Three amino acid substitutions contributing to thermostability of phosphoglucose isomerase in the Glanville fritillary butterfly.

Temperature is one of the most important environmental factors that affect organisms, especially ectotherms, due to its effects on protein stability. Understanding the general rules that govern thermostability changes in proteins to adapt high-temperature environments is crucial. Here, we report the amino acid substitutions of phosphoglucose isomerase (PGI) related to thermostability in the Glanville fritillary butterfly (Melitaea cinxia, Lepidoptera: Nymphalidae). The PGI encoded by the most common allele in M. cinxia in the Chinese population (G3-PGI), which is more thermal tolerant, is more stable under heat stress than that in the Finnish population (D1-PGI). There are 5 amino acid substitutions between G3-PGI and D1-PGI. Site-directed mutagenesis revealed that the combination of amino acid substitutions of H35Q, M49T, and I64V may increase PGI thermostability. These substitutions alter the 3D structure to increase the interaction between 2 monomers of PGI. Through molecular dynamics simulations, it was found that the amino acid at site 421 is more stable in G3-PGI, confining the motion of the α-helix 420-441 and stabilizing the interaction between 2 PGI monomers. The strategy for high-temperature adaptation through these 3 amino acid substitutions is also adopted by other butterfly species (Boloria eunomia, Aglais urticae, Colias erate, and Polycaena lua) concurrent with M. cinxia in the Tianshan Mountains of China, i.e., convergent evolution in butterflies.

RNA sequencing reveals differential thermal regulation mechanisms between sexes of Glanville fritillary butterfly in the Tianshan Mountains, China.

The Glanville fritillary butterfly (Melitaea cinxia; Nymphalidae) has been extensively studied as a model species in metapopulation ecology. We investigated in the earlier studies that female butterflies exhibit higher thermal tolerance than males in the Tianshan Mountains of China. We aim to understand the molecular mechanism of differences of thermal responses between sexes. We used RNA-seq approach and performed de novo assembly of transcriptome to compare the gene expression patterns between two sexes after heat stress. All the reads were assembled into 84,376 transcripts and 72,701 unigenes. The number of differential expressed genes (DEGs) between control and heat shock samples was 175 and 268 for males and females, respectively. Heat shock proteins genes (hsps) were up-regulated in response to heat stress in both males and females. Most of the up-regulated hsps showed higher fold changes in males than in females. Females expressed more ribosomal subunit protein genes, transcriptional elongation factor genes, and methionine-rich storage protein genes, participating in protein synthesis. It indicated that protein synthesis is needed for females to replace the damaged proteins due to heat shock. In addition, aspartate decarboxylase might contribute to thermal tolerance in females. These differences in gene expression may at least partly explain the response to high temperature stress, and the fact that females exhibit higher thermal tolerance.

Complete mitochondrial genome of a satyrid butterfly, Ninguta schrenkii (Lepidoptera: Nymphalidae).

The complete mitochondrial genome of N. schrenkii is 15,261 bp in length, containing 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), 2 ribosomal RNA genes (LrRNA and SrRNA) and 1 non-coding A + T-rich region. The nucleotide composition is significantly biased toward A + T (80.2%), similar to the known satyrid species. All PCGs utilize the typical mitochondrial start codon ATN, except for COI, which is initiated with CGA. Seven PCGs use complete stop codon (TAA), whereas ND1 and ND4 use TA as stop codon and COI, COII and ND5 end with single T. The A + T-rich region of N. schrenkii is 403 bp in length, which contains several features common to the other lepidopteran species.

The complete mitochondrial genome of Celastrina hersilia (Lepidoptera: Lycaenidae).

The complete mitochondrial genome of Celastrina hersilia (Lepidoptera: Lycaenidae) is determined in this work. The mitochondrial genome is 15,304 bp in length, which contains typical 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), 2 ribosomal RNA genes and 1 non-coding A + T-rich region. All PCGs are initiated by ATA or ATT codons, except for COI, which uses CGA as a start codon. Four PCGs (COI, COII, ND5, and ND4) terminate with incomplete termination codons TA or T, while the others use TAA as stop codons. Most of the tRNA genes can be folded into a typical cloverleaf structure. The A + T-rich region is 370 bp in length, which contains several features common to the other lepidopteran species.

The complete mitochondrial genome of Gonepteryx mahaguru (Lepidoptera: Pieridae).

