Investigation of the development of larval instars in the field

The pine bolts inoculated with the first instar were placed at three altitudinal sites of 200, 900 and 1,100 m on Mt. Hallasan, Jeju (17 July, 2018). This strategy was to provide different thermal environment for larval development, and obtain different stages of larval instars when they reached overwintering state. At each site, 33 pine bolts were treated in field cages of (120 cm × 70 cm × 60 cm) covered with a fine wire mesh (3 mm). The temperatures in the cages were also monitored using a data logger (HOBO MX2301, Onset Computer Corporation, MA, USA), for use later to calculate degree days (namely, the physiological age of larvae, see Supplementary Table ^S1 online).

The samples of pine bolts were divided into three groups, according to the investigation procedure at each site.

Nine pine bolts were collected at each altitudinal site on 22 November, 2018, to investigate the larval state that had entered into overwintering, and if there was the development of instars after overwintering in the next spring. At 200 m, the pine bolts were preliminarily examined on 21 September, to check whether the larvae made a pupal chamber (see Supplementary Table ^S2 online).

The outer bark and feces with wood shreds were clearly removed from the collected pine bolts, and kept in a plastic bag (25 cm × 33 cm), to use later for the investigation of mandibles (see below for details). Then, the pine bolts were split using a hand axe to examine the larvae inside.

The head capsule width (HCW) of alive larvae was measured across the greatest width of the head capsule, using a digital microscope (Dino-Lite, AD4113TL-MA1(R4), ANMO Electronics Co.), as the method suggested by Go et al.¹⁰. The mandibles of M. alternatus were articulated with a thickened part of the head capsule in the region of the clypeus. Thus, the length of mandible of the condyle center to incisor cusp was measured in the state where mandibles were articulated with the head capsule, according to the recommendation of Go et al.¹⁰. Also, larval weight, the diapause symptom of larvae, and the structure of pupal chamber were checked to compare the larval state among altitudinal sites (see Supplementary Table ^S2 online). The dead larvae were excluded from the measuring.

After examining the larvae, the split pine bolts were fitted with putting the larvae back inside, and wrapped using a stretch film (polyethylene chloride film, CLEANWRAP, Seoul). These pine bolts were placed in a field cage (50 cm × 60 cm × 40 cm) covered with a fine wire mesh (3 mm), installed in an experimental farm at Jeju National University. Also, temperature in the field cage was monitored using the same type of HOBO logger above.

On 18 March 2019 after sufficient days for diapause termination (> 90 days), the pine bolts were moved, and placed individually in a plastic bottle (diameter 15.6 cm, height 20 cm) in insect rearing room at (25.0 ± 1.0) °C, a photoperiod of 16:8 (L:D) h, and RH > 40%). Wet cotton pads were placed inside the cages to prevent the desiccation of pine bolts. After the emergence of adults, which was checked for 2–3 times a week, the head capsule width (HCW) and mandible length (articulated state) were measured on the larval exuviae in pupal chambers, using the same method above that was applied to live larvae. Also, the mandible length was measured on the state separated from the head capsule of larval exuviae. The body length of adults was measured as the distance from the end of vertex to the tip of elytral apex. Female and male adults were distinguished by the white band on the antennae of females⁴, and comb-like trichome on the end of female abdomen³².

When single exuviae was found in each pine bolt (namely in pupal chamber), it was assumed that the larval instar on 22 November of 2018 was directly pupated without further molting in 2019, because the pine bark and feces with wood shreds were clearly removed previously in 2018. Also, throughout all the experimental procedures, including the other experiments below, the larva and adult from the same pine bolt were traced by the same reference numbers, to examine the change of morphometric variables in each developmental stage.

The number of mandible exuviae of M. alternatus larvae were examined in the feeding gallery of pine bolts prepared previously above. The barks were carefully examined to separate all the attached feeding residues. The total feeding residue of each pine bolt was soaked in tap water in plastic container (300 ml) (approximately 1:2 of feeding residue:water), to loosen the lumps of feces and wood shreds. At a time, one teaspoon of feeding residue was transferred to a plastic petri-dish (diameter 90 mm, depth 15 mm) to find out mandibles under a stereomicroscope of (8–20) × . The found mandible length was measured using the same method above.

The number of mandible classes (strata) in size was used to determine the number of instars passing in the pine bolts¹⁰. That is, three and four strata of mandibles in the feeding gallery were regarded as the pathway of four and five instars, respectively, because the final instars were located in the pupal chamber. It was not easy to find all the successive strata of mandible in a pine bolt, since during the feeding process of M. alternatus larvae, some mandibles were probably destroyed and lost. Therefore, a missing instar was determined by the ranges of mandible length in each instar, or the relative size of mandibles calculated between two successive mandibles of instars found in a specific pine bolt.

Eleven pine bolts each at (200, 900, and 1,100) m were collected on 15 May 2019, and they were placed individually in plastic cages in the insect rearing room using the same method above. After adult emergence (daily observation was made), the mandibles in feeding residues and larval exuviae in pupal chamber were investigated using the same methods above.

The remaining pine bolts (namely each ten bolts at 200, 900, and 1100 m) were kept in the original sites, until adult emergence. Previously on 20 May 2019, the pine bolts were individually placed in a wire-mesh sleeve bag (1 mm mesh size). When the emergence of adults was made (checked 2–3 times per week), the wire-mesh sleeve bags, including pine bolt and adult, were brought to the laboratory; and they were investigated using the same method above.