The venom‐specific toxicity profiles of the mirid bug Tytthus chinensis to its prey insect Nilaparvata lugens

Abstract

BACKGROUND

Tytthus chinensis, a predatory mirid, is an effective natural enemy in rice ecosystems. Although this species utilizes venomous saliva to immobilize and digest prey, the functional specialization and insecticidal mechanisms of its salivary gland venom remain unclear.

RESULTS

In this study, we characterized the anatomy of the venom apparatus and evaluated the toxicity of its distinct salivary glands. The venom system comprises an anterior main gland (AMG), a posterior main gland (PMG) and an accessory gland (AG). Bioassays using venom extracted from each gland demonstrated that AG venom exhibited the highest toxicity, significantly reducing insect survival and eclosion rates, and also severely impairing sperm production and fecundity. AG venom elicited marked oxidative stress in insects, as evidenced by elevated antioxidant enzyme activities, increased lipid peroxidation, and decreased glutathione levels. Quantitative (q)PCR results further confirmed the high enrichment of toxin‐related genes in AG, such as venom‐like allergen 5, serine protease inhibitors and ω‐toxin‐like. By contrast, plant‐defense related genes were highly expressed in AMG and PMG tissue.

CONCLUSIONS

Our findings demonstrate a clear functional specialization among the salivary glands of T. chinensis and identify AG venom as a critical factor mediating prey paralysis, oxidative damage and reproductive suppression. This work provides a foundation for developing novel bio‐insecticides derived from predator venom components. © 2026 Society of Chemical Industry.

The salivary gland venom system of Tytthus chinensis exhibits clear functional specialization, with different glands mediating prey toxicity and plant defense modulation.

INTRODUCTION

Predatory Heteroptera are widely recognized as biological control agents that contribute to the regulation of pest populations in agricultural systems. ¹ Predatory heteropterans employ venom secreted from specialized venom glands (also referred to as salivary glands) to immobilize prey rapidly and initiate extraoral digestion. ² , ³ To date, the venom glands of >20 species of predatory heteropteran insects, including assassin bugs (family Reduviidae), ⁴ , ⁵ minute pirate bugs (family Anthocoridae), ⁶ and true water bugs (infraorder Nepomorpha), ⁷ have been investigated at multiple levels, including transcriptomics, proteomics and functional biology. The identification of venom components in predatory heteropterans is essential for elucidating their insecticidal mechanisms and evaluating their potential for pest control. Venom is a complex mixture composed of salts, small molecules, peptides and proteins. ⁸ Various venom‐associated proteins have been identified in heteropteran venoms, including serine proteases, cysteine proteases, arginine kinases and anti‐inflammatory proteins. ⁹ Additionally, disulfide‐rich peptides belonging to the Ptu1 family were detected in the venom of the assassin bug Peirates turpis and were shown to block calcium channels. ¹⁰ Venom secreted by the venom glands can rapidly induce prey paralysis, tissue liquefaction, and death, thereby enabling efficient predation and extraoral digestion. ¹¹ For example, Triatoma vitticeps (Heteroptera: Triatominae) are hematophagous species that feed exclusively on blood, and their saliva possesses local anesthetic properties, enabling stealthy feeding. ¹² For instance, the venom of the predatory stink bug Arma custos (Hemiptera: Asopinae) induces paralysis, tissue liquefaction, and death in Spodoptera litura. ¹³ Heteropteran insects constitute an ideal model group for studying venom evolution. They evolved from phytophagous ancestors, with the key biochemical adaptation of modifying salivary secretions into venom. ⁹ , ¹⁴ The evolution of venom was accompanied by the emergence of specialized delivery systems, including venom‐producing tissues (collectively referred to as the venom apparatus) and associated delivery structures. ¹⁵ The venom apparatus comprises distinct glandular components that secrete different venom fractions and typically occur in paired structures, including the anterior main gland (AMG), posterior main gland (PMG) and accessory gland (AG). ¹⁶

Numerous insect species are omnivorous, consuming resources across multiple trophic levels, from strictly herbivorous to carnivorous species, and some species exhibits both facultative zoophagy and phytophagy. ¹⁷ , ¹⁸ For example, species such as Apolygus lucorum, Nesidiocoris tenuis and Macrolophus pygmaeus exemplify this mixed feeding behavior. ¹⁹ , ²⁰ During brief periods of prey scarcity, phytophagy provides nutritional support for predatory insects or compensates for low‐quality prey, yet it cannot fully replace zoophagy. ²¹ , ²² In both zoophagous and phytophagous feeding, the salivary secretions of hemipteran insects play essential roles in extra‐oral digestion and offer insights into the underlying mechanisms. ²³ In this context, the morphology and histology of salivary glands in various predatory Hemiptera have been examined across multiple species. ²⁴ , ²⁵ Although these studies have provided valuable insights into gland morphology, the physiological processes occurring within these glands remain limited. Further investigations are required to elucidate the structural characteristics, secretory composition and biological functions of these glands.

