Panulirus longipes (Long-legged Spiny Lobster), a member of Palinuridae family, is a commercially important species and is harvested for food. This species has a very widespread distribution, as it is found from East Africa to Thailand, Taiwan, the Philippines and Indonesia1. It has a high value but has not been widely cultivated owing to restricted data about their biology, breeding and captive survival.
Many lobsters do not easily breed in captivity because they are not able to exhibit their natural spawning conduct in culture, which is a limiting factor in their culture and mass production. This species accounts for 66% of the total lobster catch in the Philippines2. High production capacity of Palinuridae family members in the wild has led many fishery researchers to cultivate them. A lobster study in Japan started in the late 1890s and evolved in the 1900s to research hatchery rearing of Palinurid lobsters for more than a century3. In New Zealand, successful Jasus edwardsii larval rearing was completed in the 1990’s. In India, it is documented that since 1977, a group of juvenile P. homarus has been raised to sexual maturity and grown in a laboratory4. In 2006, Western Australia generated the first P. ornatus post larvae grown in the world4. P. ornatus is regarded to be the best aquaculture candidate because it has the shortest stage of ocean larval development and the fastest post larval growth rate5.
Even though the crustaceans have been studied extensively, some of the female reproductive system in P. longipes species has not been described so far. Spawning behavior of this lobster has not yet been fully described, especially the exact time of spawning in the waters around Sulawesi, Indonesia.
Maintaining lobsters in captivity enables the development of gonad stages in the female reproductive system. The process of maturation can be studied by periodic checks of morphology-based maturity measures, gonad materials, as well as by conducting a gonad histology6. This is the study of tissue and cell microscopic structure. This allows interpretation of the structure and function of tissues or cell components7. De Lima and Gesteira8, studied the morphology of the spiny lobster male gonads. External body metric was used and developed for characterizing body size in morphological and sexual functionality in slipper and spiny Hawaiian lobsters6. The low incidence of inactive ovaries and the existence of ovaries at any moment of the year show that this species grows in Calatagan, Batangas in the Philippines throughout the year2. Ovarian appearance and/or weight are frequently used in reproductive research to stage the ovarian development cycle of female decapods, however, ovarian appearance or gonadosomatic index (GSI) offers restricted data about real ovarian modifications9. Histological examination, based on oocyte growth, could investigate seasonal changes in ovarian growth of P. inflatus10. However, fewer ovarian maturation phases have been defined for other species of palinurid lobsters. Therefore, research of this species is respected to contribute to the morphological and histological aspects of the reproductive system of crustaceans, particularly ovaries of the lobster P. longipes which potentially as aquatic organisms culture.
MATERIALS AND METHODS
Study site: The present study was conducted in coastal waters of South East Sulawesi, Indonesia in 2017-2018.
Sampling: A collection of 12 lobsters of variable sizes was done. Total length (TL), Total body weight (TBW) and Gonad weight (GW) of the samples were examined and recorded. Lobsters of higher body sizes were chosen for dissection in order to compare gonads of various sizes and body weight. The reproductive system was dissected by a mid-dorsal incision through the cephalothorax.
Gonad developmental analysis: The gonads were removed for determination of developmental stage and for histological study. Gonad weight and developmental stages were determined. Gonad developmental stages were classified according to description11. The gonad was divided into 3 parts to determine the consistency of oocyte growth along the ovary length. For histological assessment, a total of 12 ovaries were processed into slides. The fragments representing distinct ovarian regions were excised and immersed in formaldehyde of 10%. The samples were gradually dehydrated in an ethanol series, cleared in xylene and embedded in paraffin. Sections (at 5-6 μm thick using microtome blades) were stained with hematoxylin and eosin (H and E) in order to identify the structure. The slides were displayed at 100-400x magnification under a compound microscope. Photomicrographs were taken with Olympus P.A. 35 Camera fitted to the microscope. Throughout the study period, water quality parameters were evaluated monthly.
