The molecular evolution of visual pigments of freshwater crayfishes (Decapoda: Cambaridae)

This study examines the diverse maximum wavelength absorption (lambdamax) found in crayfishes (Decapoda: Cambaridae and Parastacidae) and the associated genetic variation in their opsin locus. We measured the wavelength absorption in the photoreceptors of six species that inhabit environments of different light intensities (i.e., burrows, streams, standing waters, and subterranean waters). Our results indicate that there is relatively little variation in lambdamax (522-530 nm) among species from different genera and families. The existing variation did not correlate with the habitat differences of the crayfishes studied. We simultaneously sequenced the rhodopsin gene to identify the amino acid replacements that affect shifts in maximum wavelength absorption. We then related these to changes that correlated with shifts in lambdamax by reconstructing ancestral character states using a maximum-likelihood approach. Using amino acid sequences obtained from five species (all were 301 amino acids in length), we identified a number of candidates for producing shifts of 4 to 8 nm in lambdamax. These amino acid replacements occurred in similar regions to those involved in spectral shifts in vertebrates.     

Spectral transmission and short-wave absorbing pigments in the fish lens--I. Phylogenetic distribution and identity

Fish lens transmission was found to vary depending on the type and concentration of short-wave absorbing compounds present within the lens. Pigments extracted from lenses of ten species were identified as mycosporine-like amino acids (mainly palythine, palythene and asterina-330, lambda maxs around 320-360 nm) which are also thought to be present in the majority of the 120 species examined here. A novel mycosporine-like pigment with lambda max 385 nm was isolated from the lens of the flying fish, Exocoetus obtusirostris, while lenses of several closely related tropical freshwater species were found to have high concentrations of the tryptophan catabolite 3-hydroxykynurenine (lambda max 370 nm). The type of lens pigment a species possesses and its concentration depends upon both the animal's phylogenetic group and its "optical niche".     

Mechanisms of spectral tuning in blue cone visual pigments. Visible and raman spectroscopy of blue-shifted rhodopsin mutants

Spectral tuning by visual pigments involves the modulation of the physical properties of the chromophore (11-cis-retinal) by amino acid side chains that compose the chromophore-binding pocket. We identified 12 amino acid residues in the human blue cone pigment that might induce the required green-to-blue opsin shift. The simultaneous substitution of nine of these sites in rhodopsin (M86L, G90S, A117G, E122L, A124T, W265Y, A292S, A295S, and A299C) shifted the absorption maximum from 500 to 438 nm, accounting for 2,830 cm-1, or 80%, of the opsin shift between rhodopsin and the blue cone pigment. Raman spectroscopy of mutant pigments shows that the dielectric character and architecture of the chromophore-binding pocket are specifically altered. An increase in the number of dipolar side chains near the protonated Schiff base of retinal increases the ground-excited state energy gap via long range dipole-dipole Coulomb interaction. In addition, the W265Y substitution causes a decrease in solvent polarizability near the chromophore ring structure. Finally, two substitutions on transmembrane helix 3 (A117G and E122L) act in combination with the other substitutions to alter the binding-pocket structure, resulting in stronger interaction of the protonated Schiff base group with the surrounding dipolar groups and the counterion. Taken together, these results identify the amino acid side chains and the underlying physical mechanisms responsible for a majority of the opsin shift in blue visual pigments.     

