The morphology of the preimaginal stages of Squamapion elongatum (Germar, 1817) (Coleoptera, Curculionoidea, Apionidae) and notes on its biology

Abstract Data on the morphology of the egg, mature larva (L3) and pupa of Squamapion elongatum (Germar, 1817) are presented. The development cycle of this species lasts 51–54 days: a 12-day egg period, a 30-day larval period, and a 12-day pupal period, on average. The larvae are attacked by parasitic hymenopterans of the superfamily Chalcidoidea.

Squamapion elongatum is a species inhabiting the southern and central part of Europe, as well as East Asia and Algeria. It inhabits lowland and submontane areas. In Poland it is recorded in the Masurian Lake District, the Wielkopolsko-Kujawska Lowland, Upper and Lower Silesia, the Krakowsko-Wieluńska Upland, the Małopolska Upland, the Świetokrzyskie Mountains, the Lublin Upland, Roztocze and Eastern Beskid (Burakowski et al. 1992). This species is characteristic of xerothermic grasslands, where it feeds on plants of the genus Salvia -S. pratensis and S. nemorosa (Cmoluch 1962). Its life cycle has not yet been described.

Material and methods
Squamapion elongatum eggs, larvae, pupae and adults were collected from two patches of xerothermic grasslands in Gródek (50°46'58.18"N, 23°56'47.04"E) near Hrubieszów and in Łęczna (51°18'9.7"N, 22°51'47.8"E) (SE Poland). The material was collected during one growing season from July to September 2011 at 3-7 day intervals between 10 a.m. and 2 p.m. Adults were collected using an entomological net in an association of Thalictro-Salvietum pratensis. To obtain the other development stages for breeding, whole plants of the genus Salvia were collected. In total 67 specimens of meadow sage were collected. A delicate cut was made along the stem and root of the plants and then they were dissected with needles to find the eggs, larvae, pupae and even adults located inside. Some of the larvae were used to begin breeding and others for microscopic slides, to be used in making drawings showing the morphology of the developmental stages. To prepare the drawings we used an OLYMPUS BX61 microscope at magnifications from 200 × to 400 × and a TESCAN VEGA3LMU scanning microscope at magnifications from 500 × to 4,500 ×. The figures were made based on the biological preparations using Corel Draw 12 software. Metric sizes are the average value of 10 measurements (Table 1).
Larval specimens in successive developmental stages were transferred in vitro to Petri dishes after the larval stadium was determined on the basis of morphological characteristics and the number of exuvia of head capsules. Breeding of larvae was carried out to the L 3 stage. Breeding of preimaginal stages was carried out according to Scherf (1964) and Łętowski (1991). Petri dishes were transferred to a breeding chamber with the following conditions: temperature during the day 30 °C, temperature at night 20 °C, humidity during the day 60%, humidity at night 80%. Adults were kept in glass containers covered with a fine mesh. As in the case of the larvae, filter paper saturated with distilled water was placed at the bottom to maintain humidity and as a possible reservoir of drinking water for the beetles. They were fed with fragments of fresh sage shoots, which were replaced on average every three days. The used stems were examined to search for eggs. Microscope slides with the developmental stages and their morphological structures were prepared according to Łętowski (1991) and Gosik et al. (2010). The terminology of Scherf (1964), May (1993May ( , 1994, Marvaldi (2003) and Wang et al. (2013) was used in the morphological description of the larva and pupa. The morphology of the egg, L 3 and pupa and the developmental cycle from egg to adult were described.
Setae of thorax and abdomen of larva (L 3 ) and pupa are described for one side only.
Pupa (Figures 6-8   Head (ventral view). Rostrum reaching ventrite V, with 1 distinct seta (rs) at midlength, in front of the antennal insertion. Antennae relatively long, club with conical papillae. Antennae sub-parallel to protibia. Frons with 1 pair of setae (vs), about as long as rostral setae, situated at the level of the hind margin of the eyes (Figs 6, 7). Pronotum length greater than width, with 5 pairs of setae (as1, as2, sls, pls1, pls2) (Figs 6-8). Setae as1and as2 long and located at the apical margin. Setae sls a bit shorter, located at the external margin in the middle of the edge. Setae pls1and pls2 as long as sls, and located close to the back margin. Mesonotum short and metanotum two times longer than mesonotum. Mesonotum with clearly visible scutellar shield, metanotum with 3 pairs of short setae, medially located. All femora with 1 long, thin seta (fes1-3) located apically (Figs 6-8). Abdomen. Abdominal tergites I-III with 7 pairs of setae arranged in two rows -2 closer to the upper edge of the segment, nearly at the external margin, 5 closer to the base. Tergites IV-VI with 5 pairs of setae arranged in two rows -1 closer to the upper edge of the segment, nearly at the external margin, 4 closer to the base. Tergite VII with 3 pairs of setae arranged in one row, VIII with one pair. Segment IX without setae. Urogomphi (pseudocerci) (pc) on abdominal segment IX, laterally parted, crescent-shaped, narrow. Segment X reduced (Figs 6-8, 14). Spiracles on abdominal segments I-VI functional, well visible, positioned longitudinally on pleura (Fig. 6). Gonotheca visibly divided in female, single in male.

