Jürgen Kusch

Structure and variability of bat social calls: implications for specificity and individual recognition

Communication sounds or ‘social calls’ of 16 European bat species (Chiroptera, Vespertilionidae) were recorded at a range of roost and foraging sites. A comparative analysis of more than 5400 individual calls for general structures and for inter- as well as intraspecific variability resulted in 50 types of calls, which differed by their specific structure and by the calling species. These types could be grouped into four different general types of calls, according to the kind and complexity of their structure, independent of the calling species. Distinct types of calls seem to have similar functions in different bat species. One general type may be used predominantly in female–infant interactions as an isolation or direction call, which serves as mutual recognition. This type of social call was also used in ‘tandem flights’ of pairs of bats, which might increase individual knowledge of roost sites and foraging success. A second type was used in mate attraction, and a further one in an aggressive context. The fourth one was used by hindered or distressed bats. The group of ‘aggressive’ calls is least variable, but the complex mating calls and isolation calls are very diverse. Species-specific sound structures were identified, which allowed a computational species distinction. The measured inter-individual variability of social calls should be significant for their functions in individual recognition. So, beyond common features concerning the frequency structure of bat social calls, interspecific differences, as well as the intraspecific variability of details of sonagraphic parameters, should elucidate the specific functions of the calls.

Phylogenetic relationships of non-mitochondrial nucleotide transport proteins in bacteria and eukaryotes

Current knowledge about the nucleotide metabolism of intracellular bacteria is very limited. Here we report on the identification of nucleotide transport proteins (NTT) of two obligate endoparasites, Caedibacter caryophila and Holospora obtusa, both alpha-proteobacteria, which reside in the vegetative macronucleus of Paramecium caudatum. For comparative studies, we also identified the first nucleotide transporter in chloroplasts of a red alga, i.e. Galdieria sulphuraria, and further homologs in plant chloroplasts. Heterologous expression of the NTT proteins from C. caryophila, H. obtusa, and G. sulphuraria in Escherichia coli demonstrate that the nucleotide influx mediated by these transporters is specific for ATP and ADP. The NTT proteins of C. caryophila and H. obtusa exhibit substantial sequence identity with their counterparts in chloroplasts and intracellular bacterial pathogens of humans, but not with the nucleotide transport system of mitochondria. Comprehensive phylogenetic analyses of bacterial and chloroplast NTT proteins showed that homologs in chloroplasts from plants, and green, red, stramenopile and glaucocystophyte algae are monophyletic. In contrast, the evolutionary relationships of the bacterial counterparts appear highly complex. In the presented phylogeny, NTT proteins of C. caryophila and H. obtusa are only distantly related to one another, although these two taxa are close relatives in 16S rRNA trees. The tree topology indicates that some bacterial NTT paralogs have arisen by gene duplications and others by horizontal transfer.

Induction of defensive morphological changes in ciliates

Freshwater ciliates of the genusEuplotes change their morphology in response to the presence of predators. The morphological transformations limit the ability of the predators to ingest the ciliates. Induction of defensive morphology by the predatorsStenostomum sphagnetorum (Turbellaria),Lembadion bullinum (Ciliata),L. magnum, andAmoeba proteus (Rhizopoda) was studied inEuplotes aediculatus andE. octocarinatus. The results suggest the possibility of natural occurrence of predator-induced defensive morphology inEuplotes. A density of 1 predator ml−1 was sufficient to induce significant changes in the morphology ofEuplotes. L. magnum was found at natural population densities of 5 individuals ml−1. Transformations can take place within 2–4 h. Morphological changes are induced by signal substances released from the predators; direct contact between prey and predators is not necessary. The extent of transformation depends on the concentration of the signal substance. The size frequency distribution of the populations only had one peak that was related to predator density. All individuals of a population ofEuplotes changed their morphology according to the predator abundance. This may reduce energy costs for the prey by prevention of unnecessary morphological changes.Amoeba proteus induces morphological changes inE. octocarinatus, but not inE. aediculatus.

Local and Temporal Distribution of Different Genotypes of Pond-Dwelling Stentor coeruleus.

