Reproductive Biology of the Thumbprint Emperor, Lethrinus harak (Forsskal 1775), using histological ultra-structural characteristic in gonads along Sudanese Coastal Waters

Lethrinus harak (Forsskal 1775) are very common species of the family Lethrinidae in Sudanese Red Sea Coast. Some aspects of the reproductive biology and histology of the gonads of Lehtrinus harak were studied between 2008 and 2009. The present study described six maturity stages for gonad development based on external features and histological study of males/females. Males of Lethrinus harak attain maturity stages at a relatively bigger size than females (at 22.5 cm for males and 21.7 cm for females). Lethrinus harak has a prolonged spawning season extending from October to April with peaks in April.


Introduction
The study of reproduction and egg production increases our knowledge about the state of a stock and improves standard assessments of many commercially valuable fish species. Lethrinus harak belongs to Family lethrinidae which is one of the commercially important fish groups along the Sudanese Red Sea Coast and widely distributed in Sudanese Red Sea Coast. There are at least four species of the genus Lethrinus common in the Sudanese waters, all of which come under the common Arabic name of Sha'oor, and are one of the most common local commercial fishes and are found throughout the year Abu Gideiri (1984).
There are no biological studies in detail such as studying reproduction and management of fishing in most fishes of Red Sea especially Sudanese Coast. Therefore, this study is an important matter. The aim of this study to collected main base line information on reproductive performance of Lethrinus harak during development in order to investigate the reproductive biology of L. harak, including its spawning season, gonad maturity stages, sex ratio, and the size at which50% of fish attain first sexual maturity which a future support mariculture and on fisheries management. Hilborn. et al. (1992) and Pauly (1994) reported that growth and mortality model parameter estimates are required for analytical fisheries management. Hunter et al., (1992) reported that description of reproductive strategies and the assessment of fecundity is fundamental topics in the study of the biology and population dynamics of fish species. Studies on reproduction, including the assessment of size at maturity, fecundity, duration of the reproductive season, daily spawning behaviour and spawning fraction, permit quantification of the reproductive capacity of individual fish. (Kraus, et al., 2002). Filho, (1989 reported that knowledge about the gonadal development of marine fish species is important information to maintain the fishery stocks. The data required to provide such information is typically gathered through the examination and classification of gonads into developmental stages so that parameters such as reproductive period, spawning frequency, size at sexual maturity and sex ratios can be determined. Many studies have been conducted on the genus Lethrinus such as some aspect of the reproductive biology of Lethrinus harak (Ntiba, 1995;Kulmiye et al., 2002and Ebisawa, 2006; histological studies on Lethrinus nebulosus (Loubens,1980); size and age at sexual maturity (Hilomen, 1997 andLassi, 2003) and some biological aspects of Lethrinus mahsena (Abu Degoon, 2005).

Collection of specimen:
Total of 358 specimens of Lethrinus harak was collected monthly and randomly from the Fish Commercial Centre at Abu Hashish Fish Central Market-Port Sudan during the period from June 2008 to May 2009 The total length (TL),was measured to the nearest 0.1 cm and the total weight (TW), and gonad weight (GW) were weighed to the nearest 0.01 grams in an electronic balance and age estimates using the scales. Fully distended with granular surface occupying almost all the abdominal cavity.

Spent
Shrunken and flaccid, walls are harder and wrinkled. No milt oozes out on pressure and blood vessels visible externally.
Ovary is not fully empty. Residual oocytes present flaccid and red in colour and Ovary wall is thick. To determine the average size at which 50% of Lethrinus harak males and females attain first sexual maturity (fish in stage 2 of gonad development and above were considered mature). Fish belonging to this species attains their first sexual maturity at 22.5 cm for males and 21.7 cm for females (Table 2 and Fig. 3).  Plate (28) transverse section through the spawning testes (oozing) the all tastes filled with spermatozoa (SZ) and some of connective tissue (CT) or blood vessels. In this stage showing reduction in size of lobules leaving space between them, decrease of the intensity of spermatozoa (SZ), and thick inter lobular connective tissue (CT). The spawning testes were observed most months of the year but peak in other from March until November for L. harak (stained with Hematoxyline and Eosin).

