From the marine environment, woodlice gradually colonized terrestrial areas benefiting from the symbiotic relationship with the bacterial community that they host. Indeed, they constitute the only group of Oniscidea suborder that ha s succeed to accomplish their lives in terrestrial even desert surfaces. Herein they play an important role in the dynamic of ecosystems and the decomposition of litter. So to enhance our understanding of the sea-land transition and other process like decomposition and digestion of detritus, we studied the bacterial community associated with 11 specimens of terrestrial isopods belonging to six species using a Culture independent approach (DGGE). Bands sequencing showed that the cosmopolitan species Porcellionides pruinosus has the most microbial diversity. Screening demonstrated the predominance of Proteobacteria followed by members from Actinobacteria, Firmicutes and Cyanobacteria phyla. In fact we detected some symbionts like Wolbachia, Mycoplasma and Spiroplasma for the first time in a terrestrial isopod species.
Woodlice are considered as important decomposers of leaf litter. Therefore, these soil detritivores contribute mainly to the ecosystem function by mineralizing organic matter [
From an aquatic environment, terrestrial isopods gradually invaded terrestrial areas taking profit from the symbiotic relationship with microorganisms. Bacterial symbionts allow their host to spread and to occupy unfilled niches [
Isopods are widespread in marine, intertidal and terrestrial habitats [
Simultaneously, bacteria colonizing the hepatopancreas contribute to the secretion of digestive enzymes and the absorption of nutrients [
To get a first impression on the microbial community associated oniscidean species we used a molecular approach. Indeed, the culture-independent fingerprinting technique DGGE was used to accomplish such purpose. DGGE analysis has been used to monitor changes in the microbial community associated environmental samples [
The aim of this study is to screen the microbial community associated with a set of terrestrial isopods species using a molecular approach. Therefore, we examine the impact of the host species on the microbial community shaping.
Sampling was carried out during spring 2013 from 9 localities in Tunisia (
Samples were rinsed with ethanol and twice with distilled water in order to avoid
Isopod species | Sampling site | DGGE bands | Closest related sequence | Id.% | Acc. No |
---|---|---|---|---|---|
P. laevis | Chebba | B20 | Paracoccus saliphilus | 90% | NR_044021 |
L. panzerii | G. Naam1 | B19 | Saccharopolyspora flava | 76% | NR_028754 |
L. panzerii | Monastir | B17 | Sinorhizobium medicae | 97% | NR_074254 |
P. laevis | Chambi | B23 | Wolbachia endosymbiont of Culex quinquefasciatus Pel | 82% | NR_074127 |
P. pruinosus | Tunis | B15 | Paracoccus bengalensis | 97% | NR_042359 |
L. panzerii | G. Naam2 | B14 | Spiroplasma montanense | 86% | NR_025709 |
A. officinalis | Chebba | B13 | Paracoccus fistulariae | 83% | NR_104533 |
A. officinalis | Beja | B11 | Pseudochrobactrum lubricantis | 93% | NR_104538 |
B12 | Pseudochrobactrum lubricantis | 98% | NR_104538 | ||
P. pruinosus | Thelepte | B6 | Calothrix sp. | 88% | NR_102891 |
B7 | Cellulomonas denverensis | 80% | NR_042936 | ||
B8 | Kineococcus xinjiangensis | 90% | NR_044522 | ||
B9 | Georgenia muralis | 89% | NR_026305 | ||
B10 | Isoptericola hypogeus | 94% | NR_042355 | ||
B21 | Wolbachia endosymbiont of Culex quinquefasciatus Pel | 82% | NR_074127 | ||
B22 | Wolbachia endosymbiont of Culex quinquefasciatus Pel | 82% | NR_074127 | ||
H. reaumurii | Satah | B3 | Paracoccus saliphilus | 96% | NR_044021 |
B4 | Niabella ginsengisoli | 84% | NR_044541 | ||
A. tunisiense | Oum Ali | B2 | Mycoplasma cynos | 83% | NR_102477 |
contamination. DNA was extracted from the whole animal using a commercial kit (Abiopure Alliance) according to the manufacturer’s procedure. DNA quality was determined using the NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies) and stored at −20˚C.
