Oviposition behaviour of four ant parasitoids (Hymenoptera, Braconidae, Euphorinae, Neoneurini and Ichneumonidae, Hybrizontinae), with the description of three new European species

Abstract The oviposition behaviour of four ant parasitoids was observed and filmed for the first time. The movies are available from YouTube (search for Elasmosoma, Hybrizon, Kollasmosoma and Neoneurus). Two of the observed species (Neoneurus vesculus sp. n. and Kollasmosoma sentum sp. n.) are new to science. A third species (Neoneurus recticalcar sp. n.) is described from Slovakia and Norway. Keys to the Palaearctic species of the genera Neoneurus and Kollasmosoma are added.

As far as the scanty biological information allows a conclusion, the Neoneurini are considered to be most likely koinobiont endoparasitoids of adult ants (Shaw and Huddleston 1991;Shaw 1992). According to Huddleston (1976) the strongly curved ovipositor, which is almost hook-shaped and forward-pointing when exserted (Figs 1,23,53), gives support to the supposition that the eggs are laid, possibly through the anus, into the metasoma of adult ant workers. With few exceptions Neoneurini have been found in association with formicine ants (Shenefelt 1969;Marsh 1979;Yu et al. 2007). These ants exude formic acid which is a powerful attractant for predatory ant species, and it seems likely that this exudate could serve also as a kairomonal stimulant to host-seeking hymenopterous parasitoids (Huddleston 1976; van Achterberg and Argaman 1993).
The scanty biological information indicates that Kollasmosoma species are associated with species of the genus Cataglyphis Foerster, 1850. One Kollasmosoma species (K. platamonense (Huddleston, 1976)) was observed to approach the formicine desert ant Cataglyphis bicolor (Fabricius, 1793) from behind and remained in contact with the tip of the metasoma of the ant for less than one second (R.D. Harkness in Huddleston 1976). Similar oviposition behaviour is reported in this paper for another Kollasmosoma species (K. sentum sp. n.) with Cataglyphis ibericus (Emery, 1906) in Spain.
For the identification of the tribe Neoneurini of the subfamily Euphorinae see van Achterberg (1990Achterberg ( , 1993Achterberg ( , 1997, for the identification of the Palaearctic genera see van Achterberg and Argaman (1993); for a key to the European Elasmosoma species see van Achterberg and Koponen (2003); for references to the genera and species, see Yu et al. (2007) and updates, and for morphological terminology see van Achterberg (1988).
The small subfamily Hybrizontinae Blanchard, 1845 (= Paxylommatinae Foerster, 1862; Wharton and van Achterberg 2000) is considered to belong to the family Ichneumonidae, but was often associated with Braconidae ( van Achterberg 1976) or considered to be a separate family (Tobias 1988). The group is treated as a subfamily of the family Ichneumonidae Latreille, 1802, by Rasnitsyn (1980) and Yu and Horstmann (1997), and indeed the structure of the connection of the second and third metasomal tergites and the venation of the hind wing seem to indicate a closer relationship with the family Ichneumonidae (Sharkey and Wahl 1987;Wahl and Sharkey 1988). From analysis of the 28S ribosomal RNA from Hybrizon it may be concluded that the Hybrizontinae are at a basal position of the Ichneumonidae-lineage (Belshaw et al. 1998;Quicke et al. 2000;Belshaw and Quicke 2002).
Until now the biology of Hybrizontinae was poorly known and based on circumstantial evidence ( van Achterberg 1999). Development was known to take place in ant-nests, from which they have been reared several times and where the naked pupae have been found among ant cocoons (Donisthorpe and Wilkinson 1930). Hybrizontinae are associated with ants of the genera Formica Linnaeus, Lasius Fabricius, 1804, Myrmica Latreille, 1804, and Tapinoma Foerster, 1850 (Yu et al. 2007). Donisthorpe (1915) and Donisthorpe and Wilkinson (1930) gave a detailed list of several species of probable hosts based on their own data and that of others (Arnold 1881;Ratzeburg 1848;Giraud 1857;Rudow 1883;Marshall 1891;Wasmann 1894Wasmann , 1899Cobelli 1906;de Gaulle 1908;Haupt 1913). According to that list Hybrizon buccatus (de Brébisson, 1825) was seen hovering over the nest entrance or over workers of Formica rufa, F. rufibarbis, F. sanguinea, Lasius alienus (Foerster, 1850), L. brunneus (Latreille, 1798), L. citrinus Emery, 1922, L. flavus (Fabricius, 1782, L. niger, Myrmica lobicornis Nylander, 1846, M. ruginodis Nylander, 1846, M. scabrinodis Nylander, 1846, and Tapinoma erraticum (Latreille, 1798. Ghilaromma fuliginosi was seen hovering over L. fuliginosus (Latreille, 1798). Eurypterna cremieri (Romand, 1838) was seen hovering over F. rufa, L. brunneus, L. fuliginosus and Camponotus herculeanus. More recently, Watanabe (1984) saw Ghilaromma fuliginosi (Wilkinson, 1930) hovering over L. fuliginosus, and Marsh (1989) refers to three specimens of H. rileyi (Ashmead, 1899) which were attracted to a disturbed nest of Lasius alienus. In The Netherlands females of Hybrizon buccatus were observed diving at Formica rufa worker ants during spring ant wars in the dunes near The Hague ( van Achterberg 1999). In total, four genera of ants (Formica, Lasius, Myrmica and Tapinoma) belonging to three subfamilies (Formicinae, Myrmicinae and Dolichoderinae) have been considered as probable hosts of H. buccatus. This large range of host-parasitoid relationships needs, beyond the mere existence of hovering behaviour, direct confirmation of ovipositions. In the case of E. cremieri, Cobelli (1906) observed 20 ovipositions on the larvae transported by workers of L. fuliginosus, and Komatsu and Konishi (2010) photographed ovipo-sition into larvae of L. nipponensis Forel, 1912. From the circumstantial evidence it was concluded that they probably are endoparasitoids of ants (Čapek 1970;van Achterberg 1976). Here we report that the egg of Hybrizon buccatus is laid in ant larva when they are transported outside of their nests. Komatsu and Konishi (2010) report oviposition by E. cremieri into the somatic cavity of the larva in less than a second. Konishi (2010) stated that the larvae are parasitized during their transport from the summer nest in a tree trunk to the winter ground nest in October; the adults of E. cremieri emerge from the summer nest in the tree trunk from late September to end of October. It remains a mystery how this large parasitoid manages to get enough food considering that the host is half the size of the parasitoid. In addition, Komatsu and Konishi (2010) described and illustrated oviposition by a new Japanese genus and species into larvae of Myrmica kotokui Forel, 1911. For the recognition of the subfamily Hybrizontinae and for the identification of European genera and species, see Tobias (1988) and van Achterberg (1999).

