Identification of Better Gene Expression Data for Mosquito Species Classification Using Radial Basis Function Network Methodology

1. INTRODUCTION

Exploration of bioinformatics has developed hastily in recent years due to the advances in molecular biology techniques. One of the most vital research interests in computational biology is sequence mining. It is more difficult to extract knowledge hidden in the sequences than to generate biological sequences. Models and data analysis has become a crucial aspect of biological sequence mining. Several tasks related to sequence mining such as pattern discovery, classification, prediction and clustering can be carried out by statistical, neural network or data mining models [1-5]. Such techniques can be used to capture the knowledge or patterns in order to predict, classify or analyze the sequence data. However, the phenomenal growth of sequence data has made the database search computationally intensive and even more forbidding. Gene sequences in DNA are augmented with huge amount of information, but the intricacy and complexity of this information makes difficult in analyzing it by using standard classical methods a challenging task.

Genetic level analysis for identification of the species in mosquito is very crucial as the species act as a vector for several diseases such as malaria, filarial, Japanese encephalitis and dengue. Among these diseases, burden of malaria is fairly high and results in heavy toll of life. Anopheles mosquito species act as a vector for malaria which is widely spread throughout the globe [6-8]. Among 3,500 species of mosquitoes, nearly 430 are Anopheles in which around 30-40 species act as a vector for malaria. Aedes is a genus of mosquito originated in tropical and subtropical zones, but it has spread through human activity to all continents with the exception of Antarctica [9]. The genus of Aedes contains over 700 species. Many of the Aedes species transmit potentially serious human diseases. Culex is a genus of mosquito in which several species serve as vectors of diseases. The genus of Culex has widespread geographical distribution [10] and consists of about 767 species. Classical methods of distinguishing species through genetic analyses have reduced dependence on error-prone morphological and anatomical bases of classification [11]. In the taxonomic classification it is quite difficult to distinguish the sibling species that bear similar morphological and anatomical features but differ at the genetic level. Therefore, for quick identification of a mosquito vector, it is always possible to target a conserved sequence at the genetic level, which is pretty species specific and shows substantial difference even among the sibling species. Internal Transcribed Spacer (ITS) region is widely used in taxonomy and molecular phylogenetics [12, 13]. The ITS2 region located between the 5.8S and 28S gene is highly conserved and species specific. This region is commonly used for DNA sequencing in mosquito genera of Anopheles, Culex, and Aedes [14]. The ITS2 region has been proven useful for differentiating between closely related species of mosquitoes [15-17]. This region has also been extensively targeted for species classification, phylogenetic and RNA structure-related analysis [18, 19].

More precise and efficient computational tools are needed to extract genetic level information in the gene sequences and to distinguish mosquito species with minimum or no misclassification. Artificial Neural Networks (ANNs), as one of the Artificial Intelligence (AI) methods, play important role in scientific investigations. ANNs have been used as prediction and classification tools for several applications in the field of bioinformatics including protein structure prediction [20, 21], DNA sequence analysis and biological pattern recognition [22-24], RNA structure related analysis [13, 16, 19] and prediction of bacterial promoter sequences [25-27]. Recently, ANNs are also used for species identification in mosquitoes [28-30]. The wide use of ANNs for prediction and identification is due to their ease of training and flexibility to process high amounts of information with good generalization ability. Although, ANN are widely used for classification and identification problems, most of the ANN models aim at finding the network parameters using a fixed network configuration. It may not be possible to train the ANN to reach a desired level of performance if the network does not have enough computational units, or if the learning algorithm fails to find the optimal network parameters. Therefore, automatic configuration of the network while establishing optimal network parameters is extremely useful in classification and identification of problems. In this work, a Radial Basis Function Network (RBFN) methodology with automatic network configuration is presented for the identification of mosquito species based on the genetic pattern information content of ITS2 ribosomal DNA sequences. A number of data sequences in varying sizes of different vectors corresponding to Anopheline, Aedes and Culex are used to develop genera specific RBFN species identifiers. Further, a comprehensive RBFN species identifier is also presented for mosquito species identification of more genera. The recall and generalization ability of the RBFN species identifiers are analyzed and further validated through bootstrap validation method.

