Aspartic proteinases in fishes and aquatic invertebrates

1. The literature on molecular properties and physiological role of aspartic proteinases in fishes and aquatic invertebrates has been reviewed. 2. Pepsins have not been detected in invertebrates, and apparently cathepsin D, as well as other cathepsins, act both as digestive and lysosomal enzymes in many of these animals. The molecular properties of invertebrate cathepsin D correspond with cathepsin D in fishes and mammalians. 3. Fishes with a true stomach have pepsinogen secretion. Fish pepsins have higher pH optimum and are less stable in strong acid conditions than mammalian pepsins. They are very efficient at low temperatures, but less thermostable than mammalian pepsins. 4. Many fishes have two significantly different pepsins: Pepsin I and Pepsin II, which digest haemoglobin at a maximal rate in the pH ranges 3-4 and 2-3 respectively. Usually the pI of Pepsin I is in the range 6.5-7, whereas pI of Pepsin II is about 4. 5. Fish Pepsin I and cathepsin D have very similar molecular properties, and a hypothesis proposing that cathepsin D is the ancestor enzyme of aspartic proteinases in higher animals is presented.     

Toward visualization of nanomachines in their native cellular environment

The cellular nanocosm is made up of numerous types of macromolecular complexes or biological nanomachines. These form functional modules that are organized into complex subcellular networks. Information on the ultra-structure of these nanomachines has mainly been obtained by analyzing isolated structures, using imaging techniques such as X-ray crystallography, NMR, or single particle electron microscopy (EM). Yet there is a strong need to image biological complexes in a native state and within a cellular environment, in order to gain a better understanding of their functions. Emerging methods in EM are now making this goal reachable. Cryo-electron tomography bypasses the need for conventional fixatives, dehydration and stains, so that a close-to-native environment is retained. As this technique is approaching macromolecular resolution, it is possible to create maps of individual macromolecular complexes. X-ray and NMR data can be ‘docked’ or fitted into the lower resolution particle density maps to create a macromolecular atlas of the cell under normal and pathological conditions. The majority of cells, however, are too thick to be imaged in an intact state and therefore methods such as ‘high pressure freezing’ with ‘freeze-substitution followed by room temperature plastic sectioning’ or ‘cryo-sectioning of unperturbed vitreous fully hydrated samples’ have been introduced for electron tomography. Here, we review methodological considerations for visualizing nanomachines in a close-to-physiological, cellular context. EM is in a renaissance, and further innovations and training in this field should be fully supported. Robert Feulgen Lecture 2009 presented at the 51st symposium of the Society for Histochemistry in Stubai, Austria, October 7–10, 2009.     

Microorganisms and the aquatic environment

The important role of bacteria in sediments is reviewed. The problems in estimating bacterial biomass in sediments are highlighted. Recent developments in studying substrate transformations in the laboratory, under conditions simulating the nutrient status occurring in nature, are also discussed.     

微囊藻毒素对水环境的影响研究进展

微囊藻毒素是富营养化淡水水体中最常见的藻类毒素,是湖泊蓝藻产生的一类肽类毒素,它的产生受到藻类的遗传和环境因素的共同影响。由于其毒性大,分布广,结构稳定,从而成为水环境中的潜在危害物质。有关微囊藻毒素性质、毒理毒性、在环境中的迁移、转化以及控制预防已成为关注热点。在总结国内外研究的基础上,综述了微囊藻毒素的性质、产生机理以及其与水环境、水生生物(水生植物、鱼类、无脊椎动物)间的相互作用,讨论了微囊藻毒素对水生生物的影响以及水生生物对微囊藻毒素的降解作用,为水体中微囊藻毒素的防治提供科学的依据。     

Colonization of an artificial stream by fishes and aquatic macro-invertebrates

We examined colonization by fishes and macro-invertebrates from permanent streams into an artificial freshwater stream simulating lotic temporary bodies of water that exist for only a limited period each year. After introducing water, invertebrates such as chironomid larvae in mud increased in numbers rapidly in the experimental stream, although they were rare in mud in the permanent streams. Eleven of 12 fish species present in the permanent streams colonized the experimental stream and preyed upon invertebrates, although fish composition differed significantly between the two streams. About 100 days after the initiation of the experiment, both species richness and diversity in the experimental stream reached almost the same level as that in the permanent streams. More diverse fishes colonized the complex section where habitat diversity was high compared to the simple section in the experimental stream. Our study strongly suggests that lotic temporary waters such as temporary streams around main rivers have unique ecological characteristics and serve as valuable foraging sites for fish.     

