﻿Morphology of immature stages, biology, and systematic position of the Violet seed weevil, Orobitiscyanea (Linnaeus, 1758) (Curculionidae, Conoderinae, Orobitiditae, Orobitidini)

﻿Abstract The mature larva of the weevil species Orobitiscyanea (Linnaeus, 1758), one of only two Palaearctic members of the supertribe Orobitiditae, is re-described, while the pupa is described for the first time. The biology of this species was studied at two sites in Germany. It was reared from seed capsules of Violacanina L. (Violaceae), and feeding holes were observed on V.riviniana Rchb. Adults of Orobitiscyanea and O.nigrina Reitter, 1885, specialists of Viola, show a well-developed escape mechanism, to which contribute a smooth surface, a rounded, nearly spherical body shape, and a seed-imitating thanatosis behaviour. The molytine weevil Leiosomacribrum (Gyllenhal, 1834), the only other known weevil specialist of Viola in Europe, has a smooth surface, also, and is the most spherical species of the genus. The unique characters of the larva and pupa of Orobitiscyanea are discussed in regard to the systematic position of this taxon.


Introduction
they noted that the final decision about their placement required further research. The treatment of Orobitidinae as a separate subfamily was also upheld by Lyal (2013). In contrast, Prena et al. (2014) reduced this subfamily to supertribe rank within the subfamily Conoderinae, while still highlighting the significant differences (both in adults and in larvae) between Orobitidinae and the other taxa grouped in Conoderinae sensu lato. The position of Orobitidinae in the supertribe rank within Conoderinae was subsequently retained by Alonso-Zarazaga et al. (2017).
In view of the difficulty in clarifying of the taxonomic position of this widespread Palaearctic species, the critical morphological differences to other Conoderinae, some important discrepancies between previously published information on the larval stage (Urban 1925;van Emden 1938), our observations concerning the biology of the Violet seed weevil, and the lack of a description of its pupa, the purpose of this contribution is to provide new morphological information on the larval stage and to describe the pupa of the taxonomically isolated genus Orobitis, that may be valuable to clarify its systematic position. In his excellent paper, van Emden (1938) listed only some of the features of Orobitis that are different from other Conoderinae.

Study sites
On 3 July 2020, Orobitis cyanea was detected in stands of Viola canina L. in nutrientpoor grassland on a military training area near the village of Scheuen in the Celle district of Lower Saxony (Niedersachsen) (Fig. 1A, B). Two other Viola species, present at the same site and in the same habitat, were Viola arvensis Murr. and Viola tricolor L. subsp. tricolor. Whereas V. arvensis occurred mainly in small numbers, V. tricolor was very common at several spots there. In 2021, this site was visited once again, and the search for larvae and pupae was repeated on 19 June 2021. On 17 July 2021, the species was found in the 'Kleines Sandtal' ('Small Sand Valley') locality in the Harz National Park in the federal state of Sachsen-Anhalt, 3.6 km south-west of Ilsenburg. The specific microhabitat lays at the foot of a well-insolated south-facing slope, mainly along the border of a ditch with Viola riviniana Rchb. (Fig. 1C). The altitude is ~ 470 m a.s.l.

Methods
Before description, all the specimens were fixed in 75% ethanol and examined under an optical stereomicroscope (Olympus SZ 60 and SZ11) with calibrated oculars. The following measurements of the larva were made: body length (BL), body width (BW) (at the third thoracic segment), head capsule width (HW) and head capsule height (HH, measured from the apex to the epistoma). The pupal measurements included body length (BL), body width (BW) (at the level of the mid-legs), head width (HW) (at the level of the eyes), length of rostrum (RL) and width of pronotum (PW). Drawings and outlines were made using a drawing tube (MNR-1), installed on a stereomicroscope (Ampliwal), and were processed with computer software (Corel Photo-Paint X7, Corel Draw X7).
Slide preparation basically followed May (1994). The larva selected for study under the microscope was cut off and clear, next the mouth parts were separated. The remaining part of the body was cleared in 10% potassium hydroxide (KOH), then rinsed in distilled water and dissected. Consequently, the head, mouthparts and body (thoracic and abdominal segments) were separated and mounted on permanent microscope slides in Faure-Berlese fluid (50 g gum arabic and 45 g chloral hydrate dissolved in 80 g distilled water and 60 cm 3 glycerol) (Hille Ris Lambers 1950).
The photographs were taken using an Olympus BX63 microscope and processed with Olympus cellSens Dimension software. The larvae selected for SEM imaging (scanning electron microscope) were first dried in absolute ethanol (99.8%), then rinsed in acetone, treated by CPD (Critical Point Drying) and finally gold-plated. TESCAN Vega 3 SEM was used to examine selected structures.

