Whole-Genome Sequencing of Theileria parva Strains Provides Insight into Parasite Migration and Diversification in the African Continent

The disease caused by the apicomplexan protozoan parasite Theileria parva, known as East Coast fever or Corridor disease, is one of the most serious cattle diseases in Eastern, Central, and Southern Africa. We performed whole-genome sequencing of nine T. parva strains, including one of the vaccine strains (Kiambu 5), field isolates from Zambia, Uganda, Tanzania, or Rwanda, and two buffalo-derived strains. Comparison with the reference Muguga genome sequence revealed 34 814–121 545 single nucleotide polymorphisms (SNPs) that were more abundant in buffalo-derived strains. High-resolution phylogenetic trees were constructed with selected informative SNPs that allowed the investigation of possible complex recombination events among ancestors of the extant strains. We further analysed the dN/dS ratio (non-synonymous substitutions per non-synonymous site divided by synonymous substitutions per synonymous site) for 4011 coding genes to estimate potential selective pressure. Genes under possible positive selection were identified that may, in turn, assist in the identification of immunogenic proteins or vaccine candidates. This study elucidated the phylogeny of T. parva strains based on genome-wide SNPs analysis with prediction of possible past recombination events, providing insight into the migration, diversification, and evolution of this parasite species in the African continent.     

Comparisons of dN/dS are time dependent for closely related bacterial genomes

The ratio of non-synonymous (dN) to synonymous (dS) changes between taxa is frequently computed to assay the strength and direction of selection. Here we note that for comparisons between closely related strains and/or species a second parameter needs to be considered, namely the time since divergence of the two sequences under scrutiny. We demonstrate that a simple time lag model provides a general, parsimonious explanation of the extensive variation in the dN/dS ratio seen when comparing closely related bacterial genomes. We explore this model through simulation and comparative genomics, and suggest a role for hitch-hiking in the accumulation of non-synonymous mutations. We also note taxon-specific differences in the change of dN/dS over time, which may indicate variation in selection, or in population genetics parameters such as population size or the rate of recombination. The effect of comparing intra-species polymorphism and inter-species substitution, and the problems associated with these concepts for asexual prokaryotes, are also discussed. We conclude that, because of the critical effect of time since divergence, inter-taxa comparisons are only possible by comparing trajectories of dN/dS over time and it is not valid to compare taxa on the basis of single time points.     

Gene expression,chromosome heterogeneity and the fast-X effect in mammals

The higher rate of non-synonymous over synonymous substitutions (dN/dS) of the X chromosome compared with autosomes is often interpreted as a consequence of X hemizygosity. However, other factors, such as gene expression, are also known to vary between X and autosomes. Analysing 4800 orthologues in six mammals, we found that gene expression levels, associated with GC content, fully account for the variation in dN/dS between X and autosomes with no detectable effect of hemizygosity. We also report an extensive variance in dN/dS and gene expression between autosomes.     

Phylogenetic relationships of the Cobitoidea (Teleostei: Cypriniformes) inferred from mitochondrial and nuclear genes with analyses of gene evolution