The total length of the collected animals ranged from 15.3-22.55 cm. The weight ranged from 86.69-327.0 g (Table 1).
Gonad weight, GSI and maturity stage of some P. longipes
TL: Total length, TBW: Total body weight, GW: Gonad weight, GSI: Gonadosomatic index
Classification of gonad maturity stages in lobster
Source: Silva and da cruz-Landim11
Macroscopic description of ovaries: Lobster ovaries are paired organs located mainly in the cephalothorax, dorsally or dorsolaterally to the digestive tract and often surrounded by hepatopancreatic lobes (Fig. 1). Each ovary is coupled to a thin, tubular oviduct linked to a gonopore opening onto the sternite of the appendage of the 6th thoracic segment, suiting to the base of the third pair of pereiopods. Gonads of female varied slightly in shape, location, size and coloration as they matured (Fig. 2).
Table 1 shows gonad weight, GSI and gonad stages of development of some female samples. The size of the lobster showing well-developed gonads and the gonads were among developmental stages 1, 2 and 3 (Stages have been categorized on t he basis of the features of Table 2). The ovaries at both ends are elongated, round, unlobed, tapering and coated with a soft membrane. The gonads end with a duct forming the urogenital duct. Immature ovaries are whitish and transparent, while mature ones, with noticeable yellowish eggs, appear orange-reddish. Specifically, for the mature stage, there was no difference in the level of maturity in gonads among different pigmentation.
Microscopic description of ovaries: Figure 2-3 shows the gonads histological analysis. Fig. 2 shows that there is a range of development stages in an individual gonad and it is indicated that this species is partial (heterochronal) spawners. Figure 3a shows a cross section of an ovary in stage 1 of gonad development. It comprises of lots of secondary oocytes or early vitellogenic oocytes (VO). Eggs were visible in batches surrounded by follicular wall or egg membrane (EM). Table 1 shows gonad weight, GSI and maturity stage of samples. Then, data were supported by microscopic descriptions. Stage 1 ovaries with gonad weighed of 0.12-0.42 g (immature female lobster, n = 7 samples) contained only early vitellogenic oocytes (Fig. 3a). Late vitellogenesis stages of vitellogenic oocytes were dominated stage 2 ovaries weighing 0.39-7.62 g (mature resting, n = 3 lobster samples) but cortical alveolar oocytes were also present (Fig. 3b). Stage 3 ovaries contained large numbers of ovarian cavity (OC) with fully grown but unripe oocytes present with gonad weighed of 3.04-18.94 g (Fig. 3c, n = 2 lobsters). The ovarian cavity (OC) comprised of degenerated the egg membrane or follicular wall. The lipid droplet (LP) egg yolk granules (YG) are visible.
Dorsolateral view of cephalothorax of P. longipes showing some external distinguishing features
so: Stomach, he: Hepatopancreas, ov: Ovary, t: Posterior digestive tract
The development of lobsters is very dependent on their cultivation techniques. During this time, various cultivation techniques have been applied to obtain maximum results. However, certain obstacles are always found that hinder hatchery activities. Limited information about the process of reproduction and life cycle is one of the main obstacles in the cultivation of this species. The reproductive process is an important part of the study of species biology12. Determination of the sex of some fish species can only be distinguished by examining the gonads if the species does not show clear sexual dimorphism. Differentiating the sexes and determining the maturity level of lobster gonads is rather difficult and requires certain techniques. More reliable microscopic techniques are needed.
In the present study, lobster reproduction was typically based on the macroscopic and microscopic examination. We compared both of them in order to complete information.
