Identification
The morphological features described by Yan are entirely comparable with the white and orange-red markings on the abdominal intersegmental membrane of the last instar larvae of Gynaephora menyuanensis, which also have black abdominal prolegs and an anterior pectoral backplane8 (Fig. S1). Furthermore, there was a significant decrease in the intraspecific genetic distances of 0–0.0012 (n = 20) and the nucleotide similarity with the reference sequences of 99.8% compared to the inter-species genetic distances of 0.03 of Lepidoptera17. The individuals acquired in our study are all members of the same species, i.e., G. menyuanensis Yan et Chou (Lepidoptera: Lymantriinae), based on molecular and morphological identifications.
Distribution and habitats
The primary distribution regions of G. menyuanensis are parts of Gansu (Minle, Sunan, and Jishishan) and the northeastern alpine meadow areas of Qinghai (Menyuan, Qilian, Haiyan, Tianjun, Datong, Huzhu, Ledu, Hualong, Xunhua, Tongren, Guide, Gonghe, Xinghai, Tongde, and Guinan) (Fig. 1A, B). The habitats have an average height of more than 3,000 m, an average annual temperature of 0.6°C, and an annual precipitation of ~ 530 mm (Fig. 1). In particular, ~ 55% of the annual precipitation falls in June, July, and August, corresponding to a typical continental plateau climate. The plant growth period spans approximately 135 days annually. According to our findings, G. menyuanensis was more abundant near streams (Fig. 1C), and (Fig. 1D) they primarily consumed high-quantity forage, including Gramineae and Cyperaceae (Fig. 1D).
Morphology of the last instar larvae
The general body morphology
Gynaephora menyuanensis larvae in their last instar are usually eruciform, with a head, thorax, and abdomen (Fig. S1A). With three pairs of thoracic legs, four pairs of abdominal prolegs, and one pair of anal prolegs, its cylindrical trunk contains 13 segments (Fig. S1B, C). There are prominent hair tufts with dense black setae covering the dorsal surface. With a pair of metathoracic spiracles and eight pairs of abdominal spiracles on A1–A8, the respiratory system is of the peripneustic type. There is a pair of funnel warts on abdominal segments VI to VII, a hallmark of the Lymantriinae family. The abdominal intersegmental membrane of G. menyuanensis has white and red speckles that can be used to identify it from other species of Gynaephora (Fig. S1B).
The head
With an inverted Y-shaped ecdysial line on the midcranium measuring 3.519 ± 0.52 mm (n = 60) in width (Fig. 2A, B, D), the hemispherical and sclerotized head turns positive red with the increase of larva instar (Fig. 2). There are a lot of bristles on the dorsal surface of the head (Fig. 2). Six pairs of protuberant stemmata were symmetrically arranged behind the antennae (Fig. 2B, C). The antennae, which are located laterally behind the mandibles, are divided into three segments: a well-developed basal scape located in the antennal fossa, a longest pedicel, and a cone-shaped and the shortest flagellum at the end of the pedicel (Fig. 3A).
The mandibulate variety of mouthparts includes a labrum, a pair of mandibles, a pair of maxillae, and a labium (Fig. 3B). The clypeus, which has six pairs of setae on its external surface, is connected to the labrum, which is a flat, medially notched structure (Fig. 3C). To assist the larvae in feeding the grass. The labrum can move back and forth. The paired mandibles have complementary heavy sclerotization and asymmetrical shapes (Fig. 3C). The cutting end has a pair of setae on the external surface and is wavy with four sharp ends for shredding food (Fig. 3D). On the labium, the paired maxillae are symmetrically positioned. Each maxilla’s primary purpose, comprising a cardo, stipes, galea, and a maxillary palp, is to grab food and facilitate chewing. The center between the paired maxillae is covered in many triangular-shaped spicules with sharp ends (Fig. 3E). A pair of labial palps and a spinneret are provided by the labium (Fig. 3F). Silk is secreted from a hole at the top of the spinneret, a cuticle-covered tubular structure that protrudes from the anterior of the labium.
Types of sensilla in the antenna and mouthparts
The mouthparts under SEM reveal the morphological traits of four distinct types of sensilla (Figs. 4, S2; Table 1):
Table 1
Morphological features of the sensilla on the antenna and mouthparts of the last instar larva of G. menyuanensis.
