Methyl Farnesoate through Feed: a Growth Manipulator in Female Crab Oziothelphusa Senex Senex

Methyl farnesoate (MF), the predominant juvenile hormone-like compound found in crustaceans. In crustaceans, MF mediated growth was reported and succeeded in the laboratory, but was not done successfully at the field level. The present investigation is aimed to test the role of dietary MF on growth of crustaceans in the semi-controlled environment. To test this, MF was supplemented through commercial shrimp pellet diet to female freshwater crab Oziothelphusa senex senex (Oss) with a concentration of 10-9, 10-8 and 10-7 moles/crab in an every alternative day for about 40 days along with eyestalk ablated (ESX) and control groups. Dietary supplementation of MF enhanced the growth of female crab by inducing molt. The molt induction frequency found in this study as MF concentration 10-8 moles/crab > 10-9 moles/crab > 10-7 moles/crab ≤ ESX and recorded molt percentage of 25%, 12.5%, 10%, 7.5% respectively. Evidencing that the dietary MF supplementation induces growth in cultured crustaceans thereby increases the yield of the culture.


Introduction
Crustacean aquaculture plays an important role in producing quality protein, but is facing difficulties for quality protein production at the end. To attain more quality protein, methods are employed to induce growth. One of such popular conventional method to induce growth is eyestalk ablation (ESX), where unilateral or bilateral eyestalk ablation experiments were carried out (Venkitraman et al., 2010 andAmer et al., 2015). Removal of eyestalk causes increase in ecdysteroid secretion from Y-organ which induces precocious molting in many decapods (Techa et al., 2015, Allayie et al., 2011Neelima et al., 2016). Though ESX induces growth, it promotes mortality due to loss of large amount of hemolymph. An effective alternative for ESX is hormonal manipulation with respect to molt regulation.
Endocrine manipulation is nothing but exogenous administration of modulators and screening or observing its effect on molting. Past to present, the manipulation of crustacean molt has led to use of several endocrine modulators including external and internal molecules (Aktas et al., 2005;Sainath et al., 2011). The molt regulatory hormones, especially positive regulators supplementation accelerates the molt and growth in crustaceans. Molting is accelerated by endogenous hormone methyl farnesoate (MF), secreted from mandibular organ (MO) and ecdysteroids synthesized and released from Y-organs. The mandibular organs located at the base of the mandibular tendon, secretes the sesquiterpene MF and farnesoic acid (FA) (Tiu et al., 2012) MF (methyl-(2E,6E,10E)-3,7,11-trimethyldodecatri-2,6,10-eneoate) is structurally similar to insect JHIII (methyl 9-(3,3-dimethyloxiranyl)-3,7-dimethyl-2,6-nonadienoate) differing only in the absence of an epoxide moiety at the terminal end. It has been identified in a large number of crustacean species including the crabs, shrimps, prawns, lobsters and crayfishes. After release from the MOs, the MF is transported in to hemolymph and finally to target tissues by binding with a protective lipoprotein known as MF-binding protein (MFBP). This plays a major role in molting Sunarti et al., 2016) and is considered an effective reproductive hormone (Laufer, 2001). Methyl farnesoate was first characterized in the spider crab Libinia emarginata (L. emarginata) (Laufer, 1987) since then identified in over 30 crustacean species. Secretion of MF from the crustacean MO is regulated through mandibular organ inhibiting hormone (MOIH) produced by the eyestalk X-organ sinus gland (Nagaraju, 2007). Though the ecdysteroids are known molt hormones, but MF is capable of inducing synthesis and release of ecdysteroids from Y-organs.
Necessitate is to identify the yield enhancers of crustacean protein at this point of time and many laboratories are working in this direction. The induction of molt by direct injection of MF was well represented in literature. We have done MF induced molt studies in crab Oss and other crustacean species at laboratory conditions Srinivasa et al., 2016]. However these reports are constrained only to the laboratory and none of these are reached to culture ponds due to its difficulty in injecting MF to each and every culturing crustacean. Though the studies are focused to induce molt by supplementing the MF through diet, Journal of FisheriesSciences.com Ramachandra Reddy, 13(1): 001-006 (2019) were in D 1 (early premolt stage) (23.08%) stage. About 38.46% mortality observed on 10 th day of experiment in ESX group. Observed most of MF 10 -9 moles/crab supplemented females were in early premolt stage D 1 (80%) and only 20% in C 4 stage on 10 th day of experiment. The animals supplemented MF 10 -8 moles/crab were in premolt stages D 1 (70%) and D 2 (30%) on day 10 of experiment. The molt stages C 4 (50%) and D 1 (50%) were observed in crabs supplemented with 10 -7 moles/crab of MF on day 10 of experiment (Table 1).
On day 30 of experiment ESX crabs were in premolt stages D 2 (22.22%), D 3 (33.33%), D 4 (11.11%) and some were molted female crabs in each. Pond one and two served as control and ESX groups and other three for 10 -9 , 10 -8 and 10 -7 moles of MF/each 100 mg pellets supplemented to each animal every alternative day. The control and ESX animals were fed with normal pellet diet. The crabs from each group are sacrificed on 10 th , 20 th , 30 th and 40 th day of experiment to check the molt stages except control group where some of them sacrificed on 0 th day.
Molt stages were determined based on the setal development in the mastigobranch of 3 rd maxillepede. The changes in the setal development are observed under phase contrast microscope (Olympus, Model BX41TF, Japan) and molt frequency was examined.

