Phylogenetic Position of the Mitochondrion-Lacking Protozoan Trichomonas tenax,Based on Amino Acid Sequences of Elongation Factors 1α and 2

Major parts of amino-acid-coding regions of elongation factor (EF)-1α and EF-2 in Trichomonas tenax were amplified by PCR from total genomic DNA and the products were cloned into a plasmid vector, pGEM-T. The three clones from each of the products of the EF-1α and EF-2 were isolated and sequenced. The insert DNAs of the clones containing EF-1α coding regions were each 1,185 bp long with the same nucleotide sequence and contained 53.1% of G + C nucleotides. Those of the clones containing EF-2 coding regions had two different sequences; one was 2,283 bp long and the other was 2,286 bp long, and their G + C contents were 52.5 and 52.9%, respectively. The copy numbers of the EF-1α and EF-2 gene per chromosome were estimated as four and two, respectively. The deduced amino acid sequences obtained by the conceptual translation were 395 residues from EF-1α and 761 and 762 residues from the EF-2s. The sequences were aligned with the other eukaryotic and archaebacterial EF-1αs and EF-2s, respectively. The phylogenetic position of T. tenax was inferred by the maximum likelihood (ML) method using the EF-1α and EF-2 data sets. The EF-1α analysis suggested that three mitochondrion-lacking protozoa, Glugea plecoglossi, Giardia lamblia, and T. tenax, respectively, diverge in this order in the very early phase of eukaryotic evolution. The EF-2 analysis also supported the divergence of T. tenax to be immediately next to G. lamblia. Received: 15 February 1996 / Accepted: 28 June 1996     

Nucleotide Sequence of the SrRNA Gene of Entamoeba gingivalis: Applications for Construction of a Species-Specific DNA Probe and Phylogenetic Analysis

The small subunit ribosomal RNA (SrRNA) gene of Entamoeba gingivalis was amplified by PCR and the product of 1.9-kbp sequence was cloned into a plasmid vector pUC18. Four clones were isolated and sequenced. The insert DNAs were 1918- to 1921-bp long and A+T rich (65.5%). The four SrRNA sequences of E. gingivalis were found to be aligned with those of nine related protozoans while searching for E. gingivalis-specific sequences. A sequence of 28 oligonucleotides was chosen, chemically synthesized, and labeled with digoxigenin for use as a DNA probe. The probe thus constructed was shown to hybridize only with either the SrRNA-coding DNAs or the cells of the two E. gingivalis strains and not with those of other protozoans or oral fungi tested. A representative SrRNA-sequence was analyzed and a phylogenetic tree was constructed by the neighbor-joining (NJ) method. Among the protists examined, E. gingivalis was placed next to Entamoeba histolytica as expected from the traditional taxonomy.     

Molecular prevalence and zoonotic potential of trichomonads from oral cavities in household dogs

This study investigated the molecular prevalence of oral trichomonads in household dogs. Of the 144 dogs, 21 (14.6%, 21/144) tested positive for oral trichomonads. The prevalence was significantly higher in dogs with severe gingivitis (gingival index 3: 30.0%, 8/26) than that in normal dogs (gingival index 0: 2.7%, 1/37). Therefore, an interaction between oral trichomonads and the development of periodontal disease is suggested. Of the 21 positive samples, 16 isolates were T. brixi, four isolates were T. tenax, and one was Tetratrichomonas sp. Considering T. tenax is recognized as a zoonotic agent, transmission between dogs and humans cannot be neglected.     

