Landesforschungsanstalt für Landwirtschaft und Fischerei Mecklenburg-Vorpommern

Single-strand-conformation polymorphism (SSCP) of DNA, a method widely used in mutation analysis, was adapted to the analysis and differentiation of cultivated pure-culture soil microorganisms and noncultivated rhizosphere microbial communities. A fragment (approximately 400 bp) of the bacterial 16S rRNA gene (V-4 and V-5 regions) was amplified by PCR with universal primers, with one primer phosphorylated at the 5' end. The phosphorylated strands of the PCR products were selectively digested with lambda exonuclease, and the remaining strands were separated by electrophoresis with an MDE polyacrylamide gel, a matrix specifically optimized for SSCP purposes. By this means, reannealing and heteroduplex formation of DNA strands during electrophoresis could be excluded, and the number of bands per organism was reduced. PCR products from 10 of 11 different bacterial type strains tested could be differentiated from each other. With template mixtures consisting of pure-culture DNAs from 5 and 10 bacterial strains, most of the single strains could be detected from such model communities after PCR and SSCP analyses. Purified bands amplified from pure cultures and model communities extracted from gels could be reamplified by PCR, but by this process, additional products were also generated, as detected by further SSCP analysis. Profiles generated with DNAs of rhizosphere bacterial communities, directly extracted from two different plant species grown in the same field site, could be clearly distinguished. This study demonstrates the potential of the selected PCR-single-stranded DNA approach for microbial community analysis.

The present study describes the seasonal changes of the fish species composition in three areas of the main channel of the Caeté River estuary, Brazil. The fish faunas of each habitat differed in density, biomass and species composition. Mean fish density and biomass for the Caeté River estuary channel was 0·25 individuals m −2 and 0·9 g m −2 respectively. Analysis of catch data showed that the number of species, total density and total biomass differed significantly between areas and seasons. For the most important species, the mean density of Cathorops spixii, Aspredinichthys filamentosus, Aspredo sp. 2, Pimelodus blochii, Pseudauchnipterus nodosus and Macrodon ancylodon , differed significantly between seasons while the mean density of Stellifer rastrifer , Stellifer microps, Aspredo aspredo , Aspredo sp. 1 and Cynoscion acoupa did not. The mean biomass of these species, with exception of S. microps and Aspredo sp. 1, also differed significantly between seasons. In the Caeté estuary seasonal salinity fluctuations appeared to be the main factor that structured the fish assemblage in the entire estuarine system. At least 85% of the species captured by the artisanal and subsistence fisheries in the Bragantine region required estuarine conditions to complete their life cycle.

A cultivation-independent technique for genetic profiling of PCR-amplified small-subunit rRNA genes (SSU rDNA) was chosen to characterize the diversity and succession of microbial communities during composting of an organic agricultural substrate. PCR amplifications were performed with DNA directly extracted from compost samples and with primers targeting either (i) the V4-V5 region of eubacterial 16S rRNA genes, (ii) the V3 region in the 16S rRNA genes of actinomycetes, or (iii) the V8-V9 region of fungal 18S rRNA genes. Homologous PCR products were converted to single-stranded DNA molecules by exonuclease digestion and were subsequently electrophoretically separated by their single-strand-conformation polymorphism (SSCP). Genetic profiles obtained by this technique showed a succession and increasing diversity of microbial populations with all primers. A total of 19 single products were isolated from the profiles by PCR reamplification and cloning. DNA sequencing of these molecular isolates showed similarities in the range of 92.3 to 100% to known gram-positive bacteria with a low or high G+C DNA content and to the SSU rDNA of gamma-Proteobacteria. The amplified 18S rRNA gene sequences were related to the respective gene regions of Candida krusei and Candida tropicalis. Specific molecular isolates could be attributed to different composting stages. The diversity of cultivated bacteria isolated from samples taken at the end of the composting process was low. A total of 290 isolates were related to only 6 different species. Two or three of these species were also detectable in the SSCP community profiles. Our study indicates that community SSCP profiles can be highly useful for the monitoring of bacterial diversity and community successions in a biotechnologically relevant process.

