The morphology of the immature stages of two rare Lixus species (Coleoptera, Curculionidae, Lixinae) and notes on their biology

Abstract The mature larvae and pupae of Lixus (Ortholixus) bituberculatus Smreczyński, 1968 and Lixus (Dilixellus) neglectus Fremuth, 1983 (Curculionidae: Lixinae: Lixini) are described and compared with known larvae of 21 other Lixus and 2 Hypolixus taxa. The mature larva and pupa of Lixus bituberculatus are the first immature stages described representing the subgenus Ortholixus. The larva of Lixus neglectus, in the subgenus Dilixellus, is distinguished from the known larvae of four species in this subgenus by having more pigmented sclerites on the larval body. All descriptions of mature larvae from the tribe Lixini, as do all known species from the tribe Cleonini, fit the diagnosis of the mature larva of the Lixinae subfamily. Furthermore, new biological information of these species in the Czech Republic, Slovakia and Romania is provided. For Lixus bituberculatus, a chicory, Cichorium intybus L. (Asteraceae), is identified as a host plant, and Lixus neglectus is found on dock Rumex thyrsiflorus Fingerh. (Polygonaceae). Both species are probably monophagous or oligophagous. Adults of Lixus bituberculatus often inhabit host plants growing in active, dry and sunny pastures with sparse patches without vegetation, being mostly active during the night in April/May and then again in September, when the highest activity levels are observed. Adults of Lixus neglectus inhabit dry grasslands on sandy soils with host plants, being active during the day from May to September, with the highest level of activity in May/June and September. The larvae of both species are borers in the stem and root of the host plant, and they pupate in root or root neck. Adults leave the pupation cells at the end of summer and do not hibernate in the host plants. Finally, Romania is a new geographic record for Lixus bituberculatus.

tect these species. In this paper, we describe the immature stages of both species and provide details of their life history based on field observations in the Czech Republic, Slovakia and Romania.

Materials and methods
The material used to describe the immature stages was collected, and field observations were conducted in the localities mentioned below: Part of the larval and pupal material was preserved in Pampel fixation liquid (4 parts glacial acetic acid, 6 parts 4% formaldehyde, 15 parts 95% ethyl alcohol and 30 parts distilled water) and used for the morphological descriptions. These specimens are now deposited in the Group Function of Invertebrate and Plant Biodiversity in Agrosystems of the Crop Research Institute (Prague, Czech Republic). Plants were identified by the collectors. Slides were prepared following May (1994) as follows: a larva was decapitated, its head was cleared in a 10% potassium hydroxide (KOH) solution and then rinsed in distilled water. After clearing, the mouth parts were separated from the head capsule. The head capsule and the mouth parts were mounted on permanent microscope slides in Euparal. The body parts (thorax and abdomen) were mounted on temporary microscope slides in 10% glycerine.

Lixus (Ortholixus) bituberculatus
The observations and measurements were made using a light microscope with calibrated oculars (Olympus BX 40 and Nikon Eclipse 80i), and the following characteristics were measured for each larva: head width, length of the body (larvae fixed in a C-shape were measured in segments), width of the widest part of the body (metathorax or abdominal segments I-IV).The length and width of the widest part of the body was measured for each pupa. The thorax and abdomen were not sclerotised, and it is unlikely that the fixation process altered the weevils' proportions; measurements of these parts are given for comparison purposes only.
Drawings were made with a drawing tube on a light microscope and processed by a computer program (Adobe Photoshop, Corel Photo-Paint 11, GIMP 2). The thoracic spiracle is located on the prothorax near the boundary of the prothorax and mesothorax, as shown in the drawing (see Figs 8, 29), but it is of mesothoracic origin (Marvaldi et al. 2002, Marvaldi 2003. The drawings show the thoracic and abdominal spiracles (see [29][30][31]. The numbers of setae are given for one side of the bilateral structures.
We used the terms and abbreviations for the setae of the mature larva and pupa studied in Scherf (1964), May (1977May ( , 1994 and Marvaldi (1998aMarvaldi ( , 1999. The count of some of the setae on the epipharynx (especially ams and mes) have not been completely resolved. According to Marvaldi (1998aMarvaldi ( , 1999, the standard status of the epipharynx in weevils is 2 ams and 3 mes, but when the position of the distal mes is very close to the anterior margin, they appear as ams. The decision was finally made 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 or Tychius Germar. We did not follow  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 , e.g., in Adosomus Faust or Sibinia Germar.