Pollen‐feeding in hover‐flies (Diptera: Syrphidae)

Analyses of the pollen contents of the crop and intestine of 11 species of New Zealand Syrphidae . showed that small, sparsely haired hover‐flies with unbranched hairs, short, simple bristles, and a short proboscis had ingested at least 99% anemophilous pollens, and that larger, more hairy hover‐flies with pollen‐collecting hairs, long, spirally grooved bristles, and elongate mouthparts had ingested pollens almost exclusively from nectar‐bearing flowers. Pollen‐feeding behaviour was studied in one hairy species, the drone‐fly Eristalis tenax, and in one sparsely‐haired species, Melanostoma fasciatum. Using granulated charcoal as a substitute for pollen, it was found that in E. tenax particles trapped among the body hairs are combed off by the front and hind tibiae and transferred to pollen‐retaining bristles on the front and hind tarsi respectively. Particles retained among the front tarsal bristles are ingested directly from the bristles. Those retained by the hind tarsi are transferred in flight by leg‐scraping movements to the front tarsi, from which they are subsequently eaten. E. tenax also eats pollen directly from anthers. In M. fasciatum apparently all the pollen ingested is taken directly from anther lobes or stigmas. The few pollen grains that adhere to the body of this species are combed off by the front and hind tibiae and transferred to the front and hind tarsi, but are not retained there because the bristles are short and simple. The mouthparts, hairs, and bristles of E. tenax and M. fasciatum are illustrated. Drawings of leg movements associated with pollen collection and ingestion, and photographs showing leg scraping in E. tenax are included. Morphological similarities between drone‐flies and honey‐bees, previously regarded as the result of mimicry, can be explained by convergent evolution in response to similar food‐gathering behaviour. Probably the majority of Syrphidae, and also the related Acroceridae, collect pollen by means of branched or curly‐tipped hairs.     

Demography and population parameters of two species of eristaline flower flies (Diptera,Syrphidae, Eristalini)

The importance of eristaline flies (Diptera, Syrphidae, Eristalini) as pollinators in natural ecosystems and for agricultural crops is well known. However, in-depth studies on the life cycle of most of these species have yet to be carried out. The aim of this research was to study the life cycle of Eristalis tenax (Linnaeus, 1758) and Eristalinus aeneus (Scopoli, 1763) in order to improve the current rearing system employed at the University of Alicante. The results were analysed using the age-stage, two-sex life table method. As one of the main results, the mean duration of the life history of E. tenax and E. aeneus was 46.06 and 65.12 days (d), respectively. The most critical step for both species was found at the beginning of the larval stage (first instar), when the highest mortality was recorded. Population parameters were also analysed and compared. The intrinsic rate of increase (r), the finite rate of increase (λ), the net reproductive rate (R₀) and the mean generation time (T) were 0.09 (d⁻¹), 1.09 (d⁻¹), 42.11 offspring and 40.97 d, in the case of E. aeneus and 0.05 (d⁻¹), 1.05 (d⁻¹), 23.13 offspring and 59.23 d, in the case of E. tenax. These results indicate that the current rearing system is more efficient for E. aeneus, which displays a faster population growth. However, some modifications need to be implemented to improve the production of E. tenax.     

Oxidation of organic compounds to CO2 with sulfur or thiosulfate as electron acceptor in the anaerobic hyperthermophilic archaea Thermoproteus tenax and Pyrobaculum islandicum proceeds via the citric acid cycle

The oxidation of organic compounds with elemental sulfur or thiosulfate as electron acceptor was studied in the anaerobic hyperthermophilic archaea Thermoproteus tenax and Pyrobaculum islandicum. T. tenax was grown on either glucose or casamino acids and sulfur; P. islandicum on peptone and either elemental sulfur or thiosulfate as electron acceptor. During exponential growth only CO₂ and H₂S rather than acetate, alanine, lactate, and succinate were detected as fermentation products of both organisms; the ratio of CO₂/H₂S formed was 1:2 with elemental sulfur and 1:1 with thiosulfate as electron acceptor. Cell extracts of T. tenax and P. islandicum contained all enzymes of the citric acid cycle in catabolic activities: citrate synthase, aconitase, isocitrate dehydrogenase (NADP⁺-reducing), oxoglutarate: benzylviologen oxidoreductase, succinyl-CoA synthetase, succinate dehydrogenase, fumarase and malate dehydrogenase (NAD⁺-reducing). Carbon monoxide dehydrogenase activity was not detected. We conclude that in T. tenax and P. islandicum organic compounds are completely oxidized to CO₂ with sulfur or thiosulfate as electron acceptor and that acetyl-CoA oxidation to CO₂ proceeds via the citric acid cycle.     