DGGE culture independent method allows the separation of amplified fragments that have the same molecular weight but with a different fusion temperature. PCR amplification of the bacterial 16S rDNA gene was performed using the primers 907R CCGTCAATTCCTTTGAG TTT and 357F TACGGGAGGCAGC AG [
After electrophoresis, the gel was stained for 30 min in ethidium bromide solution, washed with sterile distilled water and photographed on a UV transillumination table. Dominant DGGE bands were excised and eluted by overnight incubation (12 hours) in 80 µl of sterilized distilled water at 37˚C with gentle shaking. The eluted DNA fragments were reamplified using unclamped 907R and 357F primers, sequenced and deposited in the GenBank database. The DGGE band profiles were analyzed with an image analyzing system Image J software (version 1.46). PCA and CAH analysis were performed with free version of XLSTAT 752 software.
11 from 14 examined amplicons of the 16S rRNA fragments gave a positive result; Armadillidium tunisiense (Oum Ali), Porcellionides pruinosus (Thelepte), Hemilepistus reaumurii (Satah), Armadillo officinalis (Beja), Armadillo officinalis (Chebba), Porcellionides pruinosus (Tunis), Porcellio laevis (Chambi), Leptothricus panzerii (Monastir), Leptotrichus panzerii (Garat Naam1 and 2) and Porcellio laevis (Chebba) (
The most complex profiles correspond to the samples 9 and 4 relating to P. pruinosus and P. laevis from Thelepte and Chambi respectively and secondly those of the samples 3, 8, 10 and 11 relating to L. panzerii (Monastir), A. officinalis (Beja), H. reaumurii from Satah and A. tunisiense sampled from Oum Ali.
In fact, the profiles complexity reflects the microbial flora diversity. It depends on species and its local environment. Similarly, it has been shown that the microbial flora structure is closely linked to the host species and its geographical location [
The most common bands (21, 22 and 23) had a resemblance of sequences with Wolbachia. The bands 3, 13, 15 and 20 exhibited a homology with members from the bacterial genera Paracoccus. This bacterium was detected in the majority of individuals examined. It is interesting to point out that this technique can detect only 1-2% of microbial communities that represent the dominant species. Besides, bacterial symbionts are not easily detectable in arthropods [
A total of 24 DGGE bands predominant were excised from the gel to be amplified and sequenced. The sequences obtained were identified by comparison with those available in the gene bank using the BLAST (Basic Local Alignment Search Tool) software (
The sequencing of the reamplified band revealed the presence of four taxonomic groups of bacteria with a difference of dominance (
species P. scaber. In the same line Actinobacteria was the most second group detected in the gut of A. vulgare [
Sequencing revealed the presence of common bands across screened specimens. Wolbachia was detected in P. pruinosus (bands 21 and 22) and P. laevis from Chambi (band 23) In fact, the first species showed the most complex profile represented by Calothrix sp, Cellulomonas deverensis, Kineococcus xinjiangensis, Georgenia muralis and Isoptericola hypogeus apart from Wolbachia. Bands 3, 13, 20 and 15, correspond to bacteria from the genus Paracoccus. Paracoccus saliphilus was common in P. laevis (Chebba) and H. reaumurii (Satah), Paracoccus bengalensis hosted by P. pruinosus from Tunis and Paracoccus fistulariae associated with A. officinalis from Chebba.
Bands 11 and 12 detected in Armadillo officinalis from Beja showed a significant homology of sequence with Pseudobachtrum lubricantis. Band 4 extracted from the DGGE profile of H. reaumurii (Satah) has a homology of sequences with Niabella ginsengisoli. Band 2 isolated from the species A. tunisiense (Oum Ali) is closely related to the symbiont Mycoplasma cynos. Saccharopolyspora flava and Spiroplasma montanense were detected in the two samples of L. panzerii from Garat Naam. Sinorhizobium medicae (band 17) was associated with L. panzerii from Monastir.
The principal component analysis (PCA) based on the variation of the bacterial community as function to the terrestrial isopod species showed a variation of 47.41% and 11.98% according to the axes F1 and F2 according to the species and locality (
We can distinguish three groups; the first is formed by the species H. reaumurii (Satah), A. officinalis (Chebba), P. pruinosus (Tunis) and P. laevis (Chebba) which are negatively correlated with the axis F2. In fact, this group is formed by host species sharing the association with the bacterial genus Paracoccus. The second group formed by the species L. panzerii originating from Garat Naam and Monastir, A. officinalis (Beja), P. laevis (Chambi) and A. tunisiense (Oum Ali). While the third group is totally different in a function of the positive values of the F1 axis which is limited to the species P. pruinosus (Thelepte) having a bacterial profile different to the remaining specimens and which is constituted mainly by bacteria from the Actinobacteria group.