DNA fingerprints of 721 individuals of the freshwater ciliate Stentor coeruleus revealed a heterogeneous local distribution of four genotypes of this species. Different genotypes dominated in seven ponds located 0.1-400 km apart; none of the detected genotypes were detected in all of the ponds. Identities of the relative genotype frequencies in the pond populations varied from 0.00 to 0.99. These values did not correlate to the geographical distance between ponds. Several comparisons of populations showed very high genetic identity (0.99) associated with the largest geographic distance (400 km). The data suggest no strong isolation of populations, but rather relatively high levels of gene flow among several populations. In one pond, significant temporal changes in the relative genotype frequencies accompanied a decrease in the Stentor abundance during the summer of 1996. For one genotype, the changes were reversed until spring of 1997, in the others they were not. Divergent seasonal selection factors may cause such reversible shifts, but chance effects may underlie non-reversed frequency changes.

Population structure of Euplotes ciliates revealed by RAPD fingerprinting

Abstract:Random amplified polymorphic DNA fingerprinting (RAPD fingerprinting) enables the determination of inter- and intraspecific genetic diversity in Euplotes ciliates on a molecular genetic le...

ARDRA and RAPD-fingerprinting reject the sibling species concept for the ciliate Paramecium caudatum (Ciliophora, Protoctista)

Morphologically indistinguishable sibling species also known as syngens are a characteristic taxonomic feature of the ciliate genus Paramecium. This has been convincingly demonstrated for the P. aurelia species complex. For a long time this feature has also been assumed for P. caudatum. Classical morphology based techniques of taxonomic analysis are often inefficient to study sibling specie. We therefore investigated 14 P. caudatum strains of seven supposedly different syngens using random amplified polymorphic DNA (RAPD)‐fingerprinting and amplified ribosomal DNA restriction analyses (ARDRA, Riboprinting). The RAPD patterns revealed by five different random primers were similar between the different strains of the same syngen (similarity index ranging from 73 to 91%) and also between strains of supposedly different syngens (similarity index ranging from 67 to 91%). The amplified 18S rRNA‐fragments of supposedly different syngens, as well as the restriction patterns of these fragments digested by five different endonucleases, were identical for all investigated P. caudatum stains. Consequently we reject the sibling species hypothesis for P. caudatum. According to our molecular analysis, P. caudatum is not a species complex, but just one single species.

SELF-RECOGNITION AS THE ORIGINAL FUNCTION OF AN AMOEBAN DEFENSE-INDUCING KAIROMONE

A 4.5 kg/mol peptide released by an amoeban predator, Amoeba proteus, induced defensive behavioral changes in ciliates of the genus Euplotes. The interspecific signaling molecule named “A-factor” was present in amoebae culture medium at a concentration of 0.01 μg peptide/L, corresponding to 2 × 10−12 mol/L. Amoebae released ∼3 × 105 molecules of A-factor per individual per hour. Prey ciliates added to solutions containing A-factor exhibited predator-avoidance behavior after a few hours. The discovery of a second function for this peptide-kairomone indicated why the predator betrays its presence to the prey and thereby unintentionally protects it. Inhibition of phagocytosis by particle-bound kairomone revealed that it also functions as a self-recognition signal in the predator. Phagocytosis of particles by amoebae depended on the amount of A-factor on the particle surface and decreased in linear correlation to the logarithm of peptide amount. Self-recognition prevents a mutual consumption of amoebae, leading to increased survival of these asexually reproducing organisms. The importance of self-recognition apparently outweighs the disadvantages of defense induction, since only few prey species of the omnivorous predator respond in this manner.

Primary structure and origin of a predator released protein that induces defensive morphological changes in Euplotes

Summary The predatory ciliate Lembadion bullinum releases a chemical compound (L-factor) which induces morphological changes in another ciliate, Euplotes octocarinatus , and some of its relatives. The changes render Euplotes so extended that Lembadion has difficulties engulfing this prey. We have previously shown that the L-factor is a protein with a mass of 31.5 kDa. Here we report the primary structure of the L-factor deduced from cDNA, the heterologous expression of the Lembadion gene encoding the L-factor, the production of an antibody directed against this factor and the labeling of the cell surface of Lembadion with this antibody. From this and from peculiarities in the amino acid composition of the L-factor we conclude that the L-factor is a surface protein of Lembadion or at least a major part of it. To our knowledge the L-factor is the first kairomone whose origin and structure has been identified.

