Introduction
Doryctobracon areolatus is a solitary endoparasitoid, koinobiont of the family Braconidae (Hymenoptera) and native of the Neotropical region that presents a wide distribution from southern Texas to northern Argentina (Ovruski et al., Reference Ovruski, Aluja, Sivinski and Wharton2000). This species is important because it presents the highest natural parasitism on flies of the Anastrepha genus (Montoya et al., Reference Montoya, Ayala, López, Cancino, Cabrera, Cruz, Martínez, Figueroa and Liedo2016; Marinho et al., Reference Marinho, Consoli, Penteados-Dias and Zucchi2017). This dominance can be explained by the fact that females oviposit into the early stages (eggs, first, and second instar larvae) of their hosts (Nunes et al., Reference Nunes, Nava, Muller, Goncalves and Garcia2011; Murillo et al., Reference Murillo, Cabrera-Mireles, Barrera, Liedo and Montoya2015), thereby anticipating the other parasitoid species that later will attack older stages (Wang et al., Reference Wang, Bokonon-Ganta, Ramadan and Messing2004; Montoya et al., Reference Montoya, Ayala, López, Cancino, Cabrera, Cruz, Martínez, Figueroa and Liedo2016, Reference Montoya, López, Cruz, López, Cadena, Cancino and Liedo2017). A relatively small number of parasitoid species attack the eggs and early instar larvae of tephritid flies, but Fopius arisanus (Sonan) and Fopius ceratitivorous (Silvestri) (Hymenoptera: Braconidae) are also recognized for presenting high natural parasitism on Bactrocera and Ceratitis flies in the Indo-Australasian and Afro-tropical regions, respectively (Harris and Bautista, Reference Harris and Bautista1996; Bokonon et al., Reference Bokonon-Ganta, Ramadan and Messing2007). An additional advantage of parasitoid species of early tephritid stages is that the immunological systems of their hosts are not fully developed (Reed et al., Reference Reed, Luhring, Stafford, Hansen, Millar, Hanks and Paine2007; Hegazy and Khafagi, Reference Hegazy and Khafagi2008), facilitating the survival and subsequent adult emergence of the parasitoid progeny.
Biological control of fruit flies has mainly been attempted through augmentative releases of larval parasitoids such as Diachasmimorpha tryoni (Cameron) and D. longicaudata (Ashmead) (Hymenoptera: Braconidae) (Wong et al., Reference Wong, Ramadan, Mcinnis, Mochizuki, Nishimoto and Herr1991; Sivinski et al., Reference Sivinski, Calkins, Baranowski, Harris, Brambila, Diaz, Bums, Holler and Dodson1996; Montoya et al., Reference Montoya, Liedo, Benrey, Cancino, Barrera, Sivinski and Aluja2000). In most cases, the parasitism achieved has been higher than 50%, implying significant suppression of the fruit fly populations. As part of the integrated pest management of fruit fly populations including the sterile insect technique, these parasitoid releases have been performed in marginal areas surrounding commercial orchards, in order to reduce the number of wild flies that could subsequently move into the orchards (Montoya et al., Reference Montoya, Cancino, Zenil, Santiago, Gutiérrez, Vreysen, Robinson and Hendrichs2007). D. areolatus also represents an important alternative as a biological control agent against different fruit fly pests (Serra et al., Reference Serra, Ferreira, García, Santana, Castillo, Nolasco, Morales, Holler, Roda, Aluja and Sivinski2011; Murillo et al., Reference Murillo, Cabrera-Mireles, Barrera, Liedo and Montoya2015). However, it is necessary to develop an economically feasible mass rearing of this species in order to produce sufficient numbers of the parasitoids while maintaining high quality standards (van Lenteren and Tommasini, Reference van Lenteren, Tommasini and van Lenteren2003; Parra, Reference Parra and Capinera2008). One of the main tools with which to achieve efficient mass rearing of fruit fly parasitoids is the use of irradiated hosts (Hendrichs et al., Reference Hendrichs, Bloem, Hoch, Carpenter, Greany and Robinson2009). This facilitates the processes of production and the release of the natural enemy into the field since no adult emergence takes place from the irradiated hosts (Cancino et al., Reference Cancino, Ruiz, Viscarret, Sivinski and Hendrichs2012, Reference Cancino, López-Arriaga and Montoya2016).