Material and methods
Females of Hybrizon buccatus (de Brébisson) and Elasmosoma luxemburgense Wasmann were observed in Almazán (Soria, Spain) in July and August, 2010. Kollasmosoma sentum sp. n. and Neoneurus vesculus sp. n. were observed in Madrid (at the enclosed area of the Institute for Agriculture and Food Research and Technology (INIA), Carretera de La Coruña Km 7.5, Spain) in August and September, 2010. The oviposition behaviour for each species (comprising the grasping of the ant by the wasp and the insertion of the ovipositor, until departure by flight) was recorded in slow motion video, at a rate of 300 frames per second, with a Casio Exilim Pro EX-F1 digital camera and a Raynox DCR-250 Super Macro lens. The four short movies showing the oviposition behaviour of the four observed species are downloadable from YouTube (Appendices I-IV). RMNH stands for Netherlands Centre for Biodiversity Naturalis, Leiden, Netherlands; RMS for National Museums of Scotland, Edinburgh, RMSEL for Rocky Mountain Systematic Entomology Laboratory, Laramie, Wyoming, USA, and ZMUO for Zoological Museum, University of Oslo, Blindern, Oslo, Norway.

Elasmosoma luxemburgense
Oviposition behaviour. Oviposition of Elasmosoma spp. into the ants' metasoma has long been observed (Forel 1874;Olivier 1893;Pierre 1893;Wasmann 1897;Donisthorpe 1927;Kariya 1932), and adults of Elasmosoma have been reared from Formica nests on various occasions (Wasmann 1897;Watanabe 1935;Poinar 2004). Due to the very quick act of oviposition, few details are known about the accompanying behav- iour of grasping the ant or about the location of ovipositor insertion. Wasmann (1897) supposed that Elasmosoma females lay the eggs between the abdominal segments; other authors, considering the strongly curved morphology of the ovipositor, have suggested that the eggs are probably laid through the anus (Huddleston 1976;van Achterberg and Argaman 1993). Here we report new observations on the oviposition behaviour of Elasmosoma luxemburgense on Formica rufibarbis Fabricius, 1793, comprising alighting and grasping the worker ant and ovipositor insertion.
The observations were made in Almazán (Soria, Spain) in August, 2010 on a warm and calm day between 12.26 PM and 13.38 PM. A group of 30 to 40 of Formica rufibarbis workers were present surrounding a nest entrance on the ground. They were excited and aggressive, carrying materials, entering and leaving the nest. Some cadavers of another species of ant and isolated fights indicated that a more extensive battle recently occurred. Forel (1874) noted that these struggle situations attract Elasmosoma berolinense, and possibly the formic acid exuded in the course of these fights serve as a kairomon to the parasitoid wasps (Huddleston, 1976). During the 72 minutes of observation, groups of 2-3 females of E. luxemburgense could be seen hovering over and attacking the ants at a height of 1-3 cm from the ground. A total of 50 attempts at oviposition was recorded (Movie Elasmosoma, Appendix I). The ants were aware of these attacks, turning around and chasing the wasps with open mandibles. On one occasion, a worker caught a wasp while flying (Movie Elasmosoma, last sequence).
The wasp attacks always come from behind, paralleling their longitudinal axis to those of the ants. When they are less than 1 cm from an ant they dart forward and the fore legs contact the dorsal surface of the metasoma first. Meanwhile the hind legs, arranged in curved shape, are situated to brace the apex of the metasoma (Fig. 2).
Contact with the fore legs is usually followed by hitting of the parasitoid's head on the host's metasoma. At this moment the middle and hind legs grasp the metasoma and the wasp folds its wings. The site chosen by the wasp for the initial hit of the fore legs, or the head, is usually the posterior margin of the first gastral segment (T1; Fig.  3), i.e., of a total of 48 hits observed, 44 were on the posterior margin of the first gastral segment (91.7%), three on the posterior margin of the second (6.3%), and one on the posterior margin of the third (2%). When the hit occurs at the posterior margin of the second or third gastral segments, the wasp climbs onto the metasoma, changing its position to reach the posterior margin of the first gastral segment (Fig. 4).
This locational preference for alighting may be visually stimulated by the differentiated border of the posterior margin of the first gastral segment, enhanced by the characteristic dark stripe behind it. The frame analysis in the film clip suggests that the wasp's head hits the posterior margin of T1 with the mandibles opened, and that a slight deformation of the suture between T1 and T2 is produced. Presumably, the modified structure of the T1-T2 suture is used by the wasp to secure its grasp. The tarsal modifications of Elasmosoma (vestigial tarsal claws and enlarged pulvillus; Shaw 1985Shaw , 2007 may be adaptations to effect this grasping behaviour. In the final arrangement, prior to oviposition, the fore tarsi usually grasp the posterior margin of the first gastral segment, and the hind tibiae and tarsi brace the apex of the metasoma on the fourth gastral segment, with the middle legs positioned near or somewhat posterior to the hind margin of the second gastral segment (Fig. 5).    This arrangement of the legs facilitates the appropriate position of the wasp's metasoma in order to insert the ovipositor into the posterior area of the last metasomal segment, between the pygidium and the hypopygium, probably through the anus. Poinar (2004) dissected the metasoma of the ant Formica obscuriventris clivia Creighton, 1940, a host of Elasmosoma michaeli Shaw, 2007, and found for the first time the wasp egg "just under the body wall of the ant's metasoma." The precise moment of ovipositor insertion could be detected by the conspicuous downward-movement of the apex of the wasp's metasoma ( Fig. 6 and first sequence of Movie Elasmosoma). Although one single movement of the apex of the metasoma normally occurred during oviposition, in some cases 2 or 3 consecutive movements were observed. On one occasion the same wasp alighted and oviposited two consecutive times in the same ant.
