Yasser Fadlallah

Abdeldjalil Aïssa-El-Bey

Senior Member, IEEE Karim Abed-Meraim

Senior Member, IEEE Karine Amis

Member, IEEE Ramesh Pyndiah

Semi-Blind Source Separation in a multi-user transmission system with Interference Alignment

In this paper we address the decoding problem in the K-user MIMO interference channel assuming an interference alignment (IA) design. We aim to decode robustly the desired signal without having a full Channel State Information (CSI) (i.e. knowledge) at the receivers. We show the equivalency between the IA model and the Semi-Blind Source Separation model (SBSS). Then, we prove that this equivalence allows the use of techniques employed in source separation for extracting the desired signal free of interference, even though dependency exists between some components of the source signal in the SBSS model. Our simulation results illustrate a BER performance very close to the MMSE receiver with full-CSI.

I. INTRODUCTION

Interference Alignment (IA) is an interference management strategy that aims to achieve the Degrees of Freedom (DoF) of the K-user interference channel (IC). This technique results in a linearly scaling network sum-rate with the number of users sharing a common transmission medium. Its basic idea is a joint design of all transmitted signals such that interfering signals at each receiver overlap and remain distinct from the desired signal [START_REF] Cadambe | Interference Alignment and Degrees of Freedom of the K-user Interference Channel[END_REF], [START_REF] Maddah-Ali | Communication over MIMO X Channels: Interference Alignment, Decomposition, and Performance Analysis[END_REF]. In a K-user M × N MIMO system, where M and N are the number of the transmit and receive antennas, respectively, the feasibility of the IA design has been studied [START_REF] Yetis | On Feasibility of Interference Alignment in MIMO Interference Networks[END_REF], and distributed IA-achieving algorithms have been derived such as in [START_REF] Gomadam | Approaching the Capacity of Wireless Networks through Distributed Interference Alignment[END_REF]. These algorithms are based on an iterative processing at the transmitter when the full-CSI is provided, and on information exchange with the receiver when only the local CSI is provided.

In this paper, we address the decoding problem when an IA design is employed at the transmitters. That is, the interfering signals are aligned in a subspace linearly independent of the desired signal subspace. The traditional linear decoders estimate the decoding matrices, defined as the interference null space, using Least Square (LS) method essentially based on a training sequence within each frame. Our work concerning the decoding scheme pursues a different approach. The main contribution consists in separating the desired signal from the interference using higher-order cumulants, and then the desired signal is identified using a few training symbols. This approach has the advantage of allowing to decode the received signal even when the number of training symbols is low, and results in a more robust decoding scheme for a same training sequence length. We first show the equivalence between the MIMO IC model proposed herein and a determined semi Blind Source Separation (SBSS) model when the IA scheme is employed. Then, we demonstrate the ability of the SBSS problem to separate the desired signal from the interference through a joint diagonalization of the fourth-order cumulant matrices [START_REF] Cardoso | Blind Beamforming for Non-Gaussian Signals[END_REF]. The separation ability is due to the existing independence between the desired signal and the interference. The joint diagonalization is able to extract the desired streams blindly up to a permutation and scaling ambiguities. These ambiguities are revealed using a few training symbols within each transmitted frame, hence, the term semi-blind comes by.

One proposed application of our framework is the communication between K base stations (BS) and K end-users in the downlink mode assuming an IA design. The transmission is assumed in TDD mode. The channel is estimated at the BS using an N s -length uplink reference signal sent from the users assuming a reciprocal channel, that is supposed constant during one frame transmission. This strategy is employed in the TDD-uplink transmission scheme in the 3GPP-LTE network [START_REF]LTE; Evolved Universal Terrestrial Radio Access (E-UTRA); and Evolved Universal Terrestrial Radio Access Network (E-UTRAN); overall description[END_REF]. Another strategy that can be used is when each user estimates the channels connecting him with the BS, and feeds it back quantized to them BS. The reliability of this strategy depends on the channel estimator at the receiver, the channel quantizer, and the feedback link quality. Research works carried out, e.g. in [START_REF] Bolcskei | Interference alignment with limited feedback[END_REF], and have studied the IA achievability using a limited feedback link. They have defined a channel quantizer over the composite Grassmannian manifold, that achieves a full DoF in the IC when the feedback bit rate scales sufficiently fast with the SNR. In our study, we assume an IA-achieving design. Our simulation results highlight the robustness of the proposed decoding strategy when both perfect and imperfect IA design hold at the transmitters. This paper is organized as follows. In Section II, the assumed MIMO IC is described. A BSS model associated to the MIMO IC is given in Section III. In Section IV, we show the ability of the joint diagonalization technique to decode the desired signal. The simulation results are shown in Section V. Finally, Section VI concludes the paper.

Notations: Boldface upper case letters and boldface lower case letters denote matrices and vectors, respectively. For the transpose, transpose conjugate and conjugate matrix we use (.) t , (.) H and (.) * , respectively. |.| and ||.|| denote the modulus of a complex and the norm of a vector, respectively. E is the statistical expectation operator.

II. SYSTEM MODEL

We consider a K-user quasi static IC with K transmitreceive pairs with N antennas at each side of the link. A given transmitter intends to have its signal decoded by a single dedicated receiver. The received signal at the k th receiver node and at instant l is given by

y k (l) = H kk V k s k (l)+ j =k H kj V j s j (l)+z k (l), l = 0, • • • , L-1 (1)
where L represents the frame length, H kj ∈ C N ×N is the fading channel matrix between the j th transmitter and k th receiver, V j ∈ C N ×d is the precoding matrix at the j th transmitter, and z k (l) ∈ C N ×1 is the circular symmetric complex Gaussian noise vector at the k th receiver, with i.i.d. components; i.e. z k ∼ N c (0, I N ). {s j (l) ∈ M d×1 |l = 0, • • • , L-1} represents the d streams from the j th transmitter during an L-symbol duration interval. The symbols of s j are supposed i.i.d. from a finite constellation M. The decoding is over one frame, in which the channel is supposed unchanged.

The K precoders are jointly designed to satisfy the IA conditions, which can be achieved using different solutions [START_REF] Cadambe | Interference Alignment and Degrees of Freedom of the K-user Interference Channel[END_REF], [START_REF] Gomadam | Approaching the Capacity of Wireless Networks through Distributed Interference Alignment[END_REF]. At the receiver side, the intended signal can be recovered by projecting the received signal on the interference null space. The l th decoded signal vector is given by

ỹk (l) = U k H kk V k s k (l) + j =k U k H kj V j s j (l) + U k z k (l), (2)
where U k denotes the decoding matrix that we aim to estimate. To this end, we exploit the statistical independence of the transmitted streams, and we seek at each receiver for the decoding matrix that maximizes the statistical independence between the components of the mixed received signal. Then, we introduce a few training symbols in order to remove the scaling and the permutation ambiguities.

III. FROM THE FADING INTERFERENCE CHANNEL TO A BSS MODEL

This section is dedicated to the definition of a linear BSS model from the MIMO IC model, assuming an IA scheme at the transmission side. Such model is required for the application of linear BSS techniques. Before going into details, let us recall the following lemma from linear algebra Lemma 1:

Let A 1 ∈ C N ×n and A 2 ∈ C N ×n (N > n), where rank(A 1 ) = n and rank(A 2 ) = m, (m ≤ n) and span(A 2 ) ⊂ span(A 1 ). Then, for every s 2 ∈ C n×1 , ∃ s 1 ∈ C n×1 such that A 1 s 1 = A 2 s 2 1 .
In the adopted transmission model, the received signal given in (1) can be expressed as

y k (l) = Hk k s k (l) Desired signal + j =k Hk j s j (l) Interference signal +z k (l), (3)
where Hk j = H kj V j . Assuming the IA conditions are satisfied, and let Hk I ∈ C N ×(N -d) denote the matrix spanning 1 This is also valid for the case when s 1 and s 2 ∈ C N ×L the (Nd)-dimensional interference subspaces at receiver k; i.e. Hk j for j ∈ {1, • • • , K} , j = k are all spanned by Hk I . Thus, using the lemma above, the interference subspaces can be expressed in terms of Hk (4) Substituting ( 4) into (3) yields for l = 0,

• • • , L -1 y k (l) = Hk k s k (l) + Hk I sk (l) + z k (l) = Hk k Hk I [s t k (l) st k (l) t + z k (l) = B k sk (l) + z k (l), (5)
where

sk = s 1I + • • • + s (k-1)I + s (k+1)I + • • • + s KI , B k ∈ C N ×N

IV. EXTRACTION OF THE DESIRED SIGNAL USING BSS

A. Desired signal Extraction

The BSS standard model is defined as

y(l) = As(l), l = 0, • • • , L -1 (6)
where s(l) ∈ C N ×1 is the vector of N statistically independent latent variables , y(l) ∈ C N ×1 is the observation vector, and A ∈ C N ×N is a full rank unknown mixing matrix. The BSS technique seeks for the demixing matrix U that maximizes the statistical independence between the estimated components ŝ(l) = U As(l)∀l. For the sake of the simplicity, the time index will be ignored in the remaining of this section. It is shown in [START_REF] Comon | Independent Component Analysis, A New Concept?[END_REF] that, under mild assumptions, the estimated variables ŝ are similar to the original sources s up to a permutation and scaling by a constant, i.e.

U A = P Λ, ( 7
)
where P is a permutation matrix and Λ is a diagonal matrix.

