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

Abstract As yet little is known of the bionomics of weevils of the genus Cleopomiarus Pierce, 1919; current knowledge is limited to data on the morphology and biology of the preimaginal stages of certain species. This paper includes original information on the life cycle of Cleopomiarusmicros (Germar, 1821). It presents the morphology of the egg, last larva (L3) and pupa. Data on the host plant (Jasionemontana L.) and breeding plant (Campanulapatula L.) and on the oviposition and phenology of the species are updated. The anatomy of the third-stage larva of C.micros shares certain traits with other species of the tribe Mecinini Gistel, 1848. Comparison of the morphology of preimaginal stages of C.micros with those previously described for other species of the genera Cleopomiarus and Miarus Schönherr, 1826 – previously considered the same genus – reveals species differences in larval body length, colour of the body and epicranium, and chaetotaxy of head and body.


Introduction
The tribe Mecinini Gistel, 1848 is currently represented by six genera worldwide, of which five (Cleopomiarus Pierce, 1919, Gymnetron Schoenherr, 1825, Mecinus Germar, 1821, Miarus Schoenherr, 1826and Rhinusa Stephens, 1829 are known from the Palearctic fauna and one, Rhinumiarus Caldara, 2001, was discovered in the Neotropical realm (Caldara 2001, 2013, Caldara et al. 2014). This last genus was established based on analysis of the species Rhinumiarus lyali Caldara, 2001, found in Argentina, a taxon in which the external anatomy of the adult looks intermediate between species of the genera Miarus and Cleopomiarus.
The analysis of previously known adult forms and their preimaginal stages reveals a number of morphological characters common to the tribe Mecinini. In the larvae, these are as follows: head usually with 3 distinct pairs of setae (des) and with long, unbranched endocarina, which together with the epicranial suture extends 2/3 the length of the head; labial palpus usually with 1 segment -if there are 2 segments the basal segment is not distinctly separated; hypopharynx with usually 4, less often 2 or 6 epithelial anteromedial setae; premental rounded, less often pointed; sclerites of the prosternum joined or free, numbering 0, 2, 4 or 6; thoracic and abdominal spiracles uni-or bicameral depending on the genus, abdominal spiracles located laterally on the intersegmental membrane, tubes well defined from atrium (Emden 1938, Scherf 1964, Anderson 1973, May 1993, Marvaldi 2003, Jiang and Zhang 2015. Species belonging to the Mecinini tribe are poly-, oligo-or monophages of plants of the families Campanulaceae, Scrophulariaceae and Plantaginaceae (Scherf 1964, Smreczyński 1976, May 1993, Burakowski et al. 1997, Caldara 2001, Caldara et al. 2014.
The genus Cleopomiarus includes 40 species dispersed throughout the world. Palearctic species (19 spp) are associated with plants of the genera Adenophora Fisch., Campanula L., Jasione L. and Phyteuma L. (Campanulaceae, Campanuloideae), whereas beetles from southern Africa and Mexico live on plants of the genera Codonopsis Wall., Lightfootia L'Hér., Roella L. and Wahlenbergia Schrad. ex Roth from the same subfamily (Campanuloideae), as well as on representatives of the genus Lobelia L. from the subfamily Lobelioideae (Caldara 2001, 2007, Caldara et al. 2014, Caldara and Legalov 2016. In Poland, the species thus far recorded are Cleopomiarus distinctus (Boheman, 1845), C. graminis (Gyllenhal, 1813), C. plantarum (Germar, 1823) and C. micros (Germar, 1821). In the case of C. plantarum, the data come from the late 19 th and early 20 th centuries and require confirmation (Smreczyński 1976, Burakowski et al. 1997, Petryszak 2004, Wanat and Mokrzycki 2005. As yet little is known of the bionomics of insects of the genus Cleopomiarus. Certain data on the morphology and biology of preimaginal stages can be found in Scherf (1964), pertaining to C. graminis and C. micros, and in Emden (1938) on C. graminis and M. campanulae (Linnaeus, 1767) and Anderson (1973) on Cleopomiarus hispidulus (LeConte, 1876). While species identification of adult individuals poses little difficulty, systematic identification on the basis of preimaginal stages has not been possible.
In the present study the immature stages of Cleopomiarus micros are described. This is a rare, psammophilous species, found only in certain areas. It inhabits sand, dunes, ruderal communities, forest clear-cuts, and thickets. It has previously been recorded in southern, western and central Europe, as far north as northern Denmark and southern Sweden. It has also been recorded in North Africa. In the literature it has been described as a monophage living exclusively in the flowers of sheep's bit scabious, Jasione montana L. (Burakowski et al. 1997, Kubisz et al. 1998, Kuśka 1999, Łętowski and Gosik 2002, Wanat 2005, Gosik 2006, Caldara et al. 2014.
The objective of the study is to describe the morphology and biology of the preimaginal stages of Cleopomiarus micros, which is one of the rarest representatives of its genus in Poland.
Part of the individual developmental stages of these insects were fixed in 70% ethyl alcohol and a part incubated (in a ratio 1:4 -¼ to fixation, ¾ to incubation). Breeding of preimaginal stages was carried out in Petri dishes lined with filter paper, placed in a breeding chamber with constant temperature parameters (daytime minimum 25 °C, daytime maximum 35 °C, minimum at night 15 °C, maximum at night 20 °C), humidity (60%), light intensity and duration (day 16 h, night 8 h), in order to obtain all developmental stages of the beetles (egg, three larval instars, pupa and adult) and describe their biology.
Observations of the ecology of adult beetles were also conducted in the laboratory on individuals bred together with their host plants in glass insulators at 25 °C with a 14:10 photoperiod.
Two methods were used to prepare microscope slides, as described in Łętowski (1991) and Gosik et al. (2010). Photographs of the habitat and breeding plant were taken with a NIKON Coolpix B500 camera. To prepare the drawings we used an OLYMPUS SZX12 microscope with a DP72 camera at magnifications from 200× to 400× and a TESCAN VEGA3LMU scanning microscope at magnifications from 500× to 2000×. The figures were made based on the biological preparations using COREL DRAW 18 software. The terminology of Scherf (1964), Marvaldi (1998Marvaldi ( , 1999Marvaldi ( , 2003, Skuhrovec (2004), Skuhrovec and Bogusch (2016), Wang et al. (2013) and Oberprieler et al. (2014) was used in the morphological descriptions of larva and pupa, and for chaetotaxy. The distribution and number of setae are given for half of the larval body, while the chaetotaxy of the larval head and the body of the pupa is given with respect to the whole body. Measurements of the head (following decapitation) were made on the head capsule, isolated from the body, with the mandibles closed. The dimensions of the preimaginal stages are determined from two measurements of the ova, three measurements of larval instar L 1 , 4 of L 2 , 7 of L 3 and eight of the pupae.