2. DATA SELECTION AND ITS SIGNIFICANCE

Precise identification of the target species of mosquito has direct medical and practical implications, particularly in developing vector control strategies. Among the vector species of mosquito born diseases, the species belonging to the genera of Anopheles, Aedes and Culex are reported to be involved in the transmission of a variety of vector-borne diseases. Prioritizing the need for detecting the species that act as vectors for several diseases, the present work is targeted on the identification of species of mosquitoes of these three genera.

3. RBFN SPECIES IDENTIFICATION METHODOLOGY

In mosquitoes, because of the interspecific variability and intraspecific homogeneity in spacer sequences, the species cannot be easily distinguished by just looking at the sequence information. The development of an efficient species classifier is required to identify the closely related species of mosquitoes. Thus, an RBFN classifier with its automatic configuration is developed for the identification of mosquito species based on the gene sequence information contained in the conserved structure of the transcribed spacer, ITS2 region. The RBFN classifier presented in this work is used to distinguish and identify the species of Anopheles, Aedes and Culex genera. Radial Basis Function Network (RBFN) has been considered as viable tool for applications in systems modeling and state estimation [31-35]. The RBFN architecture is simple and consists of one input layer, one hidden layer and one output layer, the structure of which is shown in Fig. (1). The RBFN algorithm along with its automatic configuration is given in Appendix A.

3.1. Configuring RBFN for Species Identification

In this work, RBFN is configured for mosquito species identification using the data of ITS2 ribosomal DNA sequences corresponding to different mosquito vectors. The automatic configuration of the network is carried out by using a Hierarchically Self-Organizing Learning (HSOL) algorithm. The RBFN used for mosquito species identification has a three layered architecture with input, hidden and output layers, each layer comprising its own nodes, as shown in Fig. (2). All nodes in the input layer are connected to the hidden layer nodes, and connections exist between the hidden and output layer nodes. The input layer represents the sequence data, the hidden layer processes the nonlinear information, and the output layer depicts sequence classes. The nodes in the input layer do not perform any numerical processing; all numerical processing is done by the hidden and output layer nodes. The number of nodes in the input layer is dictated by the chosen sequence encoding schema and the number of the output layer nodes is determined by the species classes representing the network. The nodes in the hidden layer are automatically configured by the HSOL algorithm. The other network parameters that are involved in configuring the RBFN are: the learning rate η, error margin ε_m, initial nominal variance σ_o, initial effective radius r_o, lower bound on radius r_l, radius decrement rate r_d, decay factor for error gradient α, and increment rate for saturation criterion β.

4. RESULTS AND DISCUSSION

Individual RBFN models are developed for anopheles, aedes and culex genera using the input and output data corresponding to the species of these genera. The presentation of data to RBFN is briefed in the earlier section. The RBFN parameters are set as: learning rate (η) = 0.00025, error margin (ε_m) = 0.001, initial nominal variance (σ_o) = 0.0021, initial effective radius (r_o) = 5.0, lower bound on radius (r_l )= 0, radius decrement rate (r_d) = 0.995, decay factor for error gradient (α) = 1.0 x 10^-09, and increment rate for saturation criterion (β) = 1.0 x 10^-05. For network training, all the data sets representing the species inputs and outputs are sequentially employed in each iteration and iterations are performed until the network is optimally configured via HSOL algorithm. The number of iterations required for convergence, number of RBFs generated, training error evaluated and training time required for each of the networks are given in Table 3. The trained and learned RBFNs are then subjected to assess their recall and generalization abilities. In the recall phase, the prediction ability of the trained networks is evaluated by using the same input sequences as used for training. Each of these RBFNs has shown almost 100% recall ability. The generalization performance of the networks is evaluated by using the species sequences that are not involved in training. This is referred here as conventional cross validation. Thus, 15 data sequences corresponding to the species of anopheles genera, 10 data sequences corresponding to the species of each aedes and culex genera, are used to test the generalization ability of the individual RBFN models. The comparison of the model predictions with the actual output species representations in Figs. (3-5) shows the effective generalization performance of the individual RBFN species identifiers. The generalization ability of the RBFN models is further assessed in terms of Mean Squared Error (MSE) and correlation coefficient (R²) defined in Appendix B. These results in Table 4 further demonstrate the efficiency of the RBFN methodology for accurate identification of mosquito species based on the nucleotide data of ITS2 ribosomal DNA sequences.

