Appl. Entomol. Zool. 44 (4): 535–541 (2009) http://odokon.org/ Parental genetic traits in offspring from inter-specific crosses between introduced and indigenous Diadegma Foerster (Hymenoptera: Ichneumonidae): Possible effects on conservation genetics Andrew P. DAVIES,1,*,† Kenji TAKASHINO,2 Masaya WATANABE3 and Kazuki MIURA1,3 1 National Agriculture and Food Research Organization, National Agricultural Research Center for Western Region; Fukuyama, Hiroshima 721–8514, Japan 2 National Agriculture and Food Research Organization, National Agricultural Research Center for Tohoku Region; Morioka, Iwate 020–0198, Japan 3 Graduate School of Biosphere Science, Hiroshima University; Higashi-Hiroshima, Hiroshima 739–8511, Japan (Received 4 April 2009; Accepted 2 June 2009) Abstract The effects of natural enemy releases on conservation genetics within ecosystems are rarely considered. Diadegma semiclausum (Hellen) (Hymenoptera: Ichneumonidae) was introduced and continues to be released for biological con- trol of diamondback moth in Japan. Diadegma semiclausum and indigenous Diadegma fenestrale (Holmgren) (Hy- menoptera: Ichneumonidae) share geographic ranges and hosts, and produce offspring when mated under laboratory conditions. We used DNA to examine whether offspring from inter-specific one-way parental crosses (D. semiclausum / and D. fenestrale ?) were hybrid, as some Hymenoptera (e.g. Aphidius colemani Viereck (Hymenoptera: Bra- conidae)) exhibit thelytokous reproduction by gynogenesis. Molecular analyses revealed offspring mtDNA (COI) is maternally inherited, as expected, but rRNA (ITS-2) originates from both parents. Should similar hybridization occur in the field beyond the F1 generation, genetic mixing is a possible consequence that may influence biological control efficacy or pollute native population genetics. Key words: Diadegma semiclausum; D. fenestrale; hybrid; conservation genetics species’ limits and biology (Frankham et al., INTRODUCTION 2005). The application of genetics within conser- Classical biological control involves the suppres- vation biology assumes that inbreeding and loss of sion of pest populations through the importation of genetic variation increase the risk of extinction natural enemies from the pest’s place of origin for (Frankham, 1995; Frankham et al., 2005). Here we mass rearing and release (Van Driesche and Bel- reveal molecular evidence of hybridization between lows, 1996). Environmental implications associ- Diadegma Foerster parasitoids, one introduced and ated with the deliberate introduction of exotic one indigenous, which share hosts and geographic species for biological control, such as possible im- ranges in Japan, and discuss the implications of our pacts on endangered species and risks to non-target results in relation to conservation genetics. hosts, have often been ignored (Howarth, 1991; Parasitoids of the genus Diadegma incorporate Lynch and Hokkanen, 1995; Duan and Follet, 201 species (Yu and Horstmann, 1997), several of 1999). Conservation genetics utilizes genetic meth- which have been introduced for biological control ods to help preserve species as dynamic entities ca- of lepidopteran pests worldwide, most notably pable of coping with environmental change, and in- against the diamondback moth (DBM), Plutella cludes resolution of taxonomic uncertainties and xylostella (L.) (Lepidoptera: Plutellidae) (Sarfraz the use of molecular analyses in understanding et al., 2005). DBM is considered the most econom- * To whom correspondence should be addressed at: E-mail: [email protected] † Present address: USDA-ARS-CMAVE, 1600/1700 SW 23rd Drive, Gainesville, FL 32608, USA. DOI: 10.1303/aez.2009.535 535 536 A. P. DAVIES et al. ically damaging pest of cruciferous crops world- unfertilized, haploid eggs and female offspring from wide, with annual control costs estimated at one fertilized, diploid eggs. Thelytokous parthenogene- billion US dollars in the early 90’s (Talekar, 1992). sis, where females can produce daughters without Diadegma semiclausum (Hellen), a larval para- mating, is also found in the Hymenoptera. Wol- sitoid with a Palearctic distribution, has been cred- bachia Hertig are known to induce thelytoky in ited with the effective suppression of DBM in many Trichogramma Westwood (Hymenoptera: South-East Asian agro-ecosystems following its in- Trichogrammatidae) species (Stouthamer, 1989, troduction (Talekar and Shelton, 1993; Sarfraz et 1997; Stouthamer et al., 1990; Grenier et al., 1998; al., 2005). Diadegma semiclausum was likewise in- Huigens et al., 2000; Pintureau et al., 2000) where troduced to Japan, and continues to be periodically infected females produce female offspring from released for suppression of DBM in crucifer crops unfertilized eggs, while