Phylogenetic relationships among the black fly species (Diptera: Simuliidae) of Thailand based on multiple gene sequences

Simulium is a very speciose genus of the black fly family Simuliidae that includes many important pests of humans and animals. Cytotaxonomic and morphological studies have made substantial progress in Simulium systematics. 16S rRNA and ITS-1 DNA sequence studies have assisted this progress. Intensive multi-gene molecular systematic investigations will, however, be required for a comprehensive understanding of the genus’ taxonomy and evolution. Our research was conducted to investigate the relationships of Thai Simulium at the subgeneric, species group and species levels. We also examined the possibility of using mitochondrial DNA sequences to facilitate Simulium species identification. Data were collected from three mitochondrial genes (COI, ND4 and 16S rRNA) and two segments of the nuclear 28S ribosomal RNA (the D1 to D2 and the D4 expansion regions). The subgenera Simulium and Gomphostilbia were monophyletic in most analyses. Nevermannia included Montisimulium but was otherwise monophyletic in multigene analyses. In most analyses, Simulium and Nevermannia were more closely related to each other than to Gomphostilbia which was usually basal. Species groups were generally monophyletic. Within Gomphostilbia, however, the batoense species group was always paraphyletic to the other two species groups found in Thailand. Three species groups in Simulium were not monophyletic. The tendency to gill filament number reduction for some species groups in the subgenus Simulium was associated with a derived position in multigene analyses. Most species were monophyletic with two exceptions that probably represent species complexes and will present difficulties for rapid mitochondrial DNA identification.     

On the use of split moving window analysis for boundary detection in ordered dataseries from benthic communities

The capacity of Split Moving Window (SMW) boundary analysis was tested to detect the depth of the transition of a littoral to a profundal invertebrate community in two datasets that were ordered along a depth gradient. The littoral community was dominated byPolypedilum nubeculosum agg.,Potamopyrgus jenkinsi andGlyptotendipes cf.pallens. The species richness of the profundal community was much lower. Its fauna was dominated byChironomus cf.plumosus andChaoborus flavicans. The transition could be detected with small window sizes. The discontinuities are located at the same depth as the oxygen stratification. We conclude that SMW can be used to study artificial transects. Noisy datasets should be transformed to reduce the variation. The scale dependence of SMW is an advantage. Information about the nature of a discontinuity (transition, trend or local discontinuity) can be obtained by increasing the window size. The location of discontinuities that are detected at the edge of a series, with large windows, should be interpreted carefully due to the existence of a blind zone.     

Chironomidae communities of the near-shore zone in Zegrzyńsky Reservoir,Poland

The structural differences in chironomid communities of the near-shore zone in Zegrzyski Reservoir corresponded to variations in sediments composition. Sandy bottom areas covered by a thin layer of mud were inhabited mostly byPolypedilum, Cryptochironomus andGlyptotendipes larvae.Cladotanytarsus andStictochironomus were often the most numerous in the shallower zone (0.2–0.5 m). Predatory Tanypodinae were found most commonly on the sandy bottom covered by well mineralized plant debris and loose silt. Extremely high numbers and biomass ofChironomus andGlyptotendipes larvae occupied the clay bottom covered by a thick layer of mud. On the basis of developmental stage distribution it was found that at least three generations ofChironomus andGlyptotendipes occurred during the study period (April–October 1988). The combined production estimate for these two taxa exceeds any reported for Chironomidae in lentic or lotic habitats.     

