LATITUDINAL CLINE IN DROSOPHILA MELANOGASTER FOR KNOCKDOWN RESISTANCE TO ETHANOL FUMES AND FOR RATES OF RESPONSE TO SELECTION FOR FURTHER RESISTANCE

We have introduced a device for selecting Drosophila for increased resistance to very high concentrations of ethanol fumes. This device has enabled us to: 1) select quickly and easily over a thousand flies at a time, and 2) score the knockdown time of every fly in the distribution, while causing very little injury to the flies. A sample of nine west coast populations of Drosophila melanogaster showed a significant trend toward higher knockdown resistance in more northern populations. A population's level of knockdown resistance was virtually uncorrelated with its alcohol dehydrogenase (Adh) allele frequencies. Five of the above nine populations were then subjected to selection for further knockdown resistance. Each population was divided randomly into four groups of 256 flies: two lines to be selected, and two lines to remain unselected as control lines. In every generation each selected line was measured for knockdown resistance, and the last quartile of flies to be knocked down was saved to continue the selection cycle. Population sizes of the selected and unselected lines were all maintained at 256. Realized heritability, based on the responses to selection of the first four generations, was calculated for each selected line. The five populations were significantly heterogeneous for heritability estimates; the average heritability of the five populations pooled was 0.143 ± 0.019. Over the course of twelve generations, the ten selected lines increased their knockdown times by an average factor of 2.40. Before selection, the five populations were heterogeneous for knockdown resistance, and resistance was greatest among the most northern populations. The amount of change of knockdown resistance over the course of selection was also correlated with latitude: the most southern population increased its knockdown time by a factor of 2.23, and the most northern population increased it by a factor of 2.55. After ten generations of selection, the cline of knockdown resistance was about 4.5 times as steep as that before selection. Small phenotypic differences among populations before selection were thus exaggerated by the action of selection. The differences among populations in their rates of response to selection were attributed to genetic differences that existed before selection. The pattern of change of Adh frequencies over the course of selection was very inconsistent, both among and within populations. From this inconsistency of change of Adh alleles with selection, and the lack of correlation between Adh frequencies and knockdown resistance before selection, we concluded that Adh frequency changes could not have had much effect on the responses of the selected lines.     

Convergence in a distributed nervous system: parallel processing and self-organization

The present findings show that the motor system of the carnivorous sea slug Pleurobranchaea californica consists of parallel, distributed, and interconnected neuronal channels by which motor activity may emerge from the dynamics of the system rather than from "switchboard" circuitry. The findings are shown primarily through the properties of the buccal-cerebral neurons (BCNs) that extensively converge and diverge monosynaptically and polysynaptically onto brain motoneurons, providing them with drive and patterned activity. The motoneurons, some of which are electrically coupled, feed back onto the BCNs. The BCNs are functionally heterogeneous both as a group and individually. Many are multifunctional in that they take part in the generation of different behaviors, and some also appear to change their timing with respect to the phase of the pattern generator in the different motor patterns. In the buccal ganglion, the BCNs affect the characteristics of the pattern generator and may be part of the pattern generator itself. By sending axons to buccal roots and to the brain, some BCNs may act as motoneurons and also integrate the activity of brain motoneurons. Because of the effects produced by the extensive interconnections among such functionally heterogeneous and nonlinear elements, and because the "history" of activity in the system can bias subsequent activity, there is ambiguity in assessing the response properties of neurons by examining them individually or in pairs. Such an assessment requires, first, an understanding of the context of activity in which a neuron becomes coactive, and, second, because of inherent variability in the system, it is necessary to consider the temporal, nonlinear computations of the system as a whole. We discuss the findings with regard to the attractor theory that has been used to study complex mammalian systems but that does not rely on modeling of any neuronal activity. The Pleurobranchaea nervous system may provide the means for studying individual neurons within such analyses of global activity.     

Interactive effects of serotonin and acetylcholine on neurite elongation

Serotonin (5-HT) inhibits elongation of neurites of specific identified neurons. Here we report a novel, growth-enabling action of another neurotransmitter, acetylcholine (ACh). When applied simultaneously with serotonin, ACh prevents the inhibition of Helisoma neuron B19 neurite elongation that would occur in response to application of 5-HT alone. We also report that ACh prevents the rise in growth cone Ca2+ that would occur in response to application of 5-HT alone and that ACh blocks the electrical excitatory effect of 5-HT on neuron B19. These results support the hypothesis that growth cone motility and neurite elongation can be regulated by voltage-gated Ca2+ fluxes and suggest that the dynamics of neurite morphology may be complexly regulated by an array of neurotransmitters, as is functional electrical activity.     

