Antimicrobial activity in culturable gut microbial communities of springtails.

AIMS: The rise of antibiotic resistance pushes the pharmaceutical industry to continually search for substances with new structures and novel mechanisms of action. Many environmental niches are still to be explored as sources of antimicrobials. In this paper, we assess the antimicrobial potential of gut microbes of springtails, soil invertebrates which live in a microbe-dominated environment and are known to be tolerant to entomopathogenic micro-organisms. METHODS AND RESULTS: Bacteria isolated from the guts of five springtail species were tested for inhibitory activity against different microbial pathogens. We identified 46 unique isolates belonging to 17 genera and 15 families. Thirty-five of these isolates (76%) showed inhibitory activity, and 18 inhibited both bacterial and fungal pathogens. One isolate was active against all the pathogens tested. CONCLUSIONS: We demonstrated a range of antimicrobial activities in bacteria isolated from the guts of springtails, indicative of complex interactions within the gut community, possibly relating to nutrition or defence against pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results suggest that a large proportion of cultivatable microbes associated with Collembola have a potential for antimicrobial production. We propose that soil invertebrates and their associated microbes are interesting targets for drug discovery.

Transgenerational effects of nutrition are different for sons and daughters.

Food shortage is an important selective factor shaping animal life-history trajectories. Yet, despite its role, many aspects of the interaction between parental and offspring food environments remain unclear. In this study, we measured developmental plasticity in response to food availability over two generations and tested the relative contribution of paternal and maternal food availability to the performance of offspring reared under matched and mismatched food environments. We applied a cross-generational split-brood design using the springtail Orchesella cincta, which is found in the litter layer of temperate forests. The results show adverse effects of food limitation on several life-history traits and reproductive performance of both parental sexes. Food conditions of both parents contributed to the offspring phenotypic variation, providing evidence for transgenerational effects of diet. Parental diet influenced sons' age at maturity and daughters' weight at maturity. Specifically, being born to food-restricted parents allowed offspring to alleviate the adverse effects of food limitation, without reducing their performance under well-fed conditions. Thus, parents raised on a poor diet primed their offspring for a more efficient resource use. However, a mismatch between maternal and offspring food environments generated sex-specific adverse effects: female offspring born to well-fed mothers showed a decreased flexibility to deal with low-food conditions. Notably, these maternal effects of food availability were not observed in the sons. Finally, we found that the relationship between age and size at maturity differed between males and females and showed that offspring life-history strategies in O. cincta are primed differently by the parents.

Single-cell analysis of peptide expression and electrophysiology of right parietal neurons involved in male copulation behavior of a simultaneous hermaphrodite.

Male copulation is a complex behavior that requires coordinated communication between the nervous system and the peripheral reproductive organs involved in mating. In hermaphroditic animals, such as the freshwater snail Lymnaea stagnalis, this complexity increases since the animal can behave both as male and female. The performance of the sexual role as a male is coordinated via a neuronal communication regulated by many peptidergic neurons, clustered in the cerebral and pedal ganglia and dispersed in the pleural and parietal ganglia. By combining single-cell matrix-assisted laser mass spectrometry with retrograde staining and electrophysiology, we analyzed neuropeptide expression of single neurons of the right parietal ganglion and their axonal projections into the penial nerve. Based on the neuropeptide profile of these neurons, we were able to reconstruct a chemical map of the right parietal ganglion revealing a striking correlation with the earlier electrophysiological and neuroanatomical studies. Neurons can be divided into two main groups: (i) neurons that express heptapeptides and (ii) neurons that do not. The neuronal projection of the different neurons into the penial nerve reveals a pattern where (spontaneous) activity is related to branching pattern. This heterogeneity in both neurochemical anatomy and branching pattern of the parietal neurons reflects the complexity of the peptidergic neurotransmission involved in the regulation of male mating behavior in this simultaneous hermaphrodite.

Collembolan Transcriptomes Highlight Molecular Evolution of Hexapods and Provide Clues on the Adaptation to Terrestrial Life.