Biological information
According to the available literature, the period of occurrence of adults of Squamapion elongatum is April (Cmoluch 1962). During sampling they were found only on meadow sage, although woodland sage was also examined. This confirms Dieckmann's (1977) observations in Central European conditions that meadow sage is the food and breeding plant of this species. It was observed that fertilized females bit out small holes in the    nodes and internodes of the sage stems, in which they laid eggs. Often there were two or more larvae in one place, suggesting that females can lay several eggs in one hole. The oldest larvae were found on the root of the host plant, while the younger larvae were observed higher up, to about 2/3 of the plant height. After an average of 12 days (from 10 to 14 days), the larvae of the first instar (L 1 ) hatched and enlarged the egg chamber, making contact with the pith inside the stem. Here the larvae fed intensively, creating tunnels. After about seven days the larvae finished moulting, leading to L 2 . L 2 fed on average for 10 days, extending the tunnels begun by L 1 . The life span of L 3 ranged from 10 to 13 days (data based on laboratory cultures and field observations). The greatest weight gain in the larvae occurred during this period, as well as preparation for pupation. The larvae dig a tunnel 6-12 mm in length and use loose fibres to build a pupal chamber (Fig. 12). Initially the pupa was white; later it assumed a creamy shade, and as pigmentation progressed it turned grey. The eyes and rostrum took on the colour first, then the femorotibial articulations and tarsi, followed by the secondary pterothecae, and gradually the rest of the body. The pupal period lasted 12 days on average, and the development cycle of one generation from egg to imago lasted on average 51-54 days.
Larvae of this weevil were attacked by parasitic hymenopterans of the Chalcidoidea. Both internal (Entedon Dalman, 1820 sp.) and external (Trichomalus Thomson, 1878 sp.) parasites from that superfamily were observed.
The morphology of the egg does not differ substantially from the typical characteristics of the eggs of apionid beetles. The localization of eggs on the plant and the duration of this stage are also similar to those of other apionid beetles with the same lifestyle.
In general the body of larval S. elongatum does not deviate from representatives of the subfamily Apioninae described by Alonso-Zarazaga and Wanat (2014). It is very similar to that of larvae of species living inside common sainfoin described by Łętowski (1991) or Diplapion confluens (Kirby, 1808) living in the roots of Anthemis tinctoria L. (Gosik et al. 2010). The larvae of S. elongatum have a typical number of spiracles for Apionidae, as described in Emden (1938), Scherf (1964) and Łętowski (1991), the absence of spiracles on abdominal segment VIII being an important apomorphic character defining the group (as Apioninae in Marvaldi 2003). Larvae of different stages differ from one another in some characteristics, mainly changes in the distribution pattern of the setae. However, no change in the colour of the larvae was observed during growth, unless caused by internal parasitic infection. The differences in the chaetotaxy of the larval body are shown using L 3 of Squamapion elongatum, Diplapion confluens and Pseudaspidapion botanicum as examples ( Table 2).
The characteristics of the pupae of this weevil species do not differ from other representatives of Apionidae, except for the presence of a mesofemoral seta, also found in Diplapion but not in Pseudaspidapion, and the lack of a pair of setae on the 8th abdominal tergite, in contrast to one pair of setae in Pseudaspidapion and two in Diplapion. Relatively long urogomphi flared to the sides were present, as in Pseudaspidapion and Diplapion (Gosik et al. 2010;Wang et al. 2013).
The laboratory work confirmed that the food and breeding plant for this species is meadow sage (Salvia pratensis). The preimaginal development of S. elongatum occurs in the nodes and internodes of the stems, as well as in the basal part of the root.