Morphological Anatomy, Developmental Characteristics of the Reproductive System in Arhopalus rusticus (Coleoptera: Cerambycidae) and Their Impacts on the Transmission Potential of Bursaphelenchus xylophilus (Aphelenchida: Parasitaphelenchidae)

Abstract

In recent years, opinions have diverged on whether the oviposition pathway of Arhopalus rusticus (Linnaeus, 1758) (Coleoptera: Cerambycidae) can transmit Bursaphelenchus xylophilus (Steiner & Buhrer, 1934) (Aphelenchidae: Nematoda). Based on biological observations and biochemical index calculations, this study assessed the development degree of the internal reproductive system (morphological soluble total sugar and protein content) and external genital morphology of A. rusticus before and after gnawing pine needles. The study results indicate that A. rusticus developed and matured immediately after eclosion in natural conditions, and it could mate and spawn directly. However, gnawing on pine needles has no significant impact on the development of the reproductive system of both male and female A. rusticus, indicating that this behavior is not a prerequisite for reproductive maturity. Furthermore, through dissection and behavioral observations, it has been determined that the degree of ossification in the ovipositor of A. rusticus is lower than that in the ovipositor of Monochamus alternatus (Hope, 1843) (Coleoptera: Cerambycidae), and its egg-laying method involves only depositing eggs on the surface of a bark, thus confirming that the reproductive behavior of A. rusticus does not facilitate the transmission of B. xylophilus.

1. Introduction

Arhopalus rusticus (Linnaeus, 1758) (Coleoptera: Chrysomeloidae) primarily infests the base and roots of plants from within, causing the weakening and eventual death of host plants. Additionally, it can be spread over long distances via the transportation of logs and wooden packaging [1,2,3]. Bursaphelenchus xylophilus (Steiner & Buhrer, 1934) (Aphelenchidae: Nematoda), often referred to as the “cancer” of pine trees, primarily relies on vector insects for its natural dispersal and can kill infected pine trees in just over 40 days, making it one of the most severe and dangerous forest diseases in China in recent decades [4]. Currently, A. rusticus is widely considered a potential vector insect for B. xylophilus [5,6,7,8]. Although 45 species from five families of Cerambycidae have been confirmed to carry B. xylophilus, the true vectors that are capable of transmitting it in nature all belong to the genus Monochamus (Coleoptera: Cerambycidae) [9]. Some studies have reported that A. rusticus does not carry the pathogenic nematode B. xylophilus, but under natural conditions, it may carry non-pathogenic nematodes such as Bursaphelenchus mucronatus (Mamiya & Enda, 1979) (Rhabditida: Aphelenchidae) on its body surface or in its spiracles [10,11,12], with adult individuals having the highest average nematode-carrying capacity. However, the likelihood of it carrying B. xylophilus is extremely low [13]. Other studies have shown that, under laboratory conditions, A. rusticus can transmit B. xylophilus to pine branches by biting pine needles and can also introduce the pathogen into pine wood segments through oviposition in artificially created wounds [14]. Nonetheless, the role of A. rusticus as a carrier and transmitter of B. xylophilus remains controversial.

In recent years, extensive research has been conducted on the distribution and host selection [15], biological characteristics, occurrence and damage patterns, and management strategies of A. rusticus. Experiments on feeding adult A. rusticus have revealed their behavior of biting pine needles [16], as B. xylophilus typically enters the tree through notches caused by beetle feeding and oviposition. However, it is unclear whether this biting behavior serves to supplement nutrition, promotes subsequent ovarian development, or whether the female genitalia cause wounds to the tree bark.

This study, through field observations, artificial rearing, anatomical examinations, and physiological experiments, compares the reproductive system development of A. rusticus adults that have and have not bitten pine needles to determine whether biting pine needles is necessary for its reproductive behavior. Additionally, the morphological structures of female genitalia and the oviposition behavior of A. rusticus and Monochamus alternatus (Hope, 1843) (Coleoptera: Cerambycidae) were observed to assess the potential of A. rusticus oviposition behavior to transmit B. xylophilus. Clarifying whether A. rusticus is a vector insect for B. xylophilus, as well as its transmission efficiency and pathways, is of great significance for improving the precision and specificity of integrated control measures against pine wilt disease.

