HISTOLOGICAL ANALYSIS OF GONAD DEVELOPMENT IN A FRESHWATER SHRIMP, CARIDINA NILOTICA (DECAPODA: ATYIDAE) E.G. Okuthe, W.J. Muller and C.G. Palmer Institute for Water Research, Rhodes University, PO Box 94. Grahamstown. 6140. E-mail: [email protected] or [email protected] Corresponding Author: Tel: (046) 622 2428. E-mail: [email protected] ABSTRACT The use of Caridina nilotica in ecotoxicology research has been initiated. C. nilotica is a protandrous hermaphrodite. All gonads C. nilotica start differentiating as testes. Then at a specific time during the course of gonadal development, testes transform into ovaries. Gonad histopathology is a suitable effect parameter to assess the effect of toxic compounds. Therefore a detailed knowledge of the normal development is essential. This paper is an examination of gonad morphology in developing caridina nilitica. A total of 300 individuals were examined by light microscopy beginning at hatch up to 56-days post hatch (dph). Special attention was given to the period of sex transition. From hatch to 5-dph, neonate gonads contained one to two primordial germ cells (PGC’s) per cross-section. After 10-dph, the number of germ cells had increased. At 23-dph first testes containing juveniles were found. At 30-32-dph, more than 80% of the dissected juveniles displayed gonads with the morphology of an early, non-functional testis. This high percentage of juveniles containing testes was also found at 37-dph. Low percentage of juveniles with testis was found at 52-dph. No signs of inter-sex conditions were observed. The first sign of ovarian development was seen at 37-dph. Gonads with oocytes in the meiotic stage of development were identified at 52 dph. The ovaries consisted of oocytes in the first and second stage of oogenesis. INTRODUCTION During the past few years, awareness that chemicals, both man made and naturally occurring in the environment may mimic or interfere with natural regulation of the endocrine system of animals and thereby affect human health and the environment has increased. A growing number of research indicates that many industrial chemicals and pesticides may interfere with the normal functioning of human and wildlife endocrine systems. It is believed that the effects of these chemicals on endocrine function are responsible for a number of reproductive and developmental anomalies in a wide range of wildlife species from invertebrates, through fish, reptiles, birds and mammals including humans. Such effects have been documented for many wildlife species in field and laboratory studies (1, 2, 3). Successful reproduction by individual species depends on the normal ontogenesis of the gonads, a process that is marked by distinct, very predictable developmental transitional phases that begin at fertilization and continue through embryogenesis. The critical period of early gonad development is also likely to hold one key to understanding phenomena of current interest such as reproductive dysfunction caused by pesticides and other industrial chemicals with endocrine-disrupting activity as well as effects of other environmental factors. There is need for in vivo invertebrate models to improve our ability to detect those environmentally persistent compounds that adversely affect reproduction by acting as steroid hormone agonists and antagonists (4). Although early concerns over toxic chemicals focused on vertebrates, attention recently has broadened to include invertebrates because of their ecological importance. Until recently only a few investigations have been undertaken to determine the effects of chemicals on reproductive capacities in exposed aquatic invertebrates. Crustaceans are often used as Proceedings of the 2004 Water Institute of Southern Africa (WISA) Biennial Conference 2 –6 May 2004 ISBN: 1-920-01728-3 Cape Town, South Africa Produced by: Document Transformation Technologies Organised by Event Dynamics bioindicators and biomonitors in various aquatic systems (5). This is due to the fact that they are a very successful group (over 35 000 classified species) of animals, distributed in a number of different habitats including marine, freshwater and terrestrial habitats. Their success in colonization of different habitats is also reflected in the diversity of life history patterns and the reproductive strategies (6, 7). Ecologically, crustaceans play an important role in the food web and in the economy of aquatic environments. Some of the features of crustacean reproductive strategies may be useful for the interpretation of data from bioindicator studies. Thus crustacean biology is worthy of study not only from the perspective of fundamental science relevant to the advancement of knowledge in the life sciences, but also from the perspective of applied