Using wintergreen oil for mounting mosquito larvae: a safer alternative to xylene

Permanent mounting of fourth instar mosquito larvae is essential for identifying Aedes spp. This procedure requires extensive exposure to xylene, a clearing agent in the mounting process. We investigated wintergreen oil as a substitute for xylene. Five hundred larvae were mounted on slides to evaluate shrinkage or expansion of specimens after clearing using xylene or wintergreen oil. We examined the ventral brush and siphonal hair tufts for species identification and for preservation of morphological characteristics after clearing specimens in xylene or wintergreen oil. Shrinkage of the length of whole larvae and width of the head, thorax and abdomen after mounting was significantly greater after clearing with xylene than with wintergreen oil. The length of the comb scale nearest the ventral brush was similar for both clearing agents. The clarity of the specimens after mounting was improved by clearing with wintergreen oil, but the integrity of the ventral brush and siphonal hair tufts were similar for both clearing agents.     

Differential Pavlovian conditioning in the mollusc Pleurobranchaea

The present differential Pavlovian conditioning experiments on the sea slug Pleurobranchaea californica extend conditioning described in a preceding paper and provide the conditioning foundation for studies reported in another accompanying paper comparing learned behavior in whole animals with the behavior and motor patterns of electrophysiological preparations. All animals received two appetitive-conditioned stimuli (CSs), one derived from beer (Sbr) and the other derived from squid muscle (Ssq), in different temporal relationships to an electric shock unconditioned stimulus (UCS). Two groups of animals were run concurrently. One group (n = 19) received Sbr as the CS+ in close temporal pairing with the UCS, and Ssq as the CS- explicitly unpaired with the UCS (Sbr +/Ssq-). The second group (n = 20) received the opposite contingencies (Sbr-/Ssq+). All animals received only one day of conditioning involving 5 trials with an intertrial interval of 2 h. There were two replicate experiments, each involving about half of the total n, and each yielding similar results as the sum we report here. Before conditioning, animals exhibited feeding behavior (extension of the proboscis and bite-strike responses) to both stimuli at similar low thresholds. Conditioning produced long-term behavioral changes in all animals throughout the 4.5-day postconditioning observation period. However, only the Sbr+/Ssq- animals consistently exhibited the appropriate differentially conditioned food-aversion behavior which consisted of strong withdrawal and high-threshold feeding responses to Sbr, and low-threshold feeding responses to Ssq. We discuss the possibility that such differences between Sbr+/Ssq- and Sbr-/Ssq+ conditioning may arise either from inherent differences in the responses of the animals to Sbr and Ssq, or, as seems more likely to us, from training and testing effects produced by differences in the compositions of the two stimuli.     