Colouration. Head light brown or brown with a distinct pale pattern around the frontal suture (Figs 7,19). All thoracic and abdominal segments are white with a light brown, elongate stripe on the dorsum of the pronotum (Fig. 7).
Vestiture. Setae on body thin, relatively short, light yellow or orange. Head capsule (Fig. 1). Head suboval, flattened laterally, endocarinal line long more than half length of frons. Frontal sutures distinct, extended to the antennae. Single stemma (st) in the form of a slightly pigmented spot located anterolaterally on each side. Des1 and des2 located in the upper part of the central part of the epicranium, des1 near the middle part of epicranium, and des2 near the side of the epicranium, des3 located anteriorly near the frontal suture, des4 located in the central part of the epicranium, des5 located anterolaterally; all des long, subequal in length (Fig. 1). Fs1 and fs2 placed medially, fs3 located anteromedially, fs4 located anterolaterally, and fs5 located laterally, close to the epistoma; all setae relatively long, fs4 slightly longer than fs1-3 and fs5 distinctly longer than fs4 (Fig. 1). Les1-2 as long as des1; ves1-2 as long as fs3. Epicranial area with sensilla undistinct.
Antennae located at the end of the frontal suture on each side, membranous and slightly convex basal article bearing one conical triangular sensorium, relatively long; basal membranous article with 5 sensilla different in both shape and length (Fig. 4).
Clypeus (Fig. 2) approximately 2.1 times as wide as long with 2 relatively long cls, almost equal in length, localized posterolaterally and 1 sensillum; anterior margin rounded to the inside.
Thorax. Prothorax distinctly smaller than meso-and metathorax. Metathorax almost of equal length as abdominal segments I-IV. Spiracle bicameral. Prothorax  ( Fig. 8) with 10 prns unequal in length, 8 of them on weakly pigmented premental sclerite, which is subdivided medially into two triangular plates, next two prns placed below; 2 ps and 2 eus. Mesothorax ( Fig. 8) with 1 prs; 4 pds unequal in length, pds2 distinctly shorter than the remaining three setae; 1 short as; 2 short to very short ss; 1 eps; 1 ps and 2 eus. Chaetotaxy of metathorax ( Fig. 8) identical to that of mesothorax. Each pedal area of the thoracic segments well separated and pigmented, with 7 long pda, 6 of which on pigmented area, unequal in length.
Abdomen. Abdominal segments I-IV of almost equal length, subsequent abdominal segments decreasing gradually to the terminal parts of the body. Abdominal segment X reduced to four anal lobes of unequal size, the dorsal being distinctly the largest, the lateral pair equal in size, and the ventral lobe very small. Anus located terminally. Spiracles bicameral, the eight abdominal spiracles located laterally, close to   the anterior margin of abdominal segments I-VIII. Abdominal segments I-VII (Figs 9-10) with 1 prs; 6 pds, pds3 and pds5 the longest one; 2 ss of unequal length, ss1 very short, ss2 as long as pds6; 2 eps of almost equal length; 2 relatively short ps of unequal length, ps1 very short to minute, ps2 short; 1 lsts and 2 eus. Abdominal segment VIII ( Fig. 10) with 1 prs; 4 pds, pds1 and pds6 lacking, pds3 and pds5 less than half of length of the two remaining setae; 2 ss of unequal length, ss1 very short, ss2 long as pds6; 2 eps of almost equal length; 2 short ps of equal length; 1 lsts and 2 eus. Abdominal segment IX ( Fig. 10) with 4 ds (ds1 and ds3 very short, ds 2 and ds4 long); 2 short and 1 very short ps and 2 very short to micro sts. Abdominal segment X ( Fig. 10) with 2 microsetae and 1 seta (ts) on each lateral anal lobe.
