The use of recombinant protein and RNA interference approaches to study the reproductive functions of a gonad-stimulating hormone from the shrimp Metapenaeus ensis Shirley Hiu-Kwan Tiu and Siu-Ming Chan Department of Zoology, The University of Hong Kong, China Neurosecretory structures in crustacean eyestalks are known to produce the crustacean hyperglycemic hor- mone (CHH), molt-inhibiting hormone (MIH) and gonad-inhibiting hormone (GIH) of the CHH ⁄ MIH ⁄ GIH gene family. These neuropeptides can regulate a variety of physiologic processes, including molting, carbohydrate metabolism, and reproduction [1–3]. GIH is one of the most studied neuropeptides of this group because of its potential importance in shrimp aquaculture. In penaeid shrimp, GIH is produced in the X-organs and stored in the sinus glands of eye- stalks [4–7]. Although the precise mechanism is not known, GIH is postulated to inhibit reproduction by suppressing ovary growth or vitellogenesis [1,2]. Eye- stalk ablation removes the source of GIH and results in ovary growth. In contrast, when eyestalk-ablated females were injected with eyestalk extract, the gonad stimulatory effect of eyestalk ablation was abolished [1,2]. In addition to GIH, a factor found in the brain and thoracic ganglion of decapod has been implicated Keywords eyestalk neuropeptide hormone; RNA interference; shrimp; vitellogenin gene Correspondence S M. Chan, Department of Zoology, The University of Hong Kong, Pokfulam Road, Hong Kong Fax: +852 2857 4672 Tel: +852 2299 0864 E-mail: chansm@hkucc.hku.hk (Received 25 January 2007, revised 15 June 2007, accepted 2 July 2007) doi:10.1111/j.1742-4658.2007.05968.x Although the crustacean crustacean hyperglycemic hormone ⁄ molt-inhibi- ting hormone ⁄ gonad-inhibiting hormone neuropeptides have been studied extensively in the last two decades and several neuropeptides from the shrimp Metapenaeus ensis have been cloned, the functions of most of these neuropeptides remained putative. In this article, we describe the use of recombinant protein and an RNA interference approach to study the reproductive function of the previously reported molt-inhibiting hormone (MeMIH-B) in M. ensis. When hepatopancreas and ovary explants were cultured in medium containing recombinant MeMIH-B, the vitellogenin gene (MeVg1) expression level was upregulated in a dose-dependent man- ner, reaching a maximum in explants treated with 0.3 nm recombinant MeMIH-B. Shrimp injected with recombinant MeMIH-B showed an increase in vitellogenin gene expression in the hepatopancreas. Moreover, a corresponding increase in the vitellogenin-like immunoreactive protein was detected in the hemolymph and ovary of these females. Injection of MeMIH-B dsRNA into the female shrimp caused a decrease in MeMIH-B transcript level in thoracic ganglion and eyestalk. These shrimp also showed reduction of vitellogenin gene expression in the hepatopancreas and ovary. Furthermore, the hemolymph vitellogenin level was also reduced in these animals. In summary, the results from recombinant protein and RNA interference experiments have demonstrated the gonad- stimulatory function of MeMIH-B in shrimp. Abbreviations CHH, crustacean hyperglycemic hormone; GIH, gonad-inhibiting hormone; GSI, gonadosomatic index; MeVg1, Metapenaeus ensis vitellogenin gene 1; MIH, molt-inhibiting hormone; RNAi, RNA interference; si, small interfering. FEBS Journal 274 (2007) 4385–4395 ª 2007 The Authors Journal compilation ª 2007 FEBS 4385 in the stimulation of gonad maturation. Injection of protein extract from thoracic ganglion or the brain can stimulate gonad maturation [8]. In sand shrimp Metapenaeus ensis, two forms of MIH-like cDNA (i.e. MeMIH-A and MeMIH-B) have been cloned and characterized [4,5]. MeMIH-B shows only 68% amino acid s imil arity to M eMI H-A, and amino acid sequence alignment ind icates that M eMIH-B is more closely related to GIH of the lobster Homarus americanus [9] than to the mandibular organ-inhibiting hormones of the crab Cancer pagarus [10]. MeMIH-A and MeMIH-B are non-sex-specific and are expressed in the eyestalks of males and females. The expression of MeMIH-A is molt-stage-related, whereas the expres- sion of MeMIH-B is correlated with the reproductive cycle. In addition to the eyestalk, MeMIH-B is also expressed in the brain [4]. MeMIH-B transcript level is low in the initial phase of gonad maturation and increases towards the end of maturation [4]. These findings suggest that the two neuropeptides should have different functions. As they share relatively high sequence similarity, cross-bioactivity also occurs for these two neuropeptides [4]. For example, injection of recombinant MeMIH-B also delays the process of molting [4,11]. At the time when we had characterized MeMIH-B, only a few CHH type II neuropeptides were reported [2,3]. Despite its potential involvement in reproduction, no further research on the reproduc- tive function of MeMIH-B has been attempted, as there is a lack of a good bioassay system for the neu- ropeptide. The recent cloning and characterization of the gene encoding the major yolk protein, vitellogenin, may provide a potential biomarker for analysis of genes that regulate ⁄ control reproduction [12]. Concurrently, the recently developed RNA interfer- ence (RNAi) technique has been used to define the bio- logical function of many genes. This technique is based on the gene-silencing effect of dsRNA [13]. The tech- nique has revolutionized ‘reverse genetic’ research by introducing dsRNA to organisms or cells. dsRNA can knock down a gene and will produce a phenotypic loss of