Spirostomum ambiguum as a bioindicator of aquatic environment pollution

Express-methods based on functional and morphological features of the genus Spirostomum have been applied for estimation of the effect produced by chemical toxicants, γ-radiation, electromagnetic emission, and homeopathic drugs on the aquatic environment. The experiments have shown that Sp. ambiguum is a very useful indicator for the bioassay of low-intensity physical and chemical pollution in the aquatic environment.     

水环境中耐药菌污染与危害研究进展

细菌在抗菌药选择性压力下产生耐药性并可传代,通过质粒和整合子等可移动基因元件将耐药基因在相同或不同种属中广泛传播,导致细菌多重耐药,并可通过多种途径进入水体,水环境日益成为庞大的耐药基因库,为致病菌及条件致病菌提供获得大量耐药基因的机会,若多重耐药菌再次侵入人体,可能引发严重的公共卫生问题。     

Influences of native and non-native benthivorous fishes on aquatic ecosystem degradation

Invasive common carp Cyprinus carpio has long been identified as a contributor to water quality deterioration, disrupted ecosystem processes, and shifts in biological assemblage structure. In contrast, little information is available regarding the effects of native benthivorous species on aquatic systems despite their functional similarity to common carp. Effects of common carp and the native black bullhead Ameiurus melas on sediment resuspension, nutrient concentrations, macrophytes, and assemblage structure of zooplankton and macroinvertebrates, were experimentally evaluated. We observed decreased water clarity, increased nutrient concentrations, decreased macrophyte biomass, and decreased benthic macroinvertebrate abundance and biomass associated with common carp, regardless of the presence of black bullhead. In contrast, black bullhead increased total phosphorus, and chlorophyll a concentrations and copepod abundance, but had little or no effect on other measured water quality and biological variables when compared to control conditions. Overall, experimental results suggest that although black bullhead tend to be tolerant of degraded ecosystems, they are not a source of physical changes to the environment known to be responsible for perpetuating degraded water quality (e.g., stable state shifts). Therefore, increased abundance of native species, such as black bullhead, following the invasion of common carp, may serve as an indicator of ecological conditions and should not to be assumed as causative.     

An in-vitro study on a novel six-phage cocktail against multi-drug resistant-ESBL Shigella in aquatic environment

Shigella spp. are water-borne pathogens responsible for mild to severe cases bacilli dysentery all around the world known as Shigellosis. The progressively increasing of antibiotic resistance among Shigella calls for developing and establishing novel alternative therapeutic methods. The present study aimed to evaluate a novel phage cocktail of lytic phages against extended spectrum beta lactamase isolates of Shigella species in an aquatic environment. The phage cocktail containing six novel Shigella specific phages showed a broad host spectrum. The cocktail was very stable in aquatic environment. The cocktail resulted in about 99% decrease in the bacterial counts in the contaminated water by several species and strains of Shigella such as Shigella sonnei, Shigella flexneri and Shigella dysenteriae. Achieving such a high efficiency in this in-vitro study demonstrates a high potential for in-vivo and in-situ application of this phage cocktail as a bio-controlling agent against Shigella spp. contamination and infections.     

Pollution and parasitism in the aquatic environment

The studies of aquatic parasitology and of aquatic pollution effects both have experienced increasing interest during the recent fifteen years. Although considerable effort has been spent on studying the role of pollution as a cause or a trigger of anomalies, tumors and infectious diseases in aquatic organisms, the interactions between pollution and parasitism have been largely neglected by scientists.Pollution and other man-made alterations of the aquatic environment may affect a parasite community directly by acting on free-living parasite stages or on ectoparasites, or indirectly by acting on the intermediate or the definitive host population. Certain pollution conditions favour the propagation of parasites by excluding their natural predators, by reducing the resistance of their hosts or by providing improved living conditions for their intermediate hosts. In a number of experimental studies parasitized organisms have been shown to suffer from greater mortalities when exposed to high temperature, to low oxygen stress or to high levels of dissolved heavy metal salts, when compared to nonparasitized control animals. Unfortunately, field studies on synergistic effects of pollution and parasites on host populations are still scarce and seldom offer more than qualitative observations and theoretical evaluations.The complexity of the pollution-parasite-host system complicates the use of parasites as indicators of pollution effects. However, experience from aquaculture practice teaches that a number of (mostly ecto-) parasites are more susceptible to certain chemicals (used as parasiticides) and to artificial alterations of salinity, temperature or oxygen content of the water than their hosts. Accordingly, it is postulated that during future studies the composition of ectoparasitic faunas of aquatic organisms might turn out to become a useful and quickly reacting indicator for effects of certain pollution conditions, such as anthropogene oxygen deficiency, salt introduction from salines into freshwater ecosystems, and introduction of certain heavy metal salts.     