The Mexican fruit fly Anastrepha ludens (Loew) (Diptera: Tephritidae) has been used as an irradiated host for rearing different species of fruit fly parasitoids, including the exotic species D. longicaudata (Ashmead) (Hymenoptera: Braconidae) (Cancino et al., Reference Cancino, Ruíz, Gómez and Toledo2002, Reference Cancino, Ruiz, López and Sivinski2009a, Reference Cancino, Ruíz, Sivinski, Galvez and Aluja2009c; López et al., Reference López, Henaut, Cancino, Lambin, Cruz-López and Rojas2009). For this species, it has been possible to produce up to 50 million parasitized pupae per week. The use of irradiated hosts has also been implemented in the rearing of other fruit fly parasitoid species, such as Psyttalia humilis (Silvestri), P. concolor (Szépligeti), Fopius arisanus (Sonan) (Hymenoptera: Braconidae), Trybliographa daci (Weld) (Hymenoptera: Cynipidae), and Dirhinus giffardii (Silvestri) (Hymenoptera: Chalcididae) (Cancino et al., Reference Cancino, Ruiz, López and Sivinski2009a, Reference Cancino, Ruiz, Pérez and Harris2009b; Canale and Benelli, Reference Canale and Benelli2012; Yokoyama et al., Reference Yokoyama, Wang, Aldana, Cáceres, Rendón, Johnson and Daane2012; Sarwar et al., Reference Sarwar, Ahmad, Rashid and Shah2017). The doses of irradiation evaluated have ranged from 10 to 150 Gy.
Preliminary studies show that the use of A. ludens as a host for the rearing of D. areolatus represents a better option than Anastrepha obliqua Macquart (Pérez, Reference Pérez2019), since it is possible to obtain higher parasitism and a sex ratio more biased towards females. Although there is information about the use of irradiated early stages of fruit flies for rearing parasitoids (Palenchar et al., Reference Palenchar, Holler, Moses-Rowley, McGoven and Sivinski2009; Cancino et al., Reference Cancino, Ruiz, Viscarret, Sivinski and Hendrichs2012; Costa et al., Reference Costa, Pacheco, Lopes, Botteon and Mastrangelo2016), the possibility of using this method to rear D. areolatus under laboratory conditions remains unexplored. The objectives of this study were therefore to: (1) determine the effect of different radiation doses on the development of A. ludens eggs and first and second instar larvae, and (2) evaluate the viability of these irradiated hosts for the development of the parasitoid D. areolatus under laboratory rearing conditions.
Materials and methods
Study site and biological material
The study was conducted in the biological control laboratory of the Methods Development Unit of the Moscafrut Program SADER-SENASICA, located in Metapa, Chiapas, Mexico, under environmental conditions of 25 ± 2°C, RH 70 ± 10% and 12:12 h L:D. Adults of D. areolatus were provided by this laboratory, while the first and second instar larvae of A. ludens were provided as 4-day-old eggs by the Moscafrut Plant, where this species is mass reared according to Orozco et al. (Reference Orozco-Dávila, Quintero, Hernández, Solís, Artiaga, Hernández and Montoya2017). The eggs were placed in the larval diet in order to obtain the required instar larvae.
Viability of irradiated eggs and first and second instar larvae of Anastrepha ludens
The 4-day-old eggs, and the first (3 days of development in diet) and second (5 days of development in diet) instar larvae were irradiated at doses of 20, 30, 40, and 50 Gy. The control treatments comprised other non-irradiated eggs and first and second instar larvae. Radiation was applied with a Gamma Beam 127 (Nordion®Otawa, ON, Canada) panoramic irradiator with a Cobalt 60 dry storage source at an irradiation rate of 2.20 Gy min−1.
The eggs (~4000 per replicate) were irradiated in a humid chamber (Petri dish of 100 × 15 mm with moistened filter paper) and then placed on a plastic tray containing larval diet. The first and second instar larvae were irradiated on plastic trays (25 × 15 × 5 cm3) containing diet. In each case, the eggs and larvae continued with their development until reaching 9 days of age. To determine the percentages of hatching of the irradiated and non-irradiated (0 Gy) eggs, samples of 100 irradiated eggs were taken from the different doses and aligned on a piece of blue satin cloth (1 × 10 cm2), which was placed on cotton wool saturated with water in a Petri dish (9.5 cm in diameter × 0.8 cm in height), thus forming a humid chamber. The eclosion of these eggs was quantified at 48 h using a stereomicroscope (Carl Zeiss® Model Discovery V.8 Carl Zeiss, Göttingen, Germany). Fourteen replicates were evaluated for each dose of irradiation.