Sex ratio skews in relation to a variety of environmental or parental conditions have frequently been reported among mammals and, though less commonly, among birds. However, the adaptive significance of such sex ratio variation remains unclear. This has, in part, been attributed to the absence of a low-cost physiological mechanism for sex ratio manipulation by the parent. It is shown here that several recent findings in reproductive biology are suggestive of many potential pathways by which gonadotropins and steroid hormones could interfere with the sex ratio at birth. And these hormone levels are well-known to be influenced by many parameters which have been invoked in correlating with offspring sex ratios. Hence, it is argued that the significant, but inconsistent sex ratio biases reported in mammalian and avian populations are coherent with current knowledge on reproductive physiology in those species. However, whether such variations can be viewed at as a consequence of physiological constraint or as adaptive sex ratio adjustment, has still to be determined.

Genetic profiling techniques of microbial communities based on PCR-amplified signature genes, such as denaturing gradient gel electrophoresis or single-strand-conformation polymorphism (SSCP) analysis, are normally done with PCR products of less than 500-bp. The most common target for diversity analysis, the small-subunit rRNA genes, however, are larger, and thus, only partial sequences can be analyzed. Here, we compared the results obtained by PCR targeting different variable (V) regions (V2 and V3, V4 and V5, and V6 to V8) of the bacterial 16S rRNA gene with primers hybridizing to evolutionarily conserved flanking regions. SSCP analysis of single-stranded PCR products generated from 13 different bacterial species showed fewer bands with products containing V4-V5 (average, 1.7 bands per organism) than with V2-V3 (2.2 bands) and V6-V8 (2.3 bands). We found that the additional bands (>1 per organism) were caused by intraspecies operon heterogeneities or by more than one conformation of the same sequence. Community profiles, generated by PCR-SSCP from bacterial-cell consortia extracted from rhizospheres of field-grown maize (Zea mays), were analyzed by cloning and sequencing of the dominant bands. A total of 48 sequences could be attributed to 34 different strains from 10 taxonomical groups. Independent of the primer pairs, we found proteobacteria (alpha, beta, and gamma subgroups) and members of the genus Paenibacillus (low G+C gram-positive) to be the dominant organisms. Other groups, however, were only detected with single primer pairs. This study gives an example of how much the selection of different variable regions combined with different specificities of the flanking "universal" primers can affect a PCR-based microbial community analysis.

Field studies were done to assess how much of the transgenic, insecticidal protein, Cry1Ab, encoded by a truncated cry1Ab gene from Bacillus thuringiensis (Bt), was released from Bt-maize MON810 into soil and whether bacterial communities inhabiting the rhizosphere of MON810 maize were different from those of the rhizosphere of nontransgenic maize cultivars. Bacterial community structure was investigated by SSCP (single-strand conformation polymorphism) of PCR-amplified 16S rRNA genes from community DNA. Using an improved extraction and detection protocol based on a commercially available ELISA, it was possible to detect Cry1Ab protein extracted from soils to a threshold concentration of 0.07 ng/g soil. From 100 ng of purified Cry1Ab protein added per gram of soil, only an average of 37% was extractable. At both field sites investigated, the amount of Cry1Ab protein in bulk soil of MON810 field plots was always lower than in the rhizosphere, the latter ranging from 0.1 to 10 ng/g soil. Immunoreactive Cry1Ab protein was also detected at 0.21 ng/g bulk soil 7 months after harvesting, i.e. in April of the following year. At this time, however, higher values were found in residues of leaves (21 ng/g) and of roots (183 ng/g), the latter corresponding to 12% of the Cry1Ab protein present in intact roots. A sampling 2 months later indicated further degradation of the protein. Despite the detection of Cry1Ab protein in the rhizosphere of MON810 maize, the bacterial community structure was less affected by the Cry1Ab protein than by other environmental factors, i.e. the age of the plants or field heterogeneities. The persistence of Cry1Ab protein emphasizes the importance of considering post-harvest effects on nontarget organisms.

