Two new species of Oobius Trjapitzin (Hymenoptera, Encyrtidae) egg parasitoids of Agrilus spp. (Coleoptera, Buprestidae) from the USA, including a key and taxonomic notes on other congeneric Nearctic taxa

Abstract Oobius Trjapitzin (Hymenoptera, Encyrtidae) species are egg parasitoids that are important for the biological control of some Buprestidae and Cerambycidae (Coleoptera). Two species, Oobius agrili Zhang & Huang and Oobius longoi (Siscaro), were introduced into North America for classical biocontrol and have successfully established. Two new native North American species that parasitize eggs of Agrilus spp. (Buprestidae) are described and illustrated from the USA: Oobius minusculus Triapitsyn & Petrice, sp. n. (Michigan), an egg parasitoid of both Agrilus subcinctus Gory on ash (Fraxinus spp.) and Agrilus egenus Gory on black locust (Robinia pseudoacacia L.) trees, and Oobius whiteorum Triapitsyn, sp. n. (Pennsylvania), an egg parasitoid of Agrilus anxius Gory on European white birch (Betula pendula Roth). A taxonomic key and notes on the Nearctic native and introduced Oobius species are also included.


Introduction
The rather poorly known encyrtid genus Oobius Trjapitzin (Hymenoptera: Encyrtidae) currently includes 41 species worldwide, and seven are known from North America (Noyes 2014). Noyes (2010) recently described 20 of these species from Costa Rica and compared some of these new species to similar Nearctic taxa. Also, in Noyes (2010) the genera Avetianella Trjapitzin, Szelenyiola Trjapitzin, and Oophagus Liao were synonymized under Oobius.
As egg parasitoids of Buprestidae, Cerambycidae (Coleoptera; Noyes 2014) and Asilidae (Diptera; Annecke 1967), species of Oobius are important for the natural and classical biological control of some coleopteran species. Two species are being used as biological control agents in the USA where they are successfully established: O. agrili Zhang & Huang and O. longoi (Siscaro). The former was collected from China and was first released into the USA in 2007 as a biocontrol agent of the emerald ash borer, Agrilus planipennis Fairmaire (Buprestidae) (Bauer et al. in press). Agrilus planipennis is an invasive pest from Asia that attacks ash trees (Fraxinus spp.) (Haack et al. 2002;Bray et al. 2011). Releases of O. agrili are ongoing throughout infested regions of the USA. As of March 2015, establishment of O. agrili has been confirmed in Indiana, Maryland, Michigan, New York, Ohio, and Pennsylvania (Abell et al. 2014;Bauer et al. in press). Oobius longoi was introduced from Australia to California, USA, as a biocontrol agent for management of Phoracantha recurva Newman and P. semipunctata (Fabricius) (Cerambycidae), which are invasive pests of Eucalyptus trees in the USA (Hanks et al. 1995;Luhring et al. 2000).
Here two new species of Oobius are reported and a taxonomic key to the known native and introduced species of Oobius in North America is provided. One of the newly described species was reared initially from eggs of the native buprestid Agrilus subcinctus Gory in Michigan, whose larvae feed on the dead twigs of ash trees. This parasitoid was previously reported by Petrice et al. (2009) as Avetianella sp. Later, the second author of the current paper also reared this same species from eggs of A. egenus Gory on black locust trees (Robinia pseudoacacia L.) in Michigan. Agrilus egenus is a native species that oviposits on dead or dying branches of black locust (Nelson et al. 2008;MacRae 1991). The second newly described Oobius species was reared from A. anxius Gory eggs on European white birch (Betula pendula Roth) in Pennsylvania, based on the voucher specimens from the study by Loerch and Cameron (1983). Agrilus anxius is a native species that attacks both native and introduced birch trees (Betula spp.) in North America (Nelson et al. 2008).

Material and methods
Collecting and rearing new species of Oobius. Ash tree twigs with A. subcinctus eggs and black locust twigs with A. egenus eggs were collected in the field in Ingham and Clinton counties, Michigan in 2013 and 2014. Eggs were monitored in the labora-tory for parasitoid emergence. Voucher specimens of the parasitoids were preserved in 95% ethanol and sent to the senior author for identification. See Loerch and Cameron (1983) for collection of parasitoids from A. anxius.
