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Keywords:

  • cone and seed pest;
  • host range;
  • Mediterranean area;
  • natural enemy;
  • non-target species;
  • Pinus pinea

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Leptoglossus occidentalis Heidemann (Heteroptera: Coreidae) is a North American conifer seed pest that was accidentally introduced to Europe. In the Mediterranean area, it threatens the production of Pinus pinea Linnaeus seeds. The egg-parasitoid Gryon pennsylvanicum (Ashmead) (Hymenoptera: Platygastridae), the main natural enemy in the native range of L. occidentalis, was imported from British Columbia to Italy. Pre-release risk assessments were made under quarantine conditions by no-choice tests conducted with naïve and experienced G. pennsylvanicum offering single eggs of target and non-target species for varying exposure times (1, 4, 48 h). G. pennsylvanicum successfully parasitized from 75% to 100% of the target host eggs. Only one female specimen of the egg-parasitoid emerged from a non-target egg (Gonocerus juniperi Herrich-Schaeffer, Heteroptera: Coreidae). Two dead female specimens were found, one inside an egg of Coreus marginatus (Linnaeus) (Heteroptera: Coreidae) and one in an egg of Camptopus lateralis (Germar) (Heteroptera: Alydidae). All three cases occurred at the longest oviposition exposure time. Results obtained with this conservative approach suggest that the risk to non-target species of releasing G. pennsylvanicum in Italy is low.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Leptoglossus occidentalis Heidemann (Heteroptera: Coreidae) is considered a serious pest in North America, feeding on coniferous species (mainly Pinus and Pseudotsuga) causing cone and seed damage (Koerber 1963; Hedlin et al. 1981; Cibrian-Tovar et al. 1986; Bates et al. 2000, 2002; Strong et al. 2001; Strong 2006). L. occidentalis was accidentally introduced to Europe in the 1990s. In Central Italy and in Sicily, the pest causes commercially important losses to pine nuts harvested from Pinus pinea Linnaeus (the Stone Pine) (Roversi 2009; Santini 2010; Maltese 2011; Bracalini et al. 2013). In Stone Pine forests of Italy, like other natural ecosystems, the use of chemicals is constrained; therefore, biological control of L. occidentalis is desirable. Thus, a study aimed to evaluate classical biological control of L. occidentalis was started in 2010 (Roversi et al. 2011). In the native area of L. occidentalis, western North America, the main natural enemy is the solitary egg-parasitoid Gryon pennsylvanicum (Ashmead) (Hymenoptera: Platygastridae) (Bates and Borden 2004; Maltese et al. 2012). This egg-parasitoid was imported to Italy under quarantine conditions to conduct laboratory studies that assessed the possibility and feasibility of its use as a control agent. Biological parameters and life-history traits of G. pennsylvanicum on L. occidentalis were investigated in the laboratory in previous studies (Sabbatini Peverieri et al. 2012, 2013).

With regard to the introduction of beneficial organisms into new areas, recent attention has focussed on the possibility of negative effects on the native fauna (Stiling and Simberloff 2000; van Lenteren et al. 2003). Exploring the host range of an exotic natural enemy is a critical step that forms the focal point of environmental risk assessment. Various organizations have developed standards for evaluation, introduction and release of biological control agents, both parasitoids and predators (see: EPPO 2012; IPPC 2005; OECD 2004), and stepwise protocols have been described for risk assessment and non-target effects. The list of non-target species hypothetically at risk can be large and complex, and tests on non-target species can be difficult and laborious to perform. However, non-target host lists can be filtered to obtain a restricted list of species which can reasonably be tested (van Lenteren et al. 2003; Kuhlmann et al. 2006; Bigler et al. 2010).

Non-target species exploration presents several constraints and limitations in risk assessment, and laboratory tests can be less than entirely predictive, due to the extrapolation of laboratory data to field application. Moreover, introduced species can disperse and evolve (Simberloff and Stiling 1996; Briese 2005), further limiting our predictive abilities. Laboratory procedures such as no-choice host offerings and small spaces can lead to false positives, although these contribute to a more conservative risk determination (Messing and Wright 2006).