Rice (Oryza sativa) is one of the most important staple crops worldwide, serving as the primary food source for over half of the global population. ²⁶ The brown planthopper (BPH), Nilaparvata lugens, is one of the most destructive insect pests affecting rice across Asia. This pest causes substantial economic losses both directly, by feeding on rice phloem sap and weakening plants, and indirectly, by transmitting viral diseases that seriously threaten rice production. ²⁷ Owing to insecticide overapplication, BPH has emerged as a persistent and devastating pest in rice agroecosystems. ²⁸ , ²⁹ Despite the deployment of resistant rice varieties, extensive pesticide applications and integrated pest management strategies, N. lugens populations continue to thrive. ³⁰ , ³¹ Natural biological control is considered a key strategy for preventing BPH outbreaks and ensuring sustainable pest management in rice ecosystems.

Insect predators are regarded as environmentally friendly alternatives to chemical insecticides. The mirid bug Tytthus chinensis is an important natural enemy in rice ecosystems that predominantly preys on the eggs of planthoppers, and is broadly distributed across rice‐growing regions of China and Southeast Asia. ³² T. chinensis exhibits greater thermal tolerance, ecological adaptability and predatory efficiency than Cyrtorhinus lividipennis under high‐temperature conditions. ³³ The utilization of T. chinensis as a biological control resource is therefore considered critical for BPH management. To investigate the functional role of venom in T. chinensis, the AMG, PMG and AG were dissected, and their secretions were individually collected. The differential toxicities of three glandular venoms on BPH were examined, with particular focus on mortality, reproductive disruption and oxidative stress. Our findings enhance understanding of the predatory mechanisms of T. chinensis and establish a foundation for discovering bioactive molecules with potential applications in environmentally sustainable pest control. This work provides the first functional evidence of gland‐specific venom toxicity in T. chinensis and underscores its potential as a promising biocontrol agent against rice planthoppers.

MATERIAL AND METHOD

Insects

Tytthus chinensis was kindly provided by Yangzhou University in 2022. N. lugens, which was originally collected from a rice field in Nueva Ecija, the Philippines, in 2004, has since been continuously maintained in the laboratory without exposure to insecticides. Both insect species were reared on the rice variety Xiushui 11 in growth chambers maintained at 27 ± 1 °C, 65 ± 5% relative humidity (RH) and a 16 h:8 h, light:dark photoperiod. Nicotiana benthamiana seeds of the Ben's variety were provided by the Department of Plant Pathology, Nanjing Agricultural University. The growth environment temperature was 24 ± 1 °C, with RH and photoperiod conditions as described above. Plants were grown for 4–6 weeks for Agrobacterium‐mediated transient gene expression experiments.

Gland dissection

Before dissection, female T. chinensis were anesthetized on ice for 30 min within 24 h of emergence. The junction between the abdomen and thorax was gently separated with forceps to expose the salivary gland complex. The head was grasped with fine forceps to isolate the salivary gland complex, which was subsequently placed in 1× phosphate‐buffered saline (PBS) under a dissecting microscope. The salivary gland complex was divided into three main regions (PMG, AMG and AG) that were carefully isolated with dissecting needles and forceps, and immediately stored at −80 °C. The isolated tissues were used for transcriptome sequencing and quantitative real‐time (qRT)‐PCR analyses. Images were captured with a MPS 30 camera system (Leica Microsystems, Wetzlar, Germany).

Gland venom collection

The AMG, PMG and AG were dissected from female adults 2 days after the final molt following the above‐mentioned protocol. After washing with PBS more than three times, glands were transferred into a microcentrifuge tube containing 120 μL PBS. To release venom into PBS, the glands were pierced using a sterilized needle. The microcentrifuge was then centrifuged at 12000g for 10 min at 4 °C, and the crude venoms in the resulting supernatant were collected. The protein content of venom samples was determined using a Protein Assay Kit (Bio‐Rad, Hercules, CA, USA).