2. Materials and Methods

2.1. Experimental Design and Sample Collection

Adult beetles were collected from Shuangdao Forest Farm, Huancui District, Weihai City, Shandong Province, China (37°28′ N, 121°58′ E; altitude 18.84 m), in June 2021. Specifically, beetles were collected from stumps covered with nylon nets in the forest, with collections conducted twice daily to ensure all captured adults were unmated and had not laid eggs prior to capture. The collected adults were transported to the laboratory in 50 mL rearing tubes and reared in a quarantine facility of the laboratory under a permit issued by the Shandong Academy of Forestry, following the protocol described in the Supplementary Appendix of Goble et al. (2015) [16].

The following two housing regimes were implemented: (1) individual rearing: 20 females and 20 males were housed separately in individual 50 mL tubes; (2) paired rearing: 20 male–female pairs were co-housed in individual 50 mL tubes.

Indoor conditions were well ventilated and well lit, with an average temperature of 26 °C. Each housing regime was divided into two feeding treatments: one group received freshly cut black pine branches with leaves (branches replaced and tubes cleaned daily), while the other group was provided only with distilled water-soaked cotton to prevent dehydration. This experiment aimed to examine the effects of housing and feeding regimes on the reproductive performance of Arhopalus rusticus adults.

2.2. Anatomy and Image Acquisition Methods

Female and male adults were selected for dissection 1–10 days post-eclosion. The dissection protocol was performed according to the method described by Yu (2017) with minor modifications [17]. Briefly, their wings, legs, and heads were removed using dissecting scissors. An insect was fixed abdomen side up on a wax dish containing PBS buffer (pH = 7.4). Under a stereomicroscope (Leica, Wetzlar, Germany), the insect body was secured with insect pins, and the thorax and abdomen were incised with a dissecting knife. The body wall was gradually torn open, and the internal reproductive system was picked out with an insect pin. Tracheae and excess fat bodies were removed, and impurities were rinsed off with PBS buffer. The development of reproductive organs in female and male adults of different ages under various rearing conditions was observed, and images were captured using a stereomicroscope (Leica EZ4W, Leica LAS EZ software) [18]. Morphological illustrations were prepared using Procreate.

2.3. Determination of Soluble Total Sugar and Protein Content

The anthrone colorimetry method was utilized to determine the carbohydrate content [19]. Furthermore, the Bradford method was used to assess the protein content [20].

2.4. Analysis Methods

The data processing software used was IBM SPSS Statistics 25, and one-way ANOVA was conducted to analyze the significant differences in the soluble total sugar and protein content in the reproductive systems of male and female A. rusticus affected by whether they nibbled on pine needles. The significance level of the differences was p < 0.05, and highly significant differences were observed at p < 0.01.

3. Results

3.1. Morphological Anatomy of the Reproductive System of A. rusticus

3.1.1. Morphological Anatomy of the Female Reproductive System

Anatomical observations showed that the female reproductive system of A. rusticus (Figure 1) consists of one pair of ovaries, one pair of lateral oviducts, a common oviduct, a copulatory pouch, a spermatheca, a spermathecal gland, a cloaca, and a ovipositor. The ovaries are located below the digestive tracts of the first, second, and third abdominal segments, with the two central ovaries and the enlarged hindgut on the dorsal side, interspersed with Malpighian tubules. The entire ovary has many overlapping and interconnecting ovarian ducts, about 60–120 in number, containing about 50–400 mature eggs; the outside of the ovarian duct is covered with a translucent ovarian membrane, forming a suspensory band made up of the terminal filaments of the ovarian ducts, the endpoints of which are attached to the dorsal muscles of the mid-thorax. Each ovarian duct contains 3–5 large and plump eggs at the lower end, gradually decreasing in size, and 1–4 small and semi-transparent eggs at the upper end, which are immature. The stalks of the two ovarian ducts merge through the ovarian calyx into the lateral oviduct, and mature eggs are extruded from the semi-transparent lateral oviduct, which joins the common oviduct; the two lateral oviducts join in a “Y” shape located on the ventral side of the rectum, with a broad bag-shaped copulatory pouch attached to the lower end of the common oviduct, where fertilized eggs mature and are produced, the spermathecal oviduct is located at approximately one-fifth of the base of the copulatory pouch, the spermatheca being elongated, cylindrical, brown, and partially ossified, with enlargements at the base and tip, and with a semi-transparent tubular spermathecal gland at the bend of the first arcuate end, connecting downwards to the cloaca and finally opening at the cloacal aperture. One part of the gastric spiculum is connected to the ovipositor by muscles, and the other end is connected to the overlapping eighth abdominal segment. During oviposition, the ovipositor extends by the movement of the gastric spiculum, using sensory hairs on the apical process to confirm the oviposition site, and deposits eggs in the crevices on the surface of a tree bark.