The previous section shows that the MIMO IC model in ( 5) is similar to the BSS model except that some mutual dependencies exist between some components of sk = [s

t k st k ] t .
The first d components of s k are mutually independent and represent the desired streams. The (N -d)-components sk are mutually dependent and represent the interference part. This situation of dependent sources has been considered in certain recent studies, e.g. [START_REF] Castella | Blind Separation of Instantaneous Mixtures of Dependent Sources[END_REF], [START_REF] Caiafa | On the conditions for valid objective functions in blind separation of independent and dependent sources[END_REF]. Next, we show how to only extract the desired signal using the method JADE.

B. Joint Approximate diagonalization of Eigenmatrices

JADE is a well known statistical technique for solving linear determined BSS problem. It is based on the fact that the fourth order cross-cumulant of independent variables are zeros. Thus, demixing a received mixed signal as in ( 6) involves looking for the decoding matrix that makes the fourth order crosscumulant null [START_REF] Cardoso | High-order contrasts for independent component analysis[END_REF]. We summarize the steps of the algorithm JADE as follows (for further details, the reader can refer to [START_REF] Cardoso | Blind Beamforming for Non-Gaussian Signals[END_REF]) 1)

Step 1: Compute the whitening matrix W as the inverse square root of the sample covariance matrix of the received data. As shown in [START_REF] Cardoso | Blind Beamforming for Non-Gaussian Signals[END_REF], W transforms A into a unitary matrix F = W A.

2)

Step 2: Form the sample 4-th order tensor 2 Q z of the whitened data z = W y 3) Step 3: Compute the N most significant eigenpairs of

Q z : {λ n M n |n = 1, • • • , N } 4) Step 4: Perform the approximate joint diagonalization of matrices {λ n M n |n = 1, • • • , N } by a unitary matrix U 5)
Step 5: An estimate of the source vector is ŝ = U z JADE has been proved able to separate the original streams when all streams are statistically independent. Let us now demonstrate that even in the presence of some mutually dependent components, as in [START_REF] Gomadam | Approaching the Capacity of Wireless Networks through Distributed Interference Alignment[END_REF], JADE is able to separate the mutual independent streams. For our considered problem, the (N -d) interference sources are dependent in which case we can easily show 3 that the set of matrices {λ n M n |n = 1, • • • , N } are not anymore jointly diagonalizable but are jointly blockdiagonalizable. In other words, for n = 1, • • • , N , we have the following joint matrix structure:

λ n M n = F M n,1 0 0 M n,2 F H ( 8
)
where M n,1 are d × d diagonal matrices, M n,2 are (Nd) × (Nd) unstructured matrices and F = W A. In [START_REF] Bousbia-Salah | Blind separation of source signals from their convolutive mixtures: Bloc Joint Diagonalization technique based on second order statistics[END_REF], it is shown that the joint diagonalization algorithm used in the standard BSS method JADE can be used as well for the joint block diagonalization of a set of matrices. Consequently, the final transformation given by the whitening matrix and unitary transform U leads to:

U W A = D 1 0 0 D 2
where D 1 is a d×d diagonal matrix and D 2 a (N -d)×(N -d) given matrix. Hence, the first d entries of ŝ = U z represent the desired source signals while its remaining Nd entries represent linear mixtures of the (non-desired) interference signals.

Another BSS technique is the iterative FastICA. This technique is characterized by its low computational complexity and fast convergence. It also performs close to the JADE in terms of robustness [START_REF] Bingham | ICA of Complex Valued Signals: a Fast and Robust Deflationary Algorithm[END_REF] (see section V).

C. Semi-Blind separation

The ambiguities on the scale and permutation of the estimated streams are solved thanks to a few training symbols inserted within each data frame. We denote x j ∈ C 1×Ns the 2 The entries of Qz are given by Qz(i, j, k, l) = cum(z i , z * j , z k , z * l ) where cum refers to the fourth order cumulant and z * i is the complex comjugate of the ith entry of z.

3 By using the structure of Qz in [START_REF] Cardoso | Blind Beamforming for Non-Gaussian Signals[END_REF], one can directly obtain the matrix structure in [START_REF] Bingham | ICA of Complex Valued Signals: a Fast and Robust Deflationary Algorithm[END_REF]. This derivation is omitted due to space limitation. • FastICA: decoding scheme that uses FastICA as BSS technique.

• LS: the MMSE-based decoder with LS-CSI estimation.

• MMSE: the MMSE-based decoder with perfect CSI. j th training sequence for j ∈ {1, • • • , d}, and ŝi is the i th estimated independent stream with i ∈ {1, • • • , d}. We define the Normalized Minimum Mean Squared Error (NMSE) as

NMSE(ŝ i , x j ) = log 10 1 - |ŝ i x H j | 2 ||ŝ i || ||x j || . (9)
The ambiguity on the permutation order is solved by minimizing the NMSE according to the training sequence

argmin i,j∈{1,••• ,d} NMSE(ŝ i , x j ), (10)
Next, the scale ambiguity can be solved by seeking for the complex variable α that minimizes the MMSE between the estimated variables and the training sequence

MMSE = E |α ŝj -x j | 2 . (11)
The 1 × N s vector ŝi used in ( 9), [START_REF] Castella | Blind Separation of Instantaneous Mixtures of Dependent Sources[END_REF], and ( 11) is formed by the first N s estimated symbols of the stream s k .

Remarks: 1) The proposed technique requires the receiver to wait for all samples within one frame. Therefore, the authors in [START_REF] Zhiguo | HOS-Based Semi-Blind Spatial Equalization for MIMO Rayleigh Fading Channels[END_REF] have proposed an adaptative semi-blind high order separation technique. This technique can be adapted to our case since we have showed that the dependency between interference streams does not affect the desired source extraction.

2) As shown by our simulation results, the semi-blind approach results in a slight performance loss as compared to the standard (data-aided) MMSE. However, it is shown in [START_REF] Kacha | Fast adaptive blind MMSE equalizer for multichannel FIR systems[END_REF], that in such cases one can compensate for this performance loss using a decision-directed MMSE detection in a two step approach, the first step being the semi-blind approach proposed previously (see [START_REF] Kacha | Fast adaptive blind MMSE equalizer for multichannel FIR systems[END_REF] for further details).

V. SIMULATION RESULTS

In this section, we evaluate the Bit Error Rate (BER) of the JADE and FastICA in a 3-user 4 × 4 MIMO IC with an IA design. The IA scheme is achieved using the distributed iterative solution proposed in [START_REF] Gomadam | Approaching the Capacity of Wireless Networks through Distributed Interference Alignment[END_REF]. Each user sends d = 2 data streams. The symbols are QPSK modulated. The channel is supposed flat fading Rayleigh distributed, and remains constant over one frame with length L = 2000 symbols. Fig. 1 depicts the BER performance of the MMSE-based decoder with full CSI, the BSS-based decoders (JADE and FastICA), and the MMSE-based decoder with LS-CSI estimation. We use N s = 8 symbols to reveal the scale and the permutation ambiguities, and to estimate the CSI using LS. As shown, JADE and FastICA have close BER performance in the entire SNR region. They also perform close to the MMSEbased with full CSI with a gap of about 1dB -2dB over the entire SNR region. On the other hand, compared to the LS, a gain between 1dB -4dB over all SNR values is obtained.

Next, Fig. 2 describes the BER behavior of the JADE and FastICA algorithm in terms of the training sequence length. The MMSE-LS requires at least N s = 3 j=1 d j = 6 to separate the sources. The BSS techniques can separate the independent sources without any training symbols. However, to solve the scaling and the permutation problem, only N s = 1 symbol is necessary. At 20dB, the BER of both techniques improves with N s to reach 3.10 -3 for N s = 8, and remains roughly unchanged when N s increases. Comparing to the LS, an important BER gain is obtained for N s < 16. This gain decreases when N s increases and becomes negligible at N s = 32. This BER comparison is similar for SNR= 32dB.

Next, in order to perform the IA design, channel matrices have to be estimated. Therefore, we assume a TDD-uplink transmission scheme where the receiver sends to the transmitter an L s -length reference signal in a reciprocal channel for CSI estimation. This strategy is employed in the 3GPP-LTE network [START_REF]LTE; Evolved Universal Terrestrial Radio Access (E-UTRA); and Evolved Universal Terrestrial Radio Access Network (E-UTRAN); overall description[END_REF]. The estimated channel matrices are modeled as [START_REF] Tresch | Cellular Interference Alignment with Imperfect Channel Knowledge[END_REF] Hkj = H kj + E kj ; ∀ k, j

where E kj is the channel estimation error ∀ k, j. The coefficients of E kj are symmetric complex Gaussian distributed with zero mean and σ 2 e variance. In Fig. 3, the BER performance of both LS and JADE methods is illustrated in presence of a channel estimation error. It can be observed that in the region where σ 2 h /σ 2 e < 10dB, both of the decoders results in a degraded BER. However, our proposed decoder outperforms the LS: i) beyond σ 2 h /σ 2 e = 10dB when SNR=12dB, ii) beyond σ 2 h /σ 2 e = 14dB when SNR=16dB, and iii) beyond σ 2 h /σ 2 e = 21dB when SNR=20dB. VI. CONCLUSION In this paper, we have adressed the problem of detection in a MIMO IC system using IA scheme at the transmitters. The problem has been formulated as a semi-blind source separation problem, and the ability of the joint diagonalization technique (JADE) has been shown to extract the desired streams. Training sequence has been introduced to solve the permutation and scaling ambiguities. The proposed scheme performs close to full-CSI MIMO IC-IA schemes. We have also showed by simulations that it outperforms the traditional MMSE using LS for CSI estimation method with a same training sequence length.