A comprehensive RBFN model is also developed for species identification of anopheles, aedes and culex genera using the input sequence data in binary coding and output species representation in real coding as given in Table 2. The combined RBFN structure is similar to that of genera specific RBFN structure in Fig. (2) with multiple input nodes and single output node. The parameters specified for this RBFN structure are same as in genera specific model structure. The data representation as well as training of this comprehensive network is briefed in the earlier section. In contrast to the genera specific RBFN, this comprehensive RBFN model involves sequential treatment of all the input data sequences representing the species of the three genera along with their respective species output coding. Thus, the data sets in Table 2 are sequentially employed and iterated until the network is optimally configured by HSOL algorithm. The number of iterations required for convergence, number of RBFs generated, training error obtained and training time required for this comprehensive network are given in Table 3. The entire code was written and executed using C language. The computer configuration used is Intel(R) Core ™ i7-4702MQ CPU @ 2.20GHz 2.20 GHz and the system type is 64-bit Operating System, x64-based processor.

The trained and learned comprehensive RBFN is further studied through conventional cross validation to assess its recall and generalization ability. The comparison of this combined network model predictions with the actual species representations for the recall and generalization phases are shown in Figs. (6 and 7). The generalization performance of the combined network is further evaluated in terms of MSE and R² as given in last line of Table 4. These results exhibit better performance of the comprehensive model for species identification of mosquito genera.

Model validation is an important step in ascertaining the reliability of models before they can be used in decision making. The predictive performances of trained and learned RBFNs are further assessed by using bootstrapping procedure. This procedure enables to validate the full n-subject model. The RBFN parameters of individual and combined models for this approach are kept same as in earlier model configurations. According to this bootstrap, the Anopheles genera net is validated by performing 9 rounds of bootstrapping with the use of all 45 gene sequences. In each round, 40 data sequences corresponding to the species of Anopheles genera are randomly assigned for training while the remaining 5 data sequences are assigned for validation. Thus, the model in each round is assessed through training and validation. The same boot strapping procedure is employed to assess the performance of other genera specific RBFN models and the comprehensive RBFN model. The number of iterations required for convergence, number of RBFs generated, training error obtained and training time required for the genera specific networks and comprehensive network of this bootstrap procedure are found to be nearly same to that of trained and learned RBFN models used in recall and generalization phases. The model validation results of MSE and R² evaluated through the bootstrap approach to assess the predictive accuracy of the fitted models are shown in Table 4. These results confirm the predictive accuracy of the RBFN models developed in this study.