Ecology of Speciation in the Genus Bacillus

Microbial ecologists and systematists are challenged to discover the early ecological changes that drive the splitting of one bacterial population into two ecologically distinct populations. We have aimed to identify newly divergent lineages (“ecotypes”) bearing the dynamic properties attributed to species, with the rationale that discovering their ecological differences would reveal the ecological dimensions of speciation. To this end, we have sampled bacteria from the Bacillus subtilis-Bacillus licheniformis clade from sites differing in solar exposure and soil texture within a Death Valley canyon. Within this clade, we hypothesized ecotype demarcations based on DNA sequence diversity, through analysis of the clade''s evolutionary history by Ecotype Simulation (ES) and AdaptML. Ecotypes so demarcated were found to be significantly different in their associations with solar exposure and soil texture, suggesting that these and covarying environmental parameters are among the dimensions of ecological divergence for newly divergent Bacillus ecotypes. Fatty acid composition appeared to contribute to ecotype differences in temperature adaptation, since those ecotypes with more warm-adapting fatty acids were isolated more frequently from sites with greater solar exposure. The recognized species and subspecies of the B. subtilis-B. licheniformis clade were found to be nearly identical to the ecotypes demarcated by ES, with a few exceptions where a recognized taxon is split at most into three putative ecotypes. Nevertheless, the taxa recognized do not appear to encompass the full ecological diversity of the B. subtilis-B. licheniformis clade: ES and AdaptML identified several newly discovered clades as ecotypes that are distinct from any recognized taxon.The last decade has revealed unimagined levels of species diversity among the prokaryotes. Annealing experiments with environmental DNA have pushed our estimates of community diversity into the millions of species (20, 56), with a billion species estimated worldwide (15). Sequencing of the 16S rRNA gene has identified tens of thousands of species, yet to be characterized, within a single community (24). Since only about 9,000 species have been recognized and described globally, the task of characterizing the many remaining species presents a profound challenge for microbial systematists and ecologists. In particular, we need to describe the unique ecological role each species plays, the differences that allow them to coexist, and the processes by which they have originated.These key issues may all be addressed by discovering the early ecological changes that drive the splitting of one bacterial population into two ecologically distinct populations. One might expect that a comparison of the most closely related species recognized by bacterial systematics would provide insight into the first ecological differences associated with the splitting of lineages. However, the species of bacterial systematics are often extremely broadly defined, whether measured by diversity of genome content and physiology (22), by DNA sequence (53), or, most importantly, by ecology (61). So, if we are to identify the first ecological changes distinguishing newly divergent lineages, we may need to identify taxa that are more narrowly defined and more newly divergent than the named species.Our approach is to identify bacterial clades with the properties shared by many modern species concepts—that species should each be cohesive (subject to forces that constrain the diversity within the species), irreversibly separate and ecologically distinct from other species, and invented only once (13). These criteria are met by bacterial ecotypes, which are defined as ecologically homogeneous clades, whose diversity is constrained by a force of cohesion, either periodic selection or genetic drift (5, 10, 11, 29, 47, 60). In periodic selection, natural selection favoring each adaptive mutant within an ecotype can purge the ecotype''s diversity genomewide (28, 30), owing to the low rates of recombination in bacteria, occurring usually within an order of magnitude of the mutation rate (6, 7, 33, 57). Because different ecotypes are distinct in their ecological niches, periodic selection and genetic drift cannot extend beyond the limits of an ecotype. The divergence between ecotypes is thus not constrained by periodic selection or drift; also, recombination may decelerate divergence but in the end is not sufficient to prevent adaptive divergence between ecotypes (5, 10). Ecotypes therefore have the species-like properties of cohesion, as well as ecological distinctness and irreversible separateness from other ecotypes (4).Moreover, longstanding ecotypes can be discovered from patterns of DNA sequence diversity, provided that a particular model of bacterial evolution applies. In the Stable Ecotype model, ecotypes are formed only rarely, and there is recurrent purging of diversity within ecotypes, either by periodic selection or by genetic drift (11). In this case, each ecotype can accumulate a unique set of sequence mutations at every gene in the genome over the ecotype''s long history as a distinct lineage, and diversity within each ecotype is recurrently purged, allowing ecotypes to be discovered as DNA sequence clusters for any gene shared among ecotypes (9, 11, 29, 50, 61).We have employed two recently developed algorithms to hypothesize the sequence clusters that correspond to ecotypes and to confirm that the ecotypes so hypothesized are indeed ecologically distinct. AdaptML (25) and Ecotype Simulation (ES) (29) each analyze the evolutionary history of a clade to yield appropriate criteria for demarcating the significant clades corresponding to ecotypes (10). These algorithms have an advantage over the universal molecular thresholds used by bacterial systematics to identify species, because there is no evidence of a single molecular criterion that would be applicable for all bacteria (2, 10, 11), and the criteria used thus far by systematics have frequently led to overly diverse species (11, 53).The ES and AdaptML algorithms have successfully identified extremely closely related ecotypes that would otherwise have been subsumed as unrecognized entities within species (25, 29). Comparisons of the habitat associations of these ecotypes have then led to discovery of the ecological dimensions of ecotype divergence: solar exposure of soil for Bacillus (29), photic zone depth and temperature for Synechococcus from hot springs (61), host range for Legionella (9), and season and particle substrate size for marine Vibrio (25). Discovering the ecological dimensions of early divergence simultaneously addresses the evolutionary origins of bacterial diversity, the basis of niche partitioning, and the unique role each ecotype plays in its community.Here we aim to expand the set of known ecological dimensions of early divergence within the Bacillus subtilis-Bacillus licheniformis clade, which includes B. subtilis and its three subspecies (35, 41), B. vallismortis, B. mojavensis, B. atrophaeus, B. amyloliquefaciens, B. licheniformis, B. sonorensis (38), B. tequilensis (21), clades earlier described as “B. axarquiensis” and “B. malacitensis” (44) but now reclassified as B. mojavensis (59), and the clade earlier described as “B. velezensis” (43) but now reclassified as B. amyloliquefaciens (58). We collected soil following the “Evolution Canyon” paradigm developed by Eviatar Nevo and colleagues: an evolution canyon is an east-west-running canyon, which provides habitats that contrast sharply in solar exposure but are identical in geological and macroclimatic features (36). Comparisons of the biota of the two slopes of evolution canyons have revealed adaptive differences related to solar exposure in a great diversity of organisms, including bacteria, fungi, plants, arthropods, and vertebrates (36). In the present study, we collected soil from a west-running, hot-desert canyon in Death Valley, California, yielding isolates from the south-facing slope (SFS), with the most intense solar exposure; the shadier north-facing slope (NFS); and the canyon bottom''s arroyo (A), with intermediate solar exposure and more access to water than either slope. In addition, we collected soil of two texture types, a sandy soil and a badland-like silty soil, which also differed in their chemical properties and in the plants they supported. We will demonstrate that differences associated with solar exposure and soil texture are among the dimensions of ecological divergence for newly divergent Bacillus ecotypes in the Death Valley canyon.     

