Morphological Characteristics and Occurrence of an Important Stem-Boring Pest, Nassophasis sp. (Coleoptera: Rhynchophorinae )

: Dendrobium plays a key role in the orchid industry, with ornamental, medicinal, and economic value. Recently, we found a newly recorded boring pest damaging Dendrobium in Guizhou Province, China. The species is identified as Nassophasis sp. (Coleoptera: Curculionidae: Rhyncho-phorinae) by morphological and anatomical features. The occurrence and biological characteristics of this species were verified through field investigation and indoor experiments. The results showed that detailed external morphological and genitalia characters and male-female differences are described to identify Nassophasis sp. The pest produces three generations per year, with overlapping generations. The adults overwinter in the root stains of Dendrobium and emerge in mid-March of the following year. Adults feed on stems, leaves, and flowers, often laying their eggs inside the stems; larvae then bore into the stems causing decaying and hollowing until complete immature development. There are three larval instars, grouped according to their head capsule width and body length, which were measured following Dyar ’ s law and Crosby ’ s law of growth and showed a significant linear regression ( p < 0.0001). The results of this study provide a theoretical basis for the prediction and comprehensive control of the insect.


Introduction
Dendrobium is a perennial epiphytic herb of the Orchidaceae family, mainly distributed in Asia, New Guinea, and Australia, with important ornamental, medicinal, and economic values [1].The whole plant of Dendrobium can be used as a medicine.Because the plant is rich in dendrobium polysaccharides, Dendrobium alkaloids, amino acids, and other nutrients, it is now commonly used in the development and utilization of functional drugs, tea substitutes, and health care products, such as lowering blood sugar, inhibiting tumors, and improving human immunity [2].To satisfy the market demand, the artificial cultivation of the plant has developed rapidly.The planting mode is mainly based on shed cultivation, attached tree cultivation, and attached stone cultivation [1].With the frequent sales of dendrobium seedlings between provinces and the increase in planting density within local areas, the plant also faces the risk of pests spreading damage over long distances and at large scales.In particular, the occurrence of pests such as stem-boring (many kinds of weevils) and leaf-eating species (moths and slugs) is becoming severe [3,4].Among these, weevils are characterized by high concealment, untimely identification, and difficulty in control, and often have already caused severe damage to the plants when discovered [5,6].Currently, the main focus is on manual removal, control by parasitic or predatory natural enemies, and chemical control [7,8], which seriously affects the production and quality of plants.These factors restrict the development of dendrobium industries such as processing and utilization.
In 2019, our group found a species of weevil that caused extensive hollowing and wilting death of Dendrobium during a pest survey of a dendrobium plantation in Qiandongnan Prefecture; it was morphologically identified as Nassophasis sp.There are limited reports of Nassophasis sp., such as its external morphology [9], host selection, and feeding behavior [10][11][12].The life cycle, occurrence pattern, and biological characteristics of this weevil, as well as the cause of its widespread local occurrence in Guizhou, are currently unknown, which limits plant protection efforts of the dendrobium cultivation industry.
Nassophasis sp. is difficult to detect due to its boring and concealment characteristics.This species poses a great threat to the orchid industry in Southwest China.Therefore, finding effective prevention and control strategies is particularly urgent.The lack of information on the biology and life cycle hampers control strategies of Nassophasis sp.This study was conducted to determine the morphological characteristics, annual life history, and occurrence pattern of each stage of the Nassophasis sp.-through indoor and outdoor experiments related to biology and ecology-to provide an important scientific basis and technical support for avoiding large-scale infestation of this weevil.

Anatomy and Observations on the External Morphology and Genitalia of Nassophasis sp.
The morphological characteristics of Nassophasis sp. of 58 adults, 17 eggs, 133 larvae, and 34 pupae were documented and described using a stereomicroscope.The head, antennae, thorax, abdomen, sheath wings, membranous wings, feet, and external genitalia of 10 females and 10 males were dissected, photographed, and measured for each part.The genitalia were examined using the dissection methods following Valente [13] and Friedman [14].The adult was first de-winged, then the rump plate of the insect was lifted off with forceps.The reproductive system was removed with hooks and forceps, and impurities were removed with dissecting needles and placed under a microscope for observation and photography.Before dissection, the adults were placed in a 10% saline solution and immersed for 30 min until the specimen was soft enough to facilitate dissection.

