Research Article |
Corresponding author: Diána Csonka ( csonka.diana@gmail.com ) Academic editor: Katalin Szlavecz
© 2018 Diána Csonka, Katalin Halasy, Krisztina Buczkó, Elisabeth Hornung.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Csonka D, Halasy K, Buczkó K, Hornung E (2018) Morphological traits – desiccation resistance – habitat characteristics: a possible key for distribution in woodlice (Isopoda, Oniscidea). In: Hornung E, Taiti S, Szlavecz K (Eds) Isopods in a Changing World. ZooKeys 801: 481-499. https://doi.org/10.3897/zookeys.801.23088
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Terrestrial isopods, as successful colonizers of land habitats, show a great variety in species distribution patterns on a global, continental, or regional scale. On a local, within-habitat level these patterns reflect the species’ tolerance limits and the presence of suitable hiding places (shelter sites, refugia). Humidity preference reflects a species’ capability for water retention which, in turn, depends on the integumental barrier. Desiccation resistance is a key feature in isopod survival under different environmental conditions. The present study shows a correlation between cuticle thickness and desiccation resistance under three relative humidity (RH) ranges (about 30, 75 and 100% RH) in nine species, relating these to the species’ differences in meso- and microhabitat choices. Habitat preferences are also associated with differences in cuticle surface morphology. The results support our hypothesis that species distribution and desiccation resistance are associated with particular cuticular morphological traits. Phylogenetic relations seem to be less important in desiccation resistance than cuticle thickness and external morphology.
Eco-morphology, habitat preference, intrageneric comparison, mortality, sympatric species, water loss
Terrestrial isopods (Isopoda, Oniscidea) are successful colonizers of land habitats with over 3700 described species (
The sclerotized cuticle is the main barrier between oniscidean individuals and their environment. The crustacean exoskeleton is composed of four layers: the epicuticle, the procuticle (exo- and endocuticle), and the membranous layer (
It is well-known that terrestrial crustaceans lose water more rapidly than most other land arthropods due to their tegumental transpiration (
In terrestrial isopods, the epicuticle forms several surface structures such as tubercles, micro-ridges, setae, tricorns, plaques and pits (
We assumed that differences in preferred habitat type correlate with differences in desiccation resistance. We hypothesized that a key mechanism by which selection has generated the increased resistance was by increased thickness of the cuticle. We explored connections among species distribution, desiccation resistance, and morphological traits focusing on the following questions:
(i) What is the relationship between distribution and desiccation stress?
(ii) What is the relationship between microhabitat desiccation stress and species’ desiccation resistance?
(iii) What is the relationship between desiccation resistance and cuticle morphological traits?
We hypothesized that there is a higher similarity among morphological traits in epigeic species with similar ecological needs (that is sharing the same habitat) than among closely related species living under quite different environmental conditions. To test these hypotheses, we measured interspecific and intrageneric desiccation resistance and compared exoskeleton properties in a selected group of species from Central and Eastern Europe. While numerous studies have compared desiccation resistance of terrestrial isopod species (
For interspecific comparison six surface-active isopod species were tested. The species belong to different families and/or genera occurring in the same habitat: Armadillidium vulgare (Latreille, 1804), Cylisticus convexus (De Geer, 1778), Orthometopon planum (Budde-Lund, 1885), Protracheoniscus politus (C. Koch, 1841), Porcellionides pruinosus (Brandt, 1933), and Trachelipus rathkii (C. Koch, 1841). Armadillidium vulgare and T. rathkii are among the most frequent terrestrial isopod species in Hungary. The generalist A. vulgare is a frequent and widely distributed species in diverse habitat types (
The studied sympatric species (A. vulgare, C. convexus, O. planum, P. politus, P. pruinosus, T. rathkii) were hand collected in a deciduous forest of the Buda Mountains, next to the western part of Budapest, Hungary (at Solymár; 47°35.094'N, 18°57.164'E). Two mesohabitat types, different in humidity and temperature, within the sampling area were searched for woodlice: a valley (Alsó-Jegenye) along a stream (Paprikás patak) accompanied by a trail and an elevated area with an ancient deciduous forest (Felső-patak Hill). Individuals of A. zenckeri came from a marshland (Ócsa, Hungary; 47°17'39.5"N, 19°12'27.9"E), and the specimens were collected on the waterside or directly above the water, under the bark of wooden duck-boards. Specimens of A. nasatum originated from the tropical glass house of the Botanical Garden of Eötvös Loránd University (Budapest, Hungary; 47°29'05.3'N, 19°05'01.6"E) and specimens of A. versicolor were collected in the Margaret Island surrounded by the Danube river (Budapest, Hungary; 47°31'44.4"N, 19°03'06.5"E).