- 1

 1 I as Hk j s j = Hk I s jI (l), s jI (l) = Hk H I Hk I Hk H I Hk j s j (l).

  is a full rank matrix that spans the union of the desired and the interference subspaces, and sk (l) is the N × 1 source vector consisting of d desired streams and (Nd) interference streams.[START_REF] Gomadam | Approaching the Capacity of Wireless Networks through Distributed Interference Alignment[END_REF] gives the formulation of a linear determined BSS problem where an N -length source vector sk is mixed by an unknown mixing matrix B k to produce N mixture signals y k observed from N sensors. The BSS aims at separating the original streams from an observed sensor array without knowing the transmission parameters.

Fig. 1 .

 1 Fig. 1. BER performance comparison using Ns = 8 training symbols

Fig. 2 .

 2 Fig. 2. The influence of the training sequence length on the BER performance

Fig. 3 .

 3 Fig. 3. Effect of the IA imperfection on the BER performance Ns = 8


Li-Ping P Sun

Mathilde Rieu
email: rieu@emse.fr

Jean-Paul P Viricelle

Christophe Pijolat

H Zhao

Fabrication and characterization of anode-supported single chamber solid oxide fuel cell based on La 0.6 Sr 0.4 Co 0.2 Fe 0.8 O 3-δ δ δ δ -Ce 0.9 Gd 0.1 O 1.95 composite cathode

Keywords: SOFC, Single Chamber, electrolyte microstructure, LSCF-CGO cathode

    Fabrication and characterization of anode-supported single chamber solid oxide fuel cell based on La0.6Sr0.4Co0.2Fe0.8O3-δ-Ce0.9Gd0.1O1.95 composite cathode

Introduction

Single-chamber solid oxide fuel cells (SC-SOFCs) have received many attentions in recent years, due to their simplified reactor configuration and capability for quick start-up [START_REF] Yano | Recent advances in single-chamber solid oxide fuel cells: a review[END_REF][START_REF] Zhang | Improving single chamber performance of an anodesupported SOFC by impregnating anode with active nickel catalyst[END_REF][START_REF] Liu | Effects of the single chamber SOFC stack configuration on the performance of the single cells[END_REF][START_REF] Akhtar | Silver modified cathode for a micro-tubular, single-chamber solid oxide fuel cell[END_REF][START_REF] Wei | A novel design of single-chember SOFC micro-stack operated in methane-oxygen mixture[END_REF]. They have potential applications as micropower generator for portable devices and co-generation of electricity and synthesis gases [START_REF] Masaya | A singlechamber SOFC stack operating in engine exhaust[END_REF][START_REF] Shao | Electric power and synthesis gas Co-generation from methane with zero waste gas emission[END_REF]. Because they operate on fuel-air mixture, one way to minimize the gas phase reaction that would reduce the electrical efficiency is to adopt fuels with high chemical stability such as hydrocarbons with low carbon numbers, especially methane; another way is to reduce the operation temperature. Usually the operating principle of SC-SOFC is based on the selective catalytic activities of the electrodes towards the fuel/oxygen mixture.

The anode must be aimed at the partial oxidation of fuel as well as electrochemically catalytic oxidation of H 2 and CO, while the cathode must be aimed at the oxygen electrochemically catalytic reduction. During the last years, a number of studies have been reported with the results of different kinds of electrode materials used in SC-SOFC. The most commonly used anode material is cermet with nickel and the electrolyte material and it has been proven to be as a very efficient catalyst for fuel [START_REF] Van R Ln | A novel Ni-Cermet electrode based on a proton conducting electrolyte[END_REF][START_REF] Larrondo | Methane partial oxidation using Ni/Ce 0.9 Zr 0.1 O 2 catalysts[END_REF][START_REF] Hibino | High performance anodes for SOFCs operating in methane-air mixture at reduced temperatures[END_REF]. For cathode material, LSM is one of the common cathode materials used in SC-SOFC. However, it is limited to high-temperature applications (> 800 o C), at which an acceptable performance can be realized [START_REF] Jasinski | Single chamber solid oxide fuel cell-investigation of cathodes[END_REF]. In addition to LSM, mixed ionic electronic conductors (MIEC) such as SSC, LSCF, BSCF are considered to be an ideal candidate because of their extended triple phase boundaries (TPBs) [START_REF] Ruiz De Larramendi | Development of electrolyte-supported intermediate-temperature single-chamber solid oxide fuel cells using Ln 0.7 Sr 0.3 Fe 0.8 Co 0.2 O 3-δ (Ln=Pr,La,Gd) cathodes[END_REF][START_REF] Hibino | A low-operating temperature solid oxide fuel cell in hydrocarbon-air mixtures[END_REF][START_REF] Shao | A high-performance cathode for the next generation of solid oxide fuel cells[END_REF][START_REF] Kuhn | Single-chamber micro solid oxide fuel cells: Study of anode and cathode materials in coplanar electrode design[END_REF][START_REF] Song | Phase stability of Sm 0.5 Sr 0.5 CoO 3 cathodes for on-planar type, single chamber, solid oxide fuel cells[END_REF]. Our previous work has shed some lights onto the above mentioned materials under methane/air mixture [START_REF] Rembelski | Characterization and comparison of different cathode materials for SC-SOFC: LSM, BSCF, SSC, and LSCF[END_REF]. The result showed that LSM is not suitable for SC-SOFC because of its very high polarization resistance, whereas SCC and BSCF are not stable under single chamber conditions during long time use.

LSCF has suitable conductivity and good stability under methane/air mixtures so it seems to be the most promising cathode material in SC-SOFC. Electrolyte is another important component of SC-SOFC. Initially, research in the SC-SOFC is focused on the Y 0.08 Zr 0.92 O 1.96 (YSZ) electrolyte cells [START_REF] He | Electrochemical properties of cathode material La 0.75 Sr 0.25 Mn 0.5 Mn 0.5 O 3 for single chamber solid oxide fuel cell 9SC-SOFC)[END_REF][START_REF] Jacques-Bédard | Performance and ageing of an anode-supported SOFC operated in single-chamber conditions[END_REF][START_REF] Hibino | Single chamber solid oxide fuel cell constructed from an Yttria-Stabilized Zirconia electrolyte[END_REF]. However, it requires high operating temperatures to obtain high ionic conductivity, thus resulting in several problems for the cells: undesirable chemical reactions between cell components, aging of components and thermal expansion mismatch, thus accelerating the decrease of the cell performance. To reduce the operating temperature, good ionic conductor such as La 0.8 Sr 0.2 Ga 0.8 Mg 0.2 O 3 (LSGM) and ceria doped with gadolinium (CGO) or samarium (CSO) are selected as electrolyte materials [START_REF] Hibino | Singel-chamber solid oxide fuel cells at intermediate temperatures with vatious hydrocarbon-air mixtures[END_REF][START_REF] Morales | Performance and short-term stability of singlechamber solid oxide fuel cells based on La 0.9 Sr 0.1 Ga 0.8 Mg 0.2 O 3-δ electrolyte[END_REF][START_REF] Morales | Intermediate temperature single-chamber methane fed SOFC based on Gd doped ceria electrolyte and La 0.5 Sr 0.5 CoO 3-δ as cathode[END_REF]. LSGM is not widely used as electrolyte, because it may present serious chemical reactions with electrodes. CGO or CSO seems to be the most promising electrolyte for SC-SOFC operating at intermediate temperatures (500-700 o C).

Among the various geometries of SC-SOFC, many investigations have shown that anodesupported cells could provide good performance due to the reducing of the electrolyte thickness and thus the ohmic losses [START_REF] Jacques-Bédard | Performance and ageing of an anode-supported SOFC operated in single-chamber conditions[END_REF][START_REF] Ai | Performance of anode-supported singlechamber solid oxide fuel cells[END_REF][START_REF] Piñol | Low temperature anode-supported solid oxide fuel cells based on gadolinium doped ceria electrolytes[END_REF]. Anode-supported cells exhibited the highest power output reported so far in SC-SOFC technology [START_REF] Shao | High power-density single-chamber fuel cells operated on methane[END_REF] and were operated at temperatures down to 200 o C [START_REF] Tomita | Single-chamber SOFCs with a Ce 0.9 Gd 0.1 O 1.95 electrolyte film for low-temperature operation[END_REF]. Moreover, since a fuel-air mixture gas is always used in SC-SOFCs, it is reasonable to prepare porous thin film electrolyte using low-cost fabrication methods such as screen printing and tape casting. Suzuki et al. prepared a porous YSZ electrolyte film with the thickness of 18 µm on NiO-YSZ anode substrate by screen printing [START_REF] Suzuki | Performance of a porous electrolyte in single-chamber SOFCs[END_REF]. The open porosity of the YSZ electrolyte film was about 23%. The observed OCV ranged from 680 to 780 mV, depending on the gas flow rate at 600 o C. For comparison, a cell with dense film electrolyte showed OCVs of more than 800 mV under the same conditions. The authors described this difference as due to the diffusing of the formed hydrogen and CO from the anode to the cathode through the porous electrolyte. Nevertheless, the peak power density reached about 200 and 600 mW cm -2 at gas flow rate of 300 and 900 ml min -1 , respectively. In our previous work, the anode-supported fuel cells were prepared with NiO-CGO anode pellets, screen printed CGO electrolytes and LSCF-CGO (LSCF/CGO=70/30) composite cathode. The thicknesses of the obtained porous film electrolyte were varied from 20µm to 50µm respectively. At 600 o C for R mix =0.6, the maximum power density was improved from 60 to 160 mW cm -2 [START_REF] Rembelski | In situ reduction and evaluation of anode supported single chamber fuel cells[END_REF].

