.0001). The infection reduced egg to pupae duration on VIENNA 8 GSS genetic background whereas it prolonged the respective duration on the BENAKEIO flies (Fig. 3a).

Fig. 3
figure 3

Duration of Immature stage developmental period. Box-plot diagram showing the effect of (a) Wolbachia infection and medfly genotype, and (b) Wolbachia strain on the same medfly genotype, on immature developmental duration in days (egg to pupa)

Full size image

Survival analysis revealed significant differences in egg to pupae duration as well among uninfected Benakeio, and the infected 88.6 and S10.3 lines (log rank test: x2 = 82.19, P < 0.0001). Specifically, Wolbachia infection, either wCer2 or wCer4, prolonged the egg and larval developmental duration when compared to the uninfected flies (x2 = 74.115, 38.014: P < 0.0001for 88.6 and S10.3, respectively). No differences were found between the Wolbachia infected lines (x2 = 0.102, P = 0.750), (Fig. 3b).

Cox regression analysis revealed that both Wolbachia infection and sex were significant predictors of the pre-pupa duration when the GSS lines (Wolbachia infected 56S2 GSS and the uninfected Vienna 8 GSS) were compared (Wald’s t-test = 55.58 and 99.11, df = 1, P < 0.0001, respectively). The interaction between Wolbachia infection and sex was not significant (Wald’s t-test = 0.88, df = 1, P = 0.348) indicating that the bacterium affected the developmental duration of both sexes similarly in the VIENNA 8 GSS genetic background, (Fig. 4).

Fig. 4
figure 4

Immature stages development on the Genetic Sexing Strains (GSS). Box-plots showing the effect of Wolbachia infection on immature developmental duration in days (egg to pupa) of the VIENNA 8 GSS (a) females, and (b) males

Full size image

Effects of Wolbachia infection on adult sex ratio

Two-way ANOVA revealed that neither the genetic background of medfly (F = 0.046, df = 1,9, P = 0.835) nor Wolbachia infection (F = 0.793, df = 1,9, P = 0.396) and their interaction (F = 0.010, df = 1,9, P = 0.924) were significant predictors of the sex ratio (male/female) of the emerged adults. Likewise, the Wolbachia strain (wCer2 and wCer4) was not a significant predictor of the sex ratio of emerged adults (F = 0.073, df = 2,6, P = 0.931; see Additional file 2).

Effects of Wolbachia infection on adults’ longevity

Neither Wolbachia infection nor medfly genetic background were significant predictors of adult lifespan (Wald’s t-test = 1.07 and 3.75, df = 1, P = 0.300 and 0.053, respectively) in contrast, sex was significant as males outlived females (Wald’s t-test = 6.491, df = 1, P = 0.011), (Figs. 5a, b). Neither Wolbachia strain nor sex were significant predictors of adult longevity on the BENAKEIO flies (Wald’s t-test = 1.65 and 3.47, df = 1, P = 0.199 and 0.062, respectively), (Fig. 5c, d). The wCer2 infected females suffered reduced survival rates compared to uninfected and the wCer4 infected ones, but this observation was not significant (Fig. 5c).

Fig. 5
figure 5

Adult longevity. Survival curves (lx) showing the effects of Wolbachia infection and medfly genotype (a-b), and Wolbachia strain on the same medfly genotype (c-d), on females and males longevity

Full size image

Effects of Wolbachia infection on fecundity

Two-way ANOVA [on ln(x) transformed lifetime fecundity rates to achieve normality and equal variance] revealed that neither the medfly genetic background (F = 2.388, df = 1,38, P = 0.131) nor Wolbachia infection (F = 0.310, df = 1,38, P = 0.581) affected lifetime fecundity rates. Similarly, the interaction between medfly genetic background and Wolbachia infection was not significant as well (F = 0.367, df = 1,38, P = 0.548). In contrast, fecundity rates were significantly different among Wolbachia infected medfly lines S10.3, and both the 88.6 and uninfected BENAKEIO (F = 9.451, df = 2,28, P = 0.001) (Fig. 6).

