The material used to describe the immature stages was collected and field observations were conducted in Ukraine in the following localities:

1) The eastern shore of Molochnyi Estuary between the two villages of Altagir (= Bogatyr) and Radyvonivka (46°38'29"N, 35°16'59"E). Altitude: up to 20 m a. s. l. Bedrock: loess loam with herbaceous covering. Dominant plant species: Agropyron pectinatum (M. Bieb.) P. Beauv., Festuca valesiaca Schleich. ex Gaudin, Koeleria cristata (Ledeb.) Schult., Artemisia marschalliana Spreng., Ephedra distachya L., and Helichrysum arenarium (L.) Moench, 1794 (Kolomiychuk and Vynokurov 2016). Globe thistle Echinops ruthenicus M. Bieb. usually grows along the top of the slope.

2) Man-made forest near the Kamyana Mohyla Reserve (46°57'01"N, 35°28'12"E). Altitude: up to 10 m a. s. l. Bedrock: sandy chernozem. Dominant plant species: Robinia pseudoacacia L. and Morus nigra L., with herbaceous plants (i.e., Echinops ruthenicus, Centaurea adpressa Ledeb. ex Steud., Melilotus albus Medik.) in the clearings.

3) NE Cyrilivska Spit located between the Sea of Azov and Molochnyi Estuary (46°25'12"N, 35°25'09"E). Altitude: 5 m a. s. l. Bedrock: flats of quartz sand, shells and mud. Habitats: salty grasslands with dominant herbaceous species Elytrigia elongata (Host) Nevski, Puccinellia distans (Jacq.) Parl., Aeluropus littoralis (Gouan) Parl., 1850 and Juncus gerardii Loisel.; coastal communities with Phragmites australis (Cav.) Trin. ex Steud., Bolboschoenus maritimus (L.) Palla and Juncus maritimus Lam. (Kolomiychuk and Vynokurov 2016). Globe thistle Echinops ruthenicus is locally distributed between the central road and the coast of the estuary (Fig. 21).

In the above-mentioned localities, life cycle, including feeding of adults, oviposition, and early development of larvae were observed directly during the vegetation growing seasons of Echinops ruthenicus and E. sphaerocephalus L. in the time period 2012–2016.

The compound flower heads of globe thistles consist of simple capitula, each of which has only one floret. These primary capitula are aggregated in globose secondary capitula (Kadereit and Jeffrey 2007). For convenience, below, we shall name as capitula only secondary ones.

The second author collected all larvae and pupae of L. vulpes within inflorescence. Some inflorescences (n = 42) were dissected to investigate preimaginal development, and a further 250 were dissected to determine the quantity of preimaginal specimens of L. vulpes within an inflorescence. All photographs in the field were taken with digital cameras, a Nikon Coolpix 4600 and a Canon PowerShot SX500 IS.

Laboratory observations were conducted in Melitopol, Ukraine (46°50’N, 35°22’E). The dry inflorescences (n = 7) with developing mature larvae or pupae were placed into cardboard boxes. A small hole was opened in every inflorescence for possible observations of insect development. Measurements of flower head were performed with a slide caliper and ocular micrometre. The size of flower heads was determined at the greatest diameter.

Geographical distribution and phenology were studied from several entomological collections, specifically: Schmalhausen Institute of Zoology of National Academy of Sciences of Ukraine (Kyiv), TG Shevchenko Kyiv National University Zoological Museum, Zoological Institute of Russian Academy of Sciences (St. Petersburg), VN Karazin Kharkiv National University Museum of Natural History, and Igor Maltzevs’ private collection (Odessa). In total, more than 130 pinned specimens were studied.

Part of the larval and pupal material was preserved in Pampel fixation liquid (see Skuhrovec et al. 2015) and used for the morphological descriptions. These specimens are deposited in the collection of Group Function of Invertebrate and Plant Biodiversity in Agro-Ecosystems of the Crop Research Institute (Prague, Czech Republic). The collectors identified the plants. To prepare slides, we followed May (1994).

The observations and measurements were conducted using a light microscope with calibrated oculars (Olympus BX 40, SZ 11, and Nikon Eclipse 80i). The following characteristics were measured for each larva: head width, length of the body (larvae fixed in a C-shape were measured in the middle of the segments in lateral view), and width of the body in the widest place (i.e., meso- and metathorax). For the pupae, the length and the width at the widest place were measured.

Drawings were created with a drawing tube on a light microscope and edited by the programs Adobe Photoshop 10, Corel Photo-Paint X7, and GIMP 2.