function of that gene [14–16]. Although the com- plete mechanism has yet to be revealed, successful RNAi has been reported for many animal models. For example, Caenorhabditis elegans can be soaked in dsRNA or can be fed plasmids that make dsRNA and consequently exhibit RNAi effects. In many studies, dsRNA can move across cell boundaries freely. Thus, it is not necessary to inject dsRNA directly into the gonad to get progeny that exhibit RNAi effects [13]. As RNAi works in many organisms, it might also work in shrimp. Gene function analysis by RNAi may be advantageous as compared to other conventional approaches. This article describes the production of recombinant protein and dsRNA for reproduction- related eyestalk neuropeptide gene, and use of an in vitro explant culture system and an RNAi technique to demonstrate the reproductive function of MeMIH-B in M. ensis. Results Expression of MeMIH-B in shrimp Although we have previously studied the tissue distri- bution of MeMIH-B in the female shrimp, the expres- sion pattern of MeMIH-B in the central nervous system of different reproductive stages has not been fully investigated. Moreover, to ascertain that MeMIH-B expression pattern is correlated with repro- ductive developmental stages in females, we have rein- vestigated the expression pattern of MeMIH-B in the eyestalks and other nervous tissues of the adult females by northern blot analysis. MeMIH-B transcripts could be detected in the eyestalk, nerve cord, thoracic gan- glion and brain of shrimp at early to middle stages of gonad maturation (Fig. 1A). In female eyestalks, MeMIH-B transcript level was low in immature shrimp with low gonadosomatic index (i.e. GSI < 2). As gonad development was in progress, a steady increase in MeMIH-B transcript level was observed. Similarly, the expression pattern of MeMIH-B in the thoracic ganglia also followed that of the eyestalk (Fig. 1B). For example, in both eyestalk and thoracic ganglion, the highest MeMIH-B transcript level was recorded at the late maturation stage in shrimp with GSI ¼ 10. Similar to the previous results, expression of MeMIH-B is sex-nonspecific, as the males also expressed MeMIH-B (Fig. 1B). Functional study of recombinant MeMIH-B in vitro and in vivo The rMeMIH-B produced by pRSET expression was purified on an Ni 2+ -charged column. To study the function of rMeMIH-B in reproduction, hepatopan- creas explants from females at early stage of gonad maturation (GSI < 2) were used. A dose-dependent increase of MeVg1 expression was recorded when the concentration of rMeMIH-B was increased (i.e. 0.3 pm,3pm and 30 pm). The maximum increase of MeVg1 transcript level was observed in the hepatopan- creas explants treated with 0.3 nm rMeMIH-B; further increase of rMeMIH-B (i.e. 3 nm,30nm and 300 nm) resulted in a decrease in the overall MeVg1 expression level (Fig. 2A). When the ovary explants were treated Functional study of crustacean neuropeptide S. H K. Tiu and S M. Chan 4386 FEBS Journal 274 (2007) 4385–4395 ª 2007 The Authors Journal compilation ª 2007 FEBS with 0.3 nm rMeMIH-B, an increase of about 25% of MeVg1 expression was recorded (Fig. 2B). Next, we performed an in vivo injection of rMeMIH-B into females to further confirm its gonad- stimulatory effect. To demonstrate the specificity of rMeMIH-B in gonad maturation, a control group injected with rMeMIH-A was included. As compared to the NaCl ⁄ P i control, injection of an equal amount (6.6 nmol) of rMeMIH-A did not cause any change in the overall expression of vitellogenin in the hepatopan- creas and ovary (Fig. 3A,B). In contrast, injection of 6.6 nmol of rMeMIH-B stimulated an increase (2–3-fold) in MeVg1 expression by the hepatopancreas and ovary (Figs 3A,B) at 72 h, but only weakly for the 24 h time point (data not shown). It is well accepted that the vitellogenin produced in the hepatopancreas serves as an extraovarian source for the final synthesis of vitellin. The newly made vitel- logenin is expected to be secreted rapidly into the hemolymph and transported to the ovary for oocyte uptake. To demonstrate that the increase in expression of the MeVg1 gene could also result in the appearance of vitellogenin in the hemolymph for transport, we also collected hemolymph samples of these injected shrimp and analyzed the increase in vitellogenin-spe- cific protein. As shown in Fig. 3C,D, when females were injected with rMeMIH-B (i.e. 6.6 nmol), the hemolymph and ovaries of most animals contained a much higher level of vitellogenin (i.e. 148 kDa) (Fig. 3C, left panel). These vitellogenin-specific pro- teins are presumably derived from the translation of the MeVg1 gene from the hepatopancreas after rMeMIH-B stimulation. The results from SDS ⁄ PAGE and western blot analysis of the hemolymph and ovarian proteins from shrimp injected with 6.6 nmol of rMeMIH-B demonstrated an increase in the overall Es Br Tg Vn Hp Mu Ov noisserpxe evitaleR 1.0 A B 0.75 0.5 0.25 %2 %4 % 6 %7 %9 M 1.0 0.75 0.5 0.25 noisserpxe evitaleR Es MeMIH-B Tg MeMIH-B Fig. 1. Expression of MeMIH-B in different tissues of early (GSI < 2) mature females (N ¼ 5). (A) The relative expression levels of MeMIH-B in nervous tissues (ES, eyestalk; Br, brain; Tg, thoracic ganglia; Vn, ventral nerve) and non-nervous tissues (Hp, hepatopan- creas; Mu, muscle; Ov, ovary); the bar indicates the SE. (B) The expression pattern of MeMIH-B (N > 20) at different gonad matura- tion stages of the eyestalk (open bar) and thoracic ganglia (diago- nally shaded bars) of females. The percentage indicates the GSI of the females. M (B) indicates the expression pattern of MIH-B