Toxicity determination of gland venoms

Crude venom was diluted in PBS to concentrations of 0.25, 0.5, 1.0 and 2.5 μg μL⁻¹, with PBS serving as the negative control. The 3^rd‐instar N. lugens nymphs were microinjected with 200 nL of each solution using a Nanoject 4 microinjection device (MicroSyringe Pump Controller; World Precision Instruments, Sarasota, FL, USA). Five biological replicates per concentration (30 nymphs per replicate) were set up, and mortality was assessed at 5 days postinjection (dpi). Survival curve was monitored for 8 days for microinjection at 2.5 μg μL⁻¹ concentration. Morphological abnormalities were documented photographically.

Spermary dissection and sperm count analysis

Spermary (SPM) dissection and sperm count was performed according to our previous study. ³⁴ Newly emerged (<24 h) and unmated N. lugens male were anesthetized on ice for 30 min before dissection. Using fine forceps under a stereomicroscope, reproductive tissues were excised in 1× PBS. The vas deferens was carefully isolated from SPM samples, and then subjected to the following treatments: triple washing in 1× PBS, fixation in 4% paraformaldehyde (PFA) for 1 h at room temperature, three additional PBS washes, permeabilization with 0.1% Triton X‐100 (30 min), final PBS washing cycle, and nuclear staining with DAPI (10 min). Confocal imaging was performed using a TCS SP8 system (Leica Microsystems).

N. Lugens reproduction assay

One hundred treated 3^rd‐instar nymph were collected randomly from control and venom‐treated population, and reared under a 16 h:8 h, light:dark photoperiod. The emerged male and female were collected every day and paired for mating in each separate glass tubes (diameter 4 cm, height 16 cm) with fresh rice seedlings. When offspring neonates appeared, the tubes were checked every day, and the neonates were counted for 7 days after mating. The rice shoots were then checked thoroughly and the number of unhatched eggs was counted under a stereoscope. The ovary was dissected from females, and eggs retained in each ovary were counted. The number of eggs laid by a female into rice shoots, the number of eggs retained in a female ovar, and total egg number per female were calculated. Egg number was recorded in 42 females. The hatchability was recorded as offspring /total eggs. Each treatment was replicated five times.

Tissue collection, RNA sequencing and bioinformatic analysis

Salivary glands, digestive tracts and remaining body tissues were dissected from 200 adult T. chinensis individuals and immediately frozen in liquid nitrogen. Total RNA was extracted as described below, and transcriptome sequencing was performed using the BGISEQ‐500 platform (BGI, Wuhan, China). Protein domain prediction was conducted using the Pfam database. Signal peptides were predicted using signalP v5.0 and potential transmembrane domains were identified using TMHMM v2.0. Based on the transcriptome data, gene expression levels were calculated as fragments per kilobase of exon model per million mapped reads (FPKM) and visualized in tissue‐specific heatmaps. Log₂‐transformed FPKM values [log₂(FPKM +1)] were used as input for heatmap construction.

Agrobacterium‐mediated transient expression in N. benthamiana

Candidate salivary protein genes were cloned into the pBINGFP2 vector. Constructs were introduced into Agrobacterium tumefaciens strain GV3101 by electroporation. The resulting recombinant strains were grown in Luria–Bertani (LB) medium containing appropriate antibiotics at 28 °C with shaking at 200 rpm for 24 h. Cells were subsequently collected by centrifugation at 5000g for 5 min and resuspended in infiltration buffer (10 mm MES, pH 5.7, 10 mm MgCl₂ and 150 mm acetosyringone). The bacterial suspension was adjusted to an optical density at 600 nm (OD₆₀₀) of 0.6 for the cell death assay and incubated in the dark at 28 °C for 3 h. Subsequently, the suspension was infiltrated into the leaves of N. benthamiana plants using a needleless syringe for transient expression.

Quantitative real‐time (qRT)‐PCR analysis

Ten nymphs were collected per replicate, with four replicates prepared for each test. Total RNA was extracted from T. chinensis using TRIzol reagent (Invitrogen/Thermo Fisher Scientific, Waltham, MA, USA). The RNA quality and concentration were evaluated by spectrophotometric analysis using a NANODROP 2000 (Thermo Fisher Scientific). cDNA synthesis was subsequently performed with HiScript qRT SuperMix (Vazyme Biotech, Nanjing, China). qRT‐PCR analysis was conducted using ChamQ SYBR qPCR Master Mix (Vazyme Biotech, Nanjing, China) on a QuantStudio Real‐Time PCR System (Applied Biosystems/Thermo Fisher Scientific). Primers used in qRT‐PCR for the tissue‐specific expressions of seminal fluid protein genes are given in Table S2. Relative expression levels were calculated using the 2^−ΔΔCt method.