The complete mitochondrial genome of Gonepteryx mahaguru (Lepidoptera: Pieridae) is 15,221 bp in length, containing 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), 2 ribosomal RNA genes (LrRNA and SrRNA) and 1 non-coding A + T-rich region. The nucleotide composition is significantly biased toward A + T (80.9%). All PCGs are initiated by classical ATN codon, with the exception of COI, which begins with TTA codon. Nine PCGs harbor the complete stop codon TAA, whereas COI, COII, ND4 and ND5 stop with incomplete codons, single T or TA. All tRNAs can be folded into the typical cloverleaf secondary structure, except for tRNA(Ser)(AGN). The A + T content of AT-rich region is 95.2%, same to the highest one in the known species in Pieridae.

Complete mitochondrial genome of a satyrid butterfly, Lethe albolineata (Lepidoptera: Nymphalidae).

The complete mitochondrial genome of Lethe albolineata is a circular molecule of 15,248 bp in length, containing 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (LrRNA and SrRNA), and a non-coding AT-rich region. All PCGs use the standard ATN start codon, except for COI, which begins with CGA. Eight PCGs employ complete stop codon (TAA), whereas the others use a single T or TA acting as an incomplete stop codon. The AT-rich region is 413 bp in length, which contains a conserved motif common to the other lepidopteran species. Two tRNA-like pseudo-genes are found in the mitochondrial genome of L. albolineata. The phylogenetic analysis shows that L. albolineata exhibits most close relationship with L. dura.

The complete mitochondrial genome of the masked palm civet (Paguma larvata, Mammalia, Carnivora).

The complete mitochondrial genome of the masked palm civet (Paguma larvata, Mammalia, Carnivora) is a circular molecule of 16 710 bp in length, containing 22 transfer RNA genes, 13 protein-coding genes, two ribosomal RNA genes, and a control region. The features of the mitochondrial genome of the masked palm civet are similar to the other mammals. The phylogenetic analysis shows that all species from the family Viverridae cluster together, in which P. larvata exhibits the closest relationship with Genetta servalina.

Heat shock protein 70 gene family in the Glanville fritillary butterfly and their response to thermal stress.

Temperature variation in the environment is a great challenge to organisms. Induction of heat shock protein 70 (HSP70) is a common genetic mechanism to cope with thermal stress. The Glanville fritillary butterfly (Melitaea cinxia) is a model species in population and evolutionary biology, and its behavior and life history are greatly influenced by ambient temperature. We cloned and sequenced the full coding sequences of seven hsp70 genes from the Glanville fritillary. Of those genes, McHsc70-1 and McHsc70-2 were identified as heat shock cognate 70 (hsc70), of which the latter located in endoplasmic reticulum. We analyzed the expression patterns of different hsp70s under various thermal stresses using quantitative PCR. Heat shock at 40°C for 2h induced high expression of McHsp70-1, McHsp70-2 and McHsc70-2. Only McHsc70-2 had a small increase after cold shock at 0°C for 2h. Acclimation at 35°C for three days before heat shock reduced expression of McHsp70 after heat shock. The maximum mRNA level of McHsp70s was reached in the first 2h after the heat shock. This study uncovers the complexity of the hsp70 system, and provides the valuable information for further temperature-related research in the Glanville fritillary butterfly.

Phenotypic plasticity in thermal tolerance in the Glanville fritillary butterfly.

Ambient temperature is an ubiquitous environmental factor affecting all organisms. Global climate change increases temperature variation and the frequency of extreme temperatures, which may pose challenges to ectotherms. Here, we examine phenotypic plasticity to temperature and genotypic effects on thermal tolerance in the Glanville fritillary butterfly (Melitaea cinxia). We found no significant difference in heat or cold tolerance in populations originating from a continental climate in China and from Finland with moderate temperature variation. Acclimation to large-amplitude temperature variation increased heat tolerance in both populations, but decreased cold tolerance and increased hsp70-2 expression in the Chinese population only. The latter result indicates a genotypic effect in the response to temperature variation. In the Finnish population, a non-synonymous SNP in the phosphoglucose isomerase (Pgi) gene was associated with heat knock-down time.

Molecular cloning and expression in vitro of a carboxylesterase gene from the Glanville fritillary butterfly (Melitaea cinxia).