Morphology 20). Body stocky, elongated, white or yellowish. Cuticle smooth. Rostrum relatively long, approximately 2.7 to 3.0 times as long as wide, extending beyond the mesocoxae; females with slightly thinner rostrum than males. Antennae relatively long and stout. Pronotum from 1.2 to 1.3 times as wide as long. Mesonotum and metanotum of almost equal length. Abdominal segments I-III of almost equal length; abdominal segment VI semicircular and subsequent abdominal segments diminish gradually to the end of the body. Abdominal segments VII-IX distinctly smaller than other abdominal segments. Gonotheca (abdominal segment IX) in females (1 specimen) bilobed.
Chaetotaxy . Setae relatively short, unequal in length, light yellow or orange, some setae on abdominal segments III-VIII distinctly stronger and located on protuberances. Setae well visible. Head capsule includes 1 vs, 3 sos, 1 os and 4 pas. Rostrum with 2 rs, rs1 located below antenna, rs2 placed on the anterior margin. Setae on head capsule and rostrum straight, both rs and all pas distinctly shorter than the remaining setae on head, thoracic and abdominal segments. Pronotum with 2 as, 2 ds, 2 ls and 4 pls. Dorsal parts of mesothorax with 1 seta located posteromedially, 1 seta located posterolaterally and 4 setae located along its anterior margin. Chaetotaxy of metathorax identical to that of mesothorax. Each femoral apex with 2 fes. Dorsal parts of abdominal segments I-VIII each with 2 pairs of setae located posteriorly (d1, d9) and 7 pairs (d2-8) located along their anterior margins. Setae d2-3, d5 and d7 (on abdominal segments IV-VIII) short, thorn-like, located on protuberances; on abdominal segment III only setae d3 and d5. Remaining setae short to very short, hair-like; all setae very short on abdominal segments I-II. Abdominal segments I-VII with groups of 2 lateral setae and 5 pairs of ventral setae. Dorsal part of abdominal segment VIII with 1 seta located posteriorly (d9) and 7 pairs (d2-8) located along its anterior margin; d3, d5 and d7 thorn-like, located on protuberances; remaining setae elongated. Abdominal segment VIII with groups of 2 lateral setae and 5 short ventral setae. Abdominal segment IX with 2 pairs of ventral microsetae and 1 pair of short, thin setae. Urogomphi elongated, triangular.  Biology and ecology. Habitats. Adults (Fig. 14) prefer dry and sunny habitats such as dry grasslands, meadows often with grazing or mowing (Fig. 21), and road margins with specific disturbance regimes (trampling by movement of cattle or vehicles, etc.).
Adult behaviour. During the day, adults stay among the rosette leaves of the host plant (Fig. 15) near the stem base. Adults were usually observed by sweeping the host plants at night. Data were collected from April to September with the exception of July. The maximum number of records occurred in late summer.
Host plants. Adults and larvae were observed feeding on chicory Cichorium intybus L. (Asteraceae), in the studied localities (Fig. 21). Nevertheless, J. Krátký and J. Pelikán (in litt.) also found adult L. bituberculatus on Crepis sp. and Picris sp. during night sweeping and a pupa in the root of Picris sp. in Slovakia (Hajnáčka env. and Bajtava env.).
Life cycle. Lixus bituberculatus is an univoltine species. Adults feed on leaves, but larval development occurs in the basal part of the stem and in the root (Figs 18-19). Females of L. bituberculatus bite the lower part of the stem of the host plant near the ground and lay one egg in the hole (Figs 16-17). Usually, only one larva was found to occupy a plant, but rarely, there were two (one in the stem and another in the root crown). Mature larvae were found from July to August. Pupation occurs in the root neck or root (Fig. 20), and fresh adults can be found (inside plants) from middle of August. The exit hole is situated in the upper part of the pupation cell. Adults do not hibernate in the host plants. Most likely, hibernation occurs in the leaf litter, among dry plant debris or in the topsoil.
Rearing of the larvae. For laboratory breeding, 10 mature larvae were collected on August 8 th , 2014, but only three pupated under our laboratory conditions. The remaining seven larvae primarily died due to drying of the host plants. The first fresh adult hatched on September 12 th and the other two on September 15 th , 2014.