Assays of oxidative stress responses in N. lugens treated by venoms

The 3^rd‐instar nymphs were treated by different gland venom at the concentration of 1 μg μL⁻¹ or control solution. At 5 days following venom, the nymphs (in the 5^th‐instar) were collected. The oxidative stress responses were evaluated with activities of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione‐S‐transferase (GST) and glutathione peroxidase (GPx). The substance levels were determined for lipid peroxidation (LPO), glutathione (GSH), reactive oxygen species (ROS), reactive oxygen and nitrogen species (RONS) and nitric oxide (NO), RNOS (H), and NO. The assays were carried out as previous study by Yu et al. ³⁵ and Adedara et al ³⁶ Insects were homogenized in 0.1 m phosphate buffer (pH 7.4) at 4 °C and then centrifuged at 6000g for 10 min at 4 °C. The supernatant was collected and proteins were quantified using Pierce™ Bradford protein assay kit (Thermo Fisher Scientific). The lysates were diluted to a concentration of 1 mg mL⁻¹, which was then used for biochemical reaction for antioxidant enzymes. The activity of SOD was measured at 480 nm as nmol epinephrine oxidized min⁻¹ mg⁻¹ protein. The activity of CAT was measured at 240 nm as mol of H₂O₂ consumed min⁻¹ mg⁻¹ protein. The activity of GST was assayed at 340 nm as μmol CDNB‐GSH complex generated min⁻¹ mg⁻¹ protein. The activity of GPx was measured at 412 nm as μmol remaining GSH mg⁻¹ protein. The level of GSH was measured at 412 nm as unit mg⁻¹ protein. The level of LPO was measured with Lipid Peroxidation MDA Assay Kit (Abcam Trade Co. Ltd, Shanghai, China) at 532 nm as μmol MDA produced mg⁻¹ protein. For levels of ROS and RNOS, the fluorescence was measured at Ex/Em of 485/535 nm and of 488/525 nm using an Oxidative Stress Combo Assay Kit (Shanghai Jinpan Biotech Co. Ltd, China). The level of NO was spectrophotometrically measured at 540 nm as μm nitrite mg⁻¹ protein.

Statistical analysis

One‐way ANOVA analysis was used for differences among three or more groups. Results were presented as means ± SEM based on at least three independent biological replications. Data analyses were performed using prism v9.0 (https://www.graphpad.com/features).

RESULTS

Morphological structure of the salivary gland complex in T. chinensis

The salivary gland complex of T. chinensis is composed of a pair of principal salivary glands and a pair of spherical accessory glands. The glands are translucent and situated in the thoracic region, extending from the prothorax to the metathorax. The accessory glands are connected to the alimentary canal [Fig. 1(A)]. Ultrastructural observations revealed that the principal salivary glands are differentiated into a large PMG and a small AMG. A constriction is present between the two lobes of the principal gland, forming a connection between the main duct and the mouthparts. The accessory glands are generally spherical and enveloped by numerous small tubules [Fig. 1(B)]. The salivary duct is linear and projects anteriorly toward the mouthparts [Fig. 1(C)].

Figure 1.

Morphological characterization and schematic representation of the salivary glands of the mirid bug T. chinensis. (A) Anatomical view of salivary gland; (B) Isolated AG, AMG and PMG; (C) Schematic representation of salivary gland architecture.

Effects of T. chinensis salivary gland venom on N. lugens

After treatment of the 3^rd‐instar nymphs of N. lugens with crude glandular venom from T. chinensis for 8 days, the mortality of the treated groups (AMG, PMG and AG) was significantly higher than that of the control group. No significant difference in mortality was observed between the AMG‐ and PMG‐treated groups, whereas AG‐treated nymphs exhibited a significantly higher mortality rate [Fig. 2(A)]. The mortality of nymphs treated with AMG, PMG and AG venom increased markedly with rising venom concentrations. For AMG venom, the mortality rate increased significantly at 0.5 μg μL⁻¹ and reached its maximum (36.1 ± 4.1%) at 2.5 μg μL⁻¹ [Fig. 2(B)]. For PMG venom, the mortality increased significantly at 1 μg μL⁻¹ and reached a maximum of 33.2 ± 3.7% at 2.5 μg μL⁻¹ [Fig. 2(C)]. By contrast, AG venom exhibited a more pronounced toxicity, with mortality significantly increasing at 0.5 μg μL⁻¹ and gradually rising with concentration, reaching 75.3 ± 4.2% at 2.5 μg μL⁻¹ [Fig. 2(D)]. Moreover, nymphs surviving AG venom treatment displayed distinct morphological deformities, characterized by an underdeveloped left thorax, incomplete molting of the mid‐abdominal segments and retained exuviae on the right side [Fig. 2(E)].

