Anthesis Journal of Applied and Basic Sciences, Dec. 2014, 2(6): 191-203 ISSN: 2383-2002 (Online), Available online at www.ajabsci.com Copyright © 2014, Published by Anthesis Journals
Identification of Freshwater Flat Crab Species and Study of Its Sexual Maturation in Guilan, North of Iran Zahra ASHOURDAN , Somayeh ASADIAN-NARENJI*, Seyed Gholamreza HOSSEINI Department of Biology, Animal Biosystematics, Tonekabon Branch, Islamic Azad University, Tonekabon, Iran ABSTRACT: This research was carried out in order to identify freshwater flat crab species from Potamon genus in the east of Guilan province. In this study, sex organs maturation was determined and compared with carapace size. 56 mature and 18 immature crabs obtained from four rivers, Shenrud in Siahkal, Shalmanrud in Otaghvar, Kiarud in Rankuh, and Ziazrud in Kelachay, were studied during 7 months from May to November 2014. Then, we examined the gonad color and its relation with carapace width under the loop. We observed the three-part structure of vas deferens as well as the H-shaped structure of the animal’s ovaries together with the end-point bag attached. We found out in these studies that eastern Guilan’s crabs belong to the p. ibericum species. For mature crabs, testes are yellow, ovaries are dark orange, and their carapace width is more than 26 mm. Therefore, the gonad color is directly related to the carapace width. Keywords: Freshwater Crab, P. ibericum Species, Gonad, Sexual Maturation.
INTRODUCTION Freshwater crabs are among amazing parts of water ecosystem which have been neglected (Dobson, 2004). Since freshwater crabs are very conservative, it is not unexpected that information on them is very limited (Brandis et al., 2000). For this reason, a few books and articles have been published regarding identification of freshwater crabs belonging to Potamidae family, whereas this family is widely distributed. However, researchers have really tried to identify this group of crustaceans in recent years. The most widely used features in identifying the freshwater crab are attributes such as ventral and dorsal views of the carapace, jaws, and first and second structures of male genital legs. Guilan province is located in the south and southwest of Caspian Sea. It is regarded as one of the richest and most humid areas of the country owing to its good weather, abundant rainfall, and many water sources (springs, rivers, streams etc.). Despite favorable conditions, especially in mountainous regions of Guilan province, very few studies have been done that is why this province has been selected for this study. In studies done on freshwater flat crabs, the subspecies Potamon persicum elbursi from Sefidrud River, Potamon ibericum tauricum in Sefidrud opening connected to Caspian Sea, and ibericum Potamon ibericum in the coastal region of Guilan have been reported (Nasrolahzade et al., 2003). The first freshwater crab in Iran has been identified and reported by Olivier in the year 1804. As of that date, European researchers have collected many examples of Iranian crabs. English researcher named Acock introduced several other species in the southeast of Iran in 1909. Later, some German researchers, especially Bott in 1955 and Pretzmann in 1962, made several attempts to identify crabs in Iran. Pretzmann was the first person to do this for crabs in Middle East, Asia, and Africa followed by Turkey and Bauer who put a lot of effort in this area. However, different people have different classifications. For example, in a study on species of the genus Potamon, due to the absence of any reference basic study, we witnessed some differences and similarities in G2 of samples, which made it difficult to categorize. Studies on identification of crabs were first only based on their morphological characteristics, carapace structure, and having fluff, because some of these features were very prominent and visible in various populations. Also, for a population in which a lot of morphological characteristics were different, some characteristics were later Received: 17 November 2014; Accepted: 19 December 2014 Corresponding author: Somayeh ASADIAN-NARENJI (
[email protected])
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considered which have been less affected by the environment and have been more sustainable (Brandis et al., 2000). Keykhosravi and Skorbart (2013) morphologically examined the freshwater crab from the species Potamon elbursi and Potamon persicum in northern Iran. In this study, however, Potamon elbursi‟s level increased from subspecies to species. According to this research, the best distinguishing feature of these two species is the G1 structure. Nasrollah Zade et al. (2003) reported Potamon bilobatum species in Guilan Heights for the first time in a study of the freshwater crab in Lakan region. They also found that male size is bigger than female size at all stations. Shadi Khatami and Turaj Vali Nasab (2003) identified the freshwater flat crab of Jajrud River. According to taxonomic studies and by sending samples of Natural History Museum in Netherlands and receiving permission for species identification, it was found that all samples belong to the species Potamon persicum. Brandis and Apel (2000) identified a new species named Socotrapotamon nojidenss during the study of freshwater crabs in Socotra Island. However, the fundamental difference between this species and S. socotrensis is in the shape of the bottom of the first genital leg, long and strong curvature of the second genital leg, and big and narrow walking legs of the species. Yeo and Ng (1816) detected Singapore‟s freshwater crabs based on the eighth thoracic vertebra. The samples studied were collected from Historical Museum. In this research, carapace length and width were measured in millimeters. They observed that the seventh and eighth thoracic vertebrae are separated by a continuous central