Adaptive variation in offspring size in the terrestrial isopod,Armadillidium vulgare

Summary Variation in the birth size of offspring of the terrestrial isopod, Armadillidium vulgare, was observed in laboratory experiments and in field populations. In the laboratory, larger offspring were produced when the mother's food supply was reduced. In field populations, larger offspring were produced during the summer, a period of reduced food availability. Smaller offspring are produced in the spring, when food is readily available. Females may be making larger young to increase survival during the more severe conditions of the summer breeding period.     

The structure and function of the hepatopancreas of a terrestrial isopod, Armadillidium vulgare

The fine structure of the hepatopancreas of the isopod, Armadillidium vulgare was studied in intact and in experimentally treated animals. The gross anatomy of the hepatopancreas of this species is simpler than that of decapods, but microscopically the cells are similar in both. Accordingly, the function of this organ is probably the same in decapods and Armadillidium. Some phylogenetic considerations are also presented. In a previous paper Miyawaki et al. (1984), presented experimental results which indicated that the hepatopancreas of the crayfish, Procambarus clarki, had an important role in the absorption of digested food materials. These results confirmed earlier reports by Yonge (1924) and van Weel (1955) on the decapods, Nephrops norvegicus and Atya spinides, respectively. In the present study, we wished to determine whether the isopod hepatopancreas has a function like that of the decapods. A terrestrial isopod, Armadillidium vulgare collected on the University campus, and reared in the laboratory at room temperature was used. The methods of study were similar to those employed in the study with Procambarus clarki (Miyawaki et al., 1984).     

Characterization of polymorphic microsatellite loci in the terrestrial isopod Armadillidium vulgare

The common pill bug, Armadillidium vulgare, is known to harbour two distinct strains of the feminizing proteobacteria Wolbachia. In order to study the effect of the presence of Wolbachia on the evolution of A. vulgare populations, we developed and characterized a set of nine polymorphic microsatellite loci from two microsatellite‐enriched genomic libraries. We screened 48 individuals from three French populations and found high genetic variation. Locus‐specific allelic diversity ranged from four to 28 and observed heterozygosity from 0.40 to 1.00, which indicates that these markers can be used to conduct population genetic studies in A. vulgare.     

Ommochrome deficiency in an albino strain of a terrestrial isopod, Armadillidium vulgare.

In order to clarify the cause of ommochrome deficiency in an albino strain of the terrestrial isopod, Armadillidium vulgare, levels of xanthommatin, 3-hydroxykynurenine, 3-hydroxyanthranilic acid and tryptophan in whole body extracts of the albino and the wild type individuals were determined together with enzyme activities of kynurenine-3-hydroxylase, kynureninase and tryptophan-2,3-dioxygenase. Xanthommatin could not be detected in the albinos. The levels of 3-hydroxykynurenine and 3-hydroxyanthranilic acid were determined by high-performance liquid chromatography (HPLC) with electrochemical detection and were markedly low in the albinos compared with the wild type individuals. In contrast to those, the tryptophan levels determined by HPLC with fluorescence detection did not differ significantly between the two phenotypes. In the albino A. vulgare, kynurenine-3-hydroxylase activity was lower and kynureninase activity was higher than in the wild type, although the differences were not statistically significant. Tryptophan-2,3-dioxygenase activity in the albinos was less than 10% that in the wild type. Thus, ommochrome deficiency in the albino A. vulgare is considered to be caused by the extremely low activity of tryptophan-2,3-dioxygenase.     

Evidence for a new feminizing Wolbachia strain in the isopod Armadillidium vulgare: evolutionary implications

Wolbachia are intracellular maternally inherited alpha-Proteobacteria infecting a wide range of arthropods. In the common pill bug Armadillidium vulgare, the known Wolbachia strain is responsible for feminization of genetic males. We have investigated Wolbachia diversity in 20 populations of A. vulgare from west and east Europe, north Africa and north America. A new Wolbachia strain (wVulM) was identified through the variability of the wsp gene, distantly related to that previously known (wVulC) in this host species. No individual with multiple infections was detected. Inoculation experiments indicated that the new wVulM bacterial strain also induces feminization in A. vulgare. However, the wVulC strain showed a higher transmission rate than the wVulM strain and was the most geographically widespread Wolbachia in A. vulgare populations. Mitochondrial 16SrDNA gene sequencing was conducted in Wolbachia-infected individuals, revealing the occurrence of four host lineages. The comparison of bacterial strains and their respective host mitochondrial phylogenies failed to show concordance, indicating horizontal transmission of the Wolbachia strains within populations of A. vulgare.     