Discussion
Lethrinus harak was economical important fish species, beside its ease adaptability for culture an increasing interest has been given to its propagation and productivity on Red Sea Coast. The gross morphological criteria used to distinguish the six maturity stages assigned to gonad development, together with the cyclical changes shown by the gonad-index, to provide reliable evidence of spawning for Lethrinus harak in shore waters. The occurrence of high percentages of individual fishes with running gonads (stage5) in the samples in, at least, eight consecutive months from October to April and the subsequent emergence of spent fish one month after the October peak through April suggests that L. harak population at the Red Sea coast has a prolonge spawning season extending from October to April with two peaks occurring in October and February.
Unfortunately, no published data on reproduction of L. harak are available in the literature. However, the information available for other lethrinid species can be used for comparison. Wassef & Bawazeer (1992) found that the longnose emperor, L. elongatus, in the Red Sea has a protracted spawning season spanning four months (May-August). Kuo & Lee (1990) reported that the common porgy, L. nebulosus, also has a prolonged spawning season extending from September to February in the Northwestern Shelf of Australia. Nzioka (1979), examining the gonads of East African reef fishes, postulates two spawning seasons for some lethrinid species in September/ October and January/February.
The present data and histological study on length and age at first maturity for L. harak showed the male and females attain first sexual maturity (fish in stage 2 of gonad development and above were considered mature). It was found that fish lengths smaller than 19.5 cm and 18cm are always immature. The first sexual maturity at 21.5 cm for males, and 22.5 cm for females both sexes at the second year for life. These findings are in general in agreement with Ntiba., et al (1995) in study of L. harak in Kenyan water; Shakeel and Ahmed (1996) stated that, the minimum size allowed to be caught for E. areolatus in Malé, Maldives is 25 cm. McIlwain et al. (2006) in their study on L. nebulosus in the Arabian Sea, Sultanate of Oman found that, the immature individuals constituted more than 40% of the that, the age at first maturity of L. nebulosus was 1.58 year which corresponds to length 29.5 cm in the coastal areas of Mauritius. The length at first sexual maturity of L. nebulosus in Arab Emirates in front of Abu Dhabi was determined by Grandcourt et al. (2003), as 28.6 cm for males and 31.3 cm for females. While, Grandcourt et al. (2006) found that, the length at first sexual maturity of L. nebulosus in the Southern Arabian Gulf was 27.6 cm for males and 28.6 cm for females.
The Plates (from 1 to 17) show the histological changes (development) in ovaries during the reproductive cycle for the females of L. harak. Oogenesis in fish are known to undergo a sequence of external and internal changes, these changes in the oocytes development have been detected in various fish species. According to various authors, the course of development of oocytes have been divided into stages, phases or periods in order to differentiate the gradual changes in their peculiarities (Guraya et al. 1975& Matsuyama et al. 1991. It is well known that the growth of oocytes takes place at two development phases namely the primary growth phase and secondary growth phase (i.e. vitellogenic oocyte). The primary growth phase two species under the present study includes only an immature oocyte which can be divided into three subdivision or phases. Initially, the early young oocyte characterized by having a large nucleus containing one large nucleolus. The early stage of young oocyte species under the study is similar to prematuration period. These results are in agreement with Zaki et al. (1986), synapsis -period of Latif and Sandy (1973), immaturation period of Assem (1992 and1995) and chromatin -nucleolus stage of El Gamal (1997) on Cyprinus carpio study. The late immature oocyte stage of the present study characterized by increasing oocyte in size and the nucleoli mostly located towards the nuclear membrane. The late immature stage of the present study is similar to protoplasmic growth of Latif and Sandy (1973) and the perinucleolus stage of Mousa (1994 and2002) and El Gamal (1997) on Cyprinus carpio study.