The eyes of deep-sea fish. I: Lens pigmentation, tapeta and visual pigments

Deep-sea fish, defined as those living below 200 m, inhabit a most unusual photic environment, being exposed to two sources of visible radiation; very dim downwelling sunlight and bioluminescence, both of which are, in most cases, maximal at wavelengths around 450-500 nm. This paper summarises the reflective properties of the ocular tapeta often found in these animals, the pigmentation of their lenses and the absorption characteristics of their visual pigments. Deep-sea tapeta usually appear blue to the human observer, reflecting mainly shortwave radiation. However, reflection in other parts of the spectrum is not uncommon and uneven tapetal distribution across the retina is widespread. Perhaps surprisingly, given the fact that they live in a photon limited environment, the lenses of some deep-sea teleosts are bright yellow, absorbing much of the shortwave part of the spectrum. Such lenses contain a variety of biochemically distinct pigments which most likely serve to enhance the visibility of bioluminescent signals. Of the 195 different visual pigments characterised by either detergent extract or microspectrophotometry in the retinae of deep-sea fishes, ca. 87% have peak absorbances within the range 468-494 nm. Modelling shows that this is most likely an adaptation for the detection of bioluminescence. Around 13% of deep-sea fish have retinae containing more than one visual pigment. Of these, we highlight three genera of stomiid dragonfishes, which uniquely produce far red bioluminescence from suborbital photophores. Using a combination of longwave-shifted visual pigments and in one species (Malacosteus niger) a chlorophyll-related photosensitizer, these fish have evolved extreme red sensitivity enabling them to see their own bioluminescence and giving them a private spectral waveband invisible to other inhabitants of the deep-ocean.     

Evolution of the mitochondrial DNA control region in the mbuna (Cichlidae) species flock of Lake Malawi, East Africa

Considerable controversy has surrounded the application of mitochondrial DNA data to reconstruction of evolutionary relationships among the endemic cichlids of Lake Malawi. Central to this debate has been the issue of whether lineage sorting is complete, and thus whether these data actually reflect species phylogeny, or simply gene genealogy. Review of all mtDNA control region sequences available for members of one monophyletic subset of this species flock, the Malawi rockfishes, or mbuna, strongly indicates that lineage sorting is incomplete: Character-based analyses of these sequences reconstruct gene, not species, interrelationships. Analysis of the pattern of nucleotide substitutions differentiating these mtDNA alleles suggests that pyrimidine residues undergo transition substitutions more often than do purines. Estimation of the magnitude of derived sequence differentiation in light of the reconstructed gene genealogy suggests that the mbuna may be of considerably more recent vintage than previous molecular characterizations have indicated.     

Evolution of MHC class II beta chain-encoding genes in the Lake Tana barbel species flock (Barbus intermedius complex)

Major histocompatibility complex (MHC) class II protein polymorphism is maintained in allelic lineages which evolve in a trans-specific manner, passing from one species to descendant species. Selection pressure on peptide binding residues should be greatest during speciation, when organisms move into new environments and their MHC molecules encounter new pathogens. The isolation of MHC genes from teleost fishes, the most diverse group of vertebrates, has created possibilities for testing this hypothesis. The large barbels of Lake Tana have undergone an adaptive radiation within the last 5 million years, producing 14 morphotypes which inhabit different ecological niches within the lake. We studied the variability in class II beta chain-encoding genes of four of these morphotypes using polymerase chain reaction amplification and DNA sequencing. The sequences obtained were orthologous to four of the known class II genes from the common carp, from which barbels diverged approximately 32 million years ago. When subjected to phylogenetic analysis, the 48 sequences clustered into groups which represent allelic lineages. A comparison of nonsynonymous and synonymous substitutions between the peptide binding region codons and non-peptide binding region codons of these sequences revealed that they are under strong selective pressure.     

The contamination of birds with organic pollutants in the Lake Baikal region

Lake Baikal is considered to be the largest reservoir of fresh natural water in the world. Nevertheless industrial enterprises on its banks as well as river effluents contaminate this unique basin. In the present study birds' eggs (15 species) collected in the Baikal region (Selenga river estuary) have been analysed. Quantitative determination of more than 40 individual organic pollutants (polycyclic aromatic hydrocarbons, phenols, organochlorine compounds) has been carried out using GC-MS as an analytical tool. The results obtained demonstrated a wide range of toxicant concentrations (2-3 orders of magnitude) for various species. Very high levels of polycyclic aromatic hydrocarbons have been detected in the eggs of Anas platyrynchos (mallard), Tringa stagnatilis (marsh sandpiper) and Podiceps auritus (slavonian grebe). These particular species also have the highest levels of other toxicants. Taking into account high rate of metabolism of certain of these compounds in birds, it has been proposed that the major route of transfer into higher trophic levels is via water and aquatic invertebrates.     