The gut of insects may harbour one of the largest reservoirs of a yet unexplored microbial diversity. To understand how specific insects select for their own bacterial communities, the structural diversity and variability of bacteria found in the gut of different bee species was analysed. For three successive years, adults and larvae of Apis mellifera ssp. carnica (honey bee), and Bombus terrestris (bumble bee), as well as larvae of Osmia bicornis (red mason bee) were collected at a flowering oilseed rape field. Total DNA was extracted from gut material and the bacterial diversity was analysed, independent of cultivation, by genetic profiling with single-strand conformation polymorphism (SSCP) of polymerase chain reaction (PCR)-amplified partial 16S rRNA genes. The SSCP profiles were specific for all bee species and for larvae and adults. Qualitative and quantitative differences were found in the bacterial community structure of larvae and adults of A. mellifera, but differences in B. terrestris were mainly quantitative. Sequencing of the PCR products revealed a dominance of Alpha-, Beta-, and Gammaproteobacteria, Bacteroidetes, and Firmicutes in all bee species. Single-strand conformation polymorphism profiles suggested a higher abundance and diversity of lactobacilli in adults of A. mellifera than in larvae. Further phylogenetic analyses indicated common bacterial phylotypes for all three bee species, e.g. those related to Simonsiella, Serratia, and Lactobacillus. Clades related to Delftia acidovorans, Pseudomonas aeruginosa or Lactobacillus intestinalis only contained sequences from larvae. Several of the bee-specific clusters also contained identical or highly similar sequences from bacteria detected in other A. mellifera subspecies from South Africa, suggesting the existence of cosmopolitan gut bacteria in bees.

Gonad maturation in Antarctic notothenioid fish is a biennial process although spawning is likely to take place annually. However, part of the populations of Champsocephalus gunnari in the Atlantic Ocean sector do not spawn each year. Gonadosomatic index (GSI) of females is 15–40% at spawning. Apart from a few nototheniid species of the GSI of males is much less and typically only 15–20% of that of females. Length at first spawning may be from 55% of L max onwards, but in many species it is not attained until 70–80% of the maximum length. The only exception is Champsocephalus gunnari at South Georgia which may begin spawning at about 40% of L max . Most species of the Seasonal Pack-ice Zone are autumn/winter spawners, whereas in the High-Antarctic Zone more species spawn in summer and autumn. Spawning time is remarkably constant among populations of some species, in others a latitudinal shift in spawning time is apparent. Fecundity is commonly positively correlated with fish length and weight. It exceeds 100 000 eggs only in a few nototheniid species and is commonly in the order of 1000 to 15–20 000 eggs. Ova diameter varies from 0.8 to 5.0 mm. Egg size distribution among fishes of the Seasonal Pack-ice Zone is bimodal. There is a general trend in nototheniids of increasing egg size and decreasing relative fecundity towards higher latitudes. Incubation time may be up to five months. Eggs of most species are probably left unattended for the long incubation period. Nest guarding has been observed in three species but may be more common in particular among the artedidraconids. A number of reproductive strategies associated with nest guarding, egg size and the duration of the pelagic phase have been identified.

The present study investigated the assemblage of fish species in an intertidal mangrove forest during high tide in a macrotide region. We describe the seasonal changes in the fish assemblage composition in relation to biomass, density, and species number in tidal creeks of the Furo do Meio, Caet Estuary, Brazil. A total of 29 107 individuals of 49 species in 26 families were caught using a block net. Their total weight was 526 kg (total density 0.11 ind. m -2 and total biomass 2.1 g m -2 ). Analysis of the catch data showed that the number of species varied significantly between creeks, and that total fish biomass differed significantly between seasons. The densities and biomass of the 2 most important species, Cathorops pleurops and Colomesus psittacus, were significantly different between seasons. The densities and biomass of C. pleurops, Pterengraulis atherinoides, Pseudauchnipterus nodosus, and Stellifer naso showed significant temporal differences. Significant differences between creeks were observed in the density and biomass of Anchovia clupeoides and Rhinosardinia amazonica. The abundance-biomass comparison (ABC) plots for the fish fauna in the creeks of the Furo do Meio showed that the dominant species increased in number and weight at the beginning of the rainy season. As a result of increased rainfall in March and April, salinity declined to values between 6 and 8 psu. At that time, the dominant species made up more than 60% of the total biomass and density and Hill's index of diversity (N1) declined, whereas the number of species (N0) and evenness (E2) did not change. After April, rainfall decreased, and density and biomass returned to levels similar to those before the rainy season. The number of species and the density and biomass in the mangrove tidal creeks are compared with published data for other tropical and subtropical estuaries. Migration trends were inferred from the results of the seasonal fluctuations of density and biomass of the most important species in the Furo do Meio, and are compared with data from other studies in the main channel of the Caet Estuary.