in the same tissues in males (N ¼ 5). The lower panel is the northern blot analysis of MeMIH-B expression in the eyestalk (Es) and tho- racic ganglia during the gonad maturation cycle. Each lane repre- sents an RNA sample from the eyestalk or the thoracic ganglion of one shrimp. The last lane shows the RNA samples from a male. The bar indicates the SE. Relative expressionRelative expression 1.4 B A 1.2 1.0 0.8 0.6 0.4 0.2 0 Concentrations of rMeMIH-B Concentrations of rMeMIH-B * * 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0 Fig. 2. Histogram showing the relative expression levels of MeVg1 in (A) hepatopancreas and (B) ovary explants after exposure to dif- ferent concentrations (i.e. from 0.3 p M to 0.3 lM) of rMeMIH-B. The sample size (or numbers of shrimp) is 10 for the in vitro assay. Relative MeVg1 mRNA levels (A) are shown as means + SEM of 10 prawns. The shrimp that show significant differences (P<0.05) in the relative MeVg1 mRNA levels are indicated by an asterisk. S. H K. Tiu and S M. Chan Functional study of crustacean neuropeptide FEBS Journal 274 (2007) 4385–4395 ª 2007 The Authors Journal compilation ª 2007 FEBS 4387 0 0.5 1 1.5 2 2.5 3 3.5 4 A B C D treatment 0 0.5 1 1.5 2 2.5 3 rMeMIH-BCtrl rMeMIH-A Ctrl rMeMIH-A rMeMIH-B 0 2 4 6 8 12 10 0 0.5 1 1.5 2 2.5 3 Ctrl rMeMIH-A rMeMIH-B Ctrl rMeMIH-A rMeMIH-B 148 kDa Hycn 148 kDa NSP MeVg1 rRNA MeVg1 rRNA Me MIH-Actrl Me MIH-B treatment Me MIH-Actrl Me MIH-B treatment Me MIH-Actrl Me MIH-B treatment Me MIH-Actrl Me MIH-B Fold changes of Vg transcript level in hepatopancreas Fold changes of Vg transcript level in ovary Fold changes of Vg level in hemolymph Fold changes of Vg content in ovary Fig. 3. Effect of recombinant MeMIH-B and MeMIH-A on vitellogenin expression in shrimp. (A) Left: relative expression levels of MeVg1 in hepatopancreas of females (N ¼ 10) at 48 h after injecting NaCl ⁄ P i , rMIH-A and rMIH-B. Right: a typical northern blot analysis of the shrimp MeVg1 transcript level after injection of NaCl ⁄ P i , rMIH-A, and rMIH-B. (B) Left: relative expression levels of MeVg1 in ovary of females (N ¼ 10) at 48 h after injection of NaCl ⁄ P i , rMIH-A, and rMIH-B. Right: a typical northern blot analysis of the shrimp MeVg1 transcript level after injection of NaCl ⁄ P i , rMIH-A, and rMIH-B. (C) Left: relative levels of vitellogenin in hemolymph of females (N ¼ 10) at 48 h after injec- tion of NaCl ⁄ P i , rMIH-A, and rMIH-B. Right: western blot analysis (upper) of the hemolymph level of vitellogenin for shrimp injected with rMIH-B. The 148 kDa protein is one of the vitellogenin subunits recognized by the shrimp antibody to vitellogenin [27]. The lower panel shows the Coomassie blue staining of the hemocyanin (Hcy) corresponding to the same protein samples. (D) Left: relative levels of vitelloge- nin in ovary of shrimp at 48 h after injection of NaCl ⁄ P i , MIH-A, and rMIH-B. Right: western blot detection (upper) of vitellogenin (148 kDa) in ovary of shrimp injected with rMIH-B. NSP is the nonspecific protein unrelated to vitellogenin of the ovary samples. In the northern blot (or western blot) analysis, each lane represents RNA (or protein) samples collected from individual shrimps. Functional study of crustacean neuropeptide S. H K. Tiu and S M. Chan 4388 FEBS Journal 274 (2007) 4385–4395 ª 2007 The Authors Journal compilation ª 2007 FEBS vitellogenin-specific protein (Fig. 3C,D). Unlike the rMeMIH-B-injected group, shrimp injected with rMeMIH-A (6.6 nmol) did not show any changes in the overall MeVg1 transcript level in the hepatopan- creas or a significant increase in MeVg1 protein level in the hemolymph and ovary (Fig. 3A–D). Inhibition of vitellogenin expression after RNAi We have performed preliminary experiments using a nonspecific dsRNA (from Tiger frog virus), and the results show no effect on MeMIH-B gene silencing (data not shown). In the following study, individual shrimp (N ¼ 40; average GSI < 3) were injected with 3 lg of dsRNA for MeMIH-B, and RNA samples were collected after 24, 48, 72, 96 and 120 h. Northern blot results from the eyestalk (Fig. 4A) indicated no signifi- cant reduction in MeMIH-B transcript level at all time points. However, when we used RT-PCR to analyze the same samples, a significant reduction of MeMIH-B transcript was observed (Fig. 4B). In fact, by RT-PCR, the MeMIH-B dsRNA appeared to knock down most of the transcripts after 72 h of treatment (Fig. 4B). In addition, hybridization signals representing small-size RNAs were strong and persisted from 24 to 120 h after injection (Fig. 4A). This suggests that dsRNAs are very stable, as residual MeMIH-B dsRNA remained. Unlike in the eyestalk, there was a significant decrease in the MeMIH-B transcript level in the nerve cord as early as 24 h after MeMIH-B dsRNA injection. The knock- down also persisted 120 h after dsMIH-B injection (Fig. 5A). MeMIH-B transcript level was lowest in nerve cord at 72 h after injection, but started to increase afterwards (Fig. 5B). With regard to the effect of MeMIH-B dsRNA on hepatopancreas MeVg1 expression, it was observed that there was a significant drop in MeVg1 transcript level in the hepatopancreas. For example, at 24, 48 and 72 h after dsRNA treat- ment, drops of 20%, 71% and 23% of the overall MeVg1 transcript level were recorded. (Fig. 6A). Unlike in the hepatopancreas, the reduction of MeVg1 expression in ovaries of these female was small after the injection of MeMIH-B dsRNA. For example, the reduction in MeVg1 transcript level in the ovary repre- sented only 6%, 7% and 22% decreases at 24, 48 and 72 h post-dsRNA treatment (Fig. 6B). Similar SDS ⁄ PAGE analysis and western blot analy- sis were performed for these females. In the hemolymph sample of the NaCl ⁄ P i -injected control, vitellogenin-spe- cific protein could be detected using antibody to vitel- logenin. In contrast, no vitellogenin-specific protein was detected in the hemolymph of the dsRNA-injected females (Fig. 7A). In the ovary, the amount of vitelloge- nin remained relatively constant. However, only minute quantities of vitellogenin subunits (i.e. 148, 97 and 78 kDa) were detected in the ovaries of the dsRNA- injected females (Fig. 7B). These proteins were immuno- reactive to the antibody to vitellogenin of M. ensis [27]. B-HIMeM β nitca- 0 24 48 72 96 120 0 24487296120 +-+-+-+-+-Ctr 120967248240 Relative change in transcript level 100 80 60 40 20 Time after injection (h) Time after injection (h) AB Fig. 4. Effects of MeMIH-B RNAi in eyestalk of female shrimp. (A) Northern blot detection of eyestalk MeMIH-B transcript level in control (–) and dsRNA-injected (+) females from animals at different time intervals (i.e. 0, 24, 48, 72, 96 and 120 h); the arrow indicates the MIH-B transcript, and the smear indicates the residual dsRNA. (B) Top panel: RT-PCR detection of MeMIH-B gene knockdown using MIH-B-specific primers. Lower panel: relative change in MIH-B transcript level at different time intervals. The bar diagram indicates the relative transcript level of MeMIH-B after normalization with b-actin gene. S. H K. Tiu and S M. Chan Functional study of crustacean neuropeptide FEBS Journal 274 (2007) 4385–4395 ª 2007 The Authors Journal compilation ª 2007 FEBS 4389 Discussion Structure–function research on crustacean eyestalk CHH family neuropeptides remains a challenging endeavor. This is mainly due to the existence of highly similar neuropeptides in the same species [1,2]. For example, it is now known that there are at least four or five CHH-like genes in M. ensis. These genes may share high sequence similarity and ⁄ or analogous function. Some of these genes may be expressed in 0 h 24 h 48 h 72 h 96 h 120 h MIH-B β -actin +-+-+-+-+-Ctr 120967248240 Relative change in transcript level 100 80 60 40 20 120967248240 Time after injection Time after injection (h) AB Fig. 5. Effects of MeMIH-B RNAi in the ventral nerve cord of female shrimp. (A) Northern blot detection of eyestalk MeMIH-B transcript level in control (–) and dsRNA-injected (+) females from animals at different time intervals; the arrow indicates the MIH-B transcript, and the smear indicates the residual dsRNA. (B) RT-PCR detection of MeMIH-B transcript using specific primers. The bar diagram indicates the rela- tive transcript level of MeMIH-B after normalization with b-actin gene. 1gVeM ANRr 72h48h24h 72h48h24h 1g V eM ANR r yravoninoisserpxe1gVeM % decrease in MeVg I transcript level % decrease in MeVg I transcript level 30 20 10 724824 724824 100 80 60 40 20 +-+-+- +-+-+- MeVg1 expression in hepatopancreas Time (h) after injection Time (h) after injection AB Fig. 6. Expression of vitellogenin in hepatopancreas and ovary of dsRNA-injected (+) and control (–) females. (A) Upper: northern blot detec- tion of hepatopancreas MeVg1 transcript level in shrimp. Lower: bar indicates the relative decrease in expression level of MeVg1. (B) Upper: RT-PCR detection of ovary MeVg1 transcript level in shrimp. Lower: bar indicates the relative decrease in expression level of MeVg1. Functional study of crustacean neuropeptide S. H K. Tiu and S M. Chan 4390 FEBS Journal 274 (2007) 4385–4395 ª 2007 The Authors Journal compilation ª 2007 FEBS non-neuronal (or non-eyestalk) tissues [3]. Likewise, additional MIH subtypes have also been found in one species. For example, in addition to the known MIH and GIH subtypes, there is also a report suggesting the existence of novel MIH subtypes. In the GenBank data- base (http://www.ncbi.nlm.nih.gov/), there are at least four different entries for MIH-like neuropeptides in the tiger prawn Penaeus monodon. As there is a high degree of sequence similarity and structural conservation of these genes and gene products, confirmation of the true identity and function of these neuropeptides remains difficult. Unfortunately, most of these MIH-like peptides are produced in very low quantities, and they function as inhibitory regulators in a physiologic process, making it difficult to develop a good bioassay. The production of large quantities of recombinant protein for bioassay may circumvent the lack of active material for structure–function studies. However, the inhibitory nature of these hormones remains a challenge for the successful development of a bioassay system. Attempts at developing biological assays for inhibi- tory factors such as MIH have been reported, and the inhibitory function on molting has been demonstrated convincingly. Most biological assays of the gonad inhi- bition of this neuropeptide rely on its ability to inhibit ovary development and reduce oocyte size, cause a decrease in total ovary protein incorporation, and sup- press ovary total protein synthesis [11,17,18]. Biologi- cal assays using these criteria are nonspecific and provide little information on the mechanism of GIH regulation of reproduction. Previously, we have pro- duced rMeMIH-B (formerly MeeMIH-B), but little progress was made in developing a biological assay for the recombinant protein. This is mainly attributed to the lack of a biomarker for the reproductive process. With the recent cloning of the vitellogenin gene in dif- ferent crustaceans [19–23], a more precise role for GIH can be defined with the vitellogenin as