We used the terms and abbreviations for the setae of the mature larva and pupa found in Scherf (1964), May (1977, 1994) and Marvaldi (1998, 1999). The numbers of setae of the bilateral structures are given for one side.

The counts of some of the setae on the epipharynx (particularly ams and mes) have not been completely resolved. According to Marvaldi (1998, 1999), the standard status of the epipharynx in weevils is 2 ams and 3 mes; however, when the position of the distal mes is very close to the anterior margin, it appears as ams. The final decision was to add this problematic seta to the latter group (ams), and the position of this seta is similar to that in other genera, e.g., in Coniocleonus Motschulsky, 1860 or Tychius Germar, 1817. We did not follow Stejskal et al. (2014) and Skuhrovec et al. (2014) who accepted the standard status in weevils and counted the seta as mes, but we followed Trnka et al. (2015) and Skuhrovec et al. (2015), e.g., in Adosomus Faust, 1904 or Sibinia Germar, 1817. The thoracic spiracle was located on the prothorax near the boundary of the prothorax and mesothorax, as shown in the drawing (see Fig. 10), but this spiracle is of mesothoracic origin (Marvaldi et al. 2002; Marvaldi 2003).

Larvae (last instar)

The following key is based on the larvae of Larinus vulpes described in this paper and on 13 selected descriptions of larvae in the genus Larinus published previously (Lee and Morimoto 1988; Nikulina et al. 2004; Zotov 2009a, 2010; Gosik and Skuhrovec 2011; Nikulina and Gültekin 2014). Unfortunately, Lee and Morimoto (1988) only provide a general description of the genus for two species, and it was not possible to divide these two species (see aggregation of both species at point 12). Further comments are given in the previous section under Discussion.

Pupae

The following key is based on the pupa of L. vulpes described in this paper and on seven descriptions of pupae of the genus Larinus published previously (Zotov 2009a, 2010; Gosik and Skuhrovec 2011).

Habitats. Larinus vulpes occurred in the primary and degraded steppe lands, slopes, limestone and chalk cliffs of low mountains, forest edges, man-made treelines, roadsides and other ruderal plots. This weevil preferred open, sunny areas. In Iran, the weevil was recorded as high as 2580 m a. s. l. in the mountains (Gültekin and Podlussány 2012).

Adult behaviour. Adults feed on the upper surface of the leaf. As feeding was initiated, an adult raised and strongly lowered its head onto the leaf surface, which was followed by some motions of the mandibles and repeated “peck-like” motions by its rostrum. Apparently, the motion created additional pressure and helped to break through cuticle and epidermis covered with woolly hairs. Following this behaviour, an imago gnawed on mesophyll tissue, moving the head away from itself, and at one feeding, a weevil could gnaw out an irregularly shaped piece of leaf (approximately 2 × 8 mm). The translucent cuticle of the leaf downside covered with dense woolly hairs remained intact (Fig. 22). During feeding, some short pauses by the weevil were observed. After eating, the weevil cleaned the apex of its rostrum using the apexes of both tibias. Weevils moved from one flower head to another by walking; they flew very reluctantly.

Host plant. Both adults and larvae were recorded feeding exclusively on Echinops ruthenicus and E. sphaerocephalus. We never observed L. vulpes on other plant species. According to Zwölfer (1985) and Nicolas (1895), weevils feed on Echinops microcephalus Sibth. and Sm. and E. ruthenicus (as E. ritro L.). In this case, L. vulpes is oligophagous (or monophagous sensuJolivet 1992). Imagines fed on the leaves and on the apexes of the stems, and larvae gnawed tissues in the flower head. Hoffmann (1954) noted that L. vulpes were often recorded on Cirsium ferox, but whether this plant was a host for the weevil was not resolved.

Figures 21–28. 

Habitat, life cycle, and immature stages of Larinus vulpes. 21 Habitat with flowering Echinops ruthenicus (Cyrilivska Spit) 22 Adult and the damaged leaf of Echinops ruthenicus by weevil 23 When larva begins its development, upper parts of the flower head die and are visible as a light brown spot 24 Pupa cell results in deforming of flower head 25 Larva and pupa in the same inflorescence 26 Two larvae in the same flower head, right larva is younger and dead 27 Pupa in the inflorescence 28 Mature larva in a chamber outside a flower head. All photos SV Volovnik.

Life cycle. In Ukraine, adults were recorded from the end of April (usually from the end of May) onwards. The primary peak in the population of adults was reached at the end of June and then decreased. Active imagines from the new generation were observed from the beginning of August to early September. The phenology of the weevil is closely synchronized with the phenology of its host plant (Volovnik 1996). In spring, the weevils began to feed first on the rosettes of host plants and then gnawed the young stems and leaves (Fig. 22). Larinus vulpes is a univoltine species. Mating and oviposition occurred from the second part of June to the middle of July. The common scenario of oviposition and subsequent development from egg to adult were the typical for the genus Larinus (Volovnik 2016).