Oviposition attempts sometimes failed due to strong movements of the ant's metasoma, to strikes by the ant's legs, or because of defective alighting by the wasp (Fig. 7). Of a total of 50 oviposition attempts, 40 were successful (80%) and 10 failed (20%). The whole oviposition behaviour of E. luxemburgense (comprising grasping of the ant by the wasp and the insertion of the ovipositor, until taking off) lasted a mean of 0.727 seconds (95% confidence interval: 0.578-0.877; N = 38; SE = 0.074), with a median of 0.602 seconds (interquartile range: 0.480-0.900) (Fig. 79).
Key to species of the genus Kollasmosoma van Achterberg & Argaman 1 Scapus longer than pedicellus, somewhat longer than wide (Fig. 29); third antennal segment somewhat longer than fourth segment (Fig. 29); fore tarsus shorter than middle tarsus; face, frons and vertex transversely striate; hy-  (Tobias, 1986) -Scapus (excluding radix) shorter than pedicellus, wider than long (Figs 11,24,34); third antennal segment distinctly shorter than fourth segment (Figs 11,24,34); fore tarsus 1.3-1.9 times as long as middle tarsus (Fig. 39) Dorsal face of propodeum distinctly longer than metanotum, similar to posterior face (  (1)), topotypic, collected 3.ix. and 13.ix.2010; 1 ♂ (RMS), "(Spain), Granada, Orgiva, 3OS VF68, 500 m, 11241", "Leg. Jose Luis Ruiz de la Cuesta, 6.v.2009, 11241". The only known male paratype of K. platamonense from Spain probably also belongs here. Oviposition behaviour. Few observations have been made on the biology of the small Palaeartic parasitoid genus Kollasmosoma van Achterberg & Argaman, 1993. Kollasmosoma platamonense is known to approach the ant Cataglyphis bicolor from behind, briefly contacting its metasoma (R.D. Harkness in Huddleston 1976); van Achterberg and Argaman (1993) reported this species hovering over the nest of Messor semirufus, but no oviposition was observed. K. marikovskii has been reared from Formica pratensis (van Achterberg and Argaman, 1993) and, finally, no information is available on the biology of K. cubiceps (Huddleston). Here we report some observations on oviposition by Kollasmosoma sentum sp. n. in the ant Cataglyphis ibericus (Emery, 1906). The observations were made in Madrid (at the enclosed area of the Institute for Agriculture and Food Research and Technology (INIA), Carretera de La Coruña Km 7.5, Spain) during August and September, 2010. The parasitized colony of C. ibericus had a polycalic nest with three entrances on the ground, forming a triangle of about 60 cm on Figure 15. Baits were used to keep the ants quiet 1 Messor barbarus cadavers (red arrow) tied with a thread and fixed to the ground 2 honey (red arrow). Females of Kollasmosoma sentum sp. n. are indicated with a yellow arrow. each side. This area was visited daily by females of Kollasmosoma sentum during the three weeks of observation. The wasps appeared in groups of 1-3 individuals at any time between 12 PM and 15.30 PM, in the hours of highest temperature (around 35° Celsius). The visits lasted between 30 and 90 minutes. The wasps hovered over the nest entrances or looked for worker ants in the surrounding area when going out to forage or when returning to the nest carrying prey (thus, walking slowly). The wasps' attacks usually occurred during the brief and characteristic stops of Cataglyphis ants when marching. The wasp was extremely fast, flying at a height of about 1 cm over the ground. In order to observe and record the wasp's oviposition behaviour, the very speedy workers of C. ibericus were kept quiet by means of baits such as Messor barbarus cadavers -a usual prey of this species-and honey (Fig. 15).
When the wasp approaches, the ant is often aware of its presence, aggressively turning around with opened mandibles, or extending the hind or middle legs to hit the wasp even if it comes from behind ( Fig. 16). This defensive behaviour is very common and sometimes prevents the wasp from alighting and ovipositing. Kollasmosoma sentum attacks the ant from behind, and oviposition takes place into both the dorsal and ventral surface of the ant's metasoma, more rarely into its apex (Movie Kollasmosoma, Appendix II). (On one exceptional occasion, a wasp was observed attacking the ant's head). In all the cases observed (n= 22) the movements of the wasp's metasoma during oviposition, and hence the insertion of the ovipositor, followed the direction of the postero-anterior axis of the ant's metasoma, which suggests that the ovipositor may be inserted through an intersegmental membrane. Basically, two alighting strategies have been observed for achieving the postero-anterior insertion of the ovipositor; strategies that depend on the flight direction of the wasp's attack and on the inclination of the ant's metasoma, this last varying from an horizontal position to a vertical one, perpendicular to the ground surface and distinctive for the genus Cataglyphis.
1) Horizontal alighting: the wasp follows an ant with its metasoma in, or near, a horizontal position, approaches it from behind, in the direction of the longitudinal axis of the ant, and extends the fore legs until grasping the dorsal metasomal surface with its tarsi. With this grasp the wasp jumps over the ant's metasoma, lays down the middle and hind legs, and folds its wings before starting to oviposit (Fig. 17).
