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

Rafat Gosik', Peter Sprick*

| Department of Zoology and Nature Protection, Faculty of Biology and Biotechnology, Maria Curie
Sktodowska University, Akademicka 19, 20-033 Lublin, Poland 2 Curculio—Institute e.V. (CURCD),
Weckenstrafse 15, 30451 Hannover, Germany

Corresponding author: Rafat Gosik (r.gosik@poczta.umcs.lublin. pl)

Academiceditor: Miguel Alonso-Zarazaga | Received23 May 2022 | Accepted 18 August 2022 | Published 12 September2022
https://zoobank. org! C3FEAEC9-DA17-4B29-A359-54EEBLASFECF

Citation: Gosik R, Sprick P (2022) Morphology of immature stages, biology, and systematic position of the Violet
seed weevil, Orobitis cyanea (Linnaeus, 1758) (Curculionidae, Conoderinae, Orobitiditae, Orobitidini). ZooKeys
1121: 59-82. https://doi.org/10.3897/zookeys.1121.86888

Abstract

The mature larva of the weevil species Orobitis cyanea (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 Viola
canina L. (Violaceae), and feeding holes were observed on V. riviniana Rchb. Adults of Ovobitis cyanea 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 Leiosoma cribrum (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 Ovobitis cyanea are discussed in regard to the systematic position of this taxon.

Keywords

Blacus, escape mechanism, life cycle, mimicry, parasitoid, thanatosis

Copyright Rafat Gosik & Peter Sprick. This is an open access article distributed under the terms of the Creative Commons Attribution License
(CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

60 Rafal Gosik & Peter Sprick / ZooKeys 1121: 59-82 (2022)

Introduction

The subfamily Conoderinae Schoenherr, 1833, in the broad sense of some current
classifications (Prenaetal. 2014; Alonso-Zarazagaetal. 2017), is distributed worldwide
and contains four supertribes: Bariditae Schoenherr, 1836; Ceutorhynchitae Gistel,
1848; Conoderitae Schoenherr, 1833 (also known as Zygopinae Lacordaire, 1865),
and Orobitiditae C. G. Thomson, 1859. From the total number of 7571 described
Conoderinae species in 940 genera, approximately 4032 belong to Bariditae, 2164
to Conoderitae, ~ 1371 to Ceutorhynchitae, and just four to Orobitiditae (Prena
et al. 2014). The Violet Weevils (Orobitiditae) include two genera: 1) Parorobitis
Korotyaev, Konstantinov & O’Brien, 2000 with two Neotropical species: P gibbus
Korotyaev, Konstantinov & O’Brien, 2000 and P minuta Korotyaev, Konstantinov
& O’Brien, 2000; 2) Orobitis Germar, 1817 with two Palaearctic species: O. nigrina
Reitter, 1885 and O. cyanea (Linnaeus, 1758) (Korotyaev et al. 2000). Orobitiditae
are a very uniform group, owing to the extraordinarily convex body, 1.8—3.5 mm in
size, the rostrum bent at the antennal insertion, the fused meso- and metasternum,
the first ventrite no longer than the second, the claws with appendages fused in an
entire median process, and the unique structure of the stridulatory device (Lyal and
King 1996; Korotyaev et al. 2000). The distribution of Orobitis cyanea includes
Europe from the Arctic Circle to the Mediterranean, Siberia, central and east Asia
with Asia Minor (Morris 2012; Alonso-Zarazaga et al. 2017). As Orobitis cyanea has
limited dispersal abilities owing to its reduced or non-existent wings (Dieckmann
1972), it inhabits a wide range of natural or near-natural habitats: well-insolated
deciduous woodlands and forest edges, nutrient-poor grasslands, limestone
grasslands, marshes, sand dunes, and cliffs (Smreczynski 1974; Morris 2012).
Interestingly, it has also been reported as a pest species in nurseries (Dieckmann
1972), but this latter observation, dating 50 or more years ago, could indicate that
nurseries were then situated close to natural habitats or that plant material was
exchanged between them.