Pupa
The female and male morphology is externally very similar at the pupal stage, with a significant difference only in the length of the rostrum, which is longer in the female and its apex extends to the middle of the second tarsal segment of the prolegs; in the male the rostrum is short and its apex extends to the end of the first segment.
Abdomen: Tergites of abdominal segments I-VII of similar width, gradually narrowing slightly towards rear, with 4 pairs of setae pds (2 macro-and 2 micro-) arranged alternately, parallel to posterior margin of segment (Figure 9). On tergite VIII 1 pair of short microsetae. Macrosetae of tergites longer than microsetae by more than half. Sternites I-VII with 1 pair of larger msts and 2 pairs of smaller vpls, on sternite VIII 2 pairs of microsetae (Figures 7, 8). Macro-and microsetae of sternites somewhat longer than macro-and microsetae of tergites, but in similar proportions. Segment IX with short, pointed, slightly curved urogomphi (pseudocerci), and 3 pairs of setae (Figure 8). Pleu- rites of segments I-VII with 2 setae dpls (Figure 7). Spiracles (spiracle) located between tergite and pleurite, in upper part of border, clearly visible on segments I-VI, on others absent (Figures 7, 9).

Biological information
Analysis of eight plant species of the family Campanulaceae revealed that Cleopomiarus micros uses Jasione montana as a food source for the imago and Campanula patula for breeding larvae. The presence of these two plants at the same habitat provides ideal conditions for the occurrence of the species (Figure 12). Only at the study site in Nasutów were these plants present at the same time. Adults of Cleopomiarus micros hibernate in the soil and come out in late spring. The first adults were caught in the  second half of May. A total of 35 specimens were caught, but even this small number allowed for important conclusions to be formulated: -Adult insects feed on sheep's bit scabious (Jasione montana).
-Following copulation at the beginning of June, females choose a different plant species of the same family -spreading bellflower (Campanula patula) -to lay their eggs. -Single eggs are laid on the wall of the ovary (seed chamber). Unlike in other beetle species of this genus, delicate traces of oviposition appear on the plant's epiderm.
To lay an egg the female uses a natural hollow on the exterior of the seed chamber, which she gently gnaws and enlarges (Figure 13). -The L 1 larva bores a tunnel to the inside of the chamber. Subsequent larval instars (L 2 and L 3 ) eat the contents of the seed chamber. After seven days of growth the L 3 larva transforms into a pale brown pupa, and after another four days into an imago. As observed, the adult leaves the seed through cracks formed during its drying prior to germination. -One generation per year was noted in the species.
-The insect does not cause cecidia in the breeding plant.

