Getting to the start line: how bumblebees and honeybees are visually guided towards their first floral contact.

Much of the literature on foraging behaviour in bees focuses on what they learn after they have had rewarded experience with flowers. This review focuses on how honeybees and bumblebees are drawn to candidate food sources in the first place: the foundation on which learning is built. Prior to rewarded foraging experience, flower-naïve bumblebees and honeybees rely heavily on visual cues to discover their first flower. This review lists methodological issues that surround the study of flower-naïve behaviour and describes technological advances. The role of distinct visual properties of flowers in attracting bees is considered: colour, floral size, patterning and social cues. The research reviewed is multi-disciplinary and takes the perspectives of both the bees and the plants they visit. Several avenues for future research are proposed.

PCR-based identification of adriatic specimen of three scorpionfish species (Scorpaenidae, Teleostei).

The identification of three scorpionfish species, the black scorpionfish (Scorpaena porcus Linnaeus, 1758), the large-scaled scorpionfish (S. scrofa Linnaeus, 1758) and the small red scorpionfish (S. notata Rafinesque, 1810) is possible in adults by morphometry, but often problematic in juveniles due to their similar phenotypes. To develop a molecular species identification tool, first, we have analyzed the genetic similarity of the three species by a PCR-based 'blind method' that amplified bands from various locations of the genome. We found high levels of nucleotide similarity between S. porcus and S. scrofa, whereas S. notata showed a higher level of divergence from the other two species. Then, we have searched these patterns for differences between the genomes of Adriatic specimen of these three species and identified several species-specific products in two of them. For the third one a species-specific primer pair amplifying from the 16S ribosomal DNA was designed. One marker for each species was cloned, sequenced and converted into Sequence Characterized Amplified Region (SCAR) markers amplified by specific primer pairs. The SCAR markers amplified robust bands of limited variability from the target species, while no or only occasional weak products were obtained from the other two, proving that they can be used for molecular identification of these three species. These markers can help the conservation and future analysis of these three species as well as their possible selection programs for aquaculture purposes.

A standard panel of microsatellites for Asian seabass (Lates calcarifer).

Microsatellites are the most popular markers for parentage assignment and population genetic studies. To meet the demand for international comparability for genetic studies of Asian seabass, a standard panel of 28 microsatellites has been selected and characterized using the DNA of 24 individuals from Thailand, Malaysia, Indonesia and Australia. The average allele number of these markers was 10.82 +/- 0.71 (range: 6-19), and the expected heterozygosity averaged 0.76 +/- 0.02 (range: 0.63-1.00). All microsatellites showed Mendelian inheritance. In addition, eight standard size controls have been developed by cloning a set of microsatellite alleles into a pGEM-T vector to calibrate allele sizes determined by different laboratories, and are available upon request. Seven multiplex PCRs, each amplifying 3-5 markers, were optimized to accurately and rapidly genotype microsatellites. Parentage assignment using 10 microsatellites in two crosses (10 x 10 and 20 x 20) demonstrated a high power of these markers for revealing parent-sibling connections. This standard set of microsatellites will standardize genetic diversity studies of Asian seabass, and the multiplex PCR sets will facilitate parentage assignment.

Efficient transient expression system based on square pulse electroporation and in vivo luciferase assay of fertilized fish eggs.

Electroporation mediated DNA transfer into fish eggs has been improved by using a train of square pulses. Fertilized eggs of African catfish (Clarias gariepinus), zebrafish (Brachydanio rerio) and rosy barb (Barbus conchonius) were dechorionated enzymatically followed by application of pulses. Efficiency of plasmid DNA delivery was significantly increased by applying multiple pulses on dechorionated eggs. Optimization of physical parameters such as field strength, pulse width and pulse numbers resulted in reproducible transient expression in 25-50% of embryos and larvae by using the firefly luciferase and the E. coli beta-galactosidase (lacZ) genes both driven by CMV IE1 promoter. Temporal luciferase expression was assayed using both qualitative (sheet film) and quantitative (scintillation counting) methods in developing embryos and fry in vivo. Spatial expression of lacZ was assayed by histochemical staining. A number of embryos revealed foreign gene product also localised in the vegetal pole of the embryo.

Enhanced ethidium fluorescence of large DNA electrophoresed in gels submersed in an immiscible solvent.