Population genetics and structure of Buryats from the Lake Baikal Region of Siberia

Genetic polymorphisms of blood groups, serum proteins, red cell enzymes, PTC tasting, and cerumen types are reported for five Mongoloid populations of Buryats from the Lake Baikal region of Siberia (Russia). These groups are characterized by relatively high frequencies of alleles ABO*B, RH*D, cerumen D, GC*1F, ACP1*B, ESD*2, and PGD*C. Significant genetic heterogeneity between populations was demonstrated for the loci RH, MN, cerumen, PGD, ABO, GC, GLO, TF, and PGM1. Genetic distance analyses using five loci revealed a lower level of genetic microdifferentiation within the Buryat populations compared with other native Siberian groups. The distribution of gene markers in Buryats is similar to that found in neighboring Central Asian groups, such as the Yakuts and the Mongols. Intrapopulational analyses of the five Buryat subdivisions, based on R matrix and rii, indicate that one of the subdivisions is reproductively more isolated than the others and that two of the communities have received considerable gene flow. A nonlinear relationship was demonstrated between geographic and genetic distances of Buryat population subdivisions.     

Role of hydroxyl-bearing amino acids in differentially tuning the absorption spectra of the human red and green cone pigments

The human red and green cone pigments differ at either 15 or 16 amino acids, depending upon which polymorphic variants are compared. Seven of these amino acid differences involve the introduction or removal of a hydroxyl group. One of these differences, a substitution of alanine for serine at position 180, was found previously to produce a 5 nm blue shift. To determine the role of the remaining six hydroxyl group differences in tuning the absorption spectra of the human red and green pigments, we have studied six site-directed mutants in which single amino acids from the green pigment have been substituted for the corresponding residues in the red pigment. Blue shifts of 7 and 14 nm were observed upon substitution of phenylalanine for tyrosine at position 277 and alanine for threonine at position 285, respectively. Single substitutions at positions 65, 230, 233, and 309 produced spectral shifts of 1 nm or less. These data are in good agreement with a model based upon sequence comparisons among primate pigments and with the properties of site-directed mutants of bovine rhodopsin. Nonadditive effects observed in comparing the absorption spectra of red-green hybrid pigments remain to be explained.     

The molecular evolution of sperm bindin in six species of sea urchins (Echinoida: Strongylocentrotidae)

The acrosomal protein bindin attaches sperm to eggs during sea urchin fertilization. Complementary to ongoing functional biochemical studies, I take a comparative approach to explore the molecular evolution of bindin in a group of closely related free-spawning echinoid species. Two alleles of the mature bindin gene were sequenced for each of six species in the sea urchin family Strongylocentrotidae. The nucleotide sequences diverged by at least 1% per Myr at both silent and replacement sites. Two short sections flanking the conserved block show an excess of nonsynonymous substitutions. Each is homologous to a region that had been identified as a target of selection in other sea urchin comparisons. A large proportion of the bindin-coding sequence consists of a highly variable repeat region. Bindin sequences, even including the large intron, could not resolve the branching order among five of the species.     

Distribution of plasma phosphatidylcholine molecular species in rabbits fed fish oil is modulated by dietary n-6 fatty acids

Natural occurrence of Fusarium mycotoxins (trichothecenes and fumonisins) and aflatoxin B1 (AFB1) were surveyed in 32 corn samples, harvested in 1993 and randomly sampled in 1994 in several districts of Hanoi, Vietnam. Corn samples were first milled into fine powder, extracted with methanol-water (3:1) and the crude extracts obtained from the same samples were used for the simultaneous analysis of the trichothecenes such as nivalenol (NIV), deoxynivalenol (DON), and T-2 toxin (T-2) by gas chromatography/mass spectrometry (GC/NS); fumonisins B1 (FB1), B2 (FB2), and B3 (FB3) by high-performance liquid chromatography (HPLC) with a flourescence detector; and AFB1 by and ELISA kit based on a monoclonal antibody. The data revealed that 14, 8, 4, 3, and 2 out of 15 corn kernel samples were positive for AFB1, FB1, FB2, FB3, and NIV with the average levels being 28, 1, 101, 276, 232, and 858 ppb, respectively, and neither DON nor T-2 were detected. As for the other 17 samples of corn powder, 13, 15, 12, 10, 4 and 2 were positive for AFB1, FB1, FB2, FB3, DON, and NIV with the average being 30, 780, 289, 176, 3, 170, and 1,365 ppb, respectively, and T-2 was not detected. Although their positive rates and levels fell in the ranges reported elsewhere, it was found for the first time that the Fusarium toxins (NIV, DON, and fumonisins) and an Aspergillus toxin (AFB1) were naturally co-contaminated in selected samples of corn produced in north Vietnam.     

