FMRFamide tagging--how an ionotropic receptor can be used to measure peptide secretion

In this chapter, we describe a technique, FMRFamide tagging, that in principle can be used to measure the release of any sequenced neuropeptide. The method relies upon the addition of an "electrophysiologically active" tag to the prohormone that encodes the neuropeptide of interest. Secretion of the electrophysiological tag (and thus the peptide of interest) is detected by activation of the ionotropic "tag receptor." Both the tagged prohormone and the tag receptor are expressed in the cell type under investigation. Since the tag and the neuropeptide of interest are on the same prohormone they are co-secreted and thus secretion of the tag reflects the co-secretion of the neuropeptide of interest. This method can be used to detect neuropeptide secretion on a millisecond timescale.     

A novel technique that measures peptide secretion on a millisecond timescale reveals rapid changes in release

Neuropeptides are ubiquitous transmitters that have been implicated in a wide variety of physiological and pathological conditions, and it is important to understand the processes that control their secretion. We have developed a technique that measures neuropeptide secretion with high temporal resolution. This method involves placing an electrophysiological "tag" in a neuropeptide prohormone. The tagged prohormone is subsequently expressed together with an ionotropic receptor that binds the tag. Because the neuropeptide of interest and the tag enter the same population of dense core granules, neuropeptide secretion gives rise to fast, synaptic-like currents. Using this method, we show that peptide secretion can be modulated on a millisecond time scale. This technique could be readily adapted to measure the secretion of any neuropeptide.     

Non-verbal behavior as courtship signals: the role of control and choice in selecting partners

In this work, we provide evidence based on direct observation of behavior in encounters of opposite-sexed strangers, that women initiate and “control” the outcome. In the first minute of these videotaped 10-min interactions, neither female “solicitation” behavior nor “negative” behavior is strongly related to professed interest in the man, while female “affirmative” behavior at this stage modulates male verbal output in later stages (4–10 min). Although the rate of female courtship-like behavior is significantly higher in the first minute, it is only in the fourth to tenth minute that the rate of female courtship-like behavior is correlated with professed female interest. We hypothesize that this serves as a strategic dynamic reflecting sexual asymmetry in parental investment and the potential cost of male deception to women. Ambiguous protean behavioral strategies veil individuals' intentions and make their future actions unpredictable. These behavioral strategies may result in men's overestimation of female sexual interest.     

Chelicerae as male grasping organs in scorpions: sexual dimorphism and associated behaviour

Specialised structures that enable males to grasp females during sexual interactions are highly susceptible to selection and thus diverge relatively rapidly over evolutionary time. These structures are often used to test hypotheses regarding sexual selection such as sexually antagonistic co-evolution and sexual selection by female choice. In the present study, we determine whether there is a relationship between a novel record of scorpion sexual dimorphism, the sexual dimorphism of chelicerae (CSD), and the presence of the mating behaviour termed “cheliceral grip” (CG). The presence of both traits in the order Scorpiones is also reviewed from a phylogenetic perspective. The results confirm a strong relationship between CSD and the presence of CG. The morphological and behavioural patterns associated with “CSD–CG” are opposed to the predictions postulated by the hypothesis of sexually antagonistic co-evolution. However, if the female shows resistance after the deposition of the spermatophore, the possibility that the male exerts pressure as a “cryptic form” of coercion to prevent the interruption of mating cannot be ruled out completely. Female choice by “mechanical fit” could be another explanation for some aspects of the CG's contact zone. The possibility that the “CG–CSD” complex has evolved under natural selection in order to ensure sperm transfer is also considered.     

Interaction of Drosophila melanogaster prohormone convertase 2 and 7B2. Insect cell-specific processing and secretion