Fig. 6
figure 6

Fecundity rates. Effect of (a) Wolbachia infection and medfly genotype, and (b) Wolbachia strain on the same medfly genotype, on females’ egg production. White columns represent average fecundity of Wolbachia uninfected and grey columns that of Wolbachia infected lines. Columns headed with the same letter are not statistically significantly different (Tukey’s HSD test, P > 0.05)

Full size image

Effects of Wolbachia infection on male mating competitiveness

We used the RI index (Relative Index, analogous to the Relative Sterility Index (RSI), see FAO/IAEA/USDA 2014) to compare mating competitiveness of males of the five medfly lines tested against wild males in competition for wild females (Fig. 7). Overall, Wolbachia infection did not affect mating competitiveness (F = 0.553, df = 1,21, P = 0.465) in contrast to medfly genetic background (F = 45.849, df = 1,21, P < 0.0001). The interaction between medfly genetic background and Wolbachia infection was marginally significant (F = 4.636, df = 1,21, P = 0.043) indicating a rather differential impact of the Wolbachia infection on the two different medfly genetic backgrounds (Fig. 7a). One way ANOVA revealed significant differences in male mating competitiveness among BENAKEIO uninfected, S10.3 and 88.6 lines (F = 9.450, df = 1,12, P = 0.003). wCer2 and wCer4 infections reduced and increased male mating competitiveness, respectively (Fig. 7b).

Fig. 7
figure 7

Males sexual competitiveness. Effect of (a) Wolbachia infection and medfly genotype, and (b) Wolbachia strain on the same medfly genotype, on male sexual competitiveness (marks represent the Relative Index values). Marks indicated with the same letter in each graph are not statistically significantly different (Tukey’s HSD test, P > 0.05)

Full size image

Effects of Wolbachia infection on flight ability

Wolbachia infection was a significant predictor of adult flight ability (F = 70.42, df = 1,16, P < 0.0001), in contrast to the medfly genetic background (F = 0.10, df = 1,16, P = 0.754). The significant interaction between Wolbachia infection and medfly genetic background highlights the differential effect of Wolbachia infection on the two medfly genetic backgrounds resulting in positive and negative effects on flight performance on VIENNA 8 GSS and BENAKEIO flies respectively (F = 173.49, df = 1,16, P < 0.0001) (Fig. 8a).

Fig. 8
figure 8

Adults flight ability. Effect of (a) Wolbachia infection and medfly genotype, and (b) Wolbachia strain on the same medfly genotype, on flight ability. White columns represent average percent fliers of Wolbachia uninfected and grey columns that of Wolbachia infected lines. Columns headed with different letter are significantly different (Tukey’s HSD test, P < 0.05)

Full size image

Wolbachia infection, regardless of the bacteria strain, significantly reduced the flight ability of the BENAKEIO flies (F = 216.34, df = 2,12, P < 0.0001). Significant differences between the two infected lines were also observed (P < 0.05) (Fig. 8b).

Considering only the VIENNA 8 GSS medfly genetic background, two way ANOVA revealed that neither Wolbachia infection nor sex were significant predictors of the flight ability (F = 25.00, df = 1,16, P = 0.126 and F = 85.05, df = 1,16, P = 0.069, respectively). Similarly, the interaction between Wolbachia infection and adult sex was not significant (F = 0.172, df = 1,16, P = 0.684), (Fig. 9).

Fig. 9
figure 9

Adults flight ability on the Genetic Sexing Strains (GSS). Effect of Wolbachia infection on the flight ability of VIENNA 8 GSS (a) females, and (b) males. White columns represent average percent fliers of the Wolbachia uninfected and grey columns that of Wolbachia infected line. Columns headed with the same letter are not significantly different (Tukey’s HSD test, P > 0.05)

Full size image

Effects of Wolbachia infection on longevity under food and water deprivation

Medfly genetic background, Wolbachia infection and sex were significant predictors of adult longevity under food and water deprivation (Wald’s t-test = 224.17, 37.28 and 30.25, df = 1, P < 0.0001). The significant interaction between medfly genetic background and Wolbachia infection (Wald’s t-test = 39.72, df = 1, P < 0.0001) highlights the differential effect of the Wolbachia infection on the two medfly lines. Specifically, Wolbachia infection reduced VIENNA 8 GSS longevity under water and food deprivation, whereas it increased the BENAKEIO longevity under the same stress conditions (Fig. 10a, b).