Results
Description of the larva of Orobitis cyanea BL: 1.00-4.00; BH: 0.57-1.43; HW: 0.37-0.58 (all measurements are given in mm). The detailed results of measurements and the Growth Factor calculation are listed in Table 1.
General habitus and chaetotaxy. Body white, compact, almost round in outline (Figs 2B, 11A, B), partially (femora and tarsi) covered with fine asperities, rest of body smooth ( Fig. 11C-E). Rostrum elongate, almost 4× as long as wide, reaching metacoxae. Pronotum trapezoidal, 2× wider than long. Mesonotum wider than metanotum, with prominent triangular scutellar shield. Abdominal segments I-V of equal length, segments VI-VIII tapering gradually towards end of body, segment IX terminal. Gonotheca in female divided. Urogomphi (posterior processes) absent. Spiracles placed laterally on abdominal segments I-VI, functional on segments I-V, vestigial on segment VI. Chaetotaxy completely reduced, invisible even under the highest magnification.

Biological observations on host plants, life cycle, and antagonists of Orobitis cyanea
A search for immature stages at the site near Scheuen yielded several larvae and a few pupae on 19 June 2020 and 3 July 2021. They were found only in seed capsules of Viola canina (Fig. 12A). The examination of more than 20 capsules of V. tricolor did Figure 9. Orobitis cyanea mature larva, lateral view, habitus and chaetotaxy. Abbreviations: Th. I-IIIthoracic segments 1-3, Abd. I-X-abdominal segments 1-10, setae: ds-dorsal eps-epipleural, eus-eusternal, ps-pleural, pda-pedal, pds-postdorsal, prns-pronotal, prs-prodorsal, ss-spiracular, sts-sternal.  not reveal a single immature specimen or any feeding traces similar to those seen on V. canina leaves (Fig. 12B). Likewise, we did not obtain any larva or pupa at the Harz Mountains site on 17 July, but from the numerous feeding traces on the leaves and seed capsules and from the emergence holes in the capsules, the conclusion was drawn that development must have taken place in V. riviniana seed capsules, too (Fig. 13A, B).
In April and May, overwintering adults make small feeding holes in the leaves of their host plants, eating for maturation. At dry sites with early-flowering Viola species, such as V. canina L. or V. hirta L., eggs are laid mainly in April and May in the immature ovaries of the flowers. Larvae feed from young seeds, generating sufficient room to develop into the pupal stage at their feeding sites. Pupation occurred at both study sites inside the seed capsules in June and the first half of July. Adults left the seed capsules actively through feeding holes, or at the latest in July, by which time the seeds had ripened and the seed capsules burst open. Even in the Harz Mountains, the new generation had totally abandoned its place of development at the well-insolated site by mid-July, and some individuals were now occurring on their host plants; at that time, many adult weevils were present only in less exposed places. There were many feeding traces and adult weevils on the plants, but there were no more larvae or pupae inside the seed capsules. These were either still closed along the shady trench or had burst open on the sun-exposed slope.