3.1.2. Anatomy of the Male Reproductive System

Dissection observations show that the adult male reproductive system of A. rusticus (Figure 2) is distributed on both sides of the abdomen, each side containing one pair of milky-white, flat, spherical testes, which are connected to the lateral vas deferens at the central depression. The vas deferens is connected to the ejaculatory duct, which widens at its middle and lower ends into the seminal vesicle. The seminal vesicle is curved in an S-shape, is milky white in color, and contains a large number of sperm. At the base of the seminal vesicle, there is a membranous, long, and coiled big paragonia gland and a short and thick small paragonia gland. The distal end of the paragonia glands is sealed and contains a transparent content. The base of the two paragonia glands connects to the base of the seminal vesicle and the ejaculatory duct. After crossing each other, the two ejaculatory ducts enter the curved circulator, spiral inside the circulator, and join the external genitalia at the distal end. The external genitalia consists of the median lobe, the tegmen, and internal sac. In its natural state inside the body, the internal sac extends forward, but during mating, it is turned outwards, and the mating orifice of the median lobe protrudes from the body. It enters the female reproductive organ through the female reproductive orifice (as shown in Figure 2, when everted outward).

3.2. The Differences in the Structure of External Reproductive Organs and the Oviposition Methods of the Females of A. rusticus and M. alternatus

3.2.1. Structural Differences in the Female Genitalia

The ovipositor consists of the stylus, coxite lobe, coxite, valvifer, paraproct, and proctiger [21]. The ovipositor shaft of A. rusticus is slender with indistinct segments and one pair of paraproctal shafts; the paraproctal shafts are straight and make up about three-quarters of the ovipositor length; the cercus is apical; proctiger and valvifer shafts are paired; the coxite shaft is shorter than the valvifer shaft and is undivided at the base; the coxite is short and almost not ossified, with sparse setae on both sides; the stylus is apical, inverted-wedge-shaped, and almost not ossified, with sensory setae. The vaginal is membranous, and the genital plate is membranous rectangular, with a length-to-width ratio of nearly 2:1 and extremely sparse setae on both sides of the apex. In M. alternatus, the ovipositor is stout; the paraproct is extremely short, lacking a shaft, and has indistinct segments, lacking paraproctal shafts; the cercus is small and apical; the proctiger is degenerated; the coxite lobe is ossified, about 2.5 mm long; the stylus is apically ossified, semicircular, and sparsely covered with sensory setae; the vagina is long and well developed and strongly folded at the genital plate; a well-developed genital plate is hard, highly ossified, and rectangular, with a length-to-width ratio of 1:1.4, covered with unevenly short setae, which are relatively sparse, and over a large distribution area, a few very long setae are observed at the margin, with an obvious banding pattern (Figure 3).

Comparing the ovipositor structures of A. rusticus and M. alternatus, the ovipositor of A. rusticus is slender and softer than that of M. alternatus. The degree of sclerotization of the genital plate of the eighth abdominal segment is lower in A. rusticus than in M. alternatus, which prevents it from wounding the bark; thus, it lays eggs on the surface of the bark. In contrast, the ovipositor of M. alternatus uses the robust sclerotized structure of the genital plate to deposit eggs deep within grooves.