Orange-yellow colored, mature ovary in P. longipes lobes developed. Cross section of gonad in 3 parts: (a) anterior, (b) mid and (c) posterior
Histological magnification 400x
As with crabs and other species of lobster, the spiny lobster ovary is basically H-shaped with the lobes at the posterior extending over the abdomen. The male gonads of P. laevicauda was H-shaped also especially was described by De Lima and Gesteira8. Practically, macroscopic staging is sometimes used because it is conventionally reliable for analysis of sex ratios and gonad growth patterns broadly. In addition, it has velocity benefit and is most economically important, making it ideal for routine surveillance of the fish stocks exploited12. However, some errors with the use of the macroscopic staging system are unavoidable because, at times, there is difficulty distinguishing between oocyte maturation stages, in particular between late cortical alveoli and early yolk globular development stage13. There may also be mistakes in distinguishing between immature and mature females, especially when mature ovaries have lost evidence of spawning before reproductive activity begins.
Histology is the standard method to determine sex and reproductive condition. Gonadal histology can provide insights into the effects of multiple environmental stressors on reproductive health in combination with hormone and vitellogenin measurements, morphological and fecundity research14.
Cross section of P. longipes in (a) Stage 1 gonad development, arrows showing batches of early vitellogenic Oocytes, (b) Stage 2 of gonad development, arrows showing late vitellogenesis stages of vitellogenic Oocytes and (c) Stage 3 of gonad development showing ovarian cavity with fully grown but unripe Oocytes
Through this technique the information obtained from the gonad is more complete and more detailed.
Microscopic staging systems, however are expensive, time consuming and sometimes too simplistic and do not permit the collection of all the data available in histological slides13. Based on the comparison of oocyte diameter measured from the lobe's anterior, middle and posterior areas (Fig. 2) and between left and right lobes showing that ovarian growth throughout the gonad was almost distinct, it could be concluded that the spiny lobster as ovarian species in which oocytes grow and spawn as a few groups (asynchronous development). It is in line with the research result10, that every month mature female were present, suggesting a consistent pattern of reproduction. The size of the egg diameter varies according to the level of development12. This results showed that egg development is consistent throughout the ovary, depending on the degree of maturity of the ovary. The frequency distribution of oocyte diameter shows stock reserves at all levels of maturity but oocytes of larger size are only found in ovaries with higher maturity levels.
GSI value is the ratio value between gonad weight and body weight. GSI values provide helpful data to effectively differentiate mature oocytes and lead to oocyte maturation process research. GSI values also provide estimates of ovarian development stages10.
This study discovered that the lobsters with a maturity level I have a GSI of 0.2%, while lobsters with a maturity level 2 and 3, have a GSI of 2.97 and 4.19%, respectively (Table 1). Whereas in P. japonicus females with immature ovaries had gonadosomatic indexes15 were <1%. Like others researches, GSI values of P. longipes also have been effective in distinguishing between inactive, developing and mature stages.
In this research, it was found that mature gonad pigmentation was either orange or reddish. The color of the gonads seems to differ very little between the species of spiny lobster examined, although in the literature there is no other macroscopic description of the gonadal stage of P. echinatus11. In some mature fish, changes in body color and gonads occur due to atresia and tissue breakdown during and at the end of the reproductive season16.
Precise data on aspects of lobster reproduction can be used as a reference and additional information in the management of marine water resources so that lobster resources can be used appropriately and as efficiently as possible for the benefit of the community without neglecting the conservation of these lobsters and the further cultivation of this lobster in the ocean waters.
Morphologically, the female gonads of P. longipes as H-shaped, specifically for the mature stage, there was no distinction in the level of maturity of gonads with distinct pigmentation. Their ovaries histologically contained gonads in different stages of development, therefore they tend to be spawned in different groups (partial spawner).
This study found the gonad development of female lobster using macroscopic and microscopic methods that can be beneficial for reproductive health and selective breeding. This study will help the researchers to uncover broodstock selection used in aquaculture for lobster breeding that many researchers were not able to explore. Thus an efficient instrument for fishery biologists and aquaculturist on reproductive system on lobster may be arrived at.
We acknowledge funding from the Ministry of Research, Technology and Higher Education of the Republic of Indonesia (KEMENRISTEKDIKTI) which supports this work, Contract No. 056/SP2H/LT/DRPM.