Terrestrial Isopod Porcellio Scaber Research Articles

The influence of zinc on the accumulation of cadmium and copper in the terrestrial isopod Porcellio scaber (Crustacea, Isopoda)

Relative to data derived from single-metal exposure not much is known about metal kinetics in terrestrial isopods exposed to a mixture of metals. In the work presented the accumulation pattern of Zn, Cd and Cu were studied in Porcellio scaber, one of the most investigated isopods. Animals were fed with hazel leaves dosed with single metals or their binary mixtures. After twenty-one days of exposure, food consumption and metal accumulation were measured. Results revealed that the accumulation of Zn is not affected by a decreased food consumption rate or by possible interactions between the mixture constituents. In contrast, the accumulation of Cd and Cu is significantly reduced when food is also contam- inated with Zn. The lower accumulation of Cd and Cu can be ascribed to interactions with Zn that affect uptake and/or loss of metals.

Electron and ion imaging of gland cells using the FIB/SEM system

The FIB/SEM system was satisfactorily used for scanning ion (SIM) and scanning electron microscopy (SEM) of gland epithelial cells of a terrestrial isopod Porcellio scaber (Isopoda, Crustacea). The interior of cells was exposed by site-specific in situ focused ion beam (FIB) milling. Scanning ion (SI) imaging was an adequate substitution for scanning electron (SE) imaging when charging rendered SE imaging impossible. No significant differences in resolution between the SI and SE images were observed. The contrast on both the SI and SE images is a topographic. The consequences of SI imaging are, among others, introduction of Ga(+) ions on/into the samples and destruction of the imaged surface. These two characteristics of SI imaging can be used advantageously. Introduction of Ga(+) ions onto the specimen neutralizes the charge effect in the subsequent SE imaging. In addition, the destructive nature of SI imaging can be used as a tool for the gradual removal of the exposed layer of the imaged surface, uncovering the structures lying beneath. Alternative SEM and SIM in combination with site-specific in situ FIB sample sectioning made it possible to image the submicrometre structures of gland epithelium cells with reproducibility, repeatability and in the same range of magnifications as in transmission electron microscopy (TEM). At the present state of technology, ultrastructural elements imaged by the FIB/SEM system cannot be directly identified by comparison with TEM images.

Behavioral Response in the Terrestrial Isopod Porcellio scaber (Crustacea) Offered a Choice of Uncontaminated and Cadmium-Contaminated Food

The objective of this study was to find out whether Porcellio scaber discriminates against Cd-contaminated food. The foraging behavior in animals offered uncontaminated and Cd-contaminated food simultaneously was quantified for 48-h employing computer-aided video tracking. To see whether the isopods' selection of less contaminated food could diminish the influence Cd on food consumption, growth, metal assimilation, moulting and mortality, Cd-dosed food (20, 45, 200 and 450 mg kg(-1) dry weight) was offered together with untreated food for 3 weeks. Data from the video tracking experiments revealed that animals visited Cd-dosed food as often as untreated food, but spent much less time near Cd-dosed food. Discrimination against Cd-contaminated food increased with previous experience with contaminated food and/or with increased Cd body burden. In 3 weeks exposure uncontaminated food preference rose with time of exposure and cadmium concentration in food and reached a maximal preference ratio of 65% (untreated food): 35% (Cd-dosed food). The decreased consumption of Cd-dosed food was compensated by the increased consumption of control food. Cadmium body burden increased with time of exposure and cadmium concentration in food consumed, while the influence of Cd on food consumption, growth and moulting was diminished.

"Candidatus hepatoplasma crinochetorum," a new, stalk-forming lineage of Mollicutes colonizing the midgut glands of a terrestrial isopod.

Uncultivated bacteria that densely colonize the midgut glands (hepatopancreas) of the terrestrial isopod Porcellio scaber (Crustacea: Isopoda) were identified by cloning and sequencing of their 16S rRNA genes. Phylogenetic analysis revealed that these symbionts represent a novel lineage of the Mollicutes and are only distantly related (<82% sequence identity) to members of the Mycoplasmatales and Entomoplasmatales. Fluorescence in situ hybridization with a specific oligonucleotide probe confirmed that the amplified 16S rRNA gene sequences indeed originated from a homogeneous population of symbionts intimately associated with the epithelial surface of the hepatopancreas. The same probe also detected morphotypically identical symbionts in other crinochete isopods. Scanning and transmission electron microscopy revealed uniform spherical bacterial cells without a cell wall, sometimes interacting with the microvilli of the brush border by means of stalk-like cytoplasmic appendages, which also appeared to be involved in cell division through budding. Based on the isolated phylogenetic position and unique cytological properties, the provisional name "Candidatus Hepatoplasma crinochetorum" is proposed for this new taxon of Mollicutes colonizing the hepatopancreas of P. scaber.