Mitochondrial DNA diversity of Coilia mystus (Clupeiformes: Engraulidae) in three Chinese estuaries

We used sequences of mitochondrial cytb and 16SrRNA gene segments in order to clarify the genetic diversity and population structure in three Chinese estuary populations of Coilia mystus: 21 individuals from ChangJiang River (Yangtze River) estuary, 22 from MinJiang River estuary, and 22 from ZhuJiang River (Pearl River) estuary (65 individuals total). We obtained 607 base pairs of consensus cytb sequence. Thirty four distinct haplotypes were detected among the 65 cytb sequences. The indexes of nucleotide diversity (π) in these three populations were ChangJiang 0.533%, MinJiang 1.135%, and ZhuJiang 0.268%. MinJiang is the largest of the three populations. Genetic distances within the populations were between 0.3 and 1.2%, and 0.8 to 10.8% among populations. The largest genetic distance was 10.8% between the ChangJiang and ZhuJiang populations, and the smallest was 0.8% between MinJiang and ZhuJiang populations. Analysis of molecular variance (AMOVA) analysis revealed that variation among populations accounts for 90.25% of total variation, suggesting that this is the main source of total variance. We obtained 470 base pairs of consensus sequence of 16SrRNA. We detected 19 distinct haplotypes among the 65 sequences. The indexes of nucleotide diversity (π) in these three populations were ChangJiang 0.108%, MinJiang 0.843%, and ZhuJiang 0.097%. MinJiang is also the largest among these three populations. Genetic distances were between 0.1 and 0.9% within populations and 0.5 to 1.9% between populations. The largest genetic distance was the 1.9% between the ChangJiang and MinJiang populations, and the smallest was 0.5% between the MinJiang and the ZhuJiang populations. AMOVA analysis disclosed that variation among populations accounts for 74.61% of total variation, suggesting that this is the main source of total variation. The results of this study suggest that the three Coilia mystus populations, especially the most isolated Changjiang population, have developed significant genetic structure.     

Characterization of 13 newly isolated strains of anaerobic, cellulolytic, thermophilic bacteria

Characteristics of 13 newly isolated thermophilic, anaerobic, and cellulolytic strains were compared with previously described strains of Clostridium thermocellum: ATCC 27405 and JW20 (ATCC 31549). Colony morphology, antibiotic sensitivity, fermentation end-products, and cellulose degradation were documented. All 13 strains were sensitive to erythromycin (5 μg/ml) and chloramphenicol (25 μg/ml), and all strains but one were sensitive to kanamycin (20 μg/ml). Polymerase chain reaction (PCR) amplification using primers based on gene sequences from C. thermocellum ATCC 27405 was successful for all 13 strains in the case of the hydrogenase gene and 11 strains in the case of phosphotransacetylase/acetate kinase genes. Ten strains amplified a product of the expected size with primers developed to be specific for C. thermocellum 16SrRNA primers. Two of the 13 strains did not amplify any product with the PCR primers designed for the phosphotransacetylase/acetate kinase and 16SrRNA primers. A MboI-like GATC- recognizing restriction activity was present in all of the five strains examined. The results of this study have several positive implications with respect to future development of a transformation system for cellulolytic thermophiles. Journal of Industrial Microbiology & Biotechnology (2001) 27, 275–280. Received 12 September 2000/ Accepted in revised form 20 November 2000     

New species of parasitic copepods of the Genus Acusicola (Poecilostomatoida: Ergasilidae) from gill filaments of coastal and freshwater Brazilian fishes, and proposition of Acusicola rogeri n. sp. for A. tenax sensu Cressey & Collette (1970)

Four new species of Acusicola are described based on adult females found on gill filaments of several species of coastal and freshwater fishes caught in Brazilian basins. The distribution area of the genus in Brazil is significantly enlarged, since until now the previous Brazilian species had been known only from Amazonian fishes. The specimens identified as Acusicula tenax by Cressey and Collette (1970) are considered representatives of a new species, A. rogeri. A key for identification of the species of Acusicola is provided.     