Available published and unpublished information on the distribution , environment and biology of batoid fishes occurring off Brazil, Uruguay and Argentina is summarized and reviewed for sixty species. Zoogeographic provinces proposed by Lopez (1963, 1964) are considered an adequate framework to define the distribution of these species. The Magellanic fauna, which includes the Pacific Ocean coast off Chile, is a well-defined biological unit. Conversely, the northern fauna changes gradually from the temperate Bonaerensean District off northern Argentina, Uruguay and southern Brazil, to a subtropical and tropical fauna along most of the Brazilian coast. The more drastic change to a truly tropical fauna occurs off French Guiana and Surinam. Within the area studied, rajids are the dominant batoid family, with a large number of rhinobatids and myliobatoids to the north. A more detailed cluster analysis (Jaccard) of batoid distribution patterns, results in nine groups largely corresponding with biological and distributional information: Group I of Magellanic species, Group II of three Magellanic species extending into the Bonaerensean District, a small Group III formed by the deep water skates Bathyraja schroederi , Amblyraja frerichsi and Dasyatis cf . pastinaca , another small Group IV of species with uncommon distributions, Group V of Bonaerensean species, Group VI of relatively rare deep water species, Group VII of northern migrants into the Bonaerensean District, Group VIII of Brazilian species occurring in both the South Brazilian and Brazilian districts, and a completely different Group IX of Northern Brazilian species with their southern distributional limit usually at Rio de Janeiro. A large amount of information is available on many of the species, regarding depth and temperature of occurrence, patterns of distribution, and in many cases reproduction and feeding. Preliminary evaluations of abundance have been obtained for a few species only, but the risk of overfishing is clearly documented for some of them. An odd taxonomic - geographic situation is the status of D . cf. pastinaca , and a peculiar type of cloacal gestation has been described for Benthobatis (similar to that in Squatina ). Studies at community ecology level are discussed and full references provided, including many reports only published as meeting summaries.

Fourteen weeks after field release of luciferase gene-tagged Sinorhizobium meliloti L33 in field plots seeded with Medicago sativa, we found that the inoculant also occurred in bulk soil from noninoculated control plots. In rhizospheres of M. sativa plants, S. meliloti L33 could be detected in noninoculated plots 12 weeks after inoculation, indicating that growth in the rhizosphere preceded spread into bulk soil. To determine whether inoculation affected bacterial diversity, 1,119 bacteria were isolated from the rhizospheres of M. sativa and Chenopodium album, which was the dominant weed in the field plots. Amplified ribosomal DNA restriction analysis (ARDRA) revealed plant-specific fragment size frequencies. Dominant ARDRA groups were identified by 16S rRNA gene nucleotide sequencing. Database comparisons indicated that the rhizospheres contained members of the Proteobacteria (alpha, beta, and gamma subgroups), members of the Cytophaga-Flavobacterium group, and gram-positive bacteria with high G+C DNA contents. The levels of many groups were affected by the plant species and, in the case of M. sativa, by inoculation. The most abundant isolates were related to Variovorax sp., Arthrobacter ramosus, and Acinetobacter calcoaceticus. In the rhizosphere of M. sativa, inoculation reduced the numbers of cells of A. calcoaceticus and members of the genus Pseudomonas and increased the number of rhizobia. Cultivation-independent PCR-single-strand conformation polymorphism (SSCP) profiles of a 16S rRNA gene region confirmed the existence of plant-specific rhizosphere communities and the effect of the inoculant. All dominant ARDRA groups except Variovorax species could be detected. On the other hand, the SSCP profiles revealed products which could not be assigned to the dominant cultured isolates, indicating that the bacterial diversity was greater than the diversity suggested by cultivation.

The literature on the blood chemistry of rainbow trout, Salmo gairdneri Rich., is reviewed from the aspects of experimental methods, electrophoresis, normal values, environmental and endogenous factors including toxic substances and diseases.