Genetically controlled expression of surface variant antigens in free-living protozoa.

Besides parasitic protozoa some free-living ciliates have the ability to exhibit alternative types of proteins on their cell surface (Sonneborn, 1948; reviews: Schmidt, 1988; Bleyman, 1996; Schmidt, 1996). A range of these exchangeable structurally different surface proteins has been detected in species of the genera Paramecium(Ciliophora) andTetrahymena(Ciliophora). The presence of variable surface proteins is detected by immunochemical techniques. Injections of Parameciaor cells ofTetrahymenaspecies into mammals induce the production of antibodies against surface proteins of the injected cells. Therefore the proteins also are called surface antigens. Treatment of livingParameciaor of Tetrahymenacells with the homologous serum results in immobilization of the ciliates at low serum concentrations, or leads to cell death at higher concentrations. Because of the immobilizing effect of antibodies the corresponding surface antigens also are called “immobilization-antigens”, or “i-antigens.” Recent research involves the detection of mRNA specific for surface antigens by Reverse Transcriptase-PCR techniques (H.W. Breiner, H.J. Schmidt, J. Kusch, unpublished results ), with the aim to investigate single cells from field samples and a possible ecological function of these molecules. Cells that express the same type of surface antigen and therefore can be immobilized by the same antiserum belong to one serotype. Within genetically identical clones cells sometimes appear that are resistant against immobilization via antibodies. These cells induce the production of a different type of antibody after their injection into mammals. The cells represent a further serotype within the ciliate clone. In this way a range of serotypes, corresponding to different surface antigens, was observed in several species of Parameciumand of Tetrahymena(Nanney & Dubert, 1960; Koizumi, 1966; Hiwatashi, 1967; Juergensmeyer, 1969; Sonneborn, 1974; Steers & Barnett, 1982). Seven different serotypes are known forParamecium primaureliaand twelve for P. tetraurelia. Most of the known strains of these species cannot express all of the surface antigen types, e.g., types S, G and D only can be observed in most strains of P. primaurelia. Different surface antigens are generally mutually exclusive (Beale, 1957). Ciliates express only one type of surface antigen at constant environmental conditions, although genes for other surface antigens are present. A property of serotype expression is the ability to switch to another serotype. Among the stimuli for transformation are changes in temperature, pH, and the kind of growth medium, or UV radiation and proteolytic enzymes (Sonneborn, 1970). The inheritance of serotype genes follows Mendelian rules (Beale, 1957). The function of these surface proteins and the mechanism responsible for the variability of their expression are unknown. One suggestion is that they may be a buffer or defense against environmental biotic or abiotic factors (Preer, 1986). Paramecialacking variable surface proteins have never been found.

The Toxic Symbiont Caedibacter caryophila in the Cytoplasm of Paramecium novaurelia.

Endosymbiotic bacteria were observed to inhabit the cytoplasm of the freshwater ciliateParamecium novaurelia. Transmission electron microscopy and toxicity tests with sensitive paramecia showed that the endosymbionts belong to the genusCaedibacter. The bacteria conferred a killer trait to their host paramecia. The production of a proteinaceous inclusion body (“R-body”) in the bacterial cell makes them toxic to other paramecia after they become enclosed in food vacuoles. R-bodies ofCaedibacter sp were associated with phages, which are known in most otherCaedibacter species to code for the R-body proteins. The killer-effect ofP. novaurelia on sensitiveP. caudatum strains was of the “paralysis” type, which is a characteristic of the symbiont speciesCaedibacter caryophila. Until nowC. caryophila was known to inhabit the macronucleus ofParamecium caudatum only. Sequencing of the 16S rRNA-gene proved thatCaedibacter sp from the cytoplasm ofP. novaurelia belongs to the speciesC. caryophila as well. The rDNA-sequence of 1695 bp length differed in a total of only 1 bp from the corresponding gene inC. caryophila from the macronucleus ofP. caudatum. The results indicate that the infection of specific host cell compartments may depend on host genes, but not on different traits of the infecting symbiont species. The occurrence of killer and sensitive paramecia strains together in one pond is discussed with respect to the competitive advantage of the killer trait.