Essential Oil Composition and Volatile Profile of Seven Helichrysum Species Grown in Italy

Helichrysum genus consists of about 600 species widespread throughout the world, especially in South Africa and in the Mediterranean area. In this study the aroma profile (HS‐SPME) and the EO compositions of seven Helichrysum species (H. cymosum, H. odoratissimum, H. petiolare, H. fontanesii, H. saxatile, H. sanguineum, and H. tenax) were evaluated. All the plants were grown in Italy under the same growth conditions. The volatile constituents, particularly monoterpenes, depended by the plant's genotype and ecological adaptation. This study represents the first headspace evaluation on the selected plants and the results evidenced that monoterpenes represented the main class of constituents in five of the seven species analysed (from 59.2% to 95.0%). The higher content in sesquiterpene hydrocarbons was observed in the Mediterranean species of H. sanguineum (68.0%). Only H. saxatile showed relative similar abundance of monoterpenes and sesquiterpene hydrocarbons. The essential oil composition of the majority of examined species are characterised by high percentage of sesquiterpenes (especially β‐caryophyllene and δ‐cadinene) ranging from 51.3% to 92.0%, except for H. cymosum, H. tenax, and H. sanguineum leaves where monoterpenes predominated (from 51.7% to 74.7%).     

Intra-puparial development in the hoverflies Eristalinus aeneus and Eristalis tenax (Diptera: Syrphidae)

The intra-puparial development of 150 pupae of Eristalinus aeneus (Scopoli, 1763) and Eristalis tenax (Linnaeus, 1758) was analyzed. Individuals were obtained from the sixth laboratory generation kept under artificial rearing conditions at the facilities of the University of Alicante (Spain). The experiment was conducted at 25 ± 1°C temperature, 50 ± 5% relative humidity, and 12:12 hr (L:D) of photoperiod. Groups of 10 pupae were collected every 6 hr over 48 hr, after that period, pupae were collected daily until the adult emergence. They were fixed in 5% formic acid and preserved in 70% ethanol. Fixed pupae were dissected and photographed. The chronology and morphological changes that take place during the intra-puparial development in both species were analyzed and compared. Five phases were observed: prepupa, before 6 hr; cryptocephalic pupa, between 6 and 24 hr; phanerocephalic pupa, between 24 and 30 hr; pharate adult, after 30 hr; and the adult imago, restricted to the very end of the development process just before adult emergence. In total, the intra-puparial development lasted 189 ± 4 hr in E. aeneus and 192 ± 3 hr in E. tenax, with the pharate adult the longest phase (some 81% of the total developmental time). These data can be used to develop accurate cold storage protocols during artificial rearing of both pollinator species, avoiding critical events during the development and increasing survival.     

Effects of light and temperature on the attachment and development of <Emphasis Type="BoldItalic">Gloiopeltis tenax</Emphasis> and <Emphasis Type="BoldItalic">Gloiopeltis furcata</Emphasis> tetraspores

Two 60-day experiments were conducted to study the influence of photon flux density (PFD) and temperature on the attachment and development of Gloiopeltis tenax and Gloiopeltis furcata tetraspores. In the first experiment, tetraspores of the two Gloiopeltis species were incubated at five temperature ranges (8°C, 12°C, 16°C, 20°C, 24°C) under a constant PFD of 80 μmol photons m⁻² s⁻¹ with a photoperiod of 12:12. In a second experiment, tetraspores were incubated under five PFD gradients (30, 55, 80, 105, 130 μmol photons m⁻² s⁻¹) at a constant temperature of 16°C with a photoperiod of 12:12. Maximum density of attached tetraspores was observed at 16°C for both species. Maximum per cent of spore germinating into disc was recorded at 12–16°C for G. tenax and 8–12°C for G. furcata. Maximum per cent of discs producing erect axes for G. tenax and G. furcata were recorded at 24°C and 20°C, respectively. Light had no significant effect on tetraspore attachment and developing into disc, but it affected the growth, sprouting and survival of its discs. Under 30–55 μmol photons m⁻² s⁻¹, the discs of the two species of Gloiopeltis did not form thallus until the end of the experiment. Optimum PFD range for G. tenax discs was 80–105 μmol photons m⁻² s⁻¹, whilst it was 80–130 μmol photons m⁻² s⁻¹ for G. furcata. Results presented in this study are expected to assist the progress of artificial seeding of Gloiopeltis.     