The superfamily Cobitoidea of the order Cypriniformes is a diverse group of fishes, inhabiting freshwater ecosystems across Eurasia and North Africa. The phylogenetic relationships of this well-corroborated natural group and diverse clade are critical to not only informing scientific communities of the phylogeny of the order Cypriniformes, the world's largest freshwater fish order, but are key to every area of comparative biology examining the evolution of traits, functional structures, and breeding behaviors to their biogeographic histories, speciation, anagenetic divergence, and divergence time estimates. In the present study, two mitochondrial gene sequences (COI, ND4+5) and four single-copy nuclear gene segments (RH1, RAG1, EGR2B, IRBP) were used to infer the phylogenetic relationships of the Cobitoidea as reconstructed from maximum likelihood (ML) and partitioned Bayesian Analysis (BA). Analyses of the combined mitochondrial/nuclear gene datasets revealed five strongly supported monophyletic Cobitoidea families and their sister-group relationships: Botiidae+(Vaillantellidae+(Cobitidae+(Nemacheilidae+Balitoridae))). These recovered relationships are in agreement with previous systematic studies on the order Cypriniformes and/or those focusing on the superfamily Cobitoidea. Using these relationships, our analyses revealed pattern lineage- or ecological-group-specific evolution of these genes for the Cobitoidea. These observations and results corroborate the hypothesis that these group-specific-ancestral ecological characters have contributed in the diversification and/or adaptations within these groups. Positive selections were detected in RH1 of nemacheilids and in RAG1 of nemacheilids and genus Vaillantella, which indicated that evolution of RH1 (related to eye's optic sense) and RAG1 (related to immunity) genes appeared to be important for the diversification of these groups. The balitorid lineage (those species inhabiting fast-flowing riverine habitats) had, as compared with other cobitoid lineages, significantly different dN/dS, dN and dS values for ND4 and IRBP genes. These significant differences are usually indicative of weaker selection pressure, and lineage-specific evolution on genes along the balitorid lineage. Furthermore, within Cobitoidea, excluding balitorids, species living in subtropics had significantly higher dN/dS values in RAG1 and IRBP genes than those living in temperate and tropical zones. Among tropical cobitoids, genes COI, ND5, EGR2B, IRBP and RH1, had a significantly higher mean dS value than those species in subtropical and temperate groups. These findings suggest that the evolution of these genes could also be ecological-group-specific and may have played an important role in the adaptive evolution and diversification of these groups. Thus, we hypothesize that the genes included in the present study were actively involved in lineage- and/or ecological-group-specific evolutionary processes of the highly diverse Cobitoidea. These two evolutionary patterns, both subject to further testing, are hypothesized as integral in the diversification with this major clade of the world's most diverse group of freshwater fishes.     

Lineage‐specific sequence evolution and exon edge conservation partially explain the relationship between evolutionary rate and expression level in A. thaliana

Rapidly evolving proteins can aid the identification of genes underlying phenotypic adaptation across taxa, but functional and structural elements of genes can also affect evolutionary rates. In plants, the ‘edges’ of exons, flanking intron junctions, are known to contain splice enhancers and to have a higher degree of conservation compared to the remainder of the coding region. However, the extent to which these regions may be masking indicators of positive selection or account for the relationship between dN/dS and other genomic parameters is unclear. We investigate the effects of exon edge conservation on the relationship of dN/dS to various sequence characteristics and gene expression parameters in the model plant Arabidopsis thaliana. We also obtain lineage‐specific dN/dS estimates, making use of the recently sequenced genome of Thellungiella parvula, the second closest sequenced relative after the sister species Arabidopsis lyrata. Overall, we find that the effect of exon edge conservation, as well as the use of lineage‐specific substitution estimates, upon dN/dS ratios partly explains the relationship between the rates of protein evolution and expression level. Furthermore, the removal of exon edges shifts dN/dS estimates upwards, increasing the proportion of genes potentially under adaptive selection. We conclude that lineage‐specific substitutions and exon edge conservation have an important effect on dN/dS ratios and should be considered when assessing their relationship with other genomic parameters.     

Two variants of the assembly factor Surf1 target specific terminal oxidases in Paracoccus denitrificans

Biogenesis of cytochrome c oxidase (COX) relies on a large number of assembly proteins, one of them being Surf1. In humans, the loss of Surf1 function is associated with Leigh syndrome, a fatal neurodegenerative disorder. In the soil bacterium Paracoccus denitrificans, homologous genes specifying Surf1 have been identified and located in two operons of terminal oxidases: surf1q is the last gene of the qox operon (coding for a ba₃-type ubiquinol oxidase), and surf1c is found at the end of the cta operon (encoding subunits of the aa₃-type cytochrome c oxidase). We introduced chromosomal single and double deletions for both surf1 genes, leading to significantly reduced oxidase activities in membrane. Our experiments on P. denitrificans surf1 single deletion strains show that both Surf1c and Surf1q are functional and act independently for the aa₃-type cytochrome c oxidase and the ba₃-type quinol oxidase, respectively. This is the first direct experimental evidence for the involvement of a Surf1 protein in the assembly of a quinol oxidase. Analyzing the heme content of purified cytochrome c oxidase, we conclude that Surf1, though not indispensable for oxidase assembly, is involved in an early step of cofactor insertion into subunit I.     