Males prefer virgin females,even if parasitized,in the terrestrial isopod Armadillidium vulgare

In many species, males increase their reproductive success by choosing high‐quality females. In natural populations, they interact with both virgin and mated females, which can store sperm in their spermatheca. Therefore, males elaborate strategies to avoid sperm competition. In the terrestrial isopod Armadillidium vulgare, females can store sperm and produce several clutches. Moreover, this species can be parasitized by Wolbachia, which feminizes genetic males, transforming them into functional females. Our study compared attractiveness and mate choice when a male is exposed to both virgin and experienced females (i.e., females who have produced offspring and rested for 6 months), with or without Wolbachia. Our results revealed that males are more attracted to virgin females than experienced females, even if these virgin females are parasitized. Moreover, the chemical analysis highlighted different odors in females according to their reproductive and infection (Wolbachia‐free or vertically Wolbachia‐infected) status. Males attempted copulation more frequently and for longer with virgin females, even if Wolbachia‐infected, while experienced females refused further copulation. The evolutionary consequences of both male choice and female resistance on their fitness are discussed in this study.     

Partial characterization of vitellin and localization of vitellogenin production in the terrestrial isopod, Armadillidium vulgare

Vitellins were purified separately from ovaries and eggs of the isopod, Armadillidium vulgare. Ovarian vitellin consisted of at least six proteins with relative molecular masses of 205, 200, 185, 180, 122 and 112 kDa. The larger four proteins disappeared in eggs within a week after oviposition and a 59-kDa protein appeared thereafter. The amino-terminal amino acid sequences of these vitellin proteins were identical except for the ovarian 112 kDa, egg 112 kDa and 59 kDa proteins, and showed considerable similarity to those of known vitellogenins from other animals. Comparison of tryptic peptide maps of the 122 and 112 kDa proteins from eggs on reversed-phase HPLC and sequence identification of two randomly selected peaks having the same retention times indicated that two peptides were mostly similar in sequence. PCR-assisted cloning of the 5' region of a cDNA (591 bp) encoding vitellogenin revealed the presence of a signal peptide consisting of 16 amino acid residues and clarified the structural relationship among the protein components except for the ovarian 112 kDa and the egg 59 kDa proteins. Northern blot analysis revealed that the fat body is the main vitellogenin producing organ.     

Characterization and cDNA cloning of androgenic gland hormone of the terrestrial isopod Armadillidium vulgare.

The sex differentiation in crustaceans is known to be controlled by a peptide hormone called androgenic gland hormone (AGH). AGH was extracted and purified from the androgenic glands (AGs) of the male isopod Armadillidium vulgare by high-performance liquid chromatography. AGH consisted of two peptide chains and their N-terminal amino acid sequences were determined. A cDNA encoding AGH was cloned by PCR and sequenced. The cDNA had an open reading frame of 432 bp, which encoded a preproAGH consisting of a signal peptide (21 residues), B chain (44 residues), C peptide (46 residues), and A chain (29 residues). Through processing, the A and B chains might form a heterodimer interlinked by disulfide bonds. The A chain possessed a putative N-linked glycosylation site. A Northern blot analysis using the cDNA as a probe detected a hybridization signal with 0.8 kb in the RNA preparation only from the AGs.     

Feminizing Wolbachia: a transcriptomics approach with insights on the immune response genes in Armadillidium vulgare

Background

Clostridium thermocellum produces H₂ and ethanol, as well as CO₂, acetate, formate, and lactate, directly from cellulosic biomass. It is therefore an attractive model for biofuel production via consolidated bioprocessing. Optimization of end-product yields and titres is crucial for making biofuel production economically feasible. Relative protein expression profiles may provide targets for metabolic engineering, while understanding changes in protein expression and metabolism in response to carbon limitation, pH, and growth phase may aid in reactor optimization. We performed shotgun 2D-HPLC-MS/MS on closed-batch cellobiose-grown exponential phase C. thermocellum cell-free extracts to determine relative protein expression profiles of core metabolic proteins involved carbohydrate utilization, energy conservation, and end-product synthesis. iTRAQ (isobaric tag for relative and absolute quantitation) based protein quantitation was used to determine changes in core metabolic proteins in response to growth phase.

Results

Relative abundance profiles revealed differential levels of putative enzymes capable of catalyzing parallel pathways. The majority of proteins involved in pyruvate catabolism and end-product synthesis were detected with high abundance, with the exception of aldehyde dehydrogenase, ferredoxin-dependent Ech-type [NiFe]-hydrogenase, and RNF-type NADH:ferredoxin oxidoreductase. Using 4-plex 2D-HPLC-MS/MS, 24% of the 144 core metabolism proteins detected demonstrated moderate changes in expression during transition from exponential to stationary phase. Notably, proteins involved in pyruvate synthesis decreased in stationary phase, whereas proteins involved in glycogen metabolism, pyruvate catabolism, and end-product synthesis increased in stationary phase. Several proteins that may directly dictate end-product synthesis patterns, including pyruvate:ferredoxin oxidoreductases, alcohol dehydrogenases, and a putative bifurcating hydrogenase, demonstrated differential expression during transition from exponential to stationary phase.