BACKGROUND: Collembola (springtails) represent a soil-living lineage of hexapods in between insects and crustaceans. Consequently, their genomes may hold key information on the early processes leading to evolution of Hexapoda from a crustacean ancestor. METHOD: We assembled and annotated transcriptomes of the Collembola Folsomia candida and Orchesella cincta, and performed comparative analysis with protein-coding gene sequences of three crustaceans and three insects to identify adaptive signatures associated with the evolution of hexapods within the pancrustacean clade. RESULTS: Assembly of the springtail transcriptomes resulted in 37,730 transcripts with predicted open reading frames for F. candida and 32,154 for O. cincta, of which 34.2% were functionally annotated for F. candida and 38.4% for O. cincta. Subsequently, we predicted orthologous clusters among eight species and applied the branch-site test to detect episodic positive selection in the Hexapoda and Collembola lineages. A subset of 250 genes showed significant positive selection along the Hexapoda branch and 57 in the Collembola lineage. Gene Ontology categories enriched in these genes include metabolism, stress response (i.e. DNA repair, immune response), ion transport, ATP metabolism, regulation and development-related processes (i.e. eye development, neurological development). CONCLUSIONS: We suggest that the identified gene families represent processes that have played a key role in the divergence of hexapods within the pancrustacean clade that eventually evolved into the most species-rich group of all animals, the hexapods. Furthermore, some adaptive signatures in collembolans may provide valuable clues to understand evolution of hexapods on land.

Copper kinetics and internal distribution in the marbled crayfish (Procambarus sp.).

Metal pollution e.g. copper, in water bodies occurs worldwide. Although copper is an essential trace metal, at certain levels it is still considered as pollutant. The aim of this study was to investigate the effect of exposure concentration on copper bioaccumulation in marbled crayfish (Procambarus sp.) by determining uptake and elimination kinetics. Crayfish were exposed to sub-lethal copper concentrations (average measured concentrations of 0.031 and 0.38 mg Cu L(-1)) for 14 d and transferred to copper-free water for another 14 d. At different time points during the uptake and elimination phases copper concentrations were measured in five organs (exoskeleton, gills, muscle, ovaries and hepatopancreas). At 0.031 mg Cu L(-1), copper levels in the crayfish organs were not significantly increased compared to the control animals, suggesting effective regulation. Exposure to 0.38 mg Cu L(-1) did lead to not significantly increased copper levels in muscles and ovaries, while the gills and exoskeleton, which are in direct contact with the water, showed significantly higher copper concentrations. In these four organs, copper showed fast uptake kinetics with equilibrium reached within 10 d of exposure. Copper accumulation was highest in the hepatopancreas; uptake in this storage organ steadily increased with time and did not reach equilibrium within the 14-d exposure period. Copper accumulation levels in the marbled crayfish found in this study were hepatopancreas>gills>exoskeleton>muscle.

Narcotic mechanisms of acute toxicity of chlorinated anilines in Folsomia candida (Collembola) revealed by gene expression analysis.

In order to clarify the mechanisms of reproductive toxicity in a QSAR approach, the transcriptional signatures upon the 2 day exposure to the 28 days EC50 of a series of increasingly chlorinated aniline compounds and 1,2,3,4-tetrachlorobenzene were measured in Folsomia candida. In general, the transcriptional patterns associated with all compounds revealed toxicity at the cellular membranes and hence components of narcosis type I, but a principal component analysis revealed a deviating response by the pentachloroaniline and 2,3,5,6-tetrachloroaniline exposure. Moreover the expression of a subset of mainly biotransformation related genes showed a significant relationship with the logK(ow,) which stresses the presence of narcosis type I. This was confirmed by GO term enrichment at the level of cellular component. Genes involved in the three phases of xenobiotic biotransformation exhibited strict compound specific transcription patterns, which may reflect biotransformation processes in F. candida. Additional toxic mechanisms were especially observed for the 2,3,5,6-tetrachloroaniline, which possible works as an uncoupler or inhibitor of electron transport systems, which is revealed by the up-regulation of genes that encode different members of the electron transport chain. The aniline and 2,3,4-trichloroaniline exposure caused the induction of genes in the ROS defense system. Additional toxicity mechanisms were less clear, but they include the attack of microbial pathogens for the six other compounds and for 2,3,5,6-tetrachloroaniline an effect on mitochondrial protein folding.