3.2.2. The Differences in Oviposition Methods Between A. rusticus and M. alternatus

During oviposition, A. rusticus climbs along the trunk, frequently extending its ovipositor to probe the surface of the bark. When it encounters raised cracks in the bark surface, it stops and inserts its ovipositor for probing. It lays eggs mainly in bark cracks and branch scars (Figure 4). On lifting the peeled bark at the oviposition site of the host, eggs can be seen, arranged in a single layer or occasionally in a double layer in a radial pattern (Figure 5). The eggs are initially soft and then gradually harden. After 3–10 days, the larvae emerge from the eggs, crawl into deep crevices, and slowly invade the trunk (Figure 6). M. alternatus uses its antennae, labial palps, and mandibular palps to lightly touch the surface of the branch to select a suitable oviposition site. It then makes grooves in the bark with its mandibles, turns its body by 180°, flexes its abdomen, and uses its sclerotized structure to insert the ovipositor into the groove, depositing the eggs deep inside the groove [22].

3.3. The Development of the Ovaries of Adult Female A. rusticus at Different Ages

When the reproductive system of female A. rusticus was dissected at different ages, it was found that the fat content of the female body gradually decreased with increasing age. Observations made up to the death of the females showed no obvious changes in their ovaries. The number of mature eggs in their ovaries did not increase with age, regardless of whether they were feeding on pine needles and mating or not. No further development of immature eggs in the lateral oviducts of the ovaries was observed (Figure 7).

Based on anatomical observations, the morphological differences in the ovaries are related to whether mating and oviposition occur. In unmated females, the mating pouch is shrunken, and the ovary tubes are full, with 7–8 eggs in each tube. The 2–4 eggs that are closest to the oviduct are milky white, while those further away are smaller and semi-transparent. In mated females, the ovipositor becomes much larger and contains a large number of mature eggs and their content. After oviposition, the number of mature eggs in the ovary tubes decreases markedly, forming a semi-transparent cavity in the calyx region, and the 2–4 immature eggs near the terminal filaments show no further development (Figure 8).

3.4. Changes in Total Soluble Sugar and Protein Content of Ovaries Before and After Gnawing

Table 1. Concentration (mg/g) of total sugars and proteins in female and male tissues of adult A. rusticus (mean ± SD).

Tissues and Organs	Total Carbohydrates	Total Proteins
Ovary before gnawing	0.9415 ± 0.0892	24.0092 ± 18.1527
Ovary after gnawing	0.8526 ± 0.0949	19.7379 ± 7.1393
Male reproductive system before gnawing	0.2390 ± 0.1297	4.8818 ± 1.5119
Male reproductive system after gnawing	0.2420 ± 0.2623	6.4472 ± 1.7081

presents the total carbohydrate and protein content in the reproductive systems of adult female and male A. rusticus before and after pine needle consumption. The results of the between-subjects effects test via two-way ANOVA revealed that the feeding status had no significant effect on either the total carbohydrate content (F = 0.2161, p = 0.6544 > 0.05) or the total protein content (F = 0.0574, p = 0.8167 > 0.05) in the insect reproductive system. In contrast, sex exerted an extremely significant effect on the total carbohydrate content (F = 50.4297, p = 0.0001< 0.001) and a significant effect on the protein content (F = 8.1694, p = 0.0212 < 0.05) in the insect reproductive system.

4. Discussion

The spread of pine wood nematodes relies mainly on vector insects. To complete the “infected tree–insect–healthy tree” transfer parasitism, vector insects must fulfill two conditions—they must be able to carry the pine wood nematodes and transmit them to host plants. In countries such as China, Japan, and South Korea, the main vector insect for pine wood nematodes is M. alternatus; however, in northern Japan and the central and northern regions of South Korea, the main vector insect is Monochamus saltuarius Gebler. M. alternatus can significantly increase the lifespan of adults and the number of offspring [23,24] by feeding on tender pine needles as a dietary supplement [25]. M. saltuarius needs to feed on the current-year or 2-year-old pine barks after eclosion to promote ovarian maturation and successfully exhibit its reproductive behavior [26]. After mating, the female beetles of both species create ovipositor pits in dying bark, depositing eggs under the bark and leaving circular ovipositor holes in the center of the pits [27]. Female M. saltuarius also secretes a gelatinous substance to seal the burrows [28]. The feeding and egg-laying activities of the beetles create wounds that allow the pine wood nematodes they carry to enter the tree trunk, leading to the spread of pine wood nematode disease [29]. There are two main reasons for controlling A. rusticus: firstly, as a stem-boring pest, the larvae have a large appetite and cause damage by burrowing into the trunk, particularly below 1 m, weakening the tree to the point of death once infested [30]; secondly, A. rusticus has been shown to be highly capable of transmitting B. mucronatus, a close relative of pine wood nematodes [31]. In addition, both A. rusticus and the known vector insect M. alternatus [10] share similar geographical distributions and ecological niches [29], suggesting the potential role of A. rusticus as a vector of pine wood nematodes. Research has shown that A. rusticus primarily attacks weakened trees, especially those damaged by fire, but it generally does not affect healthy trees [32,33]. This study investigates whether A. rusticus has the potential to transmit pine wood nematodes by examining its reproductive system and the development and oviposition behavior of its male and female individuals.