Conclusions

Relative expression profiles demonstrate which proteins are likely utilized in carbohydrate utilization and end-product synthesis and suggest that H₂ synthesis occurs via bifurcating hydrogenases while ethanol synthesis is predominantly catalyzed by a bifunctional aldehyde/alcohol dehydrogenase. Differences in expression profiles of core metabolic proteins in response to growth phase may dictate carbon and electron flux towards energy storage compounds and end-products. Combined knowledge of relative protein expression levels and their changes in response to physiological conditions may aid in targeted metabolic engineering strategies and optimization of fermentation conditions for improvement of biofuels production.     

Cation regulation by the terrestrial isopod Armadillidium vulgare (Crustacea: Isopoda: Oniscidea) during dehydration in air

Many terrestrial arthropods display tight osmotic and ionic regulation of the hemolymph during dehydration. In this study, we sought to quantify the level of regulation of the major hemolymph cations in the terrestrial isopod Armadillidium vulgare (Isopoda, Oniscidea). Inulin space measurements showed that the hemolymph comprises 52 ± 2.2% of the hydrated water content but contributes 71 ± 9.8% of water losses during desiccation. Hemolymph concentrations of Na+, K+ and Ca2+ were measured in variably dehydrated animals using ion-selective microelectrodes and compared with predicted concentrations assuming no regulation. Na+ and Ca2+ are quite tightly regulated, showing respective concentration increases of 20.8% and 7.1% following a 50% reduction in hemolymph volume, but K+ showed no measurable regulation. The excreted cation fraction during desiccation is negligible. Sites of ion sequestration were examined by injecting 22Na and ??Ca into the hemolymph of hydrated animals and assaying tissue-specific activities following dehydration. Na+ is apparently sequestered non-specifically by an unknown mechanism. Ca2+ accumulates in the dorsal somatic tissues, possibly in the calcium pool of the cuticle. How A. vulgare avoids significant disruptions of E(m) and neuromuscular function in the absence of K+ regulation, and how it sequesters Na+, both pose intriguing challenges for future work.     

Sensitivity to photoperiod of the terrestrial isopod Armadillidium vulgare (Latreille) during photoperiodic induction of sexual rest

Summary

The characterization of photoperiodic induction of sexual rest in Armadillidium vulgare females (a cosmopolitan species) shows that the photoperiodic curves relating to the cessation of reproduction are representative of long-days type species. Experiments made on photophase maintenance or reduction show that females are sensitive not only to such reduction but also to its magnitude. Furthermore, the photophase value is relative and depends on previous conditions experienced by the female and there is evidence for the existence of a circannual endogenous cycle in A. vulgare.     

Time measurement in the photoperiodic induction of sexual rest in the terrestrial isopod Armadillidium vulgare (Latreille)

The photoperiodic control of sexual rest in Armadillidium vulgare was investigated using various experimental protocols. When reared in conditions of a Nanda-Hamner (i.e. resonance) protocol from their first parturial moult to their post experimental moult, females showed a weak resonance effect in sexual rest incidence. The transfer from a long day cycle to a symmetrical skeleton photoperiod--consisting of two equal light pulses per 24 h of continuous darkness--revealed the involvement of a circadian oscillatory system in the photoperiodic clock of this species. The data, obtained in the whole experiments, suggested that both oscillator and hourglass features are involved in the photoperiodic response controlling the sexual rest in Armadillidium vulgare. Moreover, when non-24-h light-dark cycles (with a long photophase) were applied, a mechanism responsible of arrest of reproduction also implied a photoperiodic counter which accumulated and added up the photoperiodic information within a sensitive period during post parturial intermoult.     

Organization of the large mitochondrial genome in the isopod Armadillidium vulgare.

The mitochondrial DNA (mtDNA) in animals is generally a circular molecule of approximately 15 kb, but there are many exceptions such as linear molecules and larger ones. RFLP studies indicated that the mtDNA in the terrestrial isopod Armadillidium vulgare varied from 20 to 42 kb. This variation depended on the restriction enzyme used, and on the restriction profile generated by a given enzyme. The DNA fragments had characteristic electrophoretic behaviors. Digestions with two endonucleases always generated fewer fragments than expected; denaturation of restriction profiles reduced the size of two bands by half; densitometry indicated that a number of small fragments were present in stoichiometry, which has approximately twice the expected concentration. Finally, hybridization to a 550-bp 16S rDNA probe often revealed two copies of this gene. These results cannot be due to the genetic rearrangements generally invoked to explain large mtDNA. We propose that the large A. vulgare mtDNA is produced by the tripling of a 14-kb monomer with a singular rearrangement: one monomer is linear and the other two form a circular dimer. Densitometry suggested that these two molecular structures were present in different proportions within a single individual. The absence of mutations within the dimers also suggests that replication occurs during the monomer phase.     