The evolution of mate choice in a fluctuating environment

This paper analyzes the evolutionary dynamics of a locus controlling the degree of female mating preference in a temporally fluctuating environment. Preference for mating with males with respect to their genotypes at a locus that is subject to temporally varying natural selection pressure is considered first. With weak selection and free recombination between the choice locus and the selected locus, preference for mating with heterozygotes appears to be favored. With strong selection, preference for homozygous mates may be favored. In each case, choice alleles may increase from very low initial frequencies to near fixation, in contrast to previous models of mate choice in varying environments. Linkages between the two loci has complex effects on the strength and direction of selection for mate choice. Preference for mating with males with the currently fitter genotypes at the locus under natural selection is also modelled. Provided that the environmental period is not too short, a rare allele conferring such preference may be favored and spread to fixation. Strong natural selection, tight linkage and a short environmental period may produce polymorphism for the level of mate choice.     

Patterns of recruitment of coral reef fishes in a monsoonal environment

The abundance of newly settled recruits of coral reef fishes was monitored at a total of 11 sites at two islands and two coastal locations in the central Philippines for a 20-month period (February 2008 to September 2009) that included two monsoon cycles. Recruitment occurred throughout the year. Most of the abundant species exhibited protracted recruitment seasons. This confirms the expectation of extended breeding of reef fishes at lower latitudes. The annual pattern of recruitment of reef fishes as a group was predictable. Annual fluctuations of sea surface temperature and wind strength largely explained the pattern. Rainfall, however, did not significantly influence the pattern of recruitment. Peaks in density and species richness of recruits occurred during the southwest monsoon and the second inter-monsoonal period of the year (July to October) when temperatures were highest and when most of the sites were sheltered from winds or when winds were weak. Conversely, lowest density and species richness were observed during the northeast monsoon (November to March) when temperatures were lowest and most sites were exposed to winds. The same pattern could also be seen in the recruitment of both damselfishes (Pomacentridae) and wrasses (Labridae), notwithstanding a tenfold difference in abundance of recruits between the two families. The pattern was fairly consistent across most sites, among most of the species that were examined, and between the 2 years that were sampled. This study is one of the few to provide insights into the influence of environmental factors on the recruitment patterns of fishes on Indo-Pacific coral reefs situated at lower latitudes.     

Trematode metacercariae of fishes as sentinels for a changing limnological environment

Trematode metacercaria populations infecting cichlids in Lake Kinneret (Sea of Galilee) were used as sentinels for the changing limnological environment. Parasitological data from 0+ cichlid fingerlings (Tilapia zillii, Oreochromis aureus, Sarotherodon galilaeus) were collected from the northwest shore of the lake during 1999 to 2001 and compared with data obtained from 1982 to 1984. The results show that the composition of the metacercarial populations changed significantly between the 2 samplings periods. The total number of metacercarial species found in the Kinneret cichlids was lower in 1999 to 2001 than in 1982 to 1984. Metacercariae transmitted by the snail Bulinus truncatus (Clinostomum tilapiae, Euclinostomum heterostomum, Bolbophorus levantinus and Neascus-type metacercariae--black spot and others) that were commonly found in 1982 to 1984 were absent from the fishes sampled during 1999 to 2001. The other trematode metacercariae (Centrocestus sp. and Haplorchis sp. transmitted by Melanoides tuberculata, Pygidiopsis genata transmitted by Melanopsis costata, as well as Phagicola longa, Strigeidae sp.1, an unidentified metacercaria typically found in the liver, and glochidia) increased in abundance in fishes sampled during 1999 to 2001. The calculated 'true' species richness of the habitat, extrapolated as a function of sampling effort, was significantly lower in the 1999 to 2001 versus 1982 to 1984 samples, while significantly fewer fishes needed to be sampled ('sampling effort') during 1999 to 2001 in order to reach the 'true' species richness. The higher calculated values of species richness and diversity for the 1999 to 2001 samples despite the overall decline in species number is explained by the overall increase in metacercariae prevalence.     

