Histopathological analyses of native silver catfish Rhamdia quelen (Quoy and Gaimard, 1824) immunized against and challenged with live theronts of Ichthyophthirius multifiliis

As an alternative to treating with chemicals, immunization using Ichthyophthirius multifiliis as a vaccine has been studied in fishes that were often affected with white spot diseases also to understand the possible changes to the tissue caused by the vaccine. The focus of this study was the analysis of the influence of immunization via intraperitoneal injection (i.p.) and via immersion bath (im.b.) on the histopathology of R. quelen after being challenged with live theronts of I. multifiliis distributed in: control (non‐immunized and non‐challenged); non‐immunized and challenged with 12,000 theronts/fish; non‐immunized and challenged with 22,000 theronts/fish; immunized and challenged with 12,000 theronts/fish; immunized and challenged with 22,000 theronts/fish. Water quality was measured in each assay, with 300 fingerlings distributed among 15 tanks with 20 fish in each of three replicates. Six days after challenge, samples for histopathological and parasitological analyses were collected. In both i.p. and im.b. fish the prevalence of I. multifillis in the gills was higher in the non‐immunized fish (33.33% and 27.77%, respectively). Melanomacrophages were present in 53% of the samples of i.p. non‐immunized fish. Fish im.b. immunized and challenged showed more atrophied areas in the hepatocytes. Higher numbers of melanomacrophages in the i.p. non‐immunized fish kidneys were observed compared to control. The results showed no difference in the gill lesions of either immunized or non‐immunized fish compared to control. Histological alterations in the organs of silver catfish were considered light, except in the liver that presented significant atrophy and hypertrophy of hepatocytes after immunization via i.p.     

Evaluation of Two Monitoring Approaches to Assess Effects of Waste Water Disposal on Histological Alterations in Fish

An active monitoring (caging experiment) and a passive monitoring (sampling of wild fish) were performed to investigate the effects of effluent from a sewage treatment works (STW) on brown trout (Salmo trutta) by histopathological examinations of the skin, gill, liver and kidney. Histopathological lesions were evaluated according to a standardised assessment tool, which allows calculation of indices for every organ. According to the results of both monitorings, trout exposed to river water supplemented with treated waste water from the STW Lyss showed higher histopathological indices than trout caught upstream of the discharge point of the STW or kept in river water only. These results indicate a negative effect of treated waste water from the STW on the histopathological status of the examined organs of brown trout. Both monitoring approaches revealed the liver to be the most affected organ compared with reference fish. However, data from the two monitoring approaches were not completely consistent: histologically the gills were the most sensitive organ to the effects of treated waste water in the active monitoring, but were not affected in the passive monitoring. The data provide relevant information about both the comparability and the pros and cons of the two monitoring approaches to assess effects of pollution on histopathological alterations in fish.     

Pathogenesis of mulberry leaf spot in response to meteorological aspects of Kashmir,India

In this study, the influence of climatic factors on the development of leaf spot diseases of mulberry (Morus alba L.) in Kashmir, India was studied. It was found that the highest disease incidence and severity of Goshoerami variety was found in tree type (13.86% and 1.98%) followed by dwarf (7.53% and 1.67%) and bush (3.38% and 0.99%), respectively. Due to infection of pathogen, the relative water content and chlorophyll content of leaves were consistently decreased. The order of reduction in relative water content of infected leaves was found as: bush?>?dwarf?>?tree, while as the reduction in chlorophyll content: bush?>?tree?>?dwarf. It was also found that temperature of 25–30?°C and relative humidity of above 80% favoured the development of the disease. Rapid development of disease was found during the month of June, July and August when average temperature and relative humidity was about 25–30°C and 80%, respectively.     

Histological Evidence for Different Spread of Fusarium crown rot in Barley Genotypes with Different Heights

Based on visual assessment of disease severity, previous studies reported that tall genotypes tend to be more severely affected by Fusarium crown rot (FCR) in wheat and barley. To clarify whether tall and dwarf genotypes have different susceptibility to FCR or whether it takes longer for Fusarium pathogens to infect dwarf genotypes, histological analyses were conducted with two pairs of near isogenic lines (NILs) for a semi‐dwarfing gene in barley. This analysis showed that F. pseudograminearum hyphae were detected earlier and proliferated more rapidly during the time‐course of FCR development in the tall isolines. Histological analysis showed that cell densities of the dwarf isolines were significantly higher than those of the tall isolines due to reduced lengths and widths of cells, and FCR severity was strongly correlated with cell density. An analysis with real‐time quantitative polymerase chain reaction detected a higher amount of F. pseudograminearum in the tall isolines at each of the time points assessed during FCR development. These results support the hypothesis that the increased cell density associated with dwarf genes could act as a physical barrier to the spread of FCR in cereals.     