Factors governing the spatial and temporal distribution of Chironomid larvae in the Maarsseveen lakes with special emphasis on the role of oxygen conditions

Distribution patterns of the larvae of Chironomidae are compared in three water systems in The Netherlands, which vary in trophic state and oxygen regimes. The life cycles and flying periods of some dominant chironomid species in two of the investigated lakes, Lakes Maarsseveen I and II, are determined by comparing data on the seasonal variations in larval densities with existing literature on Chironomidae in the Maarsseveen lakes. In the oligo-mesotrophic Lake Maarsseveen I (LM I), hypoxic or anoxic conditions in the hypolimnion are observed only at the end of the stratification period. A clear zonation of the chironomid fauna is present in this lake. The littoral zone is dominated byCladotanytarsus gr.mancus andStictochironomus sticticus, the littoriprofundal zone byTanytarsus bathophilus, and the profundal zone byChironomus anthracinus. In comparison with the other species in LM I,T. bathophilus larvae show the most variable distribution patterns over time. Larvae are found in all depths from July to September, but disappear from the hypolimnion as soon as oxygen conditions deteriorate. In the eutrophic Lake Maarsseveen II (LM II), oxygen depletion of the hypolimnion starts immediately after the onset of the thermal stratification in June, and continues until autumnal turnover in November. In this lake, the chironomid community consists primarily ofS. sticticus andCl. gr.mancus, and is confined to the narrow littoral zone. No chironomid larvae are found in the deeper parts of the lake. The eutrophic Lake Gijster in the Brabantse Biesbosch is a deep, man-made reservoir, that is artificially destratified during the summer. In this lakeTanytarsus bathophilus is found in the profundal sediments, whereas almost noChironomus is found in this zone. It is concluded that oxygen conditions existing in the deeper regions of the investigated lakes in large part determine the occurrence and distribution of chironomid species. The distribution ofT. bathophilus is limited by unfavorable oxygen conditions and not by the trophic state of the lake. These findings are part of a thesis (HEINIS, 1993).     

The structure of diversity in an epiphytic chironomid community

The structure of diversity in a chironomid community inhabiting submerged macrophytes was analysed, including the relationship between predation/competition and chironomid diversity. Diversity as expressed by the Shannon functionH was found to be strongly associated with equitabilityJ but not with species richnessS in this community. Chironomid species richness was correlated with the abundance of diatoms. DiversityH and equitabilityJ were significantly correlated with chironomid density through the year. Invertebrate predators were generally rare and occurred sporadically throughout the year in this habitat, with only three species (Chaetogaster diaphanus, Rhyacophila dorsalis andZavrelimyia sp.) attaining >25% habitat occupancy. Neither these predators nor non-chironomid competitors encountered in the same habitat (Stylaria lacustris, Ophidonais serpentina, Hydroptila sp.,Simulium spp. andHydropsyche siltalai) appeared to affect diversity measures of the chironomid community under study, apart from a weak tendency of highSimulium density negatively affecting the total chironomid abundance. In conjunction with other analyses, this chironomid community seemed to be stochastically dynamic and was little influenced by biotic factors such as predation and competition.     

Co-inoculation with antibiotic-producing bacteria to increase colonization and nodulation by rhizobia

A study was conducted to determine whether colonization of legume roots and nodulation byRhizobium meliloti andBradyrhizobium japonicum could be enhanced by using inocula containing microorganisms that produce antibiotics suppressing soil or rhizosphere inhabitants but not the root-nodule bacteria. An antibiotic-producing strain of Pseudomonas and one of Bacillus were isolated, and mutants ofR. meliloti andB. japonicum sp. resistant to the antibiotics were used. The colonization of the alfalfa rhizosphere and nodulation byR. meliloti were enhanced by inoculation of soil withPseudomonas sp. in soil initially containing 2.7×10⁵ R. meliloti per g. The colonization of soybean roots byB. japonicum was enhanced by inoculating soil with three cell densities ofBacillus sp., and nodulation was stimulated byBacillus sp. added at two cell densities. In some tests, the dry weights of soybeans and seed yield increased as a result of these treatments, and co-inoculation with Bacillus also increased pod formation. Inoculation of seeds withBacillus sp. and the root-nodule bacterium enhanced nodulation of soybeans and alfalfa, but colonization byB. japonicum andR. meliloti was stimulated only during the early period of plant growth. Studies were also conducted withStreptomyces griseus and isolates ofR. meliloti andB. japonicum resistant to products of the actinomycete. Nodulation of alfalfa byR. meliloti was little or not affected by the actinomycete alone; however, both nodulation and colonization were enhanced if the soil was initially amended with chitin andS. griseus was also added. Chitin itself did not affectR. meliloti. Treatments of seeds with chitin orS. griseus alone did not enhance colonization of alfalfa roots byR. meliloti or soybean roots byB. japonicum, but the early colonization of the roots by both bacterial species was promoted if the seeds received both chitin andS. griseus; this treatment also increased nodulation and dry weights of alfalfa and soybeans and the N content of alfalfa. It is suggested that co-inoculation of legumes with antibiotic-producing microorganisms and root-nodule bacteria resistant to those antibiotics is a promising means of promoting nodulation and possibly nitrogen fixation.     

