The brown planthopper (BPH) Nilaparvata lugens (Stål) (Hemiptera: Delphacidae), is a destructive insect pest that poses a significant threat to rice production in East Asia. Infestations of BPH can cause severe damage to rice plants by direct feeding and oviposition, leading to the devastating phenomenon known as “hopper burn,” where rice plants wither and wilt under dense BPH populations. Additionally, BPH plays a crucial role in the transmission of rice-ragged stunt virus (RRSV), a pathogen belonging to the Oryzavirus genus in the Reoviridae family (Chetanachit et al. 1978; Ling et al. 1977). RRSV was first identified in Indonesia and the Philippines in 1976–1977 and has since spread to various rice-growing regions in Southeast Asia and southern China, becoming one of the most significant rice pathogens in these areas (Wu et al. 2010). Leaves of infected plants typically exhibit chlorotic yellowish-white stripes, and young leaves may turn completely yellow or white. Moreover, RRSV causes stunting, darker leaf color, leaf distortion, galls on leaf veins, and suppression of flowering (Agrios 2005). The BPH horizontally transmits RRSV in a persistent propagative manner, but vertical transmission through eggs or rice seeds does not occur (Cabauatan et al. 2009). N. lugens acquire the RRSV virus when it feeds on infected rice plants. It initially enters the epithelial cells of the midgut and then progresses to the visceral muscles surrounding the midgut. Subsequently, it disperses throughout the visceral muscles of both the midgut and hindgut, ultimately reaching the salivary glands (Jia et al. 2012). During subsequent sap feeding, the virus present in the saliva is transmitted to the host rice plant (Hogenhout et al. 2008). Despite our current knowledge of the invasion route within the insect body, the precise mechanisms facilitating the transmission of this virus from its insect vector to the rice plant remain largely unexplored. Understanding the distribution of RRSV within the BPH’s foregut is crucial for elucidating vector–pathogen interactions. However, studying these foregut structures poses numerous challenges, primarily due to their scale, intricate internal complexity, and delicate nature, as they are deeply located within the insect’s head. Traditionally, imaging of the foregut and mouthparts in insects has been accomplished using scanning electron microscopy (SEM) to observe surface structures and bacterial localization in the precibarium (Almeida and Purcell 2006). Transmission electron microscopy (TEM) has also been utilized to investigate biofilm interactions with chitinous surfaces within insects (Almeida and Purcell 2006). SEM and TEM require laborious sample preparation and provide only two-dimensional images that must be reconstructed into composite images. The histological sectioning with subsequent staining and light microscopic investigation is probably better suited for studying the distribution of bacteria within a host organism than TEM because, with light microscopy, it is simpler to study larger areas/volumes. Moreover, SEM and TEM are inherently destructive, limiting visualization to a single opportunity and requiring time-consuming, technically challenging, and costly sample preparation.
X-ray computed tomography (CT) imaging has been widely used for medical imaging for decades and has recently been developed as a non-destructive diagnostic imaging tool for a wide range of biological samples. Based on the same principles as those of medical X-ray computed tomography imaging systems, micro-CT is useful for soft and mineralized tissues, with minimal sample preparation required to produce three-dimensional (3D) images with excellent contrast (Brodersen and Roddy 2016). CT has recently been applied in entomology as a means of non-destructively analyzing the internal anatomy of insects. This has been performed in studies on the head structures of Priacma serrata Leconte (Hörnschemeyer et al. 2002), the external and internal morphology of bees and ants (Hymenoptera) (Greco et al. 2008; Greco et al. 2012; Fischer et al. 2016), the wingbeats of blowflies in a synchrotron-based study performing micrometer-resolution, time-resolved microtomography (Walker et al. 2014), the structural characteristics of the earliest lineages of insects (Blanke et al. 2015), and the use of X-ray microtomography images for identification of Pheidole knowlesi species group (Metscher 2009). In recent years, synchrotron radiation X-ray tomographic microscopy (SRXTM) has emerged as a non-destructive imaging tool for a wide range of biological samples. Like medical X-ray computed tomography, SRXTM can produce high-resolution three-dimensional (3D) images with excellent contrast for soft and mineralized tissues, requiring minimal sample preparation (Brodersen and Roddy 2016). As a result, SRXTM has been successfully applied in entomology to non-destructively analyze the internal anatomy of various insect species (Betz et al. 2007; Weintraub et al. 2014), facilitating breakthroughs in the visualization of preserved specimens and real-time internal processes of living organisms (Westneat et al. 2008). In this method, the X-rays generated from the synchrotron light source exhibit significantly higher intensity across multiple orders of magnitude compared to those from X-ray tubes. Additionally, the X-rays emitted from the synchrotron light source maintain a parallel beam geometry. As a result, the detector captures an X-ray projection of the sample at the same size, due to the synchrotron light, whereas the X-ray tube, operating as a fan beam, results in a larger and less defined X-ray projection of the sample.
Herein, we employed SRXTM imaging to focus on the foregut’s cibarium and precibarium in the BPH, aiming to better characterize internal structures and provide insights into vector–pathogen interactions. We identified four key sites in the foregut: the precibarium and food meatus, the cibarium chamber’s interior, and the diaphragm. Leveraging the power of SRXTM, we used SRXTM imaging to specifically target the sites of pathogen and biofilm formation in the foregut of the BPH to better characterize the internal structures and develop a framework for studying these vector–pathogen interactions. We virtually dissected the foregut to determine the BPH’s feeding behavior, using this novel, detailed 3D structure of the entire insect, we proposed a plant phloem sap-sucking model based on the actual movement of feeding apparatuses during the feeding process. This research could open new avenues for understanding vector–pathogen interactions and contribute to more effective strategies for managing this significant agricultural pest and mitigating the impacts of RRSV on rice production in East Asia.