Figure 2.

Toxicity of venoms from different T. chinensis glands to its prey N. lugens. (A) Survival curve of the 3^rd‐instar N. lugens nymphs treated with crude venom at 2.5 μg μL⁻¹. The lowercase letters at the left site of curves indicate significant difference at 0.05 level. (B—D) Toxicity of crude venoms from AMG, PMG and AG at different concentrations against the 3^rd‐instar nymphs. The mortality was recorded in the 5 days following treatment. Data represent means ± SEM of five biological replicates, and different lowercase letters indicate significant difference at the 0.05 level. (E) Morphological deformities in surviving nymphs treated with AG crude venom. Blue arrows indicate maldeveloped thorax (left), incomplete abdominal ecdysis (middle) and retained exuviae fragment (right).

Effects of AG venom on sperm quantity in N. lugens

This test examined the effect of the AG venom from T. chinensis on sperm quantity of N. lugens. Unmated male adults were dissected, and the seminal vesicles were ruptured to release sperm. The released sperm were diluted in physiological saline to facilitate counting. The filamentous, rather than typically spherical, morphology of the sperm nucleus is a common and typical feature in insects. During spermatogenesis, chromatin undergoes extensive condensation and structural remodeling, with histones being partially or completely replaced by sperm‐specific proteins, leading to a highly compacted DNA state. Meanwhile, the nucleus elongates significantly along the longitudinal axis of the sperm, forming a slender, linear or filamentous structure. DAPI‐stained images of sperm nuclei clearly showed that the sperm count in AG venom‐treated males was significantly reduced compared with the control [Fig. 3(A)–(F)]. Following treatment with different glandular venoms, no significant changes in sperm quantity were observed in males treated with AMG or PMG venom compared with the control. However, AG venom treatment resulted in a significant reduction in sperm number [Fig. 3(G)].

Figure 3.

Sperm quantification in unmated N. lugens males and venom‐induced effects on sperm count. The sperm count was performed in the newly emerged (<24 h) unmated N. lugens male. The venom concentration was 1 μg μL⁻¹. (A, D) Bright‐field images of dispersed sperm from control (A) and AG venom‐treated (D) males after dilution. (B, E) Corresponding DAPI‐stained nuclei of sperm from control (B) and treated (E) groups. The sperm nucleus of N. lugens is elongated, exhibiting a slender linear or filamentous structure. (C, F) Merged bright‐field and fluorescence images for control (C) and treated (F) samples. (G) Quantitative analysis of venom treatment effects on sperm count. Data represent means ± SEM of eight biological replicates, and different lowercase letters indicate significant difference at the 0.05 level.

Effects of gland venoms on fecundity of N. lugens

After N. lugens females were treated with AMG, PMG or AG venom, the eggs per female were counted. No significant difference was observed in the number of eggs laid inside rice stems between the AMG‐ or PMG‐treated groups and the control. However, the number of eggs laid by AG venom‐treated females was significantly lower than that of the control group [Fig. 4(A)]. The number of retained eggs in the ovaries of AG venom‐treated females was significantly higher than that of the control, whereas AMG and PMG venom treatments showed no significant difference [Fig. 4(B)]. Regarding total egg production (laid eggs + retained eggs), no significant difference was observed between the AMG‐ or PMG‐treated groups and the control, whereas AG venom treatment significantly reduced total egg production [Fig. 4(C)]. In terms of egg hatchability, the hatching rate of eggs from AG venom‐treated females was significantly lower than that of the control group, but was not significantly different from those treated with AMG or PMG venom [Fig. 4(D)].

Figure 4.

Effect of gland venoms on the reproduction of N. lugens females. The egg number was recorded at 7 days after mating. The venom concentration was 1 μg μL⁻¹. (A) Number of eggs deposited in rice stem. (B) Retained eggs in female ovaries. (C) Total egg production (deposited + retained). In (A–C)，Data was collected from 42 females per treatment, and different lowercase letters indicate significant differences at 0.05 level. (D) Egg hatchability. Data represent means ± SEM of five biological replicates, and different lowercase letters indicate significant difference at the 0.05 level.