Gene expression microarray analysis of heat stress in the soil invertebrate Folsomia candida.

Sudden temperature changes in soil can induce stress in soil-dwelling invertebrates. Hyperthermic conditions have an impact on gene expression as one of the first steps. We use a transcriptomics approach using microarrays to identify expression changes in response to heat in the springtail Folsomia candida. An elevation of temperature (Delta 10 degrees C) altered the expression of 142 genes (116 up-, 26 down-regulated). Many up-regulated genes encoded heat shock proteins, enzymes involved in ATP synthesis, oxidative stress responsive enzymes and anion-transporting ATPases. Down-regulated were glycoside hydrolases, involved in catalysis of disaccharides. The small number of altered transcripts suggest a mild response to heat in this soil invertebrate, but further research is needed to confirm this. This study presents candidate genes for future functional studies concerning thermal stress in soil-dwelling invertebrates.

The distinction between retractor and protractor muscles of the freshwater snail's male organ has no physiological basis.

Many animals are equipped with organs that can be everted, a notable example being male copulatory organs. The ability to protrude or evert an organ generally requires protractor and retractor muscles. Male copulatory behaviour of the pond snail Lymnaea stagnalis (L.) involves eversion (protraction) and retraction of the relatively large penis-carrying organ. For this preputium, protractor and retractor muscle bands have been defined, which implies eversion and retraction through the activity of these muscle bands. However, no physiological data are available that confirm that the terms protractor and retractor are appropriate. To test whether eversion and retraction are possible without protractor and/or retractor muscle bands, lesion experiments were performed. The results show that with either one or several muscle bands lesioned, snails were still capable of everting their preputium and using it for copulation. However, the majority of animals that had six or more muscle bands lesioned were unable to retract its preputium. Hence, retractor muscle bands serve their designated function whereas protractor muscle bands do not. We therefore suggest that a different terminology is used in which all muscle bands are retractors and, based on their location, are either called distal or proximal retractors. The findings furthermore indicate that the preputium muscle bands are normally contracted, possibly in a catch state, retaining the organ inside without high-energy expenditure.

Revealing pancrustacean relationships: phylogenetic analysis of ribosomal protein genes places Collembola (springtails) in a monophyletic Hexapoda and reinforces the discrepancy between mitochondrial and nuclear DNA markers.

BACKGROUND: In recent years, several new hypotheses on phylogenetic relations among arthropods have been proposed on the basis of DNA sequences. One of the challenged hypotheses is the monophyly of hexapods. This discussion originated from analyses based on mitochondrial DNA datasets that, due to an unusual positioning of Collembola, suggested that the hexapod body plan evolved at least twice. Here, we re-evaluate the position of Collembola using ribosomal protein gene sequences. RESULTS: In total 48 ribosomal proteins were obtained for the collembolan Folsomia candida. These 48 sequences were aligned with sequence data on 35 other ecdysozoans. Each ribosomal protein gene was available for 25% to 86% of the taxa. However, the total sequence information was unequally distributed over the taxa and ranged between 4% and 100%. A concatenated dataset was constructed (5034 inferred amino acids in length), of which ~66% of the positions were filled. Phylogenetic tree reconstructions, using Maximum Likelihood, Maximum Parsimony, and Bayesian methods, resulted in a topology that supports monophyly of Hexapoda. CONCLUSION: Although ribosomal proteins in general may not evolve independently, they once more appear highly valuable for phylogenetic reconstruction. Our analyses clearly suggest that Hexapoda is monophyletic. This underpins the inconsistency between nuclear and mitochondrial datasets when analyzing pancrustacean relationships. Caution is needed when applying mitochondrial markers in deep phylogeny.