Female L. vulpes preferred to lay eggs in the larger flower heads on the side stems (Volovnik 2016). In this case, the female makes a hole in the flower head. Freshly laid, eggs were oval, milky white, glossy, 1.3–1.6 mm long and 0.7–1.2 mm wide. The eggs were laid solitarily in the deepening in the receptacle, gnawed by the female, or very close to the receptacle (maybe, the rostrum of some weevils was too short for access). The process of oviposition has been described in detail previously (Volovnik 1996). Eggs were found in inflorescences from the end of June to the end of July. Very occasionally, dry fragments of the egg and also a small larva were found in the same flower head. In other instances, the fragments of eggs were recorded in the inflorescence but without any traces of weevil larvae. These eggs were likely destroyed by parasites or abiotic factors (i.e., desiccation).

Later, after hatching of larva, the oviposition site became a visible, brownish tiny spot (Fig. 23). Apparently, this spot was a result of damage to the flower head by the larva. Larvae nibbled the receptacle of the flower head and the bases of primary capitula, with the damaged capitula fixed with a sticky liquid that flowed out of receptacle, resulting in a deformed flower head (Fig. 24). The mature larva colouration was peach-orange (Figs 26, 28) and that of the young pupa yellow-orange (Figs 25, 27). Fabre (1922) wrote that he found “six and more” larvae in the same inflorescence and that three larvae in the same flower head “frequently happens”. Our data differed greatly from the observations of Fabre (see Table 3). Apparently, two larvae could finish full development in a medium-sized flower head (Fig. 26). Larvae were recorded from the end of June to the end of August. Larval development has been described and discussed in detail by Fabre (1922). According to direct observations by Fabre, larvae feed primarily on the sap of the receptacle. Mature larvae build a pupation cell (4–5 × 7–10 mm) (Figs 29–31), with the walls significantly stronger than those of the larval cell (Figs 32–33).

After emergence, adults remained in the dry inflorescence for 5–7 days until fully sclerotised (Fig. 37), with an upper surface that was usually densely covered with a rust, pollen-like flush (Figs 34–36). Adults hibernated outside host plants, most likely in the top layer of soil or among dry plant debris.

Figures 29–37. 

Hatching, pupal cells, and adults of Larinus vulpes. 29 Dead inflorescence with pupal cell inside 30 Pupation cell and dry fragments removed from outside of the cell 31 Pupation cell with a fresh, not fully coloured adult 32, 33 Pupation cell at the beginning (left) and after finishing construction. The inner layer of the finished wall is hard and glanced 34 Adult in pupation cell 35 Adult leaving the pupa cell 36 Exit hole of adult of new generation 37 Fresh adult in pupa cell. All photos: SV Volovnik.

Preimaginal development outside of flower heads. In the second part of the summer in 2015, the second author found seven chambers on the side of the stem of Echinops ruthenicus. All were located 1–2 cm below the inflorescence. Two of the seven capsules were located on the same stem and touched one another. The largest chamber was approximately 0.7 × 1.25 cm. The walls of all chambers were solid but rather brittle and easily crushed by the fingers. The material of the walls had no taste. The external surface of the capsule wall was brown to black, rough, earthy coloured, lumpy and mat; with incrustations of elements characteristic for Echinops, i.e., wool-like coating of the stems, small leaves with spines and tips of spines (Figs 28, 38–39).

The inner surface of the wall was denser than that of the external item and was bright red-brown with yellowish spots and veins, glossy, and 0.8–1.0 mm thick. The inner surface was also smooth and appeared varnished and polished, similar to inner surface of the wall of the usual pupa chamber of L. vulpes, but without any incrustations. The contents of the chambers included the following: 2 chambers with dead imagines, 1 chamber with a living imago, one with only the head capsule of a larva, one chamber with a mature larva alive (Fig. 34), one with only pieces of a pupa and one with only parts of exuviae or cuticle remains. No excrement was observed but clearly the larvae incorporated their faeces into the wall during construction.