The characteristics of male and female reproductive organs are used in the classification of Coleoptera insects, and the adult reproductive system of A. rusticus is basically the same as that of other longhorn beetles. Specifically, insect ovarioles are classified into three main types (panoistic, meroistic polytrophic, and meroistic telotrophic) based on the presence and distribution of nurse cells, and the female A. rusticus possesses telotrophic meroistic ovaries, consisting of multiple telotrophic ovarioles, where nurse cells are concentrated at the apical germarium and connect to developing oocytes via nutritive chords to facilitate the polarized transport of cytoplasm and macromolecules, a characteristic consistent with the ovarian structure of Cerambycidae species. Unlike adult Coleoptera such as M. alternatus and Apriona germari, which require additional nutrition to develop their ovaries into a mature state after eclosion [15], there were no significant differences in the morphology and color of the female reproductive system (ovaries, spermathecae, and accessory glands) and male reproductive system (testis, accessory glands, and seminal vesicles) of A. rusticus of different ages regardless of whether they had consumed pine needles under laboratory feeding conditions. Ovarian morphological differences are related to mating and oviposition; after mating, the mating pouch of female beetles enlarges significantly with a large number of mature eggs and content, and the number of mature eggs in the ovary tubes decreases significantly after oviposition. Immature eggs near the tip of the ovary do not develop further due to age, pine needle consumption, or mating until the female beetle dies. There were also no significant differences in the total soluble sugar and protein content in the male and female reproductive systems of A. rusticus before and after pine needle consumption. By observing the development and structure of the internal and external reproductive organs of A. rusticus, and by detecting the nutritional components of the internal reproductive system, it was demonstrated that A. rusticus is sexually mature after eclosion and can mate and oviposit without the need to consume pine needles, and that they subsequently do not promote the development of immature eggs through nutritional supplementation. This is consistent with the work of other researchers [1,16] who found that A. rusticus is sexually mature after eclosion, it does not require additional nutrition for mating and oviposition, and its eggs hatch normally. Compared to the known vector insect M. saltuarius, the reproductive plate of A. rusticus is a soft membranous structure. In addition, observations made in the forest showed that A. rusticus usually lays its eggs under the outer layer of a tree bark at the base of the trunk without making grooves in the bark or damaging the inner bark; moreover, if the eggs are attached to the pine wood nematode, the nematode cannot enter the tree. Meanwhile, only a small number of adults showed feeding behavior during the experimental process, and this behavior only occurred in a relatively small and confined survival space (a 50 mL rearing tube); no feeding behavior was observed when beetles were collected in the field.

In conclusion, evidently, adult A. rusticus does not need to promote sexual maturation by feeding and does not spread pine wood nematodes by oviposition under natural conditions. This finding directly indicates that A. rusticus will not act as a transmission vector of pine wilt disease in natural environments. While previous studies have reported that the hatching rate of eggs laid by adult A. rusticus differs when adults feed on different food sources, the specific mechanisms underlying this variation remain unclear [24]. In view of this conclusion, combined with the study results, it remains to be investigated whether the feeding behavior of adult A. rusticus on pine needles is a supplementary feeding behavior. From a practical application perspective, our findings provide important implications for the prevention and control of pine wilt disease: there is no need to implement root excavation or stump crushing measures targeting A. rusticus in routine pine wilt disease management. For risk-averse scenarios, such targeted measures may be selectively applied in early-stage quarantine zones where pine wilt disease has just invaded, to achieve precise and efficient disease prevention. Looking ahead, future research could focus on conducting long-term field monitoring to verify the consistency between the laboratory-derived non-vector role of A. rusticus and its actual performance in complex forest ecosystems, which can further optimize the integrated management strategy of pine wilt disease.