a biomarker. Recently, there was a report on the effect of sinus gland extract and neuropeptide on vitellogenin gene expression in M. japonicus. In that study, the effect of a CHH peptide and two MIH-like peptides on ovary vitellogenin gene expression was investigated; the results indicated that CHH causes inhibition, whereas the MIH-like neuropeptides have no effect on vitello- genin gene epression [23]. Unlike the CHH of M. japo- nicus, MeMIH-B (a type II neuropeptide) has a stimulatory effect on vitellogenin synthesis. Taken together, the results suggest that a CHH-like (or type I) neuropeptide may be inhibitory for gonad maturation, 321M65 4 detcejni-ANRsddetcejni-SBP 321M654 detcejni-ANRsdde tcejni-SBP yravOh p mylom AB eH gni n ia ts BCgniniatsBC tolbnretseWtolbnretseW 321M654 321M 6 54 aD k 841 aDk79 aD k6 7 a D k 84 1 aD k 79 a D k67 Fig. 7. Western blot analysis of the hemolymph and ovary total protein of dsRNA-injected females. (A) Hemolymph sample of NaCl ⁄ P i - injected and dsRNA-injected females. Lanes 1–3: NaCl ⁄ P i -injected and 4–6 dsRNA-injected animals. Individual lanes represent protein sam- ples collected from injected shrimps, and the arrows indicate the vitellogenin-specific protein (148, 97 and 76 kDa) using antibody to vitellog- enin [19,27]. (B) Ovary sample of NaCl ⁄ P i -injected and dsRNA-injected females. Lanes 1–3: Ovary from the corresponding NaCl ⁄ P i -injected individual and 4–6 dsRNA-injected animals. Each lane represents protein samples collected from individuals, and the arrow indicates the vitel- logenin-specific protein determined using antibody to vitellogenin [19]. S. H K. Tiu and S M. Chan Functional study of crustacean neuropeptide FEBS Journal 274 (2007) 4385–4395 ª 2007 The Authors Journal compilation ª 2007 FEBS 4391 whereas the MIH-like neuropeptide (i.e MeMIH-B, type II) may be a vitellogenin-stimulatory factor of shrimp. Because of the existence of multiple forms of the CHH family neuropeptides, there may be a dis- crepancy in defining the function of many cDNAs cloned using a molecular biology approach. For example, although the MIH-like gene of Litopenaeus vannamei was reported [24], detailed analysis revealed that the MIH-like deduced protein was more closely related to the CHH as described in other crustaceans. Since our report of a second form of MIH subtype cDNA in M. ensis, the naming of this MeMIH-B as a GIH was based on its similarity to the lobster GIH, as other MIH subtypes in shrimp had not been reported [4]. In re-evaluating the expression pattern of MeMIH-B in the eyestalk and CNS, we further recon- firmed that its expression level increases during active vitellogenesis. Our result suggests that a high transcript level of the MeMIH-B gene (or the protein) may be needed for vitellogenin production during active vitellogenesis. The use of recombinant protein to study the func- tion of crustacean neuropeptides has been reported for a number of species [4,11,19,25]. Recombinant MeMIH-B has been produced and used in a biologi- cal assay for molt inhibition [4]. Except for the role of CHH in the increase of glycemia, the use of recombinant protein to study molt-inhibiting function and gonad maturation remains a challenge for crusta- cean endocrinologists [2,4]. The difficulty is due to the lack of a good biological marker for consistent results. As the hepatopancreas and ovary express MeVg1, explants from these tissues were used in the explant culture. Given the fact that the hepatopan- creas culture lasts for 3–4 h and active vitellogenin expression can be detected in shrimp, the explant culture system was successfully developed. This is the first demonstration of the stimulatory effects of a neuropeptide in vitellogenin gene stimulation. As rMeMIH-B can stimulate MeVg1 expression in hepa- topancreas and ovary in vitro, the result may provide information on the initial mechanism of hormone action. In other words, the in vitro results indicate that MeMIH-B may act directly on the hepatopan- creas and ovary to increase the rate of vitellogenin gene expression, indicating that both the hepatopan- creas and ovary are the targets of MeMIH-B. This result will provide the basis for identifying and characterizing the receptor for the neuropeptide. Furthermore, rMeMIH-B acted on the hepatopan- creas and ovary in a dose-dependent manner. As the optimal concentration (i.e. 30 nm in vitro and 6.6 nmol in vivo) for the stimulatation of MeVg1 expression is low, the result also suggests that rMeMIH-B is highly potent in stimulating vitellogenin gene expression. As subadult (i.e. < 15 g) and adult females also responded to rMeMIH-B in a similar dose-dependent manner, the results would be useful for us to develop a strategy to induce gonad matura- tion in shrimp aquaculture. RNAi is defined as the gene-silencing effect medi- ated by dsRNA. RNAi technology was developed in the mid-1990s, based on the antisense RNA techno- logy developed in the 1980s. RNAi can silence or knock down the expression of a gene, and the phe- nomenon appears to be universal, as it has been reported in both plants, animals, and even cultured cells. There are two major types of RNAi, with slight differences in the mechanism. They are mediated by either: (a) dsRNA; or (b) small interference (si)RNA. The longer dsRNA may generate a large population of siRNA (with 21–23 nucleotides), and the use of longer dsRNA may be advantageous over siRNA. In this study, the longer dsRNA was produced and used in the RNAi experiments. It has been reported that the longer dsRNA of approximately 600–800 nucleo- tides works best