Fig 10
figure 10

Adult survival under stress conditions. Survival curves (lx) showing the effect of Wolbachia infection and medfly genotype (a-b), and Wolbachia strain on the same medfly genotype (c-d), on females and males survival under food and water deprivation

Full size image

For the BENAKEIO flies, Cox regression analysis revealed that the Wolbachia strain significantly affected adult longevity under water and food deprivation (Wald’s t-test = 62.01, df = 2, P < 0.0001). Nevertheless, neither sex (Wald’s t-test = 2.26, df = 1, P = 0.133) nor the interaction between the different medfly lines (BENAKEIO genetic background) and sex were significant predictors of adult longevity under food and water deprivation (P > 0.05). Adults of the uninfected Benakeio showed similar stress tolerance compared to S10.3 (Wald’s t-test = 1.58, df = 1, P = 0.208) but much lower compared to the wCer2 infected ones (Wald’s t-test = 35.15, df = 1, P < 0.0001). Adults of the 88.6 line expressed longer survival rates under food and water deprivation compared to S10.3 ones (Wald’s t-test = 54.13, df = 1, P < 0.0001), (Fig. 10c, d).

Focusing on the VIENNA 8 GSS medfly genetic background, statistical analysis showed that Wolbachia infection was not a significant predictor of longevity under stress (Wald’s t-test = 2.734, df = 1, P = 0.098), in contrast to sex (Wald’s t-test = 22.52, df = 1, P < 0.0001) (Fig. 10a, b).

Discussion

Our results demonstrate that Wolbachia infection modifies several fitness components of mass-reared Mediterranean fruit flies. The outcome of the effects seems to be regulated both by medfly genetic background and Wolbachia strain resulting in a complex range of outputs. Wolbachia infection reduces fertility rates in both medfly genetic backgrounds and shortens the pre-pupa developmental duration in the GSS strain. On the other hand, regardless of the strain, Wolbachia infection does not affect either the sex ratio or the longevity of adults. wCer4 infection imposed a reduction in females’ fecundity but wCer2 did not. Males mating competitiveness, adults flight ability and longevity under water and food deprivation were affected by both medfly genetic background of medfly and the strain of Wolbachia (genotype by genotype interaction).

Effects on medfly life-history traits

Similar to earlier studies, our results point out a detrimental effect of Wolbachia infection on fertility in both medfly genetic backgrounds [33,34,35, 43, 44]. Embryonic mortality ranged from 50 to 60% and the effect of the wCer4Wolbachia strain was more pronounced when compared to wCer2. The different effects of Wolbachia strains on hatch rates have also been reported in mosquitoes [43, 44]. Our results are consistent with previous reports, which also mention a fertility advantage of wCer2 over wCer4 [33,34,35]. The amount of sperm transferred and the secretions of male accessory glands could both affect medfly female fertility [45]. Wolbachia infected Drosophila simulans males produce lower sperm quantities and transfer fewer sperm compared to uninfected ones resulting in lower fertility rates [46, 47]. Whether the reduced hatch rates reported here are the result of lower sperm production or sperm transfer because of the Wolbachia infection needs to be explored in future studies.

Wolbachia infection reduced the immature developmental duration in the Vienna 8 GSS line, whereas it seems to prolong the developmental duration in the BENAKEIO line. Working with the same medfly genotypes almost 8 years ago, Sarakatsanou et al. (2011) [35] reported that Wolbachia shortened the developmental duration of immature stages in both the VIENNA 8 and BENAKEIO flies. Considering that the transinfection into VIENNA 8 GSS genotype is more recent than into BENAKEIO genotype [33, 34] the differences recorded in the two studies might reflect the dynamic nature of the symbiotic interactions between medfly and Wolbachia as was also shown in the case of Drosophila simulans [16]. Moreover, it must be pointed out that the two studies were conducted under different rearing protocols, which could also affect the outcome of these effects. Recent studies demonstrated that the Wolbachia titer in Drosophila melanogaster and D. simulans could be nutrient-dependent, and therefore different diets may differentially modify biological traits [48]. In addition, applying different rearing methods in a given insect population could lead to continuous selection for specific characteristics resulting in the establishment of distinct laboratory colonies with slightly different biological traits [49]. Reynolds et al. (2003) [50] recorded that Wolbachia prolongs the developmental duration of immature stages in D. melanogaster, whereas Poinsot et al. (1997) [51] did not detect any effect on D. simulans. Comparing three Wolbachia-infected lines in the same Aedes albopictus genomic background, Zhang et al. (2015) [30] found that wPip accelerated immature development, whereas Islam and Dobson (2006) [52] also reported differences in developmental rates among an uninfected, a single and a superinfected Ae. albopictus line. On the other hand, Wolbachia infection did not affect the developmental rates of Aedes aegypti and Anopheles stephensi [20, 53].