Biotic interactions. A parasitic wasp, Bracon urinator (Fabricius, 1898) (Hymenoptera: Braconidae), was reared from the pupae of L. vulpes. Sometimes the capitulum dried out, cracking the walls of larval or pupal cells, and ants, Formica imitans Ruzsky, 1902, destroyed the larvae and pupae of the weevil. Feeding on the inflorescences of Echinops, the rose beetle, Protaetia metallica (Herbst, 1782) (Coleoptera: Scarabaeidae), harmed larvae of L. vulpes developing in the same inflorescence. We found cells with dead weevil larvae together and simultaneously with living larvae or adults of carnivorous bugs, Orius sp. (Heteroptera: Anthocoridae) (Volovnik 1994).

Larinus vulpes and its host-plants. Larvae of L. vulpes living in flower heads of globe thistle consumed the ovaries and unripe seeds. The prevalence of weevils in globe thistles sometimes reached 33% of inflorescences (SV, unpublished data). Some flower heads lost all their seeds, although in an overall view, the loss of some seeds may be expected. Globe thistles have a special morphological structure that separates the compound flower head into small, single primary capitula with one seed each (Mulkijanian 1951). The durable walls of the pupal cell were very strong, which might prevent the possible separation of each capitulum. Furthermore, Mulkijanian (1951) also recorded stems breaking in the wind, primarily when larvae damaged the receptacle. Most likely, the larvae of Larinus vulpes or some similar species of Larinus damaged the receptacles. Clearly, the specialisation of globe thistles to a specific environment, such as rocky slopes, also constrained the weevils, being adapted to that same environment. The chemical composition of Echinops is also somewhat specific, and some species in this genus have insecticidal, nematicidal, antifungal, bactericidal and also antiviral properities (i.e. Fokialakis et al. 2006; Zhanget al. 2009; Abdelnabby and Abdelrahman 2012; Tekwu et al. 2012; Gemechu et al. 2013; Liu et al. 2013). Therefore, the inhabitants of Echinops flower heads might also be protected from some parasites and predators because of the chemical compounds in the plant. This aspect requires further experimental investigations.

The absence of preimaginal development outside of flower heads is a characteristic of not only L. vulpes but also for the genus Larinus in general. Only six Larinus species construct analogous capsules (pupal chambers) with a sweet taste (known as “trehala”) on plant stems, namely: L. capsulatus Gültekin, 2008; L. hefenborgi Boheman, 1845; L. nidificans Guibourt, 1858; L. ruficollis Petri, 1907 (Gültekin 2008); L. trehalanus Gültekin and Shahreyary-Nejad, 2015 (Gültekin and Shahreyary-Nejad 2015); and L. onopordi (Fabricius, 1787) (Khnzoryan 1951). According to Zwölfer et al. (1971), larvae of L. vulpes sometimes construct capsules on the stems, but the authors did not provide further details for this observation. Fabre (1922) once recorded the larval chamber built in the axil of the leaf of Echinops sp. Of note, L. vulpes and the trehala building species develop on plants of the same genus, Echinops, and perhaps the construction activity outside of flower heads might be to avoid properties of the thick adhesive sap of these plants. Of the seven Larinus species that develop on Echinops spp., all construct outer pupal chambers (L. vulpes included). According to Iljin (1953), E. ruthenicus and other globe thistles contain the type of rubber that turns into a dense substance when exposed to air. Closely related to Larinus weevils, Afrolarinus moestus (Chevrolat, 1882) also develops on Echinops, but only in the flower heads (Gültekin 2013).

Two important moments in the life cycle of Larinus vulpes remain unclear: (1) why do eggs appear outside flower heads? (2) can imagines open their chambers on the stems and emerge at the proper time? According to the assumptions of Fabre (1922), an egg could fall from the inflorescence after being laid, or the female could lay an egg in an unusual place “either by inadvertence or by intention”. This event is unlikely because females preferred to lay eggs in the top of flower heads. Occasionally, females laid an egg on a lateral part but never on the bottom of flower head. Thus, falling out into the axil of a leaf is doubtful. Unlikely also was “inadvertence”, because this phenomenon is not that rare. On a relatively small patch of grassland (approximately 150 m²), seven chambers were found. Therefore, females laid eggs in an unusual place, for enigmatic reasons. In previous years, Volovnik conducted direct observations of L. vulpes in the field but never saw its capsules on plant stems. In the region of investigation, the growing season for vegetation during 2016 was abnormally dry and hot. Most of the globe thistles were short with small inflorescences and died earlier than usual (Fig. 40). It is possible that such an extreme situation resulted in an acute shortage of suitable places for normal oviposition; thus, a significant portion of eggs were laid in unconventional places.

Figures 38–40. 

Pupal chamber outside inflorescence and habitat in 2006. 38, 39 Pupal chamber in the axil of a leaf 40 Habitat in dry, hot season in 2016; flower heads were small and died early. All photos SV Volovnik.