for most genes. The GIH-specific dsRNA, however, was synthesized from the 238 bp coding region of the mature peptide. As the coding sequences of all the neuropeptides are short (< 350 nucleotides) and there are scattered repetitive sequences in the noncoding region of MeMIH-B, selection of the effective gene region to produce dsRNA is limited and restricted only to the coding region. As the siRNA produced by the endogeneous Dicer (assuming a mechanism similar to the verte- brates) is small, the siRNA has to be specific to cause an effect. For example, the RNAi will not work even with a 1–2 bp mismatch. Thus, another highly similar gene (MeMIH-A) will not be affected. This may explain why there are hybridization signals in the eye- stalk, as the eyestalk is also known to produce MeMIH-A. The apparent lack of MeMIH-B knock- down (northern blot result) may simply indicate that another very similar but abundant neuropeptide (i.e. MeMIH-A) may hybridize to the MeMIH-B probe. When the more specific RT-PCR was used, the decrease in MeMIH-B transcript level was evident. The amount of dsRNA injected in the animal may vary, depending on the expression level of the gene; a much higher dose of dsRNA was injected into the shrimp L. schmitti to silence the CHH gene [26]. In our study, the amount of dsRNA injected into each animal was about 3–5 lg for each shrimp (23–28 g). At present, the mechanism of RNAi in shrimp is not known, but we expected that these dsRNA molecules Functional study of crustacean neuropeptide S. H K. Tiu and S M. Chan 4392 FEBS Journal 274 (2007) 4385–4395 ª 2007 The Authors Journal compilation ª 2007 FEBS could circulate by way of hemolymph and would be taken up by a variety of tissues. In the target tissues that express MeMIH-B (i.e. neuronal cells of the eye- stalk and ⁄ or central nervous system), the dsRNA enters the cell to initiate gene knockdown. The dsRNA appears to be stable in the target tissues. For example, a strong hybridization signal representing the residual MeMIH-B dsRNA still remains in the eye- stalk at 120 h after injection (Figs 4A and 5A). In conclusion, the use of recombinant protein or RNAi alone may not be sufficient to confirm the func- tion of a neuropeptide. The combined use of recombi- nant protein and RNAi described in this study has provided unequivocal evidence for a stimulatory func- tion of MeMIH-B in vitellogenesis. We can also apply similar approaches to study the structure–function relationships of other CHH ⁄ MIH ⁄ GIH neuropeptide members. Experimental procedures Animals Shrimp were purchased from a local seafood market. They were acclimated in the laboratory at 25–28 °C in an indoor aquarium for 2 days before rMeMIH-B or dsRNA injec- tion. The GSI was calculated as the percentage of ovary weight per total body weight. Production of the recombinant MeMIH-B The cDNA encoding the mature peptide of the MeMIH-B was amplified by PCR using restriction enzyme-linked gene-specific primers (forward, 5¢-GACGAATTCTTCGG CCTTCGC-3¢; reverse, 5¢-AGGAGATCTAAGCTTACCA CGCTCCACCAGGG-3¢) that contained restriction sites EcoRI and BamHI, respectively. The PCR product was first digested with EcoRI and HindIII, and was then ligated into the cloning vector pRSET-B containing a T7 lac promoter site (Invitrogen, Carlsbad, CA, USA). The constructs were transformed into Escherichia coli XL1 Blue cells, and the bacteria were grown at 37 °C. The plasmids were purified by alkaline lysis DNA minipreparation. The insertion of the desired gene in the plasmid was verified by automated DNA sequencing. The clones were transformed into E. coli BL21 ⁄ DE3. For large-scale expression, bacterial cells were grown in 1 L of LB broth at 37 °C. Protein expression was induced by the addition of isopropyl 1-thio-b-d-galactopyr- anoside (Sigma, St Louis, MO, USA) to a final concentra- tion of 1 mm. The culture was allowed to continue for 4 h. The bacteria were pelleted by centrifugation (5000 g for 15 min) and resuspended in 50 mL of binding buffer (20 mm Tris ⁄ HCl, 0.5 m NaCl, and 5 mm imidazole, pH 7.9). The cells were homogenized with a polytron, and centrifuged at 5000 g for 15 min. The pellet was then resupended in 50 mL of denaturing binding buffer (8 m urea in binding buffer), sonicated, and agitated overnight at 4 °C. Follow- ing centrifugation as above, the supernatant was collected and loaded into an Ni 2+ –nitrilotriacetic acid–agarose (Qiagen, Hilden, Germany) affinity column that was pre- equilibrated with denaturing binding buffer. The column was then washed three times with washing buffer (8 m urea, 20 mm Tris ⁄ HCl, 0.5 m NaCl, and 25 mm imidazole, pH 7.9). The fusion protein was eluted with elution buffer (6 mm Tris ⁄ HCl, 0.5 m NaCl, 8 m urea, and 300 mm imid- azole, pH 7.9). The denatured recombinant protein was refolded by both dilution and dialysis. The concentration of urea present in the solubilized protein was decreased step- wise by addition of an equal volume of renaturing buffer (6 mm Tris ⁄ HCl, 0.5 m NaCl, and 300 mm imidazole, pH 7.9) for every 3 h until the concentration of urea was decreased to 1 m. The diluted recombinant protein was then dialyzed in a dialysis bag (Sigma; cut-off 6–7 kDa) in a large volume of renaturing buffer for 16 h at 4 °C, with three changes of buffer. The recombinant protein was further dialyzed in a large volume of 0.1 · NaCl ⁄ Tris over- night, and the final dialysis was against 0.1 · NaCl ⁄ P i over- night. A Bradford protein assay (Bio-Rad, Hercules, CA, USA) was performed to determine the concentration of the refolded protein. The recombinant MeMIH-A was