Biochemical and toxicopathic biomarkers assessed in smallmouth bass recovered from a polychlorinated biphenyl-contaminated river

Smallmouth bass (Micropterus dolomieu) were collected to quantify the nature and prevalence of biomarker responses, including biochemical indices, toxicopathic lesions and general health indices, among fish collected from polychlorinated biphenyl (PCB)-contaminated and nearby uncontaminated reaches of the Kalamazoo River, Michigan, USA. Blood and tissue samples (gill, liver, spleen, head kidney, trunk kidney, thyroid and gonads) were collected and preserved at necropsy for biochemical and histological analyses. The body condition factor and liver somatic index were significantly lower in fish collected from the downstream, contaminated site. Plasma vitellogenin was not detected in male fish collected from either site. Liver ethoxyresorufin-O-deethylase activity and liver and spleen superoxide dismutase activity were significantly depressed in fish collected from the downstream site. Significant toxicopathic lesions such as glycogen depletion, enhanced macrophage aggregates, hepatic foci of cellular alteration (i.e. preneoplastic lesions) and neoplasia were also detected in the liver of fish collected from the downstream site. This study indicates that many of the biochemical and histopathological biomarker responses were associated with liver and body tissue PCB concentrations. Taken together, the biomarkers of exposure and effect strongly suggest that fish within the downstream site are adversely affected by PCBs and other chemical stressors.     

Effects of landscape features and demographic history on the genetic structure of Testudo marginata populations in the southern Peloponnese and Sardinia

Testudo marginata, the largest European land tortoise, is suffering habitat degradation and destruction. Some populations, in markedly degraded habitats, are characterized by divergent morphotypes. However, the evolutionary significance of these morphotypes is of debate. Using 11 polymorphic microsatellites, we studied: (1) marginated tortoises from Sardinia that display a divergent morphotype – this population was potentially introduced from Greece; and (2) an area in the southern Peloponnese that includes a small and degraded zone in which marginated tortoises are dwarf. Genetic analyses run without any a priori assignment clearly acknowledge the specimens sampled in the territory of the dwarf form as a single group whilst Sardinian specimens are clustered with other specimens from the northern part of the area sampled in Greece. Demographic analyses suggest that Sardinian tortoises originated recently from some of the populations sampled in the northern part of the area sampled in Greece. Over locations sampled in Greece, a landscape‐genetic analysis allowed us to detect potential landscape features that may reduce gene flow between the dwarf form territory and surrounding areas. Our results suggest that the territory of the dwarf form is particularly propitious for marginated tortoises and that conservation regulations in Greece should be reinforced to protect this area from increasing impact of human activities changing from traditional agriculture to mechanization and extensive use of chemicals. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 105 , 591–606.     

How have the alpine dwarf plants in Yakushima been miniaturized? A comparative study of two alpine dwarf species in Yakushima, Blechnum niponicum (Blechnaceae) and Lysimachia japonica (Primulaceae)

Many plant species are miniaturized in the alpine region in Yakushima, Japan. To examine how these alpine dwarf plants are different from their related lowland ones of the same species, we analyzed two phylogenetically distinct species cytologically, genetically and morphologically: one is a fern species, Blechnum niponicum, and the other is an angiosperm species, Lysimachia japonica. The analysis shows that the alpine dwarf and the lowland plants in each of these species do not differ in chromosome number or genetic constitution. The organ-level comparison between the alpine dwarf and lowland plants of B. niponicum shows that the fertile leaf size correlates closely with the sterile one. By contrast, the flower size does not correlate with the leaf size in L. japonica. At the cell level, the leaf size of the alpine dwarf plants of B. niponicum consists of a smaller number of epidermal cells than that of the lowland plants of this species. On the other hand, the smaller leaf size of the alpine dwarf plants of L. japonica depends on both the smaller number and the smaller size of the epidermal cells. We conclude that plant dwarfism in Yakushima shows variation at both the organ and cell levels.     

