The morphology of the preimaginal stages of Rhinusaneta (Germar, 1821) and notes on its biology (Coleoptera, Curculionidae, Mecinini)

Abstract A detailed description of the mature larva and pupa of Rhinusaneta (Germar, 1821) and new diagnostic features of this species are presented. The development cycle of R.neta in the standard conditions lasts almost 60 days: an 11-day egg period, a 29-day larval period, and an 18-day pupal period, on average. The larvae are parasitised by hymenopterans of the superfamily Chalcidoidea. Similarities and differences with Rhinusabipustulata and other species of this genus are presented.


Introduction
The taxon Rhinusa attained the rank of the genus based on the classification made by Caldara (2001). It belongs to the tribe Mecinini and includes 40 species around the world . In Poland, ten species have thus far been recorded (Petryszak 2004;Mokrzycki and Wanat 2005), of which two, R. hispida (Brullé, 1832) and R. thapsicola (Germar, 1821), were recognised by Caldara (2013) as synonymous with fields; Lublin (51°13'39.15"N,22°38'31.43"E), lawn next to the street; Lublin (51°14'42.21"N, 22°32'12.93"E), ruderal vegetation next to tennis courts (UMCS campus); Niebrzegów (Bonów) (51°30'48.28"N,21°57'03.43"E),meadow;and Mięćmierz (51°18'22.50"N,21°54'15.80"E), unplowed strip between fields. Adults were collected from plants using a sweep net in series of 100 sweeps each time, at intervals of 3-7 days, from May to August on sunny, windless days, during the hours (10 am to 4 pm) where they are most active. To obtain preimaginal stages, the aboveground parts of Linaria vulgaris were collected for further detailed analysis in the laboratory. In total, ca. 520 specimens of the host plant were collected in the field and examined. The development stages (eggs, larvae, and pupae) were isolated from plants by making delicate cuts in the developing infructescence and extracting them from its interior. Some of the eggs and larvae (ten of both stages) were used for further breeding in the insectarium, and others were used for measurements (also ten) and microscope slides.

Breeding
Developmental stages (L 3 ) isolated from the generative parts were transferred to Petri dishes on an ongoing basis. Further breeding was carried out in accordance with recommendations by Scherf (1964) and Łętowski (1991). The Petri dishes were placed in an incubation chamber under the following conditions: temperature 25 °C during the day and 20 °C at night, humidity 40% during the day (16 h) and 50% at night (8 h). The humidity inside the Petri dishes was higher, 65-80%.

Graphics
Preparation of microscope slides of preimaginal stages and graphics. Larvae and pupae were treated with lactic acid C 3 H 6 O 3 (80%). Smaller morphological structures (mouthparts) were first immersed in a cold KOH solution (5%) before being transferred to lactic acid. Developmental stages were analysed and documented using an OLYMPUS BX61 microscope at magnifications from 200× to 400×. All graphics for the study were prepared using CorelDraw X8 software. Metric data are means from ten measurements. Drawings of the morphological structures of the larva and pupa were based on microscope slides prepared according to Łętowski (1991), Gosik (2010a), and Gosik et al. (2010). The nomenclature of larval (L 3 ) and pupal chaetotaxy was based mainly on the work of Marvaldi (2003), Oberprieler et al. (2014) and in the case unnamed structures the also on the work of Skuhrovec and Volovnik (2015). The morphology of the egg, L 3 and pupa and the developmental cycle from egg to adult were described as well. The biological material is stored in ethyl alcohol C 2 H 5 OH (70%) at the Department of Zoology, Animal Ecology and Wildlife Management, University of Life Sciences in Lublin.
Setae of the thorax and abdomen of the larva (L 3 ) and pupa are described for one side only.
Colouration. Head yellow-brown, body light yellow, covered with black, numerous setae.
Vestiture. Cuticle with strongly chitinised spots in several places on dorsal side. Asperities of body integument present on the surface of all thoracic segments and first abdominal segment covering area occupied by a group of setae prns and pds. Analogous structure presents only on the surface of the first pedal lobe.
Abdomen ( Figure 6). Abdominal segments I-VII of similar shape. Tergites I-VII with two folds, prodorsum with one seta on ridge (prs1), postdorsum with five setae: four located dorsally (pds1-3, dls1) and one, longest seta (ss) on spiracular area. Epipleurum with four setae (dpls1-3 and 1 vpls), sternum with two setae (one lsts and one msts). Segments I-VIII with unicameral spiracles, others (IX-X) without spiracles. Segment VIII with two folds, setae arranged as on segments I-VII, except for lack of seta prs. Segment IX with four setae, two on pleura (one ds and one ls) and two on sterna (one ls and one sts). Segment X with two small setae, one on pleura and one on sterna (both ts).
Colouration. Yellow-brown with distinct chaetotaxy. Head (ventral view): rostrum reaches end of mesothorax, with one short seta (drs) apically. Head with distinct eyes and one seta (sos) at their inner edge. Antennae at base of rostrum. Massive. Thorax: pronotum wider than long, trapezoid-shaped, Table 2. Differences of the pupa of Rhinusa neta and R. bipustulata based on the publications of Scherf 1964, Anderson 1973, Gosik 2010a, and on personal research.