Larval development took place under the environmental conditions described above. On the 9th day, the third instar larvae of each treatment were weighed. A 10 g sample of diet with larvae was taken, weighed on a semi-analytical balance (OHAUS® Model PA512C, Ohaus Corporation, NJ, USA), and the total number of larvae counted. These data were submitted to the following formulae: No. larvae/No. eggs sown × 100, to obtain the percentage of egg-larva transformation; and No. larvae recovered/1000 g of diet, to obtain the yield percentage as the number of larvae per 1 g of diet. Ten replicates were performed per treatment.
Larval weight was obtained by considering the individual weight (mg) of 20 larvae on an analytical balance (OHAUS® Model AP2500, Ohaus Corporation, NJ, USA), with five replicates performed per treatment. A sample of 250 larvae was taken and placed in a plastic container (9 × 45 cm2) with coconut fiber as a pupation substrate. Three days later, the percentage of pupation at 72 h was determined, and we assume that not pupating larvae at 72 h had a delay in their development. The pupal weight was obtained from ten 13-day-old pupae in each treatment. In cases where the irradiation dose did not suppress the development of flies, the percentage of emergence was obtained from a sample of 250 larvae, which were enclosed in a cylindrical plastic container (9 × 4.5 cm2) and maintained until adult emergence. Five replicates per treatment were conducted for each parameter.
Exposure of irradiated larvae to Doryctobracon areolatus
Based on our preliminary results of the previous phase, parasitoid development was evaluated in second instar larvae of A. ludens derived from eggs and first and second instar larvae that had been irradiated at 20 and 30 Gy. Another group of non-irradiated larvae of the same age was used as a control.
Both groups of larvae were exposed separately to adults of D. areolatus (5 ♀: 5 ♂) of 5 days of age for 24 h, confined in a plexiglass cage (20 × 20 × 20 cm3) and provided with water and food (mixture of bee honey and tissue paper; see Montoya et al., Reference Montoya, Pérez-Lachaud and Liedo2012). Larval exposure was conducted using near-ripe guava fruits (Psidium guajava L.) as an oviposition substrate. The pulp and seeds of these fruits were extracted and several perforations made with a dissection needle through the peel of the fruit in order to enable internal aeration. The larvae mixed with diet were placed inside these fruits, and a polygonal plastic bead (2.5 cm in diameter) was added to occupy the center of the fruit in order to ensure that the larvae remained close to the fruit surface. During the larval exposure, these oviposition substrate units were hung from the roof of the cage using a wire that passed through the center of the fruit. After exposure, the larvae were placed in a cylindrical plastic container (9 × 45 cm2) with diet in order to continue their development. At 9 days of larval age, the exposed larvae in each treatment were separated from the diet by washing with water and placed back in the same container, but this time using coconut fiber as the pupation substrate. They were maintained for 25 ± 2 days at 26 ± 2°C and 60–80% RH until emergence of the adults. Percentage of emergence and sex ratio were then calculated from the adult parasitoids. The adults emerged from each treatment were used to evaluate parameters of flight ability, survival and fecundity.
To evaluate flight ability, different quantities of adults were used as follows as they emerged from each treatment: (eggs, 0 Gy = 56 adults; eggs, 20 Gy = 47 adults; eggs, 30 Gy = 66 adults; first instar larvae, 20 Gy = 41 adults; first instar larvae, 30 Gy = 27 adults; second instar larvae, 20 Gy = 76 adults; second instar larvae, 30 Gy = 48 adults). These adults were placed into plastic containers (9 × 4.5 cm2) with no food or water. Flight ability was determined in a similar manner to that of fruit flies (FAO/IAEA/USDA, 2014), but with some adjustments. In this case, the adult parasitoids emerged from each treatment were transferred using a plastic aspirator and placed at the bottom of a black PVC tube (10 cm in diameter × 8 cm in height) with its internal wall covered in neutral talcum powder to prevent the parasitoids climbing out. The tube was placed in a cage (30 × 30 × 30 cm3), in which the number of parasitoids that left the tube by flight was determined over a period of 24 h. The percentage of flying parasitoids was then calculated from the proportional difference of adults that flew out, relative to those that did not escape the tube. The test was performed at 25 ± 2°C, 70 ± 10% RH, with a light intensity of 1500 lux (at the top of the tubes). Five replicates were conducted for each treatment.