Agarose gel electrophoretic separation of a lambda/HindIII DNA marker containing detectable fragments of 23 to 2 kb was carried out in the conventional submarine apparatus and in the horizontal gel slab apparatus of Wieme, using identical samples, agarose, gel width and procedure for ethidium bromide staining. In the Wieme apparatus, the gel on its microscope slide support is immersed in an immiscible solvent such as petroleum ether or silicone oil. Although band resolution and speed of migration are equivalent between gels run in the two systems, the relative fluorescence intensity of the ethidium bromide-stained bands is substantially more responsive to an increase in DNA length in the Wieme gels than in the submarine gels. The predicted relative fluorescence is by an average factor of 0.5 less than that observed after electrophoresis in the Wieme apparatus but is by an average factor of 3.4 more than that observed on bands derived from the submarine technique.

Epilepsy in patients with cerebral infarcts.

Out of series of 240 patients, with proved cerebral infarcts at necropsy, 14 with a clinical history of epilepsy were selected. The possible etiology of seizures was analyzed by comparing the clinical and pathological data. It appeared from this series that epileptic spells, preceding for years the occurrence of a stroke, were due to other causes than to cerebrovascular insufficiency. It was also difficult to separate apoplectic seizures from those occurring due to scar formation after a cerebral infarct, as postapoplectic epilepsy was induced either by the occurrence of a new stroke or by hemodynamic disturbances. Severe generalized or local hemodynamic and metabolic disturbances were also noted in cases of convulsions resulting from a cerebral infarct.

Rapid isolation of DNA from fresh and preserved fish scales for polymerase chain reaction.

We developed a simple and inexpensive method to extract DNA from fresh and preserved fish scales. The procedure is based on boiling the scales in 5% Chelex 100, followed by digestion with proteinase K and subsequent absorption of genomic DNA using silica. A single fresh scale from larger species (e.g., tilapia) or a few scales from smaller species (e.g., 4 scales from zebrafish) provide over 200 ng of DNA, enough for at least 40 polymerase chain reaction amplifications. The procedure is applicable for DNA isolation not only from fresh and ethanol-preserved scales, but also from dried and formaldehyde-treated samples, and thus might be useful for investigating specimens stored in museums and other collections. Since the removal of a few scales is a gentle means of sample collection, this technique will allow analysis of genetic diversity, mating systems, and parentage in populations of endangered or ornamental fish with minimal experimental influence.

Characterization of microsatellites in the IGF-2 and GH genes of Asian seabass (Lates calcarifer).

Four microsatellites were identified by screening the DNA sequences of Asian seabass (Lates calcarifer) deposited to GenBank. Two markers each are located in the growth hormone gene (GH) and in the insulin-like growth factor II gene ( IGF-2), respectively. The markers were characterized by genotyping 34 Asian seabass individuals. All 4 microsatellites showed polymorphism: the number of alleles per locus ranged from 2 to 11 (average, 5.0), while the expected heterozygosity ranged from 0.51 to 0.85 (average, 0.63) at the 4 loci. Cross-priming with all 4 primer pairs was tested in species belonging to 5 different genera, but no bands were amplified. These microsatellites are the first genomic DNA markers characterized in L. calcarifer; thus they may be valuable for research and aquaculture production of this species.

A thin-layer multistrip agarose gel electrophoresis apparatus for Ferguson plot analysis at the sub-microgram load level.

A method for the simultaneous horizontal agarose gel electrophoresis on thin-layer strips of different gel concentrations was developed for the purpose of generating Ferguson plots at the sub-microgram load level. Seven independent gel strips on a common GelBond support were formed by filling channels created by a comb-shaped spacer (polycarbonate) in a vertical multistrip cassette. Electrophoresis on a horizontal Peltier-cooled surface employed commercial apparatus (E-C Apparatus Corp.) with a modified cover which is airtight and holds anodic and cathodic voltage measurement probes for each strip. The application of the apparatus to Ferguson plot analysis in a single experiment was exemplified on the RNA-containing turnip crinkle virus (TCV) at a load of 50 ng/gel strip, using an optimized silver staining method (a modification of a procedure of FMC Corp. BioProducts) for detection. Within the range of 3.5 to 12.5 V/cm, the plot was found to be independent of field strength. Mobility is also independent of the concentration of detergent (CHAPS) up to 10 mM.