Taxonomic studies. Parasitoid specimens used in the taxonomic studies were critical point dried from ethanol and point-mounted. Selected specimens were then dissected and slide-mounted in Canada balsam, examined under a Zeiss®™ Axioskop 2 plus compound microscope using Nomarski differential interference contrast optics. Stereomicroscopic images were compiled with Auto-Montage 4.02 (Synchroscopy®™) to illustrate select specimens. Images of specimens were produced by scanning electron microscopy (SEM) and an EntoVision Imaging Suite. A Nikon®™ SMZ1500 and Leica®™ MZ 9.5 stereomicroscope with 10X oculars (Nikon C-W10X/22) and Chiu Technical Corp.®™ Lumina 1 FO-150 and fiber optic light source was used for pinned specimen observation. Mylar film was placed over the ends of the light source to reduce glare. Scanning electron microscope (SEM) images were taken with a Hi-tachi®™ TM3000 desktop unit (Tungsten source). Some specimens were manually cleaned of external debris with forceps or brushes and affixed to 12.7X 3.2 mm Leica/ Cambridge aluminum SEM stubs with carbon adhesive tabs (Electron Microscopy Sciences, #77825-12). Stub-mounted specimens were imaged uncoated or sputter coated using a Cressington Scientific 108 Auto with a gold-palladium mixture from at least three different angles to ensure complete coverage (~20-30nm coating). Color images were obtained using an EntoVision Imaging Suite, which includes a firewire JVC KY-75 3CCD digital camera mounted to a Leica M16 zoom lens via a Leica z-step microscope stand. Slides of O. buprestidis and O. dahlsteni were imaged with a Leica DMRB compound microscope fitted with Leica HCX PL "Fluotar" 5× and 10× metallurgical grade lenses. Both systems fed image data to a desktop computer where Cartograph 5.6.0 (Microvision Instruments®™, France) was used to capture a fixed number of focal planes (based on magnification); the resulting focal planes (manually captured via Archimed 5.5.0 on the DMRB) were merged into a single, in-focus composite image. Uniform lighting was achieved using a LED illumination dome with all four quadrants set to 99.6% intensity. The images were then retouched where necessary using Adobe Photoshop®™ CS4/CS6 with plates assembled using InDesign CS4/CS6. Terms used for morphological features are those of Gibson (1997). Abbreviations used are: F = antennal funicle segment; mps = multiporous plate sensillum or sensilla on the antennal flagellar segments (= longitudinal sensillum or sensilla or sensory ridge(s) of authors). Body length was measured without the exserted part of the ovipositor. Acronyms

Comments.
Oobius is a cosmopolitan genus as defined by Noyes (2010) who provided its detailed diagnosis, which is omitted here for brevity. One extralimital species, O. striatus Annecke, is also known from eggs of Asilidae (Diptera) in Montenegro and Zimbabwe (Annecke 1967;Noyes 2010Noyes , 2014.
Comments. The identity of this species remains unclear because the original description is poor and without any illustrations; unfortunately, the slide with a head and a forewing of each sex (Girault 1916) could not be found in the USNM and is presumed lost. The lectotype label affixed by B. D. Burks was not validly designated and is merely a paralectotype. To facilitate identification of this species, we provide illustrations of its scutellum (Fig. 17) and habitus of the female in dorsal view (Fig. 18).
Comments. Oobius longoi is well known as an effective biological control agent and a successfully established parasitoid of P. recurva and P. semipunctata in California and elsewhere in the world (Hanks et al. 1995;Luhring et al. 2000;Trjapitzin 2001 Description. FEMALE (holotype). Body dark brown to black except scutellum and propodeum brown; scape and pedicel brown, flagellum light brown; legs whitish or pale yellowish with wide brown bands on coxae, femora, and tibiae.
Frontovertex and mesonotum with faint mesh-like or lineolate sculpture [very difficult to see in dry-mounted specimens, best observed in slide-mounted ones (as in Fig.  25)]. Pronotum, mesoscutum, axillae, and scutellum with short, dusky setae; scutellum also with a pair of long, fine setae near posterior margin.