Faecal pellets in streams: their binding, breakdown and utilization

1. Faecal pellets of Gammarus (shredders) and Simulium larvae (suspension feeders) are bound by exopolymers. Immediately after egestion, Gammarus pellets are covered by a peritrophic membrane that breaks up within hours, although pellets remain intact because of internal binding materials. 2. Although they expand soon after egestion, the faecal pellets of Gammarus and Simulium remain intact for more than 30 days. Their internal structure is altered and the main agents of this change are bacteria that have survived passage through the gut (and become bound within pellets). 3. When disrupted physically, freshly egested (1‐ to 2‐day old) Simulium faecal pellets break up into relatively large pieces whereas freshly egested Gammarus faecal pellets break apart into much smaller pieces. Disruption of 30‐day old Simulium faecal pellets results in similar sized pieces to those from freshly egested pellets, but disruption of 30‐day old Gammarus pellets produces pieces that are two orders of magnitude larger than those resulting from disruption of freshly egested pellets. 4. Faecal pellets of Gammarus and Simulium are eaten by stream invertebrates and are sites of microbial breakdown. Faecal pellets are a source of organic matter for benthic invertebrates, bacteria and, indirectly, for plants.     

Congruence between larval ventromental plate ultrastructure and immature morphology in Yama Sublette & Martin and some Oceanian species of Chironomus Meigen (Diptera: Chironomidae)

The morphology of the larval ventromental plates of Y. tahitiensis Sublette & Martin and four Oceanian species of Chironomus Meigen, C. hawaiiensis Grimshaw, C. javanus Kieffer, C. magnivalva Kieffer and C. samoensis Edwards, has been studied by scanning electron micrography. Data on ventromental plate ultrastructure are used in phenetic and cladistic analyses of the relationships of T. tahitiensis and the Oceanian Chironomus to other species in Chironomus and additional genera. The results of these analyses support the current generic status of T. tahitiensis but indicate that Yama is less closely related to Chironomus than previously supposed, a finding that is supported by a reexamination of ‘traditional’ morphological features.     

Hemoglobins of Kiefferulus,sister genus of Chironomus (Diptera: Insecta): Evolution of the Hb VIIB cluster

A genomic clone containing hemoglobin genes was isolated from a species of the chironomid genus Kiefferulus. Eight genes, including an apparent pseudogene, were sequenced and the amino acid sequences of the putative proteins were determined. By comparison to the previously described hemoglobins in the sister-genus Chironomus, they were identified as members of the dimeric Hb VIIB group. The results indicate that the existence of clusters of hemoglobin genes may be a common feature in chironomids and not just confined to Chironomus. The Kiefferulus genes show greatest similarity of amino acid sequence to Hb VIIB-7 from the Chironomus cluster. The results suggest that the ancestral cluster contained at least two gene types, one of which gave rise to VIIB-7 and the Kiefferulus genes while the other gave rise to the other Chironomus VIIB genes. Both clusters appear to have increased in size by duplication or unequal crossing over since the separation of the genera. It also appears that an unrelated gene present in the Chironomus cluster, Hb-Y, arose from a completely independent origin with no apparent equivalent gene anywhere in the genome of Kiefferulus or some other Chironomus species. Correspondence to: J. Martin     

Herbivory and natural selection on flowering phenology in wild sunflower, Helianthus annuus