expressed using the same strategy and was used as negative control in the following in vitro and in vivo bioassay. Functional study of rMeMIH-B by explant assay and in vivo injection The functional study of rMIH-B involving a shrimp in vitro explant culture system was based on a previously developed method [20–22]. Briefly, hepatopancreas and ovary were dissected, cut into small fragments, and placed in the wells of 24-well plate containing 1.5 mL of Medium 199 (Sigma) prepared in crab saline [28]. Different concentrations of rMeMIH-B were added to the explants, and the culture was incubated at 23–25 °C for 4 h. At the end of the culture period, the tissues were collected for total RNA extraction followed by northern blot hybridization [25] or RT-PCR. For northern blot analysis, the nylon membrane was hybridized in hybridization buffer (50% formamide) containing a nonradioactive (digoxigenin) probe at 50 °C overnight. The probe (derived from partial MeVg1 cDNA) was synthesized as per kit instructions (Roche, Mannheim, Germany). After hybridization, the membrane was washed twice in 2 · NaCl ⁄ Cit (20 · NaCl ⁄ Cit: 3 m NaCl, 0.3 m Na 3 -citrate) and 0.1% SDS for 15 min, and then washed twice in 0.5 · NaCl ⁄ Cit and 0.1% SDS at 58 °C for 15 min. The signals were detected by adding the antidigoxi- genin–AP conjugate. Chemiluminescent substrate CDP-Star (Roche, Mannheim, Germany) was added, and the mem- brane was exposed to X-ray film. For PCR, the PCR mix S. H K. Tiu and S M. Chan Functional study of crustacean neuropeptide FEBS Journal 274 (2007) 4385–4395 ª 2007 The Authors Journal compilation ª 2007 FEBS 4393 (20 lL) consisted of 10 mm Tris ⁄ HCl (pH 8.0), 1.5 mm MgCl 2 ,50mm KCl, 2 mm dNTP, and 10 pmol of forward and reverse primers. The PCR conditions were one cycle of 3 min at 95 °C, followed by 35 cycles of denaturation at 95 °C for 1 min, annealing at 58 °C for 1 min, and exten- sion at 72 ° C for 1 min. In the last cycle, the PCR product was incubated at 72 °C for 10 min to allow the completion of DNA synthesis. PCR products were analyzed on a 1.5% agarose gel, and Southern Blot was performed to determine the specific amplification of the cDNA. For in vivo injection of rMIH-B, adult females in the nonreproductive stage were injected with 20 lL of either 6.6 nmol or 0.66 nmol of rMIH-B at the arthropodial membrane of the periopod and returned to the culture tanks. At 24, 48 and 72 h after injection, the hepatopan- creas and ovary of the shrimp were dissected for total RNA preparation, and the hemolymph samples were col- lected for SDS ⁄ PAGE and western blot analysis. Functional study of MeMIH-B by RNAi To prepare a DNA template for the synthesis of dsRNA, DNA corresponding to the mature peptide of MeMIH-B was amplified by PCR using T7 promoter-linked primers (forward, 5¢-TAATACGACTCACTATAGGTACTATG TATCGCATGCCAAT-3¢; reverse, 5¢-TAATACGACTC ACTATAGGTACTTTAAAGTCCCGGGTTGA-3¢). For PCR, the reaction mix consisted of 1 · Taq buffer contain- ing 1.5 mm MgCl 2 , 0.2 mm dNTP mix, 0.5 lm MIH T 7 -linked primers, and 0.25 lLofTaq DNA polymerase (Life Technologies, Carlsbad, CA, USA). PCR conditions were denaturation at 95 °C for 1 min, annealing at 55 °C for 1 min and extension at 72 ° C for 1.5 min for the first five cycles, annealing at 60 °C for another 30 cycles, and extension at 72 °C for 10 min. The resulting PCR products were analyzed by 2% agarose gel electrophoresis. The tar- get PCR product band was purified with the GENE- CLEAN II Kit (Biogene, Vista, CA, USA). For transcription of MeMIH-B dsRNA, 1–2 lg of purified T7 promoter-linked MeMIH-B DNA was used as template in an in vitro transcription reaction with the T7 Megascript RNAi Kit (Ambion, Austin, TX, USA), according to the manufacturer’s recommendations. In 20 lL of reaction mix- ture, MeMIH-B DNA template was mixed with appropriate amounts of nuclease-free water, 2 lLof10· T7 reaction buffer, 2 lL of ATP solution, 2 lL of CTP solution, 2 lL of GTP solution, 2 lL of UTP solution, and 2 lLof T7 Enzyme Mix. The mixture was incubated at 37 °C for 18 h. During the transcription, the two RNA strands were hybridized to form dsRNA. For RNAi experiments, shrimp (25–35 g) with similar GSI values were purchased from a local seafood market. They were acclimated in a culture tank overnight prior to injection. Then, 50 lL of MeMIH-B dsRNA (3 lgin 1 · NaCl ⁄ P i ) was injected into shrimp through the arthro- dial membrane of a periopod. The controls received an equal volume of NaCl ⁄ P i injection. Shrimps were returned to the tanks for culture before being killed for total RNA prepara- tion from different tissues. The relative level of MeMIH-B expression in the nerve cord was used as an indication of the RNAi effect between the treatment and control groups. Statistical analysis of northern blots and western blots Northern blot or western blot signals from the films (or blots) were scanned with the free software imagej (Image processing and analysis in Java: http://rsb.info.nih.gov/ij/) to obtain quantitative numbers representing expression lev- els of the gene or protein. The expression level was normal- ized with either the rRNA (for the northern blot) or hemocyanin (for the western blot). Either simple t-test or anova was used to perform statistical analysis. Acknowledgements This research was supported by the Research Grant Council of the Hong Kong Special Administrative Region, China (HKU 7214 ⁄ 05M) awarded to S M. Chan. References 1 Keller R (1992) Crustacean neuropeptides: structures, functions and comparative aspects. Experientia 48, 439– 448. 