Non-target host range testing has never been conducted for G. pennsylvanicum. This egg-parasitoid belongs to the floridanum group, whose species are distributed in the Nearctic and Neotropical regions and in Japan (Masner 1961, 1983; Mineo and Caleca 1987; Yasuda 1990). In its native area, G. pennsylvanicum has a polyphagous feeding habit, but is restricted to the subfamily Coreinae (Masner 1983). Recorded host species fall in three tribes: tribe Anisoscelini, including L. occidentalis, L. clypealis Heidemann, L. corculus (Say), L. gonagra (Fabricius), L. phyllopus (Linnaeus), L. fulvicornis (Westwood) and Narnia femorata Stål; tribe Hypselonotini, Anasa tristis (De Geer); and tribe Chelinideini, Chelinidea spp. (Masner 1983; Mitchell and Mitchell 1983; Yasuda 1990). In the Heteroptera fauna of Italy, as well as in the rest of Europe, the tribes including known hosts of G. pennsylvanicum are not represented; the only exception is L. gonagra, the most widespread Leptoglossus pest species in tropical and subtropical areas, exclusively present in Europe in the Canary Islands near the Atlantic coast of Morocco (Moulet 1995; MATTM 2003; Fauna Europaea 2012). In the present work, studies were performed to explore the possibility of host shifting by G. pennsylvanicum to non-target species of the fauna of Italy. We focussed on non-target heteropteran species belonging to the same family of the target species L. occidentalis and to other less closely related species, which are common to pine stands and surrounding environments. All trials were conducted in the laboratory, because G. pennsylvanicum was introduced under quarantine restrictions.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Origin and rearing of the egg-parasitoid Gryon pennsylvanicum

Gryon pennsylvanicum was originally collected in Canada (British Columbia) in 2010 and introduced to Italy for laboratory studies (Roversi et al. 2011). The strain of the egg-parasitoid (GP-BC-1, NCBI genetic sequence database GenBank code JX968492) was reared on the target host L. occidentalis in a climatic chamber (Binder KBWF 720) at 26 ± 1°C, 75 ± 10% RH and 16:8-h L : D using glass tube rearing vials and pure honey for adult food as reported by Sabbatini Peverieri et al. (2012). The length of the body of the egg-parasitoid G. pennsylvanicum was measured on 30 females and 30 males taken randomly from the colony, using a stereomicroscope Nikon SMZ 1500 provided with micrometer oculars.

Selection of the non-target species

Concepts, guidelines and methods proposed for non-target species selection and laboratory tests by Kuhlmann et al. (2006), Kuhlmann and Mason (2003), van Lenteren et al. (2003), Bigler et al. (2010), Sands and van Driesche (2003) and Toepfer et al. (2009) were adapted to the present study. Host range information based on published records of hosts of G. pennsylvanicum in the area of origin; ecological and phylogenetic affinities; and spatial, temporal and morphological attributes were used to filter the list of non-target species; ‘safeguard consideration’, for example beneficial insects, rare or endangered species, was also taken into account. The accessibility and availability of non-target species, assessed through field surveys, were the last discriminants used to build the list of non-target species. Only those species, which were collected in sufficient numbers to establish a laboratory colony, were used in the tests. This permitted the use of eggs of known taxonomic origin, known to be free of naturally occurring parasitoids or hyperparasitoids. For taxonomic hierarchy of Coreidae, data of Livermore et al. (2012) were adopted.

Rearing and egg collection of the target and non-target species

The target host L. occidentalis and the non-target species were collected from pine stands of Italy, reared in the laboratory in insect cages at + 26°C, 60 ± 10% RH, 16:8-h L : D and administered their respective natural source of food (see table 1). Food sources were provided together with a source of water (moistened cotton) and replenished with fresh material three times per week. The target host was reared using 3- to 4-year-old potted P. pinea and seeds of Pinus nigra Arnold. Eggs of tested species were collected every day from the colonies using a fine brush to remove the eggs from the substrate on which they were laid, producing fresh eggs (<24 h old) to use in the tests. Eggs of candidate host species were individually glued on a piece of cardboard (2 × 1 cm) using a fine brush and a small drop of water. Egg dimensions (length and width) of target and non-target species were taken from the tested eggs.

Table 1. List of selected non-target species (Heteroptera, Coreidae, Alydidae, Pentatomidae, Reduviidae) tested for Gryon pennsylvanicum parasitization
Family – Tribe/SpeciesHost plants/prey for rearingCollection sitesa
  1. a

    In brackets the abbreviations of Italian Provinces.