The collected individuals were kept in the lab in plastic boxes containing moist soil and litter for 14 days to ensure acclimatization. The specimens of each species were kept in 100% relative air humidity (RH) overnight to standardize the initial experimental conditions. This procedure ensured that animals replenished any possible water deficit. The isopods were without food for 24 hours, meanwhile they defecated their gut content, so this did not affect subsequent changes in body mass (
Water loss rate and mortality were studied in three different RH values in glass desiccators: an extremely dry (~30%), a relatively dry (~75%) and a humid one, nearly 100%. The humidity levels were acquired using silica gel (RH <30%), saturated sodium-chloride (RH 75%) and water (RH 100%) (
To reveal the characteristics of the exoskeleton, light microscopic investigations (LM) were applied. For this purpose, we fixed two intermolt adult specimens from each species in 4% paraformaldehyde for 7 days (we chose the greatest size category in the sampled population). The fixation was followed by rinsing in distilled water (3 × 1 h). We decalcified the tissues overnight in 8% ethylenediamine-tetraacetic acid disodium salt (EDTA). After the tissues became pliable they were dehydrated through an ascending series of ethanol (50% – 1 h, 70% – overnight, 80%, 2 × 90%, 2 × 96%, and 2 × 100% – 1 h). After dehydration, the samples were kept in xylene (2 × 1 h). Thereafter the samples were infiltrated with paraffin wax at 60 °C overnight, and embedded afterward. Histological sections (7 µm) were cut with a Reichert 2040 microtome and stained with Weighert’s hematoxylin-eosin (HE) and Periodic Acid-Schiff (PAS) reagent. With PAS reagent, we aimed to detect possible polysaccharides in tissues and on the integumental surface. The histological sections were studied and photographed with a Leica DM750 microscope.
The surface tergal ornaments were examined with a Hitachi S-2600N scanning electron microscope (SEM). For SEM we used alcohol preserved (70% ethanol) intermolt adult males and females from each studied species. The samples were dehydrated through an ascending series of ethanol (50% – 1 h, 70% – overnight, 80%, 2 × 90%, 2 × 96%, 2 × 100% – 1 h) and were air dried (
To quantify the thickness of the tergites, 100 measurements were taken for each species using light microscope (LM) cross-sections (2 specimens, 5 slides, 10 measurements/slide; Image J software) (
The relationship between mass-specific water loss, initial weight, and cuticle thickness was tested by Pearson correlation analysis (R 3.2.3 software). We analyzed the relationship between mass-specific water loss and the thickness of the epi- and procuticle separately. We used one-way ANOVA to analyze whether the variation in desiccation resistance as a function of cuticle thickness differs in the inter- and intrageneric groups (R 3.2.3 software). The comparison of the two groups was made based on the F value of the individual experiment’s ANOVA tests. An alpha value of p = 0.01 was used throughout.
Under extreme dry conditions (RH ~30%) we found high mass-specific water loss at each investigated species (Figure
Mass-specific water loss of the survived individuals (*all individuals died) at three different relative humidity values (white: ~30%, medium gray: ~75%, dark grey: ~100%). The experiment took 6 hours. [Measures: median ± first quartile and max/min; species initials: P. politus – Protracheoniscus politus, O. planum – Orthometopon planum, C. convexus – Cylisticus convexus, T. rathkii – Trachelipus rathkii, P.pruinosus – Porcellionides pruinosus, A. vulgare – Armadillidium vulgare].
At 75% relative humidity, the mass-specific water loss rates decreased in the order: C. convexus > P. pruinosus > O. planum > P. politus > T. rathkii > A. vulgare (Figure
We detected the lowest water loss (Figure
Within the genus Armadillidium, A. vulgare had the lowest and A. zenckeri had the highest water loss rates at 30% RH (Figure
Under higher humidity (75%) the order of mass-specific water loss was the same (Figure
The comparison of the desiccation resistance of the intrageneric and intergeneric groups has resulted in a lower F value for the former group, which means that there is a smaller relative variance among this group in comparison to the intergeneric group.
