Since the 19^th century, scientists have been investigating how the organs of insects are shaped and arranged. However, classic microscopy methods have struggled to image these small, delicate structures. Understanding how the organs of insects are configured could help to identify new methods for controlling pests, such as chemicals that target the mouthparts that some insects use to feed on plants.

Most insects that feed on the sap of plants suck out the nutrient via their stylet bundle – a thin, straw-like structure surrounded by a sheath called the labium. As well as drying out the plant and damaging its tissues, the stylet bundle also allows the insect to transmit viruses that cause further harm. To investigate these mouthparts in more detail, Wang, Guo et al. used a method called SBF-SEM to determine the three-dimensional structure of one of the most destructive pests of rice crops, the brown planthopper.

In this technique, a picture of the planthopper was taken every time a thin slice of its body was removed. This continuous slicing and re-imaging generated thousands of images that were compiled into a three-dimensional model of the brown planthopper’s whole body and internal organs. Previously unknown features emerged from the reconstruction, including a huge cell in the planthopper’s abdomen which is full of fungi that provide the nutrients absent in plants.

Next, Wang, Guo et al. used this technique to see how the muscles in the labium and surrounding the stylet move by imaging planthoppers that were frozen at different stages of the feeding process. This revealed that when brown planthoppers bow their heads to eat, the labium compresses and pushes out the stylet, allowing it to pierce deeper into the plant.

This is the first time that the body of such a small insect has been reconstructed three-dimensionally using SBF-SEM. Furthermore, these findings help explain how brown planthoppers and other sap-feeding insects insert their stylet and damage plants, potentially providing a stepping stone towards identifying new strategies to stop these pests from destroying millions of crops.

The morphology and arrangement of organs in the insect body have been studied as early as in the nineteenth century (e.g Lowne, 1892). These data are still used in current textbooks and atlases of insect morphology and physiology. However, the internal structures and their spatial relationship lack a direct three-dimensional (3D) representation. In recent years, morphological studies using micro-computed tomography (micro-CT) served to reveal the internal structures of some insect tissues, such as the copulating Drosophila (Mattei et al., 2015) or even of the entire organism such as the Drosophila pupa (Schoborg et al., 2019). However, for small insects and delicate structures, the resolution of micro-CT is insufficient to distinguish details. Serial block-face scanning electron microscopy (SBF-SEM) allows visualization of the insect’s internal structure at the nanometer resolution in 3D. This method relies on reiterated imaging and sectioning of resin embedded sample using a robotic ultramicrotome and digital assembly of the single images to 3D images (Denk and Horstmann, 2004). Several 3D structures of insect tissues have been resolved by SBF-SEM, such as pericardial nephrocytes in Drosophila (Kawasaki et al., 2019), and cyst-like bodies formed by Trypanosoma brucei in the anterior midgut of tsetse flies (Rose et al., 2020). Insects of small size are suitable for this technology to obtain an integrated 3D atlas of the whole body with a resolution higher than possible by any other previous method. Resolving the internal structures and their spatial relationship is of great value in understanding the relationship of structure and function, and in conducting physiological, biochemical, and molecular studies in this direction. Although reconstruction of a 3D atlas of the whole body of any insect by EM method is valuable and theoretically possible, it has not been conducted yet.

Hemipteran insects are in general small and possess delicate mouthparts with straw-like stylets that are highly specialized for penetrating plant tissues. For virus vectors from the order of Hemiptera, including small/tiny insects like planthoppers, leafhoppers, psyllids, aphids, and whiteflies, studying their phloem sap-sucking mechanism will enable us to understand the way they injure crops and inject their saliva with transmitted viruses into crop tissues. Knowledge about their internal structures such as the mouthparts and alimentary canal of the phloem sap-sucking Diaphorina citri (Hemiptera: Liviidae) (Ammar et al., 2017), Psammotettix striatus (Hemiptera: Cicadellidae) (Zhang et al., 2012), and the aphid Acyrthosiphon pisum (Hemiptera: Aphididae) (Guschinskaya et al., 2020) is restricted to studies that relied on some discrete images by either light, confocal, or electron microscopy. Study of phloem–sucking insect/pathogen interactions represents an exciting frontier of plant science (Jiang et al., 2019). When sucking in plant sap, hemipterans are proposed to protrude their straw-like stylets into the plant tissue by contracting the respective muscles (Leopold et al., 2003; Beardsley and Gonzalez, 1975), but an evidence of the muscle movement has not been provided. Indeed, the commonly assumed feeding mechanism for phloem sap-sucking insects was largely speculative instead of being based on the actual movement of muscles and the feeding apparatuses.