Colouration. Head light brown or brown (Figs 28,38). All thoracic and abdominal segments are white with a light brown elongate stripe on the dorsum of the pronotum (Fig. 28).
Vestiture. Setae on body thin, relatively long to very long, light yellow or orange. Head capsule (Fig. 22). Head suboval, flattened laterally, endocarinal line long more than half length of frons. Frontal sutures distinct, extended to the antennae. Single stemma (st) in the form of a slightly pigmented spot, located anterolaterally on each side. Des1 and des2 located in the upper part of the central part of the epicranium, des1 near the middle part of the epicranium, and des2 near the side of the epicranium, des3 located anteriorly near the frontal suture, des4 located in the central part of the epicranium, des5 located anterolaterally; all des long, subequal in length (Fig. 22). Fs1 and fs2 placed medially, fs3 located anteromedially, fs4 located anterolaterally, and fs5 located laterally, close to the epistoma; all setae long to extremely long, fs1 extremely long, fs4 and fs5 very long, distinctly longer than fs2 and fs3, but shorter than fs1 (Fig. 22). Les1-2 as long as des1; ves1-2 as long as fs3. Epicranial area with sensilla undistinct.
Antennae located at the end of the frontal suture on each side, membranous and slightly convex basal article bearing one conical triangular sensorium, relatively long; basal membranous article with 3 sensilla different in both shape and length (Fig. 25).
Clypeus (Fig. 23) approximately 2.5 times as wide as long with 2 long cls, almost equal in length, localized posterolaterally and 1 sensillum; anterior margin rounded to the inside; median part covered by thorn-shaped asperities.
Thorax. Prothorax distinctly smaller than meso-and metathorax. Metathorax almost of equal length as abdominal segments I-IV. Spiracle bicameral. Prothorax (Fig.  29) with 10 prns unequal in length, of which 8 on distinctly pigmented dorsal sclerite that is subdivided medially into two triangular plates, next two prns placed below; 2 long ps located on pigmented sclerite, and 1 eus. Mesothorax (Fig. 29) with 1 long prs; 4 long to very long pds, pds2 distinctly shorter than the remaining three setae (both on weakly pigmented sclerites); 1 very short as; 2 short ss; 1 eps; 1 ps (eps and ps on weakly pigmented sclerites) and 1 eus. Chaetotaxy of metathorax (Fig. 29) identical to that of mesothorax. Each pedal area of the thoracic segments well separated and pigmented, with 7 long pda, of which 4-6 on distinctly pigmented area, unequal in length.
Abdomen. Abdominal segments I-V of almost equal length and subsequent abdominal segments decreasing gradually to the terminal parts of the body. Abdominal  segment X reduced to four anal lobes of unequal size, the dorsal being distinctly the largest, the lateral pair equal in size, and the ventral lobe very small. Anus located terminally. Spiracles bicameral, the eight abdominal spiracles located laterally, close to the anterior margin of abdominal segments I-VIII. Abdominal segments I-VII (Figs 30-31) with 1 prs; 5 relatively short pds, pds2 and pds4 on abdominal segment VII less than half of length of the three remaining setae which are twice as long as pds on the previous six abdominal segments; 2 ss of unequal length, ss1 very short, ss2 as long as pds5; 2 eps of unequal length; 2 relatively short ps of equal length; 1 long lsts (eps, ps and lsts on weakly pigmented sclerites) and 2 relatively long eus. Abdominal segment VIII (Fig. 31) with 1 relatively long prs; 2 long to very long pds, pds1-2 and pds4 lacking; 2 ss of unequal length, ss1 very short, ss2 as long as prs; 2 eps of unequal length, eps1 relatively long, eps2 long to very long; 2 relatively short ps of equal length; 1 relatively long lsts (eps, ps and lsts on weakly pigmented sclerites) and 2 relatively long eus. Abdominal segment IX (Fig. 31) with 3 ds (ds1-2 long, ds3 short); 2 very short ps and 2 very short to micro sts. Abdominal segment X (Fig. 31) with 2 microsetae (ts), on each lateral lobe.