Identification of candidate salivary gland genes by transcriptome analysis

Illumina RNA sequencing was performed on nine samples from different tissues (salivary gland, digestive tract and remaining body), generating an average of 6.63 Gb of clean data per sample. The average mapping rate of the gene set was 83.92%. Sequencing quality assessment showed that Q20 scores exceeded 97% and Q30 scores exceeded 93%, indicating a high quality. Transcriptomic analysis identified 110 candidate salivary gland genes characterized by the presence of signal peptides, absence of transmembrane domains, FPKM>50 and high expression in salivary glands (|log₂ fold‐change| > 2). ‘TC’ is an abbreviation for T. chinensis. The candidate genes encoding salivary proteins were named TC1–TC110 based on their expression levels, ranked from high to low. Transient expression assays in N. benthamiana further revealed that four salivary proteins induced necrotic symptoms [Fig. 5(A)]. Notably, TC55 was found to suppress the defense responses in N. benthamiana [Fig. 5(C)]. Fourteen genes were selected based on known functional annotations of venom or homology, as well as their representativeness across functional categories, to explore their functional distribution in the AMG, PMG and AG [Fig. 5(B)].

Figure 5.

Identification and functional analysis of candidate salivary proteins in T. chinensis. (A) Transient expression of selected candidate salivary proteins in N. benthamiana leaves. TC1, TC7, TC19 and TC26 induced visible necrotic symptoms at the infiltration sites. INF1, an elicitor secreted by Phytophthora, was transiently expressed using the pBI121‐INF1 vector and used as a positive control to induce hypersensitive response cell death in N. benthamiana, whilst GFP was used as a negative control. (B) Heatmap showing the expression profiles of candidate salivary protein genes across different tissues. Gene expression levels were calculated based on transcriptome data and normalized as Z‐scores. Columns represent biological replicates from the salivary gland (SG), remaining body tissues (RO) and digestive tract (CD), whereas rows correspond to individual candidate genes. Hierarchical clustering was performed based on gene expression patterns. (C) TC55 suppresses the defense responses in N. benthamiana.

Functional differentiation of venom‐related genes among salivary glands

The qRT‐PCR quantification showed that several genes, including TC1, TC7, TC19, TC26 and TC11, were significantly upregulated in AMG and PMG when compared with AG [Fig. 6(A)]. Our preliminary study indicated that TC1, TC7, TC19, TC26 and TC11 were involved in regulating defense responses in tobacco. Among them, TC7 and TC19 encode apolipoproteins, TC1 and TC26 are uncharacterized proteins, and TC11 encodes a plant cell wall expansion element (Supporting information Table S1). By contrast, TC4, TC93, TC109 and TC25 were preferentially expressed in the AG compared with the AMG and PMG [Fig. 6(B)]. Specifically, TC4 encodes a venom‐like allergen 5, TC93 a serine protease inhibitor (Serpin family), TC109 an assassin bug omega toxin‐like protein and TC25 a carbonic anhydrase, most of which are associated with toxin function. Genes exhibiting comparable expression levels across all gland types included TC55, TC61, TC2, TC5 and TC35 [Fig. 6(C)]. Among these, TC61 encodes a Kazal‐type serine protease inhibitor, TC2 a chymotrypsin, TC5 a carboxypeptidase A2 and TC35 a peptidase.

Figure 6.

Gland‐specific gene expression profiles quantified by qRT‐PCR. The venom concentration was 1 μg μL⁻¹. qRT‐PCR was performed in 48 h following venom treatment. (A) Genes significantly upregulated in AMG and PMG compared to AG. (B) Genes preferentially expressed in AG relative to AMG and PMG. (C) Genes showing comparable expression levels across all gland types. Data represent means ± SEM of four biological replicates, and different lowercase letters indicate significant difference at the 0.05 level. Gene annotation is listed in Table S1, and specific primers are listed in Table S2.

Venom‐induced oxidative stress responses in N. lugens

Overall, oxidative stress responses in nymphs treated with AMG or PMG venom were similar to those in the control group, whereas significant changes were observed following AG venom treatment. In AG‐treated nymphs, activities of SOD, CAT, GST and GSH‐Px, and levels of LPO, ROS, RNOS and NO were significantly increased, whereas GSH level was significantly decreased (Fig. 7). By contrast, nymphs treated with AMG or PMG venom only exhibited significantly higher GSH‐Px activity and NO level compared with the control, whereas activities of SOD, CAT and GST, and levels of LPO, ROS, RNOS and GSH showed no significant differences [Fig. 7(A)–(I)].