The work of Sarakatsanou et al. (2011) [35] demonstrated that Wolbachia imposes a significant reduction on C. capitata fecundity and adult longevity. In the current study we found that the effect of Wolbachia on medfly fecundity is strain-specific since wCer4 and wCer2 infection induced negative and neutral effects on egg production respectively. Apart from C. capitata, many reports suggest that Wolbachia could elicit positive, negative or neutral response on hosts fecundity and life span indicating that Wolbachia effects could vary among different insect species, strains or even sexes within species [8, 11,12,13,14,15, 17,18,19,20, 29,30,31, 54, 55].

Effects on flight ability, response to food and water deprivation and male mating competitiveness

Medfly mating competitiveness against wild males for wild females is determined both by medfly genetic background of medfly and the Wolbachia strain. Wolbachia infection did not affect the performance of males of the VIENNA 8 GSS, which is the currently used medfly line in most of the SIT large scale operational programs. Previous studies on female preference for mating partners in other insect taxa (Drosophilae and Culicidae) revealed both positive and negative effects of Wolbachia infection. Wolbachia infected D. simulans and D. melanogaster males showed higher mating rates (number of copulations) compared to uninfected ones when a mixed population of infected and uninfected females were offered as mating partners [56]. Similar studies in mosquitoes revealed that Wolbachia-infected males could compete effectively with wild males of Ae. aegypti [57], Aedes polynesiensis [58] and Ae. albopictus [31, 59]. On the other hand, Wolbachia infected Anopheles stephensi males are less competitive against uninfected ones for mating [20]. Large-scale field studies should be conducted in order to fully elucidate effects of Wolbachia in medfly males.

We also investigated the effect of Wolbachia on flight ability (an index of locomotor activity) and adult longevity under food and water deprivation. Wolbachia infection increased the number of fliers on the VIENNA 8 GSS, whereas reduced the number of fliers on BENAKEIO lines. Two previous studies assessed the impact of Wolbachia infection on insects’ locomotor activity. Wolbachia-infected Aedes aegypti adult mosquitoes displayed increased locomotor activity compared to uninfected ones [60]. On the other hand, Dedeine et al. (2001) [61] following similar experimental procedure did not detect significant effects of Wolbachia infection on the locomotor activity of the parasitic wasp Asobara tabida. As far as medfly response under stress conditions is concerned, our results showed that Wolbachia infection reduced the longevity under water and food deprivation in VIENNA 8 GSS females while it did not exert any effect on VIENNA 8 GSS males. On the other hand, by testing the BENAKEIO genotype, we recorded that the wCer2 infection confers a significant improvement in longevity under the given stress conditions to both sexes. To the best of our knowledge, there are no previous data available regarding the ability of Wolbachia infected arthropods to survive under certain stress conditions.

Conclusions

In the present study we evaluated several fitness parameters of medflies comparing the same insect genotype under the presence and absence of Wolbachia. As previously noted, this is the safer path in a trial to detect ultimate benefits or detrimental effects of Wolbachia infection. This is because such an experimental approach minimizes the possibility to attribute effects caused by other factors to Wolbachia infection (e.g. curing the infection with antibiotic is a popular but questionable practice in fitness related studies) [62]. Our findings highlight the determinant role of the genotypes (insect host and Wolbachia) interaction in the expression of specific phenotypes and the potential inconsistency of certain fitness parameters over the symbiosis historic “time-line”. In general, our data reveal that Wolbachia infection could alter important life history traits of mass-reared C. capitata lines. The response of each genotype to Wolbachia infection should be considered toward ensuring the productivity of Wolbachia-infected insects under mass-rearing conditions.

Wolbachia symbiosis could be a promising tool in support of population suppression of insect pests of agricultural, veterinary and human health importance. However, this will first require the evaluation of the potential impact Wolbachia infection may have on key life history traits and particularly on those affecting rearing efficiency and male mating competitiveness of an insect line candidate for SIT and/or IIT applications. Appropriate models should also be developed including cost benefit analysis which will determine their suitability for large scale operational programs.