Allelic diversity of metallothionein in Orchesella cincta (L.): traces of natural selection by environmental pollution.

The advances made in statistical methods to detect selection from DNA sequence variation has resulted in an enormous increase in the number of studies reporting positive selection. However, a disadvantage of such statistical tests is that often no insight into the actual source of selection is obtained. Finer understanding of evolution can be obtained when those statistical tests are combined with field observations on allele frequencies. We assessed whether the metallothionein (mt) gene of Orchesella cincta (Collembola), which codes for a metal-binding protein, is subject to selection, by investigating alleles and allele frequencies among European metal-stressed and reference populations. Eight highly divergent alleles were resolved in Northwest Europe. At the nucleotide level, a total of 51 polymorphic sites (five of them implying amino-acid changes) were observed. Although statistical tests applied to the sequences alone showed no indication of selection, a G-test rejected the null hypothesis that alleles are homogeneously distributed over metal-stressed and reference populations. Analysis of molecular variance assigned a small, but significant amount of the total variance to differences between metal-stressed and non-stressed populations. In addition, it was shown that metal-stressed populations tend to be more genetically diversified at this locus than non-stressed ones. These results suggest that the mt gene and its surrounding DNA region are affected by environmental metal contamination. This study illustrates that, in addition to statistical tests, field observations on allele frequencies are needed to gain understanding of selection and adaptive evolution.

Additive genetic variation of transcriptional regulation: metallothionein expression in the soil insect Orchesella cincta.

Field-selected metal tolerance in Orchesella cincta is correlated with overexpression of the single copy cadmium (Cd) inducible metallothionein (mt). Previously, we have demonstrated large phenotypic variation in mt gene expression, and a higher frequency of high-expression phenotypes in a tolerant population. Here, we describe midparent-offspring regression analysis of mt gene expression in a laboratory culture originating from a noncontaminated natural population. Families were either not exposed (n=47) or exposed to 0.5 micromol Cd per gram dry food (n=46). Mean mt gene expressions normalized to 28S rRNA and beta-actin RNA were generated using real-time RT-PCR applied to parents and offspring RNA and subjected to regression analysis. A significant heritability (h2) for mt gene expression was estimated between 0.36 (beta-actin normalized) and 0.46 (28S normalized) in Cd exposed families. Nontreated families did not yield a significant h2 value. Restriction Fragment Length Polymorphism analysis of the metallothionein promoter sequence revealed eight promoter alleles that show structural variation. Three alleles show increased frequencies in families with high mt expression. Another gene, croquemort (isolated from a differential screening for 1 micromole Cd treatment) showed no h2 of gene expression in response to 0.5 micromol Cd. This gene codes for a receptor-protein involved in recognition of apoptotic cells and may participate in the general stress response. The present data suggest that evolution of metal tolerance in O. cincta can occur in the field by selection for high mt expression due to structural changes in mt cis-regulation.

Genetic structure in Orchesella cincta (Collembola): strong subdivision of European populations inferred from mtDNA and AFLP markers.

Population genetic structure is determined both by current processes and historical events. Current processes include gene flow, which is largely influenced by the migration capacity of a species. Historical events are, for example, glaciation periods, which have had a major impact on the distribution of many species. Species with a low capacity or tendency to move about or disperse often exhibit clear spatial genetic structures, whereas mobile species mostly show less spatial genetic differentiation. In this paper we report on the genetic structure of a small, wingless arthropod species (Orchesella cincta: Collembola) in Europe. For this purpose we used mtDNA COII sequences and AFLP markers. We show that large genetic differences exist between populations of O. cincta, as expected from O. cincta's winglessness and sedentary lifestyle. Despite the fact that most variability was observed within populations (59%), a highly significant amount of AFLP variation (25%) was observed between populations from northwestern Europe, central Europe and Italy. This suggests that gene flow among regions is extremely low, which is additionally supported by the lack of shared mtDNA alleles between regions. Based on the genetic variation and sequence differences observed we conclude that the subdivision occurred long before the last glaciation periods. Although the populations still interbreed in the lab, we assume that in the long term the genetic isolation of these regions may lead to speciation processes.