Carboxylesterase (EC 3.1.1.1) is a member of the carboxyl/cholinesterase (CCE) superfamily, which is widely distributed in animals, plants and microorganisms. This enzyme has been known to be associated with insecticide resistance and detoxification. Although CCEs have been extensively studied in insects, including lepidopterans, the research on butterflies, a major subgroup in Lepidoptera, is still poor. In the present study, we cloned a CCE gene (McCCE1) from the Glanville fritillary butterfly (Melitaea cinxia, Lepidoptera: Nymphalidae). The full-length cDNA encoding McCCE1 was 1786 bp, containing a 1641 bp open reading frame encoding 546 amino acids, a 38 bp 5'-untranslated region (5'-UTR), and a 107 bp 3'-UTR with a poly(A) tail. The functionally conserved amino acids in McCCE1 shared the 55% identity with the cytoplasmic esterase CCE017a in Helicoverpa armigera (Lepidoptera: Noctuidae), which has been associated with detoxification. Assays in vitro showed that the recombinant McCCE1 could hydrolyze α- and ß-naphthyl acetate. Thus, the present study adds to the body of knowledge concerning the detoxification of pesticides by lepidopterans.

Suppression of Rap1 impairs cardiac myofibrils and conduction system in zebrafish.

Numerous studies have revealed that Rap1 (Ras-proximate-1 or Ras-related protein 1), a small GTPase protein, plays a crucial role in mediating cAMP signaling in isolated cardiac tissues and cell lines. However, the involvement of Rap1 in the cardiac development in vivo is largely unknown. By injecting anti-sense morpholino oligonucleotides to knock down Rap1a and Rap1b in zebrafish embryos, and in combination with time-lapsed imaging, in situ hybridization, immunohistochemistry and transmission electron microscope techniques, we seek to understand the role of Rap1 in cardiac development and functions. At an optimized low dose of mixed rap1a and rap1b morpholino oligonucleotides, the heart developed essentially normally until cardiac contraction occurred. Morphant hearts showed the myocardium defect phenotypes, most likely due to disrupted myofibril assembly and alignment. In vivo heart electrocardiography revealed prolonged P-R interval and QRS duration, consistent with an adherens junction defect and reduced Connexons in cardiac myocytes of morphants. We conclude that a proper level of Rap1 is crucial for heart morphogenesis and function, and suggest that Rap1 and/or their downstream factor genes are potential candidates for genetic screening for human heart diseases.

The oral toxicity of the transgenic Bt+CpTI cotton pollen to honeybees (Apis mellifera).

Transgenic insect-resistant cotton has been planted in China in a large scale and may have adverse impacts on honeybees. Pollens from the transgenic Cry1Ac+CpTI cotton Zhong-41 and the parental cotton Zhong-23 were collected from the field and their impacts on adult worker bees were assessed. Experimental results showed that Zhong-41 pollen had no acute oral toxic effect on worker bees. No significant differences were observed in the superoxide dismutase activity or in the longevity of worker bees fed with diets containing the two cotton pollens. The main reasons for the outcome may be the low expression level of the transgenic proteins Cry1Ac and CpTI in the pollen of Zhong-41 as well as the substantial equivalence in the amounts of gross protein and soluble saccharides for the two cotton pollens. The implications of these results are discussed and further work to be carried out is put forward.

Leaf surface factors of transgenic Bt cotton associated with the feeding behaviors of cotton aphids: a case study on non-target effects.

The present paper reports case study results of the risk assessment of transgenic Bt cotton on a non-target pest, cotton aphid, Aphis gossypii. Several types of techniques, i.e., electrical penetration graph (EPG), light and electron microscopy, bioassays and chemical analysis, were applied to investigate physical and chemical leaf factors of 2 transgenic Bt cotton lines (GK12 and GK19) and their parental non-Bt cotton line (Simian3) associated with searching and feeding behaviors of cotton aphids on leaves or leaf extracts of cotton plants. EPG results showed that there were some differences among behaviors of cotton aphids on 2 Bt cotton and 1 non-Bt cotton lines. Cotton aphids performed similarly to leaf surface extracts from 3 cotton lines; and leaf surface chemicals, mainly volatiles and waxes, were almost identical in the components and concentrations among the cotton lines. However, three cotton lines were quite different from each other in the densities of certain kinds of covering trichomes. Therefore, the relationships between the physical characteristics and the searching behaviors of cotton aphids on the three cotton lines were constructed as the regression equations. Glandular trichomes and covering trichomes with 5 branches influenced the cotton aphids' searching behaviors effectively; and other trichomes with other branches affected aphids in varying ways. These results demonstrated that leaf surface physical factors of transgenic Bt cotton lines different from their parental non-Bt line could affect the penetration behaviors of non-target cotton aphids. Cotton aphids penetrate and feed more easily on two Bt cotton lines than on the non-Bt cotton line.