In the present results, the perinucleolus stage undergoes a gradual increase in the oocyte size and in number of nucleoli of the nucleus. During the late perinucleolus stage a basophilic organelle appears in the cytoplasm (yolk nucleeus). The yolk nucleus can be termed as, achroplasm, crop vitelline or Balbiani bodies (Zaki, et al., 1991).
The second growth phase (i.e. vitellogenic oocyte) includes vacuolization of cytoplasm and yolk deposition. In some other teleosts, the vitellogenic stages were divided into phases vacuolization and yolk deposition as described by Zaki et al., (1986) and Ashour et al. (1990). However, in some other fishes, there were four stages: vesicle stages, primary yolk granules stages, secondary yolk granules stage and tertiary yolk granules stage as described by (Khoo, 1979;Mousa, 1994and 2002and El Gamal 1997 on the ovary of many other fishes. On the contrary of many other fishes, the yolk depositions first appeared in the peripheral cytoplasm, thereafter scattered towards the center of ooyte as those described by (Zaki et al., 1986;Zaki and El Gharabawy 1991;El Gamal, 1997 andMousa, 2002).
Recent electron microsccopical studies revealed that the yolk nucleus was not a homogenous structure, and it was composed of various cellular organelles such as mitochondria, smooth endoplasmic reticulum, multivesicular bodies and lipid granules (Wallace and Selmon, 1981). At the end of the perinucleolus stage and at the vesicles stage the follicular epithelium appeared surrounding the oocytes. On further growth of oocytes, these follicular epithelial cells formed a layer coating the oocytes.
It was believed that the prementioned follicular cells play an important role in active transport of proteins and other nutrients from blood to oocytes during vitellogenesis as reported by Norrevang (1968). Guraya et al., (1975) claimed that follicle cells and oocytes are considered to play an important role in the cytoplasm structures was proved to synthesize sexual steroids by follicular theca envelope. In the present study the zona radiata layer contains microvillar processes pass through pore canals. The microvilli are thought to be the site of substance exchange between the follicle cells and the oocyte (Mastsuyama et al., 1991).
For the nearly ripe stage the present results revealed the presence of few spermatogonia and spermatocytes showing moderate quantity of spermatozoa. These results confirm with most teleosts as reported by Ghabrial (1990) and El-Gohary (2001) in Oreochromis nilotica. Ripe stages show a markeds dilation of seminiferous lobules containing a lot of sperms. Also the present study revealed that the spawning stage similar to the ripe stage show a decrease in the size of lobules due to discharge of considerable amounts of spermatozoa. This stage extended through the period of many months through the year for L. harak.
The complication of the process of spermatogenesis and the character of the discharge of the sexual products are relative to the asynchronism in the reproduction of the primary spermatocyte as reported by Koppel (1955). Also this asynchronism may be due to the progress of spermatogenesis wave along the different parts of the testes (Butskaya-1955). Adaptation of prolonged and continuous spawning is characterized by fractional discharge of the sperm cells. The prolonged spawning is enhanced by the presence of different individual caught at the same period exhibit different spermatogenic activities and the spermatozoa are discharged gradually from the semineferous lobules and the reduced size of testes, so the specific characteristic of spermatogenesis is related to the type of spawning depending on the character of spawning in female (Zaki et al., 1986;Assem, 1999 andEl-ghamaly, 2001).

Conclusion
The results of the first length and age of maturity for the L. harak was extremely at the 21.5 cm for males and 22.5 cm for females both sexes at second year.
The histological and morphological characters of ovaries L. harak have indicated that the oocyte pass through six successive stages of sexual maturation stage 1, immature oocyte (resting stage); stage 11, Vacuolization of the cytoplasm (preparatory stage); stage 111, beginning of yolk deposition (maturing stage); stage ıv, nearly mature (last maturing); stage v, the maturation of oocytes (spawning stage) and stage vı, egg resorption (postpawning stage).