Larval Instar Determination
The head capsule width, body length, and body width of 133 larvae were measured under an Olympus-szx7 stereomicroscope (Figure 1), and body weight was measured using an electronic balance.Histograms of frequency distributions were plotted using Origin 2021 [15].
The number of larval instars identified was verified using Dyar's rules.A Crosby index of less than 10% and a coefficient of variation of less than 20% indicate a reasonable instar classification index.Linear, quadratic, and exponential regression analyses of the two morphological measurement datasets were performed via SPSS 20.0 edition.The formulae are as follows: Coefficient of variation(%) = Standard deviation Mean value × 100%

Determination of Damage, Life History, and Spatial Distribution
Surveys were carried out in the understory forest, shed, and epiphytic cultivation areas in Anlong County.Three areas were randomly selected for survey in the attached tree and attached stone cultivation areas of the forest, and a 5 m × 5 m area was selected in each survey area for a plant-by-plant survey.Three survey sample plots were randomly selected in the greenhouse cultivation area, and three survey areas were randomly selected in each sample plot.A five-point sampling method was used for the survey, and 20 plants were surveyed in each sample plot; a total of 300 plants were surveyed.The numbers of dendrobium clumps, infested dendrobium, and each insect species in the survey area were recorded.The damaged plants were brought back to the laboratory, where the numbers and morphological characteristics of the eggs and larvae, and the infestation were recorded.At the same time, their developmental stages were recorded and identified after becoming adults.
Two aggregation index methods, Iwao regression analysis [16] and Taylor power method [17], were used to analyze the spatial distribution patterns of Nassophasis sp.Based on the field survey data, the mean population density (m), variance (S 2 ), and mean crowding degree (m*) within each survey area were calculated.The spatial distribution pattern of Nassophasis sp. was judged based on Lloyd's clustering index (m*/m), diffusion coefficient (C), clumping index (I), Cassie index (Ca), and Waters' negative binomial distribution (K-value).Each indicator was calculated using the following formulae, and the criteria for determination are shown in Table 1: Table 1.Relationship between the range of aggregation index and spatial distribution.

Distribution Pattern
Iwao's m*-m linear regression analysis is m* = α + βm; where α denotes the average crowding among individuals, and β denotes their spatial distribution type (Table 2).The Taylor power method has a functional relationship of S 2 = am b , which after logarithmic conversion is lg S 2 = lg a + b lg m (Table 3).Table 3. Spatial distribution judgment standard of Taylor power rule.

Distribution Pattern Determination Criteria
The causes of aggregation were analyzed using the population aggregation mean (λ) method proposed by Blackith [18], calculated as λ = mr/2 K, where r is the X 2 value corresponding to a degree of freedom of 2 K (p = 0.5), m is the mean insect population density, and K is the negative binomial distribution value.When λ < 2, aggregation may be caused by environmental factors; when λ ≥ 2, aggregation is caused by the insects' own aggregation habits or environmental factors.

Statistical Analysis
The data were initially collated using Excel and statistically analyzed using SPSS 26.0.Means and standard deviations were calculated for each insect stage and lengths of adult insect parts using one-way ANOVA (p < 0.05).Origin 2021 was used to generate data figures, and Photoshop 2022 was used to combine ecological pictures.