Complete mitochondrial genomes from four subspecies of common chaffinch (Fringilla coelebs): New inferences about mitochondrial rate heterogeneity,neutral theory,and phylogenetic relationships within the order Passeriformes

We describe whole mitochondrial genome sequences from four subspecies of the common chaffinch (Fringilla coelebs), and compare them to 31 publicly available mitochondrial genome sequences from other Passeriformes. Rates and patterns of mitochondrial gene evolution are analyzed at different taxonomic levels within this avian order, and evidence is adduced for and against the nearly neutral theory of molecular evolution and the role of positive selection in shaping genetic variation of this small but critical genome. We find evidence of mitochondrial rate heterogeneity in birds as in other vertebrates, likely due to differences in mutational pressure across the genome. Unlike in gadine fish and some of the human mitochondrial work we do not observe strong support for the nearly neutral theory of molecular evolution; instead evidence from molecular clocks, distribution of dN/dS ratios at different levels of the taxonomic hierarchy and in different lineages, McDonald–Kreitman tests within Fringillidae, and site-specific tests of selection within Passeriformes, all point to a role for positive selection, especially for the complex I NADH dehydrogenase genes. The protein-coding mitogenome phylogeny of the order Passeriformes is broadly consistent with previously-reported molecular findings, but provides support for a sister relationship between the superfamilies Muscicapoidea and Passeroidea on a short basal internode of the Passerida where relationships have been difficult to resolve. An unexpected placement of the Paridae (represented by Hume's groundpecker) within the Muscicapoidea was observed. Consistent with other molecular studies the mtDNA phylogeny reveals paraphyly within the Muscicapoidea and a sister relationship of Fringilla with Carduelis rather than Emberiza.     

Replacement of a terminal cytochrome c oxidase by ubiquinol oxidase during the evolution of acetic acid bacteria

The bacterial aerobic respiratory chain has a terminal oxidase of the heme-copper oxidase superfamily, comprised of cytochrome c oxidase (COX) and ubiquinol oxidase (UOX); UOX evolved from COX. Acetobacter pasteurianus, an α-Proteobacterial acetic acid bacterium (AAB), produces UOX but not COX, although it has a partial COX gene cluster, ctaBD and ctaA, in addition to the UOX operon cyaBACD. We expressed ctaB and ctaA genes of A. pasteurianus in Escherichia coli and demonstrated their function as heme O and heme A synthases. We also found that the absence of ctaD function is likely due to accumulated mutations. These COX genes are closely related to other α-Proteobacterial COX proteins. However, the UOX operons of AAB are closely related to those of the β/γ-Proteobacteria (γ-type UOX), distinct from the α/β-Proteobacterial proteins (α-type UOX), but different from the other γ-type UOX proteins by the absence of the cyoE heme O synthase. Thus, we suggest that A. pasteurianus has a functional γ-type UOX but has lost the COX genes, with the exception of ctaB and ctaA, which supply the heme O and A moieties for UOX. Our results suggest that, in AAB, COX was replaced by β/γ-Proteobacterial UOX via horizontal gene transfer, while the COX genes, except for the heme O/A synthase genes, were lost.     

Comparative genomic analysis of Mycobacterium tuberculosis clinical isolates

Background

Due to excessive antibiotic use, drug-resistant Mycobacterium tuberculosis has become a serious public health threat and a major obstacle to disease control in many countries. To better understand the evolution of drug-resistant M. tuberculosis strains, we performed whole genome sequencing for 7 M. tuberculosis clinical isolates with different antibiotic resistance profiles and conducted comparative genomic analysis of gene variations among them.