Experimental evidence of Wolbachia introgressive acquisition between terrestrial isopod subspecies

Wolbachia are the most widespread endosymbiotic bacteria in animals. In many arthropod host species, they manipulate reproduction via several mechanisms that favor their maternal transmission to offspring. Among them, cytoplasmic incompatibility (CI) promotes the spread of the symbiont by specifically decreasing the fertility of crosses involving infected males and uninfected females, via embryo mortality. These differences in reproductive efficiency may select for the avoidance of incompatible mating, a process called reinforcement, and thus contribute to population divergence. In the terrestrial isopod Porcellio dilatatus, the Wolbachia wPet strain infecting the subspecies P. d. petiti induces unidirectional CI with uninfected individuals of the subspecies P. d. dilatatus. To study the consequences of CI on P. d. dilatatus and P. d. petiti hybridization, mitochondrial haplotypes and Wolbachia infection dynamics, we used population cages seeded with different proportions of the 2 subspecies in which we monitored these genetic parameters 5 and 7 years after the initial setup. Analysis of microsatellite markers allowed evaluating the degree of hybridization between individuals of the 2 subspecies. These markers revealed an increase in P. d. dilatatus nuclear genetic signature in all mixed cages, reflecting an asymmetry in hybridization. Hybridization led to the introgressive acquisition of Wolbachia and mitochondrial haplotype from P. d. petiti into nuclear genomes dominated by alleles of P. d. dilatatus. We discuss these results with regards to Wolbachia effects on their host (CI and putative fitness cost), and to a possible reinforcement that may have led to assortative mating, as possible factors contributing to the observed results.     

The structure of a glycosylated protein hormone responsible for sex determination in the isopod, Armadillidium vulgare.

Two glycoforms (AH1 and AH2) of androgenic hormone, and its corresponding hormone precursor derived from HPLC-purified androgenic gland extract from the woodlouse Armadillidium vulgare were fully characterized by microsequencing and mass spectrometry. The amino-acid sequences of the two glycoforms were identical; they consist of two peptide chains, A and B, of 29 and 44 amino acids, respectively, with chain A carrying one N-glycosylated moiety on Asn18. The two chains are linked by two disulfide bridges. Glycoforms were only differentiated by the size and heterogeneity of the glycan chain. The androgenic hormone precursor (16.5 kDa) was shown to contain the sequence of chains A and B from the androgenic hormone, connected by a C-peptide (50 amino acids). These results were confirmed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis performed on a single hypertrophied androgenic gland. When injected into young females, both glycoforms of the androgenic hormone were able to override genetic sex-determination. In invertebrates, there is no other example where sex-differentiation is controlled by a protein hormone that is not synthesized by the gonads but by a special gland. A functional comparison with two other hormones which are believed to play a role in sex determination, i.e. ecdysone in insects and anti-Müllerian hormone in mammals, is presented. Work is in progress to clone and characterize the gene encoding androgenic hormone, moreover special attention is devoted to its regulatory regions, putative targets for the Wolbachia action.     

Mitochondrial DNA polymorphism, sex ratio distorters and population genetics in the isopod Armadillidium vulgare.

Two maternally inherited sex ratio distorters (SRD) impose female-biased sex ratios on the wood louse Armadillidium vulgare by feminizing putative males. These SRD are (i) an intracytoplasmic bacterium of the genus Wolbachia, and (ii) another non-Mendelian element of unknown nature: the f element. Mitochondrial DNA variation was investigated in A. vulgare field populations to trace the evolution of host-SRD relationships and to investigate the effect of SRD on host cytoplasmic polymorphism. The Wolbachia endosymbionts showed no polymorphism in their ITS2 sequence and were associated with two closely related mitochondrial types. This situation probably reflects a single infection event followed by a slight differentiation of mitochondria. There was no association between the f element and a given mitochondrial type, which may confirm the fact that this element can be partially paternally transmitted. The spreading of a maternally inherited SRD in a population should reduce the mitochondrial diversity by a hitchhiking process. In A. vulgare, however, a within-population mtDNA polymorphism was often found, because of the deficient spread of Wolbachia and the partial paternal inheritance of the f element. The analysis of molecular variance indicated that A. vulgare populations are genetically structured, but without isolation by distance.