Because of the complexity of the SC-SOFC, it is difficult to make a reasonable comparison with the reported results in the literature due to the discrepancy in materials, testing conditions and even cell dimensions (e.g electrode size). To further optimize the performance of the cell in our testing bench conditions, we present the recent results concerning anode-supported SC-SOFC with CGO electrolyte, Ni-CGO anode and LSCF-CGO cathode. In this study, a dense electrolyte is prepared through a dry-pressing/sintering process. The performance of the cell was compared with the one in previous work using porous electrolyte. The cathode composition, sintering parameters and testing conditions were also optimized to obtain a peak power density.

Experimental

Materials and Cell Preparation

All the materials used in the fuel cells are commercial powders from Fuel Cell Materials.

Gadolinium doped ceria Ce 0.9 Gd 0.1 O 2-δ (CGO) is used for the electrolyte, and also as one component in electrodes. La 0.6 Sr 0.4 Co 0.2 Fe 0.8 O 3-δ (LSCF) and NiO powders are selected as another composition in the electrodes.

The fuel cell employed in this study is in anode-supported thin-film electrolyte configuration.

The anode powder is a mixture of NiO (60wt%) and CGO (40wt%). A dry-pressing/sintering method was adopted in order to fabricate the anode-supported CGO bi-layer pellets. These green dual-layer pellets resulting from dry pressing were fired first at 500 o C for 2h, and then at 1200 and then homogenized and disagglomerated in a rolling mill. The resulting ink was screenprinted on the electrolyte surface of the fresh dual-layer membranes, and then calcined at 1000 o C, 1050 o C and 1100 o C for 2h, respectively. Three cathodes with effective geometric surface area of 0.36, 0.95 and 2.27 cm 2 respectively were prepared in order to evaluate the influence of the electrode area on the cell's power density. Finally, a gold mesh was screen-printed on the cathode with a commercial gold ink (ESL 8880-H) as the current collector. The typical thickness of the obtained anode-supported SC-SOFCs is about 1mm for anode, 150µm for electrolyte and 30µm for cathode.

Fuel cell performance test

The experimental set up is similar to the one that is described elsewhere [START_REF] Rembelski | In situ reduction and evaluation of anode supported single chamber fuel cells[END_REF]. An in situ reduction mode of the anode is employed in this work according to the findings by D. Rembelski et al. [START_REF] Rembelski | In situ reduction and evaluation of anode supported single chamber fuel cells[END_REF]. Generally before electrochemical performance test, 10% methane diluted in nitrogen For all the experiments performed in methane/air/nitrogen mixtures, oxygen is fixed at 10% (V/V) in order to avoid the flammability area of methane. The total gas flow is set at 30 L h -1 for cells test.

Characterization

The microstructures of the electrode and electrolyte were characterized using a scanning electron microscope (SEM, JEOL JSM-6500).

For the measurement of electrochemical properties, gold wire and gold mesh were applied as current collectors on both sides of the complete fuel cells. Polarization curves were obtained using a 4-probe technique with Keithley 2400 to eliminate the effect of the wire resistances. Two identical cells were prepared and measured separately. Each measurement was repeated three times and the mean value was used to evaluate the cell performance.

Results and discussions

Microstructure of the cell

Figure 1 shows the detail micrographs of the SC-SOFC after the measurements in the methane and oxygen mixed atmospheres. Obviously the cross-section of the cell consists of three layers, the Ni-CGO cermet anode, the CGO electrolyte and the LSCF-CGO (LSCF/CGO=70/30) composite cathode (Fig. 1a). The thickness of the electrolyte is about 150 µm, and the cathode is about 30 µm. The electrodes are well adhered on the electrolyte surface. Fig. 1b, c andd show the magnified view of the electrolyte, surface of the cathode and anode, respectively. It is observed that the CGO electrolyte prepared by dry pressing method exhibits a dense microstructure after sintered at 1400 o C for 2 hours (Fig. 1b). The morphology of LSCF powders is irregular with some agglomeration. The particle size are about 2-3 µm and the CGO particles are about 300-400nm, which are dispersed homogeneously on the LSCF particle surface (Fig. 1c). For the anode that is sintered at 1400 o C for 2 hours, a microstructure with moderate porosity and wellnecked particles was formed (Fig. 1d). The mean particle size is about 1 µm and slight aggregation of the particles was observed.

Performance of the cell

The effect of the electrolyte thickness

The test cells with cathode area of 0.36 cm 2 were investigated to optimize the electrolyte thickness. Figure 2 shows the I-V and I-P curves of the SC-SOFC cells. The measurement was performed at 600 o C under a mixing gas of R mix =2. The OCV was found to remain about 0.88 V and no significant change is observed, but the power density decreased dramatically with the increase of CGO thickness from 150 µm to 300 µm. The maximum power density of 260 mW cm -2 was obtained at CGO thickness of 150 µm. As the thickness increased to 230 and 300 µm, the maximum power density decreased to 178 and 162 mW cm -2 , respectively. As it is expected, the improvement of the SOFC performance is due to the reduction of electrolyte ohmic loss.

Currently, it is difficult to obtain an electrolyte thinner than 150 µm using the dry-pressing method. Therefore, in the following work, the thickness of the electrolyte was fixed at 150 µm and the influence of the cathode microstructure on the cell performance is further investigated.

The effect of the cathode sintering temperatures

It is well known that sintering temperature has remarkable influence on the microstructure of the electrode, which in turn has remarkable effects on the cell performance. There are two microstructure factors that govern the cell performance. One of them is the connectivity of the electrode particles. Obviously a well-developed connection among electrode particles will pave the way for electrons and oxygen ions that pass through the electrode and electrode/electrolyte interface. Porosity is another important factor, as the pores facilitate the gas diffusion and at the same time increase the number of active reaction sites. The effects of sintering temperatures on the output of cells are compared in Fig. 3. It can be seen, the power density increases with the sintering temperature of the cathode. The best performance is obtained when the sintering temperature of LSCF-CGO cathode is 1100 o C. This result can be explained by the microstructure evolution of LSCF-CGO cathodes with sintering temperature (Fig. 4). A loose agglomerated feature with inhomogeneous porous structure has been observed in the LSCF-CGO cathodes that were sintered at low temperatures such as 1000 and 1050 o C. The weak connection between particles increases the resistance of bulk/surface diffusion of oxygen species as well as electron transfer through the porous cathode. After sintering at 1100 o C for two hours, a microstructure with good connection between the electrode particles and moderate porous structure can be observed and thus results in the improved cell performance.

The effect of the cathode thickness

Three cells with respectively one, two and three layers cathode sintered at 1100 o C were prepared by screen printing method. The effect of cathode thickness on the cell performance at different current densities was investigated. The optimal power density was obtained for the cell with two layers cathode (Fig. 5). Further increase of the cathode thickness leads to the rapid decreaced of performance. Figure 6 gives the cross-section micrograph of the cells with one and two cathode layers after the test in the SC-SOFC measurement system. It can be seen that the thickness of one and two layers are about 15µm and 30µm, respectively. Unfortunately, the cathode with three layers was peered off after the test. Considering the good repeatability of screen-printing technique, we propose that the thickness of the three layers cathode is about 45 µm. Considering the combined effect of the number of reaction active site (RAS, which is propotional to the cathode thickness) and the gas diffusion depth in the porous cathode, a cathode with suitable thickness must ensure enough RAS, and at the same time to make sure that the reaction gas can penetrate through the cathode. With the increase of the cathode thickness however, the ohm resistance of the cathode increases, and the gas diffusion becomes difficult [START_REF] Kivi | Influence of cathode thickness on the oxygen reduction kinetics at the intermediate temperature SOFC cathodes[END_REF]. The combined effects result in optimized cell performance when the cathode thickness is ∼30 µm.

The effect of cathode composition

Figure 7 shows the OCV and power density of the cells with various cathode compositions as a function of current density. The power density increases with the CGO content, reaches a maximum at 30 wt% CGO, and then decreases with the further increase of CGO content. The OCV is about 0.9V and shows no dependance on the CGO composition. The effect of CGO contents on cell performance can be understood, considering the catalytic properties of the composite cathode. An increase of CGO content in LSCF may provide additional oxygen ions transfer path in the composite cathode, which inturn reduce the ion transfer resistance and improve the oxygen reduction reaction (ORR) [START_REF] Muffay | Electrochemical performance of (La,Sr)(Co,Fe)O 3 -Ce,Gd)O 3 composite cathodes[END_REF][START_REF] Dusastre | Optimization of composite cathodes for intermediate temperature SOFC applications[END_REF][START_REF] Qiang | Characterization of electrical properties of GDC doped A-site deficient LSCF based composite cathode using impedance spectroscopy[END_REF]. An further increase of CGO content however will interrupt the connection between the LSCF particles and decrease the conductivity [START_REF] Leng | Development of LSCF-CGO composite cathodes for lowtemperature solid oxide fuel cells with thin film GDC electrolyte[END_REF]. As a result, the performance begin to decrease as the CGO content is over 30wt% in the composite cathode.

