Parasitoids of Monochamus galloprovincialis (Coleoptera, Cerambycidae), vector of the pine wood nematode, with identification key for the Palaearctic region

Abstract The parasitoid complex associated with Monochamus galloprovincialis (Olivier), vector of the pine wood nematode, is discussed. Four species of the family Braconidae and one Ichneumonidae were found associated with Monochamus galloprovincialis in Portugal, namely Atanycolus denigrator (Linnaeus), Atanycolus ivanowi (Kokujev), Cyanopterus flavator (Fabricius), Doryctes striatellus (Nees) (Braconidae), and Xorides depressus (Holmgren) (Ichneumonidae). Atanycolus ivanowi, Atanycolus denigrator, Doryctes striatellus and Xorides depressus are new species for Portugal fauna, and Monochamus galloprovincialis is recorded as a new host of Xorides depressus. A key for determination of the ichneumonoid parasitoids of the pine sawyer is provided for the Palaearctic fauna.


Introduction
Worldwide, beetles of the genus Monochamus Dejean (1821) (Coleoptera; Cerambycidae) are the most important vectors of the pine wood nematode Bursaphelenchus xylophilus (Steiner and Buhrer 1934) (Nematoda, Tylenchida, Aphelenchoididae) (Linit 1988, 1990, Kishi 1995. This nematode is native to North America, where it's not considered a primary pathogen of indigenous pines, although in countries where it has been introduced it is an important agent of mortality for susceptible pines. In Portugal, where the nematode has been present for over a decade now, the pine sawyer Monochamus galloprovincialis (Olivier 1795) is it's sole vector (Sousa et al. 2001).
The pine sawyer M. galloprovincialis is widely distributed in Europe (except in the United Kingdom, Ireland and Cyprus), and is also present in Caucasus, Russia, North Africa, China and Mongolia (Hellrigl 1971, Francardi andPennacchio 1996). The Monochamus beetles do not breed on healthy trees, and are attracted only to stressed, dying or recently killed trees and freshly felled timber for egg laying (Linsley 1959, Hellrigl 1971, Linit 1987, Hanks 1999. Before the introduction of B. xylophilus in Portugal, M. galloprovincialis was considered a secondary forest pest and nothing was known about the most important aspects of its biology and ecology. In the rest of Europe there is also an absence of detailed studies on its biology, with the exception of the classic paper of Hellrigl (1971).
The most efficient way to control wilt disease is to decrease the population levels of the vector Monochamus beetles. However, the different methods to control these insects usually have success only in localized, small-dimension areas, but are difficult to implement at low cost and have reduced efficiency over large forested areas. In Portugal, the most important management and control strategy consists in the elimination of symptomatic trees during late autumn, winter and early spring, while the insect vector is inside the host as late-instar larvae or pupae. The vector's populations can also be diminished during the beetle´s flight season with the use of traps baited with attractive lures (Naves et al. 2008).
Specific and efficient natural enemies (bio-control) would be an interesting and environmental-friendly option, but until now there are no adequate species for such control program (Naves et al. 2005). A few studies have already dealt with the parasitoids of this pine pest (Francardi and Pennacchio 1996, Francardi et al. 1998, Naves et al. 2005, although the information is scarce and disperse. In this paper we report on the diversity of parasitoids associated with M. galloprovincialis in Portugal, their frequency and revise all previous information on ichneumonoids parasitoids of M. galloprovincialis in the Palaearctic Region, resulting in a key for their identification.

Material and methods
For the parasitoid surveys, two different approaches were employed: In each location two dead Pinus pinaster Aiton, trees were felled, the wood sections colonized by M. galloprovincialis were divided into 60 cm logs, and taken to the INIAV (Instituto Nacional de Investigação Agrária e Veterinária) laboratories in Oeiras to be stored in wood boxes prepared with a wire mesh. The boxes were completely covered with a black plastic, leaving two holes with transparent containers to collect emerged insects. Boxes were analyzed every two days for insects, which were collected and stored in alcohol for posterior identification. Additionally, the boxes were opened frequently for the evaluation of the content and to collect other emerged insects. The logs were kept inside the boxes until no emergences were registered for a period of two months, and then debarked and opened (with a vertical chain saw) to detect the hosts and life stages attacked by the parasitoids.
II -Complementary to the previous approach, artificial trap-trees were also prepared, consisting of living maritime pine trees which were cut into logs and given to adult M. galloprovincialis to lay eggs under laboratory controlled conditions. Each log had a medium length of 40 cm and a medium diameter of 10 cm. subsequently, the logs were taken to the terrain to be colonized by parasitoids, and in each log a hole was made to allow passing a rope to hang them in the trees, at the branches height.