Glacial effects on sequence divergence of mitochondrial COII of Polyura eudamippus (Lepidoptera: Nymphalidae) in China.

Sequence divergence of mitochondrial COII was analyzed in 50 specimens belonging to five subspecies of Polyura eudamippus (Lepidoptera: Nymphalidae) collected from southern China. There were nine haplotypes in the 405 bp of partial COII sequence. Distribution of the five subspecies was primarily consistent with the spatial distribution of haplotypes. The K (st) statistic showed genetic differentiation among these subspecies, except between the pair of P. e. kuangtungensis and P. e. formosana, which were separated by the Taiwan Strait. This is consistent with the 10,000-year history of the Taiwan Strait, not long enough for detectable differentiation. The present distribution pattern of COII haplotypes of P. eudamippus should be shaped by the alteration of Pleistocene glaciations, and Yunnan might be the refugium of P. eudamippus in the ice age, judging from the abundant haplotypes remaining. There were two routes for P. eudamippus in the postglacial expansion, one northward to Sichuan, Chongqing, and Hubei and another eastward to the southeastern coast of mainland China and Taiwan Island. Because the haplotype of butterflies on Hainan Island (P. e. whiteheadi) was completely different from that of mainland China, it was estimated that butterflies on Hainan Island might be from the Indo-China Peninsula rather than from mainland China.

Distribution and variations of three 1,4-benzoxazin-3-ones in maize induced by the Asian corn borer, Ostrinia furnacalis (Guenée).

Contents of three 1,4-benzoxazin-3-ones in tissue samples from different parts (young leaf, second leaf, old leaf, stem and root) of young maize plants of 4-leaves stage, fed by the third instar larvae of the Asian corn borer, Ostrinia furnacalis (Guenée), were analyzed by high-performance liquid chromatography-mass spectroscopy (HPLC-MS). Samples were taken immediately (set A) or 48 h (set B) after larvae had fed on the second leaf for 48 h. The three 1,4-benzoxazin-3-ones investigated in our experiments were 2,4-dihydroxy-7-methoxy-1,4(2H)-benzoxazin-3-one (DIMBOA), 2,4-dihydroxy-1,4(2H)-benzoxazin-3-one (DIBOA) and 2-hydroxy-7-methoxy-1,4(2H)-benzoxazin-3-one (HMBOA). In samples of set A, the levels of DIMBOA and HMBOA were significantly lifted in the old leaf (L3) and young leaf (L1), respectively, while amounts of these two chemicals in other plant parts were not significantly different between larvae-fed plants and intact plants. Concentrations of DIBOA in each plant part remained unchanged. In samples of set B, no concentration differences for any of these three 1,4-benzoxazin-3-ones between larvae-fed plants and controls were observed in any plant part. The feeding of the Asian corn borer seems to have limited effects on induction of these three 1,4-benzoxazin-3-ones in young maize plants of the variety investigated.

Identification of potential nuclear reprogramming and differentiation factors by a novel selection method for cloning chromatin-binding proteins.

Nuclear reprogramming is critical for animal cloning and stem cell creation through nuclear transfer, which requires extensive remodeling of chromosomal architecture involving dramatic changes in chromatin-binding proteins. To understand the mechanism of nuclear reprogramming, it is critical to identify chromatin-binding factors specify the reprogramming process. In this report, we have developed a high-throughput selection method, based on T7 phage display and chromatin immunoprecipitation, to isolate chromatin-binding factors expressed in mouse embryonic stem cells using primary mouse embryonic fibroblast chromatin. Seven chromatin-binding proteins have been isolated by this method. We have also isolated several chromatin-binding proteins involved in hepatocyte differentiation. Our method provides a powerful tool to rapidly and selectively identify chromatin-binding proteins. The method can be used to study epigenetic modification of chromatin during nuclear reprogramming, cell differentiation, and transdifferentiation.