Bioavailability of contaminants estimated from uptake rates into soil invertebrates.

It is often argued that the concentration of a pollutant inside an organism is a good indicator of its bioavailability, however, we show that the rate of uptake, not the concentration itself, is the superior predictor. In a study on zinc accumulation and toxicity to isopods (Porcellio scaber) the dietary EC(50) for the effect on body growth was rather constant and reproducible, while the internal EC(50) varied depending on the accumulation history of the animals. From the data a critical value for zinc accumulation in P. scaber was estimated as 53 microg/g/wk. We review toxicokinetic models applicable to time-series measurements of concentrations in invertebrates. The initial slope of the uptake curve is proposed as an indicator of bioavailability. To apply the dynamic concept of bioavailability in risk assessment, a set of representative organisms should be chosen and standardized protocols developed for exposure assays by which suspect soils can be evaluated.

Population substructures in the soil invertebrate Orchesella cincta, as revealed by microsatellite and TE-AFLP markers.

Microsatellite and three enzyme-amplified fragment length polymorphism (TE-AFLP) DNA markers were used to describe the population genetic structure in the soil dwelling collembolan Orchesella cincta (L.). Two forests were sampled according to a three-level nested hierarchical design, with fixed distances among samples within a parcel and among parcels within a forest. The largest component of variation was found at the smallest scale, within parcels (77-97%), while the smallest component of variation was found between forests. The two different methods to study population structure indicated a similar allocation of variance. Population genetic substructuring was revealed between samples on a scale of 50 m; the degree of substructuring however, varied between parcels and forests. One forest showed a high degree of structure as revealed by microsatellites, while another showed a low degree of structure. A significant deviation from random-mating (average FIS = 0.23) over the two forests was detected. Two of 18 samples showed a difference in population genetic structure between males and females. We discuss the fact that the population genetic structure of O. cincta is significantly affected by long-range dispersal, even though it is a small and wingless insect. This interpretation is supported by observations on tree-climbing behaviour in this species that may facilitate air dispersal. As a consequence, the assumption that migration a priori may be neglected in demographic analysis of O. cincta is incorrect.

Two metal-binding peptides from the insect Orchesella cincta (Collembola) as a result of metallothionein cleavage.

Metallothionein (MT) is an ubiquitous heavy metal-binding protein which has been identified in animals, plants, protists, fungi and bacteria. In insects, primary structures of MTs are known only for Drosophila and the collembolan, Orchesella cincta. The MT cDNA from O. cincta encodes a 77 amino acid protein with 19 cysteines. Isolations of the protein itself have demonstrated the presence of two smaller metal-binding peptides, whose amino acid sequences correspond to parts of the cDNA, and which apparently result from cleavage of the native protein. The present study was undertaken to complete the picture of cleavage sites within the MT protein by applying protein isolation techniques in combination with mass spectrometry and N-terminal sequence analysis. Further, recombinant expression allowed us to study the intrinsic stability of the MT and to perform in vitro cleavage studies. The results show that the MT from O. cincta is specifically cleaved at two sites, both after the amino acid sequence Thr-Gln (TQ). One of these sites is located in the N-terminal region and the other in the linker region between two putative metal-binding clusters. When expressed in Escherichia coli, the recombinant O. cincta MT can be isolated in an uncleaved form; however, this protein can be cleaved in vitro by the proteolytic activity of O. cincta. In combination with other studies, the results suggest that the length of the linker region is important for the stability of MT as a two domain metal-binding protein.

Characterization of enzymes involved in biotransformation of polycyclic aromatic hydrocarbons in terrestrial isopods.