Based on the average cuticle thickness the species can be sorted in decreasing order: A. vulgare > C. convexus > P. politus > T. rathkii > P. pruinosus > O. planum (Figure
Within the Armadillidium genus, A. vulgare had the thickest tergal cuticle, while A. zenckeri had the thinnest cuticle (Figure
On the LM micrographs we found tricorn exteroreceptors in connection with neural processes (Figure
Light microscope micrographs of the studied species’ tergites. Armadillidium vulgare (A), Cylisticus convexus (B), Orthometopon planum (C), Protracheoniscus politus (D), Porcellionides pruinosus (E), Trachelipus rathkii (F). Abbreviations: ec – epicuticle, pc – procuticle, p – polysaccharide spheres, t – tricorn receptor, n – nerve; x 63. Staining: hematoxylin-eosin (HE) – A, E, F; Periodic Acid-Schiff (PAS) – B, C, D. Scale bars: 50 µm.
The dorsal surface of the studied sympatric terrestrial isopod species. Armadillidium vulgare (A), Cylisticus convexus (B), Orthometopon planum (C), Protracheoniscus politus (D), Porcellionides pruinosus (E), Trachelipus rathkii (F). Abbreviations: pl – plaques, p – polysaccharide spheres, t – tricorn receptor. Scale bars: 50 µm.
Within the Armadillidium genus the SEM micrograph showed interspecific differences. There were squat tricorns on the tergites of A. versicolor (Figure
Scanning electron (A, C, E, G) and light microscope (B, D, F, H) micrographs on the studied Armadillidium species’ tergites. Armadillidium vulgare (A, B), A. versicolor (C, D), A. nasatum (E, F), A. zenckeri (G, H). Abbreviations: pl – plaques, t – tricorn receptor. Staining: hematoxylin-eosin (HE) – B, D, F, H. Scale bars: 50 µm.
In the present study we compared intergeneric (A. vulgare, C. convexus, O. planum, P. politus, P. pruinosus, T. rathkii) and intrageneric (A. vulgare, A. versicolor, A. nasatum, A. zenckeri) desiccation resistance of terrestrial isopods under three humidity ranges. As the applied experimental setup did not allow air to circulate, the calculated water loss might be underestimated. We assumed that differences in tolerance limits are connected to morphological characters such as tergal thickness and surface ornaments of the cuticle, which might be related to their habitat preferences.
Several studies showed that body shape and cuticle permeability are also significant factors in water loss rate.
In the present study individuals which died during the experiment lost more water than the surviving ones.
The cuticle of isopods is more permeable than that of the most terrestrial arthropods, and transpiration through the exoskeleton is a major part of water loss (
Despite the relatively thick tergal cuticle P. politus did not survive under extreme dry conditions. The survival of oniscideans in natural habitats is critically dependent not only on habitat but also on daily activity patterns (
In the case of O. planum and P. pruinosus we found rather thin exoskeleton covered by polysaccharide spheres that might also reduce water loss. The composition and function of these structures is unknown.
Previous studies showed that ancestral terrestrial isopod species had lower desiccation resistance.
Nevertheless, the desiccation resistance could not be explained by only phylogenetic relationship. This is further supported in the present study by the different water loss rate of Armadillidium species under dry conditions. Desiccation resistance of the four investigated species is in accordance with their cuticle thickness and habitat preference.
Resistance against desiccation in terrestrial isopod species was significantly associated with the two investigated morphological traits: body mass (size) and thickness of tergal cuticle. Species with the smallest mass-specific water loss rate were larger and possessed thicker tergal cuticle. Significant variation in both desiccation resistance and morphological traits was observed among the four Armadillidium species, despite their close phylogenetic relatedness.
The authors would like to thank Anikó Keszőcze (University of Veterinary Medicine Budapest) for her help in microscopic preparations. We are grateful to the anonymous referee and the subject editor (Katalin Szlavecz) for their language suggestions and their useful and valued advice which improved the quality of the manuscript. This work was supported by a PhD grant (DC) from the University of Veterinary Medicine Budapest. This publication was supported by the 17896-4/2018/FEKUTSTRAT grant of the Hungarian Ministry of Human Capacities.
The non-significant results of the ANOVA tests