Morphology (Figs 32-34, 39). Body stocky, elongated, white or yellowish. Cuticle smooth. Rostrum relatively long, approximately 3.1 to 3.5 times as long as wide and extending beyond the mesocoxae. Antennae relatively long and stout. Pronotum from 1.2 to 1.3 times as wide as long. Mesonotum and metanotum of almost equal length. Abdominal segments I-V of almost equal length; abdominal segment VI semicircular and subsequent abdominal segments diminish gradually to the end of the body. Ab- dominal segments VII-IX distinctly smaller than other abdominal segments. Gonotheca (abdominal segment IX) in females (1 specimen) bilobed.
Chaetotaxy (Figs 32-34). Setae relatively short, unequal in length, light yellow or orange, some setae on abdominal segments III-VIII distinctly stronger and located on protuberances. Setae well visible. Head capsule includes 1 vs, 3 sos, 1 os and 4 pas. Rostrum with 2 rs, rs1 placed below antenna, rs2 on the anterior margin. Setae on head capsule and rostrum straight, both rs and all pas distinctly shorter than the remaining setae on head, thoracic and abdominal segments. Pronotum with 2 as, 2 ds, 2 ls and 4 pls, and 1 short seta on ventral side (probably pls5). Dorsal parts of mesothorax with 1 seta located posteromedially, 1 seta located posterolaterally and 4 setae located along the anterior margin. Chaetotaxy of metathorax identical to that of mesothorax. Coxa with 1 very short cs. Each femoral apex with 2 fes. Dorsal parts of abdominal segments I-VIII each with 2 pairs of setae located posteriorly (d1, d9) and 7 pairs (d2-8) located along the anterior margins. Setae d2-7 (on abdominal segments III-V) and setae d2-5 and d7 (on abdominal segments VI-VII) short, thorn-like, located on protuberances. Protuberances on abdominal segment VII distinctly prolongated. Remaining setae relatively short, hair-like. Abdominal segments I-VII with groups of 2 lateral setae and 5 pairs of  ventral setae. Dorsal part of abdominal segment VIII with 1 seta located posteriorly (d9) and 5 pairs (d3-7) located along its anterior margin; d7 thorn-like, located on protuberances; remaining setae short. Abdominal segment VIII with groups of 2 lateral setae and 5 short ventral setae. Abdominal segment IX with 2 pairs of ventral microsetae and 1 pair of short, thin setae. Urogomphi distinctly elongated, hooked, triangular.

Biology and ecology
Habitats. Adults live in dry grasslands and meadows with sandy substrates (wind-blown river sand) (Fig. 41).The meadows are often managed for hay production. Numerous specimens were also found on grassy embankments along roads.
Adult behaviour. Adult beetles (Fig. 35) exhibit diurnal as well as nocturnal activity. During sunny days, they spend almost all of their time hiding among the leaves of the host plant near the ground (Figs 36,42) and rarely climb to the higher parts of the plant.
Life cycle. Lixus neglectus is an univoltine species. Adults feed on leaves, and larvae are stem and root borers (Fig. 38). Females of L. neglectus bite the lower part of the stem and lay one egg in the hole (Fig. 37). Larvae feeding in the root produce orange feeding frass (Fig. 37), which is thrown out of the host plant, and its presence is a reliable indication that the plant root is occupied by a larva. One plant is most likely occupied by only one larva. Mature larvae were found from July to August. Pupation takes place in the root neck (Fig. 39), and freshly hatched individuals can be found (inside plants) beginning in the middle of August (Fig. 40). Adults do not hibernate in the host plants, so hibernation most likely occurs in the leaf litter, among dry plant debris or in the topsoil.
Rearing of the larvae. For laboratory breeding, 15 mature larvae were collected on July 13 th , 2014, but only two of them pupated in our laboratory conditions. The remaining larvae died primarily due to drying of the host plants. Both of the adults hatched on July 30 th .