Little is known about the capacity of terrestrial invertebrates to transform organic soil pollutants such as polycyclic aromatic hydrocarbons (PAHs). Studies were designed to characterize microsomal mixed function oxygenase and accompanying conjugation enzymes from the hepatopancreas of the terrestrial isopods Porcellio scaber and Oniscus asellus using pyrene and 1-hydroxypyrene as model substrates. The hydroxylation of pyrene and the formation of pyreneglucoside and pyrenesulfate appeared to be sensitive measures for the activity of cytochrome P450 aryl hydrocarbon hydroxylase (AHH), uridinediphosphateglucosyltransferase (UDPGT), and aryl sulfotransferase (ST), respectively. Treatment with the antibiotic riphampicine demonstrated that the enzyme activities originate from the animals themselves and not from symbiotic microflora present in the hepatopancreas and the gut. In both species, ST has a very high affinity for 1-hydroxypyrene with Km values two orders of magnitude lower than that of UDPGT. The Vmax values of UDPGT, however, are 10- to 20-fold higher than that of ST. Taking the P450 activities into consideration, both species are expected to transform PAHs in an equally effective way. When the isopods were fed with food containing benz[a]pyrene and 3-methyl-cholanthrene, none of the enzyme activities appeared to be inducible except for a small enhancement of UDPGT in O. asellus. Our findings indicate that terrestrial isopods have a high, noninducible capacity for biotransformation of PAHs and that the sulfate conjugation pathway is as important as the carbohydrate conjugation pathway. This conclusion is consistent with the low body residues of parent PAHs found in the field.

A review of the effects of multiple stressors on aquatic organisms and analysis of uncertainty factors for use in risk assessment.

Risk assessment procedures use toxicity tests in which organisms are subjected to chemicals under otherwise constant and favorable experimental conditions. Because variable and suboptimal environmental conditions are common aspects of natural ecosystems, the hazard of underestimation of risk arises. Therefore, an uncertainty factor is used in the extrapolation of results of standard toxicity tests to field situations. The choice for these uncertainty factors is based on little ecological evidence. This review discusses studies on the toxicity of various chemicals to aquatic organisms, modified by temperature, nutritional state and salinity, excluding papers on changes in bioavailability of compounds. Collected data were analyzed quantitatively to evaluate the validity of toxicity data obtained from standard toxicity tests in the laboratory under field conditions. Generally, organisms living under conditions close to their environmental tolerance limits appeared to be more vulnerable to additional chemical stress. Usually, increasing temperature and decreasing food or nutrient level raised toxicity. The influence of salinity was less clear; metal toxicity increased with decreasing salinity, toxicity of organophosphate insecticides increased with higher salinity, while for other chemicals no clear relationship between toxicity and salinity was observed. The interactions can be explained by several physical and physiological processes, acting on factors such as bioavailability, toxicokinetics, and sensitivity of organisms. Quantitative analysis of data indicated that an uncertainty factor for the laboratory to field extrapolation should be smaller than one for an ecosystem in a temperate region, while a factor greater than one would be appropriate for systems nearby discharge points of cooling water. The factor should be greater than one when varying nutritional state is concerned, but smaller than one with respect to salinity. Dependent on the effect parameter used, the differences in toxicity between laboratory and relevant field situations ranged from a factor of 2.6 to 130 and 1.7 to 15 for the two temperature conditions and 1.2 to 10 for nutritional state. A salinity increase from freshwater to marine water decreased toxicity by a factor of 2.1. However, as less extreme salinity changes are more relevant under field conditions, the change in toxicity is probably much smaller. To obtain uncertainty factors that sufficiently protect natural systems without being overprotective, additional research is required.

Toxicokinetics and toxicity of zinc under time-varying exposure in the guppy (Poecilia reticulata).