2) Vertical alighting: the wasp follows an ant having its metasoma arranged in vertical position, or forming an angle bigger than 45 degrees with the ground surface. It approaches the ant from behind, sometimes following a direction deviating from the longitudinal axis of the ant, and extends its fore legs until grasping the ventral metasomal surface with the tarsi. Now, with this grasp, the wasp accomplishes two kinds of rotational movements, which vary according to both the flight direction of the wasp and the inclination of the ant's metasoma. An example of this surprising pirouette, that fully involves the two rotations, occurs when the wasp, in horizontal flight, approaches an ant's metasoma placed in a vertical position ( Fig. 18 and the first two sequences of Movie Kollasmosoma). After grasping the ant's metasoma with the tarsi, and being perpendicularly aligned with respect to it, the wasp starts a 180° rotation around its longitudinal axis. At the same time, the wasp rotates vertically, approaching the metasoma. As a result of both rotational movements, the wasp alights downwards, allowing it to insert the ovipositor following the direction of the postero-anterior axis of the ant's metasoma.
It is interesting that during the rotation movements of the wasp, its fore tarsi (Fig. 19) keep permanent contact with the ant's metasoma. To achieve rotation around Figure 17. Horizontal alighting. 1 female of Kollasmosoma sentum sp. n. approaches an ant with the metasoma in horizontal position 2 extends the fore legs (yellow arrow) and grasps the metasoma with the tarsi 3 jumps over the metasoma placing the rest of its legs on it, and folds its wings.  1 The wasp (the red arrow points the apex of its metasoma) approaches the ant's metasoma (blue arrow) and extends its fore legs (yellow arrow) 2 the right tarsus is placed over the left one 3 the wasp starts its counter clockwise rotation (yellow arrow points to separation between the fore legs) 4 the wasp alights downwards; at that moment the hind and middle legs (yellow arrow) grasp the ant's metasoma, and the fore legs move forwards. Figure 21. Arrangement of the fore legs of the female of Kollasmosoma sentum sp. n. for the rotation movement. 1 the wasp (the red arrow points the apex of the metasoma) approaches the ant's metasoma and extends its fore legs 2 the right tarsus (yellow arrow) is placed over the left one 3 and 4 the wasp starts its counter clockwise rotation around its longitudinal axis 5 the wasp is in profile and the right fore leg hides the left one 6 the wasp alights downwards on the ant's metasoma. tarsus is placed over the right one, the rotation is clockwise. This longitudinal disposition of the wasp's tarsi on the ant's metasoma is, therefore, a behavioural adaptation to enable the necessary rotation of the body before oviposition.
The rapid insertion of the ovipositor follows a uniform behavioural pattern. When alighting, the wasp grasps the ant's metasoma with its three pairs of legs and folds its wings. Immediately, the wasp moves gradually backwards toward a perpendicular position with respect to the metasoma surface, the apex of its metasoma remaining over the ant's metasoma. A good example is offered during horizontal alighting (Fig. 22): the body of the wasp goes back tending to the vertical position. Before reaching the vertical, the apex of the wasp's metasoma moves down, presumably inserting the ovipositor into the ant's metasoma. At the vertical position, the apex of the wasp's metasoma presses down on the ant's metasoma, completely attaching to it. The wasp continues leaning backwards some way beyond the vertical and, finally, takes flight backwards.
Regarding the oviposition behaviour of Kollasmosoma sentum sp. n., the probable function of the ventral spine, peculiar to this species, located on the fifth sternite (anterior to the hypopygium; Fig. 23) needs mention. Since the rapid insertion of the ovipositor occurs when the wasp is in or near a perpendicular position with respect to the surface of the ant's metasoma (most likely with the fore legs detached from it), the ventral spine could serve to fix the wasp's position and act as a supporting point for the oviposition movements of the wasp's metasoma. Diagnosis. Outer spur of hind tibia of female normal and apically acute (Fig. 13); fifth metasomal sternite of female with an apical spine (Figs 14, 23); face strongly convex (Fig. 9); height of eye about 3.6 times width of temple in lateral view (Fig. 9); dorsal face of propodeum shorter than metanotum (Fig. 10); pedicellus of female distinctly protruding (Fig. 11); fore tarsus of female about 1.9 times as long as middle tarsus.
Head. Length of third segment of antenna 0.5 times fourth segment, length of third, fourth and penultimate segments 0.5, 0.8 and 1.0 times their width, respectively, and basal segments with distinct setae; pedicellus distinctly protruding and larger than scapus; face strongly convex and densely setose (Fig. 9), without facial tubercles and bristles; length of eye 2.4 times temple in dorsal view; height of eye about 3.6 times width of temple in lateral view (Fig. 9); vertex superficially granulate and having a satin sheen; temples roundly narrowed behind eyes; OOL:diameter of ocellus:POL = 5:4:20; length of malar space 0.05 times height of eye, eye nearly touching base of mandible.
Mesosoma. Length of mesosoma 1.1 times its height; mesoscutum evenly granulate; scutellum granulate and distinctly convex; precoxal sulcus absent; mesopleuron superficially granulate, but speculum shiny and largely smooth; mesosternal sulcus narrow and micro-crenulate; metanotum without a median carina and longer than dorsal face of propodeum; propodeum finely rugulose, dorsal face much shorter than posterior face, with satin sheen, without a median carina and no medial areola and its spiracle small and far in front of middle of propodeum.