Coreidae – Coreini
 Coreus marginatus Rumex spp., Cardus spp., Corylus avellana (seeds)Impruneta (FI), Firenze (FI), M.te S. Michele (FI), M.te Senario (FI)
Coreidae – Gonocerini
 Gonocerus acuteangulatus Buxus sempervirens, Cupressus spp., C. avellana (seeds)Cascine del Riccio (FI), M.te S. Michele (FI), M.te Senario (FI)
 Gonocerus insidiator Arbutus unedo, C. avellana (seeds)Torre del Lago (LU), Marina di Vecchiano (PI)
 Gonocerus juniperi Juniperus spp., Cupressus spp., C. avellana (seeds)Cascine del Riccio (FI), M.te S. Michele (FI), S. Rossore (PI), Torre del Lago (LU), Gavorrano (GR)
Alydidae
 Camptopus lateralis Inula viscosa, Spartium junceum, C. avellana (seeds)M.te Senario (FI)
Pentatomidae – Pentatomini
 Nezara viridula Phaseolus vulgaris, Glycine max, Arachis hypogaea (seeds)Cascine del Riccio (FI)
Reduviidae – Harpactorini
 Rhynocoris erythropus Acanthoscelides obtectus (adults), N. viridula (nymphs)S. Rossore (PI)
 Sphedanolestes cingulatus Mylabris variabilis (adults), N. viridula (nymphs)M.te S. Michele (FI)

No-choice test with inexperienced Gryon pennsylvanicum females

In the experiments using inexperienced females, newly emerged specimens of G. pennsylvanicum were taken from the colony, housed individually in glass tubes (15 cm long and 2 cm dia., closed on both ends by a plastic net of 250 μm mesh and provisioned with honey) and coupled with a male for 24 h. To each 1-day-old female, a single egg of one of the tested non-target species or of L. occidentalis was offered for parasitization. Three different parasitization exposure times were tested: 1, 4 and 48 h. In 1-h tests, direct observation of female behaviour was conducted at laboratory conditions (45 ± 10% RH, 26 ± 1°C) under a constant cold light source. In the 4 and 48-h tests, trials were performed in a climatic chamber at 26 ± 1°C, 75 ± 5% RH with a 16:8-h L : D cycle with no direct observations on female behaviour.

Female behaviour, observed in the 1-h tests, was classified into the following sequential steps: drumming, drilling (including ovipositing, because in Gryon the boundary between drilling and oviposition cannot be identified by any change in behaviour) and marking (Strand and Vinson 1983; Romeis et al. 2000; Hirose et al. 2003; Wiedemann et al. 2003). Acceptance of the egg, evidenced by marking behaviour after egg drilling, was considered to indicate successful oviposition (Strand and Vinson 1983; Wiedemann et al. 2003). We calculated the host acceptance rate, defined as the ratio between the number of eggs accepted (marked) and the number of eggs tested, and the host suitability rate on the basis of the proportion of tested eggs from which an egg-parasitoid successfully emerged.

At the end of each test, eggs exposed to G. pennsylvanicum were individually stored in a climatic chamber and checked daily until egg-parasitoids emerged or unparasitized eggs hatched. After 30 days, all host eggs that neither hatched nor produced an egg-parasitoid were dissected under a stereomicroscope and examined for signs of parasitization. Tests were replicated 10–20 times for each tested species and for each exposure time, depending on the number of eggs produced by the laboratory colonies and available for the tests.

No-choice test with experienced Gryon pennsylvanicum females

In the experiments using experienced females, tests were conducted using specimens that oviposited in eggs of the target host L. occidentalis before testing with non-target species. One-day-old females, as used in the previous experiment, were individually housed in glass tubes containing a single L. occidentalis egg. Females were observed for 1 h, and occurrence of parasitization was assessed by marking behaviour, after drilling of the egg. Females that did not parasitize the host egg during this time period were not used in the successive tests. At the second day, eggs of non-target species or of L. occidentalis were offered to the experienced females for parasitization opportunity with the same procedures described above in the experiment with inexperienced females.

