The origin of terrestrial isopods (Crustacea: Isopoda: Oniscidea)

Living isopods of the suborder Oniscidea (commonly called woodlice) are the only group of Crustacea almost entirely composed of terrestrial forms. Furthermore, woodlice are completely independent from the aquatic environment from which they originally arose. From marine ancestors, woodlice are a key taxon to study the conquest of the land among arthropods because of their interesting gradation of morphological, physiological and behavioral adaptations for terrestriality. However, the origin and evolution of this model group are still poorly known. Herein, we provide a synthesis of the oniscidean fossil record to replace this group in a deep-time context. Because members of the Oniscidea are difficult to fossilize, their fossil record alone is undoubtedly fragmentary and not representative of their complete evolutionary history, but it maintains an important relevance by providing reference points. To date, the first attested occurrences of Oniscidea are recorded from the Early Cretaceous. At this time, woodlice were already widely distributed (from Western Europe to Eastern Asia) with several species. By evaluating phylogenetic studies, palaeobiogeographic context of fossil specimens and current biological considerations, we discuss and support a pre-Pangaean origin of the Oniscidea, in the Late Paleozoic—most likely during the Carboniferous.     

The Best Timing of Mate Search in Armadillidium vulgare (Isopoda,Oniscidea)

Mate choice is mediated by many components with the criteria varying across the animal kingdom. Chemical cues used for mate attractiveness can also reflect mate quality. Regarding the gregarious species Armadillidium vulgare (isopod crustacean), we tested whether individuals can discriminate conspecifics at two different levels (between sex and physiological status) based on olfactory perception. Tested conspecifics were individuals of the same or opposite sex, with the females at different moult stages. We found that the attractiveness of individuals was mediated by short-distance chemical cues and tested individuals were able to discriminate and prefer individuals of the opposite sex. Moreover, male preference to female increased during their moulting status as they matured. Males were particularly more attracted by females with appearing white calcium plates, which corresponds to the beginning of their higher receptivity period. These differences in attractiveness due to sex and physiological status are likely to shape the composition of aggregates and facilitate mate finding and optimize the reproductive success for both males and females. Thus aggregation pheromones could be linked to sex pheromones in terrestrial isopods.     

Ecotypic differentiation and phenotypic plasticity in reproductive traits of Armadillidium vulgare (Isopoda: Oniscidea)

Armadillidium vulgare differed in growth and survivorship on two field sites. Growth rates were higher at a site with consistently higher quality food than at the other site where less high-quality food was produced and which was less predictably accessible. Survivorship was higher at the second site where temperature fluctuations were consistently smaller. Individuals from the two populations were kept for 6 months under the same food and temperature conditions and patterns of resource allocation to reproductive traits analysed. Members of the population from the site with good growth conditions had significantly higher reproductive allocation, by 13.5%, and larger broods, by 9.1%, than those from the site with poor growth conditions. Contrary to theoretical predictions, they also had significantly larger offspring, by 7.5% dry mass. Larger offspring survived better than small ones. This differential survivorship, by 20% for a 3.4% difference in live mass, was much more pronounced under conditions of moisture stress and under fluctuating temperature regimes. Larger offspring would therefore be at a selective advantage on the site with more severe temperature fluctuations. Phenotypic plasticity in reproductive traits in response to experimental changes in food quality, temperature and crowding were monitored. Reproductive allocation was increased by 20.8% under conditions of higher food quality, by 14.7% at higher temperatures, and by 12.5% under less crowded conditions. Brood size, but not offspring dry mass, increased when food quality increased. When crowding increased by 25.0%, the size of broods remained the same but the dry mass of individual offspring decreased by 11.2%. Members of the population from the site with more variable access to high-quality food showed more plasticity in reproductive traits in response to changes in food supply than members of the population from the site with the more predictable food supply. Members of the population from the site with more stable temperatures showed less plasticity to temperature changes than members of the population from the site with greater temperature fluctuations. It is concluded that the observed microevolutionary processes and phenotypic plasticity have adaptative value as responses to spatial and temporal heterogeneity in environmental conditions.     

Description of the postmarsupial manca stages of Armadillidium granulatum (Crustacea,Isopoda, Oniscidea)

The development of the postmarsupial manca stages of Armadillidium granulatum Brandt, 1833 was studied in detail by morphological analysis. Ovigerous females were reared separately under controlled conditions, allowing us to follow the stages of development from release from the marsupium until the appearance of the first juvenile stage, identified by the full development of the seventh pair of pereopods. Each newborn was followed to record the subsequent moults that identify the three postmarsupial manca stages. Manca stage M I had a mean duration of 6 h, manca stage M II 15 days, and manca stage M III 32 days. The cephalothorax width was measured to provide a growth measure for each stage. The mean values of the cephalothorax width were: 0.512 mm (± 0.083 SD) for M I, 0.677 mm (± 0.058 SD) for M II and 1.194 mm (± 0.079 SD) for M III. The morphological modifications in the three postmarsupial manca stages were described, the body parts illustrated, and SEM images taken. The distinguishing characteristics among mancas were discussed, and comparisons made with manca stages of other terrestrial isopod species.     