The rice sap-sucking brown planthopper (BPH), Nilaparvata lugens (Stål) (Hemiptera: Fulgoroidea: Delphacidae), is the most important pest of the rice plant. They damage rice not only by directly feeding and ovipositing on it but also by transmitting two rice viruses, rice rugged stunt virus and rice grassy stunt virus. Dense BPH infestations can cause complete drying and wilting of rice plants, a condition known as ‘hopperburn’. Since the late 1960s, BPH outbreaks have been re-occurring approximately every 3 years in Asian countries, with their annual outbreak area amounting to approximately 10–20 million hectares of rice, resulting in millions of tons of losses. BPHs seasonally migrate from their overwintering rice fields in tropical Asia northward to temperate areas in China, Korea, Japan, and northern India when rice becomes available, inflicting damage in an even larger area across Asia (Perfect and Cook, 1994). BPH has been a model insect for ecological research and a model insect for hemipteran insects in biological research in recent years. In this study, we applied SBF-SEM to investigate the structure of various systems involved in food ingestion, digestion, locomotion, gas exchange and perception, and muscle movement during the feeding process of the nymph. A detailed 3D structure for a whole insect is presented for the first time, and a plant phloem sap-sucking model based on the actual movement of muscles and all feeding apparatuses during feeding process is proposed.

The readers are encouraged to read the results along with the interactive 3D images in the 3D PDF file, in which the readers can rotate the 3D, zoom in/out, hide or show any interested internal part of the insect in detail.

3D structures of the whole insect body

The length of the insect measures approximately 600 µm (Figure 1). We reconstructed the 3D structure of the whole insect body (Figure 1, Figure 1—video 1, Figure 1—video 3, interactive 3D PDF file), including the organ systems of food ingestion and digestion, locomotion, gas exchange, and perception. We discover several novel and fascinating internal structures in BPH, such as 24 neuropils in the central nerve system (Figure 1—figure supplement 1), three four-way rings connecting the spiracles in adjacent segments to facilitate efficient gas exchange (Figure 2D,H–J), and fungal endosymbionts in a single huge mycetocyte occupying 22% of the abdominal volume to enable the host to thrive on a low-nutrient diet provided solely by rice (Figure 2G, Figure 2—figure supplement 2D, Table 2).

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The alimentary canal starts with specialized mouthparts and includes the esophagus, the anterior diverticulum (AD) (Figure 2—figure supplement 1D), the midgut (Figure 2—figure supplement 1E), and the hindgut (Figure 2—figure supplement 1F) and ends with the anus (Figure 2—figure supplement 1G). The anterior diverticulum (AD) extends anteriorly into the head (Figure 2A,B, Interactive 3D-PDF). The midgut is divided into an anterior sac and a midgut loop (Figure 2—figure supplement 1A,E). The midgut loop is a narrow tube coiling into a cluster of loops. The anterior part of the loop coils into the inner loop, and the outer loop has its posterior end sticking into the center of the cluster. After that, the midgut descends posteriorly to fuse with the Malpighian tubules (Figure 2B). The inner surface of the whole midgut is covered by a dense layer of microvilli, especially in the loop region where the gut lumen is almost constricted (Figure 2—figure supplement 1E). There is no membrane or sheath enveloping the midgut loop, and the filter chamber, which is a common structure in sap-sucking Hemipteran insects, is not found (Figure 2—figure supplement 4). The anterior midgut contacts the end of the loop region at one point where the epithelia of the anterior midgut become thinner than in the neighboring area (Figure 2—figure supplement 1E). The midgut loop at position ‘3’ is connected to the hindgut.

The 3D reconstruction reveals the distribution of three structurally different endosymbionts in the nymph. The most generally distributed endosymbiont is the Ascomycete fungus Entomomyces delphacidicola (Fan et al., 2015), also known as the yeast-like symbionts (YLSs), which have an ellipsoidal shape from a few microns to over 10 microns (Figure 2G, Figure 2—figure supplement 2D). Most of the YLS reside in a huge fat body mycetocyte that surrounds the midgut, taking up 22% of the abdominal volume and 4.2% of the total body volume (Table 2). A few YLSs are dispersed in the body cavity including the head and legs. Another two fat body mycetocytes adjacent to the midgut (Figure 2C,F) are full of thread-like bacterial endosymbionts. The anterior diverticulum lumen connects the midgut lumen though a narrow region (Figure 2—figure supplement 1B). Anterior to this narrow region, in the wall we found 10 hollow cells that each accommodates numerous rod-like bacterial endosymbionts (Figure 2C,E, Figure 2—figure supplement 2A,B), whereas in the anterior diverticulum lumen there are no endosymbionts. These cells seem to be the specialized structures to host the symbiont bacteria. 16S rDNA sequence of the AD showed that 95% of the symbionts are Arsenophonus species (Figure 2—figure supplement 2E).

