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Identification of platyhelminth parasites of the wild bullseye pufferfish Sphoeroides annulatus Jenyns, using invariant digital color correlation. Marine fish culture in Mexico was less than 7. The bullseye puffer fish, Sphoeroides annulatus Jenyns, , is an important species in local fisheries in the East coast of the Mexican Pacific. Recent studies show that this puffer fish is a species with potential to aquaculture development Duncan et al.
The need to identify helminth parasites is increasing since the development of aquaculture in the last few decades has been seriously affected by parasite problems that decrease production Kennedy Systematic parasitology has made significant contributions in tropical medicine, public health and studies of biodiversity and evolutionary biology. Systematists are key to parasitology and associated disciplines but despite this, fewer expert morphologists are being trained and the number of active systematists decreases yearly Brooks The specific identification of parasites using morphological data can be slow and time consuming, requiring quality preparations so that each taxonomically important structure can be clearly observed.
Analysis of these structures, comparison with keys, consultation of relevant literature, and recording of morphometric data are all required for parasite identification using established techniques. The application of new techniques in biosystematics is a real possibility. Accurate molecular techniques have been developed for instance, to identify some species of helminth parasites Mattiuci et al.
Others studies show that image processing is a technique that can give reliable results. Gubanyi and Shinn et al. Work in this area is concentrating on developing techniques that will allow systematists to identify parasite species. Zavala-Hamz et al. Pech-Pacheco et al. Recent studies undertaken by Fajer et al. These studies provided whole mount specimens necessary to examine a new method of parasites identification. The proposed aims were to discriminate among six helminth species found in the wild bullseye by invariant digital color correlation.
Our results suggest that this technique can provide a rapid method for the identification of parasites by systematists. Each fish was examined for helminth ectoparasites on gills and fins, and from body surface scrapes, as well as for endoparasites from the internal organs, e. For this study, we selected a number of parasites identified by morphological methods.
Parasites included the monogeneans Heterobothrium ecuadori He and Neobenedenia melleni Nm , ectoparasites from the gills and skin, respectively, and the digeneans Lintonium vibex Lv and Phyllodistomum mirandai Pm , endoparasites from the air sacs and urinary bladder, respectively, and Bianium plicitum Bm and Homalometron longisinosum Hl from the intestines.
T; Pm cat. T; Bm cat. T; Hl cat. Our method of invariant correlation uses species-specific composite filters which includes information of morphological variations of each target species. The composite filter was built by several images of the same species. The number of images depends of the morphological complexity. In order to test the species-specific composite filters, we used images, 9 images for Hl, 31 images for Bp, 48 images for He, 46 images for Lv, 46 images for Nm and 13 images for Pm.
The method for obtaining the composite filters is presented in Fig. Since parasites have variable morphologies e. This provides, in a single matrix, all the frequency information related to the morphology of the species to be recognized. In Step 3, it is not necessary to use a uniform scale and orientation for the images since the final product is scale and orientation invariant. The high frequencies are then enhanced using high-pass filtering parabolic function.
This process is what differentiates the scale transform from the Mellin-transform. After these steps, the Cartesian coordinates were mapped to polar coordinates to obtain invariance to rotation Step 4.
In Step 5, a bilinear interpolation of the first coordinate conversion data is introduced for more details see Pech-Pacheco et al. The resulting image Step 6 is the species-specific composite filter which will be used in the invariant correlation with data arising from images of individual helminth specimens. S POF is the name used for the species-specific composite phase only filter obtained in Step 6.
Steps 3, 4, 5, and 6 from Fig. Because color images, one species-specific composite filter was made for each RGB channel. The explanation for Fig. A fuller discussion of this technique considering a single filter only is given in Alvarez-Borrego et al. To discriminate among parasite species, images of each individual to be recognized were transformed as indicated in Fig.
In Step 6, we can see the results obtained using the same steps as Fig. In Step 7 of Fig. The recognition of particular species of parasite is complex.
Color can be an important discriminative feature, which needs to be included for successful identification. Since our analysis was digital, it was possible to separate the color image in 3 channels RGB using pixel view. Thus, the process shown in Fig.
In each color channel, the composite filter to be used is matched to the corresponding component of the target.
So, an object is detected as the target if it simultaneously produces a correlation peak in the 3 channels. The final result will be the product of the correlation for each of the RGB channels multiplied. The algorithm allows the different steps showed in Figs. The parasites studied are shown in Fig. In order to illustrate the method used in this study, six parasite species were analysed. Figure 3. Correlation values are normalized.
Figures represent the mean correlation for multiple individuals of each species. Figure 4. C orrelation results for a Homalometron longisinosum Hl and b Bianium plicitum Bp. Boxplots show mean correlation for product of 3 color channels RGB Figura 4. Figure 5. C orrelation results for a Heterobothrium ecuadori He and b Lintonium vibex Lv.
Boxplots show mean correlation for product of 3 color channels RGB Figura 5. Figure 6. C orrelation results for a Neobenedenia melleni Nm and b Phyllodistomum mirandai Pm. Boxplots show mean correlation for product of 3 color channels RGB Figura 6. The results for Hl and for Bp are shown in Fig. For both species the figure shows the product of multiplying RGB channels.
In each figure, the X-axis shows the filter for each species-specific composite and the Y-axis gives us the mean invariant correlation value obtained for test individuals of a given species. In Fig. A mean value of 0. These values are completely separated from the values of the other parasites species. Fig s. The correlation values for Heterobothrium ecuadori with the He filter had a mean value of 0. The correlation for Lintonium vibex with the Lv filter had a mean of 0.
These values were also separated from the mean value obtained for the other parasite species. The mean correlation value obtained for Neobenedenia melleni with the Nm filter and the mean value for Phyllodistomum mirandai with the Pm filter were 0.
The time used in to identify or not an image of a parasite with this digital system is less than 8 seconds. The results obtained from the techniques employed in the present report are very good, considering the complexity of the morphology of parasitic platyhelminths. Different numbers of worm images were used to build and test the species-specific composite filters, because the morphology and the number of samples are different for each species.
The best way is to use few images 10, in practical sense because with each image added increase noise in the identification process. However it is necessary to consider some pitfalls involved in this mathematical technique used to discriminate platyhelminth images. One of them is the problem of imaging soft bodied organisms. Another problem can be the coloration in the sample, but if the sample is well sealed in balsam, the color will not change for a number of years.
However, for these parasites the most important stage for identification on cover stopped slides is in the adult age only, because the sexual organs are developed. In helminths, the morphology of several organs are important for their identification. In summary, a successful method for the discrimination of a selected set of platyhelminth species has now been developed. However, the generation of an image library is necessary, which should include digital images of the main platyhelminth parasite species in order to apply the technique and to continue with this research.
Detection of IHHN virus in shrimp tissue by digital color correlation. Aquaculture Invariant recognition of polychromatic images of Vibrio cholerae Optical Engineering Parasite systematics in the 21 st Century: Opportunities and obstacles.
Colour invariant character recognition and character-background colour identification by multichannel matched filter. The scale representation. Institute of Electrical and Electronics Engineers, Inc.