Captive maturation studies in Penaeus monodon by GIH silencing using constitutively expressed long hairpin RNA

Highlights

• GIH silencing by RNAi did not prove to be superior to eyestalk ablation.

• Constitutive expression of anti-GIH lhRNA caused mass molting and mortality.

• GIH silencing resulted in up-regulation of maturation indicator transcripts.

Abstract

Captive maturation and spawning in Penaeus monodon are currently induced by unilateral eyestalk ablation, a method that removes the gonad-inhibiting hormone (GIH) secreted from the eyestalk. However, unilateral eyestalk ablation creates physiological complications in the brooder due to the unintended removal of other hormones secreted by the eyestalks. Here we studied the effect of gih silencing by long hairpin RNA on the reproductive physiology of P. monodon. We observed 3–5 fold enhanced expression of the androgenic gland hormone (AGH) transcript in males, but no effect on vitellogenin expression in females. In the destalked animals, however, positive effect on the maturation indicator transcripts was seen in both the sexes, and surpassed the efficiency of the silencing treatment. There were indications of active spermatogenesis in histological sections of both the gih silenced and destalked males, but no changes at histological level in the females. We also observed a significantly higher rate of molting and associated mortality in the gih silenced animals at the end of the experiment. Our results suggest that eyestalk ablation is still the most efficient technique to induce maturation in P. monodon. Although gih silencing could be a potential alternative to eyestalk ablation, further research is needed to enhance its efficiency over eyestalk ablation by using tissue specific and/or inducible promoter for lhRNA expression. It is also evident that females need to be fairly large in size for these interventions to succeed.

Statement of relevance

Induction of captive maturation has been a challenge for shrimp industry. The seed production is often hampered by the low abundance and high price of naturally collected broodstock. Collection of natural brooders also has the intrinsic problem of WSSV infection. Also, genetic improvement of this highly preferred species is difficult unless the reproductive cycle is closed. For all these reasons it is important to study the reproductive physiology and endocrinology of this species and ways to manipulate it in order to induce captive maturation. The present study utilizes the RNA interference mechanism to silence the gonad-inhibiting hormone gene to see its effect on maturation.

Introduction

Induction of maturity and spawning of Penaeus monodon in captivity has been a topic of intense research for several decades. Current captive maturation techniques are highly capital intensive and require specialized rearing facilities (Hoang, 2009) and diets (Coman et al., 2007, Kian et al., 2004, Meunpol et al., 2005, Wouters et al., 2001). These systems are successful and eco-friendly, but economically challenging for Indian farmers. To meet the demand of post-larvae in India (around 10 billion per year), it is estimated that 0.2 million wild brooders would have to be acquired every year (FAO, 2007), which is likely to affect recruitment to the natural population and pose difficulties for health management.

Unilateral eyestalk ablation is practiced at commercial level for inducing captive maturation in P. monodon females. The technique is based on the principle of removing the neurohemal medullar terminallis X organ (MTXO) system which is the source of gonad inhibiting hormone (GIH) that interferes with the maturation process of the animal. Unilateral eyestalk ablation was also demonstrated to improve the quality of spermatophores in Litopenaeus vannamei (Alfaro and Lozano, 1993, Leung-Trujillo and Lawrence, 1985, Salvador et al., 1988). However, the eyestalk of shrimp also houses organs that produce a variety of other hormones involved in a wide range of metabolic processes (Huberman, 2000). As a result, eyestalk ablation produces detrimental effects on the overall physiology of the animals and has been associated with subsequent depletion in egg quality and quantity, as well as exhaustion and death of the brooders (Benzie, 1998). The injury inflicted during the eyestalk removal also renders the animal susceptible to infections.

Specific silencing of gih gene by RNA interference (RNAi) was attempted by Treerattrakool et al. (2011) who used this technique to induce maturation in wild and captive P. monodon females. The average age of reproductive maturity in male and female P. monodon is around 6 months (Jiang et al., 2009), though there are reports of successful vitellogenesis in females only five months old (Primavera, 1978). First mating in wild is known to occur at around 4–5 months of age and completion of ovarian maturation and spawning occurs at around 10 months of age (Motoh, 1981). Treerattrakool et al. (2011) carried out gih silencing in wild and farm bred previtellogenic P. monodon females by injecting anti-GIH double stranded (ds)RNA produced in vitro. Here, captive maturation in P. monodon adults was induced through gih silencing by a long-hairpin RNA constitutively expressed in vivo from a DNA construct. The treatment was compared with unilateral eyestalk ablation and impact of both treatments on the expression of relevant neuropeptides and gonadal maturation was analyzed.