The volume of total tracheae in the first instar nymph was 24,000 µm³, accounting only for about 0.1% of the body volume (Table 2). Two pairs of thoracic spiracles (ts1 and ts2), eight pairs of abdominal spiracles (as1–as8), and tracheae extending from the spiracles make up the network (Figure 2D). Every abdominal spiracle is embedded in a pitting called the atrium with two valves, one of which separated the trachea and the atrium, while the other one divides the atrium into two parts (Figure 2—figure supplement 3). All the spiracles are connected by dorsal longitudinal trunks (dlt) (Figure 2D). At the ventral side, there are no longitudinal trunks. Three four-way tracheal rings at the ventral side connect the adjacent two spiracles and their counterparts at the other side of the body, like a traffic circle (Figure 2D,H–J). The ts2–as1 four-way tracheal ring and the as7–as8 four-way tracheal ring connect the spiracles in adjacent segments, as well as the left and right spiracles in the same segment. The as1 four-way tracheal ring connects two tracheae branches from the ts1 spiracle and their symmetrical counterparts at the opposite side.

In addition, we applied the SBF-SEM technique to explore the mechanism of phloem sap-sucking of N. lugens. Here, we focus on the sap-sucking process and the organs involved in food uptake.

Mouthparts

The external morphology of the mouthparts in different Hemiptera is roughly identical. The beak is formed of the labium under a conical labrum (Figure 3E, Figure 3—figure supplement 1C, Figure 3—figure supplement 2A,B). The labium is 130 μm in length with three segments, but only the second (L2) and third segments (L3) are seen from the ventral side, as the first segment (L1) is covered by the labrum (Figure 4A, Figure 3—figure supplement 2A,B). A longitudinal groove on the ventral surface of the labium accommodates the stylet bundle, resulting in a longitudinal lumen. The stylet bundle is in this lumen (Figure 3F). During the feeding process, the stylet bundle protrudes from the apex of the labium (Figure 4B, Figure 3—figure supplement 2A–C).

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Figure 4—source data 1

Measured lengths of the beaks and stylets.

Related to Figure 4C.

https://cdn.elifesciences.org/articles/62875/elife-62875-fig4-data1-v3.xlsx

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The muscles in the labium include three pairs of retractors of the third segment (RT1–RT3), four pairs of lockers of the stylets (LS1–LS4), two pairs of rotators of the second segment (RS1 and RS2), and a pair of rotators of the first segment (RF) (Figure 3E, Interactive 3D PDF). RT1 originates from the lateral region of the L2 and inserts into the floor of the labium groove in the L3. RT2 and RT3 both insert into the apodeme of the L3. RT2 originates on the dorsal wall of the L2, while RT3 originates from the lateral wall of the L2. The four pairs of lockers of the stylets have their origins in the dorsal region of the second labium and insert into the floor of the labium groove on the same segment. RS1 and RS2 originate from the dorsal and lateral regions of the L1, respectively, and both insert into the apodeme of L2 (Figure 3E, Figure 3—figure supplement 1C).

The stylet bundle consists of two mandibular stylets and two maxillary stylets. They emerge from the stylet bases (Figure 3D). As the stylets enter the groove, they become adherent to one another forming a compact bundle. The two maxillary stylets are interlocked by longitudinal ridges and grooves (Figure 3F). The food canal and the saliva canal are formed where the two maxillary stylets meet. The thicker mandibular stylets lie laterally to the maxillary stylets (Figure 3F, Figure 3—figure supplement 2C,D). They encircle the maxillary stylets for tight packaging and at the same time allowing themselves to slide along the longitudinal axis.

Every stylet base is enclosed by a cuticular pouch (MdPC and MxPC) that partly wraps the sheath (Figure 3A,C,D, Figure 3—figure supplement 1A). The mandibular pouch produces two long arms called the mandibular levers (MdL) (Figure 3A,C,D). One of the levers proceeds dorsally, and at the end of the lever are the insertion of two retractor muscles. One (RMdA) originates from the dorsal wall of the head capsule, while the other one (RMdB) extends anteriorly. The other lever extends anteriorly to the mandibular plate. This lever supports most of its length a wide and thin epidermal inflection, on which the protractor of the mandibular stylet (PMd) is inserted (Figure 3A,C, Figure 3—figure supplement 1A,B). The protractor of the mandibular stylet originates in the inner surface of the mandibular plate. The maxillary stylet arises from the maxillary pouch, which is dorsal to its mandibular counterpart. The retractor of the maxillary stylet (RMx) is inserted in the dorsal end of the pouch and has its origin on the dorsal wall of the head capsule, positioned lower than the origin of the RMd (Figure 3C). On the lateral side of the pouch, we discerned the insertion point of the protractor of the maxillary stylet (PMx), which originates on the inner surface of the maxillary plate (Figure 3A).

