Semanotus bifasciatus Motschulsky (Coleoptera: Cerambycidae) is an important borer of cypress and pine trees (e.g., Sabina spp., Chamaecyparis spp., Platycladus spp., and Cunninghamia lanceolata) (Gao and Gong 2007; Zhu et al. 2018). It is widely distributed in China, Korea, and Japan (Kim and Park 1984; Zhang and Zhao 1996) and primarily attacks unhealthy trees, wilted trees, and newly cultivated woods. In most of its distribution, S. bifasciatus exhibits one or two generations per year and overwinters as an adult in the sapwood of tree trunks. In late February to early May of the following year, a large number of adults will feed on the bark of the trunk where they lay eggs 2 m above the soil surface. Initially, the larvae feed on the phloem and form an irregular tunnel on the surface of the xylem; they then feed on the xylem, causing damage. From mid-July to mid-October, larvae pupate, emerge as adults, and survive the winter from late August (Meng et al. 2016; Zhu et al. 2018). However, probably owing to its biology characteristics and the larvae remaining concealed in the trunks of host trees (Li et al. 2017), it is difficult to fully comprehend the biology and ecology of field populations, including the identification of larval instars according to the timing of molts. The accurate identification of the developmental stages of larvae can provide important information for pest management (Daly 1985; Gosik et al. 2019). The best-known model for determining larval instars of insects is Dyar's rule, which states that sclerotized structures of individual insects usually remain constant in size during any given stadia (Daly 1985). Frequency distributions of body measurements have been used to determine the number of instars (Bleiker and Régnière 2014; Daly 1985; Dyar 1890). The most suitable age structures are validated by mean difference analysis and regression analysis. Indexes of sclerotized body parts used to determine insect stages include antenna spacing, mandible width, head capsule (HC) width, ocellus spacing, and pronotum width (Cazado et al. 2014; Cen et al. 2015; Daly 1985; Dyar 1890; Luo et al. 2016; Morales-Ramos et al. 2015). Dyar's rule indicates that the HC width of insect larvae increases according to a certain geometric sequence; that is, the change in HC width is related to the larval instar and can, therefore, be used as the basis for the determination of instars (Daly 1985). Though body width and body length are often less reliable for determining insect instars, they are convenient indices of age in field investigations. Thus, the objectives of this study were to identify instars of field-collected larvae and to develop optimal morphometric variables for the determination of S. bifasciatus instars.

Materials and Methods

Larvae collection. To ensure the collection of first-instar larvae, adults were collected from the Summer Palace in Beijing (N 39.9°, E 116.6°) in early March 2016 and May 2018. Each pair of male and female adults was matched in a plastic cup for mating and oviposition. Then, eggs were collected and placed in plastic Petri dishes with moistened cotton until they hatched. Other instars were obtained from Platycladus orientalis (L.) Franco in the Summer Palace and the Temple of Heaven Park in Beijing. To cover all instar stages, larvae were collected from damaged trunks every 10–15 d from late March to August 2016 and early May to August 2018. All larvae were preserved in 70% ethyl alcohol.

Measurement of six morphological variables. Six morphological variables were measured: HC width, pronotum width, mandible length and width, and body length and width (Fig. 1). All measurements were made with the aid of a fluorescence stereo zoom microscope (M205FA; Leica, Xi'an, Shaanxi, China).

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Statistical analyses. A frequency distribution analysis of the six morphological variables was performed using SPSS (Statistical Package for the Social Sciences, Version 21.0, Chicago, IL), and Origin (2018, www.originlab.com) was used to plot a histogram of the frequency distributions (Fig. 1) (Zhou et al. 2015). Based on these analyses, the lowest point between two adjacent peaks and the separation points between instars can be determined (Li et al. 2015). Curves were generated based on measurements of the sclerotized structures to evaluate the relationship between larval stages and each variable (SPSS 21.0 for Windows). The mean, standard error (SE), and coefficient of variation (CV) can be calculated for each variable. The exponential growth model was used to analyze the relationship between the HC width, pronotum width, and instar of S. bifasciatus larvae based on Dyar's rule (Crosby 1973), Brooks' ratios (Gaines and Campbell 1935), and linear regression (Loerch and Cameron 1983; Logan et al. 1998). According to the Crosby growth rule, a difference of 10% or less between Brooks' ratios indicates the correct grouping (Loerch and Cameron 1983). Based on the Crosby growth rule and regression analysis, differences in the six morphological variables between successive instars were evaluated and the best criteria for instar division were determined (Crosby 1973). A Crosby index of greater than 10% indicated that the grouping of age-dependent indicators was not reasonable.