Levels of toxic substances released into the environment are often highly variable and fluctuate over time. The present study deals with a simple type of time-variable exposure, diluted pulse. We determined toxicokinetic parameters of zinc in guppy fish (Poecilia reticulata) and evaluated the applicability of a toxicokinetics-based survival model developed earlier. In the toxicokinetics experiment, zinc was rapidly taken up and released again; the half-life in fish was only 1.5 d. In the toxicity experiments with diluted-pulse exposure, survival leveled off to a baseline level, which in accordance with the model is explained by the dilution of zinc. The model fitted reasonably well for the lower initial concentrations but tended to overestimate survival rates at the higher concentrations. Toxicokinetic parameters estimated from changes in survival were close to values estimated from measured zinc concentrations in water. Elimination rates during pulse exposure varied from 0.434 to 0.488 d(-1) and corresponded very well to the elimination rate during constant exposure (0.463 d(-1)). Ultimate LC50 values were estimated as 6.40 and 9.10 mg/L. These results suggest that toxicity experiments with a simple, time-varying exposure can be used as an alternative to conventional, constant-exposure experiments. Toxicokinetic parameters and toxicological endpoints can still be estimated in static bioassays with decreasing exposure if the concentration in the medium is measured. At the same time, diluted-pulse experiments may simulate exposure from discharges in the field better than constant-exposure experiments.

Spatial distribution of trace metals in sediments from urban streams of Semarang, Central Java, Indonesia.

Elevated environmental concentrations of metals are usually associated with the impact of urbanization. The present study is focused on metal contamination in urban sediments. A field survey was carried out to determine the distribution of four metals, i.e., cadmium (Cd), lead (Pb), copper (Cu), and zinc (Zn), in the coastal urban area of Semarang, Central Java, Indonesia. Sediment samples were collected from 101 grids of 2x2 km. To map the spatial distribution of these metals, concentrations of each metal were plotted against the corresponding grid coordinate. Cd was below the detection limit (<0.03 microg/g) in all samples, whereas concentrations of Pb, Zn, and Cu fell into a wide range. Frequency distributions of Pb, Zn, and Cu concentrations indicated a similar pattern, in which the major proportion of the sites had a low metal concentration. Some sites, however, had extremely high metal concentrations, Zn up to 1257 microg/g, Pb up to 2666 microg/g, and Cu up to 448 microg/g. The data were used to define background concentrations for sediments in coastal zones of Indonesia ("reference values"). The proposed reference values are 25.6 microg/g, 132.2 microg/g, and 40.7 microg/g, respectively, for Pb, Zn, and Cu. The degree of metal contamination of each individual site was classified according to the calculated value of a combined pollution index, W. Four categories of the degree of metal contamination were proposed, i.e., unpolluted, slightly polluted, polluted, and heavily polluted. Based on this classification, from 101 sites investigated in the greater Semarang area, 51 are unpolluted, 36 slightly polluted, 9 polluted, and 5 heavily polluted.

Associations between trace metals in sediment, water, and guppy, Poecilia reticulata (Peters), from urban streams of Semarang, Indonesia.

The present study aims to answer the question whether the amount of metal in aquatic biota reflects the concentrations in the sediment and water, and whether the physico-chemical properties of the water and sediment have any influence on the suspected relationship. A study was made of 101 small streams in the city of Semarang, Central Java, Indonesia. Data on fish occurrence in 63 streams of the greater Semarang indicated that the guppies did not avoid the highly polluted sites. No significant difference in body weight between sites was found. Significant differences were found in metal body concentrations (Pb and Zn) between fish collected from sites with different degrees of pollution. A significant declining trend of Pb concentrations with increasing organism size was observed, whereas for two other metals, Zn and Cu, the concentrations did not depend on the body weight. Apparently, body concentrations of these two metals are regulated and maintained at a certain concentration. For the relationships between metal concentrations in water, sediment and fish, water and sediment parameters, and fish dry weight, the presence of significant multiple correlations and bivariate correlation (in 17 of 91 pairs of variables) indicated that, in general, abiotic parameters and body size had no influence on the metal flux from sediment to water, and into fish. Results of partial correlation analyses further suggested that metal concentrations in the sediments were the most important factor governing the metal body concentrations of fish. The present study indicates that the guppy Poecilia reticulata from urban streams is a potential bioindicator for urban metal pollution, especially with respect to their (1) spatial distribution over sites of all pollution regimes and (2) variation in metal accumulation levels reflecting the degree of pollution.