The prohormone convertases (PCs) are an evolutionarily ancient group of proteases required for the maturation of neuropeptide and peptide hormone precursors. In Drosophila melanogaster, the homolog of prohormone convertase 2, dPC2 (amontillado), is required for normal hatching behavior, and immunoblotting data indicate that flies express 80- and 75-kDa forms of this protein. Because mouse PC2 (mPC2) requires 7B2, a helper protein for productive maturation, we searched the fly data base for the 7B2 signature motif PPNPCP and identified an expressed sequence tag clone encoding the entire open reading frame for this protein. dPC2 and d7B2 cDNAs were subcloned into expression vectors for transfection into HEK-293 cells; mPC2 and rat 7B2 were used as controls. Although active mPC2 was detected in medium in the presence of either d7B2 or r7B2, dPC2 showed no proteolytic activity upon coexpression of either d7B2 or r7B2. Labeling experiments showed that dPC2 was synthesized but not secreted from HEK-293 cells. However, when dPC2 and either d7B2 or r7B2 were coexpressed in Drosophila S2 cells, abundant immunoreactive dPC2 was secreted into the medium, coincident with the appearance of PC2 activity. Expression and secretion of dPC2 enzyme activity thus appears to require insect cell-specific posttranslational processing events. The significant differences in the cell biology of the insect and mammalian enzymes, with 7B2 absolutely required for secretion of dPC2 and zymogen conversion occurring intracellularly in the case of dPC2 but not mPC2, support the idea that the Drosophila enzyme has specific requirements for maturation and secretion that can be met only in insect cells.     

Rapid monitoring of cell size, vitality and lipid droplet development in the oleaginous yeast Waltomyces lipofer

The aim of this work was the development of rapid methods suitable for monitoring the growth of the oleaginous yeast Waltomyces lipofer by means of cell size, vitality and the development of internal lipid droplets throughout different growth phases. Oleaginous yeasts are of interest for the industrial production of lipids and therefore precise monitoring of growth characteristics is needed.This paper provides information about both the method development as well as about examples for their use in monitoring applications. Cell size and shape were determined using FPIA (Flow Particle Image Analysis). Vitality and internal lipid droplets were measured using two independent staining methods for Flow Cytometry. Double staining with cFDA & PI was used for the distinction between “vital”, “sublethal” and “dead” subpopulations, whereas Nile Red allowed the monitoring of lipid accumulation. In this approach the method for vitality measurement was optimized focussing on the staining buffer. An addition of 25 mM citric acid and pH 4.8 revealed to be optimal. The cells in the growth experiment showed a constantly high vitality, which was always above 90%, but slowly decreasing over time. In the course of lipid droplet development it could be seen that the cell size and the Nile Red fluorescence intensity increased. It was demonstrated that the tested method combination provides a powerful tool for rapid fermentation monitoring of the oleaginous yeast W. lipofer, which allows gaining information about the desired growth characteristics in less than 45 min. Further applications for the two methods will be discussed in this article.     

Rethinking ‘style’ for historians and philosophers of science: converging lessons from sexuality, translation, and East Asian studies

Historians and philosophers of science have furnished a wide array of theoretical-historiographical terms to emphasize the discontinuities among different systems of knowledge. Some of the most famous include Thomas Kuhn’s “paradigm”, Michel Foucault’s “episteme”, and the notion of “styles of reasoning” more recently developed by Ian Hacking and Arnold Davidson. This paper takes up this theoretical-historiographical thread by assessing the values and limitations of the notion of “style” for the historical and philosophical study of science. Specifically, reflecting on various methodological and theoretical concerns prompted by sexuality, translation, and East Asian studies, this paper argues that the heretofore ways in which historians and philosophers of science have used the notion of “style” are severely restricted in terms of its mere applicability to the intellectual history of Western science. The particular example of the translation of “homosexuality” into Chinese during the May Fourth era reveals that the notion of “style” has the potential of carrying a much more dynamic conceptual weight, as when used in “styles of argumentation”. The paper also engages briefly with the historiography of scientific “national styles” and ends with some concluding remarks on the limitations of “social histories from below” and the under appreciated importance of “epistemological histories of possibilities”.     