Results

We observed that all 7 M. tuberculosis clinical isolates with different levels of drug resistance harbored similar numbers of SNPs, ranging from 1409–1464. The numbers of insertion/deletions (Indels) identified in the 7 isolates were also similar, ranging from 56 to 101. A total of 39 types of mutations were identified in drug resistance-associated loci, including 14 previously reported ones and 25 newly identified ones. Sixteen of the identified large Indels spanned PE-PPE-PGRS genes, which represents a major source of antigenic variability. Aside from SNPs and Indels, a CRISPR locus with varied spacers was observed in all 7 clinical isolates, suggesting that they might play an important role in plasticity of the M. tuberculosis genome. The nucleotide diversity (Л value) and selection intensity (dN/dS value) of the whole genome sequences of the 7 isolates were similar. The dN/dS values were less than 1 for all 7 isolates (range from 0.608885 to 0.637365), supporting the notion that M. tuberculosis genomes undergo purifying selection. The Л values and dN/dS values were comparable between drug-susceptible and drug-resistant strains.

Conclusions

In this study, we show that clinical M. tuberculosis isolates exhibit distinct variations in terms of the distribution of SNP, Indels, CRISPR-cas locus, as well as the nucleotide diversity and selection intensity, but there are no generalizable differences between drug-susceptible and drug-resistant isolates on the genomic scale. Our study provides evidence strengthening the notion that the evolution of drug resistance among clinical M. tuberculosis isolates is clearly a complex and diversified process.

Electronic supplementary material

The online version of this article (doi: 10.1186/1471-2164-15-469) contains supplementary material, which is available to authorized users.     

Saline Irrigation Affects Belonolaimus longicaudatus and Hoplolaimus galeatus on Seashore Paspalum

Seashore paspalum (Paspalum vaginatum) has great potential for use in salt-affected turfgrass sites. Use of this grass on golf courses, athletic fields, and lawns in subtropical coastal areas may aid in conservation of freshwater resources. Belonolaimus longicaudatus and Hoplolaimus galeatus are considered among the most damaging root pathogens of turfgrasses in Florida. Glasshouse experiments were performed in 2002 and 2003 to examine the effects of increasing levels of irrigation salinity on B. longicaudatus and H. galeatus. Irrigation treatments were formulated by concentrating deionized water to six salinity levels (0, 5, 10, 15, 20, and 25 dS/m). Final population densities of H. galeatus followed a negative linear regression (r² = 0.92 and 0.83; P <= 0.01) with increasing salinity levels. Final population densities of B. longicaudatus were quadratically (r² = 0.72 and 0.78; P <= 0.01) related to increasing salinity levels from 0 to 25 dS/m. An increase in population densities of B. longicaudatus was observed at moderate salinity levels (10 and 15 dS/m) compared to 0 dS/m. Root-length comparisons revealed that B. longicaudatus caused root stunting at low salinity levels, 0 to 10 dS/m, but roots were not affected at 15 to 25 dS/m. These results indicate that the ability of B. longicaudatus to feed and stunt root growth was negatively affected at salinity levels of 15 dS/m and above.     

Apparent Selection Intensity for the Cytochrome Oxidase Subunit I Gene Varies with Mode of Reproduction in Echinoderms

When most amino acid substitutions in protein-coding genes are slightly deleterious rather than selectively neutral, life history differences can potentially modify the effective population size or the selective regime, resulting in altered ratios of non-synonymous to synonymous substitutions among taxa. We studied substitution patterns for the mitochondrial cytochrome oxidase subunit I (COI) gene in a sea star genus (Leptasterias spp.) with an obligate brood-protecting mode of reproduction and small-scale population genetic subdivision, and compared the results to available COI sequences in nine other genera of echinoderms with pelagic larvae: three sea stars, five sea urchins and one brittle star. We predicted that this life history difference would be associated with differences in the ratio of non-synonymous (dN) to synonymous (dS) substitution rates. Leptasterias had a significantly greater dN/dS ratio (both between species and within species), a significantly smaller transition/transversion rate ratio, and a significantly lower average nucleotide diversity within species, than did the non-brooding genera. Other explanations for the results, such as altered mutation rates or selective sweeps, were not supported by the data analysis. These findings highlight the potential influence of reproductive traits and other life history factors on patterns of nucleotide substitution within and between species.     