The effect of testing temperature

The working temperature has significant effect on the cell performance. In the case of CGO electrolyte, for T > 700 o C, a sizeable electronic conductivity takes place. This causes a decrease of the OCV, thus reduce the power density. Considering these factors, a moderate testing temperature range 500-600 o C was chosen. In our experiment, the testing temperature refers to the furnace temperature (T f ). Due to the exothermic effect of methane oxidation reaction, as reported by Rembelski et al. [START_REF] Rembelski | In situ reduction and evaluation of anode supported single chamber fuel cells[END_REF] and Hibino and al. [START_REF] Hibino | A Solid oxide fuel cell using an exothermic reaction as the heat source[END_REF], the actual temperature on the electrode (T e ) is different from T f . Fig. 8 shows overheat temperatures (∆T= T e -T f ) on the anode cermet side and cathode side for different R mix gas system at set (furnace) temperatures. A 35-50 o C overheating is found for anode cermet as shown in Fig. 8a. At high temperatures (above 550 o C), the tendency of increasing overheat temperature with R mix is maintained. The overheating effect on cathode in Fig. 8b is smaller than that on cermet anode, and exhibits opposite tendency with R mix above 550 o C. At 500 o C, the ∆T on the cermet and cathode exhibits no obvious change with R mix .

The influence of the test temperature on the cell performance is shown in Fig. 9. It can be observed that the maximum power density increases from 121 to 260 mW cm -2 as the furnace temperature increases from 500 to 600 o C. At the same time, there is an apparent decrease in the OCV with the increase of the temperature. At 500 o C an OCV of 0.94V can be obtained. Upon raising the furnace temperature to 600 o C, the OCV decreases to 0.88V. This is due to the deteriorated selectivity of the electrodes and the promoted catalytic activity of the cathode towards methane chemical oxidation reaction with the increase temperatures.

The effect of gas composition

The effect of gas composition on the cell performance was studied under different operating temperatures from 500-600 o C. Fig. 10 shows the typical discharge profile of the cell prepared in optimal conditions and tested at furnace temperature of 600 o C under different ratios of methane to oxygen. A common trend has been observed in which R mix =2 gives the best and R mix =0.6

gives the worst cell performance. The similar results were obtained at 550 o C and 500 o C. The influence of the gas composition on the OCV is presented in Figure 11. At 500 o C, the OCV is almost unchanged with R mix . With the increase of the temperature, a tendency of slight increase of OCV with R mix can be observed. The maximum value of OCV is obtained at R mix =2, independent on furnace temperatures. As we know, R mix =2 corresponds to the stoichiometric composition of the gas mixture for the partial oxidation of methane according to the following reaction:

CH 4 + 1/2O 2 2H 2 + CO
This reaction is expected to occur on the anode catalyst followed by the complete electrooxidation of the syngas (H 2 +CO). With the decrease of R mix , the oxygen-rich mixture will enhance the non-electrochemical combustion of methane, reduce the oxygen partial pressure differential between the electrodes and hence the OCV. Similarly, fuel-rich mixtures will decrease the oxygen partial pressure at the cathode and therefore the cell voltage. Our observation (maximum OCV values appear at R mix =2) is consistent with previous studies of anode-supported SC-SOFCs including Ni-La 0.75 Sr 0.25 Cr 0.5 Fe 0.5 O 3-δ -YSZ/YSZ/LSM cells [START_REF] Zhu | Fabrication and evaluation of a Ni/La 0[END_REF],

Ni-YSZ/YSZ/LSM cells [START_REF] Jacques-Bédard | Performance and ageing of an anode-supported SOFC operated in single-chamber conditions[END_REF][START_REF] Napporn | Operation conditions of a singlechamber SOFC[END_REF], Pd-Ce 0.8 Sm 0.2 O 1.9 -Ni/ Ce 0.8 Sm 0.2 O 1.9 /Sm 0.5 Sr 0.5 CoO 3 cells [START_REF] Hibino | High performance anodes for SOFCs operating in methane-air mixture at reduced temperatures[END_REF],

and Ni-YSZ/YSZ/LSM-SDC cells [START_REF] Wei | Enhanced performance of a singlechamber solid oxide fuel cell with an SDC-impregnated cathode[END_REF]. This result is contrast to some reports in the literature where the porous electrolyte was used in SC-SOFCs [START_REF] Rembelski | In situ reduction and evaluation of anode supported single chamber fuel cells[END_REF]. It was proposed that the diffusion of hydrogen from the anode to the cathode through the pores electrolyte could lower the oxygen partial pressure at the cathode side and thus decrease the OCV. Therefore in their cases, OCV increases when R mix is reduced because of less hydrogen diffusion.

The effect of cathode area

Another important factor that has dramatic effect on the cell performance is the area ratio of cathode to anode. However few researchers take into account of this factor in the literature.

Chung and al. and Suzuki et al. reported that using a cathode with a geometric area significantly less than that of the anode can enhance the power density [START_REF] Chung | Influence of electrode configuration on the performance of electrode-supported solid oxide fuel cells[END_REF][START_REF] Suzuki | Anode supported single chamber solid oxide fuel cell in CH 4 -Air mixture[END_REF]. To illustrate the effect of cathode area, several test cells with different area ratio of cathode to anode have been prepared.

The anode area is fixed at 2.83 cm 2 and the cathode area (S) was 0.36 cm 2 , 0.95 cm 2 and 2.27 cm 2 , respectively. The ratio of cathode area to anode area (r c /r a ) is 0.13, 0.34 and 0.80. As it can be seen in Fig. 12, the power density increases gently with the decrease of r c /r a . The peak power density varies from 260 mW cm -2 to 210 mW cm -2 when the cathode area increases from 0.36 cm 2 to 2.27 cm 2 . This result is consistent with the relevant research of Chung et al. and Suzuki et al., but less severely attenuation of the power density was found in our cells as that observed in literature [START_REF] Chung | Influence of electrode configuration on the performance of electrode-supported solid oxide fuel cells[END_REF][START_REF] Suzuki | Anode supported single chamber solid oxide fuel cell in CH 4 -Air mixture[END_REF]. This can be attributed to the well optimized processing technology and the careful control of electrode microstructure, thus the effect of the area ratio on the normalized power density enhancement is less pronounced. Considering the real application in a stack, the area ratio of the cathode to anode should be as closer as to 1. Further comparison was made with our previous work using the r c /r a = 0.80. In the previous work, a test cell was prepared with porous electrolyte in 50 µm thickness (with cathode area 2.27 cm 2 ), and the peak power density reached 160 mW cm -2 at 600 o C for R mix =0.6. The current result shows an increase of 50 mW in the power density and the OCV improves from 0.67V to 0.88V. Although it is not obliged to use densified electrolyte in SC-SOFC (as it is always to be high-lighted as one of the merits in SC-SOFC), our result indicates that the electrolyte density is beneficial for improving the cell performance. This may be due to the improved ion conductivity in the densified electrolyte and at the same time the blocking effect of the direct diffusion of syngas from anode to cathode.

Further decrease in the thickness of the electrolyte in current cell would lead to much promising results.

Conclusions

Anode-supported fuel cells with NiO-CGO (60:40 wt%) anode, CGO electrolyte and LSCF-CGO cathode were fabricated and tested under methane-air mixtures. Preparation condition of the cathode, electrolyte thickness, and testing conditions were found to play strong roles in fuel cells overall behavior. In this work, the best processing condition is LSCF-CGO (70:30 wt%) cathode with 30 µm thickness and sintered at 1100 o C for 2h. A R mix = 2 gas mixture leads to improved performance and the maximum power density is 260 mW cm -2 at 600 o C. Electrolyte thickness is another important parameter for the SC-SOFC. The maximum power density increases from 162 mW cm -2 to 260 mW cm -2 when the electrolyte thickness decreases from 300µm to 150µm.

Figure captions

oC

  for 4h, 1400 o C for additional 2h in air, to achieve the densification of the electrolyte layer and good connection between the dual layers. The thickness of the electrolyte is controlled by adding different amount of CGO powders during pressing. The cathode powders (LSCF + CGO) with different compositions (50:50, 60:40, 70:30, and 80:20 wt%) were first mixed with a binder (ESL V400, 0.5g per gram of powder) and solvent (ESL T404, eight drops per gram of powder)

( 10 :

 10 90) is introduced into the reactor for 3 min and then switched to a mixed atmosphere of CH 4 -O 2 -N 2 (20:10:70) with R mix =2 for 15 min at 700 o C, to get the fully reduction of the NiO in the anode. The performances of the single cell are measured at furnace temperature between 500 and 600 o C. The ratio of methane to oxygen (R mix ) is varied from 0.6 to 2.0 as controlled by mass flow controllers (BROOKS Smart ⅡMass Flow).

Fig. 1 :

 1 Fig. 1: SEM images of cross-section view of Ni-CGO/CGO/LSCF-CGO cell (a), magnified CGO

2 Fig. 2 :Fig. 3 :

 223 Fig. 2: I-V and I-P curves for cells with different thickness of electrolyte test at 600 o C for

Fig. 4 :Fig. 5 :Fig. 6 :Fig. 7 : 2 Fig. 8 :Fig. 9 : 2 Fig. 10 : 2 Fig. 11 : 2 Fig. 12 :

 4567289210211212 Fig. 4: SEM images of top-view of LSCF-CGO composite cathode sintered at different temperatures for 2 hours: (a)1000 o C, (b) 1050 o C, (c)1100 o C .

Acknowledgement:

The project was supported by La Région Rhône-Alpes "CMIRA 2012 Accueil Pro" and National Natural Science Foundation of China (51302069 and 51372073), Natural Science foundation of Heilongjiang Province in China (JC201211, B201107).