Salivary glands

Sap-sucking requires the function of the salivary glands that are associated with the mouthparts. These paired organ lies in the anterior part of the thorax, lateral to the alimentary tract (Figure 1A,B). On each side, a principle gland is connected with an accessary gland by the accessary duct (Figure 5A). The efferent salivary ducts arise from the two principle glands and converge anteriorly to form the common salivary duct that transports saliva into the salivary syringe (Figure 5D–I).

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The principle gland is acinar, with nine kinds of follicles (A–I). It is digitate with the nuclei at the periphery of the cell. Follicles C–I are composed of several compact cells with no clear boundary or interspace between them (Figure 5—figure supplement 1). Therefore, cells are difficult to distinguish and count. In follicles A and B, by contrast, the boundaries between the cells are clear. Both include six binucleate cells (A1–A6 and B1–B6) (Figure 5B). The follicles are classified according to the density, size, and electron densities of the vesicles in the cells. Among the nine kinds of follicles, follicle D contains the smallest and the highest number of vesicles, while follicle E contains the lowest number of vesicles (Figure 5—figure supplement 1). Each one of the accessary glands is composed of two symmetrical follicles, and each follicle contains translucent vesicles (Figure 5—figure supplement 1J). Different cell types suggest they may secrete different components of saliva. Ductules from each cell join up into the efferent salivary gland (Figure 5—figure supplement 1L). Follicle H is on a separate branch from other follicles. The two branches merge with the accessary duct into the efferent salivary duct, which is a single layer of cells lined with a thin cuticle (Figure 5—figure supplement 1K). A nerve bundle protrudes from the ventral pharyngeal sensory center, bypasses the accessary duct, penetrates follicles G and A successively, and then bifurcates to two branches at follicle A, finally they terminates in follicle H (Figure 5B).

The salivary syringe lies at the base of the hypopharynx (Figure 5A). Its core component is a pocket-like structure, the salivarium (Baptist, 1941), which connects the common salivary duct, the reservoir, and the anterior salivary duct. A pair of dilators of the salivary syringe are attached on the lateral side of the reservoir.

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 1–5.

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Author details

1. Xin-Qiu Wang

   Institute of Insect Science, Zhejiang University, Hangzhou, China

   Contribution

   Formal analysis, Investigation, Writing - original draft

   Contributed equally with

   Jian-sheng Guo

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-2961-1211

2. Jian-sheng Guo

   Department of Pathology of Sir Run Run Shaw Hospital, and Center of Cryo-Electron Microscopy, School of Medicine, Zhejiang University, Hangzhou, China

   Contribution

   Conceptualization, Investigation, Methodology, Writing - review and editing

   Contributed equally with

   Xin-Qiu Wang

   Competing interests

   No competing interests declared

3. Dan-Ting Li

   1. Institute of Insect Science, Zhejiang University, Hangzhou, China
   2. State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-Products, Key Laboratory of Biotechnology in Plant Protection of Ministry of Agriculture and Zhejiang Province, Institute of Plant Virology, Ningbo University, Ningbo, China

   Contribution

   Investigation

   Competing interests

   No competing interests declared

4. Yang Yu

   Carl Zeiss (Shanghai) Co., Ltd.60 Meiyue Road, China (Shanghai) Pilot Free Trade Zone, Shanghai, China

   Contribution

   Methodology

   Competing interests

   Yang Yu is affiliated with Carl Zeiss (Shanghai) Co., Ltd. The author has no financial interests to declare

5. Jaco Hagoort

   Department of Medical Biology, Amsterdam University Medical Centers, University of Amsterdam, Amsterdam, Netherlands

   Contribution

   Software, Methodology

   Competing interests

   No competing interests declared

6. Bernard Moussian

   Université Côte d’Azur, CNRS, Université Côte d’Azur, Institute of Biology Valrose, Parc Valrose, Inserm, France

   Contribution

   Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-2854-9500

7. Chuan-Xi Zhang

   State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-Products, Key Laboratory of Biotechnology in Plant Protection of Ministry of Agriculture and Zhejiang Province, Institute of Plant Virology, Ningbo University, Ningbo, China

   Contribution

   Conceptualization, Supervision, Funding acquisition, Writing - review and editing

   For correspondence

   chxzhang@zju.edu.cn

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-7784-1188

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