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.     

Embryo tolerance and maternal control of the marsupial environment in Armadillidium vulgare (Isopoda: Oniscidea).

Marsupial development in terrestrial isopods subjects embryos to potential physiological stresses, including desiccation, osmotic variation, and high ammonia concentrations. In this study, we investigated tolerance of osmotic extremes, total ammonia, and pH in developmental stages of Armadillidium vulgare cultured in vitro. Marsupial stages were classified as stage 1 (chorionated eggs), stage 2 (having shed the chorion), and stage 3 (mancas). All stages showed wide but differing tolerance ranges. Stage 1 eggs possess the greatest ammonia tolerance, with high 7-d survival in 150 mM total ammonia, and a wide pH tolerance range. Mancas show the widest osmotic tolerance (100-1,400 mosm x kg(-1)) and display proficient hemolymph osmoregulation over this range. Stage 2 eggs reveal the narrowest tolerance ranges for all three parameters but still qualify as eurytopic. Silver staining revealed two distinct ion-transporting tissues in the developmental stages: a median band on the vitelline membrane of stage 1 and stage 2 eggs, corresponding in location to the embryonic dorsal organ, and the posterior three pairs of pleopodal endopodites in mancas. Gravid females do not downregulate ammonia but show efficient regulation of marsupial fluid pH and downregulation of osmolality during dehydration, both of which will provide additional protection to the marsupial young.     

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.     

Eco-morphological studies on pleopodal lungs and cuticle in Armadillidium species (Crustacea,Isopoda, Oniscidea)

Terrestrial isopods (Crustacea, Isopoda, Oniscidea) have adapted to land life by diverse morphological, physiological and behavioral changes. Woodlice species exhibit a large variety in this respect, their preferences ranging from moist to dry habitats. These moisture preference values are related to various morphological adaptations, rendering terrestrial isopods amenable to studying morphological adaptations to terrestrial life. We performed a comparison of four Armadillidium species (Armadillidium zenckeri, Armadillidium nasatum, Armadillidium versicolor, Armadillidium vulgare), by quantifying two morphological traits: the extent of the interfacial endothelium between the respiratory space and the hemolymph within pleopodal lungs and the thickness of tergite cuticle, which are ‘key factors’ in determining protection from desiccation. These values were measured from light micrographs of cross-sectioned lungs. The cosmopolitan A. vulgare, as a habitat generalist, seems to be the most resistant against desiccation and other environmental conditions, while A. zenckeri is the most sensitive one. Light microscopic studies revealed that the four species can be ordered similarly, if we compare them by the extension of the endothelial interface and cuticle thickness, suggesting that these morphological traits are important determinants of their distribution on habitat, microhabitat scales and through the existence of suitable habitats – together with many other factors – the geographical pattern of species occurence.     

A morphometric approach to the geographic variation of the terrestrial isopod species Armadillo tuberculatus (Isopoda: Oniscidea)

The terrestrial isopod species Armadillo tuberculatus Vogl, 1876 (Crustacea, Isopoda, Oniscidea) is a widely polymorphic species distributed in the south-central Aegean region (Greece) with a different morph on each island. Variation consists in coloration, size of cuticular tubercules, shape of telson and the shape of the male first pleopod exopodite (secondary sexual character of taxonomic importance). We studied the allometric growth of a cuticular tubercule in 17 populations (for both male and female individuals) and the shape variation of the first male pleopod exopodite in 10 populations using Elliptic Fourier Analysis, in order to test for patterns of intraspecific variation and possible relationships between morphs. In addition, Thin Plate Spline analysis was used for the calculation of the minimum bending energy between different exopodite shapes, which was then used for estimating the minimum spanning network (MSN) connecting them. The different allometric growth rates of the tubercule among island groups were significantly related to island latitude and climatic factors. On the other hand, the clustering of islands and the MSN based on male exopodite shape differences were not related to the palaeogeography of the Aegean region or to the present geographic distances of islands. These results are interpreted as evidence for non-adaptive radiation of the morphs.     

Morphology and evolution of respiratory structures in the pleopod exopodites of terrestrial Isopoda (Crustacea, Isopoda, Oniscidea)

The morphology of the respiratory structures located in the pleopod exopodites of terrestrial Isopoda is described for representative species of different genera. Special emphasis is set on the evolution of these lungs in the context of phylogenetic relationships as revealed by other morphological characters. In the most primitive species of the Oniscidea, and still of subordinate taxa of the Crinocheta, respiration takes place in the thin ventral integument of the exopodites. The evolution of dorsal respiratory fields with a weakly wrinkled surface occurred at least six times within the Crinocheta. In five of these cases, a further development can be observed. The evolution of a partly covered respiratory field with strongly wrinkled surface may have taken place six times. It is assumed that completely internalized lungs with spiracles surrounded by a water‐repellent surface microsculpture, evolved at least six times independently within the Oniscidea: in the Tylidae, Actaecia, Aphiloscia, the Eubelidae, the Armadillidae and in a taxon probably comprising Porcellionidae plus Armadillidiidae.     