Use of genital inspection and female urine tests to detect oestrus in captive Asian elephants

Captive Asian elephant (Elephas maximus) populations are decreasing due to low birth rates compared to wild elephants. Improving oestrous detection in female elephants is required to ensure successful mating in captive and semi-captive herds. Responsive behaviours of eight semi-captive bull elephants to the uro-genital area (genital inspection test) or urinary pheromones (urine test) of 14 female elephants throughout the oestrous cycle were evaluated. Weekly blood samples were collected for 27 consecutive months (14 months for the genital inspection test and 13 months for the urine test) from female elephants to characterize the patterns of circulating progestagen. Responsive behaviours of bulls were compared between females in the follicular versus the luteal phase of the cycle. The sensitivity and specificity of the genital inspection test were 65% and 68%, while those of the urine test were 52% and 61%, respectively. The bulls showed significantly higher “genital inspection”, “flehmen from genital area” and “trunk on back” behaviours during the genital inspection test, and “flehmen” behaviours during the urine test in oestrous than in non-oestrous females. In sum, this study showed that monitoring sexual behaviours of Asian elephant bulls towards females or their urine can be used to detect the oestrous period. Although the sensitivity and specificity of both tests were not as high as expected, still, these methods appear to be more efficient at detecting oestrous than traditional methods based on mahout estimations of female receptivity. The use of genital inspection and urine tests may lead to more successful matings and thus to creating self-sustaining populations of captive elephants in range countries.     

Signs, communication, and language

Noting the vagueness and confusion in the literature on animal behavior with regard to the phenomenon of communication, an attempt is made to define “sign” and “communication” from the point of view of cybernetics, drawing also on Susanne Langer's philosophy of symbols. The author argues that neither communication nor language can be successfully investigated as long as the dogmatic behaviorist's taboo against goal-directed or purposive activities is maintained. In accord with recent developments in feedback theory, he shows that the cyberneticist's concept of purpose can be used quite legitimately in the scientific analysis of behavior and that it makes possible a viable classification of signs as well as the discrimination of communication from other types of interaction. In conclusion some of Hockett's “design features” of language are discussed and an attempt is made to formulate criteria for the form of communcation that should be called linguistic.     

Comparison of two mass-selective detectors with special reference to the analysis of residues of anabolics

In the analysis of residues of anabolic agents the use of combined techniques, especially gas chromatography—mass spectrometry, is of increasing interest. When this method is used for the analysis of residues of anabolics it is very important to have a knowledge of the characteristics of the different types of apparatus on the market and to be sure that the results are not “apparatus-dependent”. In this study two mass-selective detectors, the Hewlett-Packard 5970 and the ion trap detector 800, were compared by injecting the same samples of derivatized anabolics (standard solutions) into the two systems.     

A stochastic model for chemosensory cell movement: application to neutrophil and macrophage persistence and orientation

Two central features of leukocyte chemosensory movement behavior demand fundamental theoretical understanding. In uniform concentrations of chemoattractant, these cells exhibit a persistent random walk, with a characteristic “persistence time” between significant changes in direction. In chemoattractant concentration gradients, they demonstrate a biased random walk, with an “orientation bias” characterizing the fraction of cells moving up the gradient. A coherent picture of cell-movement responses to chemoattractant requires that both the persistence time and the orientation bias be explained within a unifying framework. In this paper we offer the possibility that “noise” in the cellular signal perception/response mechanism can simultaneously account for these two key phenomena. In particular, we report on a stochastic mathematical model for cell locomotion based on kinetic fluctuations in chemoattractant receptor binding. This model proves to be capable of stimulating cell paths similar to those observed experimentally for two cell types examined to date: neutrophils and alveolar macrophages, under conditions of uniform chemoattractant concentrations as well as chemoattractant concentration gradients. Further, this model can quantitatively predict both cell persistence time and dependence of orientation bias on gradient size. The model also successfully predicts that an increase in persistence time is associated with a decrease in orientation for typical system parameter values, as is observed for alveolar macrophages in comparison to neutrophils. Thus, the concept of signal “noise” can quantitatively unify the major characteristics of leukocyte random motility and chemotaxis. The same level of noise large enough to account for the observed frequency of turning in uniform environments is simultaneously small enough to allow for the observed degree of directional bias in gradients. This suggests that chemosensory cell movement behavior may be based on a “usefully” imperfect integrated signal response system, which allows both random and directed searches under appropriate conditions.     