Wings. Fore wing: parastigma comparatively large (Fig. 8); vein SR distinctly pigmented; basal half of wing much less densely setose than its distal half. Hind wing: wing membrane sparsely setose basally.  Legs. Hind coxa partly superficially micro-granulate, nearly smooth and with satin sheen; fore coxa nearly flat ventrally; all tarsal claws slender and simple; length of femur, tibia and basitarsus of hind leg 2.9, 4.5 and 4.0 times their width, respectively; fore femur moderately curved in dorsal view, compressed and apically without tooth; fore tibia without protuberances and evenly densely setose, its length 6.3 times its maximum width in lateral view; fore tarsus 1.9 times as long as middle tarsus and 1.6 times as long as fore tibia; fore tibial spur slightly curved and 0.7 times as long as fore basitarsus and 0.4 times fore tibia (Fig. 12); spurs of hind tibia acute apically, their length 1.1 and 1.0 times hind basitarsus.
Metasoma. Length of first tergite 0.6 times its apical width, its surface with satin sheen, granulate, basally and medially flat, and its spiracles not protruding and near apex of tergite; second and third tergites superficially granulate; second metasomal suture obsolescent; remainder of metasoma largely smooth and depressed; fifth sternite with a large and acute apical spine (Fig. 14); setae of metasoma spread and short; second tergite with sharp lateral crease; length of ovipositor sheath 0.05 times fore wing.
Etymology. From "sentus" (Latin for "thorny, spiny"), because of the unique thorn-like spine of the fifth sternite of the female.

Key to Palaearctic species of the genus Neoneurus Haliday 1
Females: third and following antennal segments with short and adpressed setae; fore tibia widened subbasally (Figs 46,49); metasoma apically with a strongly downcurved ovipositor and a short and elliptical ovipositor sheath (Fig. 53)  Fore femur straight in dorsal view (Fig. 45) and comparatively narrow in lateral view (Fig. 46); face without facial tubercles and bristles (Fig. 48); fore tibia without protuberances (Fig. 46); fore basitarsus 0.7-0.9 times as long as fore tibia (Fig. 46); [fore tibial spur 0.3-0.5 times hind basitarsus and its apical half narrow (Fig. 46) (Fig. 58) ...5 4 Fore tibial spur nearly straight and 0.7-0.8 times as long as fore basitarsus (Fig. 55); facial tubercles minute, with slender bristle and distance between bristles 1.2-1.3 times width of scapus (Fig. 57); apical tooth of fore femur medium-sized (Fig. 56); fore tibia 4.5-5.0 times as long as wide (Fig. 57) Fore tibial spur strongly curved and 0.8-0.9 times as long as fore basitarsus (Fig. 49); facial tubercles medium-sized, with robust bristle and distance between bristles about equal to width of scapus (Fig. 52); apical tooth of fore femur minute (Fig. 51); fore tibia about 6 times as long as wide (Fig. 49) Pair of facial bristles minute, 0.2 times as long as pedicellus and distance between bristles about 1.2 times width of scapus (Fig. 60); posterior subbasal tooth of fore tibia small, narrow (Fig. 58)  -Pair of facial bristles medium-sized, about as long as pedicellus and distance between bristles about equal to width of scapus (Fig. 67); posterior subbasal tooth of fore tibia medium-sized, wide triangular or falcate (Fig. 68) Length of fore tarsus 1.8-2.0 times fore tibia and tibia widened apically (Fig. 47); fore tibial spur straight or nearly so and with medium-sized setae (Fig. 47) Giraud, 1871, syn. n. (examined). According to Shaw (1992) the holotype of N. clypeatus is a male and has the third and fourth antennal segments shorter and less densely setose and the hind tibial spurs and hind tarsal segments shorter than in the male holotype of E. viennense; the body, hind coxa and the antenna of the holotype of N. clypeatus are much paler than those of E. viennense. E. viennense has the tegulae pale yellowish; dark brown or brown, the clypeus yellowish-white, yellowish-brown or brown and dorsally darkened, the hind coxa yellowish; largely black or dark brown. However, the holotype of N. clypeatus is coloured as a typical female and it may be either just an anomaly or a female. In general, Foerster types are paler by ageing than types from other collections of similar age, including Giraud types and limited weight should be given to colour differences in general.

Synonyms. Elasmosoma viennense
Notes. If the scapus and tegulae of a male are pale yellowish or whitish, the hind coxa largely yellowish-brown and the vertex finely granulate, the specimen may represent the unknown male of N. vesculus or N. recticalcar, the latter probably has a slenderer fore femur and tibia than the former. Oviposition behaviour. Unknown. Diagnosis. Fore tibia of female about 4.8 times as long as wide, slightly narrowed basally, with short carina and below it bristly setose and with a small anterior subbasal tooth (Fig. 55); mesosoma extensively marked with pale yellowish patches (Fig. 54); metasoma brownish-yellow, with first tergite entirely blackish and most tergites basally and apically dark brown, fore femur curved in dorsal view; fore spur straight and moderately wide. It does not run in the key by Shaw (1992) to any species because of having the long spur of the fore tibia combined with short facial spines. As indicated in our key it is similar to N. clypeatus, but easily to separate by the nearly straight fore tibial spur and minute facial tubercles.
Head. Length of third segment of antenna 1.1 times fourth segment, length of third, fourth and penultimate segments 5.0, 4.7 and 7.0 times their width, respectively and basal segments without distinct setae; facial tubercles small and facial bristles 0.4 times as long as pedicellus, distance between bristles about 1.3 times width of scapus (Fig. 57); length of eye 3.2 times temple in dorsal view; vertex superficially granulate, with few superficial punctures and a satin sheen; temples directly narrowed behind eyes; OOL:diameter of ocellus:POL = 11:5:12; length of malar space 0.10 times height of eye.
Mesosoma. Length of mesosoma 1.4 times its height; mesoscutum superficially punctulate-granulate, but medio-posteriorly densely granulate; precoxal sulcus medially slightly impressed and with a few rugae; mesopleuron superficially granulate, but postero-dorsally shiny and largely smooth; mesosternal sulcus finely crenulate, rather narrow and moderately impressed; metanotum with a median carina, moderately protruding dorsally; propodeum finely granulate and with some rugulae, dorsal face about as long as posterior face, with satin sheen, with complete median carina and no medial areola, flat antero-medially and its spiracle small and far in front of middle of propodeum.