A total of 96 logs were divided according to the insect´s life stages (eggs, phloem larvae, xylem larvae and pupae), with 24 replicates each. Trials were made in Monção, Marinha Grande (Leiria), Comporta and Vimeiro, Alcobaça (Lat: 39.477811, Lon: -9.022316). In each location, six trap-trees were taken to the terrain four times during the year (pupae: April; eggs: July; phloem larvae: August; xylem larvae: October), in synchrony with the natural life cycle of the insect, and hanged in a healthy adult pine tree for a period of ten days. Subsequently, the logs were taken to the INIAV laboratory, and kept in wooden boxes (similarly to the previous experiment) to allow for emergencies. All emerged insects were identified and prepared to be photographed in the stereomicroscopy and environmental scanning electron microscopy (Serveis Científico-Tècnics de la Universitat de Barcelona). The field-emission gun environmental scanning electron microscope (FEI Quanta 200 ESEM) was used for high-resolution imaging without gold-coating with the purpose of not damaging the specimens.
All the collected material was stored in INRB Forestry Entomology Collection, Oeiras, Portugal. The insects collected with the trap-tree were labeled "Artificial" and the ones from the dead trees were labeled "Natural". Terminology employed in the key for morphological features, sculpture and measurements as well as wing venation nomenclature follows Belokobylskij and Maeto (2009).
No parasitoids emerged from the trap trees with eggs, xylem larvae and pupae of M. galloprovincialis. Parasitism was only found in the sub-cortical larvae, corresponding to the host´s first instars. A total of 27 specimens, belonging to five species, were recovered solely from Marinha Grande and Vale Feitoso, seven of which (all Cyanopterus flavator) from the trap-trees, while the remaining species were all obtained from the dead trees. Cyanopterus flavator (Fabricius) and Atanycolus ivanowi (Kokujev) were found in Vale Feitoso, and in Marinha Grande the following ichneumonids and braconids were recovered: Atanycolus denigrator (L.); C. flavator; Doryctes striatellus (Nees) and Xorides depressus (Holmgren).
By far, Cyanopterus flavator was the most abundant species with a total of 15 specimens from Marinha Grande and Vale Feitoso. Cocoons of this species were found in the xylem galleries of M. galloprovincialis, alongside with mandibles of the dead larvae.
The other cocoons found were in the inner bark associated with the larval galleries of the pine sawyer. The number of cocoons found matches exactly the number of parasitoids obtained from this surveys, and no other cocoons were found parasitizing any of the species previously mentioned. The parasitized species emerged between May and September under laboratory conditions and the precise dates are recorded in the labels of each specimen.
The following hymenoptera emerged from the wood, with both Braconidae (4) and Ichneumonidae (1):
Biology. Atanycolus denigrator is an ectoparasitoid of M. galloprovincialis attacking Pinus pinaster. The species was found parasitizing the first larval instars under the bark of the tree.
Biology. Atanycolus ivanowi was found to be an ectoparasitoid of first larval stages of M. galloprovincialis living under the bark of P. pinaster.
Biology. The biology of this parasitoid is poorly known, but in this study all the specimens emerged from cocoons from the xylemic galleries of M. galloprovincialis, which were not completely sealed with frass, as it is normal. Considering the length of the ovipositor of C. flavator, it is apparent that only first larval instars of M. galloprovincialis (found beneath the bark) are parasitized, which subsequently enter the wood carrying the parasitoid. Only the mandibles of the host larvae were found in galleries with cocoons.
Remark. Monochamus galloprovincialis as a host of C. flavator was already recorded by Campadelli and Scaramozzino (1994) for Italy and Naves et al. (2005) for Portugal.
Remark. This species was already recorded in Italy on the name D. mutillator (Thunberg) as parasitoid of M. galloprovincialis (Campadelli and Scaramozzino 1994). Distribution. Palaearctic: Austria, former Czechoslovakia, Finland, France, Germany, Hungary, Latvia, Poland, Romania, Russia, Spain, Sweden (Yu et al. 2005). This species is here recorded for Portugal for the first time.