The difference in microbial pattern between samples could be attributed to the impact of species habitat as reported in [
Data analysis of the bacterial diversity in terms of species of terrestrial isopod by hierarchical clustering (HCA) (
Indeed, a high level of dissimilarity separate P. pruinosus from Thelepte from the remaining species. Dissimilarity less marked, divide the rest of the samples into two groups, the first one is formed by the specie H. reaumurii (Satah), A. officinalis (Chebba), P. laevis (Chebba) and P. pruinosus from Tunis sharing the bacterial genus Paracoccus. The second group is formed by the species L. panzerii (Monastir) and (Garat Naam 1 and 2), P. laevis (Chambi), A. Tunisiense (Oum Ali) and A. officinalis (Beja) reflecting a mosaic of bacterial flora which might be acquired from the environment through ingested food.
Three sequenced bands showed a homology with the famous bacteria Wolbachia. Indeed, it was detected in two different species; P. laevis and P. Pruinosus. These two species are known as the host for this bacterium in Tunisia [
In our study, the estimated values of the sex ratio for both species harboring Wolbachia P. pruinosus from Thelepte and P. laevis from Chambi was strongly biased in favor of females (over 60%) which raise the probability of feminizing activity of Wolbachia in these species.
The band (14) from DGGE profile of L. panzerii has a sequences homology to Spiroplasma montanense. This bacteria affects a wide range of arthropods, it can be symbiotic or pathogen [
The bacterial symbiotic of the genus Spiroplasma has long been associated with arthropods but never in isopods. In fact, [
The DGGE profile of the species A. officinalis showed a band which sequences are homologous to that of Mycoplasma symbiotic of arthropods. Indeed, it has been shown that this bacterium is endowed with a tissue specificity in arthropods especially among semi-terrestrial isopods like Ligia genus where it was associated with the hepatopancreas [
Since the gut constitutes the best suited exchange organ with the environment of terrestrial isopod [
Cellulomonas is known as an aerobic microorganism which can be found in water and soil. It is capable to hydrolyze cellulose and produce a large quantity of enzymes degrading cellulose [
In fact, Paracoccus bacteria are known as symbiotic for pulses food. They are associated with soil and plants where they allow the reduction of nitrate [
Several bacteria detected in this study are simple passengers which are acquired from soil and plants through ingested nutrients and eliminated later through feces. Indeed, [
Sequencing results reflect a mosaic of bacteria transmitted via a horizontal or vertical path and which are variable depending on the species and sampling sites. The most relevant example is the different bacterial profiles between P. pruinosus from Tunis and Thelepte. This finding corroborates previous studies showing that the bacterial prevalence varied greatly depending on environment even within the same species of terrestrial isopod [
The microbial community associated with a set of Oniscidean species was targeted based on 16S rDNA-DGGE fingerprinting. 16S rDNA sequencing revealed that the microbial community was composed by members of Cyanobacteria, Actinobacteria, Protobacteria and Mollicutes. Furthermore, we detected the reproductive manipulator Wolbachia which is known to affect the whole biology, behavior and physiology of arthropods. Wolbachia has been previously studied in many species of terrestrial isopods in Tunisia like P. pruinosus, A. sulcatum, P. albinus and P. laevis. Mycoplasma was reported scarcely in some species like P. scaber. Finally Spiroplasma that is present has never been demonstrated previously within terrestrial isopods.
Although other microbes were detected, their role remains elusive. It is quite possible that the presence of these bacteria is transient rather than permanent. Further studies are needed to determine their interaction with the host species.
The authors would like to thank Professor Maher Gtari from the Laboratory of Microorganisms and Active Biomolecules and to those who contributed to the field and laboratory components of this work. This work was undertaken within the framework of the research activities of the laboratory of “Bio-Ecology and Evolutionary Systematics” financed by the Tunisian Ministry of Higher Education, Scientific Research.
The authors declare no conflicts of interest regarding the publication of this paper.
Majed, D., Wahiba, Z., Fadhel, B.M. and Sghaier, A.M. (2018) Characterization of the Dominant Bacterial Communities Associated with Terrestrial Isopod Species Based on 16S rDNA Analysis by PCR-DGGE. Open Journal of Ecology, 8, 495-509. https://doi.org/10.4236/oje.2018.89030