Paul Alain Nana

Zéphyrin Fokam

Serge Hubert

Zébazé Togouet

Gideon Aghaindum Ajeagah

Geneviève Aghaindum

Philippe Bricheux

Télesphore Bouchard

Pierre Ngassam

Geneviève Bricheux

Philippe Bouchard

Télesphore Sime-Ngando

Distribution of Astomatia Schewiakoff, 1896 and Hysterocinetidae Diesing, 1866 (Ciliophora, Oligohymenophora) along the digestive tract of Alma emini (Oligochaete, Glossoscolecidae) is correlated with physico-chemical parameters

Introduction

Oligochaeta represent a major component of the soil macrofauna. They are grouped into three ecological categories: epigeic, anecic and endogeic [START_REF] Bouché | Lombriciens de France: ecologie et systématique[END_REF][START_REF] Bouché | Strategies lombriciennes. In: Soil organism as components of ecosystems[END_REF]. Alma emini which measures 51 cm on average and weighs 3.8 g is an anecic species belonging to the family of Glossoscolecidae. This fairly pigmented worm is found in the wet soil, near the less polluted rivers. Like all Oligochaeta, it is a hermaphrodite and creates more or less deep galleries, probably, in response to various constraints such as the content of food and water, the temperature or the degree of oxygenation [START_REF] Jégou | Burrowing activity of the earthworms Lumbricus terrestris and Aporrectodea giardi and consequences on transfers in soil[END_REF]. These galleries increase soil macroporosity and, consequently, contribute to its aeration [START_REF] Lavelle | Faunal activities and soil processes: adaptive strategies that determine ecosystem function[END_REF] and to water infiltration. They also facilitate root soaking in the soil as well as the movements of invertebrates [START_REF] Jégou | Impact of soil compaction on earthworm burrow systems using X-ray computed tomography: preliminary study[END_REF]. The role of A. emini in the formation, dynamics and fertility of soil has been long known [START_REF] Darwin | The formation of vegetable mould, through the action of worms, with observations on their habits[END_REF]. Besides its role of "the engineer" of the soil, A. emini is regarded as a microhabitat as its digestive tract lodges an important microbial fauna (protozoa, bacteria, and viruses). The protozoa are mainly represented by ciliates belonging to Heterotrichida Stein, 1859 [START_REF] Albaret | Etude systématique et cytologique sur les ciliés hétérotriches endocommensaux[END_REF][START_REF] Albaret | Description de cinq espèces nouvelles de ciliés hétérotriches des genres Metanyctotherus Albaret, Pronyctotherus n. gen. et Plagiotoma Dujardin[END_REF], Hysterocinetidae Diesing, 1866 [START_REF] Njiné | Morphogenèse de bipartition de deux espèces de ciliés Hysterocinetidae du genre Ptychostomum Stein, 1890, endocommensaux d'oligochètes Glossoscolecidae[END_REF][START_REF] Ngassam | Contribution à l'étude des ciliés hystérocinetidés : le genre Preptychostomum de Puytorac[END_REF][START_REF] Ngassam | Trois espèces nouvelles de ciliés astomes des genres : Almophrya de Puytorac et Dragesco, 1968, Maupasella Cépède[END_REF]Grain, 1997, 2000) and Astomatia Schewiakoff, 1896 (de [START_REF] Puytorac | Structure et ultra structure de quatre nouvelles espèces de ciliés Hysterocinetidae Diesing[END_REF]Puytorac, , 1969;;de Puytorac andDragesco, 1969a, 1969b;[START_REF] Ngassam | Trois espèces nouvelles de ciliés astomes des genres : Almophrya de Puytorac et Dragesco, 1968, Maupasella Cépède[END_REF][START_REF] Fokam | Trois espèces nouvelles de Coelophrya, ciliés astomes endocommensaux d'Alma nilotica (oligochète terricole) du Cameroun[END_REF][START_REF] Fokam | Révision de la sous-famille des Metaracoelophryinae de Puytorac, 1972 (Oligohymenophora: Hoplytophryida: Hoplytophryidae), ciliés astomes du tube digestif d'oligochètes terricoles d'Afrique: description de cinq espèces nouvelles[END_REF].

These studies demonstrated that several species of ciliates may be found simultaneously in the same worm, each of them living in a given compartment favorable to its development. Up to now, the reason of this stratification still remains unclear. Very few data were known on the living conditions of these endocommensal ciliates from the digestive tract of their host.

The aim of this study is to assess whether physico-chemical parameters (Hydrogen potential; Electric Conductivity; Total Dissolved Substance and Water Content) may influence the distribution considered as a set of natural microhabitats in which certain physico-chemical factors generate ecological niches suitable for one or another group of species.

Key words: Alma emini, Astomatia, endocommensal ciliates, electric conductivity, intestinal liquid, hydrogen potential, Hysterocinetidae, total dissolved substance, water content and abundance of ciliate species along the digestive tract of Alma emini.

Material and methods

COLLECTION AND IDENTIFICATION OF EARTHWORMS

Earthworms were collected on Sanaga River bank in Ebebda village, located between 11°30' and 11°50' of Eastern longitude and 4°00' and 4°30' of Northern latitude, 60 km north of Yaoundé-Cameroon (Central Africa) (Fig. 1). Worms were then identified according to the keys described by [START_REF] Sims | Earthworm[END_REF]. These worms were randomly divided into two batches for the assessment of physico-chemical parameters and abundance of ciliate species present in their digestive tract.

MEASUREMENTS OF PHYSICO-CHEMICAL PARAMETERS OF THE EARTHWORM'S INTESTINAL LIQUID

Once in the laboratory, the first batch of earthworms was carefully washed with tap water, and then fixed using formalin (10%). The digestive tract of each of these worms was then separated from the rest of its body and stretched on a filter paper. Once the blood and the coelomic liquid dried up, the intestine of worms was divided into three equivalent portions (fore-, mid-and hindgut) (Fig. 2). The content of each portion of the digestive tract was emptied in an earthenware dish by applying a slight pressure to the walls of the intestine, moving from the middle towards the extremities. The intestinal content was placed on a glass with very fine meshes (1-2 µm). The yellowish liquid was aspirated using a vacuum pump and collected in a flask. This technique, developed by [START_REF] Puytorac | Détermination de certaines des conditions écologiques propres aux différents Ciliés parasites du tube Paul Alain Nana et al. Address for correspondence[END_REF], is fast and allows the collection of three to four drops of the intestinal liquid deprived of any particles. In order to collect sufficient amount of the earthworm's digestive liquid for direct measurements of physico-chemical parameters, 15 earthworms were used for each series of experiment. Thirty tree series of identical experiments were performed during the whole study.

Paul Alain Nana et al.

Four physico-chemical parameters were assessed for each compartment of the digestive tract. The pH and Electric Conductivity (EC) were measured respectively by introducing the electrodes of a portable pH-meter (Shott Gerate CG 812, England) and electrodes of a portable conductimeter (Hanna series HT 8733, Germany) in a flask containing the intestinal liquid. The values of pH were expressed in conventional units and EC in micro Siemens per centimeter (µS/cm). The Total Dissolved Substace (TDS) and Water Content (WC) were evaluated before and after a complete evaporation of samples at 80°C in an oven (Gallenkamp, Germany). Weighs were recorded using a balance (Sartorius, France). Note that we measured WC of the total intestinal content and not of the intestinal liquid only, 33 earthworms were used during the study.

IDENTIFICATION AND ENUMERATION OF CILIATES

Worms of the second batch were cut alive in three compartments from the prostomium to the pygidium as above (fore-, mid-, and hindgut) (Fig. 2). Each portion was dilacerated in a petri dish (10 cm in diameter) containing 10-15 ml of mineral water (Volvic™, France). Ciliates found in these different portions of the earthworms were identified according to the keys previously described (de [START_REF] Puytorac | Structure et ultra structure de quatre nouvelles espèces de ciliés Hysterocinetidae Diesing[END_REF]Puytorac , 1969;;de Puytorac andDragesco 1969a, 1969b;[START_REF] Ngassam | Trois espèces nouvelles de ciliés astomes des genres : Almophrya de Puytorac et Dragesco, 1968, Maupasella Cépède[END_REF][START_REF] Njiné | Morphogenèse de bipartition de deux espèces de ciliés Hysterocinetidae du genre Ptychostomum Stein, 1890, endocommensaux d'oligochètes Glossoscolecidae[END_REF][START_REF] Ngassam | Contribution à l'étude des ciliés hystérocinetidés : le genre Preptychostomum de Puytorac[END_REF][START_REF] Ngassam | Trois espèces nouvelles de ciliés astomes des genres : Almophrya de Puytorac et Dragesco, 1968, Maupasella Cépède[END_REF]Grain, 1997, 2000;[START_REF] Fokam | Trois espèces nouvelles de Coelophrya, ciliés astomes endocommensaux d'Alma nilotica (oligochète terricole) du Cameroun[END_REF][START_REF] Fokam | Révision de la sous-famille des Metaracoelophryinae de Puytorac, 1972 (Oligohymenophora: Hoplytophryida: Hoplytophryidae), ciliés astomes du tube digestif d'oligochètes terricoles d'Afrique: description de cinq espèces nouvelles[END_REF]. They were sorted and counted under a binocular dissecting scope Wild M5 (Heerbrugg, Germany) at 250× magnification. This experiment was performed on 33 earthworms.

STATISTICAL ANALYSES

Correlation tests were used to assess the degree of binding between the physico-chemical parameters and ciliate abundance in different portions of digestive tract. Since our variables do not follow a normal distribution, we applied correlation test 'r' of Spearman to analyze our data. P-values were used to assess the degree of significance of correlation between physico-chemical parameters and ciliate abundance. P less than 0.05 were set as significant.

The means of various physico-chemical parameters in different portions of the digestive tract were compared using the Kruskal Wallis 'H' test. The 'U' Mann-Whitney test was used to compare the means of each parameter two by two. The criterion for significance was set at P<0.05. Values presented in the tables and figures are the mean ± standard deviation of the mean (sdm, n = 33).