Characterization of the complete mitochondrial genome of Tryporyza incertulas, in comparison with seven other Pyraloidea moths

The complete 15,223-bp mitochondrial genome (mitogenome) of Tryporyza incertulas (Walker) (Lepidoptera: Pyraloidea: Crambidae) was determined, characterized and compared with seven other species of superfamily Pyraloidea. The order of 37 genes was typical of insect mitochondrial DNA sequences described to date. Compared with other moths of Pyraloidea, the A + T biased (77.0%) of T. incertulas was the lowest. Eleven protein-coding genes (PCGs) utilized the standard ATN, but cox1 used CGA and nad4 used AAT as the initiation codons. Ten protein-coding genes had the common stop codon TAA, except nad3 having TAG as the stop codon, and cox2, nad4 using T, TA as the incomplete stop codons, respectively. All of the tRNA genes had typical cloverleaf secondary structures except trnS1(AGN), in which the dihydrouridine (DHU) arm did not form a stable stem-loop structure. There was a spacer between trnQ and nad2, which was common in Lepidoptera moths. A 6-bp motif ‘ATACTA’ between trnS2(UCN) and nad1, a 7-bp motif “AGC(T)CTTA” between trnW and trnC and a 6-bp motif “ATGATA” of overlapping region between atp8 and atp6 were found in Pyraloidea moths. The A + T-rich region contained an ‘ATAGT(A)’-like motif followed by a poly-T stretch. In addition, two potential stem-loop structures, a duplicated 19-bp repeat element, and two microsatellites ‘(TA)₁₂’ and ‘(TA)₉’ were observed in the A + T-rich region of T. incertulas mitogenome. Finally, the phylogenetic relationships of Pyraloidea species were constructed based on amino acid sequences of 13 PCGs of mitogenomes using Bayesian inference (BI) and maximum likelihood (ML) methods. These molecular-based phylogenies supported the morphological classification on relationships within Pyraloidea species.     

Characterization of the complete mitochondrial genome of Chilo auricilius and comparison with three other rice stem borers

The mitogenome of Chilo auricilius (Lepidoptera: Pyraloidea: Crambidae) was a circular molecule made up of 15,367 bp. Sesamia inferens, Chilo suppressalis, Tryporyza incertulas, and C. auricilius, are closely related, well known rice stem borers that are widely distributed in the main rice-growing regions of China. The gene order and orientation of all four stem borers were similar to that of other insect mitogenomes. Among the four stem borers, all AT contents were below 83%, while all AT contents of tRNA genes were above 80%. The genomes were compact, with only 121–257 bp of non-coding intergenic spacer. There are 56 or 62-bp overlapping nucleotides in Crambidae moths, but were only 25-bp overlapping nucleotides in the noctuid moth S. inferens. There was a conserved motif ‘ATACTAAA’ between trnS2 (UCN) and nad1 in Crambidae moths, but this same region was ‘ATCATA’ in the noctuid S. inferens. And there was a 6-bp motif ‘ATGATAA’ of overlapping nucleotides, which was conserved in Lepidoptera, and a 14-bp motif ‘TAAGCTATTTAAAT’ conserved in the three Crambidae moths (C. suppressalis, C. auricilius and T. incertulas), but not in the noctuid. Finally, there were no stem-and-loop structures in the two Chilo moths.     