Statistical analysis

Data of G. pennsylvanicum parasitization were analysed using the χ2 test (P < 0.05), to compare non-target species vs. the target species L. occidentalis.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Selection of the non-target species

Candidate non-target coreid species were selected from the subfamily Coreinae represented in Italy, although no candidates were from the known host tribes. The family Coreidae in Italy is comprised of 37 taxa, of which 17 species belong to the subfamily Coreinae. Considering that all known host species of G. pennsylvanicum have a common egg structural feature, the presence of a pseudoperculum, the list was reduced, deleting the species of the tribes Prionotylini, Phyllomorphini and those of the genus Syromastus (tribe Coreini), which do not have pseudopercolated eggs (see: Beard 1940; Brailovski et al. 1994; Koerber 1963; Moulet 1995). The remaining 13 candidates all belong to six genera, found in two tribes: Coreini (Centrocoris, Coreus, Enoplops, Haploprocta, Spathocera) and Gonocerini (Gonocerus). Furthermore, egg length of the known host species ranged from approximately 1.5 to 2 mm, while body length of the tested strain of G. pennsylvanicum is about 1.8 mm long (females: 1.82 ± 0.06 mm; males: 1.71 ± 0.06 mm). Thus, from the candidate list, the species of the genus Spathocera were removed because eggs were considered to be too small for hosting the egg-parasitoid (eggs are less than 1.1 mm in length) (Moulet 1995). This left a list of 9 non-target candidate species.

Field surveys were then conducted over a 2-year period on trees, shrubs and herbaceous plants of pine woods of Italy and their bordering environments. We collected adults of five species included in the non-target list: Gonocerus acuteangulatus (Goeze), Gonocerus juniperi Herrich-Schaeffer, Gonocerus insidiator (Fabricius), Coreus marginatus (Linnaeus) and Centrocoris spiniger (Fabricius). For all of these Coreidae, the number of collected adults was sufficient to establish a laboratory colony, with the exception of C. spiniger, that finally was not tested here (table 1). During the field surveys, other heteropterans were collected which either share the habitat of the target species or were temporary visitors from neighbouring areas (table 1). Of these, it was possible to rear in the laboratory four taxa, which were added to the non-target candidate list: Camptopus lateralis (Germar) (Heteroptera: Alydidae) belongs to the superfamily Coreoidea, which is closely related to Coreidae; Nezara viridula (Linnaeus) (Heteroptera: Pentatomidae), belongs to the same infraorder as that of the known host species (the Pentatomomorpha) and thus represents a more distantly related non-target species; Rhynocoris erythropus (Linnaeus) (Heteroptera: Reduviidae) and Sphedanolestes cingulatus (Fieber) (Heteroptera: Reduviidae) were also included as representatives of beneficial organisms, being generalist predators of various plant-feeding insects. Moreover, S. cingulatus is considered an endemic Italian species (Gobbi 1973).

No coreids native to Italy present eggs and egg clusters with size and morphological features similar to those of the target host L. occidentalis: eggs are pseudopercolated, semi-cylindrical, of about 1.9 mm in length and 1.3 mm wide and are laid in a row on pine needles forming clusters of one up to a dozen or more eggs. However, the selected coreid non-target species listed in table 1 lay eggs similar to other known hosts of G. pennsylvanicum in its native area. These eggs are also pseudopercolated, semi-ellipsoidal in shape (dome-shaped or ovoidal with a flattened base), approximately 1.6–1.7 mm long and 1.0–1.2 mm wide and are laid on the plant foliage individually and widely distributed, or tightly clustered with a uniform spacing arrangement in the egg masses. Other less closely related non-target species listed in table 1 present eggs with different features: eggs of C. lateralis are not pseudopercolated, ellipsoidal in shape with a flattened top and approximately 1.4 mm long and 0.9 mm wide, while eggs of N. viridula are pseudopercolated, subspherical and approximately 1 mm wide; eggs of R. erythropus and S. cingulatus are not pseudopercolated, subcylindrical, with a dimension of 2 mm and 1.4 mm in length and 0.7 mm and 0.6 mm wide, respectively.

No-choice test with inexperienced Gryon pennsylvanicum females

In the experiments using inexperienced specimens, in the 1-h observation tests G. pennsylvanicum females showed a high host acceptance rate (75% of marked eggs) and a high host suitability rate (75%) on the target species L. occidentalis (table 2). Compared to the target species, the number of drummed eggs was significantly lower for G. insidiator and R. erythropus2 = 10.67, d.f. = 1, P = 0.0011; χ2 = 14.73, d.f. = 1, P = 0.0001, respectively), while no eggs of N. viridula were drummed (χ2 = 18.91, d.f. = 1, P < 0.0001). The number of drilled eggs of L. occidentalis was significantly higher than those of C. marginatus, G. acuteangulatus, G. insidiator and C. lateralis2 = 12.52, d.f.  = 1, P = 0.0005; χ2 = 19.65, d.f.  = 1, P = 0.0001; χ2 = 22.73, d.f.  = 1, P = 0.0001; χ2 = 14.44, d.f. = 1, P = 0.0001, respectively), while eggs of G. juniperi2 = 26.19, d.f. = 1, P < 0.0001), R. erythropus2 = 26.19, d.f.  = 1, P < 0.0001) and S. cingulatus2 = 16.31, d.f. = 1, P = 0.0001) were not drilled. Despite the fact that some eggs of C. marginatus, G. acuteangulatus, G. insidiator and C. lateralis (for all these species: χ2 = 20.91, d.f.  = 1, P < 0.0001) were drilled by the females of G. pennsylvanicum, they did not accept the eggs as hosts, as evidenced by the lack of marking behaviour, and no egg-parasitoid offspring were produced from those eggs. Moreover, drilling did not affect hatching of the heteropteran eggs as first instars hatched successfully from 100% of these tested eggs.