Water temperature sensitivity of EOD waveform in Brachyhypopomus pinnicaudatus

The mechanisms that trigger the onset of the breeding season depend on geographical latitude. At the edge of Gymnotiform distribution in America, variations in day length and water temperature are likely cues to initiate breeding. In this study we aim to clarify the role of temperature and the interaction between temperature and hormonal state upon electric organ discharge waveform. In breeding ponds, we measured naturally occurring changes of water temperature and of electric organ discharge waveform during two 48-h periods in a sample of identified mature males and females of Brachyhypopomus pinnicaudatus. Water temperature, day-night cycle, and sexual maturity each modified electric organ discharge waveform. Temperature sensitivity was also evaluated in the laboratory in adult sexually-differentiated individuals, adult non-differentiated fish, juveniles, and testosterone-treated fish. Our data strongly suggest an interaction between the effects of temperature and steroid hormones upon electric organ discharge waveform. High temperature (30 °C) induced a significant decay of head negative phase amplitude in temperature-sensitive fish. This sensitivity was observed in physiological conditions that coincide with low levels of steroid hormones: juveniles and adult fish kept in captivity at 20–21 °C. Conversely, temperature resistance was observed in mature fish in the breeding habitat and was induced by testosterone treatment and by captivity at 27–28 °C. Accepted: 23 May 1999     

Double evaluations of chemicals using a cocktail of fused recombinant receptors

Some chemicals have multipotential as endocrine-disrupting chemicals (EDCs). For example, some chemicals act as both estrogens and antiandrogens. Numbers of such chemicals should be evaluated from many aspects; however, labor and expenses are generally limited. We have developed two expression systems for the wild type of human estrogen receptor alpha and the wild type of human androgen receptor fused with a maltose binding protein. They are soluble and have binding activities. They showed dose-responses to natural hormones and well-known potential EDCs. After we established each assay condition for a competitive binding assay using each receptor, we found that two assay systems can be carried out simultaneously under limited and harmonized conditions. Under harmonized conditions using a cocktail of two types of receptors, we could estimate natural hormones and potential EDCs. Interference between two assay systems was not observed under these conditions. We believe that some competitive binding assays can be carried out using a cocktail of receptors at the same time if interference among different assay systems can be avoided by choosing ideal conditions.     

The use of histopathological indicators to evaluate contaminant-related stress in fish

As a component of a large research program toevaluate the effects of contaminants on fish healthin the field, histopathological studies have beenconducted to help establish causal relationshipsbetween contaminant exposure and various biologicalresponses. Brown trout (Salmo trutta f. fario)and loach (Barbatula barbatula) were exposedto water diverted from polluted streams undersemi-field conditions at various times during theyear. The histopathological studies revealedseasonal differences in the types and severity oforgan lesions between fish of the two streams. Bothtoxicant-induced alterations and organ lesionsresulting from natural stressors (physicochemicaland limnological water parameters) and secondarystress effects of pollution (diseases) could bedetected. In evaluating the general health ofexperimental and control fish, the use ofhistopathological studies are recommended for makingmore reliable assessments of biochemical responsesin fish exposed to a variety of environmentalstressors. Stereological analysis providesquantitative data on pathological lesions whichhelps to establish correlation with other biomarkersthereby increasing the probability of identifyingcause (stressor) and effect (biomarker) relationships.     

Sediment-associated contaminants and liver diseases in bottom-dwelling fish

High concentrations of chemicals have been found in sediments from urban areas of Puget Sound. Hundreds, of organic chemicals (including certain aromatic hydrocarbons [AHs] and various chlorinated compounds) were identified. Statistical methods were used to evaluate possible relationships between the chemistry data and fish diseases. Positive correlations were found between the frequencies of liver neoplasms (e.g., hepatocellular carcinoma) and other liver lesions in English sole (Parophrys vetulus) and concentrations of AHs in sediment; such correlations were not found with chlorinated hydrocarbons. Strong evidence was also obtained to show that many organic chemicals in sediment are bioavailable to bottom-dwelling fish. Stomach contents (consisting mainly of benthic invertebrates) from English sole had concentrations of a number of AHs similar to those in the sediment from which the fish were taken. In these same fish, metabolites of many aromatic compounds were found in bile using a procedure combining HPLC with fluorescence detection. Further, the concentrations of certain xenobiotic metabolites in bile were correlated positively with the occurrence of liver neoplasms in English sole.     