Legs. Hind coxa largely superficially micro-granulate; fore coxa flat ventrally; all tarsal claws slender and simple; length of femur, tibia and basitarsus of hind leg 3.7, 7.6 and 7.0 times their width, respectively; fore femur curved in dorsal view, compressed and apically with medium-sized tooth; anterior subbasal tubercle of fore tibia small (Fig. 55) and longitudinal carina of tibia at basal 0.3, bearing a small posterior subbasal tooth, followed by bristly setae, area of tibia in between subbasal teeth concave (Fig. 56); fore tibia 4.7 times longer than its maximum width in lateral view; fore tibial spur straight, comparatively slender and about as long as fore basitarsus and 0.5 times fore tibia (Fig. 55); spurs of hind tibia acute apically, their length 0.8 and 0.7 times hind basitarsus.
Metasoma. Length of first tergite 1.6 times its apical width, its surface with satin sheen, granulate with some rugulae posteriorly, basally flat, medially convex and its spiracles slightly protruding and near middle of tergite; second tergite superficially granulate and anteriorly with some rugulae; second metasomal suture distinct but shallow; remainder of metasoma largely smooth and compressed; setae of metasoma spread, short, but second tergite and anterior half of third tergite glabrous; second tergite with sharp lateral crease; length of ovipositor sheath 0.06 times fore wing.
Etymology. From "rectus" (Latin for "straight") and "calcar" (Latin for "spur"), because of the straight spur of the fore tibia.  Oviposition behaviour. In recent years the oviposition behaviour of the genus Neoneurus was studied for the first time (Shaw 1992(Shaw , 1993 with Neoneurus mantis Shaw, 1992. Shaw proposed a "raptorial hypothesis" to explain the greatly modified morphology of Neoneurus fore legs (compression of the fore femur, robustness and shortening of the fore tibia, enlargement of the tibial spur, development of a tibial carina often associated with sharp tubercles and spines, shortening of the tarsus and enlargement of the fore pulvillus). These features, together with the two peculiar spinules situated in the frontal area of the head, could serve to grasp the ant before oviposition. Here we confirm for Neoneurus vesculus sp. n. Shaw's raptorial hypothesis. While this author mentions the metasoma of Formica pozdzolica as the location for alighting and oviposition for N. mantis, our observations show that Neoneurus vesculus sp. n. alights and probably oviposits in the mesosoma of Formica cunicularia Latreille, 1789.

Neoneurus vesculus
The observations were made in Madrid (at the enclosed area of the Institute for Agriculture and Food Research and Technology (INIA), Carretera de La Coruña Km 7.5, Spain) during August and September, 2010, in three colonies of F. cunicularia situated in the base of Atlas cedar trees (Cedrus atlantica). Neoneurus vesculus sp. n. could be seen around the nest entrances in the morning and in the afternoon, with a peak activity of oviposition attacks between 4-7 PM. Two strategies were observed (Fig. 62): a) the perching behaviour as described by Shaw (1993), the wasp standing on a grass stem, on a tree trunk (in both cases at a height less than 5 cm), or on the ground, until an ant approaches; a moment later the wasp takes flight and begins its attack (Movie Neoneurus, first sequence, Appendix III); b) the hovering behaviour -at a distance of about 1 cm-over ants leaving the nest entrance and going up the tree trunk at a height of 3 or 4 cm from the ground. Oviposition attacks following hovering behaviour (Movie Neoneurus, second sequence) increased in the afternoon, being then predominant over the perching behaviour.
When the ant moves up, the wasp approaches it from behind and waits until the ant's body is in a vertical position. Then, the wasp head hits the ant's mesonotum while the fore legs dart forward and brace the mesopleuron. The frame analysis reveals that the tibia are the part of the legs that firmly hold the mesopleuron (Figs 63-65).
After contact, the wasp's head is separated from the ant's body, the wasp's metasoma is placed vertically and its wings are folded. Then ovipositor insertion begins, during which time the middle legs can be seen to be sometimes holding the posterior part of the ant's mesosoma (Fig. 64). The wasp's metasoma is bent towards the posterior lower part of the ant's mesosoma, going between the metasoma and the hind leg of the ant. This occurred in a surprisingly asymmetric fashion: of 29 ovipositions observed, the wasp always bent its metasoma between the left hind leg of the ant and the left side of its metasoma. This suggests some asymmetrical morphology of the ovipositor system. According to the frame analysis, the ovipositor was inserted near the posterior coxal cavities, perhaps into the coxal cavities of the middle or hind legs or in the area between them (Fig. 66).
Oviposition was not always fully successful. Of a total of 25 attempts observed, 17 were completed, 4 were initiated but ended with the wasp and the ant -still joined- falling to the ground, and in the other 4 cases the wasp failed to grasp the ant and flew away immediately. Hence, the grasping of the ant appears to be a critical moment of the oviposition process. Sometimes the wasp's head hit on the ant's pronotum instead of its mesonotum, or the wasp attacked an ant that was not in a vertical position. In these circumstances it had more difficulty holding the ant, whose vigorous movements usually resulted in oviposition failure. Other times the first hit of the wasp's head, together with the strong grasping of its fore legs, caused the ant to detach from the surface and fall down with the wasp.
The whole oviposition behaviour of Neoneurus vesculus sp. n. (comprising the grasping of the ant by the wasp and the insertion of the ovipositor, until taking flight) lasted a mean of 2.023 seconds (95% confidence interval: 1.352-2.694; N = 17; SE = 0.317), with a median of 1.507 seconds (interquartile range: 1.377-1.927; Fig. 79). Three outlier observations corresponded to ovipositions lasting more than 3 seconds due to the ant's vigorous movements which made it difficult for the wasps to bend their metasomata towards the postero-lower part of the ants' mesosomata.