2 De Kleijn DP & Van Herp F (1995) Molecular biology of neurohormone precursors in the eyestalk of Crusta- cea. Comp Biochem Physiol B Biochem Mol Biol 112, 573–579. 3 Chan SM, Gu PL, Chu KH & Tobe SS (2003) Crusta- cean neuropeptide genes of the CHH ⁄ MIH ⁄ GIH family: implications from molecular studies. Gen Comp Endo- crinol 134, 214–219. 4 Gu PL, Tobe SS, Chow BK, Chu KH, He JG & Chan SM (2002) Characterization of an additional molt inhibi- ting hormone-like neuropeptide from the shrimp Metapenaeus ensis. Peptides 23, 1875–1883. 5 Gu PL & Chan SM (1998) Cloning of a cDNA encod- ing a putative molt-inhibiting hormone from the eye- stalk of the sand shrimp Metapenaeus ensis. Mol Mar Biol Biotechnol 7, 214–220. 6 Gu PL & Chan S-M (1998) The shrimp hyperglycemic hormone-like neuropeptide is encoded by multiple copies of genes arranged in a cluster. FEBS Lett 441, 397–403. 7 Gu PL, Yu KL & Chan SM (2000) Molecular charac- terization of an additional shrimp hyperglycemic hor- mone: cDNA cloning, gene organization, expression Functional study of crustacean neuropeptide S. H K. Tiu and S M. Chan 4394 FEBS Journal 274 (2007) 4385–4395 ª 2007 The Authors Journal compilation ª 2007 FEBS [...]... N, Saido-Sakanaka H, Yang WJ, Jayasankar V, Jasmani S, Okuno A, Ohira T, Okumura T, Aida K & Wilder MN (2004) Molecular characterization of a cDNA encoding vitellogenin in the coonstriped shrimp, Pandalus hypsinotus and site of vitellogenin mRNA expression J Exp Zool A Comp Exp Biol 301, 802–814 Tiu SHK, Hui JHL, Mak ASC, He J & Chan S-M (2006) Equal contribution of hepatopancreas and ovary to the. .. the eyestalk and brain of the white shrimp Penaeus vannamei Mol Mar Biol Biotechnol 4, 262–268 Maniatis T, Sambrook J & Fritsch EF (1989) Molecular Cloning: a Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY Lugo JM, Morera Y, Rodriguez T, Huberman A, Ramos L & Estrada MP (2006) Molecular cloning and characterization of the crustacean hyperglycemic hormone cDNA from Litopenaeus... silencing across kingdoms Genes Dev 10, 638–643 Blandin S, Moita LF, Kocher T, Wilm M, Kafatos FC & Levashina EA (2002) Reverse genetics in mosquito Anopheles gambiae: targeted disruption of the defensin gene EMBO Rep 9, 852–856 Ohira T, Tsutsui N, Nagasawa H & Wilder MN (2006) Preparation of two recombinant crustacean hyperglycemic hormones from the giant freshwater prawn, Macrobrachium rosenbergii, and their... H.-K Tiu and S.-M Chan 8 9 10 11 12 13 14 15 16 17 18 and biological assay of recombinant proteins FEBS Lett 472, 122–128 Fingerman M (1997) Roles of neurotransmitters in regulating reproductive hormone release and gonadal maturation in decapod crustaceans Invertebr Reprod Dev 31, 47–54 de Kleijn DP, Janssen KP, Waddy SL, Hegeman R, Lai WY, Martens GJ & Van Herp F (1998) Expression of the crustacean hyperglycaemic... Chan SM (2001) Bacterial expression of the shrimp molt-inhibiting hormone (MIH): antibody production, immunocytochemical study and biological assay Cell Tissue Res 303, 129–136 Tsang WS, Quackenbush LS, Chow BK, Tiu SH, He JG & Chan SM (2003) Organization of the shrimp vitellogenin gene: evidence of multiple genes and tissue specific expression by the ovary and hepatopancreas Gene 303, 99–109 Fire A, ... the crustacean hyperglycaemic hormones and the gonad-inhibiting hormone during the reproductive cycle of the female American lobster Homarus americanus J Endocrinol 156, 291–298 Lu W, Wainwright G, Webster SG, Rees HH & Turner PC (2000) Clustering of mandibular organ-inhibiting hormone and moult-inhibiting hormone genes in the crab, Cancer pagurus, and implications for regulation of expression Gene 253,... hyperglycemic activities Zool Sci 23, 383–391 Ohira T, Okumura T, Suzuki M, Yajima Y, Tsutsui N, Wilder MN & Nagasawa H (2006) Production and characterization of recombinant vitellogenesis-inhibiting Functional study of crustacean neuropeptide 19 20 21 22 23 24 25 26 27 28 hormone from the American lobster Homarus americanus Peptides 27, 1251–1258 Tiu SH, Hui JH, He JG, Tobe SS & Chan S-M (2006) Characterization... production of vitellogenin (PmVg1) transcripts in the tiger shrimp, Penaeus monodon Aquaculture 254, 666–674 Tsutsui N, Katayama H, Ohira T, Nagawasawa H, Wilder M & Katsumi A (2005) The effects of crustacean hyperglycemic hormone- family peptides on vitellogenin gene expression in the kuruma prawn, Marsupenaeus japonicus Gen Comp Endocrinol 144, 232–239 Sun PS (1995) Expression of the molt-inhibiting hormone- like... Litopenaeus schmitti Functional analysis by double-stranded RNA interference technique FEBS J 273, 5669–5677 Kung SY, Chan SM, Hui JH, Tsang WS, Mak A, He JG (2004) Vitellogenesis in the sand shrimp, Metapenaeus ensis: the contribution from the hepatopancreasspecific vitellogenin gene (MeVg2) Biol Reprod 71, 863–870 Duan S & Cooke IM (1999) Selective inhibition of transient K+ current by La3+ in crab peptide-secretory... & Chan S-M (2006) Characterization of vitellogenin in the shrimp Metapenaeus ensis: expression studies and hormonal regulation of MeVg1 transcription in vitro Mol Reprod Dev 73, 424–436 Mak AS, Choi CL, Tiu SH, Hui JH, He JG, Tobe SS, Chan SM (2005) Vitellogenesis in the red crab Charybdis feriatus: hepatopancreas-specific expression and farnesoic acid stimulation of vitellogenin gene expression Mol . The use of recombinant protein and RNA interference approaches to study the reproductive functions of a gonad-stimulating hormone from the shrimp Metapenaeus. primers (forward, 5¢-TAATACGACTCACTATAGGTACTATG TATCGCATGCCAAT-3¢; reverse, 5¢-TAATACGACTC ACTATAGGTACTTTAAAGTCCCGGGTTGA-3¢). For PCR, the reaction mix consisted of