Computer-aided analysis of DNA curves on transverse gradient gels.

Transverse pore gradient polyacrylamide gel electrophoresis of DNA restriction fragments was used to generate gel patterns describing migration distance as a function of gel concentration (Ferguson curves). These Ferguson curves were digitized, traced and analyzed with the aid of a personal computer. The traced curves were plotted semi-logarithmically and the plots were subjected to least-squares linear regression analysis to yield values of the slope (KR) and the intercept at %T = 0 (YO). These values are highly precise since they are based on approx. 100 measurements per curve. The computerized method reduces the errors due to manual measurements of migration distances and is time and labor saving. The method is still limited to intra-experimental comparison of Ferguson curves, since it does not as yet comprise a determination of gel concentration. At present, curve tracing remains semi-automated, requiring manual intervention when Ferguson curves cross or approach one another. Potentially, the importance of the computerized analysis of transverse pore gradient gels lies in the rapid quantitative interpretation of Ferguson curves for detection of anomalously migrating DNA species. Potentially, that application provides a more sensitive and informative mode of detection than either the mere visual observation of crossing Ferguson curves or of a shift in mobility at a single gel concentration.

Periodic electroencephalographic compleses and subacute sclerosing panencephalitis. A case report and a survey of literature.

The authors report the case of a child with a presumable diagnosis of subacute sclerosing panencephalitis (SSPE). But pathological examination of the brain showed only a moderate neuronal loss in the cortical and subcortical grey matter. From this case and from the data in the literature it is concluded that the electroencephalogram in stage II of SSPE is characterized by the appearance of paroxysmal wave complexes which recur pseudo-periodically against a disorganized basic activity. The criteria for these complexes are not as strict and the periodicity is not as regular as formerly believed. Such a tracing is not pathognomonic of SSPE and may also occur-although rarely-in other brain diseases. The theory that the periodic discharges originate from a diffuse alteration of cortical and subcortical grey matter seems the most plausible.

[Occurrence of triphasic waves in two cases of thyrotoxic crisis (author's transl)]. / Ondes lentes triphasiques dans deux cas d'encéphalopathie thyréotoxique

An electroencephalogram taken on two patients suffering from a thyrotoxic crisis, showed the presence of typical triphasic waves. This pattern was first described in 1950 by Foley et al., who believed that it occurred specifically in patients with hepatic encephalopathy. Since then, however, it has been shown that triphasic waves can be identified in other (e.g. renal) metabolic brain diseases and, less commonly, in a variety of either organic or functional cerebral disorders.

Acute prolonged ictal confusion (resembling petit mal status) presenting "de novo" in later life.

Two female patients, aged respectively 52 and 70 years, with no past history of epilepsy, presented an acute prolonged confusional state. The electroencephalograms showed generalized slow spike and wave discharges with frontal emphasis. No structural neurological lesion nor progressive neurological disorder was found. Both patients responded to intravenous administration of clonazepam and were placed on regular anti-epileptic treatment. On follow-up both remained well. These cases do not fit in the classical category of petit mal status but appear to belong to a distinct entity.

The effect of an organophosphorus pesticide on the enzymes of carp (Cyprinus carpio L.).

1. The effect of 1.5, 2.0 and 5.0 ppm methidation was studied on the ASAT, ALAT, LDH and AChE enzymes, as well as the blood sugar and adrenaline levels in carp. 2. According to our studies methidation implies a potential hazard on the normal biochemical processes of fishes: causing tissue necrosis indicated by the increase ASAT, ALAT and LDH activities, inducing continual stress effect reflected by the increased blood sugar and adrenaline levels, inhibiting AChE activity in the various organs, the consequence under acute effects being fish kill.

The first transcriptome and genetic linkage map for Asian arowana.