Figure 7.

Peroxidation stress in N. lugens nymphs induced by gland venom treatment. The venom concentration was 1 μg μL⁻¹. The measure was performed in 5 days following venom treatment at the 3^rd‐instar nymphs. The activity was determined for SOD (A), CAT (B), GST (C) and GSH‐Px (D). Substance level was determined for LPO (E), GSH (F), ROS (G), RNOS (H) and NO (I). Data represent means ± SEM of four biological replicates, and different lowercase letters indicate significant difference at the 0.05 level.

DISCUSSION

The venom apparatus of T. chinensis comprises a bilobed principal gland and a pair of accessory glands, an architecture common among hemipterans. A narrow region, termed the hilum, separates the anterior and posterior lobes, and the overall architecture resembles the glandular organization in species such as Poecilocapsus lineatus (Fabricius) and Scaptocoris castanea. ³⁷ , ³⁸ In some species, muscle cells or valvular openings are present within the hilum. ³⁹ In the salivary glands of Euschistus heros, epithelial cells in the hilum associate with a muscle layer that may regulate the release of secretions from different glandular compartments. ⁴⁰ Likewise, the predatory hemipteran Rhynocoris marginatus features a valvular hilum that controls the flow of secretions from both the principal and accessory glands. ⁴¹ The salivary ducts from distinct lobes of the principal gland converge within the head region, forming a single duct that opens into the stylet bundle. ⁴² Although the PMG and AMG of T. chinensis share anatomical features with the lobed glands of other hemipterans, its AG exhibits a notable morphological distinction. In T. chinensis, the AG is spherical and directly connected to the mouthparts. However, in many hemipterans, including Eocanthecona furcellata and Sycanus croceovittatus, the AG is linked to the principal gland. ¹⁶ The morphological diversity of salivary glands is likely to reflect evolutionary adaptations tied to diversity of feeding strategies within the Hemiptera. In our study, the AMG is larger than the PMG, and a potential reason for this difference may be attributed to the fact that T. chinensis is a less aggressive predator, preying on nymphs and eggs of N. lugens. It does not require a powerful venom to kill its prey but instead relies on rapid salivary secretion to control prey as part of its predation strategy, rather than aggressive venom attacks. By contrast, predatory stink bugs are more aggressive predators, preying on larger and more mobile targets, which require the instant killing of the prey. Thus, the PMG in these predators is likely to possess strong proteolytic and tissue liquefaction abilities. However, comparative studies on mirid salivary glands remain limited, and further research is needed to elucidate the structure–function relationships underlying the feeding ecology in this family.

Beyond its structural uniqueness, the AG of T. chinensis also serves as the source of its most potent venom, which profoundly impairs survival and reproduction of prey insects. As a major rice pest with high reproductive potential, the brown planthopper (BPH, N. lugens) threatens global food security, ⁴³ and targeting its reproductive capacity offers a promising pest management strategy. Understanding the mechanisms regulating insect reproduction is therefore critical for the development of novel pest management strategies. In this study, the AG venom of T. chinensis was found to significantly impair BPH reproduction with a marked reduction in sperm generation in males and egg production in females, which was not observed with the AMG or PMG venom. These findings highlight the potential of AG‐specific components as novel biocontrol agents that could suppress pest populations by compromising their reproductive fitness. To date, few studies have documented the antireproductive effects of heteropteran AG venoms. Future work should focus on identifying the specific AG‐derived molecules responsible for this activity.

The functional differentiation among glandular venoms is further illustrated by their distinct bioactivities. The bioactivities of AMG, PMG and AG venom from T. chinensis differed significantly. AG venom not only led to the highest mortality in BPH, but also caused severe developmental deformities, including thoracic malformations, incomplete abdominal ecdysis and partial retention of exuviae. This suggests that AG venom may disrupt key physiological processes such as neuro‐muscular coordination and hormone‐regulated molting. Comparative evidence from other predatory heteropterans supports the notion of functional specialization. In S. croceovittatus, only PMG venom has been reported to digest prey tissues, whereas AG venom exhibits acute toxicity, rapidly inducing prey death. ¹⁶ In Platymeris rhadamanthus, both PMG and AMG venoms demonstrate paralytic activity, whereas PMG‐secreted venom is primarily responsible for prey digestion. ⁴⁴ Likewise, AMG venom from assassin bugs Haematorrhophus nigroviolaceus and Peirates affinis induces rapid prey paralysis, whereas PMG venom results in delayed mortality. ⁴⁵ , ⁴⁶ Collectively, the compartmentalization of venom components in T. chinensis appears to facilitate a functional division of labor and context‐dependent deployment. Such functional differentiation is likely to necessitate compositional heterogeneity among gland‐derived venoms to accomplish diverse ecological and physiological roles.