Pyrene biotransformation and kinetics in the hepatopancreas of the isopod Porcellio scaber.

Various techniques exist for polycyclic aromatic hydrocarbon (PAH) determination in environmental samples, but an adequate risk assessment of PAHs should include aspects such as bioavailability of the contaminant and biotransformation capacity of the species under investigation. In this study, we provided an analysis of the kinetics of pyrene in the terrestrial isopod Porcellio scaber. Isopods were exposed to pyrene in their food (10 microg/g d/w) for 7 days followed by an elimination period of 7 days. The animals were dissected, and the hepatopancreases were analyzed for pyrene biotransformation products; nonmetabolised pyrene in the gut was also monitored. Concentrations of 1-hydroxypyrene in the hepatopancreas were very low. Almost all of the pyrene was found as three conjugates: pyrene-1-glucoside, pyrene-1-sulfate, and a third unknown 1-hydroxypyrene conjugate. Concentrations of the metabolites were extremely variable between individuals because of variable feeding activity. An apparent steady state was reached already after 24 hours of exposure, whereas elimination was complete 48 hours after ending the exposure. This rapid response to changes in the exposure concentration shows that terrestrial isopods have a high biotransformation capacity for PAHs. The data show that concentrations of parent PAHs will not provide a good indication of exposure in rapidly metabolizing invertebrates such as isopods; instead, pyrene metabolites may be considered a promising biomarker for bioavailability of PAH contamination in the field.

‘Candidatus Rhabdochlamydia porcellionis’, an intracellular bacterium from the hepatopancreas of the terrestrial isopod Porcellio scaber (Crustacea: Isopoda)

Intracellular bacteria were observed in the hepatopancreas of the terrestrial isopod Porcellio scaber. Comparative 16S rRNA gene sequence analysis and electron microscopic observations were used to determine the taxonomic position of these intracellular bacteria. Phylogenetic analysis and a complex developmental cycle affiliate these bacteria to the order Chlamydiales, within which they form a distinctive lineage, close to the family Simkaniaceae. They share <92 % 16S rRNA gene sequence similarity with their closest relative and <88 % similarity with other members of the order Chlamydiales. A specific signature oligonucleotide sequence was identified and used as a probe, enabling the identification of intracellular bacteria in infected hepatopancreatic tissue. According to the distinctive morphology of their elementary bodies, which are rod-shaped rather than spherical and contain translucent oblong structures, their genomic properties and their crustacean host, the name 'Candidatus Rhabdochlamydia porcellionis' is proposed for intracellular bacteria in the hepatopancreas of P. scaber.

The glycosylated androgenic hormone of the terrestrial isopod Porcellio scaber (Crustacea)

It appears from grafting experiments that the androgenic hormone (AH) of terrestrial isopods has a narrow species-specificity [J. Crust. Biol. 19 (1999) 684], even if AH of different species shared common epitopes [Gen. Comp. Endocrinol. 125 (2002) 218]. To date only the glycosylated AH of the woodlouse Armadillidium vulgare has been deciphered by us [Ann. New York Acad. Sci. 839 (1998) 111; Eur. J. Biochem. 262 (1999) 727] and [Biochem. Biophys. Res. Commun. 264 (1999) 419] have confirmed the primary structure of this protein. We reported in the present paper the characterization of the AH in another species Porcellio scaber by a combination of microsequencing, mass spectrometry, and molecular cloning. We found only one glycoform which consisted of two peptide chains, A and B, of 31 and 44 amino acids, respectively, with A chain carrying on Asn 18 a N-glycosylated moiety, size of which has been determined by MALDI-MS measurements. The expected structure of the glycosylation was proposed. The deduced amino acid sequence of the AH precursor was mainly identical to the one obtained independently by another group [Zool. Sci. 20 (2003) 75]. We also showed that AH gene is exclusively expressed in androgenic glands. Sequence comparison with A. vulgare and P. scaber (population of Japan) AH was discussed.