To date, the systematic placement of Orobitidinae has been changed many times.
Van Emden (1938) accepted the status of Orobitinae (sic) in the rank of a subfamily.
At the same time, taking into account the morphology of the larval stage, he drew
attention to their fundamental distinctiveness from Ceutorhynchinae (originally
Ceutorrhynchinae), and he noticed some similarities between Orobitinae, Apioninae,
and Gonatoceri. Dieckmann (1967, 1972), Lohse (1983), and Smreczynski (1974)
included Orobitidinae in Ceutorhynchinae, whereas Zherikhin and Gratshev (1995)
placed Ovobitis in an enlarged concept of Barididae.

However, Alonso-Zarazaga and Lyal (1999) extracted them as a separate subfam-
ily. Also, Korotyaev et al. (2000), based on a very detailed morphological analysis
of the adult stage of Ceutorhynchinae, Zygopinae (i.e., Conoderinae sensu stricto),
Baridinae, and Orobitidinae, left all these groups in the rank of subfamilies, emphasis-
ing especially the distinctiveness of Orobitidinae from the others. At the same time,

Immatures, biology and taxonomy of the Violet seed weevil 61

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 sub-
family 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 sub-
sequently 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 Ovobitis that are different from other Conoderinae.

Materials and methods

Study sites

On 3 July 2020, Orobitis cyanea was detected in stands of Viola canina L. in nutrient-
poor 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 ma.s.l.

Material studied

Larvae: 10 exx. 03.07.2020, Scheuen (Celle), military training area, dry, nutrient-
poor grassland on sandy soil, in Viola canina seed capsules (Fig. 2A).

Pupae 9: 1 ex. 03.07.2020, 2 exx. 19.06.2021, Scheuen (Celle), military train-
ing area, dry, nutrient-poor grassland on sandy soil, in Viola canina seed capsules

(Fig. 2B).

62 Rafat Gosik & Peter Sprick / ZooKeys 1121: 59-82 (2022)

Figure |. Biotopes of Orobitis cyanea A, B dry, nutrient-poor grassland on sandy soil near Scheuen

C clearing on a well-insolated south—facing slope in the Harz National Park, 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). Draw-
ings and outlines were made using a drawing tube (MNR-—1), installed on a stereomi-
croscope (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 un-

der the microscope was cut off and clear, next the mouth parts were separated. ‘The

Immatures, biology and taxonomy of the Violet seed weevil 63

Figure 2. Orobitis cyanea A larva and seeds in a fruit of Viola canina B pupa in seed capsules of V. canina.

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 micro-
scope slides in Faure—Berlese fluid (50 g gum arabic and 45 g chloral hydrate dissolved
in 80 g distilled water and 60 cm? 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.

The general terminology and chaetotaxy follow Anderson (1947), May (1994),
Marvaldi (1999, 2003), and Skuhrovec et al. (2015); the terminology for the antennae
follows Chaika and Tomkovich (1997). Larval instar determination and calculation of

the Growth Factor (GF) are based on Willis (1964) and Gosik et al. (2019).

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. Live larva pure white, with yellow head capsule
(Fig. 2A). All spiracles unicameral; thoracic (Fig. 3A) placed laterally between pro- and
mesothorax; abdominal spiracles (Fig. 3B) placed medio-laterally on segments I—VII.
Body rather elongate, curved, rounded in cross section.

64 Rafat Gosik & Peter Sprick / ZooKeys 1121: 59-82 (2022)

Table 1. Measurements and Growth Factor calculation in Orobitis cyanea larvae (measurements are
given in mm, "—number of specimens; HW is relevant to GF calculation; abbreviations: BL—body length,

BW-body width, HW-—head width; HH-head height).

Instar HW HH BL BH GF
1 instar Ore 0858) 0:393-055) 1.00'; 1.05! 0.57'; 0.60!
24 instar 0.467; 0.47! 0.407; 0.42! 3.007; 3.16! 0.837; 1.00! 1.23
3"4 instar 0.577; 0.58?; 0.467; 0.507; 0.53! 3.00!; 3.667; 4.007 —-1.00!; 1.16!; 1.337; 1.43); 1.24
(mature)

Figure 3. Ovobitis cyanea mature larva, spiracles A spiracle of prothorax B spiracle of abdominal segment I.