unfertilized, uninfected despite the presence of indigenous D. fenestrale eggs normally develop into males. This phenome- (Holmgren), as the latter is not considered effective non is not restricted to Trichogramma and has been alone (K. Miura, personal communication). found in a wide range of hymenopteran taxa An ongoing problem associated with the contin- (Stouthamer, 1997). Although not tested, it is un- ued use of Diadegma in biological control is the likely that offspring produced from previous confusion over species status within the genus (Fit- crosses between D. semiclausum and D. fenestrale ton and Walker, 1992). In an attempt to alleviate in the laboratory (Hardy, 1938) were induced this problem, Azidah et al. (2000) conducted mor- parthenogenetically due to Wolbachia infection as phometric analyses of Diadegma attacking DBM both parents were obtained from sexual popula- and reported seven distinct morphospecies, how- tions and, to date, Wolbachia infection has only ever their results did not definitively separate mor- been reported in Diadegma insulare (Cresson) phologically similar D. semiclausum and D. fenes- (Hymenoptera: Ichneumonidae) (Jeyaprakash and trale, among others. The authors gave three possi- Hoy, 2000) and Diadegma chrysostictos (Gmelin) ble explanations for this: high intraspecific varia- (Hymenoptera: Ichneumonidae) (Cook and Butcher, tion, missing values due to damaged specimens, 1999) where it is not known to induce thelytoky. and possible hybridization, which had been ob- Laboratory crosses between arrhenotokous D. served previously (see Hardy, 1938). Azidah et al. semiclausum and D. fenestrale have produced off- (2000) concluded, “Poorly resolved taxonomy is spring possessing morphological features charac- still a major factor in limiting exploitation of teristic to each parent (Hardy, 1938), but hybridiza- Diadegma species in biological control of Plutella tion has yet to be confirmed by molecular methods. xylostella”. For example, crosses between geographically sepa- Recent molecular analyses utilizing PCR-RFLP rated sub-species of the parasitoid Aphidius cole- of the ITS-2 (rRNA) region effectively discrimi- mani have induced thelytoky following failed cop- nated between seven Diadegma species attacking P. ulation attempts, termed gynogenesis (Tardieux xylostella, including D. semiclausum and D. fenes- and Rabasse, 1988), and this may be occurring trale, with the CfoI enzyme and one new species with Diadegma. We analyzed phylogenetic relation- was reported (Wagener et al., 2004). Later phylo- ships between a D. semiclausum mother crossed genetic studies found D. semiclausum and D. fenes- with D. fenestrale fathers and their offspring using trale had 3.9 and 8.0% nucleotide diversities of COI and ITS-2 fragment sequences in an attempt COI (mtDNA) and ITS-2 sequences, respectively, to detect molecular evidence of hybridization, and and the two species were placed in distinct phylo- so discount the possibility that gynogenesis is oc- genetic clades (Wagener et al., 2006). Although curring. these molecular studies provide effective tools for discrimination of Diadegma species, they do not MATERIALS AND METHODS address problems associated with possible hybridiza- tion between D. semiclausum and D. fenestrale. Diadegma culturing and hybridization. Sepa- Diadegma, like most Hymenoptera, reproduce rate D. semiclausum and D. fenestrale adult cul- parthenogenetically, the most common form being tures were maintained in the laboratory at 15Ϯ1°C, arrhenotoky, in which male offspring arise from ambient R.H. and under 16L8D light conditions. Genetics of Diadegma Hybridization 537 Adult parasitoids were supplied with third instars 1 min 30 s. COI fragments were amplified by the of their natural host, DBM, for parasitization ad li- same method, except for the annealing step con- bitum, and a constant honey supply for sustenance. ducted at 55°C with alternate primers as follows: Both parasitized and unparasitized DBM larvae C1-J-1718 (5Ј-GGA GGA TTT GGA AAT TGA were reared in plastic containers on radish seedlings TTA GTT CC-3Ј) and C1-N-2329 (5Ј-ACT GTA at 20Ϯ1°C, ambient R.H. and under 16L8D. AAT ATA TGA TGT GCT CA-3Ј) (ex Simon et For hybridization, one virgin female D. semi- al., 1994). All PCRs included a negative control clausum and five D. fenestrale males were placed (sterile water instead of DNA) to detect DNA con- in an enclosed plastic cup, approximately 90 mm in tamination. PCR products were resolved on 2% diameter and 40 mm in height, and observed under agarose gels, stained with ethidium bromide and ambient laboratory conditions. Once copulated, the visualized under a UV transilluminator. female was immediately moved to a separate plas- PCR products from both parents and one ran- tic container,