Histological studies on the marsupium of two terrestrial isopods (Crustacea,Isopoda, Oniscidea)

The marsupium, a brood pouch in peracarid crustaceans (Crustacea, Malacostraca) has evolved in terrestrial environment for providing nutrition and optimal conditions for embryogenesis. In the present study we give details on the histology and ultrastructure of its constituting elements such as oostegites and cotyledons. Marsupia of two different eco-morphological types of woodlice, namely the non-conglobating species Trachelipus rathkii Brandt, 1833 and the conglobating species Cylisticus convexus De Geer, 1778 were investigated. Light microscopic (LM) studies showed some differences in the main structure of the two species’ brood pouch: in Trachelipus rathkii, a ‘clinger’ type woodlice, the oostegites bend outwards during brood incubation as growing offspring require more space, while in Cylisticus convexus, a ‘roller’ type isopod, the sternites arch into the body cavity to ensure space for developing offspring and still allowing conglobation of the gravid females. The quantitative analysis of the oostegites’ cuticle proved that the outer part is about 2.5 - 3 times thicker compared to the inner part in both species. Electron microscopic (TEM) examinations show only small histological differences in the oostegites and cotyledon structure of the two species. Cellular elements and moderately electron dense fleecy precipitate are found in the hemolymph space between the two cuticles of oostegites. The cells contain PAS positive polysaccharide areas. TEM studies revealed some differences in the cotyledon ultrastructure of the two species. Cotyledons of Trachelipus rathkii consist of cells with cristate mitochondria and granular endoplasmic reticulum with cisterns. Cotyledons of Cylisticus convexus consist of cells with densely cristate mitochondria and ribosomes attached to vesicular membrane structures. In both species cells with electron dense bodies were observed. We conclude that - besides the differences in marsupial shapes - the fine structure of the oostegites and cotyledons is hardly affected by the eco-morphological type, specifically the conglobating or non-conglobating character of the studied species.     

Genetic variation and differentiation in Armadillidium vulgare (Isopoda: Oniscoidea)

Genetic variation was examined with polyacrylamide and starch gel electrophoresis at 22 loci in 8 populations of Armadillidium vulgare. Polymorphism was observed at 9 loci, and the average heterozygosity was 3.0%. Heterozygosity was low when compared to most invertebrates but was similar to that reported for certain decapods. A significant heterozygote deficiency occurred in most populations of A. vulgare and considerable genetic differentiation was observed among isopod populations.     

Shelter site use and secondary sex ratios in the woodlice Armadillidium vulgare and Porcellio scaber (Crustacea: Isopoda)

In grassland populations of the woodlice Armadillidium vulgare and Porcellio scaber (Latreille) the sex ratio in hand collections from beneath wooden offcuts and roofing tiles varied through the breeding season. The proportion of males was higher in these collections from surface refugia than in independent estimates of the population sex ratio taken from soil cores. Collections became female-biased in June and July when most females were carrying broods, most notably in P. scaber . It is suggested that males use surface shelter between foraging events. A. vulgare males are particularly abundant under boards in May as they become active on the soil surface in order to locate mates. Females may find the higher and more equable temperatures in refugia attractive because they reduce brood development time but the cost of this advantage is the risk of failure to balance moisture requirements. These patterns of habitat use by the sexes have important implications for several population processes, the evolution of life-history tactics and the development of mating systems in these species.     

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).     

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.     

The immune cellular effectors of terrestrial isopod Armadillidium vulgare: meeting with their invaders, Wolbachia

Background

Most of crustacean immune responses are well described for the aquatic forms whereas almost nothing is known for the isopods that evolved a terrestrial lifestyle. The latter are also infected at a high prevalence with Wolbachia, an endosymbiotic bacterium which affects the host immune system, possibly to improve its transmission. In contrast with insect models, the isopod Armadillidium vulgare is known to harbor Wolbachia inside the haemocytes.

Methodology/Principal Findings

In A. vulgare we characterized three haemocyte types (TEM, flow cytometry): the hyaline and semi-granular haemocytes were phagocytes, while semi-granular and granular haemocytes performed encapsulation. They were produced in the haematopoietic organs, from central stem cells, maturing as they moved toward the edge (TEM). In infected individuals, live Wolbachia (FISH) colonized 38% of the haemocytes but with low, variable densities (6.45±0.46 Wolbachia on average). So far they were not found in hyaline haemocytes (TEM). The haematopoietic organs contained 7.6±0.7×10³ Wolbachia, both in stem cells and differentiating cells (FISH). While infected and uninfected one-year-old individuals had the same haemocyte density, in infected animals the proportion of granular haemocytes in particular decreased by one third (flow cytometry, Pearson''s test = 12 822.98, df = 2, p<0.001).

Conclusions/Significance

The characteristics of the isopod immune system fell within the range of those known from aquatic crustaceans. The colonization of the haemocytes by Wolbachia seemed to stand from the haematopoietic organs, which may act as a reservoir to discharge Wolbachia in the haemolymph, a known route for horizontal transfer. Wolbachia infection did not affect the haemocyte density, but the quantity of granular haemocytes decreased by one third. This may account for the reduced prophenoloxidase activity observed previously in these animals.     

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.