Characterization of the Dominant Bacterial Communities Associated with Terrestrial Isopod Species Based on 16S rDNA Analysis by PCR-DGGE

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 has succeed to accomplish their lives in terrestrial even desert surfaces. Here-in 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 predo-minance of Proteobacteria followed by members from Actinobacteria, Fir-micutes and Cyanobacteria phyla. In fact we detected some symbionts like Wolbachia, Mycoplasma and Spiroplasma for the first time in a terrestrial isopod species.


Introduction
Woodlice are considered as important decomposers of leaf litter. Therefore, these soil detritivores contribute mainly to the ecosystem function by mineraliz-mercury [4] [5].
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 [6] [7].
Therefore, Oniscidean isopods are the only one of ten isopod suborders where the majority of species have become independent of the aquatic environment. In this sense, it has been evidenced that arthropods such as isopods are known to house a wide range of bacteria that can affect the biology of their host by the manipulation of reproduction (as the bacteria Wolbachia) [8], locomotion [9], diet [10] and interspecific competition [11]. On the other hand, [12] established that microbes can cause profound effects on the nutritional ecology, defensive interactions, reproduction, development and genome evolution of their hosts.
Symbionts are found in multiple compartments within Oniscidea. Indeed they colonize the genital tract, the head, the abdominal cavity, the cytoplasm and the digestive tissues.
Isopods are widespread in marine, intertidal and terrestrial habitats [6] and this may partly due to the advantages provided by the bacterial partner. In fact, nutritional symbiote improves the digestion process by hydrolysis of cellulose and the oxidation of phenol and lignin via the enzymatic activity within the arthropod gut [13] [14]. Besides, microbial activity permits the providing of essential nutrients such as amino acids and vitamins [15].
Simultaneously, bacteria colonizing the hepatopancreas contribute to the secretion of digestive enzymes and the absorption of nutrients [16] [17]. Given the lack of baseline information on the dynamics of microbial flora associated with terrestrial isopods [18] and the focusing interest primarily on the presence of Wolbachia infections in Oniscidea [19], it is of importance to deepening our knowledge concerning the total microbial communities associated with terrestrial isopod species. Hence terrestrial isopods provide an excellent model to study the evolutionary transition from sea to land from an adaptive point of view.
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 [20]. This molecular tool allows to separate DNA fragments that are identical in length based on their melting characteristics in a gradient of chemical denaturant [21].
The aim of this study is to screen the microbial community associated with a

Extraction of Total DNA
Samples were rinsed with ethanol and twice with distilled water in order to avoid  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.

Culture-Independent Approach: DGGE Analysis
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 [25]. The amplified sequences correspond to the V3-V5 hypervariable region of the 16S rDNA gene. A 40 bp GC clamp was added to primer 357F to avoid complete denaturing and separation of the DNA strands while migrating in denaturant conditions [21]. PCR products were resolved on 7% (w/v) polyacryla-   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.

Bacterial Diversity in Terrestrial Isopods
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 [26].
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.
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 (Table 1).

Sequencing of Bands
The sequencing of the reamplified band revealed the presence of four taxonomic groups of bacteria with a difference of dominance ( Figure 3). Indeed profiling gave 42% of Proteobacteria (bands 3, 4, 11, 12, 13, 15, 17, 21, 22 and 23), 35% of Actinobacteria (bands 7, 8, 9, 10 and 19), 14% of Mollicutes (bands 2 and 14) and 8% of Cyanobacteria through the strip 6. Otherwise, Lapanje et al. 2010 [28] showed that Actinobacteria is one of the most detected isolated phyla from the gut of Porcellio scaber originating from a polluted area. Hence it has been suggested that this class is involved in the physiological resilience of the cited species. It has been mentioned that representatives of Actinobacteria have a crucial role in improving the quality of nutrients poor in nitrogen within the Onicidean Open Journal of Ecology species P. scaber. In the same line Actinobacteria was the most second group detected in the gut of A. vulgare [26].
Sequencing revealed the presence of common bands across screened speci-

Distribution of Bacterial Community Based on Species and Its Locality
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 (Figure 4).
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 [26] and [29] which established that the diversity of intestinal microflora is a function of soil and local environment.