External Morphological Characteristics
The eggs were about 2.05 ± 0.09 mm long, oval in shape, with a smooth surface, light yellow at both ends of development, and yellow in the middle that gradually deepened in color (Figure 2A) The larvae were soft, 9.84 ± 2.14 mm in length, roughly rounded and curved, with a distinctive depression in the middle of the cranial suture, reduced antenna size, no caudal whiskers, and reduced thoracic legs, typical of weevil larvae (Figure 2B).When the larvae molted, the epidermis of the head splits opened first, and the molted skin was gradually pushed downwards, finally falling off from the end of the abdomen, with the newly molted larvae in a lighter color that gradually deepened (Figure 2C).
The pupa was exarate, 10.37 ± 1.63 mm long, and had a yellow body; its appendages and wings were not fixed to the body and thus able to move freely (Figure 2D).The dorsal plate of the prothorax was a cross-shaped fleshy projection, the head tube was bent towards the thorax, and the wings were sandwiched between the mid and hind legs.Three pairs of setae united the head and rostrum.There were a pair of caudal setae at the end of the abdomen and several pairs of short, small hairs on the dorsal side of the abdomen.The body color changed gradually from yellow to black-brown in the pre-feathering period.
The adult was 12.39 ± 1.15 mm long and 4-5 mm wide, in a reddish-brown color at first, which changed to black covered with incised dots (Figure 2E,F).The anterior end of the head extended into a rostrum, which was hard and apically tetragonal and awnshaped, averaging 4.5 mm.The compound eyes were black, long, and oval, with an average distance of 0.1 mm between them.The antennae were hammer-shaped, attached at approximately 1/3 of the rostrum, with the end of the flagellum dilated and the antennae averaging 4.0 mm.The thorax was ellipsoidal, 6.6 mm long, and covered with uneven yellow spots of varying sizes.The abdomen was five-segmented and oblate, with the middle three segments equal in width and the middle ventral plate fused, averaging 4.2 mm.The sheath wings were hard, and peltate, with four symmetrical yellow spots on the anterior and posterior margins.The membranous wings were transparent, and the wing veins were clear.The legs were orange-red, clawed, and covered with small incised spots.The incised spots were sunken, the transepithecae were black, and the tarsal segments ended with brownish-yellow tomentum.

Morphological Differences between Male and Female Adults
The male and female adults were extremely similar in appearance and difficult to distinguish from each other (Figure 3A).Females were 12.84 ± 0.62 mm long and 4.69 ± 0.42 mm wide, while males were 12.63 ± 0.76 mm long and 4.36 ± 0.17 mm wide.Although females were larger than males, there were no significant differences in body length (t = 1.046, p = 0.192) and width (t = 1.277, p = 0.378).The differences between males and females were mainly found in the abdomen, where the base of the abdomen was more elevated in females and more depressed in males (Figure 3B).

Morphological Characteristics of Male and Female External Genitalia
Male external genitalia consisted of a phallus base and a phallus, the top of which was enclosed within the phallus base, connected by an intersegmental membrane in a ring-like configuration (Figure 4A,B).The phallus base was U-shaped, with the anterior and its margins flanked by setae and the posterior margin translucent (Figure 4E,F).The phallus had two elongated phallic endostyles, curved in an arc, slightly transparent at the base, and duck-tongued at the tip (Figure 4C,D), which were nested in the ring-like midstem.
Female external genitalia comprised the ovipositor sheath, spines, and bony needles (Figure 4G-I).The ovipositor sheath was V-shaped with setae on either side of the anterior end, the ovipositor outer wall was ossified with spines at the end, and a membranous structure connected the ovipositor and ovipositor sheath.

Larval Instar Determination
Nassophasis sp.larvae varied in body length from 4500 to 14,100 μm, body width from 990 to 5000 μm, headshell width from 1000 to 2300 μm, and body weight from 0.03 to 0.16 g (Figure 5).Each peak represented an instar frequency analysis of the measured value; results showed that the larvae had three instar groups.The coefficients of variation for body length and head capsule width were less than 20% for all larval ages.However, only the Crosby index was less than 10% (Table 4), indicating that it was reasonable to divide the larvae of Nassophasis sp.into three instars.As the Crosby indices for both body width and body weight were greater than 10%, they were not suitable as indicators for the instar classification of Nassophasis sp.larva.Note: Data are mean ± SE, and different lowercase letters following the data indicate significant differences in the same variable between instars at the 0.01 level by one-way ANOVA test.
Regression analyses with head capsule width and body length against larval instars showed that each larval instar was well-fitted to the measurements (p＜0.0001), with the exponential fit being the best (Table 5).6), with overlapping generations, taking 30-90 days to complete one generation (Figure 6).The overwintering adults gather at the roots of the plant and emerge in mid-March the following year.When the temperature rises, the overwintering adults start to move.It takes about a week for eggs to hatch and the larval periods take 20-70 days.The second generation begins to pupate in mid-July, with a pupal period of 7-9 days.The larvae of the overwintering generation pupate in early September and the adults enter the overwintering stage in mid-November.