Food selection as a means of Cu intake reduction in the terrestrial isopod Porcellio scaber (Crustacea, Isopoda)

The idea that terrestrial isopods regulate copper intake through the state of their copper stores by selection of an optimal copper diet has never been experimentally assessed. We investigated discrimination between untreated and Cu-enriched diets in the isopod Porcellio scaber in relation to their copper stores. Animals were pre-exposed to untreated or Cu-enriched poplar leaves. After 14 and 28 days of pre-exposure, two-way food choice experiments were performed for 14 days. Food consumption rates and the amount of copper accumulated due to the copper content of the food and the duration of pre-exposure were compared. Food selection and rejection as ways by which P. scaber may reduce or avoid the toxic effects of copper were evaluated. P. scaber offered both Cu-enriched food and untreated food preferred the latter, irrespective of the amount of assimilated copper. Copper body burdens increased with copper concentration in the food. In animals offered both Cu-enriched and untreated food, copper body burden also increased, but to a lower extent than in animals fed only a Cu-enriched diet. Therefore, food selection is a way of regulating copper intake, but only to a limited extent. The mortality of experimental animals increased significantly after longer exposure to highly Cu-enriched food. We conclude that in P. scaber, the possibility of selecting food of different copper content may mitigate but cannot prevent the toxic effects of copper in copper-polluted environments.

Nano-architecture and structural changes in the sternal CaCO3 deposits of the terrestrial isopod Porcellio scaber during the molt cycle

Nano-architecture and structural changes in the sternal CaCO₃ deposits of the terrestrial isopod Porcellio scaber during the molt cycle

Intake and assimilation of zinc, copper, and cadmium in the terrestrial isopod Porcellio scaber Latr. (Crustacea, Isopoda).

Intake and assimilation of zinc, copper, and cadmium in the terrestrial isopod Porcellio scaber Latr. (Crustacea, Isopoda).

Analysis of CaCO 3 deposit formation and degradation during the molt cycle of the terrestrial isopod Porcellio scaber (Crustacea, Isopoda)

Terrestrial isopods store cuticular calcium in large sternal deposits composed of an amorphous CaCO 3 compound. A large part of the deposits consists of numerous small spherules that increase the exposed surface to facilitate resorption of CaCO 3 during cuticle mineralization. It is not known how these spherules are formed and how they are dissolved. This paper presents for the first time an analysis of ultrastructural changes occurring in the sternal CaCO 3 deposits of a terrestrial isopod during their formation and degradation. Our results indicate that formation of the spherules takes place in a specialized aggregation zone, in which 10- to 30-nm-thick granules form agglomerations that then increase in size to form spherules that reveal a concentric growth pattern. Degradation of the deposits occurs in a manner that exposes a maximum of surface area on all levels of their structural organization.

Ultrastructural alterations of the hepatopancreas in Porcellio scaber under stress

Cellular ultrastructure varies in accordance with physiological processes, also reflecting responses to environmental stress factors. Ultrastructural changes of the hepatopancreatic cells in the terrestrial isopod Porcellio scaber exposed to sublethal concentrations of zinc or cadmium in their food were identified by transmission electron microscopy. The exclusive structural characteristic of the hepatopancreas of animals exposed to metal-dosed food was grain-like electrondense deposits (EDD) observed in the intercellular spaces and in vesicles of B cells. In addition, hepatopancreatic cells of metal-exposed animals displayed non-specific, stress-indicating alterations such as cellular disintegration, the reduction of energetic reserves (lipid droplets, glycogen), electron dense cytoplasm, ultrastructural alterations of granular endoplasmic reticulum (GER), the Golgi complex and mitochondria.

Transformations of mercury in the terrestrial isopod Porcellio scaber (Crustacea)

The biological cycle of mercury in the terrestrial isopod Porcellio scaber was investigated. Testing the possibility of in vivo Hg 2+ methylation was divided into two methodologically different parts. Firstly, concentrations of total mercury and MeHg in isopods P. scaber and their environment from a Hg-unpolluted area were measured by the use of validated methods (CV AAS, CV AFS). The data obtained show that the percentage of MeHg in leaves, soil and faeces was less than 1%. In contrast, the percentage of MeHg in gut and hepatopancreas was increased to 14 and 77%, respectively, indicating methylation of Hg 2+ in the gut and its further accumulation in glands. To confirm this assumption, the second methodology was applied—a radiotracer technique with 203Hg 2+ of high specific activity. There are few radiotracer techniques for Hg-methylation assays; for our work we chose the method of Czuba et al. which includes alkaline leaching of Hg species, their extraction into dithizone–toluene, followed by specific separation of Hg dithizonates by thin-layer chromatography and gamma counting. All steps of the analytical protocol were checked and optimised by the use of aqueous solutions of 203Hg 2+ and Me 203Hg +. The most important finding was that cleaning-up the extract through a florisil column is not appropriate, because the column retains different percentages of Hg 2+ and MeHg + and consequently affects the accuracy of the final result. This optimised protocol was then applied to Hg transformation studies in the terrestrial isopod P. scaber. Leaching Hg species from P. scaber fed with 203Hg 2+ or Me 203Hg + dosed food was completely efficient only at elevated temperatures. Preliminary results of methylation/demethlytion studies are rather variable but they show that both processes (Hg 2+↔MeHg +) take place in the isopod P. scaber. Additionally, an assessment of the mass balance of Hg in isopods P. scaber exposed to 203Hg 2+ indicates that volatile Hg species are also formed.