Polarity of microtubular structures in manchette-like formations: Possible role of the “11th filament”

Bundles of microtubular structures appear in the cytoplasm of spermatids of the African frog Dicroglossus occipitalis. They are observed in the vicinity of axonemes. Natural tubulin polymerization leads to the formation of hooks on microtubular structures. They can be related to experimentally induced tubulin hooks. The direction of curvature of the hooks allows us to define the polarity of the bundles. This is opposite to the polarity of axonemal microtubules: Bundles and axonemes are antiparallel. Under colchicine action, arch-like microtubular structures are shown to open in the same direction as they lock. This enables us to characterize their opening and locking site: It corresponds to the place of the “11th filament” described in microtubular structures such axonemes. The “11th filament” is thus demonstrated to be the most susceptible to natural opening and to the action of colchicine in microtubular structures.     

How is the individuality of a face recognized?

A familiar face is instantly recognized in a crowd. This cannot be achieved through a feature by feature comparison of the observed face with either an average face (norm-based model of face recognition) or with a set of similarly constructed faces stored in memory (exemplar-based model of face recognition). A modified norm-based model is thus proposed. Instead of memorizing an average face, the normal variations for each facial feature are used to construct a multidimensional volume of face-space devoid of unusual features, here defined as features whose metrics lie below the 5th or above the 95th percentiles for that feature. A face consisting of 100 independently variable features will thus have, on average, 10 unusual features. Face identification then becomes exception-reporting. It requires only 10 such rare features to render a given face a one in 10¹³ faces (P=0.05¹⁰=9.8×10⁻¹⁴). In a world containing 6.7×10⁹ people, such a face would be unique. Faces remembered in this way can have their unusual features exaggerated or attenuated without loss of identity. This is the basis of caricatures and anti-caricatures. It also means that individuals belonging to a foreign race, possessing several features with modes beyond the “usual range” of the own-race population, will all look alike. Features that render a face unique in the own-race population are now shared by everyone in the foreign race.Average faces are more beautiful than the faces used in the averaging process. This makes evolutionary sense. Natural selection increases the frequency of fit features at the expense of maladaptive features. “Usual features” are therefore fitter than “unusual features”, and play an important role in mate selection. Such an existing fundamental sexual attribute could easily have been harnessed for the fast and efficient recognition of individuals in the community.     

Praon Haliday (Hymenoptera: Braconidae: Aphidiinae) of Southeastern Europe: key, host range and phylogenetic relationships

A review of species in the genus Praon Haliday, 1833 is presented. Twenty described species are keyed and illustrated with scanning electron micrographs and line drawings. The Praon species presented in this work have been identified from 67 aphid taxa occurring on 120 plant taxa. Furthermore, 87 original parasitoid–aphid–plant associations of the species mentioned in the key are presented. Phylogenetic relationships among Praon species are reconstructed using parsimony and cladistic distance methods. Praon abjectum is the sister taxon to the remaining Praon species. We recognized three species group: “Parapraon”, “dorsale-yomenae” and “rosaecola”. Monophyly is suggested for “Parapraon” species group and paraphyly for “dorsale-yomenae” group. Finally, by phylogenetic reconstruction, a close phylogenetic relationship between “Parapraon” and “dorsale-yomenae” species group was found.     

Some remarks on the interpretation of Penrose's size and shape components. Craniometrical similarities of African groups

The present paper deals with cranial data of different African races and shows how far the components “size” and “shape” of the Penrose distance express biological distances as well as the kind of changes occurring when different groups of variables are used. The stability of the distances between the populations was further studied by exchanging the reference group on a racial and subracial level. The analyses supply a differentiated picture according to the affinities, especially between the reference populations of the major races. Europoid and Negroid populations can often be separated only by shape distance, while the size distance is more suitable for separating Bushmen and Hottentots.     

“Exolysosomes,” Enzyme-Containing Vesicles in the Ecdysial Space of Molting Crabs

Free vesicle-like bodies (VLBs) present in the ecdysial space of cuticle regions undergoing degradation during preecdysis of the Atlantic shore crabCarcinus maenashave been interpreted either as infectious organisms or as secretion structures associated with degradation of the old cuticle. Ultrastructural, cytochemical, and immunocytological investigations were performed to test these hypotheses and to see whether VLBs are peculiar to this crab species. Similar VLBs were systematically found in two other preecdysial crabs,Cancer pagurusandMacropipus puber.InCar. maenas,they originate during early premolt inside Golgi buddings and are often gathered into large vacuoles in epidermal cells. The histochemical azo-dye technique and a cerium-based cytochemical method revealed acid phosphatase activity in both the ecdysial space and the VLBs, while Feulgen's method and immunocytological labeling always failed to reveal any DNA or RNA in either the ecdysial space or the VLBs. We conclude that VLBs are not infectious organisms but “extracellular” cuticle-degrading organelles of lysosomal origin and propose to coin them “exolysosomes.”     

