Automated identification of Monogeneans using digital image processing and K-nearest neighbour approaches.

BACKGROUND: Monogeneans are flatworms (Platyhelminthes) that are primarily found on gills and skin of fishes. Monogenean parasites have attachment appendages at their haptoral regions that help them to move about the body surface and feed on skin and gill debris. Haptoral attachment organs consist of sclerotized hard parts such as hooks, anchors and marginal hooks. Monogenean species are differentiated based on their haptoral bars, anchors, marginal hooks, reproductive parts' (male and female copulatory organs) morphological characters and soft anatomical parts. The complex structure of these diagnostic organs and also their overlapping in microscopic digital images are impediments for developing fully automated identification system for monogeneans (LNCS 7666:256-263, 2012), (ISDA; 457-462, 2011), (J Zoolog Syst Evol Res 52(2): 95-99. 2013;). In this study images of hard parts of the haptoral organs such as bars and anchors are used to develop a fully automated identification technique for monogenean species identification by implementing image processing techniques and machine learning methods. RESULT: Images of four monogenean species namely Sinodiplectanotrema malayanus, Trianchoratus pahangensis, Metahaliotrema mizellei and Metahaliotrema sp. (undescribed) were used to develop an automated technique for identification. K-nearest neighbour (KNN) was applied to classify the monogenean specimens based on the extracted features. 50% of the dataset was used for training and the other 50% was used as testing for system evaluation. Our approach demonstrated overall classification accuracy of 90%. In this study Leave One Out (LOO) cross validation is used for validation of our system and the accuracy is 91.25%. CONCLUSIONS: The methods presented in this study facilitate fast and accurate fully automated classification of monogeneans at the species level. In future studies more classes will be included in the model, the time to capture the monogenean images will be reduced and improvements in extraction and selection of features will be implemented.

Monogenean anchor morphometry: systematic value, phylogenetic signal, and evolution.

Background. Anchors are one of the important attachment appendages for monogenean parasites. Common descent and evolutionary processes have left their mark on anchor morphometry, in the form of patterns of shape and size variation useful for systematic and evolutionary studies. When combined with morphological and molecular data, analysis of anchor morphometry can potentially answer a wide range of biological questions. Materials and Methods. We used data from anchor morphometry, body size and morphology of 13 Ligophorus (Monogenea: Ancyrocephalidae) species infecting two marine mugilid (Teleostei: Mugilidae) fish hosts: Moolgarda buchanani (Bleeker) and Liza subviridis (Valenciennes) from Malaysia. Anchor shape and size data (n = 530) were generated using methods of geometric morphometrics. We used 28S rRNA, 18S rRNA, and ITS1 sequence data to infer a maximum likelihood phylogeny. We discriminated species using principal component and cluster analysis of shape data. Adams's K mult was used to detect phylogenetic signal in anchor shape. Phylogeny-correlated size and shape changes were investigated using continuous character mapping and directional statistics, respectively. We assessed morphological constraints in anchor morphometry using phylogenetic regression of anchor shape against body size and anchor size. Anchor morphological integration was studied using partial least squares method. The association between copulatory organ morphology and anchor shape and size in phylomorphospace was used to test the Rohde-Hobbs hypothesis. We created monogeneaGM, a new R package that integrates analyses of monogenean anchor geometric morphometric data with morphological and phylogenetic data. Results. We discriminated 12 of the 13 Ligophorus species using anchor shape data. Significant phylogenetic signal was detected in anchor shape. Thus, we discovered new morphological characters based on anchor shaft shape, the length between the inner root point and the outer root point, and the length between the inner root point and the dent point. The species on M. buchanani evolved larger, more robust anchors; those on L. subviridis evolved smaller, more delicate anchors. Anchor shape and size were significantly correlated, suggesting constraints in anchor evolution. Tight integration between the root and the point compartments within anchors confirms the anchor as a single, fully integrated module. The correlation between male copulatory organ morphology and size with anchor shape was consistent with predictions from the Rohde-Hobbs hypothesis. Conclusions. Monogenean anchors are tightly integrated structures, and their shape variation correlates strongly with phylogeny, thus underscoring their value for systematic and evolutionary biology studies. Our MonogeneaGM R package provides tools for researchers to mine biological insights from geometric morphometric data of speciose monogenean genera.