Transcriptomics Reveals the Killing Mechanism by Which Entomopathogenic Fungi Manipulate the RNA Expression Profiles of Termites and Provides Inspiration for Green Pest Management.

As chemical pesticides have caused serious environmental pollution, fungus-based biological control has become a developing alternative to chemical control. Here, we aimed to determine the molecular mechanism underlying how Metarhizium anisopliae facilitated invasive infection. We found that the fungus increased its virulence by downregulating glutathione S-transferase (GST) and superoxide dismutase (SOD) throughout termite bodies. Among 13 fungus-induced microRNAs throughout termite bodies, miR-7885-5p and miR-252b upregulation significantly downregulated several mRNAs in response to toxic substances to increase the fungal virulence [e.g., phosphoenolpyruvate carboxykinase (GTP) and heat shock protein homologue SSE1]. In addition, nanodelivered small interfering RNA of GST and SOD and miR-7885-5p and miR-252b mimics increased the virulence of the fungus. These findings provide new insights into the killing mechanism of entomopathogens and their utilization of the host miRNA machinery to reduce host defenses, laying the groundwork to enhance virulence of biocontrol agents for green pest management.

Discovery of novel whitefly vector proteins that interact with a virus capsid component mediating virion retention and transmission.

Specificity and efficiency of plant virus transmission depend largely on protein-protein interactions of vectors and viruses. Cucurbit chlorotic yellows virus (CCYV), transmitted specifically by tobacco whitefly, Bemisia tabaci, in a semi-persistent manner, has caused serious damage on cucurbit and vegetable crops around the world. However, the molecular mechanism of interaction during CCYV retention and transmission are still lacking. CCYV was proven to bind particularly to the whitefly foregut, and here, we confirmed that the minor coat protein (CPm) of CCYV is participated in the interaction with the vector. In order to identify proteins of B. tabaci that interact directly with CPm of CCYV, the immunoprecipitation (IP) assay and DUALmembrane cDNA library screening technology were applied. The cytochrome c oxidase subunit 5A (COX), tubulin beta chain (TUB) and keratin, type I cytoskeletal 9-like (KRT) of B. tabaci shown strong interactions with CPm and are closely associated with the retention within the vector and transmission of CCYV. These findings on whitefly protein-CCYV CPm interactions are crucial for a much better understanding the mechanism of semi-persistent plant virus transmission by insect vectors, as well as for implement new strategies for effective management of plant viruses and their vector insects.

Identification and Physicochemical Properties of the Novel Hemolysin(s) From Oral Secretions of Helicoverpa armigera (Lepidoptera: Noctuidae).

Hemolysins cause the lysis of invading organisms, representing major humoral immunity used by invertebrates. Hemolysins have been discovered in hemolymph of Helicoverpa armigera larvae as immune factors. As oral immunity is great important to clear general pathogens, we presumed that hemolysins may be present in oral secretions (OS). To confirm this hypothesis, we conducted four testing methods to identify hemolysin(s) in larval OS of H. armigera, and analyzed physicochemical properties of the hemolysin in comparison with hemolytic melittin of Apis mellifera (L.) (Hymenoptera: Apidae) venom. We found hemolysin(s) from OS of H. armigera for the first time, and further identified in other lepidopteran herbivores. It could be precipitated by ammonium sulfate, which demonstrates that the hemolytic factor is proteinaceous. Labial gland showed significantly higher hemolytic activity than gut tissues, suggesting that hemolysin of OS is mainly derived from saliva secreted by labial glands. Physicochemical properties of hemolysin in caterpillar's OS were different from bee venom. It was noteworthy that hemolytic activity of OS was only partially inhibited even at 100°C. Hemolytic activity of OS was not inhibited by nine tested carbohydrates contrary to bee venom melittin. Moreover, effects of metal ions on hemolytic activity were different between OS and bee venom. We conclude that there is at least a novel hemolysin in OS of herbivorous insects with proposed antibacterial function, and its hemolytic mechanism may be different from melittin. Our study enriches understanding of the potential role of hemolysins in insect immunity and provides useful data to the field of herbivorous insect-pathogen research.

Characteristics of morphology, electrophysiology, and central projections of two sensilla styloconica in Helicoverpa assulta larvae.