Type of sensillum | Length/µm | Base width/µm | Tip | Wall |
Sensilla trichodea I (Str I) | 106.72 ± 9.97 | 11.11 ± 1.36 | Trichoid | Smooth |
Sensilla trichodea II (Str II) | 205.55 ± 19.42 | 25.81 ± 1.97 | Trichoid | Smooth |
Sensilla chaetica I (Sch I) | 39.97 ± 3.25 | 13.01 ± 3.87 | Sharp | Smooth |
Sensilla chaetica II (Sch II) | 16.33 ± 2.23 | 6.41 ± 1.21 | Sharp | Smooth |
Sensilla basiconica I (Sb I) | 5.63 ± 2.08 | 4.51 ± 0.38 | Blunt | Oblique striation, Porous |
Sensilla basiconica II (Sb II) | 31.07 ± 2.83 | 15.22 ± 0.42 | Blunt | Smooth |
Sensilla styloconica I (Ss I) | 24.60 ± 1.34 | 12.86 ± 0.85 | Blunt | Smooth |
Sensilla styloconica II (Ss II) | 19.98 ± 2.65 | 13.40 ± 1.76 | Blunt | Smooth, Porous |
Sensilla trichodea (Str, two subtypes: Str I, Str II); Sensilla chaetica (Sch, two subtypes: Sch I, Sch II); Sensilla styloconica (Ss, two subtypes: Ss I, Ss II); and Sensilla basiconica (Sb, two subtypes: Sb I, Sb II).
Located on the mouthparts of G. menyuanensis larvae in their last instar, Str are the most prevalent sensilla. With an end that resembles hair, the Str I are located near the apex of the antennal pedicle. Str II are more prolonged than Str I and located on the surface of the maxillae and apex of the antennal pedicle (Fig. 4A; Table 1). At the midpoint, the Str II are helical.
Sch have two kinds, primarily located on the ventral side of the larval antennae: long Sch (Sch I) and short Sch (Sch II). Sch I are found near the base of the antennal flagellum and maxillary galea, and they progressively taper to the blunt tip with smooth cuticles. The Sch II are nearly perpendicular to the antennal surface and are gathered on the antennal pedicle’s surface (Fig. 4B). Sch I and Sch II have mean lengths of 39.97 ± 3.25 µm and 16.33 ± 2.23 µm, respectively, and the basal widths of 13.01 ± 3.87 µm and 6.41 ± 1.21 µm, respectively (Table 1).
Ss can be divided into two types based on their shapes. Ss I have a smooth surface without any visible wall pores at the top of the antennal flagellum and a thin and pointed cone protrusion at the blunt distal points. Ss II have a smooth surface on the maxillary galea and a blunt tip papillary protrusion (Fig. 4C). The basal widths of Ss are 12.86 ± 0.85 µm and 13.40 ± 1.76 µm, respectively, and the mean lengths are 24.60 ± 1.34 µm and 19.98 ± 2.65 µm (Table 1).
The apex of the Sb is rounded and blunt. Sb I are found on the antennal flagellum’s surface and the maxillary palp’s apex. The columnar Sb I have oblique striations and several tiny, uneven, wrinkle-shaped pores at their blunt tips and hidden sockets. Longer and more robust than Sb I, Sb II is primarily located in the maxillary basal fossa and antennal pedicel. The Sb II has sharp tips and smooth walls without detectable depressions (Fig. 4D; Table 1).
Thoracic legs and prolegs
Three thoracic legs (Fig. 5A, B), each of the thoracic leg is composed with a coxa, femur, tibia, tarsus, and pretarsus (Fig. 5C). The thickest coxa is covered with dense hair. The femur and longest tibia have several setae on their lateral surfaces, whereas their anterior surfaces are usually glabrous. The tarsus is subconical in shape and has two short setae, five short sensilla chaetica, and a terminally curved, pointed pretarsus (Fig. 5D).
The unsegmented abdominal prolegs consist of proximal and distal bases and are located on abdominal segments III and VI (Figs. 5E, S3). Several setae cover the lateral and mesal surfaces of the proximal base (Figs. 5E, 6A, S3). Mesally, the planta are covered in dense microtrichia (Fig. 6D). The apical planta of prolegs have 17–25 uniform crochets arranged in a mesal penellipse (Figs. 6A–D, S4). Except for the number of crochets, the anal and abdominal prolegs have similar structures.
Setae on the trunk
The grass caterpillar larvae are covered by dense hair tufts, on where several setae insert (Fig. 7A, E). On the last instar larvae, there are three main types of setae: needle-shaped, spiral-shaped, and penniform (Fig. 7B, C, D). Compared to spiral-shaped setae, penniform setae are longer and have dense microtrichia. When touched, the hollow setae can break readily and irritate the skin (Fig. 7F). In addition to helping the larvae crawl and spread, the setae can be used to identify the taxonomic status of the larvae based on the number and arrangement of their hair tufts.
Funnel warts
On abdominal segments VI to VII, the larval funnel warts display two distinct colors: red (♂: n = 29a, ♀: n = 27a) and creamy yellow (♂: n = 20a, ♀: n = 21a) (Fig. 8A–D). The findings showed no correlation between the gender of G. menyuanensis and the intraspecific color differences of funnel warts (χ2 = 0.003, P = 0.956). The funnel warts have an oval, volcano-shaped aperture. At the proximal opening of the funnel, furrows and a few secretions are visible (Fig. 8C, D). When viewed from within the larva, prominent muscle insertion points may be seen at the bottom of the funnel, and many trichomes are visible only inside. The larvae’s body will curl into a ball, and their versible osmeterium will expand outward to form a papillary bulge in response to external stimulation. According to the ultrastructure, many well-developed secretory cells are connected to the hollow column-shaped organ dispersed across the gland’s surface (Fig. 8E, F).
