Sequencing, Analysis and Organization of the Complete Genome of a Novel Baculovirus Calliteara abietis Nucleopolyhedrovirus (CaabNPV).

Baculoviridae, a virus family characterized by a single large double stranded DNA, encompasses the majority of viral bioinsecticides, representing a highly promising and environmentally friendly pesticide approach to insect control. This study focuses on the characterization of a baculovirus isolated from larvae of Calliteara abietis (Erebidae, Lymantriidae) collected in Mongolian pinaceae forests. This new isolate was called Calliteara abietis nucleopolyhedrovirus (CaabNPV). CaabNPV exhibits an irregular polyhedron shape, and significant variation in the diameter of its occlusion bodies (OBs) was observed. Nucleotide distance calculations confirmed CaabNPV as a novel baculovirus. The CaabNPV genome spans 177,161 bp with a G+C content of 45.12% and harbors 150 potential open reading frames (ORFs), including 38 core genes. A comprehensive genomic analysis categorizes CaabNPV within Group II alphabaculovirus, revealing a close phylogenetic relationship with Alphabaculovirus orleucostigmae (OrleNPV). Additionally, repeat sequence analysis identified three highly repetitive sequences consisting of 112 bp repeat units, known as homologous regions (hrs). This research contributes valuable insights into CaabNPV's phylogenetic placement, genomic structure, and its potential applications in insect biocontrol.

Transcriptome and Metabolome Analyses of Thitarodes xiaojinensis in Response to Ophiocordyceps sinensis Infection.

Ophiocordyceps sinensis exhibits more than 5 months of vegetative growth in Thitarodes xiaojinensis hemocoel. The peculiar development process of O. sinensis has been elucidated through morphological observation and omics technology; however, little information has been reported regarding the changes that occur in the host T. xiaojinensis. The RNA sequencing data showed that when O. sinensis blastospores were in the proliferative stage, the greatest change in the infected larval fat body was the selectively upregulated immune recognition and antimicrobial peptide genes. When O. sinensis blastospores were in the stationary stage, the immune pathways of T. xiaojinensis reverted to normal levels, which coincides with the successful settlement of O. sinensis. Pathway enrichment analysis showed a higher expression of genes involved in energy metabolism pathway in this stage. Metabolomic analyses revealed a reduction of amino acids and lipids in hemolymph, but an upregulation of lipids in the fat body of the host larvae after O. sinensis infection. We present the first transcriptome integrated with the metabolome study of T. xiaojinensis infected by O. sinensis. It will improve our understanding of the interaction mechanisms between the host and entomopathogenic fungi, and facilitate future functional studies of genes and pathways involved in these interactions.

The invasion process of the entomopathogenic fungus Ophiocordyceps sinensis into the larvae of ghost moths (Thitarodes xiaojinensis) using a GFP-labeled strain.

Chinese cordyceps is a well-known and valuable traditional Chinese medicine that forms after Ophiocordyceps sinensis parasitizes ghost moth larvae. The low natural infection rate of O. sinensis limits large-scale artificial cultivation of Chinese cordyceps, and the invasion process is unclear. To investigate the temporal and spatial regulation when O. sinensis enters ghost moths, we constructed an O. sinensis transformant that stably expresses green fluorescent protein (GFP). Inoculating Thitarodes xiaojinensis larvae with a high concentration of GFP-labeled O. sinensis, we observed that O. sinensis conidia could adhere to the host cuticle within 2 days, germinate penetration pegs within 4 days, and produce blastospores in the host hemocoel within 6 days. The reconstructed three-dimensional (3D) structures of the invasion sites showed that penetration pegs germinated directly from O. sinensis conidia at the joining site with the larval cuticle. Differentiated appressoria or hyphae along the host epicuticle are not required for O. sinensis to invade ghost moths. Overall, the specific invasion process of O. sinensis into its host is clarified, and we provided a new perspective on the invasion process of entomopathogenic fungi.

A Greenhouse Test to Explore and Evaluate Light-Emitting Diode (LED) Insect Traps in the Monitoring and Control of Trialeurodes vaporariorum.