Head (as in Fig. 15, collapsed when air-dried) with ocelli in an obtuse triangle, posterior ocellus a little less than its diameter away from eye margin. Transfacial and inner orbital sutures present. Mandible 3-dentate, the inner tooth with two denticles; maxillary palpus 4-segmented, labial palpus 1-segmented (i.e., palpal formula 4-1). Antenna (Fig. 12) inserted below lower eye margin. Radicle about 0.3× total scape length, rest of scape slender, 4.5× as long as wide, a little wider in the middle, with faint longitudinal sculpture. Pedicel longer than any funicle segment; F1-F5 slightly transverse, F1-F4 subequal in length, F5 a little longer and slightly wider than long; F6 the longest funicle segment, longer than wide; F1-F5 without mps, and F6 with 2 mps. Clava 3-segmented, about 2.3× as long as wide and almost as long as funicle; first claval segment with 1 mps, second and third segments each with 3 mps.
Mesosoma a little shorter than gaster (Fig. 13). Mesoscutum about 1.7× as wide as long. Scutellum a little wider than long, a little shorter than mesoscutum; scutellar placoid sensilla closer to the posterior margin of scutellum and close to each other. Wings (Fig. 31) not abbreviated, forewing extending far beyond apex of gaster. Forewing 2.1× as long as wide, hyaline; marginal setae very short; disc densely setose, linea calva interrupted posteriorly by an irregular row of setae, filum spinosum present. Hindwing 4.2× as long as wide, hyaline; longest marginal seta 0.3× maximum wing width.
Mesotibial spur a little longer than mesobasitarsus.
Ovipositor occupying a little more than 0.5× length of gaster, exserted markedly beyond gastral apex (by 0.2× own length) (Fig. 13) Fig. 33) or 0.46-0.53 mm (critical-point dried specimens from A. egenus, Fig. 34). In the latter specimens, legs (except tarsi) are somewhat darker (mostly brown), scape (minus radicle) of the female antenna is about 5.0× as long as wide, and clava is about 2.5× as long as wide. Mandibles are identical for specimens reared from both host species, and there is no doubt that they are conspecific. In all specimens, F6 is sometimes slightly paler than other flagellomeres but not contrastingly, still almost concolorous or often concolorous. MALE (paratype from A. subcinctus). Head dark brown, mesosoma and gaster dark brown to black except mesoscutum with a brownish tinge, base of gaster whitish; antenna with scape and pedicel brown to dark brown, flagellum light brown. Antenna (Fig. 16) with scape minus radicle 2.9× as long as wide; F2-F4 more or less subequal in length, F1 and F5 slightly longer, F6 the longest funicle segment; F2-F4 without mps, F1, F5, F6 and clava with mps; flagellar segments with very long setae (slightly longer than each funicle segment's width); clava entire, 2.6× as long as wide, a little wider than funicle segments. Mesosoma (Fig. 25) about as long as gaster. Forewing 2.0× as long as wide, hyaline. Genitalia (Fig. 14) typical for the genus.
Diagnosis. This species is similar to the European O. zahaikevitshi Trjapitzin (Figs 19,30), whose type locality is Zhuravlivka, Vinnytsia Oblast, Ukraine, where it was reared from eggs of Agrilus viridis (Linnaeus) on Carpinus betulus (Trjapitzin 1963). Oobius zahaikevitshi was recently well illustrated by Gumovsky et al. (2013). It was recorded from several European countries and Agrilus spp. hosts, listed by Volkovitsh (2011) andNoyes (2014). However, we are not absolutely confident that all these records are correct: it is quite possible that they might represent a complex of more than one cryptic species that are difficult to distinguish without supporting molecular data and thorough morphological studies based on good quality slide-mounted specimens. Proportions of funicle segments of the female antenna seem to be somewhat different between the specimens of O. zahaikevitshi from Ukraine illustrated by Trjapitzin (1963) and Gumovsky et al. (2013), in which F5 is about as long as wide, and the examined specimens from Volgograd Province of Russia, in which F5 is a little wider than long (Fig. 30).
Oobius minusculus differs from O. zahaikevitshi in having the palpal formula 4-1, a relatively smaller F5 of the female antenna and also by F6 being longer than wide and almost concolorous or often concolorous with other flagellomeres (Fig. 12). In contrast, the palpal formula for O. zahaikevitshi is 3-1, F5 is relatively larger, and F6 is about as long as wide and contrastingly lighter than other flagellomeres (Fig. 30), as also described and illustrated in Trjapitzin (1963) and Gumovsky et al. (2013).
