Description of the Mature Larva of Synergus filicornis (Hymenoptera: Cynipidae: Synergini), with Notes on the Immature Larvae

Abstract 
 The final instar larva of Synergus filicornis (Cameron) (Hymenoptera: Cynipidae: Synergini) is described and illustrated. Morphological structures of a diagnostic value are discussed. The most remarkable character states shown by the mature larva of this species lie in the integument, which unlike the rest of described cynipoid mature larvae, presents tegumental differentiations. Additionally the antennal orbits, unlike those of other described Synergini mature larvae, did not present one seta on each antennal orbit. The immature larvae can be differentiated from the mature larva by the following characters: a) number and location of sensorial tegumental differentiations on the head, b) number of sensilla on the maxillary palpi, and c) morphology of the mandibles. The determination of the mandibles size, in this species, permits the estimation of the larval stages number.

This study addresses the larval morphology of a species belonging to the tribe Synergini, Synergus filicornis (Cameron) (= Synergus furnessana, Weld). The insects had been reared from galls of Andricus quercuslaurinus Melika & Pujade-Villar (Cynipidae: Cynipini) recently described from Mexico as a serious pest on Quercus laurina Humb. & Bonpl. (Fagales: Fagaceae) in the Hidalgo state (Melika et al. 2009;Pujade-Villar et al. 2012;Pujade-Villar 2013) and the presence of the inquiline, S. filicornis, increases the problem in the areas devastated by A. quercuslaurinus (Cebrian, unpublished data). Synergus filicornis was described from Guatemala by Cameron (1883); later Weld (1913) described a new species from Mexico (S. furnessana) and that later was synonymised to S. filicornis (Weld 1930). In Mexico this species develops in woody galls induce by Callirhytis furnessae 'sensu Weld' and A. quercuslaurinus. Regarding the relevant biological features of Synergus filicorniss, this species does not seem a lethal inquiline. However, this possibility cannot be completely ruled out, because although there is no evidence that the inquiline larva kills the inducer (Andricus quercuslaurina), it is possible that-on laying their eggs-the first females of S. filicornis, which oviposit after the gall is induced, could kill the egg of the inducer. It is also possible that the first instar inquiline larva might emerge from the egg before the inducer and kill it. This situation cannot occur later, since Andricus quercuslaurina is never found at the ends of the galls. The tribe Synergini, includes Synergus Hartig genus with 109 known species worldwide (Penzés et al. 2012). Previous to this study descriptions of the pre-imaginal stages of only 3 Synergus species were carried out: the Nearctic Synergus pacificus McCracken and Egbert by Evans (1965), and the mature larvae of the Western Palaearctic Synergus incrassatus Hartig and S. clandestinus Eady by Nieves-Aldrey et al. (2005). The aims of this study are to describe the final instar larva of Synergus filicornis, to increase knowledge of this immature stage of inquilines, and to compare its morphology with that of other 3 known species.

mATeriALs AnD meThoDs
Pre-imaginal stages of Synergus filicornis were obtained from the ends of the galls of Andricus quercuslaurina, collected from branches of Quercus laurina in Mexico (State of Hidalgo, Acaxochitlán township, La Victoria, [N 20° 10'16" and W 98° 11'25"], 2,120 m asl.). In this respect, it should be noted that the inquiline, S. filicornis, deposits its eggs on the developing gall of the host, A. quercuslaurina, and therefore the gall grows at the end points, at which new chambers form in which the inquiline develops alone. Only this kind of inquiline comes from this type of gall.
A total of 52 mature larvae and 67 immature larvae were cut out of galls on 25 Apr 2008 and preserved in 70% ethanol. Descriptions below, however, are based on a thorough study of several specimens only.