To evaluate adult survival, different quantities of adults/pairs (1 ♀: 1 ♂) were used as they emerged from each treatment (eggs, 0 Gy = 53 pairs; eggs, 20 Gy = 48 pairs; eggs, 30 Gy = 47 pairs; first instar larvae, 20 Gy = 46 pairs; first instar larvae, 30 Gy = 41 pairs; second instar larvae, 20 Gy = 52 pairs; second instar larvae 30 Gy = 55 pairs). These were placed in plastic containers (9 × 4.5 cm2) without food and water. The total number of dead adults per treatment was recorded daily until total mortality was achieved.
Fecundity was evaluated for each treatment using 20 pairs (1 ♀: 1 ♂) of 5-day-old adults, which were maintained in a plexiglass cage (20 × 20 × 20 cm3) with water and food as described above. Ten larvae were exposed daily to each parasitoid pair using the oviposition substrate described above. The exposed larvae were removed and maintained as described above until obtaining the offspring. This was carried out for 5 consecutive days (from 5 to 9 days of age). The number of male and female offspring per female per day was registered.
Statistical analysis
The percentage of hatched eggs was analyzed using a simple ANOVA. The percentage of egg-larva transformation, larval yield, larva and pupa weight, and percentage of pupation were analyzed with a two-way ANOVA, considering the developmental instar and irradiation dose as factors. In the evaluations with parasitoids, larval weight, percentage of emergence, sex ratio, percentage of flying adults and fecundity were analyzed with a simple ANOVA, in both cases using the Tukey's test to compare means. Prior to analysis, the assumptions of equal variances were verified and data were transformed using Arcsine, log 10, and box-cox options. Due to the lack of normality, the data of fly emergence was analyzed using a generalized linear model with a binomial response and logit function. Differences among the slopes of the survival data of emerged adults in each treatment were analyzed with a Log Rank test based on a χ2 distribution. All analyses were conducted with the statistical software JMP version (5.0.1).
Results
Viability of irradiated eggs and first and second instar larvae of Anastrepha ludens
No significant difference was found in the percentage of eggs hatched at 48 h among the different radiation doses applied (F = 1.18; df = 4, 65; P = 0.32) (Table 1). Similarly, the percentage of egg-larval transformation did not differ statistically among the eggs and the different instar larvae (F = 0.57; df = 2, 135; P = 0.56), or at the different doses (F = 0.39; df = 4, 135; P = 0.81). No significant interaction was found between doses and host instars (F = 0.56; df = 8, 135; P = 0.80). The yield of larvae (number of larva per g of diet) did not differ statistically between the eggs and larvae (F = 0.52; df = 2, 135; P = 0.59), or at the different doses (F = 0.37; df = 4, 135; P = 0.82). No significant interaction was found among doses and eggs and the different instar larvae (F = 0.52; df = 8, 135; P = 0.83).
Table 1. Mean (±SE) percentage of egg hatch and egg-larva transformation; larval yield, larval weight, pupal weight, percentage of pupation at 72 h and emergence of A. ludens irradiated as eggs and as first and second instar larvae, at different irradiation doses
Values followed by different letter among columns denote significant differences. Egg hatching: ANOVA and Tukey's HSD; egg larva transformation, larval yield, larval weight, pupal weight, and pupation: two-way ANOVA; emergence: generalized lineal model (P < 0.05).
Larval weight decreased as the dose of radiation increased (F = 73.58; df = 4, 1485, P < 0.001) (Table 1). The subsequent larval weight of A. ludens subjected to irradiation at doses of 40 and 50 Gy at the egg stage was significantly lower than that obtained when irradiation was applied to the first and second instar larvae (F = 212.22; df = 2, 1485; P < 0.0001). There was a significant interaction between doses and developmental instars (F = 18.43; df = 8, 1485; P < 0.001). The 13-day-old pupal weight of the control differed significantly to that of the eggs and larvae that had been exposed to radiation (F = 46.77; df = 2, 735; P < 0.0001). There was a significant effect on the pupal weight of the different radiation doses applied (F = 182.1; df = 4, 735; P < 0.0001), and a significant interaction was observed between doses and developmental instars (F = 9.37; df = 8, 735; P < 0.0001). The percentage of pupation at 72 h decreased significantly with increased radiation doses applied to the eggs (F = 28.63; df = 4, 135; P < 0.0001). This decrease was not observed in either the irradiated first or second instar larvae, apart from a difference found between these and the control (F = 9.63; df = 2, 135; P < 0.0001). There was a significant interaction between radiation dose and the developmental instars (F = 3.15; df = 8, 135; P = 0.002).