Results and Discussion

We measured 589 larvae. The frequency histograms for HC width and pronotum width showed eight distinct peaks, indicating that the larval development of S. bifasciatus can be divided into eight instars (Figs. 2, 3). However, the frequency histograms for the other four variables failed to show conspicuous peaks and regular variation (Figs. 4–7), indicating that body length, body width, mandible length, and mandible width were not reliable indicators for identifying larval instars of S. bifasciatus.

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We calculated the Crosby ratios to evaluate the reliability of the results (Table 1). The Crosby ratios and CV (%) for the two traits were less than 10%, and the Brooks' ratio ranged from 1.1649 to 1.6804, indicating that HC width and pronotum width effectively group the S. bifasciatus larvae into eight instars. Linear regression indicated significant relationships for HC width and pronotum width (P < 0.0001, R² > 0.95). This was consistent with the exponential relationship based on the Brooks' ratios, further validating the division of larvae into eight instars. The regression equation for the relationship between the HC width and the instar number was Y = 0.5919^0.2360x (R² = 0.9640, P < 0.0001, regression coefficient = 0.9691) and that for the relationship between pronotum width and instar number was Y = 0.5930^0.2442x (R² = 0.9549, P < 0.0001, regression coefficient = 0.9614) (Table 2). Accordingly, both the HC width and pronotum width are the best variables for larval instar determination (Figs. 8, 9).

Table 1 Measurements and summary statistics for head capsule width and pronotum width of Semanotus bifasciatus larvae for the determination of larval instars.^A

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Table 2 Linear regression results for the number of larval instars and phenotypic variables for Semanotus bifasciatus.

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Statistical techniques based on the normal distribution are adopted in most studies of instar determination for insects (Hunt and Chapman 2001; Lionel et al. 2010; Logan et al. 1998). HC width was considered the most reliable indicator to determine larval instar, but various additional characters are used to identify instars (McClelan and Logan 1994; Velásquez and Valoria 2010). In most Cerambycidae species, HC width is the best morphological variable for larval instar identification. For Dorysthenes granulosus (Thomson) and Monochamus alternatus Hope, the HC width, with the most significant regression result, is the best indicator of larval instars (Yu et al. 2012). In Arhopalus rusticus (L.), Apriona germari (Hope), and Xylotrechus rusticus (L.), besides the HC width, the pronotum width can also be used as an indicator for larval instar determination (Go et al. 2019; Li et al. 2012; Pan et al. 2015). For other coleopterans, and potentially for all insects, instars are determined based on the HC width, pronotum width and length, mandible length and width, and body length (Cazado et al. 2014; Loerch and Cameron 1983; Luo et al. 2016;, Novák et al. 2018). Although the body length and width were not stable parameters for determining instars, we provide ranges and average values of these traits for each instar (Table 3) to serve as a reference for field observations.

Table 3 Mean values for body length and width of S. bifasciatus larvae at different ages.

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The determination of the larval instar number can provide a basis for further understanding the species. The number of instars is often considered to be constant within species (Esperk et al. 2007). However, various factors, such as the growth environment (e.g., temperature, humidity, photoperiod, food quality, and host species) and genetic factors, impact the number of instars in insects (Dyar 1890; Langor et al. 1990; Morales-Ramos et al. 2015). Guo et al (2016) reported that larvae of the long-horned beetle Anoplophora glabripennis (Motschulsky) can be grouped into five instars whereas Keena and Moore (2010) detected nine instars. Opisina arenosella Walker has 5 larval instars in field populations and 5–8 larval instars under laboratory conditions (Yang et al. 2015). However, M. alternatus larvae sampled from Zhejiang province and Sichuan province were both identified as having five instars (Huang et al. 2018; Xu et al. 2009). This situation also happened in Dendroctonus ponderosae Hopkins; larvae samples from southern and northwestern British Columbia and northcentral Alberta were identically four instars (Bleiker and Régnière 2014). Because of the hidden life of the larvae in Cerambycidae species, the larval instars within species could be most affected by the host plant rather than other environment factors.

In this study, the results indicate that S. bifasciatus larvae sampled in Beijing has eight instars. Though S. bifasciatus is widely distributed in China (Yang et al. 2015), it has a narrow host range: mainly hosted on Platycladus orientalis (L.) Franco and occasionally feeding on other cypress trees such as trees in genera of Sabina and Chamaecyparis (Liang et al. 2002). Moreover, the life history of S. bifasciatus, almost one generation per year in China, is relatively short compared with other Cerambycidae species. Based on its biology and ecology traits we infer that the larvae instars of this species may be identical in different geographic populations. However, it should be tested by collecting samples from different distributions if factors such as hosts or geographic conditions can impact on the number of larvae instar of S. bifasciatus.