The length of the mandibles is defined as the maximum length measured from the head's articulation point to the tip of the basal tooth. For light microscopic preparation of larval stages, the methods described by Tormos et al. (2003Tormos et al. ( , 2004Tormos et al. ( , 2007Tormos et al. ( , 2009a were employed. Photos and measurements to the nearest 0.01 mm of different structures of the pre-imaginal stages were taken under a Leica MI65C microscope, equipped with a Leica EC3 camera using the Application Suite Version 3.6.O (Imagine software Integrates, Leica Microsystems Imaging Solutions) (Instituto Valenciano de Investigaciones Agrarias, Valencia, Spain). The sketches were made with a drawing tube and use of the Adobe Illustrator CS5 application to create and manipulate the vector drawings. For scanning electron microscopy, the live samples were frozen in slush N2 and attached to the specimen holder of a CT-1000C Cryo-transfer system (Oxford Instruments, Oxford, United Kingdom) interfaced with a JEOL JSM-5410 scanning electron microscope (SEM) (Universidad Politécnica de Valencia, Valencia, Spain). The samples were then transferred from cryostage to the microscope sample stage, where the condensed surface water was sublimed by controlled warming to -90° C. Then, the samples were transferred again to the cryostage and were sputter-coated with gold. Finally the samples were returned to the microscope sample stages for visualization at an accelerating voltage of 15 KeV. Images were also taken by using Oxford Instruments.
In the descriptions we essentially employed the terminology and organization used by Nieves-Aldrey et al. (2005) and Tormos et al. (2009a, b). In all cases the term "seta" is used in the meaning of setigerous sensilla. Data are presented as range and mean values ± standard deviation (SD). In order to determine the possible underlying implicit grouping in the larval instars, with respect to mandible length, a cluster analysis was performed by using the hierarchical clustering method. The statistical analysis was performed using the SPSS (v15.0) package. Voucher specimens are deposited at the Fundación Entomológica "Torres-Sala" (València, Spain).
Description of the Immature Larvae Numerous larvae were obtained with size [l = 1.25-2.18 mm (x -± SD = 1.67 ± 0.11, n = 58 ), maximum w = 0.46-1.25 mm (x -± SD = 0.88 ± 0.22, n = 51)] and with a morphology similar to the mature larvae: general aspect fusiform (Fig. 4a); integument with tegumental differentiations (Fig. 4b); spiracles with peritreme, asperities differentiated and closing apparatus adjacent to atrium (Fig. 5c), and head wider than high (Fig. 5d), narrower than the first thoracic segment, very weakly indented dorsally along its middle line, very weakly sclerotized except for apical half of mandibles, with pa-rietal bands absent, and antennal orbits circular with 2 sensilla at the centre, and labial palpi with 2 sensilla. Nevertheless, the immature larvae are distinguished from the mature larva in the following character states: a) number and situation of sensorial tegumental differentiations of the head, b) maxillary palpi with 2 sensilla, and c) mandibles with different morphology. In this respect, in the meconium 3 different mandibular models were identified (Fig. 8): a) one first model [l = 21-28 µm ( x -± SD = 25.52 ± 2.20, n = 22)] with only one tooth and slightly sclerotized, b) one second model [l = 33-41 µm (x -± SD = 32.96 ± 1.62, n = 27) with a well differentiated tooth and others lightly outlined and well sclerotized, and c) a third model [l = 61-66 µm (x -± SD = 63.35 ± 1.72, n = 31)] with 3 well developed and well sclerotized teeth (Figures 4d y 5c' show this latter model in an immature larva). These 3 models could correspond with different larval instars. The cluster analysis (Fig. 6) carried out using the variable for mandible-length of the immature and mature larvae revealed a perfect delimitation of fourth groups, probably corresponding to larval instars present in the larval phase of Synergus filicornis. Therefore, the length and morphology of the mandibles of the larval stage allows the supposition of the existence in S. filicornis of at least 4 larval instars.