Head and antenna. Head capsule (Fig. 4A—C) almost rounded; endocarina reach-
es 4/5 of the frons; frontal sutures distinct along entire length up to antennae; stem-
mata (st) invisible. Hypopharyngeal bracon without median sclerome. Setae of head
minute, only des. and setae on frons short, hair—like. Cranial setae: des, placed medi-
ally, des, placed posterolaterally, des, and des, placed suture on epicranium away from
frontal suture, des, placed anterolaterally, fs, placed medially, fs, placed anteromedially,
fs, placed anterolaterally, close to epistome, /es, and /es, placed close to des,, postepic-
ranial area with one pes. Antennae (Fig. 4D) placed on each side at anterior margin of
head; membranous basal segment convex, semi—spherical, bearing conical, distinctly
elongated sensorium and nine sensilla: five basiconica (sb) and three styloconica (ss).

Mouthparts. Clypeus (Fig. 5A) ~ 4.5x wider than long, with single c/s medium
in size, placed posteromedially, sensillum (clss) posterolaterally. Anterior margin of
clypeus distinctly concave. Labrum (Fig. 5A, B) - 2x wider than long, anterior margin
sinuated; /rs, medium, placed anteromedially, /s, absent and lrs, medium, placed
posterolaterally. Epipharynx (Fig. 5C) with two ads and one ams, all semi-circular, mes
absent. Labral rods (Ir) absent as such but five sclerotisations like ribs distinct between
the ams and als (Fig. 5D). Clypeus and labrum distinct, with transverse, median furrow.

Immatures, biology and taxonomy of the Violet seed weevil 65

SEM HV: 30.00 kV WD: 18.09 mm | SEM HV: 30.00 kV WD: 29.02 mm

View field: 642.0 ym Det: SE 200 pm View field: 642.0 um Det: SE

SEM MAG: 450 x Date(midly): 01/28/22 SEM MAG: 450x = Date(m/d/y): 01/31/22

des,O
C

SEM HV: 30.00 kV WD: 8.404 mm

= : : } SEM HV: 30.00 kV WD: 8.854 mm
View field: 722.3 pm Det: SE View field: 28.89 ym Det: SE
_ SEM MAG: 400x _ Date(midiy): 01/31/22 SEM MAG: 10.00 kx Date(m/dfy): 01/31/22

Figure 4. Ovobitis cyanea mature larva, head and antenna (SEM micrograph) A frontal view B lateral view
C ventral view D antenna. Abbreviations: at — antenna, sb — sensillum basiconicum, Se — sensorium, ss

—sensillum styloconicum, setae: des — dorsal epicranial, fs — frontal, /es — lateral epicranial, pes — postepicranial.

Mandible (Fig. 6) with two apical teeth of almost equal height, the inner one subapical
and slightly smaller; cutting edge smooth, without additional protuberance; setae:
mds, and mds, minute, both placed medially in shallow pits. Maxillolabial complex:
(Figs 7A, B, 8A) stipes with a medium s¢ps, two short pfs, and one minute mbs plus

66 Rafal Gosik & Peter Sprick / ZooKeys 1121: 59-82 (2022)

A B fd
SEM HV: 30.00 kV WD: 17.88 mm | | SEM HV: 30.00 kV Wo: 8.538 mm |
View field: 240.8 pm Det: SE 50 pm View field: 144.5 pm Det: SE (20 ym

SEM MAG: 1.20 kx | Date(m/d/y): 01/28/22 . SEM MAG: 2.00 kx Date(m/dfy): 01/31/22 |

Figure 5. Orobitis cyanea mature larva, clypeus, labrum, epipharynx and mandible A, B clypeus and
labrum (SEM micrographs) C clypeus, labrum and epipharynx D epipharynx with ribs. Abbreviations:

clss — clypeal sensorium, setae: ams — anteromedial, a/s — anterolateral, c/s — clypeal, /rs — labral.