Diversity and molecular evolution of the RPS2 resistance gene in Arabidopsis thaliana

The RPS2 gene in Arabidopsis thaliana governs resistance to strains of the bacterial pathogen, Pseudomonas syringae pv. tomato, that express the avrRpt2 gene. The two loci are involved in a gene-for-gene interaction. Seventeen accessions of A. thaliana were sequenced to explore the diversity present in the coding region of the RPS2 locus. An unusually high level of nucleotide polymorphisms was found (1.26%), with nearly half of the observed polymorphisms resulting in amino acid changes in the RPS2 protein. Seven haplotypes (alleles) were identified and their evolutionary relationships deduced. Several of the alleles conferring resistance were found to be closely related, whereas susceptibility to disease was conferred by widely divergent alleles. The possibility of selection at the RPS2 locus is discussed.     

Adrenergic signaling and second messenger production in hepatocytes of two fish species

The activation of cAMP and inositol 1,4,5-trisphosphate (IP3) transduction pathways by epinephrine (EPI) has been studied in hepatocytes and hepatic membranes of two teleost species, the American eel (Anguilla rostrata) and the black bullhead (Ictalurus melas). EPI at 10 microM increased both cAMP and IP3 in a dose- and species-dependent manner. The activation of both systems was the greatest in the eel, and the IP3 system was activated at lower EPI concentrations than the cAMP system. The individual transduction pathways were identified by the EPI-induced increase in cAMP being blocked by propranolol (PROP) but not phentolamine (PHT) and the increase in IP3 by PHT but not PROP. alpha1-Adrenoceptors were characterized by the specific binding of [3H]prazosin (PRZ) to purified hepatic membranes: Kd and Bmax values were 3.0 and 2.2 nM and 108 and 178 fmol x mg-1 protein for eel and bullhead, respectively. PRZ binding was displaced by nonradioactive PRZ and PHT but not by PROP. [3H]IP3 bound specifically to microsomal membranes from hepatic tissues of both species and this binding could best be explained by a two-site binding model; significant differences in binding characteristics were seen between eel and bullhead membranes. This study demonstrates that EPI may act on hepatocytes isolated from the American eel and black bullhead through both signal transduction pathways, inducing changes in glucose production and Ca2+ mobilization previously reported in these species. The quantitative differences between the two species in aspects of this system may reflect differences at the level of the coupling between receptor and messenger production, or at the degradation of the messenger, aspects of the transduction pathways which may be either true species differences or related to the conditions under which the species were held.     

Neural substrates for species recognition in the time-coding electrosensory pathway of mormyrid electric fish

Mormyrid electric fish have species- and sex-typical electric organ discharges (EODs). One class of tuberous electroreceptors, the knollenorgans, plays a critical role in electric communication; one function is species recognition of EOD waveforms. In this paper, we describe cell types in the knollenorgan central pathway, which appear responsible for analysis of the temporal patterns of spikes encoded by the knollenorgans in response to EOD stimuli. Secondary sensory neurons in the nucleus of the electrosensory lateral line lobe (NELL) act as relays of peripheral responses. They fire a single phase-locked spike to an outside positive-going voltage step. Axons from the NELL project to the toral nucleus exterolateralis pars anterior (ELa). Immediately after they enter the ELa, they send collaterals to terminate on one to three ELa large cells and then continue in a lengthy neuronal pathway that traverses the ELa several times. After a path length of up to 5 mm, the NELL axon terminates on as many as 70 ELa small cells. Thus the large cells appear to be excited first, followed by the small cells, with the intervening length of the axon serving as a delay line. The large cells also respond with phase-locked spikes to voltage steps. Large cell axons extend for approximately 1 mm and terminate on several small cells within the ELa. The terminals are known to be GABAergic inputs and are presumed inhibitory. We propose that small cells receive direct inhibition from large cells and delayed excitation from NELL axons. The small cells may act as anti-co-incidence detectors to analyze the temporal structure of the EOD waveform.     

