Application of noninvasive sampling technique in mitochondrial genome intraspecific phylogeny of the endangered butterfly, Teinopalpus aureus (Lepidoptera: Papilionidae).

The butterfly genus of Teinopalpus, endemic to Asia, embodies a distinct species of mountain-dwelling butterflies with specific habitat requirements. These species are rare in the wild and hold high conservation and research value. Similar to other protected species, the genetic analysis of the rare Teinopalpus aureus poses challenges due to the complexity of sampling. In this study, we successfully extracted DNA and amplified mitochondrial genomic DNA from various noninvasive sources such as larval feces, larval exuviae, larval head capsules, pupal exuviaes, and filamentous gland secretions, all integral parts of butterfly metamorphosis. This was conducted as part of a research initiative focused on the artificial conservation of T. aureus population in Jinggang Shan Nature Reserve. Our findings illustrated the successful extraction of DNA from multiple noninvasive sources, achieved through modified DNA extraction methodologies. Although the DNA concentration obtained from noninvasive samples was lower than that from muscle tissues of newly dead larvae during rearing, all samples met the requirements for PCR amplification and sequencing, yielding complete circular sequences. These sequences are pivotal for both interspecific and intraspecific genetic relationship analysis. Our methods can be extended to other insects, especially scarce species.

Mitogen-activated protein kinase inhibition enhances the antitumor effects of sporamin in human pancreatic cancer cells.

Sporamin, a sweet potato tuber storage protein, is a Kunitz-type trypsin inhibitor (TI) that has exhibited antitumor activity through poorly defined mechanisms in a number of types of tumor cells. The present study aimed to analyze the combined effects of sporamin and three mitogen-activated protein kinase (MAPK) inhibitors, PD98059, SP600125 and SB203580, on the pancreatic cancer cell line, PANC-1. Cell proliferation activity was assessed using a 3H-thymidine incorporation assay, and cell viability was analyzed using an MTT assay. Apoptosis was assayed by flow cytometry and fluorescence microscopy. Protein expression levels in PANC-1 cells were determined by western blotting. The results of this analysis demonstrated that sporamin induced a temporary increase in the phosphorylation of MAPKs, including phosphorylated extracellular signal regulated-kinase 1/2, phosphorylated c-Jun amino-terminal protein kinase 1/2 and phosphorylated p38-MAPK, in a concentration-dependent manner. However, treatment with MAPK inhibitors promoted the inhibition of cell proliferation and viability, and the induction of apoptosis in sporamin-treated PANC-1 cells. In conclusion, the present study demonstrated that MAPK inhibition enhanced the antitumor activity of sporamin in PANC-1 cells.

Sporamin suppresses growth of human esophageal squamous cell carcinoma cells by inhibition of NF­κB via an AKT­independent pathway.

The aim of the present study was to determine whether sporamin, a trypsin inhibitor, suppresses the growth of human esophageal squamous cell carcinoma (ESCC) cells in vitro. Sporamin treatment led to the suppression of viability and proliferation of human ESCC cell lines, EC9706 and EC109, as determined by MTT and [3H] thymidine incorporation assays, respectively. Flow cytometry and fluorescence microscopy demonstrated that sporamin significantly induced apoptosis in EC9706 and EC109 cells. Western blotting demonstrated that sporamin downregulated the expression of Bcl­2 and Bcl­2 like 1, and upregulated the expression of Bcl­2­associated X in EC9706 and EC109 cells. In addition, marked inhibition of nuclear factor (NF)­κB activation was observed in sporamin­treated EC9706 and EC109 cells by an electrophoretic mobility shift assay. Sporamin treatment also resulted in reduced expression levels of phosphorylated (p)­NF­κB inhibitor α and nuclear NF­κB p65. However, the expression levels of p­protein inase (AKT) and its downstream target, p­p70 S6 kinase, were not markedly altered following sporamin treatment. In conclusion, sporamin may suppress the growth of human ESCC cells via NF­κB­dependent and AKT­independent mechanisms and may act as a promising natural therapeutic agent for the treatment of human ESCC.

Complete mitochondrial genomes of Teinopalpus imperialis (Lepidoptera: Papilionidae) and phylogenetic relationships analyses.

In this study, we sequenced the complete mitochondrial genome of Teinopalpus imperialis, which is a national butterfly of India, and a grade-II protected species in China. The complete mtDNA from T. imperialis was 15 299 base pairs in length and contained 13 protein-coding genes (PCGs), 22 tRNA genes, two rRNA genes, and 401 bp non-coding region. The T. imperialis genes were highly similar to those of sequenced mitogenomes of other lepidopteran species in the order and orientation. Twelve PCGs (ND2, ATP8, ND3, COII, ATP6, COIII, ND4, ND4L, CytB, ND1, ND5, and ND6) start with a typical ATN codon, only the COI gene starts with CGA codon. Eight PCGs (ND2, COI, ATP8, ATP6, COIII, ND5, ND6, and Cyt B) terminate in the common stop codon TAA, three PCGs (ND4L, ND3, and ND1) terminate in the stop codon TAG, and two PCGs (COII and ND4) terminate in a single T residue. The phylogenetic relationships were reconstructed with the concatenated sequences of the 13 PCGs of the mitochondrial genome, and phylogenetic results showed that Danaidae, Satyridae, Libytheidae, Nymphalidae, Acraeidae, Pieridae, Hesperiidae, Riodinidae, and Lycaenidae are monophyletic clades.

Effect of needle damage on the release rate of Masson pine (Pinus massoniana) volatiles.

The effect of needle damage on the release rate of Masson pine (Pinus massoniana Lamb.) volatiles was examined. Needles were continuously damaged by mechanical damage (MDP) or by feeding of pine caterpillar (Dendrolimus punctatus) larvae (LFP); undamaged pine was used as a control (UDP). Volatiles were collected before damage, and at 16, 24, 40, 48, 64, 72, 88 and 96 h post-damage, and analyzed. The analyses revealed that 19 compounds identified as constitutive volatiles from UDP were terpenes and green leaf odors. The release rate of volatiles from MDP or LFP was higher than that from UDP. At 96 h post-damage, emission from MDP or LFP returned to the same level as that of UDP. Some volatiles, including sabinene, ocimene, limonene-1,2-epoxide, linalool, linalool acetate, germacrene D: -4-ol, farnesol, and (E)-4,8-dimethyl-1,3,7-nonatriene were induced by mechanical damage and/or larval attack. Furthermore, the release rate of linalool acetate, farnesol, or (E)-4,8-dimethyl-1,3,7-nonatriene from LFP was higher than that from MDP. Based on an exact estimation of the proportion of damaged pine needles, a significant linear correlation between the release rate of total volatiles identified and the proportion of damaged needles was found in the case of LFP but not MDP.