sciences relevant to management of aquatic resources. In the present study, gonad histology of a freshwater caridean shrimp, Caridina nilotica will be investigated. C. nilotica belongs to the Decapoda group in the class Malacostraca. The majority of these groups are marine, but some are freshwater forms (8). Caridina nilotica is an inhabitant of freshwater environments both in tropical and temperate regions (9). In Southern Africa, C. nilotica is the most common freshwater caridean shrimp and forms an important component of the invertebrate community (10). It is a characteristic occupant of lowland streams, where it is usually found amongst vegetation (10). They are detritivores, feeding mainly on detritus, algal films on rocks and on bacteria (11, 12). Testing protocols to measure the effect of toxicants and the utility of C. nilotica have recently been initiated. However, little is known about the reproductive biology of C. nilotica compared with other caridean species. Histological changes accompanying development of the gonads have been described in mostly marine species. The majority of previous reports in the literature on the reproductive characteristics of shrimps have focused on the gonadal development at the time of sexual maturation (13, 14). The vitellogenic phase of oogenesis, for example, is characterized by a remarkable increase in size of the oocyte primarily due to the accumulation of yolk. Thus vitellogenesis has been a focus in the study of ovarian development of most species studied (15, 16, 17, 18). Consequently these studies are mostly concerned with special details, which encompass only a section of gametogenesis rather than providing accounts of gametogenesis throughout development. In order to gain insights into events that lead to the formation of mature gametes in C. nilotica, studies should be conducted at the earlier stages of gonad development. It is precisely during these stages that important processes occur connected with sex differentiation, the establishment of individual fecundity, and the species characteristics or the reproductive function of the organism (19). The process of sex differentiation, which occurs early in ontogeny, is crucial for the formation of an ovary or testes, and sex related processes occurring at the beginning of development such as sex differentiation of the brain and associated behavioral responses. Aims and Objectives of Study To describe the histological development of gonads of C. nilotica in order to provide appropriate developmental endpoints, which can be used as indicators of exposure to chemicals, which, disrupt the normal developmental processes and may have subsequent ecological consequences. MATERIAL AND METHODS Collection of Samples Newly hatched neonates, were obtained from stock bred over 2 years at the Unilever Center for Environmental Water Quality laboratory (IWR, Rhodes University). Spawning females were kept at 24 ° C in glass aquaria each with a volume of 70 liters. Three females and nine male C. nilotica were placed in breeding traps and left until fertilization. Following fertilization, males were removed from traps and females left until they release neonates from the brood pouch (at hatch). Alternatively, gravid females were collected from hatchery tanks and enclosed individually in breeding traps and until neonates were released. After hatching, females were removed and larvae left in breeding traps and sampled at specified times for histological examination. Neonates and juveniles were sampled from hatchery tanks at hatch, and days, 5, 10, 15, 20, 23, 30, 37, 52 post hatch. Organisms were killed by exposing to an overdose of MS 222 (Sigma Chemicals). They were then individually measured for total length (TL) from the tip of the carapace to the tip of the telson. The carapaces of the bigger shrimps were punctured and telson trimmed before fixation and smaller neonates processed whole. Sample Fixation and Sectioning Tissue samples were fixed in Davidson’s AFA fixative (20) for 24 hours transferred directly to 50% ethanol and stored in 70% ethanol. Samples were then dehydrated through ethanol series, cleared in chloroform and embedded in Paraplast. Paraplast blocks were then trimmed and sectioned at 7 µm, placed on glass slides pre-treated with 2 % aminopropyltriethoxy-saline in acetone and stained with and routinely stained with Mayer’s modified haematoxylin and eosin (21). Sections were thereafter examined and photographed using an Olympus 4040 zoom digital camera, Olympus American Inc. USA. RESULTS Gonad Development Between day 0 (hatch) to 5 days post hatch (dph), the undifferentiated gonads are situated at the anterior part of the heart tissue and slightly above the gut (intestine). The gonads contain one to two germ cells