Detection of Wolbachia
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 [30] [31].
Wolbachia is an obligate intracellular bacterium affecting a wide range of arthropods including 60% of terrestrial isopods [32]. Wolbachia is a reproductive parasite that manipulates the reproduction of its host serving its own maternally transmission. It is responsible for a set of phenotypes including cytoplasmic incompatibility, male killing, thelytokous parthenogenesis and feminization of males [19]. In addition, [33] suggested that Wolbachia may confer several reproductive advantages like fertility and longevity in infected females for example in the species A. vulgare, the presence of Wolbachia enhances the immunocompetence by causing an increase in natural septicemia.
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.

Detection of Spiroplasma
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 [34]. Some species of Spiroplasma genus are vertically transmitted to the progeny. In some cases, Spiroplasma can modify the sex ratio by killing infected males then ultimately leading to an exclusively female population [35]. In fact, it has been shown that this bacterium shares with Wolbachia the induction of male killing during embryogenesis in Drosophila [36]. In arthropods, Spiroplasma can colonize the digestive system where it can play a role in the digestion process of the host and in the decomposition of plant material. In plants, this bacterium can be found on the surface, in particular flowers, and phloem.
The bacterial symbiotic of the genus Spiroplasma has long been associated with arthropods but never in isopods. In fact, [37] showed that this bacterium infects 6% and 23.1% of insects and spider respectively but until now no study has revealed an association relationship between Spiroplasma and terrestrial iso-

Mycoplasma Detection
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 [38], as well as in the terrestrial isopod P. scaber [39]. Its presence is correlated with the surviving of terrestrial isopods exposed to a diet of poor quality for 90 days. Controversially, it acts negatively on longevity whatever the diet quality [17]. It has been shown that Mycoplasma is involved in the cellulose digestion and the oxidation of phenol. This symbiont of hepatopancreas can be acquired from the environment [40]. Apart from Wolbachia, the terrestrial isopod P. pruinosus has a diverse microbial community marked by soil microbes or plant symbionts that colonize the digestive tissues through digested nutrient. For example, the bacteria Calothrix sp. can be harbored by plants, algae, stones and wood substrates in wetland [41].
Since the gut constitutes the best suited exchange organ with the environment of terrestrial isopod [14], the microbial pattern in P. pruinosus reflects the environment of Thelepte characterized by a mountain area belonging to a semi-arid bioclimatic with cool winters. In addition, the diversity of microbial flora found in this species could explain its ecological plasticity and consequently the wide distribution. In this line, a metagenomic analysis of the digestive tissues of this species showed a great diversity of bacteria including hepatopancreatic symbionts, cellulolytic bacteria and reproductive parasites like Wolbachia and Serratia (Unpublished data). Indeed [26] showed that the host protection against fluctuation of environmental conditions and the resistance to environmental stress depend on the microbial flora diversity. Besides, the bacterial symbionts allow their host to spread and to occupy unfilled niches through the adaptive capabilities especially nutritive ones as shown by [7]. Therefore, symbionts diversity provide several advantages to the host like the genetic differentiation, the adaptation to a wide range of food plants and the sensitivity to thermal stress and other environmental factors [12] [42] [43]. It has been shown that the amount of biofilm colonizing the leaf litter affects the life history of the terrestrial isopod P. Scaber [44]. 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 [45]. The genus Paracoccus is the most represented in the studied samples. Indeed four species are associated with this microorganism; H. reaumurii (Satah) and P. laevis (Chebba) harbor the same species, A. officinalis (Chebba) and P. pruinosus (Tunis). 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 [46]. This bacterium was introduced as a bioindicator of pollution. Hence, [28] showed that this microorganism confers resistance to its host P. scaber against pollution. Paracoccus is the most common bacterial genus in individuals collected from the contaminated area. It has been proposed that it is responsible for the resistance of the terrestrial isopod species to pollutant. Consistently, the species harboring Paracoccus were P. laevis and A. officinalis sampled from Chebba marked by a salt area and H. reaumurii collected from Satah characterized by phosphate mines.
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, [47] showed a rapid elimination of non-indigenous pathogenic bacteria by scorpion after each meal.
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 [17] [26] [40].

Conclusions
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.