Table 2. Parasitization of Gryon pennsylvanicum females on target and non-target species during 1 h of egg exposure time
Tested speciesnDrummed eggs (%)Drilled eggs (%)Marked eggs (%)Host suitability (%)
  1. Among columns, separately for inexperienced and experienced females, asterisk (*) indicates a statistically significant difference between non-target and the target species (χ2 test procedure, Yates correction, P < 0.05); ‘–’ not possible to test because the sequential steps of the parasitization process (drumming, drilling, marking) were not completed by the females at the related previous step, resulting in the non-host acceptance/suitability.

Inexperienced females
 L. occidentalis (target)2090857575
 C. marginatus 207025*0*
 G. acuteangulatus 206010*0*
 G. juniperi 20600*
 G. insidiator 2035*5*0*
 C. lateralis 206520*0*
 N. viridula 100*
 R. erythropus 2025*0*
 S. cingulatus 10900*
Experienced females
 L. occidentalis (target)201001008080
 C. marginatus 2035*5*0*
 G. acuteangulatus 2035*0*
 G. juniperi 2030*10*0*
 G. insidiator 2030*5*0*
 C. lateralis 2025*5*0*
 N. viridula 1010*0*
 R. erythropus 200*
 S. cingulatus 10900*

In the 4 and 48-h tests, G. pennsylvanicum host suitability of the target species L. occidentalis was high, respectively 80% and 100%. No non-target species tested were suitable hosts, producing no egg-parasitoid offspring, with the exception of a single egg of G. juniperi in the 48-h test (5% of the total eggs tested), from which one female G. pennsylvanicum successfully emerged. The difference between the target and non-target species was statistically significant (χ2 = 32.48, d.f. = 1, P = 0.0001). In two other cases, and only in the 48-h tests, one egg of C. marginatus and one of C. lateralis were accepted by G. pennsylvanicum, but they were not suitable as host because no adult G. pennsylvanicum emerged successfully. In the case of the parasitized egg of C. marginatus, the female egg-parasitoid died during emergence: the exit hole chewed by the female did not coincide with the pseudoperculum, but was oriented at the opposite side of the egg, and the specimen could not create a sufficiently large hole to escape. In the case of parasitization of the egg of C. lateralis, after egg dissection, a dead female G. pennsylvanicum was detected in the egg, and no chew marks were observed on the chorion.

No-choice test with experienced Gryon pennsylvanicum females

In the 1-h observation tests using experienced females, 80% host acceptance (marked eggs) and 80% host suitability of the target species L. occidentalis were recorded (table 2). Drumming on non-target eggs by experienced females of G. pennsylvanicum was significantly lower in all tested non-target species compared to the target host, with the exception of S. cingulatus2 = 16.41, d.f.  = 1, P = 0.0001 for C. marginatus and G. acuteangulatus; χ2 = 18.57, d.f. = 1, P = 0.0001 for G. juniperi and G. insidiator; χ2 = 20.91, d.f. = 1, P = 0.0001 for C. lateralis; χ2 = 21.61, d.f. = 1, P = 0.0001 for N. viridula). Eggs of R. erythropus were never drummed (χ2 = 36.10, d.f. = 1, P < 0.0001). Few non-target eggs were drilled, belonging only to the species C. marginatus, G. juniperi, G. insidiator and C. lateralis; drilling was significantly lower than on the target host L. occidentalis2 = 32.48, d.f. = 1, P = 0.0001 for C. marginatus, G. insidiator and C. lateralis; χ2 = 29.19, d.f. = 1, P = 0.0001 for G. juniperi). No eggs of G. acuteangulatus2 = 36.10, d.f. = 1, P < 0.0001), N. viridula and S. cingulatus were drilled (for both species: χ2 = 25.67, d.f. = 1, P < 0.0001). However, once again, none of the drilled eggs of C. marginatus, G. juniperi, G. insidiator and C. lateralis were marked (for all the species: χ2 = 23.44 d.f. = 1, P < 0.0001). Thus, females of G. pennsylvanicum did not oviposit in non-target eggs and heteropterans successfully hatched from 100% of the eggs that were drilled.