Short-range Navigation of the Weakly Electric Fish,Gnathonemus petersii L. (Mormyridae,Teleostei), in Novel and Familiar Environments

We investigated the electrolocation performance of the weakly electric fish, Gnathonemus petersii, in novel and familiar environments. By selectively interfering with the fish's sensory input, we determined the sensory channels necessary for navigation and orientation. The fish's task was to locate a circular aperture (diameter: 64 mm) in a wall dividing a 200–1 aquarium into two equal compartments. To assess the fish's performance, we measured (1) the time it took the fish to locate the aperture, (2) the height at which it contacted the divider, (3) its electric organ discharge rate, and (4) the frequency of divider crossings. In the first experiment (novel environment), 50 naive G. petersii assigned to five groups of 10 fish each (intact, blind, electrically “silent,” blind and “silent,” and shamoperated animals) were tested with the aperture presented randomly in one of three positions (aperture center: 7.6, 17.7, 27.8 cm from the bottom). In a novel environment, G. petersii depend on active electrolocation. Despite the changing aperture position, over the 15 trials, fish with a functioning electric organ found the aperture, whereas those without one did not. The electric organ discharge rate was inversely correlated with the amount of time spent searching for the aperture. In a second experiment (familiar environment) 20 intact fish learned the position of a fixed aperture. When we subsequently denervated the electric organ in 10 of these animals, their performance did not differ significantly from that of their conspecifics. Thus, once the fish were familiar with the aperture's position, they no longer depended on active electrolocation. We interpret and discuss this behavior as evidence for a “central expectation” and discuss its possible role in electronavigation.     

Diversification, Sympatric Speciation, and Trophic Polymorphism of Arctic Charr, Salvelinus Alpinus Complex, in Transbaikalia

In northern Transbaikalia, independently evolving landlocked populations of Arctic charr are found in mountain lakes. To assess the diversity of charr in this region, speciation modes involved in the evolution of charr forms, and the role of trophic polymorphism in their divergence, we studied the morphology and feeding of dwarf, small, and large forms of Arctic charr from a number of Transbaikalian lakes. Meristic data on charr from five lakes support the earlier conclusion that the three forms do not represent separate lineages but have independently diverged in sympatry in each of the lakes. In 10 lakes, the dwarf form showed varying degrees of differentiation from normal (small and large) charr in meristic characters (up to morphologically distinct and presumably reproductively isolated groupings), which is viewed as various levels of sympatric divergence. Indexes of gill raker length in fish from 20 lakes vary among populations of both dwarf and normal charr, with forms having short and long rakers being sympatric in some of these lakes. However, the index can be used only for comparing charr of different forms up to about 32cm fork length (FL) because it is strongly negatively correlated with size in larger fish. The study of charr diets in 21 lakes indicates that large charr are piscivorous whereas dwarf and small charr feed on a wide range of invertebrates, partitioning these resources in different ways. Planktivores, including very specialized ones, and non-planktivores (benthic feeders, insectivores) can be identified within the small and dwarf forms. The proportion of plankton in the diets of dwarf and small charr is positively correlated with the number and length of gill rakers while the proportion of benthos is negatively correlated. Allopatric planktivorous and non-planktivorous small charr differ in body proportions; parallel emergence of such morphotypes in different parts of the range is a characteristic feature of the Salvelinus alpinus complex.     

Variability of the electric organ discharge interval duration in resting Gymnotus carapo

 We recorded the electric organ discharges of resting Gymnotus carapo specimens. We analyzed the time series formed by the sequence of interdischarge intervals. Nonlinear prediction, false nearest neighbor analyses, and comparison between the performance of nonlinear and linear autoregressive models fitted to the data indicated that nonlinear correlations between intervals were absent, or were present to a minor extent only. Following these analyses, we showed that linear autoregressive models with combined Gaussian and shot noise reproduced the variability and correlations of the resting discharge pattern. We discuss the implications of our findings for the mechanisms underlying the timing of electric organ discharge generation. We also argue that autoregressive models can be used to evaluate the changes arising during a wide variety of behaviors, such as the modification in the discharge intervals during interaction between fish pairs. Received: 14 March 2000 / Accepted in revised form: 9 October 2000     