Comments and inferences regarding the host plants, biology and parasitoids of Orobitis cyanea
Our observations regarding the feeding and development of Orobitis cyanea on Viola riviniana seed capsules are in accordance with those of Scherf (1964) The only species to add from our own observations is V. hirta L., from which O. cyanea was swept on one occasion in Luxembourg. But as this was only a singular finding without observation of feeding traces, the host plant status of V. hirta for O. cyanea has still to be confirmed. All these Viola species are violet in colour, and none are pansy species like V. arvensis, V. tricolor or V. × wittrockiana Gams ex Neuenb. & Buttl., which are also members of the genus Viola. Dieckmann (1972) described the development of O. cyanea in seed capsules, but he did not list any particular Viola species. His statement that O. cyanea was found to be a pest on pansy species has therefore to be regarded as doubtful. According to our observations, O. cyanea is related to natural habitats, but Morris (2012) reported it also from cultivated areas like pastures and gardens.
On the other hand, we confirm the information given by Dieckmann (1972) regarding the phenology and pupation of Orobitis cyanea in a cocoon, in the immediate vicinity of the feeding place or directly there. Our observations are contrary to those of Urban (1925), who reported a late start of development in the season, and violet seed capsules with larvae, pupae, fresh and fully coloured adults of O. cyanea that were studied in September. As Urban (1925) did not supply any data on either site or host plants, it can only be assumed that he studied the development of this weevil in moist, shady or cool sites with late-flowering host plant species, such as Viola epipsila or V. palustris. There, the first adults of the new generation should occur considerably later, in July and August, and the latest adults may hibernate at the pupation sites inside the capsules. Obviously, the activity of this oligophagous weevil is closely linked to plant development and may even differ from one locality to another as a result of microclimatic differences, as observed at the study site in the Harz Mts.
Some specimens of a parasitoid wasp, Blacus sp. (Fig. 14), were found in the samples of seed capsules containing Orobitis cyanea larvae from Scheuen, taken to the laboratory for rearing. Wasps of the genus Blacus Nees, 1818 (Blacini, Braconidae) are common parasitoids of weevil larvae and are frequently reported from weevil genera like Scolytus, Stereonychus, Gymnetron and Barynotus (Belokobylskii 1995;Farahani and Talebi 2013).

Larval instar determination and Growth Factor calculation
The method of larval instar determination worked out by Dyar (1890) has been widely accepted (Leibee et al. 1980;Rowe and Kok 1985). It was ultimately popularised under the name of Growth Factor (GF) (Sprick and Gosik 2014;Gosik et al. 2019), which is in fact Dyar's ratio -1 , which bears a closer relationship with natural development. The results of measurements and the Growth Factor calculation indicates three larval instars in Orobitis cyanea (Table 1) and GF values of 1.23 and 1.24 from the first to the second, and from the second to the third instar, respectively.
The number of larval instars in weevils is correlated primarily with the body size of a species. Thus, small species (head width of the mature larva below ~ 0.65 mm) usually have only three larval instars (Dosdall et al. 2007;Gosik et al. 2020;Skuhrovec et al. 2022), whereas larger species can have up to seven instars (Skuhrovec et al. 2022).
In some previously studied Entiminae species, GF usually varied between 1.38 and 1.44 (Leibee et al. 1980;. There is only one GF value each for a Ceutorhynchinae and Lixinae species: in Ceutorhynchus subpubescens LeConte, 1876 it was calculated at 1.43 (Dosdall et al. 2007) and in Rhinocyllus conicus (Frölich, 1792) at 1.538 (Rowe and Kok 1985). In the case of Orobitis cyanea, the significantly low GF value is probably correlated with the relatively big size of the first larval instar. There is usually only one larva per seed capsule (Urban 1925). Taking into consideration the limited dispersion capability and the close host plant affinities of O. cyanea, females invest more in the size than in the number of eggs.