Plant fitness is strongly affected by flowering phenology, and there are several ecological factors that are thought to shape the distribution of flowering times. One relatively underexamined factor is the timing and intensity of attack by herbivores that feed on flowers or developing seeds. This study tests the hypothesis that herbivores that feed on developing seeds of wild sunflower, Helianthus annuus (Asteraceae), impose selection on flowering phenology. First, the study population was found to contain genetic variation for mean date of flowering, so this trait could evolve if natural selection were operating. Next, the phenological pattern of abundance of five seed-feeding herbivores was documented. Damage by three herbivores, Haplorhynchites aeneus (Cucurlionidae), the head-clipping weevil, Homoeosoma electellum (Lepidoptera: Pyralidae), the sunflower moth, and Suleima helianthana (Lepidoptera: Tortricidae), the sunflower bud moth, was highest early in the flowering season, and declined as the season progressed. Damage by one herbivore, the seed fly Gymnocarena diffusa (Diptera: Tephrididae), was lowest early in the flowering season and increased as the season progressed. Finally, damage by two seed weevils, Smicronyx fulvus and S. sordidus (Curculionidae), whose damage was not distinguished, was constant through the flowering period. Third, damage by Haplorhynchites, Homoeosoma, and Suleima was found to be detrimental to plant fitness, suggesting that plants that flower when these herbivores are not abundant should have higher fitness. Finally, two phenotypic selection analyses were performed. The first included damage by Homoeosoma and Suleima, as well as flowering date, leaf area, and inflorescence diameter, as characters predicting plant fitness. In this analysis directional selection was found to act to decrease damage by the two herbivores, but did not act on flowering date. The second selection analysis was identical except that damage by the two herbivores was not included. In this analysis significant directional selection was found to favor later-flowering plants. Comparison of these two analyses suggests that all selection on flowering phenology is attributable to damage by Homoeosoma and Suleima: plants that flower later avoid damage by these two herbivores. While other influences on flowering phenology, such as pollination, mate availability, and seasonality, have been well documented, this study is one of few to demonstrate natural selection on flowering phenology that is a direct consequence of insect attack. Received: 17 November 1998 / Accepted: 18 July 1999     

A new genus of theActinoplanaceae: Dactylosporangium,gen. nov.

Summary Two aerobic mesophilic species of a new genus belonging to the familyActinoplanaceae are described under the nameDactylosporangium (D. aurantiacum strainD/748 type species andD. thailandensis strainD/449). The new genus is characterized by the production of finger shaped sporangia emerging directly from the vegetative mycelium.The motile sporangiospores, three to four in number are arranged in a single straight row inside the sporangium.The genusActinoplanes of the familyActinoplanaceae was described in 1950 byCouch and is characterized by the bacteria-like, flagellated spores formed in sporangia. Other members of the familyActinoplanaceae have been studied byKarling (1954),Rothwell (1957) andCross et al. (1963) in the United States, byGaertner (1955) in Germany, byVan Brummelen andWent (1957) in Holland, byNonomura andOhara (1960) in Japan, byTaig et al. (1962),Tsyganov et al. (1963), andKoniev et al. (1965) in Russia. Except for the organisms studied byKarling and byRothwell, which undoubtedly belonged to theActinoplanes but were not studied in pure culture, the organisms studied by most of the other authors belonged to the genusStreptosporangium.Three new genera having motile spores were described more recently:Ampullariella andSpirillospora described byCouch (1963, 1964), andPlanomonospora byThiemann et al. (1967b).     

An orphan gyrB in the Mycobacterium smegmatis genome uncovered by comparative genomics

DNA gyrase is an essential topoisomerase found in all bacteria. It is encoded bygyrB andgyrA genes. These genes are organized differently in different bacteria. Direct comparison ofMycobacterium tuberculosis andMycobacterium smegmatis genomes reveals presence of an additionalgyrB inM. smegmatis flanked by novel genes. Analysis of the amino acid sequence of GyrB from different organisms suggests that the orphan GyrB inM. smegmatis may have an important cellular role.     