sensillum; mala with row of four dms various in shape and size (first semi-circular,
second and third elongated, pointed, fourth short, blunt) and a group of four digitate,
medium vms; maxillary palpi bi-segmented; basal palpomere distinctly wider and
shorter than distal one; length ratio of basal and distal palpomeres 2:1; basal palpomere
with medium short mps and one pore, distal palpomere (Fig. 8B) with one digitiform
sensillum (ds) and a group of 13 apical sensilla (ampullacea) on terminal receptive
area (tra) (Fig. 8C); dorsal parts of mala partially covered with fine asperities; labium
with cup-shaped prementum, with one medium prms placed medially (Fig. 7A);
ligula divided, with two minute /igs, at margin covered with prominent asperities
(Fig. 8B, E, F); premental sclerite C-shaped; postmentum rather elongate, and narrow,
membranous, triangular, with two medium pms: pms, situated posterolaterally and
pms, mediolaterally; labial palpi one-segmented; each palpus with single pore, distal
palpomere with a group of 12 apical sensilla (ampullacea) on terminal receptive area
(Fig. 8D); surface of labium smooth.

Immatures, biology and taxonomy of the Violet seed weevil 67

Figure 6. Orobitis cyanea mature larva, left mandible. Abbreviation: mds — mandibular setae.

Body. Prothorax small, pronotal shield not pigmented; mesothorax slightly smaller
than metathorax. Meso- and metathorax each divided dorsally into two lobes (pro-
dorsal and postdorsal lobes almost equal in size). Pedal lobes of thoracic segments
isolated, conical, prominent. Abdominal segments I-III of similar size, slightly smaller
than metathorax (Figs 9, 10A). Segments [V—IX tapering towards posterior body end.
Abdominal segments I—-VII each with weakly developed prodorsal fold and promi-
nent, undivided postdorsal lobe (Figs 9, 10B). Segments VIH—IX dorsally undivided.
Epipleural lobes of segments I—VII slightly conical, on segments VIII and IX almost
invisible. Laterosternal and eusternal lobes of segments I—VIII conical, weakly isolated
(Figs 9, 10C). Abdominal segment X divided into three lobes, dorsal small, lateral
lobes prominent, of almost equal size. Anus situated terminally. Body cuticle with
asperities forming rows and circles (Fig. 9D). Lateral part of prothorax densely covered
with thorn-like asperities, arranged in vertical rows (Fig. 9E, F).

Chaetotaxy: distinctly reduced, most setae minute, thorn—like, only on dorsal part
of abdominal segment IX very short, hair—like. Thorax (Fig. 9A): prothorax with seven
equal in size prns, two ps, and one eus. Meso- and metathorax each with one prs and one

68 Rafat Gosik & Peter Sprick / ZooKeys 1121: 59-82 (2022)

Figure 7. Orobitis cyanea mature larva, maxillolabial complex and apical part of maxilla A maxillolabial

complex, ventral aspect B apical part of left maxilla, photo. Abbreviations: setae: dms — dorsal malar,
ligs — ligular, mbs — malar basiventral, mps — maxillary palp, pfs — palpiferal, prms — prelabial, pms — post-

labial, stps — stipal, vms — ventral malar.

pas, two ss, one eps, one ps and one eus. Pedal areas of thoracic segments each with three
pda. Abdomen (Fig. 9B, C): segments I-VI with one prs, one pds, one ss, one eps, one
lsts, and one eus. Abdominal segments VI and VIII with one pds, one ss, one eps, one
lsts, and two eus. Abdominal segment IX with two ds, two ps, and two sts. Abdominal
segment X without setae.

