In fl uence of natal host on parasitism by Spalangia cameroni ( Hymenoptera : Pteromalidae )

Spalangia cameroni Perkins (Hymenoptera: Pteromalidae) is sold commercially as a biocontrol agent of fi lth fl ies, including the house fl y, Musca domestica L. (Diptera: Muscidae). For this reason, S. cameroni is mass-reared for inundative releases to control harmful fl ies. However, the mass-rearing protocols include very little information on the infl uence of natal host on subsequent host selection by parasitoids with more than one potential host as in the genus Spalangia. Here, we report on the use of S. cameroni against M. domestica. The S. cameroni were reared using Ceratitis capitata Wiedemann (Diptera: Tephritidae) (natal host) pupae for several generations. Freeze-killed fl y pupae were used in assays to determine the fecundity, number of adult progeny and sex-ratio of this parasitoid. Realized fecundity and number of adult progeny were greater when provided with house fl y pupae than Mediterranean fruit fl y pupae. Thus S. cameroni parasitized more house fl y pupae than C. capitata pupae, even though the parasitoid was reared on Mediterranean fruit fl y for many generations. These results indicate that S. cameroni reared on C. capitata can be successfully used in inundative releases against both fruit fl ies (agriculture) and house fl ies (livestock farming).


INTRODUCTION
At the Valencian Institute of Agrarian Research (Instituto Valenciano de Investigaciones Agrarias, IVIA, Valencia, Spain) Spalangia cameroni Perkins (Hymenoptera: Pteromalidae) has been reared for use in biological control against the Mediterranean fruit fl y since 2003. During this time the Medfl y Ceratitis capitata (Wiedemann) was used as the host (Tormos et al., 2010(Tormos et al., , 2014. Recently, we decided to use S. cameroni to control Diptera that are common pests of feedlot cattle and in animal breeding facilities, such as Musca domestica L. and the stable fl y, Stomoxys calcitrans (L.) (Diptera: Muscidae).
In biological control, the host used for rearing parasitoids is important as according to Hopkins' Host-Selection Principle it may infl uence their subsequent choice of host and percentage parasitism (Davis & Stamps, 2004). Currently, little is known about conditioning and host selection in parasitoids of muscoid fl ies, but in other species the host in which the female parasitoid is reared has a signifi cant effect on their host-seeking behaviour and oviposition (Ferrero, 2008). In this study, therefore, we determined wheth- Eur. J. Entomol. 113: 99-103, 2016 doi: 10.14411/eje.2016.012

ORIGINAL ARTICLE
nated with honey and a pair (male and female) of 4-5 day-old parasitoids, which had been kept together since their emergence. Each treatment lasted for 3 consecutive weeks and, therefore, each replicate lasted one week. At the beginning of each week, each pair of parasitoids was confi ned with 10 host pupae for 24 h on three occasions over the course of a week. Different pairs of parasitoids were used in each replicate. At the end of each week we determined the total number of eggs laid per female (realized fecundity), total number of adult offspring produced per female (adult progeny), incidence of superparasitism (presence of > 1 egg per pupa) and sex ratio. In order to determine realized fecundity and superparasitism, 450 pupae from each treatment were dissected under a binocular microscope (Leica MZ8), with the aid of mounted needles and soft tweezers (Tormos et al., 2009). The remaining 450 pupae of each treatment were allowed to develop in order to determine the number of adult progeny that emerged S. cameroni is one of the most commonly used parasitoids in the biological control of the house fl y, M. domestica, and stable fl y, S. calcitrans (pest of equine facilities, see Machtinger & Geden, 2015). Inundative releases of several species of Spalangia are now being used in several countries. Under some conditions, these releases have resulted in levels of parasitism suffi cient to control populations of these fl ies and keep them below economic thresholds (Morgan & Patterson, 1990;Geden et al., 1992;Crespo et al., 1998;Kaufmann et al., 2001;Steenberg et al., 2001;Skovgård & Nachman, 2004;Geden & Hogsette, 2006).
Currently there is information that supports the effectiveness of using parasitoids, in particular S. cameroni, to control harmful fl ies on farms. Consequently S. cameroni, reared on house-fl y pupae (Kaufman & Geden, 2009) are used against fi lth fl ies in several countries. In this study we reared S. cameroni on C. capitata with the following objective: to assess the potential use of S. cameroni reared on C. capitata to control M. domestica.

Study center and insects *
This study was carried out at the Valencian Institute of Agrarian Research (IVIA, Valencia, Spain). Freeze-killed host pupae were used because previous studies indicate these pupae are suitable hosts for S. cameroni (Tormos et al., 2010;Ogawa et al., 2012). Pupae of the Mediterranean fruit fl y and adults of S. cameroni were obtained from cultures reared following the standard protocol used at IVIA, where they have been reared for over 6 years. The laboratory conditions were 21-24°C, RH 55-80% and a 16L : 8D photoperiod. Live pupae of the house fl y provided by BioFlyTech UA (Alicante, Spain) were used to determine the minimum time it took house fl y pupae to die at -20°C. The time required to kill Mediterranean fruit fl y pupae at -20°C was previously determined by Tormos et al. (2010). Both Mediterranean fruit fl y and house fl y pupae used in the experiments were 2 day old when freeze killed. In both cases, after being frozen, they were stored at 4-5°C for a period of 6-7 days prior to being confi ned with parasitoids for 24 h.