A critical review of bark anatomical terms is undertaken to stimulate the discussion on bark terminology. Suggestions are made for a standardised usage of the corresponding terms for facilitating the communication between people working on bark anatomy. A tentative glossary of terms is given at the end of the paper.

Production of the microphytobenthos community of a near-shore reef in the Great Barrier Reef system was measured on 7 occasions over 13 mo using in situ respirometry with dome chambers.

A cultivation-independent approach based on polymerase chain reaction (PCR)-amplified partial small subunit rRNA genes and genetic profiling by single-strand conformation polymorphism (SSCP) was used to characterize the bacterial diversity inhabiting the rhizosphere of maize plants grown on an agricultural field. The community structures of two cultivars, a genetically engineered and a nonengineered variety, different herbicide regimes and soil tillage were compared with each other at two sampling dates. SSCP-profiles were generated with DNA from bacterial cell consortia with primers hybridizing to evolutionarily highly conserved rRNA gene regions. On silver-stained gels, each profile consisted of approx. 50 distinguishable bands. Similarity analyses of patterns recorded by digital image analyses could not detect any difference between cultivars or treatments that was greater than the variability between replicates. A total of 54 sequences recovered from different bands were identified and grouped into operational taxonomical units (OTUs). Surprisingly, only five of 40 OTUs contained sequences of both samplings. Three different bands from a profile were selected to test whether this small overlap was due to an incomplete recovery of sequences. From a faint band, two different OTUs were found when 12 clones were analysed, and from two strong bands 24 and 22 OTUs were detected from a total of 26 and 36 clones, respectively. The OTUs belonged to phylogenetically different groups of bacteria. Gene probes that were developed to target different bands of the profiles, however, indicated in Southern blot analyses that patterns between treatments, replicates and samplings, and even from two different growing seasons were highly conserved. Our study demonstrates that community profiles can consist of more sequences than detectable by staining and that gene probes in Southern blot can be a useful control to investigate the composition of microbial communities by genetic profiles.

Abstract Studies on the nutritional physiology of larval fish should provide the basis for defining the length of the larval period and for understanding the quantitative and the qualitative feed requirements of the larvae. For these purposes, it is necessary to perform both descriptive investigations on the ontogenesis of structures and functions as well as experimental investigations on adaptive strategies of the larvae under changing feeding regimes. In the present communication, examples of both approaches are discussed comparing three species: African catfish Clarias gariepinus , whitefish Coregonus lavaretus , and turbot Scophthalmus maximus . At the onset of exogenous feeding, the digestive system of all three species is sufficiently developed to ensure efficient utilization of live food, but not of dry food. A major event during the subsequent development is the differentiation of the stomach. Evidence exists that for turbot and catfish, a functional stomach is necessary to utilize dry feeds as efficiently as live feeds. Therefore, from a nutritional point of view, in those two species the larval period, during which a special larval diet has to be given, ends with the completion of stomach differentiation. The capacity of the larvae to acclimate physiologically to different nutritional conditions seems to be limited. Using general nutritional indices such as protease activity, RNA/DNA ratio, midgut cell height or nuclear diameter of hepatocytes, larvae of the three species show partly starvation symptoms when reared on dry food. This effect can be explained to some extent by quantitative considerations, i.e., lower food consumption and digestibility is less for dry diets than for live diets. The contribution of the qualitative factors involved in the different performance of larvae reared on dry or live food is presently not well understood. Future studies should: 1) investigate why utilization of dry diets depends on presence of the stomach; 2) define more precisely the quantitative feed requirements of larvae; and 3) search those diet‐induced qualitative differences of larval metabolism which affect growth performance.