Discussion
Considering the literature data and information presented in this study, the following key identifies the species of parasitoids attacking this pine sawyer in the Palaearctic Region. Only species that are reliably confirmed as parasitoids of M. galloprovincialis were considered for the key. Some species of parasitoids were excluded from this list and discussion about this decision is present in the final section. A key to species of Ichneumonidae and Braconidae parasitoids of Monochamus galloprovincialis is presented: 1 Second recurrent vein of fore wing present (Figure 1a). In hind wing, second longitudinal cubital vein always present and arising near middle of nervellus. Second and third metasomal tergites movable, not fused (Figures 1c, 2g) (Fam. Ichneumonidae). -Spiracles of first metasomal tergite placed on or before its middle (Figure 2e-f)  First metasomal sternite distinctly separated from tergite, this tergite with glymma (Figure 2e), and/or propodeum without transverse basal carina (Figure 2a). Claws of leg in female with teeth or basal lobe (Figure 2c) (Pimplinae) -Second metasomal tergite with pair of oblique furrows running from almost middle of its base to spiracles (Figure 2g). Lower valva of ovipositor apically with lateral lobes covered partly upper valva; dorsal lobe of lower valva with six-seven furrows. Ovipositor sheath 1.2-1.3 times longer than body. Body entirely black (including corner of pronotum); tegula brownish yellow; pterostigma dark; legs red, hind tibia and tarsus brownish red. -Occipital and prepectal carinae absent (Figure 4c). First tergite without dorsope and with median area delineated by furrows (Figures 1d, 5b Pedicel of antenna almost as long as first flagellar segment. First and second flagellar segments not longer than median segments of flagellum and concave below (Figure 6a). Second metasomal tergite without mediobasal triangle area (Figure 5c). -Ovipositor short, its sheath 1.0-1.3 times as long as metasoma, 0.60-0.65 times as long as fore wing. Second metasomal tergite about as long as third tergite, without or with fine oblique lateral furrows ( Figure  5c) With the exception of A. ivanowi collected from Vale Feitoso, all the other species were collected in Marinha Grande. This location is near Portugal´s oldest managed pine forest, in a pine stand with about 1700 square km and which was first planted in the XIII century. This stable and managed environment may have created favorable conditions for the establishment of a diverse entomofauna in the region. In fact, the larger number of parasitoids found in the region, and the low population levels of the vector insect suggest that M. galloprovincialis may be locally well controlled by its natural enemies. Further studies in the Leiria pine stand should confirm this hypothesis.
There is no obvious reason for the absence of parasitoids in the other sampled locations, although factors such as the local density of M. galloprovincialis (and other insect hosts), and differences in the local edapho-climatic conditions may explain the absence of the natural enemies.
Despite Atanycolus genus being the most diverse, Cyanopterus is the genus were the most specimens were found. Each parasitoid was reared from one specific location, except Cyanopterus specimens which were found in two very distanced sites, which present completely different edapho-climatic conditions. According to Watanabe (1937), the cocoons of C. flavator occurred in the trunk of Picea jezoensis Siebold et Zuccarini shut in by a thick corky lid at the end of the tunnel made by the larva of M. rosenmulleri, a conclusion which completely supports the suggested hypothesis for the parasitizing activity in Portugal.
Worldwide and including this study, there is now a total of 14 species of parasitoids associated with M. galloprovincialis, being six Ichneumonidae and eight Braconidae. Previous reliable records (confirmed rearing from the larvae of M. galloprovincialis) in the literature include references from Portugal (Naves et al. 2005), Italy (Campadelli and Scaramozzino 1994), and Siberia (Tobias and Belokobylskij 2000), among other locations Hilszczanski 2004, Tobias et al. 1986;Yu et al. 2005). Although not detected in this study, other groups, such as the braconids of the subfamily Helconinae (namely species of Helcon Nees 1812 and Helconidea Viereck 1914), will also likely parasitized larvae of M. galloprovincialis as they have been found to develop in larvae of other Monochamus species (Tobias et al. 1986, Yu et al. 2005. Other records are more dubious and need further confirmation. Among these, three records of Ichneumonidae are possibly erroneous, namely Rhyssa persuasoria (Linnaeus) (Pimplinae), Perithous divinator (Rossi) (Pimplinae) and Stenarella domator (Poda) (Cryptinae). The first species is a specialized parasitoid of Siricidae larvae (Yu et al. 2005), and its rearing from Cerambycidae is probably inaccurate. Likewise, the other two species are specialized parasitoids of vespoid and sphecoid wasps (Kasparyan 2010), and their associations with Cerambycidae is quite doubtful. Therefore, in the identification key only three ichneumonids were included, namely Odontocolon quercinum (Thomson), Xorides depressus (Holmgren) and Dolichomitus tuberculatus (Geoffroy). On the other hand, as all species of Braconidae were directly reared from M. galloprovincialis, they were included in our key.
Despite the relatively high diversity of parasitoids associated with M. galloprovincialis worldwide, all species are mainly idiobiont ectoparasitoids (except M. corax) and seem to be generalists attacking a vast array of other insects living in dead and dying trees. Cyanopterus flavator, which had already been found parasitizing young larval stages (Naves et al. 2005), appears to be the most frequent and promising candidate for studies aiming the biological control of the pine sawyer, despite its generalist habits. As mentioned, the disperse distribution of Cyanopterus can be considered as a major adaptation two the diverse edapho-climatic conditions characteristic for Portugal. Other options, such as the introduction of exotic natural enemies would create new parasitehost interactions, which usually offer greater changes of success for biological control than the promotion of already established associations (Hokkanen and Pimentel 1984). Nevertheless, such measures require rigorous pre-release risk assessment of the economic and environmental costs and benefits of the introduction, to evaluate its potential effectiveness, host specificity, acclimatisation and viability for mass-production (van Lenteren et al. 2006).
Detailed studies on the effect of the parasitoid guild found in Portugal on the pine sawyer´s population and the suitability of the species for biological control are being planned, with the final objective of eventually establishing an integrated bio-control program against the vector of the pine wilt disease in Europe.