Lin Miao

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 cell line from Spodoptera exigua (Lepidoptera: Noctuidae) and its differentially expressed genes

A new cell line, designated IOZCAS‐Spex XI, was established from the pupal ovaries of Spodoptera exigua (Lepidoptera: Noctuidae) in TNM‐FH medium containing 10% foetal bovine serum. The spherical cells were predominant among the various cell types. The population‐doubling time during the logarithmic phase of growth was 81.7 h. It was confirmed that the cell line originated from S. exigua by DAF‐PCR technique. Analysis of susceptibility to baculovirus showed that the new cell line was susceptible to S. exigua nucleopolyhedrovirus (SeNPV), Autographa californica multiple NPV (AcMNPV) and slightly susceptible to S. litura NPV (SpltNPV), while not permissive to Helicoverpa armigera NPV and Hyphantria cunea NPV (HcNPV). Real‐Time PCR analysis was carried out to compare some differentially expressed genes between the cell line and the primary culture. The result showed that marked significant differences were observed in the expression of the genes of SUMO‐1 activating enzyme, BCCIP‐like protein, 10 kDa HSP, CypA, receptor for activated PKC, PDI‐like protein ERp57, ALDH, DEAD box ATP‐dependent RNA helicase‐like protein (P < 0.01), while a significant difference was obtained in the expression of GST gene between the cell line and the primary culture (P < 0.05).

A NEW CELL LINE FROM LARVAL FAT BODIES OF THE BOLLWORM, HELICOVERPA ARMIGERA (LEPIDOPTERA: NOCTUIDAE)

SummaryA new cell line, designated IOZCAS-Ha-I, was initiated from the fat body of larvae of Helicoverpa armigera (Lepidoptera: Noctuidae) in TNM-FH medium containing 10% fetal bovine serum. Spherical cells were predominant among the various cell types. The cell line showed a typical lepidopteran chromosome pattern ranging from 58 to 239 chromosomes in the majority of the cells, it was confirmed to have originated from the H. armigera by the DNA amplification-fingerprinting polymerase chain reaction (DAF-PCR) technique. The new cell line was only slightly susceptible to the multiple nucleocapsid nuclear polyhedrosis viruses (NPV) from H. armigera.

TTAGG-repeat telomeres and characterization of telomerase in the beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae)

Telomeres are maintained usually by telomerase, a specialized reverse transcriptase that adds this sequence to chromosome ends. In this study, telomerase activity was detected in the in different somatic tissues, such as midgut and fat bodies, by the telomeric repeat amplification protocol (TRAP) in Spodoptera exigua. The structure of the telomeres of S. exigua was evaluated by sequence analysis of the TRAP products, revealing that the telomerase synthesized a (TTAGG)n repeat. The presence of a telomerase reverse transcriptase (TERT) subunit coding gene has been cloned, sequenced and expressed in vitro successively. Notably, the S. exigua telomerase (SpexTERT) gene structure lacks the N‐terminal GQ motif. Telomerase contains a large RNA subunit, TER, and a protein catalytic subunit, TERT. Here we report an in vitro system that was reconstructed by all components of the telomerase complex, a purified recombinant SpexTERT without a N‐terminal GQ motif and a mutant human telomerase RNA (TER), showed telomerase activity. Together, these results suggest the GQ motif is not essential for telomerase catalysis.

Obstacles and approaches in artificial cultivation of Chinese cordyceps

Chinese cordyceps, also known as “Dong Chong Xia Cao” in Chinese, which means being worm in winter and herb in summer, is the fruiting body of Ophiocordyceps sinensis developed from the cadaver mummy (sclerotium) of infected ghost moth larva distributed in the Qinghai–Tibetan Plateau around the altitude of 3000–5000 m (Zhang et al. 2012). It is a well-known traditional Chinese medicine mostly because of its legendary miraculous beneficial effects to human health and its very high prices in the market, which reached as high as USD 60,000 per kg in 2015 (Lei et al. 2015). Almost all the Chinese cordyceps in the market are harvested in the alpine pasture of the Qinghai–Tibetan Plateau during the period of May to July every year, which severely damage the pasture vegetation and ecology of the plateau. A lot of institutions and companies have been investing huge resources to explore artificial cultivation of Chinese cordyceps indoor or in its natural habitat for a long time, but failed until recent because of difficulties of artificially completing both life cycles of host insect and fungal infection in the host involved in the cultivation. It is known that several institutions and companies have been successful in artificial cultivation of Chinese cordyceps. However, there is no formal scientific report on the details of this success, which is probably because of huge commercial interests behind the area and technical know-hows in which developers do not want to share with others. In this short note, we briefly present some technical bottlenecks retarding the progress of artificial cultivation of Chinese cordyceps empirically and list some examples of the success from personal communications. It is a need to illustrate that the information provided below is more empirical than scientific. 1. Long life cycle makes it difficult to rear the host insect artificially indoors on large scale

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

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.