There was a significant effect of radiation dose on the emergence of parasitoids (χ2 = 31,269.49; df = 4; P < 0.0001), but emergence was similar among eggs and different instar larvae (χ2 = 0.0002; df = 2; P = 0.99). There was a significant interaction between doses and developmental instars (χ2 = 685.82; df = 8; P < 0.0001). No emergence was recorded from first and second instar larvae in any of the tested irradiation doses, unlike in their respective control treatments. However, at 20 Gy, exposed eggs produced 25.52% of emerged fly adults, which differed statistically from the control (Table 1).
Exposure of larvae to Doryctobracon areolatus
Emergence of adult parasitoids developed from second larval instar exposed to D. areolatus after irradiated at the egg stage and as first and second instar larvae, presented no significant difference (F = 0.71; df = 6, 28; P = 0.63) (Table 2). The applied radiation had no influence on the sex ratio of the adult parasitoids that developed from eggs or from first and second instar larvae (F = 0.83; df = 6, 28; P = 0.545) (Table 2). Similarly, the attributes of flight ability, survival and fecundity did not differ significantly among treatments (flight ability: F = 1.71; df = 6, 28; P = 0.15, survival: Log Rank χ2 = 0.21; df = 6; P = 0.99, fecundity: F = 1.0; df = 6, 133; P = 0.40) (figs 1–3).
Table 2. Mean (±SE) percentage emergence and sex ratio of D. areolatus obtained from exposure to second instar larvae of A. ludens, irradiated as eggs and as first and second instar larvae at 20 and 30 Gy
Values followed by a different letter among columns denote significant differences, ANOVA and Tukey's HSD (P < 0.05).
Figure 1. Percentage of adult flying D. areolatus emerged from eggs and from first and second instar larvae irradiated at 20 and 30 Gy.
Figure 2. Fecundity of female D. areolatus emerged from eggs and from first and second instar larvae irradiated at 20 and 30 Gy.
Figure 3. Survival of adult D. areolatus emerged from second instar larvae of A. ludens irradiated as eggs and as first and second instar larvae at 20 and 30 Gy.
Discussion
Irradiation with gamma rays applied to the eggs and intermediate instar larvae of A. ludens may represent a feasible and convenient strategy for rearing D. areolatus, since the adults that emerged from these immature stages did not present significant differences from non-irradiated individuals in terms of the aptitude parameters evaluated. Irradiation of hosts prior to their exposure to parasitoids in the context of mass rearing acts to prevent the emergence of adult flies, eliminating the risk of their liberation into the field during releases of the natural enemy (Sivinski and Smittle, Reference Sivinski and Smittle1990).
Our results indicate that it is possible to use second instar larvae (irradiated as eggs or as first instar larvae) for rearing D. areolatus. According to Costa et al. (Reference Costa, Pacheco, Lopes, Botteon and Mastrangelo2016) and Cai et al. (Reference Cai, Gu, Yao, Zhang, Huang, Idress, Ji, Chen and Yang2017), the use of such early stages offers the advantage of simplifying the irradiation procedure. However, it is important to highlight that young developmental stages of the insects are also more susceptible to the negative effects of radiation (Benschoter and Telich, Reference Benschoter and Telich1964) since the organisms experience several important metabolic changes during these stages (Hallman and Worley, Reference Hallman and Worley1999; Han et al., Reference Han, Song, Yun and Yi2006; Mastrangelo and Walder, Reference Mastrangelo, Walder and Singh2011). Although no difference in the aptitude parameters of the emerged parasitoid was observed using early instars, the results indicated that both percentage of pupation at 72 h and pupal weight were lower at doses of up to 30 Gy in irradiated eggs. Mastrangelo and Walder (Reference Mastrangelo, Walder and Singh2011) previously suggested that radiation of earlier stages could be associated with reduced development and thus lower survival of the irradiated hosts, which would decrease the efficiency of any mass rearing process. Painthankar et al. (Reference Painthankar, Deeksha and Patil2017) reported negative effects on the pupation capacity of Drosophila melanogaster when larvae were irradiated at intermediate instar; Cai et al. (Reference Cai, Gu, Yao, Zhang, Huang, Idress, Ji, Chen and Yang2017) reported that doses above 20 Gy in eggs of Bactrocera dorsalis (Hendel) caused reduced pupal weight.