Homologies to plastid DNA in the nuclear and mitochondrial genomes of potato

Summary Potato plastid DNA clones, representing onefourth of the potato plastome complexity and containing sequences of the 16SrRNA, rps16, atpA, atpE, psaA, psaB, trnK, trnV, and trnG genes, were used as hybridization probes on nuclear- and mitochondrial-enriched DNAs. Each probe hybridized to multiple nuclear restriction fragments distinct from the plastid cleavage products generated by the same endonucleases. The nuclear hybridizable fragments are highly methylated at their Hpall target sequences (C/CGG). In some instances, the transfer seemed to involve plastid regions of several kilobase pairs, as reflected by the co-integration in the nucleus of restriction sites that are distant in the plastome. Three clones hybridized additionally to distinct mitochondrial fragments. These results indicate that extensive DNA transfers did occur between plastids and other organelles in potato.     

An in-depth study of the larval head skeleton and the external feeding structures related with the ingestion of food particles by the eristaline flower flies Eristalis tenax and Eristalinus aeneus

There is an important lack of knowledge regarding the mechanisms and morphology of the structures involved in the feeding process of the bee-mimicking eristaline flower fly species (Diptera: Syrphidae). In order to look more deeply into their larval feeding biology, a morphological study of the head skeleton and the external feeding structures of the three larval instars of Eristalis tenax (L.) and Eristalinus aeneus (Scopoli), important pollinators and bioindicators of water quality, was carried out, using scanning electron microscopy and confocal laser scanning microscopy. Additionally, an analysis of the ingested particle size was conducted, using various pollen grains and fungal spores. The main differences, found between the third instars of both species, consisted of the more restrictive mandibular lobes of E. tenax, which prevent the ingestion of bigger particles, and a wider filtering area in the cibarium of E. aeneus, which increases the number of particles that can be retained and ingested. Eristalinus aeneus is able to ingest bigger particles (80–100 μm) than E. tenax (70–80 μm). These results suggest that the structures involved in the feeding process of E. aeneus are less restrictive, thus increasing its survival chances when, under artificial rearing conditions, the same medium is used for both species. This study may contribute to the improvement in artificial rearing for future studies on pollination and decomposition.     

Structure of genes and an insertion element in the methane producing archaebacterium Methanobrevibacter smithii

Summary DNA fragments cloned from the methanogenic archaebacterium Methanobrevibacter smithii which complement mutations in the purE and proC genes of E. coli have been sequenced. Sequence analyses, transposon mutagenesis and expression in E. coli minicells indicate that purE and proC complementations result from the synthesis of M. smithii polypeptides with molecular weights of 36,697 and 27,836 respectively. The encoding genes appear to be located in operons. The M. smithii genome contains 69% A/T basepairs (bp) which is reflected in unusual codon usages and intergenic regions containing approximately 85% A/T bp. An insertion element, designated ISM1, was found within the cloned M. smithii DNA located adjacent to the proC complementing region. ISM1 is 1381 bp in length, has 29 bp terminal inverted repeat sequences and contains one major ORF encoded in 87% of the ISM1 sequence. ISM1 is mobile, present in approximately 10 copies per genome and integration duplicates 8 bp at the site of insertion. The duplicated sequences show homology with sequences within the 29bp terminal repeat sequence of ISM1. Comparison of our data with sequences from halophilic archaebacteria suggests that 5GAANTTTCA and 5TTTTAATATAAA may be consensus promoter sequences for archaebacteria. These sequences closely resemble the consensus sequences which precede Drosophila heatshock genes (Pelham 1982; Davidson et al. 1983). Methanogens appear to employ the eubacterial system of mRNA: 16SrRNA hybridization to ensure initiation of translation; the consensus ribosome binding sequence is 5AGGTGA.     