Rhinusa neta
Rhinusa bipustulata Body length 3.35 mm (mean), width (between the apex of mesofemora)1.92 mm (mean), yellow-brown length 2.9-5.0 mm, width (between the apex of mesofemora) 1.50-2.60 mm, white or yellowish Head rostrum with 1 seta (drs), head with 1 seta (sos) rostrum with 3 setae -2 drs, 1 es, head with 2 setae (brs) Thorax pronotum: 8 setae: 2 aps, 3 lps, 2 bps, 1 dps, all femora with 1 long seta (fes) pronotum: 9 setae: 2 aps, 3 lps, 2 bps, 2 dps, all femora with 2 long setae (fes) Abdomen dorsal part of segments I-VIII with 4 setae of unequal length in one row and 2 of setae located laterally on pleural area III-VIII dorsal part of segments I-VIII with 4 setae of unequal length in one group (3) and 1 seta located laterally and 1 seta on pleural area I-VIII ventral part of segments I-VIII with 4 of setae, of which 1 medial is longer and 3 shorter, arranged in row ventral part of segments I-VII with 5 short setae distributed in regular lines abdominal segment VIII with 4 setae of unequal length located dorsally, 1 lateral slightly shorter and 4 short setae located ventrally in regular line abdominal segment VIII with 2 microsetae located dorsally and 2 short, thin setae located ventrally abdominal segment IX without setae abdominal segment IX with 3 microsetae located ventrally pseudocerci (urogomphi) longer, clearly visible pseudocerci (urogomphi) very short, poorly visible with two distinct, highly sclerotised, bare tubercles at anterior margin, with eight long setae: aps1-2, lps1-3, dps2, and bps1-2 (Figs 7-9). Mesonotum longer than metanotum. Latter with two clearly visible scutellar shields posteriorly. Dorsal part of meso-and metanotum with three setae (msns, mtns) of unequal length located laterally. All femora with one long, thin seta (fes) located apically. Abdomen: tergites I-VII with four setae in one row slightly beyond midpoint of segment. Longest located near lateral outer margin, on segments I-V below spiracle entrance, on others (VI-VII) centrally. Of three remaining setae, two short -centrally located and third from middle of segment, second from centre somewhat shorter, but clearly shorter than outer seta. Segment VIII with four setae, shortest located medially, longest approx. three times longer than first. Segment IX with one distinct urogomphi, darker, bent back, highly chitinised (pseudocerci -pc) (Figs 7, 8). Sternites: segments I-VIII with four setae arranged in one row, of similar length except longest pair located medially (Figs 7,9). Spiracles on abdominal segments I-V placed laterally, functional. Pleurites (III-VIII) with seta of equal length, slightly shorter and curved on segment VIII, pleurites (I-II) without setae (Figure 7). Biological information. After overwintering, adults emerge in May and June, depending on weather conditions in the year (Burakowski et al. 1997;Wilson et. al 2005). Initially, they feed on the vegetative parts of plants, and after the inflorescence has been formed they feed on the generative parts. After copulation, the females lays eggs into channels bored in the seed capsules and then uses excrement to seal the hole. One or two larvae were usually observed in one seed capsule. Approximately 10-12 days after eggs were laid eggs in the laboratory, L 1 larvae appeared, which fed on the seeds without causing them to swell. The full larval stage lasted on average 29 days, followed by pupation. This stage lasted approximately 18 days. The full development Figure 7. Pupa, lateral view: aps -apical pronotal seta, bps -basal pronotal seta, dps -discal pronotal seta, drs -distrirostral seta, fes -femoral seta, lps -lateral pronotal seta, msns -mesonotal seta, mtns -metanotal seta, pds -postdorsal seta, pls -pleural seta, pc -pseudocerci, sos -supraorbital seta, sp -spiracle. Figure 8. Pupa, dorsal view: aps -apical pronotal seta, bps -basal pronotal seta, dps -discal pronotal seta, fes -femoral seta, lps -lateral pronotal seta, msns -mesonotal seta, mtns -metanotal seta, pds -postdorsal seta, sp -spiracle, cycle of the beetle in laboratory conditions averaged 58 days. Some larvae died in the seed capsules, having been attacked by parasitic hymenopterans of the superfamily Chalcidoidea, with parasitism reaching 20%. Species complete one generation per year and the new generation of adults emerge in August and September. Figure 9. Pupa, ventral view: aps -apical pronotal seta, bps -basal pronotal seta, dps -discal pronotal seta, drs -distrirostral seta, fes -femoral seta, lps -lateral pronotal seta, pc -pseudocerci, sos -supraorbital seta.