Population control of small sucking insects has been challenging, and alternative control methods are constantly being sought. Visual traps have long been used to monitor and control pests. Colored sticky cards are widely used for diurnal pests, but their effects are influenced by environmental light conditions. Artificial light traps are mostly used for nocturnal pests. Here, we explored and evaluated light-emitting diode (LED) traps for the monitoring and control of small diurnal sucking insects using greenhouse tests targeting the greenhouse whitefly Trialeurodes vaporariorum. We tested the trapping efficacy of the LED water pan trap, assessed the most attractive LED light and analyzed its efficacy under different weather conditions. The results showed that the LED water pan trap was too inefficient to be useful. Green LEDs were more attractive than yellow LEDs, UV LEDs and green-UV combinations. Regardless of sunny or cloudy conditions, the green LED trap caught more than twice the number of whiteflies than the yellow sticky card alone under summer shading conditions. Our study suggests that LED traps have a significant field application value in whitefly mass trapping and may also be efficient for other diurnal insects. The design of LED traps specific for diurnal insects is discussed.

Metabolomics reveals the key role of oxygen metabolism in heat susceptibility of an alpine-dwelling ghost moth, Thitarodes xiaojinensis (Lepidoptera: Hepialidae).

Ghost moths inhabiting the alpine meadows of the Tibetan Plateau are cold-adapted stenothermal organisms that are susceptible to heat (dead within 7 days at 27 °C exposure). Exploring the metabolic basis of their heat susceptibility would extend our understanding of the thermal biology of alpine-dwelling invertebrates. Here, gas chromatography-mass spectrometry-based metabolomics was combined with physiological and transcriptional approaches to determine the metabolic mechanisms of heat susceptibility in Thitarodes xiaojinensis larvae. The metabolomics results showed that 27 °C heat stress impaired the Krebs cycle and lipolysis in T. xiaojinensis larvae, as demonstrated by the accumulation of intermediary metabolites. In addition, carbohydrate reserves were highly and exclusively consumed, and an anaerobic product, lactate, accumulated. This evidence suggested a strong reliance on glycolysis to anaerobically generate energy. The respiration rate and enzymatic activity test results indicated a deficiency in O2 metabolism; in addition, the Krebs cycle capacity was not decreased, and the metabolic flux through aerobic pathways was limited. These findings were further supported by the occurrence of hypoxia symptoms in midgut mitochondria (vacuolation and swelling) and increased transcription of hypoxia-induced factor 1-α. Overall, heat stress caused O2 limitation and depressed the overall intensity of aerobic metabolism in ghost moths, and less efficient anaerobic glycolysis was activated to sustain their energy supply. As carbohydrates were depleted, the energy supply became deficient. Our study presents a comprehensive metabolic explanation for the heat susceptibility of ghost moths and reveals the relationship between O2 metabolism and heat susceptibility in these larvae.

Metabolic insights into the cold survival strategy and overwintering of the common cutworm, Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae).

The common cutworm, Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae), is a destructive pest in Asia. Although overwintering in the field has not been reported for this species, their larvae are capable of long-term survival in fluctuating temperatures, i.e., 5°C (12h) plus 13°C (12h), if food is available. With an increase in climate change due to global warming and the widespread use of greenhouses, further understanding of their cold survival strategy is needed to predict and control their population in the future. In this study, metabolomics was performed to analyze the metabolic features of S. litura larvae exposed to two typical low temperatures: 15°C and 4°C, at which the development, locomotion and feeding activities are maintained or halted, respectively. The results showed that the strategies that regulate lipid and amino acid metabolism were similar at 15°C and 4°C. Cold exposure induced a metabolic shift of energy from carbohydrate to lipid and decreased free amino acids level. Biosynthesis likely contributed to the decrease in amino acids levels even at 4°C, a non-feeding temperature, suggesting an insufficient suppression of anabolism. This explains why food and high temperature pulses are necessary for their long-term cold survival. Glycometabolism was different between 15°C and 4°C. Carbohydrates were used rapidly at 15°C, while trehalose accumulated at 4°C. Interestingly, abundant trehalose and serine are prominent features of Spodoptera exigua larvae, an overwintering species, when compared to S. litura larvae. Exposure to 4°C also induced up-regulation of carbohydrase and protease in the guts of S. litura. Therefore, it is likely that concurrence of food supplement and fluctuating temperatures could facilitate the cold survival of S. litura larvae. We also found that exposure to 4°C could activate the mevalonate pathway in S. litura larvae, which might be related to glycometabolism at 4°C. Overall, our study describes systematically the responses of a cold susceptible insect, S. litura, to low temperatures and explains how fluctuating temperatures facilitate their long-term cold survival indicating the possibility for overwintering of S. litura larvae with global warming and agricultural reforms.