Ribosomal DNA gene and phylogenetic relationships of Diplura and lower Hexapods

The monophyly of Diplura and its phylogenetic relationship with other hexapods are important for understanding the phylogeny of Hexapoda. The complete 18SrRNAgene and partial 28SrRNA gene (D3-D5 region) from 2 dipluran species (Campodeidae and Japygidae), 2 proturan species, 3 collembolan species, and 1 locust species were sequenced. Combining related sequences in GenBank, phylogenetic trees of Hexapoda were constructed by MP method using a crustaceanArtemia salina as an outgroup. The results indicated that: (i) the integrated data of 18SrDNA and 28SrDNA could provide better phylogenetic information, which well supported the monophyly of Diplura; (ii) Diplura had a close phylogenetic relationship to Protura with high bootstrap support.     

Temporal patterns of genetic and phenotypic variation in the epidemiologically important drone fly, Eristalis tenax

Eristalis tenax L. (Diptera: Syrphidae) is commonly known as the drone fly (adult) or rat‐tailed maggot (immature). Both adults and immature stages are identified as potential mechanical vectors of mycobacterial pathogens, and early‐stage maggots cause accidental myiasis. We compared four samples from Mount Fru?ka Gora, Serbia, with the aim of obtaining insights into the temporal variations and sexual dimorphism in the species. This integrative approach was based on allozyme loci, morphometric wing parameters (shape and size) and abdominal colour patterns. Consistent sexual dimorphism was observed, indicating that male specimens had lighter abdomens and smaller and narrower wings than females. The distribution of genetic diversity at polymorphic loci indicated genetic divergence among collection dates. Landmark‐based geometric morphometrics revealed, contrary to the lack of divergence in wing size, significant wing shape variation throughout the year. In addition, temporal changes in the frequencies of the abdominal patterns observed are likely to relate to the biology of the species and ecological factors in the locality. Hence, the present study expands our knowledge of the genetic diversity and phenotypic plasticity of E. tenax. The quantification of such variability represents a step towards the evaluation of the adaptive potential of this species of medical and epidemiological importance.     

三重PCR检测鱼类致病性嗜水气单胞菌

[目的]建立一种能够快速准确地检测致病性嗜水气单胞菌的PCR.方法.[方法]根据嗜水气单胞菌的16S rRNA、气溶素基因(aer)和丝氨酸蛋白酶基因(ahp)的保守序列设计了3对引物,然后进行了PCR反应条件的优化、特异性和敏感性的检测并与普通的细菌分离鉴定进行了临床样本和人工攻毒样本检出率的比较.[结果]该方法特异性好,只对致病性嗜水气单胞菌呈阳性扩增;敏感性高,最低可检测100fg的细菌DNA模版.对临床疑似黄鳝(Monopterus albus)样本的检出率为81.8%,高于细菌分离的40.9%;对人工攻毒鲫鱼(Carassius auratus)样本的检出率为87.5%,高于细菌分离的67.5%.[结论]本方法的成功建立,实现在同一反应管中同时对16SrRNA、aer和ahp的检测,避免了只针对aer或ahp单个毒力基因的PCR检测方法可能存在的漏检和误检,为致病性嗜水气单胞菌的诊断、大规模检疫、流行病学调查等提供了一种快速、准确而有效的检测方法.     

The relationship between precursor and mature forms of the 23 S ribosomal RNA

Summary Oligonucleotide fingerprinting shows the precursor form of the 23S ribosomal RNA fromBacillus megaterium to be larger than its mature counterpart, by some 8 percent, or approximately 250 nucleotides. It can further be shown that the 23SrRNA precursor doesnot contain the 5SrRNA sequence, as had been previously suggested.