Methods for facilitating web-based participatory research informatics

Scientific datasets are often generated and used by “communities”, or groups of users and creators. These communities, today often virtual, can include researchers, decision-makers, and the general public; participants in these communities may contribute different components during various iterations in the data generation process, and have different needs for resulting data products. As this “participatory research informatics” model, which includes elements of collaborative research, public participation, and digital data libraries, becomes the norm, new approaches to dynamic data management will be important. Researchers commonly use the web as a medium for research collaboration among colleagues, as well as to disseminate results to decision-makers and the public. In addition, the web can be used to facilitate dynamic collaboration by allowing research-in-progress to be accessed and edited by multiple participants. Indeed, one key benefit of using the web to facilitate research projects is that data can undergo revisions and corrections while being displayed in some form over the web. However, it can be a challenge to keep track of multiple edits to files while maintaining the integrity of a web-interface. Here we describe a system for promoting collaboration in the creation, maintenance, and use of dynamic data over the web. Using simple Microsoft Excel spreadsheets combined with traditional relational databases to facilitate dynamic data generation and updates and maintain a data schema, we are able to facilitate efficient collaboration within and among participant communities. This approach allows for the separation of web display and content, which in turn allows participants to be responsible for their own content. This separation simplifies the interaction between those responsible for the content (researchers and other participants), those responsible for the data display (web designers), and those responsible for data management, storage, and retrieval (web programmers). The approach described here is particularly well suited to large, spatially-specific ecological datasets because it can be used to facilitate the “real-time” editing and display of visual data such as web-maps, as well as to encourage multiple participants to both contribute to and interact with “beta” data and content.     

Turbulence, watermass stratification and harmful algal blooms: an alternative view and frontal zones as “pelagic seed banks”

Watermass stratification has been considered the essential physical condition that dinoflagellates require to bloom because of their relative inability, unlike diatoms, to tolerate the elevated shear-stress associated with water-column mixing, turbulence and high velocity, coastal currents. The swimming speeds of 71 flagellate taxa, with a focus on dinoflagellates, are compared to the turbulence fields and vertical velocities that develop during representative wind conditions, upwelling and at frontal zones. The results suggest that the classical stratification–dinoflagellate bloom paradigm needs revision. Tolerance of turbulence, growth within well-mixed watermasses and survival and dispersal while entrained within current systems are well developed capacities among dinoflagellates. Their secretion of mucous, often copious during blooms, is suggested to be an environmental engineering strategy to dampen turbulence. Biophysical tolerance of turbulence by dinoflagellates is often accompanied by high swimming speeds. Motility speeds of many species exceed in situ vertical current velocities; this also allows diel migrational patterns and other motility-based behavior to persist. Species belonging to “mixing-drift” life-form assemblages can increase their swimming speeds through chain formation, which helps to compensate for the increased turbulence and vertical water-column velocities of their habitats. The ability of dinoflagellate species to tolerate the vertical velocities of offshore, frontal zones, where abundant populations often develop, suggests that fronts may serve as “pelagic seed banks”, occurring as pelagic analogues of nearshore seed beds, from which seed stock is dispersed. The different ecologies associated with the hypothesized, “pelagic seed banks” of vegetative cells and the “seed beds” of resting stage cells deposited onto sediments are discussed. There is a contradiction in the stratification–HAB paradigm: the quiescent conditions of a stratified watermass, with its characteristic nutrient-poor conditions are expected to promote stasis of the population, rather than growth and blooms. The analyses suggest that dinoflagellate blooms do not preponderate in stratified watermasses because the bloom species are biophysically intolerant of the higher velocities and turbulence of more mixed watermasses. The watermass stratification that often accompanies flagellate blooms is probably a secondary, parallel event and less essential than some other factor(s) in triggering the observed bloom.