Supporting this functional divergence, our transcriptomic data revealed gland‐specific gene expression profiles. Salivary gland‐associated venom genes in T. chinensis displayed distinct expression profiles across AMG, PMG and AG, indicating potential tissue‐specific functional specialization. Genes associated with plant‐defense regulation were predominantly enriched in AMG and PMG, whereas several toxin‐related genes were specifically enriched in AG. This expression pattern may reflect an evolutionary transition of feeding behaviors within Heteroptera. The retention of plant‐interaction genes in AMG/PMG could be a vestige of the phytophagous ancestry, whereas the acquisition of toxin genes in the AG may be tied to the evolution of zoophagy. ⁴⁷ The presence and elevated expression of plant‐interaction‐related genes in predatory insects is likely to represent evolutionary remnants retained from their phytophagous ancestors. Previous studies have reported high expression of numerous serine protease inhibitor (SPI) genes in the salivary glands of Sycanus croceovittatus, particularly in the AG, ⁴⁸ similar to our present finding. We propose that the AG serves as the primary ‘weapon’ gland in prey paralysis and lethality, whereas the AMG and PMG may retain ancestral roles related to plant interaction or digestion. Miridae possess complex feeding strategies, with distinct salivary glands fulfilling specialized functional roles.

A key finding of this study is that AG venom induces significant oxidative stress in BPH, as evidenced by increased activities of antioxidant enzymes, enhanced LPO, and decreased GSH levels. Oxidative stress has been shown to induce protein damage and LPO, resulting in long‐term effects, including decreased survival and compromised reproduction in insects. ⁴⁹ GSH is a critical nonenzymatic antioxidant that scavenges free radicals via reactive thiol groups, thereby maintaining intracellular redox homeostasis and mitigating oxidative damage. ³⁶ To date, oxidative stress responses induced by AG venom from heteropteran predators have not been reported in pest insects. These findings provide the first direct evidence that AG venom induces oxidative stress in insect pests, although further toxicological studies are needed to clarify the underlying molecular mechanisms.

Tytthus chinensis is a widely distributed predatory mirid and a key natural enemy in rice ecosystems, where it contributes to the suppression of planthopper populations. This study characterized its venom apparatus and revealed that venom from the AG exhibited pronounced insecticidal activity against planthoppers, markedly decreasing survival, sperm counts and egg production, and inducing oxidative stress. These results underscore the diverse biological activities of T. chinensis venom and its potential to impair both survival and reproductive performance in BPH. This study provides novel insights into the biological control mechanisms of predatory mirids and indicates that AG venom components may serve as promising candidates for the development of bio‐insecticides. Developing formulations of venom components is a critical step in their application as biocontrol agents. This can be achieved through biosynthetic approaches, such as using genetically engineered microorganisms to produce venom components, followed by extraction and purification to isolate the active components. Additionally, biodegradable delivery systems, such as microcapsules or nanocarriers, can be developed to enhance the environmental stability of venom components and enable their sustained release. The developed biocontrol agents should be subsequently evaluated through field trials to assess their efficacy and stability under different environmental conditions. This includes studies on dose optimization, application frequency and application methods. Furthermore, long‐term monitoring is necessary to ensure that their use does not adversely affect ecosystems or nontarget organisms.

AUTHOR CONTRIBUTIONS

Ruifang Chen: conceptualization, methodology, writing review, final paper review. Qing Zhao: investigation, methodology, visual chart making. Huihui Zhang: literature research, data analysis. Xiaowei Yuan provided experimental materials and implemented the experiment. Rui Wang: conceptualization, methodology. Guangjun yuan: investigation, methodology. Haowen Peng: literature research, conceptualization, writing review. Zewen Liu: conceptualization, methodology, final paper review, funding acquisition.

CONFLICT OF INTEREST

The author declares no competing financial interests.

Supporting information

Table S1. Annotation of genes for qRT‐PCR determination.

Table S2. Specific primers for qRT‐PCR.

DATA AVAILABILITY STATEMENT

The data that supports the findings of this study are available in the supplementary material of this article.