Immatures, biology and taxonomy of the Violet seed weevil 69

y 4
A i
SEM HV: 30.00 kv WD: 8.400 mm ! SEM HV: 30.00 kV WD: 8.424 mm ial |
View field: 288.9 pm Det: SE 50 pm View field: 144.5 pm Det: SE 20 um
SEM MAG: 1.00 kx Date(m/diy): 01/31/22 SEM MAG: 2.00 kx Date(m/d/y): 01/31/22

Cc )
SEM HV: 30.00 kV WD: 7.999 mm ia SEM HV: 30.00 kV WD: 8.012 mm | |VEGA3 TESCAN
View field: 16.05 pm Det: SE Sum

SEM MAG: 18.00 kx Date(m/d/y): 01/28/22

View field: 24.08 pm Det: SE Sum
SEM MAG: 12.00 kx | Date(m/d/y): 01/28/22 |

F

SEM HV: 30.00 kV WD: 17.96 mm | | VEGA3 TESCAN SEM HV: 30.00 kV WD: 47.97 mm
View field: 28.89 pm Det: SE View field: 28.89 um Det: SE
SEM MAG: 10.00 kx Date(m/d/y): 01/28/22 SEM MAG: 10.00 kx | Date(m/diy): 01/28/22

Figure 8. Orobitis cyanea mature larva, maxillolabial complex (SEM micrographs) A maxillolabial com-
plex, ventral aspect B prementum, ventral aspect C apical part of distal maxillary palp D apical part of
labial palpomere E, F surface of ligulae. Abbreviations: ds—digitiform sensillum, sa — sensillum ampul-
laceum, tra — terminal receptive area, setae: dms — dorsal malar, /igs — ligular, mbs — malar basiventral,
mps — maxillary palp, pfs — palpiferal, prms — prelabial, pms — postlabial, stps — stipal, vms — ventral malar.

70 Rafal Gosik & Peter Sprick / ZooKeys 1121: 59-82 (2022)

Abd. III
Abd. II —~ — ~ Abd. IV
| ~
| \ =~
} f
7b | \
Abd.1 \ aN
a \ 4 oa ; \ Abd. N
THA MRO RISD p + Ne
A \ ; . \ re — = SN iE a = ;
ypy\ 4 \ / Me /
THA » aN See Lay, : KN /
/ | \ fo q /
\ OY % Ce Pe.
/ 4 { eps oe ; Abd. VI
S pds “ \ is \
spr | —
[x 2 \ *
Th. | tog ae 1
| - - sq a SOx \
a, Y Ists S NN \ \
SSi.2 \\ \ We ~* . \ : \ ss
er oe Ne he Ah \A¥Xeps Wh \ Aba. vi
1-7 . \ er Lan F \rr
ee CPS IAN Ae Se S08; jee oh \1 | \ pds*
sf f \
f . af al \ ] \. oe
/ \- ps) | Ist.
| ae Na J eus,., wh ; 1 % 7 . Abd. VIII
‘ N\, Ne |
PS.» Noe R
i Cen <a ‘= pda, \ /
eus ~—~— f
| ps,
pda,., hr! a ‘ds...
sts,. i
. Abd. IX
Abd. X

Figure 9. Ovobitis cyanea mature larva, lateral view, habitus and chaetotaxy. Abbreviations: Th. I-IH-
thoracic segments 1-3, Abd. I-X—abdominal segments 1-10, setae: ds—dorsal eps—epipleural, eus—euster-
nal, ps—pleural, pda—pedal, pds—postdorsal, prns—pronotal, prs—prodorsal, ss—spiracular, sts—sternal.

Description of the pupa of Orobitis cyanea

Female: BL: 2.00!; 2.16'; 2.20'; BW: 2.16'; 2.33!; HW: 0.55'; 0.577; RL: 1.00'; 1.05';
1.10'; PW: 1.23'; 1.30! (one pupa partially deformed). "-number of specimens.
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 4x as long as wide, reaching meta-
coxae. Pronotum trapezoidal, 2x 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. Gon-
otheca in female divided. Urogomphi (posterior processes) absent. Spiracles placed lat-
erally 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