The precise general description of the larvae of the genus Lixus, which can be summarized by 19 character sets, has been presented by May (1994) and more in detail by Nikulina (2001) (for details, see Nikulina 2001 andWanat 2014). The larvae of L. bituberculatus and L. neglectus possess all of these characters, with only a few exceptions that partly result from differences in terminology (for details, see Gosik and Wanat 2014). Nikulina (2001) also published the only comprehensive and known larval key for this genus. The larva of L. bituberculatus has the closest affinity to the larva of L. kiritshenkoi Ter-Minasian, 1985 (abdominal segment X with two setae on the ventral side, lateral sides without setae, and dorsal side with less than three setae, see Table 1). The main differences are as follows (see Table 1): prothoracic tergite with 10 prn (vs. 13 prn); maxilla stipes with short 1 mbs (vs. without mbs, which could also be easily overlooked because it is very often minute); prodorsum on abdominal segment IX with only 4 ds (vs. 3 ds), and 3 short ps (vs. 1 ps). The larva of L. bituberculatus is the first of the immature stages described for the subgenus Ortholixus, but the larva of L. neglectus does not fit the description of any larva in the key (Nikulina 2001) because the first step provides no option for abdominal segment X to be without setae on the dorsal side (see Fig. 31). An interesting characteristic of L. neglectus is the presence of more pigmented sclerites on the larval body (see Figs 28-31, Table 1), which is similar to the description of L. filiformis (Nikulina and Gültekin 2011). In the subgenus Dilixellus, to which L. neglectus belongs, the larvae of four species have already been described: L. bardanae (Fabricius, 1787) Table 1). The creation of a precise key and a detailed subgeneric study of the genus Lixus is currently limited due to the lack of knowledge of the immature stages (see Table 1). The main problem is that we are unable to divide with certainty the morphological characteristics of this group into (i) characteristics that are useful for phylogenetics and (ii) characteristics that are useful only for species identification. Once this categorization is complete, it will be possible to apply it for future cladistics analysis, which are planned for the near future. All these morphological data should be compared and correlated with known biological data of the host plant families for the different groups of Lixus and also related genera, because some subgenera of Lixus are probably composed of different probably unrelated groups developing on quite different families of plants (Skuhrovec, Gosik, Stejskal, Trnka, Volovnik, Gültekin, unpublished data). May (1993) considered the increased number of pds on the meso-and metathorax and abdominal segments I-VII and the increased number of setae on the epipharyngeal lining (als) (i.e., higher than the most frequent number of setae in weevils) as diagnostic of the mature larva of the Lixinae subfamily, and the descriptions of mature larvae from the tribe Lixini (Larinus species : Zotov 2009a: Zotov , 2010Gosik and Skuhrovec 2011;Lixus species: Scherf 1964;Lee and Morimoto 1988;May 1994;Nikulina 2001Nikulina , 2007Zotov 2009a, b;Nikulina and Gültekin 2011;Gosik and Wanat 2014;Skuhrovec and Volovnik 2015;Rhinocyllus conicus: May 1994) fit this diagnosis, as do all known Table 1. Differential diagnosis of mature larvae of both described species and the most similar or relative species. species from the tribe Cleonini , Trnka et al. 2015. Currently, the comparison of both tribes, including key and detailed generic studies, is impossible due to our limited knowledge of the immature stages. A categorization of the morphological characteristics of Cleonini and a comparison of both tribes is planned following a detailed study of the genus Lixus (see the previous paragraph). The presence of 5 vms in the maxillary mala observed in all Lixinae and also most curculionids could be helpful as differential character from the root feeder larvae in Entiminae which have only 4 vms (Marvaldi 1998a, b).

Biology and ecology
The biology and development of these two Lixus species are very similar as both species are stem and root (crown) borers. In the genus Lixus, root borers, such as L.  (Dieckmann 1983) have not been identified as frequent. The prevailing nocturnal activity of the adults and the species hidden life habits are probably the main reasons why these species have not been found elsewhere, and it is very difficult to confirm them at a locality. The majority of the Central European Lixinae species require a specific habitat disturbance regime that results in sparse vegetation cover Trnka 2013, 2014). In both species, it seems that adults and the immature stages prefer places with pasture vegetation or meadows that are managed for hay production. This is the first report of L. bituberculatus from Romania, and it probably has a larger area of distribution including Bulgaria, Hungary, Romania and Slovakia (Gültekin and Fremuth 2013;. Based on our observations L. bituberculatus appears to be oligophagous on Asteraceae; all of our specimens were only found on Cichorium intybus, which originates from the Mediterranean but has been introduced to North America, southern Africa and New Zealand (Dvořáková 2005). It seems that this weevil is not specialized on only one plant species because it was found on Crepis sp. as well as Picris sp. in Slovakia, so its suitability as a candidate for the biological control of C. intybus is questionable. Only a host plant choice test can help us to determine all of the host plant species and the preferences of this species.