On one occasion a strange behaviour was observed. One Neoneurus hovering over the nest entrance alighted on the tree trunk, turning and resting, 2 cm away from a worker. The ant approached and touched the apex of the wasp's metasoma with its antenna. Then the wasp curved its metasoma inward extruding the ovipositor. Finally, the ant attacked the wasp, held it by the wings, and transported it into the nest.
Discussion. The described oviposition behaviour of Neoneurus vesculus sp. n. fits well with the raptorial function predicted by Shaw for the modified morphology of the genus Neoneurus. The head spinules may fix the position of the wasp when its head hits the ant's mesonotum and the robust tibia are suitable for grasping the ant's mesosoma by the mesopleura. The location of the wasp when alighting on the ant, and the final arrangement of its body, allow the insertion of the ovipositor into the postero-lower part of the ant's mesosoma. These facts call for a re-examination, with high speed photography or video, of the oviposition behaviour of Neoneurus mantis in order to confirm the alighting and oviposition of this species in the ant metasoma, as mentioned by Shaw (1993). It may be remarked that this author several times dissected the ant's metasoma following the wasp's oviposition, and could not find the wasp's eggs. The possibility is open that N. mantis, and other species of the genus, have a similar oviposition behaviour to that of Neoneurus vesculus sp. n., and hence that the eggs are laid in the ant's mesosoma. where the wasp's head will hit, and the mesopleuron (red arrow) that will be braced by the wasp's legs 3 after the first hit, the wasp's metasoma is positioned vertically and its wings are folded.

Diagnosis.
Fore tibia of female about 4.0 times as long as wide, distinctly narrowed basally, with long carina and below it a double row of small pegs and with a wide triangular anterior subbasal tooth; mesosoma extensively marked with pale yellowish patches; metasoma brownish-yellow, with first tergite entirely blackish and most tergites basally and apically dark brown, fore femur curved in dorsal view; fore spur nearly straight and robust; facial tubercles small and facial bristles 0.2 times as long as pedicellus, distance between bristles about 1.2 times width of scapus. Runs in the key by Shaw (1992) to N. pallidus Shaw, 1992, from Canada (Ontario) and USA (Maryland, Michigan, North Carolina, Virginia and Colorado), but the new species has the third and fourth antennal segments pale yellowish (dark brown (except base of third segment) in N. pallidus); first metasomal tergite entirely blackish (dark yellowish-brown and medially irregularly black), apex and base of second-sixth tergites dark brown (entirely pale yellowish-brown except dark yellowish-brown base of second tergite), fore tibia with row of small pegs below carina (below carina largely smooth, but a few small pegs near apex), fore tibia of female 4.0 times longer than its maximum width in lateral view (4.5 times) and fore tibia of female distinctly narrowed basally (slightly narrowed; Fig. 4 in Shaw 1992).
Head. Length of third segment of antenna 1.1 times fourth segment, length of third, fourth and penultimate segments 5.3, 4.8 and 2.5 times their width, respectively and basal segments without distinct setae; facial tubercles small and facial bristles 0.2 times as long as pedicellus, distance between bristles about 1.2 times width of scapus (Fig. 60); length of eye 1.5 times temple in dorsal view; vertex superficially granulate, with few superficial punctures and a satin sheen; temples directly narrowed behind eyes; OOL:diameter of ocellus:POL = 6:3:7; length of malar space 0.13 times height of eye.
Mesosoma. Length of mesosoma 1.4 times its height; mesoscutum superficially punctulate-granulate, but medio-posteriorly densely granulate; precoxal sulcus only medially impressed and with a few rugae; mesopleuron superficially granulate, but postero-dorsally shiny and largely smooth; mesosternal sulcus finely crenulate, narrow and rather shallow; metanotum with a median carina, not protruding dorsally; propodeum finely granulate and with some rugulae, dorsal face longer than posterior face, with satin sheen, only dorsally with a median carina and no medial areola, flat anteromedially and its spiracle small and far in front of middle of propodeum.
Legs. Hind coxa nearly smooth, dorsally partly superficially micro-granulate; fore coxa flat ventrally; all tarsal claws slender and simple; length of femur, tibia and basitarsus of hind leg 3.9, 9.2 and 5.3 times their width, respectively; fore femur curved in dorsal view, compressed and apically with small tooth; anterior subbasal tubercle of fore tibia wide triangular (Fig. 58) and longitudinal carina of tibia at basal 0.6, bearing a small posterior subbasal tooth and apical half curved, followed by a row of small slender pegs, area of tibia in between subbasal teeth concave (Fig. 59); fore tibia 4.0 times longer than its maximum width in lateral view; fore tibial spur nearly straight and 0.9 times as long as fore basitarsus and 0.5 times fore tibia (Fig. 58); spurs of hind tibia acute apically, their length 0.7 and 0.6 times hind basitarsus.
Metasoma. Length of first tergite 1.4 times its apical width, its surface with satin sheen, granulate with some rugulae posteriorly, basally flat, medially convex and its spiracles slightly protruding and near middle of tergite; second tergite superficially granulate and anteriorly with some oblique rugulae; second metasomal suture obsolescent; remainder of metasoma largely smooth and compressed; setae of metasoma spread, short, but tergites glabrous anteriorly; second tergite with sharp lateral crease; length of ovipositor sheath 0.05 times fore wing.
Variation. Length of body 2.6-3.0 mm, of fore wing 1.8-1.9 mm, all females have 16 antennal segments; mesoscutum medially, mesopleuron antero-medially and scutellum may be dark brown; third and fourth antennal segments pale yellow or brownish.