Homology of Balbiani rings among chironomid species and localization of a new mobile element on the polytene chromosomes

The results ofin situ cross-hybridization of the cloned DNA fragments of BRa, BRb and BRc fromChironomus thummi to the polytene chromosomes of 14Chironomus species andCamptochironomus tentans are presented. BRs of the species studied were demonstrated to contain the homologus DNA sequences. The cloned fragment from the BRa ofC. thummi hybridized with the BRa ofC. piger and with a region on the arm G ofC. tentans andC. plumosus, the latter species had no extra BR. The clone 16 from the BRb ofC. thummi hybridized only with the developed BR on the arm G of all species studied. The sequence from the BRc ofC. thummi was located in the BRc ofC. piger and in the developed BR ofC. plumosus andC. nuditarsis, which were located at the most distal end of arm G. These clones can be used as markers of homologous BRs. The new mobile elements C6.10 fromC. thummi genome were localized on the polytene chromosomes of 10Chironomus species andCamptochironomus tentans. The species of the generaLipiniella, Cryptochironomus andGlyptotendipes did not contain the sequences homologous to ME C6.10.     

Synergistic utilization of dichloroethylene as sole carbon source by bacterial consortia isolated from contaminated sites in Africa

The widespread use and distribution of chloroethylene organic compounds is of serious concern owing to their carcinogenicity and toxicity to humans and wildlife. In an effort to develop active bacterial consortia that could be useful for bioremediation of chloroethylenecontaminated sites in Africa, 16 combinations of 5 dichloroethylene (DCE)-utilizing bacteria, isolated from South Africa and Nigeria, were assessed for their ability to degradecis- andtrans-DCEs as the sole carbon source. Three combinations of these isolates were able to remove up to 72% of the compounds within 7 days. Specific growth rate constants of the bacterial consortia ranged between 0.465 and 0.716 d⁻¹ while the degradation rate constants ranged between 0.184 and 0.205 d⁻¹, with 86.36–93.53 and 87.47–97.12% of the stoichiometric-expected chloride released during growth of the bacterial consortia, incis- andtrans-DCE, respectively. Succession studies of the individual isolates present in the consortium revealed that the biodegradation process was initially dominated byAchromobacter xylosoxidans and subsequently byAcinetobacter sp. andBacillus sp., respectively. The results of this study suggest that consortia of bacteria are more efficient than monocultures in the aerobic biodegradation of DCEs, degrading the compounds to levels that are up to 60% below the maximum allowable limits in drinking water.     

Synthesis of glucosyl-inositol using a CGTase,isolation and characterization of the positional isomers,and assimilation profiles for intestinal bacteria

A novel disaccharide, glucosyl-inositol, was obtained by glucoamylase digestion of the oligoglucosylinositols synthesized frommyo-inositol as an acceptor and -cyclodextrin as a donor by transglucosylation of CGT ase fromBacillus ohbensis. The glucosyl-inositol fraction was separated by ion-exchange column chromatography and two positional isomers contained in the fraction were isolated by crystallization and HPLC on a graphitized carbon column. The structure of one of the two isomers isolated was fully determined as 1L(D)-5-O--D-glucopyranosyl-myo-inositol and another one was presumed as 1D-4-O--D-glucopyranosyl-myo-inositol from physicochemical data, H-H and C-H COSY NMR analyses and FAB-MS spectra. The disaccharide was assimilated byBifidobacterium adolescentis, B. breve, B. infantis andB. longum. On the other hand, it was not utilized byE. coli, Clostridium butyricum, C. clostridiiforme andKlebsiella pneumoniae.     