longitudinal line. Salih and Barwary (2010) studied the genital anatomy of reproductive organs of male freshwater crabs from the species Potamon magnum in Tigris. In the study, a longitudinal incision was made at the posterior end towards the anterior end. Male animals‟ testicles were examined. They found that the male genital system includes a pair of deferens, ejaculatory duct end, a pair of genital organs and gonopods. Each deferens consists of three anterior, middle, and posterior parts. In the present research, freshwater flat crabs will be identified by studying the height above sea in rivers of the east of the province and their sexual maturity will be determined and compared to carapace size. MATERIALS AND METHODS In order to conduct this research, first and after specifying heights of areas with the help of the internet software Google Earth and observing places of interest, sampling was done at places with a distance of 100 meters from each other. Due to the animal‟s habit of secrecy, samples were prepared from pristine areas and places not frequented by humans. Crabs were collected from rivers, their lateral margins, and under large stones. In the following, in order for further investigation, sampling continued in every one of geographical areas to distances of several hundred meters and 2 kilometers at most. Incomplete samples (damaged forks and walking legs or lack of one leg or both) were, of course, released. On rainy days, rivers flooded and crabs were not detectable, although it was occasionally possible to see crabs in the narrow waterway related to the river. For species identification and sexual maturation study, geographical characteristics of areas were investigated by GPS and four areas of interest were marked on the map of Google Earth. The areas include: Shenrud River which is a high river located at a distance of 20 km from Siahkal; Shalmanrud River which is a not-so-high one situated at a distance of 11 km form Otaghvar- a town in Langerud; Kiarud River that is a not-so-high river at a distance of 5 km from Rankuh- a town in Rudsar; and high Ziazrud River at a distance of 14 km from Rahim Abad in Rudsar. Sampling was done around the cities of Siahkal, Langerud, Rudsar, and Kelachay from May to October 2014. This should be done quietly because in case of being alarmed crabs hide so that they are no more available. Crabs adapt to living in different freshwater habitats. In this study, samples were selected from places such as rivers, brooks, and under large stone and transferred to plastic buckets. In order to measure the distribution of crabs at different heights, stations were chosen in terms of height,
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distance, substrate type, and availability. Carapace width for crabs which were put under the biometric study was more than 29 mm. different geographical regions have been shown on Map 1-1.
Fig. 1, Google Earth map of the study area
For sample size in each station, 7 male and 7 female mature crabs were randomly collected from rivers of the study area in order for bio-systematic investigation and sexual dimorphism study. In the following, with the identification of the species type in four different geographical regions, 18 immature crabs in 3 different sizes were randomly caught from different stations so that we had three males and three females of each size. Some samples were fixed in 10% formalin from the first and some other samples, which were to be described in order to study the gonads color, were frozen at -20°C. At the same time, the sample collector‟s name, date, location, and height of sampling were recorded. This information was also labeled on the container. Male and female crabs were separated by ventral appearance (triangular in males and curvilinear in females). Each station‟s name and number was written on labels using an especial pencil. Numbers 1 to 7 and 8 to 14 were allocated to males and females, respectively. To prevent the penetration of water into the label, it was covered by a piece of tape. Labels were attached to samples by nylon strings. Samples together with labels were placed their own containers.
Fig. 2, Samples‟ labels
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In order to identify freshwater crabs of four regions of Eastern Guilan (Siahkal, Langerud, Rudsar, and Kelachay), three male crabs were gathered and frozen from every one of the four sampling places. Firstly, two pairs of genital legs of male crabs were separated and kept in special tubes containing 70% ethanol for the biometric study. Early identification of samples was done by using valid identification key (Brandis et al., 2000) based on morphological characters such as the carapace shape, the presence of teeth on the edge of the carapace, low width of the forehead, the appearance of the lateral anterior part, jaws, and the third jaw. For a complete recognition of species type under the loop, the placement of the end and sub-end of the animal‟s genital legs, especially the flexible area between the end and sub-end, was investigated. Thus, primary identification was done at family and genus levels. Then, some photos of samples and genital legs related to them were provided with the help of a camera and the description loop. The color of productive organs of frozen samples was checked under the loop. Later, the color of the gonad of each crab was compared to the size of that crab‟s carapace and the logical relationship between these two factors was found by studying mature and immature samples. Immature samples were not chosen for morphometric measurements and statistical analyses.
Fig. 3, A description of P. ibericum crab. (a) male crab, (b) female crab, (c) gonopods description. Finally, we used SPSS22 and tests such as PCA (Principal Component Analysis), DA (Discriminal Function Analysis) and T-Test. In all these statistical tests, P