Morphological adaptations and behaviour
When disturbed, Orobitis cyanea shows death-feigning or thanatosis behaviour and appears to imitate a Viola seed, which may be a form of mimicry: the dark part of the weevil may imitate the main part of the seed, the light part the elaiosome (Morris 2012;Kutzelnigg 2013). Thanatosis, the spherical shape and the colouring prove to be effective components of a shelter mechanism when escaping from danger, as they allow rapid down-rolling and concealment in vegetation or leaf litter below the plants, possibly among seeds or dark soil particles at the same time (Fig. 13B), especially as the smooth leaves of most Viola species support this escape mechanism. In addition, the behavioural data relating to Orobitis cyanea are presumably applicable to the closely related O. nigrina Reitter, 1885, which lives on Viola biflora L. in the Alps (Penecke 1922). Escape mechanisms similar to those described here for Orobitis species from Viola are widespread in weevils or even other beetles. Apparently, they have developed independently many times.
It seems worth mentioning that the only other European Viola-inhabiting weevil specialist, Leiosoma cribrum (Molytinae), occupies the top position in spherical body shape among all available Leiosoma species, where this could be tested (Table 2). In 21 of ~ 44 Leiosoma species, available as adult specimen or as habitus photo from different sources, e.g., Pedroni (2010Pedroni ( , 2012 or Bahr (2021), we determined the length-width ratio of the body to demonstrate the degree of spherical body shape (Table 2).
Leiosoma cribrum is the most rounded, shortest, and smallest species of this genus, approximating mostly the nearly perfect spherical shape of both Orobitis species. All other Leiosoma species are more elongate, closest are L. reitteri Bedel, 1884, L. apionides (Wollaston, 1864) (both ~ 1.88:1), and L. deflexum (Panzer, 1795) (1.91:1). The bulk of the species ranges between ~ 2.00:1 in L. diottii Pedroni, 2018, L. osellai Diotti & Caldara, 2020, and L. senex Pedroni, 2018:1 in L. hernicum Pedroni, 2012 andL. komovicum Pedroni, 2018. Even if only for a small part of Leiosoma species the host plant species are known (Sprick and Krämer-Klement, in press), it is noticeable that there should be some selection pressure to weevil specialists that live on Viola species, which are unable to fly, to improve the escape mechanisms by falling down, rolling away or imitate biotic or abiotic structures of the environment in which they live, e.g., seed in Orobitis, or soil or underground in Leiosoma. Night activity of Leiosoma species may be another behavioural adaptation to reduce the possible loss of adult weevils by unspecific predators.