Asian arowana or dragonfish (Scleropages formosus) is an important fish species due to its unusual breeding biology and high economic value in the ornamental fish markets. In the present study, we aimed to (i) create the first transcriptome by Roche 454 pyrosequencing of Asian arowana brain and gonad samples; (ii) identify differentially expressed genes between the two sexes and develop microsatellite (SSR) markers; and (iii) construct a first-generation SSR-based genetic linkage map. A total of over 1.3 million reads were obtained from the brain and gonad of adult Asian arowana individuals through pyrosequencing. These reads were assembled into 16,242 contigs that were further grouped into 13,639 isogroups. BLASTX annotation identified a total of 8316 unique proteins from this data set. Many genes with sexually dimorphic expression levels and some putatively involved in sex development were identified. A total of 316 EST-SSRs and 356 new genomic-SSRs were developed by screening through the current transcriptome data set and SSR-enriched genomic libraries. The first genetic linkage map of the species was constructed based on these markers. Linkage analysis allowed for mapping of 308 markers to 28 linkage groups (LGs), ranging in size from 14.9 to 160.6 cM. The potentially sex-associated gene sox9 was mapped to LG4 on the consensus linkage map. Pairwise putative conserved syntenies between the Asian arowana, zebrafish, and three-spined stickleback were also established. These resources will help the conservation of the species through better understanding of its phylogenetics, genomics and biology, and comparative genome analysis within the Osteoglossidae family.

Mapping QTL for an adaptive trait: the length of caudal fin in Lates calcarifer.

The caudal fin represents a fundamental design feature of fishes and plays an important role in locomotor dynamics in fishes. The shape of caudal is an important parameter in traditional systematics. However, little is known about genes involved in the development of different forms of caudal fins. This study was conducted to identify and map quantitative trait loci (QTL) affecting the length of caudal fin and the ratio between tail length and standard body length in Asian seabass (Lates calcarifer). One F1 family containing 380 offspring was generated by crossing two unrelated individuals. One hundred and seventeen microsatellites almost evenly distributed along the whole genome were genotyped. Length of caudal fin at 90 days post-hatch was measured. QTL analysis detected six significant (genome-wide significant) and two suggestive (linkage-group-wide significant) QTL on seven linkage groups. The six significant QTL explained 5.5-16.6% of the phenotypic variance, suggesting these traits were controlled by multiple genes. Comparative genomics analysis identified several potential candidate genes for the length of caudal fin. The QTL for the length of caudal fin detected for the first time in marine fish may provide a starting point for the future identification of genes involved in the development of different forms of caudal fins in fishes.

Anti-Müllerian hormone and 11 beta-hydroxylase show reciprocal expression to that of aromatase in the transforming gonad of zebrafish males.

During development all zebrafish males first develop a "juvenile ovary" that later degenerates and transforms into a testis. In this study, individuals undergoing gonadal transformation were identified from a vas::egfp transgenic line and used for gene expression analysis of anti-Müllerian hormone (amh), ovarian aromatase (cyp19a1a) and 11 beta-hydroxylase (cyp11b, also known as P450(11 beta)) by real-time polymerase chain reaction and in situ hybridization. In the "normal (i.e., nontransforming) juvenile ovary" cyp19a1a was expressed around the oocytes, but cyp11b and amh could not be detected. During gonadal transformation cyp19a1a was down-regulated and could not be detected anymore; in contrast amh was up-regulated and highly expressed at similar regions where cyp19a1a had been expressed earlier. Furthermore, the normalized transcript levels of cyp19a1a and amh showed a reciprocal picture, i.e., the higher was the level of amh, the lower was that of cyp19a1a. Expression of cyp11b was also up-regulated but later than amh, and its localization was not related to the position of degenerating oocytes. These data indicate that amh is a candidate gene down-regulating cyp19a1a, leading to "juvenile ovary-to-testis" transformation. Whereas, cyp11b or its product, 11-ketotestosterone, is unlikely to be the inducer of zebrafish gonad transformation, as proposed earlier for some protogynous hermaphroditic fish species.

Mutation rate and pattern of microsatellites in common carp (Cyprinus carpio L.).

Microsatellites are popular molecular markers in genetic and evolutionary studies. Their mutational dynamics have been extensively studied in humans and fruit flies, but few data were available in fish. By genotyping 55 individuals of a F1 pedigree, we investigated the mutation rates and patterns of 49 microsatellites in one of the most important fresh water fish species, the common carp (Cyprinus carpio L.). The overall mutation rate of the 49 loci was 5.56 x 10(-4)/locus/generation (95% confidence interval 1.52 x 10(-4) and 1.63 x 10(-3)). The change of allele size was between +2 to -5 repeat units, assuming that the mutation allele arose from the parental allele most similar in size to the mutant.