M L E M L E M L E M L E M L E M L E M L E M L E M L E M L
Overwintering generation Note: •: Egg; -: Larva; ▲: Pupa; +: Adult; (+): Overwintering adult; and E, M, and L: the early, middle, and the last ten days of a month, respectively.

Infestation Traits and Spatial
Distribution of the Nassophasis sp.

Infestation Traits
Nassophasis sp.adult can feed on dendrobium stems and leaves (Figure 7A,B).After feeding, only the leaf veins remain.During heavy feeding, it can feed on the whole leaf and bore its beak into the stem to feed on the flesh of the leaves, causing pit-like wounds.The mating behavior of Nassophasis sp.adults occurs mainly during the daytime.The adults lay their eggs on the stems of the plant (Figure 7C).Females usually choose to lay eggs near the base of young dendrobium stems (Figure 7D), generally laying only one egg within a stem, rarely two or three, and may lay eggs 2-3 times.Immediately after spawning, it covers the cavity with secretions from the ovipositor.After eggs hatch, the larvae feed and develop inside the stems.At early feeding, it is difficult to recognize an affected plant with the naked eye (Figure 8D(b)).As the level of infestation increases, the leaves of the plant begin to yellow (Figure 8D(c,d)).Then the leaves fall off, and the affected stem becomes hollow (Figure 8D(e)) and dries out over time (Figure 8D(f)).At this point, the larvae can switch plants and cause the whole plant to die in severe cases (Figure 8A).The mature larvae use plant fibers and cavities to build pupal chambers in the stems or rootstocks of the plant for pupation (Figure 8B,C) until the adults have fledged and burrowed out to feed and lay eggs on the nearby plants.

Spatial Distribution
The data revealed aggregation distribution (Table 7).The mean crowding degree ranged from 821.45 to 1443.59 and 340.08 to 1474.37 in forest and shed, respectively.The Lloyd's clustering index and diffusion coefficient (C) were greater than 1.0, which also indicated the aggregation distribution of Nassophasis sp.The clumping index (I) values and cassie index (CA) were all positive, which confirmed the aggregation nature of Nassophasis sp.The mean population density (m) and mean crowding (m*) of the joint sample, according to the Iwao method, were m* = 957.99m + 9.54 and m* = 725.62m -82.94 for Nassophasis sp. in forest and shed, respectively (Figure 9A).The index of basic contagion (α) was positive in the forest planting pattern and negative in the shed planting pattern; the positive value indicated that the populations of Nassophasis sp. in the forest were mutually attracted, and the negative value indicated that the populations of Nassophasis sp. in the shed were mutually exclusion.The slope value β is greater than 1, indicating that populations of Nassophasis sp. are aggregated in both the shed and the forest.According to Taylor's power rule, the linear regression equations for the variance and mean density of Nassophasis sp.samples in the forest and shed were log S 2 = 1.8 log m + 2.95 and log S 2 = 1.66 log m + 2.82, respectively (Figure 9B), where lg a > 0 and b > 1 indicate that Nassophasis sp. were aggregated at all densities under different cropping patterns.The intensity of aggregation increased with the population density.The r values of −0.49 for both the shed and the forest were obtained by the proportional interpolation method, where λ < 2 indicates that the aggregation of Nassophasis sp. was caused by external environmental factors such as plant cultivation mode and climate.