Influence of molecular resolution on sequence-based discovery of ecological diversity among Synechococcus populations in an alkaline siliceous hot spring microbial mat

Previous research has shown that sequences of 16S rRNA genes and 16S-23S rRNA internal transcribed spacer regions may not have enough genetic resolution to define all ecologically distinct Synechococcus populations (ecotypes) inhabiting alkaline, siliceous hot spring microbial mats. To achieve higher molecular resolution, we studied sequence variation in three protein-encoding loci sampled by PCR from 60°C and 65°C sites in the Mushroom Spring mat (Yellowstone National Park, WY). Sequences were analyzed using the ecotype simulation (ES) and AdaptML algorithms to identify putative ecotypes. Between 4 and 14 times more putative ecotypes were predicted from variation in protein-encoding locus sequences than from variation in 16S rRNA and 16S-23S rRNA internal transcribed spacer sequences. The number of putative ecotypes predicted depended on the number of sequences sampled and the molecular resolution of the locus. Chao estimates of diversity indicated that few rare ecotypes were missed. Many ecotypes hypothesized by sequence analyses were different in their habitat specificities, suggesting different adaptations to temperature or other parameters that vary along the flow channel.     

A high-speed multispectral spinning-disk confocal microscope system for fluorescent speckle microscopy of living cells

Fluorescent speckle microscopy (FSM) uses a small fraction of fluorescently labeled subunits to give macromolecular assemblies such as the cytoskeleton fluorescence image properties that allow quantitative analysis of movement and subunit turnover. We describe a multispectral microscope system to analyze the dynamics of multiple cellular structures labeled with spectrally distinct fluorophores relative to one another over time in living cells. This required a high-resolution, highly sensitive, low-noise, and stable imaging system to visualize the small number of fluorophores making up each fluorescent speckle, a means by which to switch between excitation wavelengths rapidly, and a computer-based system to integrate image acquisition and illumination functions and to allow a convenient interface for viewing multispectral time-lapse data. To reduce out-of-focus fluorescence that degrades speckle contrast, we incorporated the optical sectioning capabilities of a dual-spinning-disk confocal scanner. The real-time, full-field scanning allows the use of a low-noise, fast, high-dynamic-range, and quantum-efficient cooled charge-coupled device (CCD) as a detector as opposed to the more noisy photomultiplier tubes used in laser-scanning confocal systems. For illumination, our system uses a 2.5-W Kr/Ar laser with 100-300mW of power at several convenient wavelengths for excitation of few fluorophores in dim FSM specimens and a four-channel polychromatic acousto-optical modulator fiberoptically coupled to the confocal to allow switching between illumination wavelengths and intensity control in a few microseconds. We present recent applications of this system for imaging the cytoskeleton in migrating tissue cells and neurons.