No significant effects of radiation were observed on the development of immature fly stages, suggesting that the use of irradiated A. ludens larvae for rearing D. areolatus offers operational advantages. Doses of 30, 40, and 50 Gy, applied to eggs and to first and second instar larvae, were effective in suppressing the emergence of adult flies, and the delay in host pupation observed at 72 h was only significant when radiation was applied to eggs. This effect was also reported on third instar larvae of B. dorsalis (Balock et al., Reference Balock, Burditt and Christenson1963; Chang et al., Reference Chang, Goodma, Ringbauer, Geib and Stanley2016), with no negative repercussions for the development of parasitoids (Yulo-Nazarea and Marroto, Reference Yulo-Nazarea and Marroto1993; Cancino et al., Reference Cancino, Ruiz, Pérez and Harris2009b). Irradiation affects the pupal development of the host, but is independent of the physiology of the parasitoid (Nation et al., Reference Nation, Smittle, Milne and Dykstra1995). The emergence of adult parasitoids from the second instar larvae of A. ludens was similar regardless of which host developmental instar was irradiated and did not differ from the control. Palenchar et al. (Reference Palenchar, Holler, Moses-Rowley, McGoven and Sivinski2009) also reported that emergence of D. areolatus in irradiated second instar larvae (70 Gy) of Anastrepha suspensa (Loew) did not differ from that of the non-irradiated hosts.
With applied irradiation doses of 20 and 30 Gy, the emergence, sex ratio, survival, flight ability and fecundity of the emerged parasitoids were similar among the different treatments, probably because of the relatively small doses of radiation involved and because the range of variation among these doses was narrow. Different studies (e.g., Sivinski and Smittle, Reference Sivinski and Smittle1990; Cancino et al., Reference Cancino, Ruiz, Viscarret, Sivinski and Hendrichs2012) have documented that irradiation applied to inhibit the emergence of non-parasitized hosts has no influence on the fecundity and longevity of the parasitoids that emerge from the parasitized hosts.
Cancino et al. (Reference Cancino, Ruiz, Viscarret, Sivinski and Hendrichs2012) recommend the use of higher irradiation doses once the rearing of parasitoid species has been scaled up to millions per week. This because in large volumes of biological material, the gamma rays undergo greater dispersion thus increasing the risk of applying a lower dose than required to ensure the non-emergence of adult hosts. For instance, irradiation of third instar A. ludens larvae at 40 Gy is routinely applied in the mass production of Diachasmimorpha longicaudata (Ashmead) (Hymenoptera: Braconidae), an exotic parasitoid species of fruit fly pests (Cancino et al., Reference Cancino, Ruiz, López and Sivinski2009a), although this dose also works with third instar larvae of Anastrepha serpentina (Wiedemann) and A. obliqua as alternative hosts.
The results obtained in this study suggest that second instar larvae irradiated at 30 Gy can be used for the rearing of D. areolatus, since these larvae are less susceptible to the inevitable manipulation during the rearing process. Once the rearing of D. areolatus is extended to levels of millions per week, it may be necessary to reassess the appropriate irradiation dose in order to ensure the non-emergence of adult flies, as in the case with D. longicaudata production (Cancino et al., Reference Cancino, Ruiz, Viscarret, Sivinski and Hendrichs2012; Montoya et al., Reference Montoya, Pérez-Lachaud and Liedo2012).
Acknowledgements
We thank Patricia López, Amanda Ayala, Erik Flores, and Velisario Ribera of the Department of Biological Control of the Moscafrut Program SADER-SENASICA for technical support. Javier Valle-Mora (ECOSUR) provided advice regarding the statistical analyses. The Moscafrut Plant provided the biological material, equipment, and technical support for irradiation.