The taxonomic placement of Orobitiditae -based on morphological studies of immature stages
In his comprehensive work, van Emden (1938) omitted several notable features that are probably almost impossible to notice in first-instar larvae, especially if only a light microscope is available. Above all, these are the deeply divided ligula, the T-shaped anus, the extreme reduction in head bristle size, the lack of stemmata, and the clypeus divided by a transverse furrow.
The larva of Orobitis cyanea is easily recognised by the following features: 1) postdorsal folds of abdominal segments I-VII undivided; 2) abdominal segments VIII and IX without prodorsal folds; 3) anus T-shaped, with dorsal and lateral lobes; 4) body cuticle with asperities forming rows and circles; 5) all spiracles unicameral; 6) epicranial Table 2. Comparison of length-width ratio of the body of Viola-inhabiting species (A) and from species with other or unknown host plants (B). Measured from the front margin of the eyes to the apex of elytra and at the widest part of the elytra. *Groups defined by Pedroni (2010Pedroni ( , 2012Pedroni ( , 2018 setae minute; 7) stemmata absent; 8) extremely elongate endocarina, almost reaching the epistome; 9) antennal sensorium elongate; 10) clypeus with prominent median depression and curved to inside anterior margin; 11) labrum extremely narrow with anterior margin deeply rounded inwards (concave); 12) clypeus with only one pair of cls; 13) labrum with two pairs of lrm; 14) labrum with one pair of ams, two pairs of als but no mes; 15) labral rods absent but presence of multiple rib like sclerotisations; 16) postlabium with two pairs of pms; 17) labial palpi uni-segmented; and 18) ligula divided. Knowledge of the immatures of the various Conoderinae supertribes is uneven. This is mainly because the supertribes of this subfamily differ in species numbers, distribution, individual abundance and economic importance. Bariditae and Ceutorhynchitae have been relatively well studied (Scherf 1964;Pakaluk 1993Pakaluk , 1994Prena et al. 2014). Conoderitae, on the other hand, have a rather small number of species with the preimaginal stages described (Gosik et al. 2021). Nevertheless, the available material is sufficient to discover the characteristics of each group. Since the larva of Orobitis cyanea is the only one in the genus Orobitis described so far and there are only two genera in the entire supertribe Orobitiditae, the above-mentioned features can be considered diagnostic of this suprageneric taxon, although it may change with further studies. Finding features common to all known larvae that would be diagnostic of Conoderinae sensu lato is not possible. Some larval characteristics present in all the supertribes belonging to this subfamily are in fact common to the family Curculionidae (May 1994), and then cannot be diagnostic of such a subfamily, such as 1) the numbers of des and les; 2) the numbers of some thoracic setae, i.e., two ps, one eus, one prs, one eps, one ps; 3) the numbers of some abdominal setae: one lsts, two eus, two ds, two sts, two ps; and 4) the numbers of mandibular and maxillary setae. Even though larvae from the subfamily Conoderinae Schoenherr, 1833 constitute a group that is morphologically very diverse, it is still possible to find larval features common to all of them, so long as Orobitiditae are excluded. But again, these common features are typical of the family Curculionidae: 1) abdominal segments I-VII with well-separated prodorsal folds and always divided postdorsal folds; 2) anal lobes divided into four X-shaped lobes; 3) thoracic and abdominal spiracles bicameral; 4) epicranial setae elongate; 5) endocarina absent or extending to mid-length of frons; 6) ocelli present; 7) antennal sensorium short, conical; 8) clypeus trapezium-shaped with two pairs of cls; 9) labrum semi-circular with rounded or slightly sinuate anterior margin, always with three pairs of lrs; 10) labral rods elongate, well visible; 11) labrum with three pairs of ams, three pairs of als and two mes; 12) labial palpi bi-segmented; 13) postlabium with three pairs of lrm; and 14) premental sclerite tridental, with elongate posterior extension.
The features given by Prena et al. (2014) as characteristic of Conoderinae pupae are very general and are widespread in other weevil subfamilies, also. Among those mentioned by these authors, there is not a single feature unique to Conoderinae. Moreover, many of the cited features are additionally annotated "present or absent".
In the pupae, the differences between Orobitiditae and other Conoderinae are more clearly visible: setae on head, rostrum, pronotum and abdomen always clearly visible vs no setae entirely; pupal urogomphi, which are more or less developed or reduced in Conoderinae, are completely absent in Orobitiditae.
In general, endophagous larvae have significantly shorter segmental setae than exophagous larvae (Gosik et al. 2016). However, in the case of the head bristles, the difference between the two groups is not so obvious. On the other hand, one of the characteristics of Orobitis cyanea larvae is the complete absence of long and mediumlength setae on both body and head. The longest bristles are considered at best to be microsetae. The others are almost indistinguishable from cuticular asperities.
It is worth noting that a structure similar to "ligula with depression in middle" has been described as characteristic of the larva of only one species of Baridini, namely Aulacobaris johanni (Korotyaev, 1988) (Nikulina 2013). However, it is difficult to draw any further conclusions about the relationship between these species from this feature.
Both larva and pupa of Orobitis cyanea display many diagnostic features and at the same time differences from other Conoderinae species that it is difficult to find arguments supporting the current systematic position of this species. We consider, therefore, that there is ample justification for retaining Orobitiditae as a separate subfamily (as suggested by Korotyaev et al. 2000).
The study of immatures of the two Neotropical Orobitiditae species could well provide new data, but at this stage, the placement of Orobitiditae within an enlarged concept of Conoderinae is not supported. Finding features unique to immatures of Orobitis is rather easy, but associating them with any other Curculionidae group is problematic. Therefore, leaving Orobitiditae as the subfamily Orobitidinae, as suggested by Alonso-Zarazaga and Lyal (1999) and Korotyaev et al. (2000), is best supported by our results.