Cold adaptation mechanisms in the ghost moth Hepialus xiaojinensis: Metabolic regulation and thermal compensation.

Ghost moths (Lepidoptera: Hepialidae) are cold-adapted stenothermal species inhabiting alpine meadows on the Tibetan Plateau. They have an optimal developmental temperature of 12-16 °C but can maintain feeding and growth at 0 °C. Their survival strategies have received little attention, but these insects are a promising model for environmental adaptation. Here, biochemical adaptations and energy metabolism in response to cold were investigated in larvae of the ghost moth Hepialus xiaojinensis. Metabolic rate and respiratory quotient decreased dramatically with decreasing temperature (15-4 °C), suggesting that the energy metabolism of ghost moths, especially glycometabolism, was sensitive to cold. However, the metabolic rate at 4 °C increased with the duration of cold exposure, indicating thermal compensation to sustain energy budgets under cold conditions. Underlying regulation strategies were studied by analyzing metabolic differences between cold-acclimated (4 °C for 48 h) and control larvae (15 °C). In cold-acclimated larvae, the energy generating pathways of carbohydrates, instead of the overall consumption of carbohydrates, was compensated in the fat body by improving the transcription of related enzymes. The mobilization of lipids was also promoted, with higher diacylglycerol, monoacylglycerol and free fatty acid content in hemolymph. These results indicated that cold acclimation induced a reorganization on metabolic structure to prioritise energy metabolism. Within the aerobic process, flux throughout the tricarboxylic acid (TCA) cycle was encouraged in the fat body, and the activity of α-ketoglutarate dehydrogenase was the likely compensation target. Increased mitochondrial cristae density was observed in the midgut of cold-acclimated larvae. The thermal compensation strategies in this ghost moth span the entire process of energy metabolism, including degration of metabolic substrate, TCA cycle and oxidative phosphorylation, and from an energy budget perspective explains how ghost moths sustain physiological activity in cold environments.

A new insect cell line from pupal ovary of Spodoptera exigua established by stimulation with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG).

A continuous cell line derived from the pupal ovary of Spodoptera exigua was established by treating primary cells with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Three days after treating cells with 3.0 µg/ml of MNNG, the cells formed a monolayer and were initially subcultured 60 d after the MNNG was removed, followed by subculturing for 30 passages. The established cell line, designated IOZCAS-Spex 12, consisted of a mixture of three types of cells, including spherical, spindle-shaped, and oval cells. The population doubling time of the cell line during its logarithmic growth phase was found to be 71 h. DNA amplification fingerprinting polymerase chain reaction analysis confirmed that the new cell line originated from S. exigua. Susceptibility of IOZCAS-Spex 12 cells to infection by certain nucleopolyhedroviruses was investigated. The results showed that the cell line was highly susceptible to infection by S. exigua nucleopolyhedrovirus and Autographa californica multiple nucleopolyhedrovirus, slightly susceptible to infection by Spodoptera litura nucleopolyhedrovirus, and not susceptible to infection by Helicoverpa armigera nucleopolyhedroviruses or Hyphantria cunea nucleopolyhedroviruses. The results of this study suggest that MNNG treatment may overcome existing limitations to obtaining continually proliferating cells and may open up the possibilities for immortalizing isolated insect cells.