HIV-1 Tat protein directly induces mitochondrial membrane permeabilization and inactivates cytochrome c oxidase

The Trans-activator protein (Tat) of human immunodeficiency virus (HIV) is a pleiotropic protein involved in different aspects of AIDS pathogenesis. As a number of viral proteins Tat is suspected to disturb mitochondrial function. We prepared pure synthetic full-length Tat by native chemical ligation (NCL), and Tat peptides, to evaluate their direct effects on isolated mitochondria. Submicromolar doses of synthetic Tat cause a rapid dissipation of the mitochondrial transmembrane potential (ΔΨ_m) as well as cytochrome c release in mitochondria isolated from mouse liver, heart, and brain. Accordingly, Tat decreases substrate oxidation by mitochondria isolated from these tissues, with oxygen uptake being initially restored by adding cytochrome c. The anion-channel inhibitor 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) protects isolated mitochondria against Tat-induced mitochondrial membrane permeabilization (MMP), whereas ruthenium red, a ryanodine receptor blocker, does not. Pharmacologic inhibitors of the permeability transition pore, Bax/Bak inhibitors, and recombinant Bcl-2 and Bcl-XL proteins do not reduce Tat-induced MMP. We finally observed that Tat inhibits cytochrome c oxidase (COX) activity in disrupted mitochondria isolated from liver, heart, and brain of both mouse and human samples, making it the first described viral protein to be a potential COX inhibitor.     

Testing the neutral theory of molecular evolution using genomic data: a comparison of the human and bovine transcriptome

Despite growing evidence of rapid evolution in protein coding genes, the contribution of positive selection to intra- and interspecific differences in protein coding regions of the genome is unclear. We attempted to see if genes coding for secreted proteins and genes with narrow expression, specifically those preferentially expressed in the mammary gland, have diverged at a faster rate between domestic cattle (Bos taurus) and humans (Homo sapiens) than other genes and whether positive selection is responsible. Using a large data set, we identified groups of genes based on secretion and expression patterns and compared them for the rate of nonsynonymous (dN) and synonymous (dS) substitutions per site and the number of radical (Dr) and conservative (Dc) amino acid substitutions. We found evidence of rapid evolution in genes with narrow expression, especially for those expressed in the liver and mammary gland and for genes coding for secreted proteins. We compared common human polymorphism data with human-cattle divergence and found that genes with high evolutionary rates in human-cattle divergence also had a large number of common human polymorphisms. This argues against positive selection causing rapid divergence in these groups of genes. In most cases dN/dS ratios were lower in human-cattle divergence than in common human polymorphism presumably due to differences in the effectiveness of purifying selection between long-term divergence and short-term polymorphism.     

Identification,genotyping, and molecular evolution analysis of duck circovirus

Duck circovirus (DuCV) is a contagious immunosuppressive virus affecting many duck species, which is responsible for multiple outbreaks in poultry industries worldwide. In this study, the first DuCV isolate GH01 was identified in Sichuan by PCR, which shared a high level of nucleotide identity (81.8–99.4%) with sequences of other DuCV isolates available in GenBank. Comparative phylogenetic and pairwise sequence comparison analyses indicated that DuCV could be divided into two genotypes (DuCV-1 and DuCV-2) and six subtypes (1a, 1b, 1c, 2a, 2b and 2c) based on the complete genome sequence. The results revealed that both DuCV-1 and DuCV-2 had evolved from the same ancestor but undergone divergent evolution. Interestingly, phylogenetic analyses indicated that three isolates were classified into a cluster DuCV-2a using complete DuCV genome sequence and cap gene, except rep gene. Recombination analyses revealed that DuCV-2a arose from recombination between DuCV-1a and DuCV-2b isolates within the rep genes, and the recombination events mainly occur both in non-structural protein coding region and structural protein coding region. In addition, the mechanisms of recombination supporting the genetic variability in DuCV isolates were investigated. Likewise, selective pressure indicated that purifying selection had been a major driving force in maintaining diversity among the DuCV isolates. Because eradicating the virus from commercial ducks is impossible, it is necessary to take effective control measures and implement them throughout the world.     