A study by Emden (1938) includes key characters by which the larvae of R. neta can be distinguished from R. antirrhini and R. collina. R. neta has a lighter, brownishyellow head with a coarsely granulate frons. In addition, this species has 2-3 basal bristles of the maxillary mala that are more prominent than the others ( Figure 5). Scherf (1964) presents only few data on the biology of R. neta larvae, regarding its spectrum of host plants, feeding site, and the shape of the cecidia it causes. A work by Anderson (1973) presents graphics illustrating the frons of R. neta with an identical arrangement of setae (but unnumbered and unnamed) as in the present study, as well as characteristic, transverse asperities on its surface, making the L 3 larvae easily distinguishable from Rhinusa antirrhini, which also feeds on the seeds of Linaria sp. (in which the frons of the L 3 larva is smooth). Another distinctive character of R. neta is the presence of a long endocarina extending 4/5 of the length of the frons. On the epipleuron and pleuron of the abdominal segments (I-VIII) of the L 3 larvae, there is a group of setae dpls1-3 and vpls1, which differs from the typical arrangement found in weevils (dpls1-2 and vpls1-2), but is confirmed in the key by Anderson (1973), according to which the three epipleural setae dpls distinguish R. neta from R. antirrhini, which has only two.
The number of setae on the dorsal surface of mandible within the Curculionidae family usually varies from 0 to 2. On this surface, there are also sensillae in the number of 0 to 3. The mandible of the discussed taxon generally resembles the system present in Ceutorhynchinae in terms of the setae and sensillae system (Scherf 1964), except that R. neta has three setae of similar length arranged in one line. The arrangement of the three setae at the mandible surface discussed in the paper is unique within this family.
In regard to the characters contained in the key by Marvaldi (2003) describing the length of a few setae on the frons (fs4, des5, and les), they are also well visible in this species, but setae fs5, des3, and les2 are well developed as well. On the other hand, the lengths of setae fs4 and fs5, which in Curculionidae sensu lato should be at least of similar length (or fs4 should be longer), are not in agreement. In R. neta the pattern is reversed, as fs5 is longer than fs4. A similar relationship between the lengths of these two setae is also reported in a study on R. bipustulata (Gosik 2010a), where fs5 is designated as fs3.
As regards the biology of species, in addition to Rhinusa taxa, data on R. pilosa have been published as well (Gassmann et al. 2014). Phylogenetic research by Caldara et al. (2010), based on morphological data and host plants, indicates that the taxa most closely related to R. neta are R. collina (which is also confirmed in the key by Emden (1938)), R. eversmanni, R. canescens and R. soluta, but their preimaginal stages have not yet been described in detail. The similarities and differences between the larvae and pupae of R. neta and R. bipustulata, which has been studied in the greatest detail by Gosik (2010a), are presented below. The most significant differences between these species in the case of the L 3 larval instars and pupae are shown in bold in Tables 1 and 2. The information given in the two tables, grouping all available differences in the morphological structure of the L 3 larva and pupa, can be used to prepare more detailed keys, both between the taxa given above and at the level of the tribe.
In the case of the pupae of the two taxa, the differences are in the number and location of the pairs of setae on the rostrum and head, number of pairs of setae on the prothorax, number of setae on the femora, location of the setae on the tergites, number of setae on the pleurites, number of setae on the sternites of segments I-IX, and length of the pseudocerci (or urogomphi).
Data presented by Wilson et al. (2005) indicate that in Canada the development cycle of R. neta lasts ca. 40-60 days, including 20-30 days for the larval stages and 10-15 days for the pupal stage.