In the 4 and 48-h tests, none of the non-target species were parasitized by G. pennsylvanicum, while the target species was parasitized at a high rate (95% and 90%, respectively, for the two exposure times).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Our results show that G. pennsylvanicum cannot utilize eggs of any tested non-target species, at least in tests when the eggs were offered for 1–4 h, while the target species was parasitized at a high rate. In the 1-h tests, females of G. pennsylvanicum adopted the drilling posture on some non-target eggs, but oviposition did not occur, as evidenced by a failure to mark after drilling and no egg-parasitoid offspring were produced. This would suggest that physical or chemical composition of the chorion or egg contents plays a role in egg rejection. In the 1-h tests, drilling behaviour was only observed in the Coreinae species (same subfamily of the target host L. occidentalis) and in the Alydidae C. lateralis; the last is probably due to the close taxonomic similarity to Coreidae. However, female drilling of the non-target eggs did not compromise the hatching of the eggs to heteropteran nymphs.

In the tests with 48-h exposure time, parasitization of the target host was also high. On non-target species, despite G. pennsylvanicum females accepting one egg of both C. marginatus and C. lateralis for oviposition (evidenced by the presence of dead specimens inside the egg), successful parasitization with the emergence of an egg-parasitoid occurred only in one egg of the non-target species G. juniperi.

Thus, G. pennsylvanicum parasitization on eggs of non-target species in laboratory tests can occur but at a very low rate. However, the confinement with a non-target egg for an extended period does not reflect a field situation, in which contact with a non-target egg would be very transitory; therefore, this laboratory result is unlikely to occur in the field.

Gryon pennsylvanicum's known host range is restricted to host species whose eggs feature a pseudoperculum. The emergence hole of adult G. pennsylvanicum on eggs of L. occidentalis is produced by chewing along the line of the structural weakness which delimits the pseudoperculum; successful emergence from a different point of the egg shell has never been observed (G. Sabbatini Peverieri, personal observation). This behaviour was also observed in the single case of successful parasitization of the non-target species G. juniperi. On the other hand, in the two cases of unsuccessful parasitization, the parasitoids failed to emerge from C. marginatus because the exit hole produced by the adult did not coincide with the pseudoperculum, and failed to emerge from C. lateralis because the egg did not feature a pseudoperculum. This evidence suggests that the presence of a pseudoperculum and the ability for a correct positioning of the adult inside the host egg (probably occurring during larval development) are crucial. This may play a significant role in host suitability, even if the females of the egg-parasitoid accept non-target eggs for oviposition.

The lack of successful parasitism in 1 and 4-h tests and the very low rate of parasitism in the 48-h tests show that non-target Heteroptera are either non-hosts or very poor hosts. This high specificity would reduce the risk of non-target effects following release of G. pennsylvanicum into pine forests of Italy. Moreover, the no-choice laboratory tests overestimate the likelihood of successful parasitism in the wild, where non-target parasitization is unlikely to occur except possibly in the case of complete host deprivation. For these reasons, negative results in no-choice tests are considered very robust (van Driesche and Murray 2004). Therefore, our results present a conservative estimate of the risk of release in the wild.

The high apparent host specificity allows us to set the risk to non-target species to the lowest levels on the risk scale proposed by Bigler et al. (2010) and van Lenteren et al. (2003). However, considering the wide range of territory colonized by L. occidentalis across Europe (see EPPO 2010), it would be beneficial to explore in future the non-target host range in other Mediterranean areas where Stone Pine stands are widely distributed.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We are grateful to Beatrice Carletti for the species determination of heteropterans, to Agostino Strangi for updating the GenBank code of the strain of G. pennsylvanicum collected in British Columbia (Canada) and to Daniele Benassai for maintaining the laboratory populations of L. occidentalis. This work was supported by the Italian Ministry of Agricultural, Food and Forestry national project ‘PINITALY – The restart of the Pine Nuts production in Italy by new pest control strategies’ (DM 256⁄7303⁄2007).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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