The photoreceptors and visual pigments of the garter snake (Thamnophis sirtalis): a microspectrophotometric, scanning electron microscopic and immunocytochemical study

Scanning electron microscopy, immunocytochemistry, and single cell microspectrophotometry were employed to characterize the photoreceptors and visual pigments in the retina of the garter snake, Thamnophis sirtalis. The photoreceptor population was found to be comprised entirely of cones, of which four distinct types were identified. About 45.5% of the photoreceptors are double cones consisting of a large principal member joined near the outer segment with a much smaller accessory member. About 40% of the photoreceptors are large single cones, and about 14.5% are small single cones forming two subtypes. The outer segments of the large single cones and both the principal and accessory members of the doubles contain the same visual pigment, one with peak absorbance near 554 nm. The small single cones contain either a visual pigment with peak absorbance near 482 nm or one with peak absorbance near 360 nm. Two classes of small single cones could be distinguished also by immunocytochemistry and scanning electron microscopy. The small single cones with the 360-nm pigment provide the garter snake with selective sensitivity to light in the near ultraviolet region of the spectrum. This ultraviolet sensitivity might be important in localization of pheromone trails.     

Difference in molecular properties between chicken green and rhodopsin as related to the functional difference between cone and rod photoreceptor cells

Using low-temperature spectroscopy, we have investigated the photobleaching process of chicken green, a green-sensitive cone visual pigment present in chicken retina, and compared it to that of rhodopsin, a rod visual pigment. Like rhodopsin, chicken green converts to all-trans-retinal and opsin through batho, lumi, and meta I, II, and III intermediates. However, all of the intermediates of chicken green except lumi, are less stable than the corresponding intermediates of rhodopsin. While early intermediates, batho and lumi are similar in absorption maxima between chicken green and rhodopsin, the meta intermediates of chicken green are about 20 nm blue shifted from those of rhodopsin. Low-temperature time-resolved spectroscopy was applied to estimate the thermodynamic properties of meta intermediates, and it indicated that the less stable properties of meta II and III intermediates of chicken green originate from the smaller activation enthalpies. The decay of the meta II intermediate of chicken green is greatly suppressed when a chicken green sample is irradiated at alkaline conditions while the net charge becomes similar to that of rhodopsin at neutral conditions. These results strongly suggest that the functional properties of chicken green that are different from those of rhodopsin are regulated by the dissociative amino acid residue(s).     

Different patterns of molecular evolution of influenza A viruses in avian and human population

Patterns of molecular evolution of the influenza virus proteins and genes are discussed. The subsets of all viral genes corresponding to statistically significant clusters on dendrogram were shown to fall into two distinct groups. The first group was characterized by the presence of an exact linear relationship between the year of the strain isolation and the evolutionary distance. The subsets of human influenza virus genes belong to this group. A method for eliminating the "frozen" strains from the subsets and for calculating the evolutionary rates without construction of phylogenetic trees has been elaborated. The substitution rates calculated according to this technique agreed with the data obtained previously. A linear relationship was not observed in the second group. This group was predominantly composed of avian influenza virus genes. The lack of linear correlation pointed to the cocirculation of a large amount of different influenza virus genomic segments in the avian population. An approach for an examination of the role of intragenic recombination in the development of the antigenic subtypes of hemagglutinin is suggested. Our results suggest that recombination did not play a considerable role in this process, and that all modern subtypes of this protein were probably formed before the introduction of the influenza viruses into the human population. These findings are consistent with the hypothesis that influenza viruses penetrated into human population from their pools in avian populations.