Oobius minusculus is the only described native Nearctic species of Oobius s. str., as characterised by Noyes (2010) in having the outer plate of the ovipositor being relatively short and apically rounded with paired subapical setae (one long and one short), in which this new taxon fits well. In the key by Trjapitzin and Volkovitsh (2011) to the world species of Oobius (s. str.), it keys to O. zahaikevitshi. In Noyes (2010), O. minusculus tentatively keys (although it really does not key to any of the included Neotropical species) to the same couplet with O. xochipili Noyes and O. zagan Noyes from Costa Rica, from both of which it differs by F5 of the female antenna being much less transverse, just slightly wider than long (Fig. 12) whereas in O. xochipili and O. zagan F5 is anelliform, much wider than long (Noyes 2010).
Etymology. The name of this new taxon is an adjective referring to its small size.
Notes on biology. Originally reported by Petrice et al. (2009) as Avetianella sp. that parasitized A. subcinctus eggs. The second author has never found this parasitoid to overwinter in A. subcinctus eggs. However, collections of A. egenus eggs found overwintering O. minusculus larvae in eggs. This species likely attacks other Agrilus spp. in North America, and has multiple generations per year.
Mesotibial spur as long as mesobasitarsus.
Measurements of the holotype (mm, as length or length:width). Body (of the dry-mounted specimen prior to slide-mounting Variation (paratypes). Body (Fig. 35) length 0.66-0.75 mm (dry-mounted specimens). MALE (paratype). Body length (of the dry-mounted specimen prior to slidemounting) 0.66 mm. Head and mesosoma dark brown, gaster brown; scape and pedicel brown, flagellum light brown; legs light brown to brown. Antenna (Fig. 32) with scape minus radicle 3.2× as long as wide; funicle segments longer than wide, more or less subequal in length (F5 and particularly F6 slightly longer), F1 and F2 without mps, F3 with or without mps, F4-F6 and clava with mps; flagellar segments with very long setae (slightly longer than each funicle segment's width and about as long as width of clava); clava entire, 2.6-2.7× as long as wide, a little wider than funicle segments. Mesosoma about as long as gaster. Forewing 1.9× as long as wide, hyaline. Hindwing 3.5× as long as wide, hyaline. Genitalia (Fig. 29) typical for the genus.
Diagnosis. Among the Nearctic species of Oobius, O. whiteorum is most similar to O. dahlsteni, from which it differs by the proportions of the funicle segments of the female antenna, as indicated in the key. In Trjapitzin's (2001) key to the world species of the former genus Avetianella (s. str.) in which this new species mostly fits, as characterised by Noyes (2010) in having the outer plate of the ovipositor being conspicuously distally elongate and ribbon-like and always with only a single subapical seta, it keys to O. dahlsteni. Oobius whiteorum differs from O. depressus, to which it is also somewhat similar, by a relatively less flattened body and by the much smaller body size in females; according to Girault (1916), the body length of the latter species is 1.15 mm. Oobius whiteorum differs from the North American species, but Neotropical species O. hasmik (Trjapitzin), known from Mexico (Trjapitzin 2001) and also Costa Rica (Noyes 2010), by the "closed" linea calva (Fig. 27) on the forewing ("open", not interrupted, in O. hasmik, Fig. 21) and also by the different proportions of the scape of the female antenna (Figs 26 and 20, respectively). In Noyes (2010), O. whiteorum keys to the same couplet with O. lutron Noyes from Costa Rica and Brazil, from which it differs by each of F4-F6 of the female antenna being of different length and longer than wide (Fig. 26) whereas in O. lutron F4-F6 are subequal and each quadrate or hardly longer than broad (Noyes 2010).
Etymology. This species is named in honor of Lisa and Michael White of Chicago, Illinois, USA, good friends of the author's family.
Comments. According to Loerch and Cameron (1983), additional voucher specimens of the egg parasitoids of A. anxius were deposited by them in PSUC; any of them belonging to this species are non-type specimens. Unfortunately, due to a renovation of the museum, point-mounted specimens in that collection are now inaccessible (A. Deans, personal communication).
Comments. One female [UANL] of this undescribed species from Mexico, which has no host information, was mentioned by Trjapitzin and Volkovitsh (2011); however, they did not indicate the collecting locality so it is unknown from which part of that country it was found (Nearctic or Neotropical).