Bacterial communities in rhizospheres of transgenic maize (Zea mays, with the pat-gene conferring resistance to the herbicide glufosinate; syn. l-phosphinothricin) were compared to its isogenic, non-transgenic cultivar. Total DNA was extracted from bacterial cell consortia collected from rhizospheres of plants grown in an agricultural field. With the use of three different primer pairs binding to evolutionarily conserved regions of the bacterial 16S rRNA gene, partial sequences were amplified by polymerase chain reaction (PCR). The PCR products were subjected to single-strand conformation polymorphism (SSCP) to generate genetic profiles which corresponded to the diversity of the amplified sequences. Genetic profiles of rhizospheres consisted of 40-60 distinguishable bands depending on the chosen primer pairs, and the variability between independent replicates was very low. Neither the genetic modification nor the use of the herbicide Liberty (syn. Basta; active ingredient: glufosinate) affected the SSCP profiles as investigated with digital image analysis. In contrast, PCR-SSCP profiles of bacterial communities from rhizospheres of sugar beet, grown in the same field as a control crop, were clearly different. A less pronounced but significant difference was also observed with rhizosphere samples from fine roots of maize plants collected 35 and 70 days after sowing. Sequencing of the dominant 30 products from one typical SSCP profile generated from transgenic maize rhizospheres indicated the presence of typical soil and rhizosphere bacteria: half of the bands could be attributed to Proteobacteria, mainly of the alpha- and beta-subgroups. Other SSCP bands could be assigned to members of the following phylogenetic groups: Cytophaga-Flavobacterium-Bacteroides, Chlamydiales-Verrucomicrobium, Planctomyces, Holophaga and to Gram-positive bacteria with a high G+C DNA content.

Abstract A comparison was made of 15 different techniques which are used in assessing soil microbial populations and/or biomasses. These include direct observations (fungal standing crop, fluorescein diacetate active mycelia, acridine orange stained bacteria), cultural methods (bacterial plate counts), physiological methods (total microbial, bacterial and fungal biomasses, O 2 ‐uptake), soil enzyme analyses (dehydrogenase, catalase, alkaline and acid phosphatase, protease, amylase), and ATP‐analyses. The various techniques were applied to six soils known to have different microbial characteristics. The results are discussed with respect to the convertability of counts and measurements into microbial biomasses, the variability of the techniques, the correlations within comparable groups of methods, and the practical limitations in application of individual methods to different soils.

Wolbachia are obligatory, cytoplasmatically inherited alpha-Proteobacteria which are known for infecting the reproductive tissues of many arthropods. Their prevalence in the large group of Collembola, however, is not known, except for PCR detection in the parthenogenetically reproducing species Folsomia candida (Order: Entomobryomorpha; Family: Isotomidae). In this study, fluorescence in situ hybridization on microscopic sections of F. candida specimens indicated that Wolbachia-related bacteria were restricted to tissues of the ovary and brain. PCR with primers designed to detect 16S rRNA genes of Wolbachia were positive with specimens from all of five geographically independent F. candida breeding stocks and with three parthenogenetic species from another order (Poduromorpha; Family Tullbergiidae), i.e. Mesaphorura italica, M. macrochaeta and Paratullbergia callipygos. In contrast, negative results were obtained with the two sexually reproducing species, Isotoma viridis (Isotomidae) and Protaphorura fimata (Poduromorpha; Onychiuridae). The ftsZ gene of Wolbachia could be PCR-amplified from all Wolbachia-positive hosts with the exception of M. macrochaeta. The phylogenetic distances of the ftsZ and 16S rRNA gene sequences reflected the phylogenetic distances of the host organisms but the sequences of Wolbachia were relatively closely related, indicating that Wolbachia infections took place after the Collembola had diversified. Our study confirms a monophyletic branch (supergroup E) of Collembola colonizing Wolbachia and indicates that this group is a sister group of supergroup A, the latter harbouring a high diversity of host organisms within the group of insects.

Carbon fixed in organic form in plants by photosynthesis must be returned to the atmosphere as carbon dioxide for use by future generations of plants and animals. This is accomplished primarily by biodegradation of plant residues in soils and waters. This chapter first discusses the effect of clays on degradation of plant lignins and the effect of clays on biodegradation and stabilization of 14C-Iabeled organic substrate carbons over extended incubation periods. It then explains the effect of clays with and without iron and aluminum oxide surface coatings on biodegradation and stabilization of specific substrate carbons. The chapter also presents comparative decomposition and stabilization studies of specific 14C-Iabeled substrates intimately mixed or complexed with pure clays, metal ions, metal ion-clay complexes, and metal ion-clay-humic colloid complexes. For these studies, incubation times should be 1 yr or longer.