Result
The molt frequency was determined on 10 th , 20 th , 30 th and 40 th day of experiment in all experimental groups along with ESX and controls. No mortalities were recorded in experimental groups except in ESX animals. Throughout the experimental period the control crabs are in C 4 (intermolt) stage only. In case of ESX, on day 10 observed majority are in C 4 stage (38.46%) and some   (11.11%). In the same group observed 22.22% mortality on this day. In case of MF 10 -9 moles/crab supplemented were found in the molt stage D 2 (30%), D 3 (20%), D 4 (30%) and some were molted (20%). observed crabs supplemented MF 10 -8 moles/crab in premolt stages D 3 (20%) and D 4 (50%), and molted (30%). About 20% molted, 40% D 3 and 40% D 4 were observed in crabs supplemented 10 -7 moles/crab of MF on 30 th day of experiment.

Discussion
Molt induction was reported in ESX female crabs in the present study. Similar reports was noted in many crustacean species like Macrobrachium rosenbergii (Sanjeevraj et al.,1997), Aphanomyces astacus (Gydemon et al., 1988), Procambarus clarkii (Chen et al., 1995), Mangelia dobsoni (Venkitraman et al., 2004), L. emarginata (Rotllant et al.,2000) and Scylla serrate (S. serrata) through eyestalk ablation technique. Eyestal ablation removes molt regulatory principles occurs in the eyestalks. The two major hormones of eyestalk are molt-inhibiting hormone (MIH) and mandibular organ inhibiting hormone (MOIH). Molt inhibition is the primary function of MIH and MOIH in crustaceans. Eyestalk is a key organ for molt regulation due to presence of inhibitory principles (Hosamani et al., 2017). It is clear from the present study that ESX leads to removal of MIH and MOIH the inhibitory principle of molt. However, ablation of eyestalk is failed due to loss of hemolymph by cautery at the base of eyestalk and loss of several other physiological factors including loss of eyestalk hormones in spiny lobster Panulirus argus (Quackenbush et al., 1981) and in other crustacean species. This is in agreement with the results of present study where 17.5% mortality reported in eyestalk ablated group.
The role of dietary supplementation of MF and its minimum effective concentration in inducing molt were determined in the present study in crab Oss maintained in semi-controlled environment. Out of three concentrations of MF tested, 10 -8 moles/ crab dietary supplemented is found more effective molt inducer in female crab Oss. Moreover, the dietary MF at 10 -9 and 10 -7 moles/ crab are also inducing molt in females. A number of studies were proved the molt induction capacity of MF injection in many crustacean species. In Oss molt induced studies were done with MF injections in laboratory (controlled) conditions . It is reported in the same study that MF reduces the molt interval to 14 days i.e., within 2 weeks in both in males and females crabs. Besides Oss MF induced molt was studied in crustaceans like Cherax quadrcarinatus (Abdu et al., 2001), Cancer magister (Tamone et al., 1993) Litopenaeus vannamei (Tariq et al., 2014) L. emarginata (Hosamani et al.,2019), S. serrate (Girish et al., 2015), Portunus trituberculatus (Xie et al., 2015) , Neocaridina denticulate (Sin et al., 2015), Metapenaeus ensis (Gunawardene et al., 2002), Homarus americanus (Homola et al.,1997), Penaeus monodon (Suneetha et al., 2010), Chionectes opolio (Marilyn et al., 2014), and Scylla olivacea (Akbar et al., 2016). On the other hand ecdysteroids from Y-organs are also released by MF thereby in molt induction. However, it is proved from the present study that dietary MF supplementation induced the molt in female crab. But the clear mechanistic action of MF on molt induction is not clear. It is predicted to be by direct induction or by releasing the ecdysteroids or both.
In the present experiment it is clear that the molt induction frequency of MF at 10 -8 moles/crab supplemented through diet is showing high than other MF treatment groups in female crabs. The function of MF is to promote the molt cycle through induction of MH synthesis and release thereby growth in crustaceans. It is clear that dietary supplementation of MF induces molt effectively with reduced molt cycle durations than positive control ESX group in crab Oss. Moreover the MF 10 -8 moles/crab supplemented crabs also showing reasonably good numbers with molt induction. This report is a base for implementing growth of crustaceans at the farm level. However, no recorded studies are available on dietary supplementation of MF for testing growth at semi-natural aquatic environment (Hosamani et al., 2017).
The present investigation is giving a base to increase the yield of crustacean protein at the pond level by supplementing MF through pellet diet. Since the dietary MF supplementation induces the molt by reducing the length of natural molt cycle in the crab Oss grown in semi-controlled environment, it may directed to the semi-controlled crustacean cultures and gradually to the open pond system. This study provides a base to initiate and test the dietary supplementation of other endocrine manipulators for growth in cultured crustacean species at the pond level. Moreover,