Lixus neglectus has been found exclusively on Rumex thyrsiflorus, but the distribution of its host plant covers the majority of Europe (Kubát 1990). However, this beetle is only known from a very small and specific area. The sorrel R. thyrsiflorus is considered to be a naturalized neophyte in Central Europe (Danihelka et al. 2012), but its origin remains unknown (Kubát 1990). This Lixus species seems to be monophagous, and its only host plant, R. thyrsiflorus, is not considered to be harmful and has not yet been introduced to other continents or countries. Therefore, it is not necessary to utilize this weevil to regulate its host plant despite its suitability as a biological control agent, but this must be validated by future studies. The distribution of L. neglectus has to be confirmed, especially across the wider area of distribution of R. thyrsiflorus. This weevil can be easily overlooked due to its cryptic way of life, but the easiest way to confirm its presence in the field is to search for frass at the root crown of the host plant. This frass is a very unique behaviour within the genus Lixus. To date, this weevil has only been recorded from R. acetosa (e.g., Fremuth 1983;Koch 1992;Böhme 2001), which is likely due to misidentification as occurred in our case . It is possible that L. neglectus historically lived on R. acetosa but has recently come to occupy a new available ecological niche on expanding R. thyrsiflorus (from ca 15th century). However, because each of the host plants prefer different habitats, this explanation is quite unlikely. The common sorrel, R. acetosa, prefers wet habitats, whereas the compact dock, R. thyrsiflorus, prefers dry ones (Kubát 1990). Both sorrels are also known as host plants for some other weevils, but there does not seem to be any competition among the species. Development on R. acetosa is also known in one other Lixus species, L. bardanae (Fabricius, 1787), but this weevil is a typical stem borer. Thus there is no competition between it and L. neglectus (Dieckmann 1983). A similar situation occurs in the development of some Apionids (e.g., Apion cruentatum Walton, 1844 or Perapion oblongum (Gyllenhal, 1839)), whose larvae also feed only in the stems (Dieckmann 1977). Furthermore, both plants host the oligophagous weevil species Marmaropus besseri Gyllenhal, 1837 (Dieckmann 1972) in their stems and roots, and this species has been recently expanding along with R. thyrsiflorus (Rheinheimer and Hassler 2010). Both of these weevils belong to the same guild (stem and root (crown) borers), so there could be some competition. However, this requires more information on the timing of their development as well as some of the other abiotic and/or biotic effects inside the plant. Thus, the complete switch in host plant by L. neglectus seems unlikely, mainly due to its rarity despite the recent expansion of its host plant. It is more probable that its preference for R. thyrsiflorus as a host plant is recent, but this could only be resolved through a host plant choice test.
Knowledge of the immature stages and life histories of insects can help protect endangered species (including the species presented here) more effectively. The detailed descriptions of the larva and pupa and their comparison with known descriptions reported here demonstrates the possibility of identifying species in their immature stages. Future detailed biological and morphological studies can yield unique information on the factors determining host specificity in this insect group and will provide useful background information for planning efficient biocontrol of invasive plant species. The issue of using some insects as biological control agents is a key topic in both the basic and applied research on invasive plants. Our results will significantly contribute to basic research but will also have practical implications for conservation biology and/ or biological control.
the Czech Ministry of Agriculture (Mze ČR) RO0416. We thank Adriana Marvaldi (Argentina) and Rafal Gosik (Poland) for commenting on the manuscript and for valuable discussions about larval and pupal chaetotaxy. We are sincerely grateful to Pavel Špryňar (Prague, Czech Republic) for notes about Rumex thyrsiflorus and to Martin Dančák (Olomouc, Czech Republic) for the identification of this species. We also thank Jiří Krátký and Jan Pelikán (both of Hradec Králové, Czech Republic) for the notes on the biology of L. bituberculatus at the Slovakian sites. The manuscript was edited for English language by American Journal Experts.