DisCUssion
The mature larva of Synergus filicornis is similar in general appearance to other Cynipoidea (Evans 1987;Nieves-Aldrey et al. 2005). The general aspect is hymenopteriform, with 13 segments and yellowish color. Nevertheless, the integument, unlike the rest of described cynipoid mature larvae, presents tegumental differentiations. It shares a number of character states with the mature larvae of the inquiline cynipids  Table 1), among which we highlight the following: 1) body generally ventrally curved, fusiform, 2) head quite large relative to the body, 3) vertex generally incised, 4) antennal area small and inconspicuous, 5) maxillae triangular, 6) sensorial tegumental differentiations on: a) each side lateral to the antennal area (one), b) clypeus (1 pair), b) labrum (2 pairs), c) each gena (one), d) each maxilla (2) and e) each side of the labium (2); 7) maxillary and labial palpi large, conspicuous, and more or less protruding, 8) mandible tridentate with a broad second tooth, and with striations on the base. Additionally, in the Synergus species the labrum does not cover at all the apices of the mandibles. Nevertheless, unlike the rest of mature larvae of Synergini described, Synergus filicornis    Larval body: 1. Gradually tapering towards the posterior end (0) (Fig. 1a,b, b', c)/abruptly tapering towards posterior end (1). 2. With a fusiform appearance (0) (Fig. 1a, b', c)/with other appearances. 3. Fourth body segment (ventral view) is: not narrower than the 5th segment (0) (Fig. 1a)/clearly narrower than the 5th segment. 4. Head width (ventral view): clearly more than half the width of the first body segment (0) (Fig. 1a, b)/less than half the width of the first body segment (1). 5. Anterior and posterior margins of ventral part of first body segment: are more or less parallel, side forming an even edge posterior to the head or converge slightly to a thinner ventral area (0) (Fig. 1a)/are almost touching at the ventralmost point. Head region: 6. Vertex: rounded (0)/incised medially (1)  17. Mandible: more or less covered by labrum (0)/exposed (1) (Fig. 3b) Fig. 3 (6), c]/ not marked (0). 23. Labial palpi: clearly present (0) [ Fig. 3 (6), c]/absent or inconspicuous (1). 24. Left and right mandibles: more or less symmetrical (0) (Fig. 3b)/strongly asymmetrical (1). 25. Structure of mandibular incisor: sharp, thin and slender/strong and blunt (1) (Fig. 3b). 26. Number of teeth on left mandible: 3 or more (0) (Fig. 3b)/less than three. 27. Structure of apical tooth: straight, even inner margin (0) (Fig. 3b)/ serrated inner margin. 28. Second tooth of right mandible: reaching at least to the middle of the apical tooth (0) (Fig. 3b)/clearly shorter than half the length of the apical tooth (1). 29. Shape of apex of second tooth of the right mandible/rounded (0) (Fig. 3b)/other morphology (1). 30. Deep and wide gap separating the second and the third tooth: absent (0)/present (1) (Fig. 3b). 31. Surface of mandible: more or less smooth/sculptured at the base of the teeth with strong longitudinal and/or transversal striations (1) (Fig. 3c, see arrow).  (Evans 1965) does not allow for a in depth comparative analysis, and thus we can only state that this species shares with Synergus filicornis all character states mentioned in its description. Synergus filicornis presents the 2 autapomorphies that allow characterizing the mature larvae of the tribe Synergini (Nieves-Aldrey et al. 2005): a) second mandibular tooth broader and blunter than incisor, and b) presence of the sculpture on the base of the mandible.
The immature larvae are similar to those already described for Synergus pacificus McCracken and Egbert (Evans 1965). In Synergus filicornis, different mandibles lengths corresponds to different larval instars, which confirms that so far, as happens in parasitoids (Onagbola & Fadamiro 2008;Tormos et al. 2009a, b) this variable can also be used to corroborate the number of states of the larval phase, in certain inquilines. Unlike of other species of Cynipidae (Viggiani & Nugnes 2010), in the different larval instars of this species were not observed mandibular asymmetry nor variation in number and position of the spiracles.
With this description the authors concur with the statement by Nieves-Aldrey et al. (2005): "Undoubtedly, further study of immature stages of Cynipoidea will be rewarding from the perspectives of both phylogeny and larval identification. In particular, there is an urgent need to study a better sample of the larval phase of the insect parasitic cynipoids. Even for the phytophagous gall-inducers and inquilines, larvae of many important taxa remain to be described in detail". referenCes CiTeD Fig. 6. Synergus filicornis: Dendrogram from the cluster analysis using the mandible length as clustering variable (a, b, c, d correspond to the probable larval instars).