The medial and lateral styloconic sensilla, constituting the main taste organs of lepidopterous caterpillars, were investigated in the oligophagous species, Helicoverpa assulta (Guenée) (Lepidoptera: Noctuidae). In this paper, the two sensilla were morphologically and physiologically characterized by scanning electron microscopy and tip recordings, respectively. The central projections of their respective sensory neurons were mapped by anterograde staining experiments combined with confocal laser scanning microscopy. The results showed that the two sensilla are in general morphologically similar. However, the size of the peg on the medial sensillum is significantly greater than that of the lateral. Tobacco leaf saps, sinigrin, and nicotine elicited strong responses from neurons housed by the medial sensillum, whereas sucrose activated primarily the lateral sensillum. All stained neurons in either sensillum showed a projection pattern involving axons entering the subesophageal ganglion through the ipsilateral maxillary and passing further on through the ipsilateral circumesophageal connective to the tritocerebrum of the brain. In the subesophageal ganglion, the axons targeted two areas: the ventrolateral section and the region near the neuromere midline. One distinction between the staining patterns originating from the two sensilla, however, is that axons arising from the medial sensillum, and not the lateral, give off some additional neural branches in the subesophageal ganglion including a few arborizations surrounding a tract, plus a long process extending posteriorly along the midline. Differences in the central projections derived from the two sensilla styloconica have not been reported previously.

Central projections of gustatory receptor neurons in the medial and the lateral sensilla styloconica of Helicoverpa armigera larvae.

Food selection behavior of lepidopteran larvae is predominantly governed by the activation of taste neurons present in two sensilla styloconica located on the galea of the maxilla. In this study, we present the ultrastructure of the sensilla styloconica and the central projection pattern of their associated receptor neurons in larvae of the heliothine moth, Helicoverpa armigera. By means of light microscopy and scanning electron microscopy, the previous findings of two morphologically fairly similar sensilla comprising a socketed conic tip inserted into a large peg were confirmed. However, the peg size of the medial sensillum was found to be significantly bigger than that of the lateral sensillum. The sensory neurons derived from each sensillum styloconicum were mapped separately using anterograde staining experiments combined with confocal laser-scanning microscopy. For determining the afferents' target regions relative to each other, we reconstructed the labeled axons and placed them into a common reference framework. The sensory axons from both sensilla projected via the ipsilateral maxillary nerve to the suboesophageal ganglion and further through the ipsilateral circumoesophageal connective to the brain. In the suboesophageal ganglion, the sensory projections targeted two areas of the ipsilateral maxillary neuropil, one located in the ventrolateral neuromere and the other adjacent to the neuromere midline. In the brain, the axon terminals targeted the dorso-anterior area of the ipsilateral tritocerebrum. As confirmed by the three-dimensional reconstructions, the target regions of the neural projections originating from each of the two sensilla styloconica were identical.

Fine structure and primary sensory projections of sensilla located in the labial-palp pit organ of Helicoverpa armigera (Insecta).

The fine structure and primary sensory projections of sensilla located in the labial-palp pit organ of the cotton bollworm Helicoverpa armigera (Insecta, Lepidoptera) are investigated by scanning electron and transmission electron microscopy combined with confocal laser scanning microscopy. The pit organ located on the third segment of the labial palp is about 300 µm deep with a 60-µm-wide opening, each structure containing about 1200 sensilla. Two sensillum types have been found, namely hair-shaped and club-shaped sensilla, located on the upper and lower half of the pit, respectively. Most sensilla possess a single dendrite. The dendrite housed by the club-shaped sensilla is often split into several branches or becomes lamellated in the outer segment. As reported previously, the sensory axons of the sensilla in the labial pit organ form a bundle entering the ipsilateral side of the subesophageal ganglion via the labial palp nerve and project to three distinct areas: the labial pit organ glomerulus in each antennal lobe, the subesophageal ganglion and the ventral nerve cord. In the antennal lobe, the labial pit organ glomerulus is innervated by sensory axons from the labial pit organ only; no antennal afferents target this unit. One neuron has been found extending fine processes into the subesophageal ganglion and innervating the labial palp via one branch passing at the base of the labial palp nerve. The soma of this assumed motor neuron is located in the ipsilateral cell body layer of the subesophageal ganglion. Our results provide valuable knowledge concerning the neural circuit encoding information about carbon dioxide and should stimulate further investigations directed at controlling pest species such as H. armigera.