Discussion and conclusions
The results of the study, the first morphological description of the preimaginal stages of the species Cleopomiarus micros, serve to verify and supplement previous scant data on the biology of the species. Previous data on the morphology of the preimaginal stages of representatives of the genus Cleopomiarus concern only C. graminis, C. hispidulus, and Miarus campanulae, which in the old taxonomic system belonged to the genus Miarus (Emden 1938, Scherf 1964, Anderson 1973, Caldara and Legalov 2016. The morphology of the L 3 larvae and pupa of C. micros does not differ from the typical characters of preimaginal stages of weevils of the tribe Mecinini (Emden 1938, Scherf 1964, Anderson 1973, Marvaldi 2003, Marvaldi and Lanteri 2005, Gosik 2010, Jiang and Zhang 2015. The morphology of mature C. micros larva is similar to the description of the characters of the third-stage larvae of the tribe presented by Emden (1938), including pronounced convexity of the body, a long, unbranched endocarina, which together with the epicranial suture extends two-thirds the length of the head, and an intersegmental thoracic spiracle.
Certain anatomical traits of C. micros are common to species of Mecinini tribe, but less frequent. As mentioned in the Introduction, the head of species of this tribe usually has 3 pairs of des. In C. micros there are 4 pairs, as in Rhinusa bipustulata (Rossi, 1792) or Gymnetron miyoshii Miyoshi, 1922(Gosik 2010, Jiang and Zhang 2015. In Mecinini, the labial palpus usually has a single segment; if there are two segments the basal segment is not distinctly separated. This is the case with C. micros, which has a two-segmented palpus. The presence of a two-segmented labial palpus seems to be a character common to species of the genus Cleopomiarus. This is confirmed by species such as Cleopomiarus distinctus and C. graminis (the authors' unpublished data). A twosegmented labial palpus is also present in species of the genus Miarus (M. campanulae and M. ajugae) (Scherf 1964, the authors' unpublished data). Another character present in C. micros, which is common to species of the Mecinini tribe but occurs less frequently, is the presence of two anteromedial setae (ams) on the epipharynx. The same number of ams is found in Gymnetron antirrhini, G. lineariae andG. villosulum (Emden 1938, Scherf 1964). Analysis of the anatomical characters of C. micros in comparison with other species of the Mecinini tribe suggests that it is most similar to species of the genus Miarus (Emden 1938, Scherf 1964, Anderson 1973. Comparison of the morphology of C. micros with previously described preimaginal stages (L 3 and pupa) of species of the genera Cleopomiarus and Miarus (previously the same genus) on the basis of previously described features reveals species differences in larval body length, the colour of the body and epicranium, and the chaetotaxy of the head and body (Emden 1938, Scherf 1964. Among the species compared, the larva of C. micros has the shortest body (on average 3.41 mm), and C. graminis has the longest (up to 5 mm). In M. campanulae, as distinct from M. ajugae (Herbst, 1795), the average larval length is 4.75 mm (our own data). The colour of the larvae ranges from white in M. campanulae to cream-coloured in C. micros to yellowish-white in C. graminis. The head in individual species is of various shades of brown: yellowish-brown in C. graminis, dark brown in C. micros and black-brown in M. campanulae (Scherf 1964). The differences in the chaetotaxy of the body and the form of the mouthparts are presented in Table 1. The present research made it possible to discover, supplement and describe the chaetotaxy of many parts of the body of C. micros.
Differences between the species analysed were also noted in the size and colour of the pupal body. In C. micros the body is 2.95-3.53 mm long and dark brown, whereas the yellow-white pupa of C. graminis reaches a length of 3.4-5.0 mm and the body of the M. campanulae pupa is 2.5-3.0 mm long and white (Scherf 1964, Anderson 1973. The differences in the chaetotaxy of the body and the shape of the pseudocerci are presented in Table 2. The development of the Mecinini species is correlated with the phenology of the breeding and host plants and is strongly dependent on the environmental conditions prevailing at a given site. In the case of C. micros, a necessary condition for the presence of the beetle is the co-occurrence of plants of the species Campanula patula, in which it develops, and Jasione montana, which constitutes the food base for adults. This was confirmed by the observations in the field and experiments in the laboratory. The species was not found when only one of the listed plant species was present in the environment. Among the species from genus of Miarus and Cleopomiarus so far studied, only C. micros develops in such a way -development in a one species of plant and feeding on another. This indicates that this species is oligophagous. Until now, it would have been described as a monophag (Scherf 1964). Probably, the development in Campanula montana is conditioned by the appropriate size of the ovary and the abundance of seeds.
Understanding of the morphology of the adult larva of the described species allows for the recognition of potential generic features for the labial palp of Cleopomiarus spe-  cies. They are: the ratio of length to its width -the labial palp is longer than wider, the apex is clearly longer than wider, and the basic segment is pronounced. Morphological descriptions of the next species of the genera Miarus and Cleopomiarus will probably allow other generic features to be distinguished, and, in turn, for the production of a key to identify mature larvae. The data presented herein provide new information on the biology and ecology of the species. Previous data covered only its living environment, host plant and the number of generations per year (Scherf 1964, Burakowski et al. 1997.