Results

During this study, a total of 561 earthworms were dissected: 528 worms were used for measurements of physico-chemical parameters and 33 for studies of biodiversity of ciliates along the digestive tract.

PHYSICO-CHEMICAL VARIABLES

The pH varied from 6.22 ± 0.43 in the foregut, 7.13 ± 0.17 in the midgut, and 7.35 ± 0.16 in the hindgut (Fig. 3A). Thus, the acid pH in foregut of the digestive tract of earthworms became alkaline in its mid and the hind portions. The average pH of the whole intestinal liquid of the worm was close to neutrality (6.90 ± 0.25).

The mean value of EC in the fore-, mid-and hindgut were 97.51 ± 11.18 µS/cm, 84.55 ± 12.94 µS/cm, and 66.22 ± 8.60 µS/cm respectively (Fig. 3B). Then, the greatest ionic concentration was obtained in the foregut.

The TDS was on average 11.38 ± 2.41% in the foregut, 16.20 ± 3.46% in the midgut, and 10.75 ± 3.76% in the hindgut (Fig. 3C). Globally, the mean value of this parameters 12.77 ± 3.21%.

The WC decreased gradually from the foregut to the hindgut, with average values of 46.94 ± 7.77% in the foregut, 39.27 ± 5.05% in the midgut, and 28.84 ± 3.97% in the hindgut (Fig. 3D).

The Kruskal Wallis 'H' test appeared significant for the whole of the measured parameters (P<0.05) (Table 1). Thus, for each variable, the Mann Whitney 'U' test showed a significant difference among the three portions of the digestive tract of A. emini taken two by two: foregut and midgut (P<0.05), foregut and hindgut (P<0.05), midgut and hindgut (P<0.05) (Table 2).

CILIATE BIODIVERSITY

Twenty three species belonging to nine genera of ciliates were found during this study. Twelve species belonged to the Subclass of Astomatia: Almophryra bivacuolata de Puytorac and Dragesco, 1969b; Almophryra mediovacuolata [START_REF] Ngassam | Trois espèces nouvelles de ciliés astomes des genres : Almophrya de Puytorac et Dragesco, 1968, Maupasella Cépède[END_REF]; Almophrya laterovacuolata de Puytorac and Dragesco, 1969b; Dicoelophrya almae de Puytorac and Dragesco, 1969b Among the 762 specimens of Astomatia recorded in the digestive tract of Alma emini, 462 were found in their foregut, 264 in their midgut and 36 in their hindgut. The abundance of Astomatia then significantly decreased gradually along the digestive tract of earthworms (Table 3).

The Hysterocinetidae ciliates were mostly found in the hindgut (128 specimen), while they were absent in the foregut and only 86 were found in the midgut. We noted however, the existence of a buffer medium in the midgut where Hysterocinetidae and Astomatia (Dicoelophrya almae, Dicoelophrya mediovacuolata) ciliates dwelled together. In addition, we noted an effective cohabitation among species of the same genus (Almophryra bivacuolata, Almophryra mediovacuolata and Almophryra laterovacuolata; Coelophrya ebebdensis and Coelophrya roquei; Ptychostomum prolixus and Ptychostomum commune) (Table 3).

CORRELATION BETWEEN THE RELATIVE ABUNDANCE OF CILIATES AND THE PHYSICO-CHEMICAL PARAMETERS OF THEIR HOST

Table 4 displays the relationship between the ciliate abundance and physico-chemical parameters of the three portions of the digestive tract of their host.

In the foregut, a positive and significant correlation was found between the abundance of the ciliates Metaracoelophrya intermedia (r = 0.694; Notes: * -correlation is signifi cant at the 0.05 level; ** -correlation is signifi cant at the 0.01 level; "-" -no value; EC -electric conductivity; pH -Hydrogen potential; TDS -total dissolved substances; WC -water content. P<0.05),Coelophrya roquei (r = 0.628; P<0.05) and WC.

In the midgut, a negative and significant correlation was observed between the number of the ciliates Metaptychostomum ebebdae (r = -0.717; P<0.05), Ptychostomum macrostomum (r = -0.725; P<0.05) and EC. In the same compartment, a negative and significant correlation was also observed between the abundance of Ptychostomum macrostomum and TDS (r = -0.619; P<0.05).

In the hindgut, a positive and very significant correlation was found between the values of the pH and Ptychostomum macrostomum abundance (r = 0.755; P<0.01). A significant and positive correlation was also recorded between the number of Ptychostomum commune counted in the hindgut and the WC of the digestive tract of the latter (r = 0.682; P<0.05), while the correlation between the abundance of Coelophrya roquei and the pH was negative and significant (r = -0.669; P<0.05).

Discussion

The present paper aimed to assess whether the physico-chemical parameters (pH, EC, TDS and WC) of Alma emini's digestive tract may influence the distribution and abundance of ciliate species.

The pH of the digestive tract of A. emini, collected in acidic soil of Sanaga River bank [START_REF] Fokam | Biodiversité des ciliés astomes, endocommensaux d'oligochètes terricoles de la région de Yaoundé[END_REF], increased from the foregut to the hindgut. These results are in accordance with previous experiments carried out on the oligichaetes Allolobophora savignyi and Lumbricus herculeus collected in alkaline environment where similar variation of the pH of the digestive tract was observed [START_REF] Simm | Verdauungsvorgange bei reifen und knospenden Würmern der Guttung Chaetogaster[END_REF][START_REF] Krieg | Die Ernithrung des Regenwurms[END_REF][START_REF] Puh | Digestive enzyme actions in the gut of the earthworm[END_REF][START_REF] Puytorac | Détermination de certaines des conditions écologiques propres aux différents Ciliés parasites du tube Paul Alain Nana et al. Address for correspondence[END_REF], suggesting that the pH of the habitat of the earthworm might not affect this parameter.

The gradual reduction in the total content of ions (TDS and EC) in the digestive tract of A. emini might be due to the action of deionizing bacteria present in the digestive tract. Indeed, some authors reported the presence of a strong and much diversified bacterial community in the digestive tract of oligochaetes [START_REF] Hyun-Jung | Analysis of aerobic and culturable bacterial community structure in earthworm (Eisenia fetida) intestine[END_REF][START_REF] Brito-Vega | Bacterial diversity in the digestive tract of earthworms (Oligochaeta)[END_REF]. The progressive decrease of the values of EC observed is in good accordance with the maximum concentration of ions (chloride, sulfate, calcium, potassium, and sodium) observed in the foregut [START_REF] Maluf | Osmo and volume regulation in the earthworm with special reference to the alimentary tract[END_REF] and detection of a great number of denitrifying organisms in the digestive tract of Lumbricus rubellus, L. terrestris and Aporrectodea caliginosa [START_REF] Depkat-Jakob | Effect of earthworm feeding guilds on ingested dissimilatory nitrate reducers and denitrifiers in the alimentary canal of the earthworm[END_REF].

Regarding TDS in the three compartments of the digestive tract of A. emini, it seems to be concentrated in the first two parts (fore-and midgut), and to be assimilated in the hindgut, thus explaining its reduction in this latter portion of the intestine as suggested by [START_REF] Puytorac | Détermination de certaines des conditions écologiques propres aux différents Ciliés parasites du tube Paul Alain Nana et al. Address for correspondence[END_REF].

We observed a progressive decrease, from the foregut to the hindgut, in WC. Such gradient of fluidity had already been reported by [START_REF] Maluf | Osmo and volume regulation in the earthworm with special reference to the alimentary tract[END_REF], and then by [START_REF] Puytorac | Détermination de certaines des conditions écologiques propres aux différents Ciliés parasites du tube Paul Alain Nana et al. Address for correspondence[END_REF] in Lumbricus terrestris and Allolobophora savignyi collected in various fields. This parameter seems to be influenced by the habitat or environment (porosity of the soil), and the physiological status of the worm, as previously reported by [START_REF] Edwards | Earthworm ecology[END_REF] and [START_REF] Lavelle | Soil ecology[END_REF].

In the digestive tract of A. emini, astomes, in their great majority, proliferated in the acid foregut, rich in mineral elements and in fluid. Besides, we noted that the ciliates inhabiting the anterior part of the digestive tract were very mobile. In contrast, in the posterior portion, populated by Hysterocinetidae and characterized by alkaline pH and low content of mineral substances and water, ciliates were attached to the intestinal wall by their sucker. Along the same lines, de [START_REF] Puytorac | Détermination de certaines des conditions écologiques propres aux différents Ciliés parasites du tube Paul Alain Nana et al. Address for correspondence[END_REF] had already shown stratification of Allolobophora savignyi related to the physico-chemical variables. These results reveal high affinity between ciliates and physicochemical variables prevailing in their respective biotope. In general, the ciliate abundance in each portion of the digestive tract of A. emini was variable, according to the conditions of the medium.

It is important to notice that the stratification of cells is observed not only in the digestive tract of earthworms. [START_REF] Gohre | Untersuchungen uber den plasmatischen Feinbau der Gregarinen mit besonderer Berucksichtitung der Sexualitatsverhaltnisse[END_REF] reported similar trend in three species of the genus Gregarina, parasites of the mealworm (Tenebrio molitor). [START_REF] Adam | The quality and distribution of the ciliate protozoa in the large intestine of the horse[END_REF] also showed that two successive ciliate fauna can respectively be found in the coecum and the rectum of the large intestine of horses.

Therefore, it appears that the stratification of ciliate species in the digestive tract of a given host might be largely associated with the physicochemical parameters of this environment and to a lesser extent with the biotic conditions of the habitat of these hosts.