Etymology. From "vesculus" (Latin for "weak, little, poor") because this new species has poorly developed facial bristles. Oviposition behaviour. Donisthorpe and Wilkinson (1930) found naked pupae of H. buccatus among the cocoons of the ant Lasius alienus, and concluded that H. buccatus is likely to be parasitoid of adult ants, as are the wasps of the genus Elasmosoma. Although authors such as Watanabe (1984) and Marsh (1989) suggested that Hybrizon species may be endoparasitoids of ant larvae, the adult-parasitism hypothesis has remained, being included in general revisions dealing with ants (Hölldobler and Wilson 1990;Schmid-Hempel 1998). Here we report larval-parasitism of Hybrizon buccatus of the ant Lasius grandis Forel, 1909. The observations were made in Almazán (Soria, Spain) during July and August, 2010, on a permanent vertical trail of L. grandis, situated on a wall 60 cm high. The ants walked up and down, day and night, between two nest entrances of the same colony, one placed in the base and the other at the top of the wall (Fig. 73).

Ichneumonidae latreille, 1802
During the 3 weeks of observations, especially between 5-8 PM, one or two females of H. buccatus could be seen hovering over the trail, at 1 cm or less from the ants, usually in the lower part of the trail (less than 15 cm from the base). They could remain almost stationary in the air for more than 5 minutes. Even in the absence of ants on the trail for a period of time, specimens of H. buccatus found the precise location of the trail and stayed hovering over it. Location of the trail may involve olfactory or visual clues or both. After a long set of video recording, and hundreds of workers passing through the trail, no oviposition of the wasp could be observed on adult ants. Sometimes the wasp followed and approached an ant with a very quick movement, even touching the ant metasoma with its fore legs, but without oviposition (Fig. 74). This rapid approaching behaviour must be the one referred to Giraud (1857) and Donisthorpe (1910), respectively, as "pounces" and "striking" at the ants, behaviour that led Donisthorpe and Wilkinson 1930) to conclude adult-parasitism for H. buccatus.
The analyse of video frames revealed oviposition of H. buccatus into the final instar larvae of L. grandis while being transported by worker ants. Two cases were recorded, one with the worker going upward, and the other with the worker going downward (Movie Hybrizon, Appendix IV). In the first case (Fig. 75) the wasp grasped the larva    with its fore legs and placed its body in a vertical position over the adult ant. When the metasoma began to bend toward the larva, the middle legs seized the adult ant's head, and the wings were folded until oviposition finished. Throughout the process the hind legs remained in the air. The whole behaviour, comprising the grasping of the larva and the insertion of the ovipositor, until flying off, lasted 0.40 seconds.
In the second case (Fig. 76), contact of the fore legs with the larva can be seen, while the ovipositor is exserted. The middle legs are probably used to grasp the larva during the bending of the metasoma and oviposition. Again, the hind legs hang in the air. The whole behaviour lasted 0.58 seconds.
Specimens of H. buccatus twice ignored smaller larvae transported by workers of L. grandis (Fig. 77), which may indicate that only final instar larvae are selected for oviposition.
An unexplained aberrant behaviour was observed in Madrid (at the enclosed area of the Institute for Agriculture and Food Research and Technology (INIA), Carretera de La Coruña Km 7.5, Spain) in September, 2010, when a female of H. buccatus was hovering near a nest of L. grandis located at the base of an Atlas cedar tree (Cedrus   atlantica). First, the wasp held the apex of a grass stem with its fore legs, and then grabbed it with all legs, bending the metasoma and folding the wings (last sequence of Movie Hybrizon and Fig. 78). The frame analysis revealed the movement of the apex of the wasp metasoma touching the stem. The whole behaviour lasted 0.30 seconds.

General conclusions
From the observations here recorded on the oviposition behaviour of four European ant parasitoid wasps, some general conclusions are offered. The grasping of the ant (or the larva, in the case of Hybrizon buccatus) appears to be a critical phase of the wasps' oviposition. In all four species the wings are folded after alighting on the ant and during the insertion of the ovipositor. The legs are used to grasp the ant's body, following different strategies according to the species. H. buccatus uses the fore and middle legs to hold the ant larva; Neoneurus vesculus sp. n. has especially adapted fore legs to grasp the ant mesosoma firmly, making secondary use of the middle and, probably, hind legs. Elasmosoma luxemburgense and Kollasmosoma sentum sp. n. use all three pairs of legs.
In all four species the fore legs are the first to grasp the ant (or the larva, in the case of H. buccatus). Neoneurus vesculus sp. n., and usually E. luxemburgense, hit the ant's body with their heads when alighting.
In order to grasp the host, the visual perception of these ant parasitoids seems highly developed, especially considering the extremely short time elapsing during the oviposition sequence (Fig. 79). Hybrizon buccatus detects the ant larvae transported by workers along the trails, most probably selecting the mature instars. Neoneurus vesculus sp. n. directly hits with its head on the ant's mesonotum. Elasmosoma luxemburgense tends to alight by fixing attention on the posterior margin of the first gastral segment of the ant. Kollasmosoma sentum sp. n. has different alighting strategies corresponding to the inclination of the host's (Cataglyphis) metasoma, always aligning itself with the longitudinal axis of the ant's metasoma.
The location of the oviposition insertion varies in the four species, each presenting particular situations. H. buccatus lays the egg into an ant larva, apparently without any locational preference, but with the difficulty of dealing with the moving transporter worker. Neoneurus vesculus lays the egg in the postero-ventral part of the ant's mesosoma, bending its metasoma between the hind leg of the ant and the metasoma, certainly the longer and most complex of the ovipositions observed. Elasmosoma luxemburgense lays an egg into the posterior area of the last metasomal segment, probably through the anus. Kollasmosoma sentum lays the egg in any location of the ant's metasomal surface, probably through an intersegmental membrane; its extremely fast oviposition seems well adapted to the very speedy workers of Cataglyphis ibericus, which usually march with the metasoma held in a vertical position. Regarding the oviposition behaviour of the three neoneurines, the persistent defensive behaviour displayed by the ants is also significant. The ants are usually aware of the presence of the wasps, to which they turn towards with opened mandibles and sometimes catch them. Oviposition is also frequently impeded by the hits and movements of the ant's legs.