Immatures, biology and taxonomy of the Violet seed weevil Fall

eus pda, .
SEM HV: 30.00 kv WD: 28.70 mm SEM HV: 30.00 kV ‘WD: 28.70 mm |

View field: 1.44 mm Det: SE 200 um View field: 1.44 mm Det: SE 200 ym
SEM MAG: 200x — Date(m/d/y): 01/31/22 SEM MAG: 200x — Date(mid/y): 01/31/22

Abd. VIII

© pas

SEM HV; 30.00 kV WD: 28.97 mm SEM HV: 30.00 kV WD: 28.59 mm i i VEGA3 TESCAN|
View field: 963.1 pm Det: SE 200 ym View field: 321.0 pm Det: SE 100 pm
SEM MAG: 300x _ Date(m/d/y): 01/31/22 _ ff SEMMAG: 900x — Date(midiy): 01/31/22 _

SEM HV: 30.00 kV WD: 19.09 mm | WD: 8.675 mm
View field: 72.23 ym Det: SE 20 ym View field: 144.5 ym Det: SE 20 pm
SEM MAG: 4.00 kx = Date(m/diy): 01/28/22 | SEM MAG: 2.00 kx Date(m/dfy): 01/31/22

Figure 10. Orobitis cyanea mature larva, habitus and cuticle (SEM micrographs) A lateral view of head and
thorax B lateral view of abdominal segments I-V C lateral view of abdominal segments VII-IX D lateral
view of abdominal segment V (magnification) E structure of cuticle of dorsolateral part of prodorsum
F structure of cuticle of ventrolateral part of prodorsum. Abbreviations: setae: ds—dorsal eps—epipleural,

eus—eusternal, ps—pleural, pda—pedal, pds—postdorsal, prns—pronotal, prs—prodorsal, ss—spiracular, s¢s—sternal.

72 Rafal Gosik & Peter Sprick / ZooKeys 1121: 59-82 (2022)

not reveal a single immature specimen or any feeding traces similar to those seen on
V. canina \eaves (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 spe-
cies, 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.

Discussion

Comments and inferences regarding the host plants, biology and parasitoids
of Orobitis cyanea

Our observations regarding the feeding and development of Ovobitis cyanea on Viola
riviniana seed capsules are in accordance with those of Scherf (1964), who listed
the following Viola species as host plants of this weevil: V. canina, V. epipsila Ledeb..,
V. odorata ., V. palustris L., V. reichenbachiana Jord. ex Bor. (as V. silvatica Fr. ex
Hartm.) and V. riviniana. In addition, Urban (1925) listed Viola canina (once under
this name and once as V. stricta Hornem.), V. odorata, V. palustris, V. pumila Chaix (as
V. pratensis Mert. & Koch), and V. reichenbachiana Jord. ex Bor. (as V. silvestris auct.).
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. x 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.

Immatures, biology and taxonomy of the Violet seed weevil a

Cc

SEM HV: 30,00KV | WD! 28.44 mm
View field: 1.44 mm Det: SE
SEM MAG; 200x — Date(midiy): 01/34/22

SEM HV: 30.00 kV WD: 29.10 mm
View field: 963.1 pm Det: SE
SEM MAG: 300 x — Date(mid/y): 01/31/22

SEM HV: 30.00 kV WD: 28.62 mm in
View field: 192.6 um Det: SE
SEM MAG; 1.50 kx Date(mid/y); 01/31/22

Figure | 1. Ovoditis cyanea pupa, habitus and structure of cuticle A ventral view B dorsal view, scheme
C head and rostrum, frontal view D abdomen, ventral view E tarsi of first pairs of fore legs, magnification

(SEM micrographs).

On the other hand, we confirm the information given by Dieckmann (1972) re-
garding 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

74 Rafal Gosik & Peter Sprick / ZooKeys 1121: 59-82 (2022)

pie: r

Figure 12. Viola canina A flowering host plant B host plant with some feeding holes after flowering.