Expression of Ca 2+-ATPase and Na +/Ca 2+-exchanger is upregulated during epithelial Ca 2+ transport in hypodermal cells of the isopod Porcellio scaber

It is thought that a plasma membrane Ca 2+-transport ATPase (PMCA) and a Na +/Ca 2+-exchange (NCE) mechanism are involved in epithelial Ca 2+ transport (ECT) in a variety of crustacean epithelia. The sternal epithelium of the terrestrial isopod Porcellio scaber was used as a model for the analysis of Ca 2+-extrusion mechanisms in the hypodermal epithelium. Using RT-PCR, we amplified a cDNA fragment of 1173 bp that encodes a protein sequence possessing 72% identity to the PMCA from Drosophila melanogaster and a cDNA fragment of 791 bp encoding a protein sequence with 50% identity to the NCE from Loligo opalescens. Semiquantitative RT-PCR revealed that the expression of both mRNAs increases from the non-Ca 2+-transporting condition to the stages of CaCO 3 deposit formation and degradation. During Ca 2+-transporting stages, the expression of PMCA and NCE was larger in the anterior sternal epithelium (ASE) than in the posterior sternal epithelium (PSE). The results demonstrate for the first time the expression of a PMCA and a NCE in the hypodermal epithelium of a crustacean and indicate a contribution of these transport mechanisms in ECT.

Analysis of Ca(2+) uptake into the smooth endoplasmic reticulum of permeabilised sternal epithelial cells during the moulting cycle of the terrestrial isopod Porcellio scaber.

In terrestrial isopods, large amounts of Ca(2+) are transported across anterior sternal epithelial cells during moult-related deposition and resorption of CaCO(3) deposits. Because of its toxicity and function as a second messenger, resting cytosolic Ca(2+) levels must be maintained below critical concentrations during epithelial Ca(2+) transport, raising the possibility that organelles play a role during Ca(2+) transit. We therefore studied the uptake of Ca(2+) into Ca(2+)-sequestering organelles by monitoring the formation of birefringent calcium oxalate crystals in permeabilised anterior and posterior sternal epithelium cells of Porcellio scaber during Ca(2+)-transporting and non-transporting stages of the moulting cycle using polarised-light microscopy. The results indicate ATP-dependent uptake of Ca(2+) into organelles. Half-maximal crystal growth at a Ca(2+) activity, a(Ca), of 0.4 micromol l(-1) and blockade by cyclopiazonic acid suggest Ca(2+) uptake into the smooth endoplasmic reticulum by the smooth endoplasmic reticulum Ca(2+)-ATPase. Analytical electron microscopical techniques support this interpretation by revealing the accumulation of Ca(2+)-containing crystals in smooth membranous intracellular compartments. A comparison of different moulting stages demonstrated a virtual lack of crystal formation in the early premoult stage and a significant fivefold increase between mid premoult and the Ca(2+)-transporting stages of late premoult and intramoult. These results suggest a contribution of the smooth endoplasmic reticulum as a transient Ca(2+) store during intracellular Ca(2+) transit.

Genetic diversity of bacteria associated with the hindgut of the terrestrial crustacean Porcellio scaber (Crustacea: Isopoda)

Molecular approaches were used to examine the genetic diversity of bacteria associated with the gut wall of the terrestrial isopod Porcellio scaber and to determine whether an autochthonous microflora exists in the P. scaber hindgut. 16S ribosomal genes were amplified from the total DNA isolated from thoroughly washed papillate regions of the hindgut, where the highest concentrations of bacteria are commonly found. The amplified genes were cloned, sequenced and phylogenetically analysed. The results implied an unexpectedly large diversity of microflora associated with the cuticle of the hindgut. Almost half of the retrieved sequences were found to be less than 80% homologous with any of the known sequences available at DNA data banks. Most of these sequences were clustered in one of three groups, and were clearly distant from the sequences of other bacterial taxa, indicating that they could represent novel bacterial species or even genera. More than two thirds of the sequences were found to be phylogenetically related to sequences from bacteria typically isolated from human and animal intestines, e.g. streptococci, enterococci, and members of the genus Bacteroides. The majority of the remaining sequences were most closely related to typical soil bacteria, e.g. bacilli and pseudomonads. The facts that a large proportion of the retrieved sequences was related to the sequences of bacteria, which are autochthonous to intestinal ecosystems, and that bacteria, specifically attached to the cuticular spines, were observed, indicate that truly autochthonous bacteria may well be present in the hindgut of P. scaber.