It is also important to notice that the parameters assessed in our study might not be the only factors affecting the abundance of ciliates. Some authors have not detected enzymes (cellulase and chitinase) in the pharynx, the esophagus, the jabot and the gizzard of many worms, but registered them in the anterior part of the intestine [START_REF] Tracy | Cellulase and chitinase of earthworms[END_REF][START_REF] Laverack | The physiology of earthworms[END_REF][START_REF] Urbasek | Cellulase activity in the gut of some earthworms[END_REF]. If Hysterocinetidae (Ptychostomum Paul Alain Nana et al.

prolixus, Ptychostomum commune, Ptychostomum macrostomum, Ptychostomum elongatum, Ptychostomum variabilis, Proptychostomum commune, Proptychostomum simplex) thus seem to be able to spare from such diastases, it is possible that Astomatia (Almophryra bivacuolata, Almophryra mediovacuolata, Almophrya laterovacuolata, Coelophrya ovales) have reversely great need for them. A similar case has been demonstrated in Diplodinium and Entodinium species of the paunch of ruminants. The first have cellulase and cellobiase which enables them to transform cellulose into glucose whereas the second are deprived of it [START_REF] Tracy | Cellulase and chitinase of earthworms[END_REF].

Variation of physico-chemical parameters and ciliate species along the digestive tract of earthworms suggests that passage of soil in digestive tract would influence not only physical and chemical properties of soil, but also its microbial biomass, as was previously suggested by [START_REF] Pedersen | Effect of passage through the intestinal tract of detritivore earthworms (Lumbricus spp) on the number of selected Gram-negative and total bacteria[END_REF][START_REF] Fischer | In situ analysis of the bacterial community in the gut of the earthworm Lumbricus terrestris L. by whole-cell hybridization[END_REF][START_REF] Lavelle | Faunal activities and soil processes: adaptive strategies that determine ecosystem function[END_REF], [START_REF] Houjian | Fate of Protozoa transiting the digestive tract of the earthworm Lumbricus terrestris L[END_REF] and [START_REF] Depkat-Jakob | Effect of earthworm feeding guilds on ingested dissimilatory nitrate reducers and denitrifiers in the alimentary canal of the earthworm[END_REF]. The density of soil nematodes, protozoa and coliformes also changes after its transit through the intestine of the epigeic earthworms, providing further evidence in favor of this hypothesis [START_REF] Monroy | Changes in density of nematodes, protozoa and total coliforms after transit through the gut of four epigeic earthworms (Oligochaeta)[END_REF]. Falling into the line, the passage of soil through the digestive tract of earthworms could stimulate [START_REF] Brito-Vega | Bacterial diversity in the digestive tract of earthworms (Oligochaeta)[END_REF] or inhibit [START_REF] Byzov | Fate of soil bacteria and fungi in the gut of earthworms[END_REF] the growth of microorganisms and mineralizing bacteria.

Conclusion

The present study reveals that the digestive tract of oligochaetes in general and that of A. emini in particular is a set of biotopes with specific physical and chemical parameters responsible for ecological conditions which can favor the development of a particular ciliated fauna. Contrary to Hysterocinetidae, Astomatia mostly proliferate in the acid foregut, rich in mineral elements and fluid. Despite the quite relevant information provided by our study, other factors influencing the distribution of ciliates in their host still remain to be assessed. The importance of various parameters of the medium differs and depends on the particular organism or a group of organisms (its sensitivity to the particular factor) and the amplitude of variations these factors undergo. In the case of endocommensal ciliates of A. emini, each parameter has certainly an essential and primordial action. The greatest concentration of cells of each group (Astomatia and Hysterocinetidae) seems to occur in the fore and hindgut, correspondingly. Nevertheless, specimens of these two groups can be observed in the midgut, qualified as a buffer medium, where tolerant species occur. Each of these species is probably more sensitive to the quality and quantity of substances present in the medium. It is also necessary to consider predation, rate of oxygen, osmotic pressure, nutrients and ions, specific enzymes, interaction with ciliates, bacteria and viruses in digestive tract of A. emini in further research.

Fig. 1 .

 1 Fig. 1. A map showing collection site for earthworms.

Fig. 2 .

 2 Fig. 2. Diagram of the digestive tract of earthworms (from Horn et al., 2003, modified).

Fig. 3 .

 3 Fig. 3. Variation of physico-chemical parameters along the digestive tract of Alma emini. A -Hydrogen potential (pH), B -electric conductivity (EC), C -total contents in dissolved substances (TDS), Dwater content (WC).

  Notes: m -mean; sd -standard deviation. Paul Alain Nana et al. Correlation between ciliate abundance and physico-chemical parameters in the different portions of the digestive tract.

Table 1 .

 1 Overall comparison of the different physicochemical parameters in the different portions of the digestive tract*.

	Parameters	pH	EC	TDS	WC
	P-value	0.010	0.047	0.022	0.034

Notes: * -correlation is signifi cant at the 0.05 level; EC -electric conductivity; pH -Hydrogen potential; TDS -total dissolved substances; WC -water content.

;

  Dicoelophrya mediovacuolata[START_REF] Fokam | Révision de la sous-famille des Metaracoelophryinae de Puytorac, 1972 (Oligohymenophora: Hoplytophryida: Hoplytophryidae), ciliés astomes du tube digestif d'oligochètes terricoles d'Afrique: description de cinq espèces nouvelles[END_REF]; Paracoelophrya intermediade Puytorac, 1969; Paracoelophrya polymorphus Fokam et al., 2012; Paracoelophrya ebebdensis Fokam et al., 2012; Metaracoelophrya intermediade Puytorac and Dragesco, 1969a; Coelophrya roqueide Puytorac and Dragesco, 1969b; Coelophrya ovales Fokam et al., 2008; Coelophrya ebebdensis Fokam et al.,

Table 2 .

 2 Values of the 'U' Mann-Whitney test*.

	Parameters	Foregut	Midgut	Hindgut
	pH	0.033	0.037	0.030
	EC	0.024	0.020	0.019
	TDS	0.027	0.035	0.031
	WC	0.021	0.032	0.021

Notes: * -correlation is signifi cant at the 0.05 level; EC -electric conductivity; pH -Hydrogen potential; TDS -total dissolved substances; WC -water content. • 197 Protistology 2008, while the eleven others were Hysterocinetidae: Metaptychostomum ebebdae Ngassam and Grain, 1997; Metaptychostomum pirimorphus Ngassam and Grain, 2000; Ptychostomum sanagae Ngassam and Grain, 2000; Ptychostomum prolixus

[START_REF] Njiné | Morphogenèse de bipartition de deux espèces de ciliés Hysterocinetidae du genre Ptychostomum Stein, 1890, endocommensaux d'oligochètes Glossoscolecidae[END_REF] Ptychostomum commune de Puytorac, 1968

; Ptychostomum macrostomum

[START_REF] Njiné | Morphogenèse de bipartition de deux espèces de ciliés Hysterocinetidae du genre Ptychostomum Stein, 1890, endocommensaux d'oligochètes Glossoscolecidae[END_REF]

; Ptychostomum elongatum

[START_REF] Njiné | Morphogenèse de bipartition de deux espèces de ciliés Hysterocinetidae du genre Ptychostomum Stein, 1890, endocommensaux d'oligochètes Glossoscolecidae[END_REF] Ptychostomum variabilis Ngassam and Grain, 2000; Proptychostomum commune Ngassam and Grain, 1997; Proptychostomum simplex Ngassam and Grain, 1997; Preptychostomum microstomum Ngassam et al., 1993.

Table 3 .

 3 Species richness and variation of ciliates abundance along the digestive tract of A. emini.

				Digestive tract
		Species
			Foregut (m ± sd)	Midgut (m ± sd)	Hindgut (m ± sd)
		Almophryra bivacuolata	58 ± 11	25 ± 4	0
		Almophryra mediovacuolata	73 ± 10	28 ± 5	0
		Almophrya laterovacuolata	14 ± 3	7 ± 1	0
	Astomatia	Dicoelophrya almae Dicoelophrya mediovacuolata Paracoelophrya intermedia Paracoelophrya polymorphus	0 0 48 ± 8 33 ± 6	23 ± 7 20 ± 5 27 ± 5 19 ± 4	5 ± 2 7 ± 2 3 ± 1 1 ± 0
		Paracoelophrya ebebdensis	56 ± 7	34 ± 7	18 ± 7
		Metaracoelophrya. intermedia	32 ± 6	18 ± 8	1 ± 0
		Coelophrya roquei	62 ± 4	24 ± 3	1 ± 0
		Coelophrya ovales	27 ± 4	16 ± 3	0
		Coelophrya ebebdensis	59 ± 9	23 ± 6	0
		Metaptychostomum ebebdae	0	17 ± 3	6 ± 2
		Metaptychostomum pirimorphus	0	14 ± 4	7 ± 2
	Hysterocinetidae	Ptychostomum sanagae Ptychostomum prolixus Ptychostomum commune Ptychostomum macrostomum Ptychostomum elongatum	0 0 0 0 0	13 ± 4 2 ± 1 3 ± 1 10 ± 2 0	2 ± 1 12 ± 4 17 ± 5 13 ± 3 12 ± 3
		Ptychostomum variabilis	0	4 ± 2	20 ± 6
		Proptychostomum commune	0	0	11 ± 3
		Proptychostomum simplex	0	4 ± 2	22 ± 3
		Preptychostomum microstomum	0	19 ± 6	6 ± 3

Acknowledgements

We thank Dr. Arnaud Kengmo Tchoupa (University of Constance, Germany) and Dr. Marie Alfrede Mvondo (University of Dschang, Cameroon), for the critical revision of this manuscript.