Recent studies have shown the effectiveness of intelligent computational tools such as Artificial Neural Networks (ANN) for rapid and reliable identification of mosquito species. The genetic data base of mosquitoes has been exploited by such methods to differentiate even closely related species of mosquitoes. Banerjee et al (2008) [29] presented a multilayered feed-forward neural network model structure for classification and identification of mosquito species based on the Internal Transcribed Spacer 2 (ITS2) data of ribosomal DNA string. Two network models, namely, a Multi-Input Single-Output Neural Network (MISONN) and Multi-Input Multi-Output Neural Network (MIMONN) have been configured in which the MISONN was found to provide effective performance in distinguishing and identification of mosquito species. However, the above reported work was confined to the analysis of species of single mosquito genera only based on the data of 18 species of Anopheles mosquito vectors. The above neural network modeling approach has been further extended by Venkateswarlu et al. (2012) [30] for classification and identification of species of more mosquito genera by presenting a Hierarchical Artificial Neural System (HANS) in two levels, in which the first level has a single network that serves as a genera classifier and the second level has multiple networks that perform as species identifiers. Ribosomal DNA sequence data of 34 species of Anopheles, Aedes, and Culex genera, each species with 6 sequences were used for training and validation. The method of HANS was found to provide accurate genera classification and rapid species identification. However, the genera net and species nets involved in HANS were found to require more training efforts. Very recently, Lorenz et al. (2015) [36] employed ANN for identification and classification of mosquitoes based on the wing shape characters of the species. A data base of 32 principal components representing the wing shape characteristics of 17 mosquito species of Anopheles, Aedes and Culex genera were analyzed by using a multi layer perception ANN classifier. Species identification by this method was found to be accurate enough. Although the ANN technique of Lorenzo et al. can handle large amount of data, unique variations in wing shape data is required for correct analysis of mosquitoes. Though the above reported ANN configurations are found effective for classification and identification of mosquito species, their major limitation is the heuristic configuration of the network parameters and computational units which may restrict them in achieving the desired performance. The RBFN methodology of this work overcomes such a limitation by automatically configuring the network with optimal selection of computational units.

This work differs from the earlier reported works with respect species involved for analysis as well as the method of species identification. The analysis of the results exhibits accurate identification of mosquito species by genera specific RBFN models, but their application is limited to the identification of species of individual genera. Even though, the genera specific RBFN species identifiers of this work provides marginally improved performance over the Multi-Input Single-Output Neural Networks (MISONN) of earlier reported works, the major advantage comes from the comprehensive RBFN of this work. The training effort as well as the training time required for this comprehensive RBFN with automatic learning, is found to be much lower to the above reported neural network configurations. This method is found to exhibit better species identification ability and it can be advantageously extended for identification of species of more mosquito genera.

This is the first study for proposing and using such an automatically configured RBFN for classification and identification of mosquito species based on ITS2 ribosomal DNA sequence data. Though the comprehensive RBFN model of this study is quite useful for accurate identification of mosquito species of different genera, the computational effort required for automatic configuration of RBFs and interconnection weights increases with the increase of number of genera and species. The methodology can be applied with similar success to identify the other species of Anopheles, Aedes and Culex genera as well as the species of other geographical varieties, if the network is trained properly by involving analogous species of such genera.

CONCLUSION

Rapid and accurate identification of mosquito species are of paramount importance for taking control measures against deadly diseases like malaria, filariasis, encephalitis, dengue and so on. Genetic identification is the conformation for any kind of biological classification. The data sequences of ITS2 region considered for mosquito species identification in this work are widely used to extract the phylogenetic relation due to their well-conserved nature in a particular species. In this work, a Radial Basis Function Network (RBFN) methodology with automatic network configuration is presented for identification of mosquito species based on the genetic pattern information content of ITS2 ribosomal DNA sequences. A number of data sequences in varying sizes of different vectors corresponding to Anopheline, Aedes and Culex are used to develop genera specific RBFN species identifiers and a comprehensive RBFN species identifier. The recall and generalization ability of the RBFN species identifiers are analyzed and further validated through bootstrap validation method. The genera specific RBFN models are found to provide accurate identification of mosquito species of individual genera. The comprehensive RBFN species identifier is found to exhibit better species identification ability and it has the added advantage of identifying species of more mosquito genera. Since the RBFN computational method designed for species classification involving reported NCBI data base has shown effective classification efficiency, the method is expected to perform well for other data sequences if their analogues are involved in training.

REFERENCES