Genetic diversity of bacteria associated with the hindgut of the terrestrial crustacean Porcellio scaber (Crustacea: Isopoda)

Molecular approaches were used to examine the genetic diversity of bacteria associated with the gut wall of the terrestrial isopod Porcellio scaber and to determine whether an autochthonous microflora exists in the P. scaber hindgut. 16S ribosomal genes were amplified from the total DNA isolated from thoroughly washed papillate regions of the hindgut, where the highest concentrations of bacteria are commonly found. The amplified genes were cloned, sequenced and phylogenetically analysed. The results implied an unexpectedly large diversity of microflora associated with the cuticle of the hindgut. Almost half of the retrieved sequences were found to be less than 80% homologous with any of the known sequences available at DNA data banks. Most of these sequences were clustered in one of three groups, and were clearly distant from the sequences of other bacterial taxa, indicating that they could represent novel bacterial species or even genera. More than two thirds of the sequences were found to be phylogenetically related to sequences from bacteria typically isolated from human and animal intestines, e.g. streptococci, enterococci, and members of the genus Bacteroides. The majority of the remaining sequences were most closely related to typical soil bacteria, e.g. bacilli and pseudomonads. The facts that a large proportion of the retrieved sequences was related to the sequences of bacteria, which are autochthonous to intestinal ecosystems, and that bacteria, specifically attached to the cuticular spines, were observed, indicate that truly autochthonous bacteria may well be present in the hindgut of P. scaber.

Isopod gut microflora parameters as endpoints in toxicity studies.

Terrestrial isopods Porcellio scaber (Crustacea) were fed for five weeks on food contaminated by 250, 500, or 1,000 microg of Cd/g or for 10 d on diets with 50 or 250 microg Cd/g food. In both experiments, fecal production rate and colony forming units (CFUs) in the guts were determined. In addition, at the end of 10 d, each distinct colony morphotype obtained in gut samples was purified and characterized. Isolates were separated into 25 groups based on morphological and biochemical characteristics. These bacterial groups were used as units for calculating Shannon equitability indices (J) for each gut. The relative frequencies of the 25 bacterial units were determined in both cadmium groups (50 or 250 microg Cd/g food) and in the control. Cadmium-induced perturbations observed in the gut microbial communities were (1) increased number of morphologically distinct bacterial isolates in the group fed low-cadmium-dosed food (50 microg Cd/g) and reduced number of morphologically distinct bacterial isolates in the group fed high-cadmium-dosed food (250 microg Cd/g) compared with the control, (2) increased or decreased relative frequencies of almost all 25 bacterial units provoked by cadmium-contaminated food, (3) time-dependent increased numbers of gut CFUs in cadmium-fed animals (dose dependence was not observed), and (4) significant changes in community structure described by Shannon equitability indices at lower levels of food contamination (50 microg Cd/g) only. Gut microflora parameters are proposed as additional endpoints in the standardized single-species toxicity test with the terrestrial isopod P. scaber as a means of increasing the ecological relevance of the results.

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.

Dietary effects on growth, reproduction, body composition and stress resistance in the terrestrial isopods Oniscus asellus and Porcellio scaber

Summary The effect of differences in food composition on growth, body composition and cold tolerance of the isopods Porcellio scaber (Latreille) and Oniscus asellus (L.) has been studied. The effect on reproduction of P. scaber was included. Total lipid and protein content remained the same in O. asellus, given food with a nitrogen content of 2.4, 2.5, 2.8 or 3.1% for 8 weeks. Those fed on 3.1% N gained significantly more weight than those fed on 2.4, 2.5 or 2.8% N. Cold tolerance after a period of low temperature acclimation could not be tested, due to high mortality at the subzero treatment. Haemolymph osmolality increased after the winter acclimation for all food treatments. Juvenile P. scaber, given food of 1.8, 2.5 or 3.1% N, showed no difference in growth over 18 weeks, nor in body composition over 14 weeks. Diet did not influence cold tolerance ability and haemolymph osmolality remained the same with changing cold tolerance of the animals. Gravid females of P. scaber were separated to examine the effect of diet on reproduction. Diet did not influence the number of juveniles, nor their weight. In all food treatments, a significant correlation was found between the weight of the female and the number and weight of her offspring.