Experimental design
To determine the minimum time necessary to kill house fl y pupae by freezing them, 2-day-old pupae were divided into 5 groups each of 65 pupae, each group of which was placed in a glass Petri dish (5 × 2 cm). The pupae were exposed to -20°C for 15, 30, 60, 90 or 120 min (Table 1). The age of the pupae, temperature, duration of exposure and number of repetitions was based on previous experiments using other hosts of S. cameroni (Geden & Kaufman, 2007;Tormos et al., 2010Tormos et al., , 2014. The treated pupae were then transferred to a climatic chamber at 25 ± 1°C, RH 55-85% and a 16L : 8D photoperiod. Untreated pupae (n = 65), were also placed in the climatic chamber and used as controls.
The effect of host species (C. capitata or M. domestica) on the reproductive behaviour of the parasitoid was recorded in two treatments performed simultaneously (Fig. 1). Each treatment consisted of three replicates in each of which 30 pairs of parasitoids were confi ned individually in a container with a Petri dish containing pupae (90 in total). In each container (transparent plastic, 15 cm length × 10 width × 10 height) there was an uncovered Petri dish base (Ø 3 cm) containing a group of host pupae, a Petri dish top containing sugar, a bowl of water, blotting paper impreg-* Experiments comply with current Spanish legislation. and their sex ratio. Throughout this study, the containers were kept in a Sanyo climatic chamber (MLR350; Sartorius, Barcelona, Spain) at 24 ± 1°C, 55-65% relative humidity and a photoperiod of 16 h per day.

Statistical tests and analyses
A two-way factorial ANOVA was used to test the effect of the fi xed factor: Mediterranean fruit fl y pupa/house fl y pupa, and a random factor: time period (week) in which the pairs of parasitoids were kept with pupae on the variables: (1) realized fecundity; (2) number of pupae parasitized; (3) superparasitism; (4) number of adult progeny and (5) proportion that were females. Chi squared tests (χ 2 ) was also used to analyze the sex-ratios. Values are reported as means ± SE. Analyses were performed using the IBM SPSS statistical software package (IBM, Spain; v20; critical p value used 0.05).

RESULTS
Temperature and minimum exposure time resulting in 100% mortality of M. domestica pupae was -20°C for 120 min (Table 1).

DISCUSSION AND CONCLUSIONS
It is reported that the pupae of M. domestica are killed if kept at -80°C for 10 min (Geden & Kaufman, 2007) and those of C. capitata if kept at -20°C for 60 min (Tormos et al., 2010).
S. cameroni is an idiobiont pupal parasitoid with a broad range of dipterous hosts, harmful to both crops and livestock (Birkemoe et al., 2009;Tormos et al., 2014). Consequently, since 2006, when S. cameroni was fi rst identifi ed as a parasitoid of C. capitata (Falcó et al., 2006), the Valencian Institute for Agricultural Research (IVIA) in Spain started rearing S. cameroni on C. capitata for controlling the Mediterranean fruit fl y. This parasitoid continued to be bred on the fruit fl y pupae for the following reasons: (a) according to Hopkins' Host-Selection Principle, as a parasitoid develops inside a host it is conditioned visually and olfactorily, which enables the adult parasitoid to locate the same type of host in which to lay its eggs (Craighead, 1921;Smith & Cornell, 1979;Davis & Stamps, 2004). Little is known about conditioning and host selection in parasitoids of muscoid fl ies; however, in Trichopria nigra (Nees) (Hymenoptera: Diapriidae), a pupal endoparasitoid of several common species of fl ies, the host in which the female parasitoid is reared has a signifi cant effect on their host-seeking behaviour and oviposition, with approximately twice as many females choosing their natal host over a non-natal host (Ferrero, 2008). In addition, (b) laboratory data on fecundity, number of adult progeny, host-induced mortality and sex ratio of S. cameroni reared on C. capitata are acceptable (Pérez-Hinarejos & Beitia, 2008;Tormos, 2010Tormos, , 2014. Recently, problems caused by M. domestica in animal breeding facilities in the region of Valencia (Spain) (authors' pers. observ.) led us to determine the effectiveness of S. cameroni, reared for several generations on C. capitata pupae in parasitizing M. domestica pupae. Little is known about the effects of the host in which Spalangia is reared on its subsequent choice of host (Mandeville & Mullen, 1990); however, our results demonstrate that a parasitoid reared for many generations on C. capitata is a more effective parasite of M. domestica. Measures of biotic potential, such as realized fecundity, number of pupae parasitized and number adult progeny emerging, were higher when freeze-killed pupae of M. domestica were used as hosts for S. cameroni than those of C. capitata. The sex ratio of the parasitoids emerging from both hosts was almost the same and the incidence of superparasitism was higher when M. domestica was the host. The higher level of parasitism of the house fl y could be due to the greater size of M. domestica pupae or other characteristics (for example odour), making them appear more benefi cial or attractive to ovipositing females of S. cameroni (King, 2002;Machtinger & Geden, 2015).
Regarding the use of S. cameroni obtained from pupae of C. capitata for controlling M. domestica we have to take into account the possibility of natal conditioning when this parasite is used as a biocontrol agent where Mediterranean  Mandeville & Mullens (1990). In their study, the strong innate preference of M. zaraptor for house fl y over F. canicularis pupae shifted in favour of the latter species after two days of ovipositing on the latter. This shift occurred in spite of the fact that M. zaraptor is substantially more successful when it parasitizes M. domestica than F. canicularis. S. cameroni reared on C. capitata can be used, in inundative releases, for the biological control of fruit fl ies (agriculture) and house fl ies (livestock farming). Studies carried out on dairy and pig farms show that S. cameroni is effective in controlling M. domestica, as it can significantly reduce the numbers of house fl ies and decrease levels of discomfort (Skovgård & Nachman, 2004). In addition, companies such as Productos Biológicos Perkins Ltda (http://perkinsltda.com.co/) have optimized protocols for the use of S. cameroni as a biocontrol agent and sell them commercially for use against fl ies present in feedlots, stables, amongst pasture cattle and in piggeries.