Discussion
There are 92 dendrobium species in China, distributed in 19 provinces [19].Dendrobium is one of the twelve special industries in Guizhou, ranking first in the country regarding the area of imitating wild forests and trees, production, and the output value of its cultivation [20].The large area and high density of dendrobium cultivation have facilitated the gathering, short-distance dispersal, and mating activities of Nassophasis sp.Dendrobium is sold to many places and is very easy to cause the long-distance transmission of Nassophasis sp.during seedling transmission.Therefore, attention should be paid to the introduction of dendrobium seedlings to strictly check whether the seedlings carry eggs, larvae, or pupae, to eliminate the trans-regional dispersion of Nassophasis sp.
The study of the biological characteristics and life history of Nassophasis sp.enriched the theoretical knowledge and provided the basis for its identification and control (Sun et al. 2021).In this study, we conducted field surveys and indoor rearing of Nassophasis sp. in Guizhou Province and evaluated the occurrence and damage of Nassophasis sp. in dendrobium plantations in Guizhou.Nassophasis sp.overwinter as adults, probably due to the susceptibility of eggs, larvae, and pupa to low temperatures.Overwintering adults emerge in late March, consistent with the host's growth period.The occurrence of Nassophasis sp. in Guizhou Province is slightly different from that reported by Gao et al. [9].Firstly, the size of each insect stage of Nassophasis sp. in Guizhou Province is smaller than that of Yunnan.Secondly, the developmental time is shortened, which leads to the difference in the occurrence of generations.It is assumed that Nassophasis sp. has reduced its body size and shortened its developmental period to maximize its population size, thus ensuring its population reproduction.Regarding distribution of Nassophasis sp., the insects were aggregated in the forest and shed, and the intensity of aggregation increased with increasing population density.This distribution pattern is the same as Lissorhoptrus oryzophilus [21], Anthonomus grandis [22], and Cosmopolites sordidus [23].Upon analysis, the aggregation of Nassophasis sp. may be caused by external factors.Many weevils prefer young tissue of the host [24,25], so we hypothesized that the aggregation behavior might be related to the degree of plant development.Nassophasis sp.adults are found to aggregate during overwintering, so we assume that temperature may be another reason for the aggregation behavior.
Larvae were divided into three instars by body length and head capsule width in the current study.According to Dyar's law, there is a certain geometric relationship between the head capsule width of the larvae during each instar; this measured value was used for calculating the Crosby index.The classification of the larvae of Nassophasis sp.into three instars is consistent with Dyar's law.The head capsule width was also considered to be the most suitable instar determination in weevils such as Heilipus lauri [26], Hypothenemus hampei [27], Conotrachelus perseae [28], and Pissodes yunnanensis [29].
There is no effective control strategy for Nassophasis sp., and control measures for most stem-boring pests are usually by injecting chemical reagents such as insecticides into the stem [30,31].However, chemical control is not recommended for pest control in Dendrobium as its flowers, leaves, stems and even whole plants can be used as medicine and food.Moreover, the weevil larvae tend to hide in the stems of Dendrobium; thus, it is hard to detect damage in the early stage.Therefore, understanding the life history cycle of Nassophasis sp. can seize the critical period of control, regulate the egg-laying behavior of the adult weevil, and reduce the number of larval boring.The adults are highly mobile and can fly short distances.In the attachment-tree-cultivation mode, dendrobiums are planted close together, facilitating the breeding and activity of Nassophasis sp.adults.Through indoor and outdoor experiments, we can determine the relationship between Nassophasis sp. and the host plant.Based on observed phenomena of host search and location, we speculate that there is chemical communication between them.The follow-up study will be carried out through research methods in chemical ecology.

Figure 3 .
Figure 3. Morphological differences among females and males of Nassophasis sp.(A) External morphology of male and female.(B) abdomen of male and female.Left: Male; Right: Female.Red boxes indicate male and female differences.

Figure 5 .
Figure 5. Frequency distribution of the (A) body length, (B) body width, (C) head capsule width, and (D) body weight of Nassophasis sp.larvae.

Figure 7 .
Figure 7. Damage symptoms of Nassophasis sp.adult.(A) Adult feeding on stems.(B) Adult feeding on leaves.(C) Adult spawning.(D) Spawning site (red arrow indicates the spawning site).

Figure 8 .
Figure 8. Damage symptoms of Nassophasis sp.larva.(A) Complete drying of dendrobium in an advanced stage of infestation.(B) Larvae infesting stems.(C) Pupal chamber of the mature larva.(D) Dendrobium stems with different levels of larval infestation: (a) healthy stem and (b-f) damaged stem.

Figure 9 .
Figure 9. Linear regression models of the spatial distribution of Nassophasis sp.under different planting patterns.(A) Iwao m*-m regression equation.(B) Taylor's power law.

Table 2 .
Spatial distribution judgment standard of Iwao regression equation.

Table 4 .
Measurements and statistics of instar division of Nassophasis sp.larva.

Table 5 .
The regression equation and coefficient of two morphological variables of Nassophasis sp.
Nassophasis sp.reproduces three generations a year in Guizhou (Table

Table 6 .
Life history of Nassophasis sp.

Table 7 .
Aggregation indices of the spatial distribution of Nassophasis sp.