Acid-base regulation in fishes: cellular and molecular mechanisms

The mechanisms underlying acid-base transfers across the branchial epithelium of fishes have been studied for more than 70 years. These animals are able to compensate for changes to internal pH following a wide range of acid-base challenges, and the gill epithelium is the primary site of acid-base transfers to the water. This paper reviews recent molecular, immunohistochemical, and functional studies that have begun to define the protein transporters involved in the acid-base relevant ion transfers. Both Na(+)/H(+) exchange (NHE) and vacuolar-type H(+)-ATPase transport H(+) from the fish to the environment. While NHEs have been thought to carry out this function mainly in seawater-adapted animals, these proteins have now been localized to mitochondrial-rich cells in the gill epithelium of both fresh and saltwater-adapted fishes. NHEs have been found in the gill epithelium of elasmobranchs, teleosts, and an agnathan. In several species, apical isoforms (NHE2 and NHE3) appear to be up-regulated following acidosis. In freshwater teleosts, H(+)-ATPase drives H(+) excretion and is indirectly coupled to Na(+) uptake (via Na(+) channels). It has been localized to respiratory pavement cells and chloride cells of the gill epithelium. In the marine elasmobranch, both branchial NHE and H(+)-ATPase have been identified, suggesting that a combination of these mechanisms may be utilized by marine elasmobranchs for acid-base regulation. An apically located Cl(-)/HCO(3)(-) anion exchanger in chloride cells may be responsible for base excretion in fresh and seawater-adapted fishes. While only a few species have been examined to date, new molecular approaches applied to a wider range of fishes will continue to improve our understanding of the roles of the various gill membrane transport processes in acid-base balance.     

Reproductive strategies of Amazonian stream fishes and their fine‐scale use of habitat are ordered along a hydrological gradient

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Fluorous-tagged compound: a viable scaffold to prime oligosaccharide synthesis by cellular enzymes

Fluorous-tagged saccharide primers could be viable scaffolds for the synthesis of oligosaccharides. This research demonstrates that a fluorine-containing saccharide derivative could actually be taken up by the cell, the saccharide chain elongated by cellular enzymes, and the elongated product released by the cells to the culture medium. A fluorous-tagged lactoside primer, 6-(perfluorohexyl)hexyl-4-O-(beta-D-galactopyranosyl)-beta-D-glucopyranoside, was chemically synthesized and introduced in mouse B16 cells to prime oligosaccharide synthesis. Uptake of the primer by B16 cells resulted in the sialylation of the terminal galactose residue to afford an oligosaccharide with the same glycan structure as ganglioside GM3. The presence of many fluorine atoms did not have any adverse effects to the cells. Moreover, the number of fluorine atoms did not pose a steric barrier and instead, their presence possibly increased the hydrophobicity of the primer and enhanced membrane permeability. This strategy of using a fluorous-tagged primer and cells can pave the way for an easier way of preparing oligosaccharides via an environment-friendly approach that eliminates the use of large amounts of organic solvents.     

Development of a risk assessment for BSE in the aquatic environment

Bovine spongiform encephalopathy (BSE) is believed to be transmitted by the ingestion of proteinaceous agents called prions which accumulate in the brain and spinal cord of infected bovines. Concern has been expressed about the risks of transmission of BSE to humans through BSE prions discharged to the aquatic environment from rendering plants, abattoirs and landfills. The disease-related form of the prion protein is relatively resistant to degradation, and infectivity decays rather slowly in the environment. Levels of disinfection used for drinking water treatment would have little effect. This paper presents the assumptions which were used to model the risks from a rendering plant disposing of cull cattle carcasses in the catchment of a chalk aquifer which is used for a drinking water abstraction. The risk assessment approach focused on identifying the hydrogeological and physical barriers which would contribute to preventing BSE infectivity gaining entry to the aquifer. These barriers included inactivation of BSE agent by the rendering process, removal from the effluent by treatment at the plant, filtration and adsorption in the clay and chalk, and dilution in the ground water. The importance in environmental risk assessment of the cow-to-man species barrier is considered. Two key conclusions about the environmental behaviour of the BSE agent are that prion proteins are 'sticky' and bind to particulates, and that the millions of BSE prion molecules comprising a human oral ID₅₀ are subject to some degree of dispersion and hence dilution in the environment. Assuming the rendering plant processes 2000 cull cattle carcasses per week, the risks to drinking water consumers were estimated to be remote. Indeed, even using worst case assumptions an individual would have to consume 2 l d⁻¹ of tap water for 45 million years to have a 50% chance of infection through drinking water drawn from the aquifer.