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 in-
side 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 sam-
ples 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 un-
der the name of Growth Factor (GF) (Sprick and Gosik 2014; Gosik et al. 2019),
which is in fact Dyar’s ratio, which bears a closer relationship with natural develop-
ment. 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) usu-
ally 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; Sprick et al. 2022). There is only one GF value each for a
Ceutorhynchinae and Lixinae species: in Ceutorhynchus subpubescens LeConte, 1876 it

Immatures, biology and taxonomy of the Violet seed weevil aS

Figure 13. Orobitis cyanea on Viola riviniana A adult on seed capsule with feeding traces and emergence

hole B adult exhibiting thanatosis.

was calculated at 1.43 (Dosdall et al. 2007) and in Rhinocyllus conicus (Frélich, 1792)
at 1.538 (Rowe and Kok 1985). In the case of Ovobitis 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

76 Rafal Gosik & Peter Sprick / ZooKeys 1121: 59-82 (2022)

of ~ 44 Leiosoma species, available as adult specimen or as habitus photo from different
sources, e.g., Pedroni (2010, 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 Ovoditis 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, and 2.42 — 2.43:1 in L. hernicum Pedroni,
2012 and L. komovicum Pedroni, 2018.

Even if only for a small part of Leiosoma species the host plant species are known
(Sprick and Kramer-Klement, in press), it is noticeable that there should be some se-
lection 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 Ovobitis, or soil
or underground in Leiosoma. Night activity of Leiosoma species may be another behav-
ioural 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) post-
dorsal folds of abdominal segments I-VI 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 (2010, 2012, 2018).

Host Species/Species group* Ratio Data source

A Orobitis cyanea ites Bal own data
O. nigrina 1.58:1 own data
Leiosoma cribrum L721 own data

B Leiosoma cribrum group (five further species) 1:99:31 —2:24:1 Pedroni (2018) Bahr (2021)
Leiosoma oblongulum group (four species) 2.00:1 — 2.16:1 Pedroni (2010), Diotti and Caldara

(2017, 2020); own data

Leiosoma scrobiferum group (six species) 2.22:1 — 2.43:1 Pedroni (2012)
Species from undefined species groups: L. apionides, 1.88:1—2.28:1 — Stiiben (2011), Sabaljuev (2013),
L. bosnicum, L. deflexum, L. kirschii, L. reitteri Pedroni (2016), own data (coll. J.

Messutat, coll. M. Stern)

Immatures, biology and taxonomy of the Violet seed weevil ria

Figure 14. Orobitis cyanea larva and the parasitoid wasp.

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 an-
terior margin deeply rounded inwards (concave); 12) clypeus with only one pair of cls;
13) labrum with two pairs of /rm; 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) post-
labium 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, distri-
bution, individual abundance and economic importance. Bariditae and Ceutorhynchi-
tae have been relatively well studied (Scherf 1964; Pakaluk 1993, 1994; Prena 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 di-
agnostic of this suprageneric taxon, although it may change with further studies.

78 Rafal Gosik & Peter Sprick / ZooKeys 1121: 59-82 (2022)

Finding features common to all known larvae that would be diagnostic of Cono-
derinae sensu lato is not possible. Some larval characteristics present in all the super-
tribes 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 num-
bers of des and /es; 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 /sts, 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 morpho-
logically 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 c/s; 9) labrum
semi-circular with rounded or slightly sinuate anterior margin, always with three pairs
of /rs; 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 /rm; and 14) premental sclerite tridental, with elongate posterior extension.

In addition, the GF measurements indicate that there are three larval stages in
Orobitis cyanea, as in Ceutorhynchitae (Scherf 1964; Dosdall et al. 2007), but not in
Conoderitae (5 instars) (Gosik et al. 2021).

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 men-
tioned 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 ex-
ophagous 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 medium—
length 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,

Immatures, biology and taxonomy of the Violet seed weevil 79

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 sug-
gested by Alonso-Zarazaga and Lyal (1999) and Korotyaev et al. (2000), is best sup-
ported by our results.

Acknowledgements

We thank Andreas Marten for his assistance with the fieldwork, Marek Wanat and
Michael Morris for their help with the literature queries. We would like to express
our cordial thanks to Werner Ulrich for his help in identifying the parasitoid. We are
indebted to Peter Senn for the linguistic editing of the text.

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