Structural genesvirC1 andvirC2 of the host range determiningvirC operon are determinants of virulence inAgrobacterium tumefaciens

The host range determiningvir C operon ofAgrobacterium tumefaciens is known to consist of two open rea’ding frames designatedvirC1 andvirC2. Earlier work that employed insertional mutations invirC1 andvirC2 established the role of thevirC2 component in the determination of virulence. In this work a plasmid with an internal deletion invirCl was constructed. This deletion derivative restored virulence to bacteria carrying a mutation in thevirC2 region but not to bacteria carrying avirC1 mutation. This evidence establishes that bothvirC1 andvirC2 are required for efficient host plant transformation byAgrobacterium tumefaciens.     

Interaction of various bacteriocins withKlebsiella edwardsii var.edwardsii

The interaction of four different bacteriocins produced byKlebsiella pneumoniae andCitrobacter freundii strains with cells ofKlebsiella edwardsii var.edwardsii has been studied. All four bacteriocins have different activity spectra. The existence of multi-tolerant and multi-receptor-negative mutants supports the hypothesis that the specific receptor sites for these bacteriocins on sensitive bacteria have some components in common.Bacteriocins S6 and S8, produced byKlebsiella pneumoniae strains inhibit protein biosynthesis. Colicin A, produced byCitrobacter freundii inhibits all macromolecular synthesis, but pre-treatment of sensitive cells with colicin A had no influence on the production of ATP by oxidative phosphorylation in cell homogenates. Bacteriocin G196, also produced byCitrobacter freundii inhibits protein and RNA synthesis, with little effect on DNA synthesis. Homogenates of cells pre-treated with bacteriocin G196, show a substantial phosphorylating activity.The authors wish to thank Dr. W. de Vries for performing P:O measurements. The skilful technical assistance of Miss E. A. Spanjaerdt Speckman and Miss W. M. C. Kapteijn is gratefully acknowledged.The investigations were supported (in part) by the Netherlands Foundation for Chemical Research (SON) with financial aid from the Netherlands Organization for the Advancement of Pure Research (ZWO).     

Investigations concerning the symbiosis of bacteria inTriatoma Infestans (KLUG)

Summary 1. From the proventriculus ofTriatoma infestans aPseudomonas species could always be obtained in pure culture by simple means.2. The bacteria isolated out of the ventriculus of various bugs appeared to agree, not only morphologically but also biochemically and agglutinatorically.3. Further evidence for the identity of the intracellular and the lumen form of this bacterium could be furnished by serological means.4. The assumption expressed byWigglesworth that the infection of the young bug would occur already in the egg and his view on the means by which the egg is infected are very much open to question.5. It does not appear probable that the bacterium, isolated byWigglesworth fromRhodnus prolixus is identical with the proventriculus bacterium ofTriatoma infestans.6. The intracellular bacteria set free into the lumen of the proventriculus have in virtue of their haemodigestive capacity, probably a function in the digestion of the blood sucked by the bug.     

Changes in organic matter composition of forest soil treated with a large amount of urea to promote ammonia fungi and the abilities of these fungi to decompose organic matter

Organic matter composition (lignin, holocellulose, 50% (v/v) methanol extract, water-soluble carbohydrate (WSC) and phenolics (WSP), petroleum ether extract, and ash) of A₀ layer soil treated with 700 g/m² of urea to promote ammonia fungi was investigated in a Japanese red pine (Pinus densiflora) forest. Nine species of fungi were found during the study period of 18 months after the treatment. Of these, seven species belong to the ammonia fungi. WSC content of the treated soil was lower than that of the control. Methanol extract content increased initially after the treatment, then decreased to below the control level. There were no consistent differences in other components between the treated plot and the control. The abilities to decompose cellulose, lignin, chitin, protein and lipid in 18 strains in 10 species of the ammonia fungi were also screened. Cellulose was not lysed byPseudombrophila deerata, Hebeloma spp. andLaccaria bicolor. Strong lignolytic activity was shown byLyophyllum tylicolor, Coprinus echinosporus andP. deerata. Chitin was decomposed byAmblyosporium botrytis, L. tylicolor, C. echinosporus andHebeloma vinosophyllum. All strains possessed proteolytic and lipolytic activities. Supply of glucose to the culture media resulted in weaker enzyme activities except for lignolytic ability.