Neuropathology of mitochondrial diseases

The term “mitochondrial diseases” (MD) refers to a group of disorders related to respiratory chain dysfunction. Clinical features are usually extremely heterogeneous because MD may involve several tissues with different degrees of severity. Muscle and brain are mostly affected, probably because of their high dependence on oxidative metabolism. Muscle can be the only affected tissue or involved as a part of a multi-system disease; ragged red fibers, accumulation of structurally altered mitochondria and cytochrome-c-oxidase (COX) negative fibers are the main pathological features. In mitochondrial encephalopathies, central nervous system (CNS) structures are affected according to different patterns of distribution and severity. Characteristic lesions are neuronal loss, vasculo-necrotic changes, gliosis, demyelination and spongy degeneration. In accordance with either grey matter or white matter involvement two main groups of diseases may be distinguished. Neuronal loss and vasculo-necrotic multifocal lesions are the common features of grey matter involvement; demyelination and spongy degeneration occur when white matter is affected, often associated with less severe lesions of the grey structures. Grey matter lesions are prevalent in MERRF, MELAS, Alpers and Leigh syndromes. White matter involvement is always seen in Kearns-Sayre syndrome and was recently described in mtDNA depletion syndrome linked to dGK mutations and in the rare conditions associated with complex I and II deficiency. In this review we describe the main histopathological features of muscle and CNS lesions in mitochondrial diseases.     

Validation of a simple and well‐suited chemical cleaning method for fish otoliths

In marine biology, many research fields are based on use of fish otoliths. All the studies dealing with otoliths need as starting point a perfectly clean otolith. Dissection is difficult when working on small or highly jagged otoliths. A common problem is that during otolith preparation some fish tissue may remain stuck to it, even after a mechanical cleaning. Then, supplementary cleaning with chemicals is needed. Classical methods are known to possibly alter otolith's structure and/or composition. Here, we present a chemical cleaning method using only sodium hydroxide. We have validated the method on two different fish species, Oblada melanura and Dicentrarchus labrax. Observation and analyses of morphological measurements have confirmed significant suppression of residual tissues. We propose this method as a good alternative to previously published mono‐ or multichemical methods.     

Characterization of chemical defenses in ranid tadpoles against a fish predator

Tadpoles of some ranid species appear to possess chemical defenses against fish predators, but the chemicals have not been characterized. Here, we evaluated the vulnerability of three Japanese anuran tadpole species (Glandirana rugosa, Pelophylax nigromaculatus, and Hyla japonica) to a fish (Gnathopogon elongatus elongatus) and analyzed the defensive chemicals extracted from the unpalatable tadpoles. Additionally, we examined the defensive behavior of unpalatable tadpoles in response to fish chemical cues. The fish rejected both G. rugosa (83%) and P. nigromaculatus (48%), but not H. japonica (0%). Many of the rejected tadpoles survived (60–80%). Possible defensive chemicals were extracted by methanol from the skin of G. rugosa, but were not identifiable by gas chromatography–mass spectrometry because of small quantities. The chemicals have high polarity and non-volatility. When exposed to fish chemical cues, P. nigromaculatus decreased activity presumably as a defensive behavior, but G. rugosa did not. We demonstrated the presence of chemical defenses in at least two of these species and revealed that G. rugosa releases more effective or greater amounts of defense chemicals than P. nigromaculatus with respect to this fish predator. The increased efficacy of chemical defenses may correlate with decreasing defensive behavior.     

Biodegradation of endocrine-disrupting phenolic compounds using laccase followed by activated sludge treatment

Endocrine-disrupting phenolic compounds in the water were degraded by laccase fromTrametes sp. followed by activated sludge treatment. The effect of temperature on the degradation of phenolic compounds and the production of organic compounds were investigated using endocrine-disrupting chemicals such as bisphenol A, 2,4-dichlorophenol, and diethyl phthalate. Bisphenol A and 2,4-dichlorophenol disappeared completely after the laccase treatment, but no disappearance of diethyl phthalate was observed. The Michaelis-Menten type equation was proposed to represent the degradation rate of bisphenol A by the lacasse under various temperatures. After the laccase treatment of endocrine-disrupting chemicals, the activated sludge treatment was attempted and it could convert about 85 and 75% of organic compounds produced from bisphenol A and 2,4-dichlorophenol into H₂O and CO₂, respectively.     

Behavioral repertoire of biparental care in Apistogramma trifasciata (Pisces: Cichlidae)

Cichlid fish exhibit a complex behavioral repertoire, mainly regarding social and reproductive behaviors. Nevertheless, although dwarf cichlids exhibit a remarkable diversity of behaviors, only a few studies have been conducted with these fish. Apistogramma trifasciata is a common dwarf cichlid in the Neotropics and widely used as ornamental fish. Both males and females are territorial and engage in biparental care. Here we observed the behavior of A. trifasciata males and females to construct an ethogram. We registered the parental care of a couple since the spawn until the moment when the offspring became independent. Striking behaviors were observed, such as male-male contests involving circling displays and mouth wrestling, and parental care, which involved mouth brooding and parental color change. Finally, we suggest that our results may be used to establish this species as a model for parental care and territoriality studies.