The morphology of the immature stages of Squamapion atomarium (Kirby, 1808) (Coleoptera, Brentidae) and notes on its life cycle

Abstract The immature stages (egg, mature larva and pupa) of Squamapion atomarium (Kirby, 1808), as well as its development cycle and the phenology of its developmental stages, are described for the first time. The larva and pupa of S. atomarium have typical morphological features of the subfamily Apioninae. Morphological data on the immature stages were compared with the only fully described Squamapion species, S. elongatum (Germar, 1817). The larvae of the two species differ in body size and shape, head shape, setae length, the chaetotaxy of the mouthparts, and individual types of setae on the pronotum and thorax. In the case of the pupa, there are also differences in body size and in the type of setae and chaetotaxy of the head, pronotum, metanotum and abdomen.

The biology of only one species and the morphology of its immature stages are known -Squamapion elongatum (Germar, 1817) (Łętowski et al. 2015).
This study is a continuation of research on representatives of this genus found in Poland. The authors describe the morphology of the third larval instar and pupa as well as issues concerning the development and ecology of Squamapion atomarium (Kirby, 1808).
According to the literature, this species prefers warm, sandy areas and is usually found in xerothermic grasslands (Burakowski et al. 1992). Its host plants are Breckland thyme (Thymus serpyllum L.) and broad-leaved thyme (T. pulegioides L.). As regards its biology, S. atomarium feeds on the upper part of the stem of these plants, causing oval cecidia 2-4 mm long and 2 mm wide (Burakowski et al. 1992).

Breeding
Adult specimens were placed in plastic containers covered with mesh -separately for T. serpyllum L. and T. pulegioides L. Wet filter paper was placed on the bottom of the containers to maintain a suitable moisture level, together with thyme. The stems were searched for signs of oviposition and eggs about every three days. Then immature stages were grown in Petri dishes in a growth chamber, in the following conditions: daytime minimum 25 °C, daytime maximum 35 °C, minimum at night 15 °C, maximum at night 20 °C, humidity (60%), light duration -day 14 h, night 10 h. Immature stages were also grown in 125 ml plastic containers stored under room conditions (25 °C with a 14:10 photoperiod). Filter paper soaked in water was placed on the bottom of the container to maintain moisture, together with thyme stems with galls. The closed containers were monitored daily for mould. This method produced better results in terms of larvae survival than the use of the Petri dishes proposed by Scherf (1964). In order to track development and acquire larval stages, 5 stems were randomly selected, the galls were cut open, and developmental stages were isolated from them.

Morphological descriptions
The immature stages obtained by the methods described above were preserved in 70% ethyl alcohol. Two methods were used to prepare microscope slides, as described by Łętowski (1991) and Gosik et al. (2010). To prepare the drawings, we used an OLYM-PUS SZX12 and DP72 microscope at magnifications from 200× to 400× and a TES-CAN VEGA3LMU scanning electron microscope (SEM) at magnifications from 500× to 2000×. The larvae for SEM images were subjected to critical point drying (CPD). Drawings based on the slides were made using Corel Draw 18.
The terminology of Marvaldi (1999Marvaldi ( , 2003 and Oberprieler et al. (2014) was used in the morphological descriptions of the larva and pupa for chaetotaxy, and the terminology of Zacharuk (1985) and Marvaldi (1998) for antennae. The number and distribution of setae are given for one side. Measurements of the head (following decapitation) were made on the head capsule, isolated from the body, with the mandibles closed. Measurements were made of 10 L 1 , 4 L 2 , 15 L 3 and 10 pupae. The larvae were not separated by gender for the measurements. The mean and standard deviation for each parameter were calculated using Excel.
An analysis was made of the growth of the heads of individual larval instars based on Dyar's law (1890), and the growth rate (GF) was determined based on Bednarz (1953).
Labium cup-shaped (Fig. 6). Base of prementum rounded. Postmentum with 3 pairs postmental setae (pms1-3), distributed evenly, one over the other, closer to outer part of postmentum, more or less parallel to its edges. First pair setae (pms1) situated closest to lower margin, shortest of all pms. Above it pms2, very long and longest of pms, thick, narrowing only at apex. Setae pms3 situated at 2/3 height of labium, similar in structure to pms2 but half their length. Labium with Y-shaped premental sclerite situated centrally. 1 pair sensilla at base of arms of this structure. At height of premental sclerite, dorsally, chitinized inverted comma-shaped labial rods with uneven edges.  Labium with 1 pair simple palpi (lbp), with 7 palpillae apically, 1 inner seta at base and 1 outer sensillum. In front of palpi 1 pair long premental setae (prms). Behind palpi 2 pairs very short ligular setae (lgs) and 1 pair sensilla (Fig. 6).
Abdomen. Chaetotaxy very sparse. Each segment with 1 short dorsal seta located close to lateral margin. Each of lateral parts of abdominal segments I-VII with 1 pair Figures 10-15. Squamapion atomarium 10 pupa 11 occurrence environment 12 a gall 13 the most common place to lay eggs at the root collar 14 place for laying eggs 15 larva in prepupal stage for pupation. minute lateral setae. Spiracles located between tergites and pleurites, clearly visible on segments I-VI, on others absent (Fig. 9). Segment IX terminally with 1 pair urogomphi (ur) with characteristic ends in form of flattened bifurcation (Fig. 7). Figs 11-17 Host plant. The life cycle of S. atomarium was described based on field data and laboratory observations. Thymus serpyllum and T. pulegioides were confirmed as host plants (Fig. 11).