Chaetotaxy of the first-instar larvae
For the left side of the body, the chaetotaxy of the first larval trunk is shown (Figs. 9, S5). Chaetotaxy on tathorax (T1–T3) and the abdominal segments 1–10 (A1–A10) show similar setal arrangement.
Prothorax (T1)
The prothorax bears an enormous prothoracic shield, on which are three setigerous tubercles: dorsal cluster (D), subdorsal cluster (SD), and lateral cluster (L). With 22 setae, the dorsal cluster (D) is anterior to the subdorsal cluster (SD). There are 20 setae on the lateral cluster (L) and 24 on the subventral cluster (Table 2; Figs. 9, S5). Each seta is a single, equal-length stiff filament that rises directly from the cluster.
Table 2
The number of setae in each segment and the setigerous tubercle of the first instar larva of G. menyuanensis.
Segments | PD | D | SD | L | SV | V | CV |
T1 | / | 22 | 24 | 20 | / | / | / |
T2-T3 | / | 22 | 12 | 14 | 9 | / | / |
A1-A2 | 3 | 18 | 18 | 11 | 8 | 3 | 1 |
A3-A4 | 3 | 18 | 18 | 11 | 8 | / | / |
A5-A6 | / | 18 | 18 | 11 | 8 | / | / |
A7 | / | 18 | 16 | 11 | 8 | 3 | 1 |
A8 | 3 | 18 | 16 | 11 | / | 3 | 1 |
A9 | / | 18 | 16 | 8 | / | 3 | 1 |
A10 | / | 7 | / | 3 | / | / | / |
Meso- and meta- thorax (T2, T3)
In chaetotaxy, the meso- and meta-thorax are identical. The single bristle inserts on the dorsal cluster (D), subdorsal cluster (SD), lateral cluster (L), and subventral cluster (SV). With 22 setae, the setae rising from the dorsal cluster (D) are significantly more noticeable than the rest. The subdorsal cluster (SD) and lateral cluster (L) have 12 and 14 bristles, respectively. The subventral cluster (SV) has the smallest number of setae, with 9 bristles (Table 2; Figs. 9, S5).
Abdominal segments 1 and 2 (A1, A2)
Regarding chaetotaxy, the first two abdominal segments are the same in that they lack any appendages. Very noticeable setae rise from the dorsal cluster (D) and subdorsal cluster (SD). With 3 setae, the predorsal cluster (PD) is located anterolateral to the abdominal prolegs. With 11 setae, the lateral cluster (L) is anterior to the prothoracic spiracle. There are 3 shorter setae in the ventral cluster (V) and 8 in the subventral cluster (SV). On these segments, the central ventral seta (CV) is the shortest (Table 2; Figs. 9, S5).
Abdominal segments 3 and 6 (A3–A6)
Except for the absence of the V and CV clusters, the morphology of the A3 and A4 segments is identical, with five different types of clusters (PD, D, SD, L, and SV) that follow the same pattern as the A1 and A2 segments. A5 and A6 segments had the identical morphology as A3 and A4, with four types of clusters (D, SD, L, and SV), except for the absence of predorsal cluster (PD) (Table 2; Figs. 9, S5).
Abdominal segment 7 (A7)
Segment A7, with six types of clusters (D, SD, L, SV, V, and CV), is similar to that in segments A1 and A2, other than the absence of a predorsal cluster (PD) (Table 2; Figs. 9, S5).
Abdominal segment 8 (A8)
The setal arrangement and number of bristles originating from the predorsal cluster (PD), dorsal cluster (D), subdorsal cluster (SD), lateral cluster (L), ventral cluster (V), and central ventral cluster (CV) on A8 are the same as those on A1 and A2, except for the lack of a subventral cluster (SV) (Table 2; Figs. 9G, S5).
Abdominal segment 9 (A9)
The setae on A9 are comparably shorter, and the number of setae ascending from dorsal cluster (D), subdorsal cluster (SD), ventral cluster (V), and central ventral cluster (CV) are identical to that on A8 except for the lateral cluster (L: 8) (Table 2). The predorsal cluster (PD) and the spiracle are lacking compared to the setae layout on A8 (Figs. 9G, S5).
Abdominal segment 10 (A10)
The dorsal cluster (D) and the lateral cluster (L) are located on the dorsal sclerite of A10. Although the dorsal cluster (D) has a large surface area, it only has seven bristles. The lateral seta (L) is located ventral to the anal proleg with three setae (Figs. 9G, S5F).