Investigation of cytocrom c oxidase gene subunits expression on the Multiple sclerosis

INTRODUCTION:

Multiple sclerosis (MS) is an autoimmune inflamatory disease, which affects the (Central Nervous System) and leads to the destruction of myelin and atrophy of the axons. Genetic factors, in addition to environmental ones, seem to play a role in MS. Numerous studies have reported mitochondrial defects including a reduction in cytochrome c oxidase (COX) complex function related to the reduction of mitochondrial genes expression in the cortex tissue of patients with MS have been reported.

MATERIALS AND METHODS:

This study aimed to assess COX5B and COX2 genes expression in MS patients and compare it with normal subjects. We determine expression levels of genes COX5B and COX2, and also gene reference ß-actine using real–time polymerase chain reaction (RT-PCR) method. Data were obtained and obtained and standardized with the gene reference and were analyzed using independent sample t-test with SPSS and Excel programs.

RESULT AND DISCUSSION:

The resultshowed COX5B gene expression reduced significant in MS patients compared to normal subjects (P < −0.05) whereas, there was no significant difference in the COX2 gene expression between normal subjects and patients. Thus, it can be claimed that down-regulation of mitochondrial electron transport chain genes supported the hypothesis that hypoxia-like tissue injury in MS may be due to mitochondrial genes, different expression impairment.     

Genetic compatibility between Anopheles lesteri from Korea and Anopheles paraliae from Thailand

To assess differentiation and relationships between Anopheles lesteri and Anopheles paraliae we established three and five iso-female lines of An. lesteri from Korea and An. paraliae from Thailand, respectively. These isolines were used to investigate the genetic relationships between the two taxa by crossing experiments and by comparing DNA sequences of ribosomal DNA second internal transcribed spacer (ITS2) and mitochondrial DNA cytochrome c oxidase subunit I (COI) and subunit II (COII). Results of reciprocal and F₁-hybrid crosses between An. lesteri and An. paraliae indicated that they were compatible genetically producing viable progenies and complete synaptic salivary gland polytene chromosomes without inversion loops in all chromosome arms. The pairwise genetic distances of ITS2, COI and COII between these morphological species were 0.040, 0.007-0.017 and 0.008-0.011, respectively. The specific species status of An. paraliae in Thailand and/or other parts of the continent are discussed.     

Differential expression, phosphorylation of COX subunit 1 and COX activity during diapause phase in the cotton bollworm, Helicoverpa armigera

Helicoverpa armigera (Lepidoptera, Noctuidae) is an important agricultural pest with a pupal diapause. Cytochrome c oxidase (COX) is a key speed-limited enzyme of oxidative phosphorylation in mitochondria for ATP production. A differentially expressed cDNA fragment encoding COX subunit 1 (cox1) was cloned by differential display-PCR from the pupal brain at diapause termination with an injection of ecdysone. We then obtained the full length of H. armigera cox1 (Hea-cox1) cDNA which has an open reading frame of 1530 nucleotides encoding a putative protein of 510 amino acid residues, with CGA as a start codon. To evaluate the response to different energy demands during pupal development and at diapause termination, we assessed the expression of Hea-cox1 mRNA and protein, COX activity and its phosphorylation. The results show that Hea-cox1 expression at the mRNA and protein levels is associated with COX activity, and high levels of Hea-cox1 expression and COX activity are present in nondiapause pupae, suggesting that low energy metabolism provided by oxidative phosphorylation in mitochondria in diapause individuals is necessary. After diapause is broken by injection of 20-hydroxyecdysone, expression of Hea-cox1 mRNA and protein increases gradually and COX activity increases significantly. Furthermore, Hea-cox1 phosphorylation is closely correlated with COX activity, suggesting that reversible protein phosphorylation may play a key role in insect diapause by suppressing the rate of energy production.