Notes on biology and life cycle
Life cycle. Adults, following overwintering and maturation feeding, begin copulation and egg laying in the first half of May. Increased egg laying was observed at the end of May, and single eggs were still noted in early June. Adults usually feed in the evening, by gnawing round holes in the leaf that do not exceed 1 mm in diameter. The fertilized female gnaws a cavity in the stem and lays one egg in it (Fig. 12). Oviposition takes place primarily at the root collar, but it was also observed up to the fourth or fifth node, in both nodes and internodes (Fig. 13). After laying the egg the female does not seal the site with any secretion. The first instar larva (L 1 ) hatches on average 4 days after the egg is laid and moults after 10-12 days. The L 1 instar was observed as early as mid-May, but these were isolated specimens. Maximum emergence was observed from the second third of May. L 1 larvae were found until mid-June. The second larval instar (L 2 ) appeared at the end of May. The activity of L 2 larvae causes distinct galls about 1.32 mm long and about 0.75 mm wide. Furthermore, L 2 gnaws out an opening for oviposition on the opposite side of the groove, but does not gnaw through the skin. The second larval instar lasts on average 10 days, and then the larva moults again. L 3 larvae appeared as early as the last third of June and were noted until mid-July. The average duration of this stage is about 11 days. This stage continues feeding and the gall grows, reaching on average about ca 2.31 in length and ca 1.70 mm in width (Fig. 14). The third larval instar enlarges the opening in the stem. Then pupation takes place (Fig. 15). The pupal stage lasts 2-3 days on average. The first pupae appeared at the end of June. Finally, at a maximum 40 days after the egg is laid, adult individuals appear. An increase in the emergence of adults took place from mid-July. The entire life cycle of S. atomarium is presented in the diagram in Figure 16.
Parasitoids. In the second half of July, endoparasitic hymenopterans of the superfamily Chalcidoidea were very active, which is manifested by the high level of parasitism of L 3 larvae. On average 7 of 10 third-instar larvae exhibited symptoms of parasite infection: dark red discolouration on the thoracic tergites and pleurites and swelling of the ab-   dominal segments caused by the growth of the intruder larvae (Fig. 17). The mature larva of the parasitoid usually occupied the space from the second or third thoracic segment to the eighth abdominal segment. The adult larva of the parasite is ca 0.75 mm long and ca 0.56 mm wide. The body of the pupa of the parasitoid is black with a metallic sheen and well chitinized. The parasites brought about the death of L 3 of S. atomarium.

Head growth of larval instars and growth factor (GF) Figs 18, 19
Deviations of the mean dimensions of the heads of individual larval stages from the theoretical dimensions are shown in Figures 18, 19. Analysis of the ratios of the head sizes of larval instars does not clearly result in a single growth factor. GF between L 1 and L 2 is 1.43 and between L 2 and L 3 it is 1.75.

Discussion
Among species of the genus Squamapion, only S. elongatum (Germar, 1817) has previously been described, and the existing data on S. atomarium concern only its habitat and host plants, with an equal role ascribed to T. serpyllum and T. pulegioides (Burakowski et al. 1992;Łętowski et al. 2015). The present study has shown that the preferred plant species is broad-leaved thyme (T. pulegioides), on which more galls were observed. This is most likely linked to the environment inhabited by S. atomarium, where this species of thyme is more common. Another new observation is the site of oviposition and galls. According to Burakowski et al. (1992), the larva feeds on the upper part of the stem. In the present study, the eggs were usually laid in the lower part of the stem.
The morphology of the L 3 larva and pupa of S. atomarium does not differ from the typical characters of the subfamily Apioninae (Alonso-Zarazaga and Wanat 2014). These features are the strongly convex and C-shaped body, colour, subglobose head, coronal suture and endocarinal line, clearly visible stemmata close to the frontal suture; numbers of des, les and fs; transverse and trapeziform clypeus with one pair of cls and one pair of clss; chaetotaxy of the labrum and epipharynx; mandible chaetotaxy; morphology and chaetotaxy of the maxilla and labium; thoracic segments with a prodorsum and postdorsum; very small prodorsum of the pronotum; morphology and chaetotaxy of the pro-, meso-and metanotum, except the number of as, with three pairs in S. atomarium; mesothoracic spiracles on the membrane between the pro-and mesothorax; and the abdominal morphology and chaetotaxy, except for the presence of lsts on the 8 th abdominal segment in S. atomarium.
In the comparative analysis of the egg and L 3 larva of S. atomarium and S. elongatum, the two species are distinguished by differences in the size of both the egg and the L 3 larvae -in S. atomarium they are about half the size as in S. elongatum (Łętowski et al. 2015). Similar differences are found in the width of the epicranium of the two species, the shape of the head, and some features of their chaetotaxy. The differences are presented in Table 1.
The case of the pupa is similar. There are clearly visible differences between species in body size and chaetotaxy. The body of the pupa of S. atomarium is shorter than that of S. elongatum (1.5-2.0 times) and has far fewer abdominal setae (Table 2). There are also minor differences in body colour. Similar proportions of body length are found in adults.
The study and descriptions of additional species of the genus Squamapion will make it possible to distinguish and describe its generic characters.  Analysis of the growth rate and the ratio of actual and theoretical average head sizes produced some discrepancies that may have been influenced by the fact that the individuals were not divided by sex or collection site, and thus may have represented different populations.