Báo cáo khoa học: Molecular cloning and characterization of the crustacean hyperglycemic hormone cDNA from Litopenaeus schmitti Functional analysis by double-stranded RNA interference technique pot

Molecular cloning and characterization of the crustacean hyperglycemic hormone cDNA from Litopenaeus schmitti Functional analysis by double-stranded RNA interference technique Juana M. Lugo 1 , Yuliet Morera 2 , Tania Rodrı ´guez 2 , Alberto Huberman 3 , Laida Ramos 2 and Mario P. Estrada 1 1 Aquatic Biotechnology Department, Animal Biotechnology Division, Center for Genetic Engineering and Biotechnology, Havana, Cuba 2 Marine Research Institute, Havana University, Cuba 3 Nacional Nutrition Institute, Salvador Zuriban, Mexico DF, Mexico In crustaceans, the X-organ–sinus gland complex in the eyestalk is a major neuroendocrine system in which a variety of neuropeptides have been identified [1]. A neuropeptide family comprising crustacean hyperglyce- mic hormone (CHH), molt inhibiting hormone (MIH), mandibular organ inhibiting hormone (MIOH), vitello- genesis ⁄ gonad inhibiting hormone (VIH ⁄ GIH) was recently identified and referred to as the CHH family [2]. The CHH is the most abundant component of the sinus gland and the one which gives the name to the family [3]. The main CHH activity is to elevate glucose concen- tration in the hemolymph by a process of glycogen de- gradation in the hepatopancreas [4]. Besides its primary role in energetic regulation, CHH has been demonstra- ted to be pleiotropic [5]. It also participates in reproduc- tion [6], molting [7–9], digestion [10], osmoregulation [10,11] and lipid metabolism [12] in different species. Although the X-organ–sinus gland complex is con- sidered the main source of CHH production, there are other sites in the organs of crustaceans where CHH peptide has been observed [13]. CHH has been detec- ted by radioimmunoassay in the pericardial organs [14], in the second roots of the thoracic ganglia and in the subesophagic ganglion of Homarus americanus [15,16]. It is also detected in the retina of the crayfish Procambarus clarkii [17]. The cloning and molecular characterization of CHH family peptides have been reported in different species of lobster, crab, crayfish and shrimp [3,5,18]. In the lobster H. americanus, at least two forms of CHH Keywords cDNA amplification; CHH; double-stranded RNA; Litopenaeus schmitti; shrimp Correspondence M. P. Estrada, Aquatic Biotechnology Project, Animal Biotechnology Division, Center for Genetic Engineering and Biotechnology, PO Box 6162, Havana 10600, Cuba Fax: +53 7 2731779 Tel: +53 7 2716022 Ext. 5154 E-mail: mario.pablo@cigb.edu.cu (Received 23 March 2006, revised 20 Octo- ber 2006, accepted 25 October 2006) doi:10.1111/j.1742-4658.2006.05555.x The crustacean hyperglycemic hormone (CHH) plays an important role in the regulation of hemolymph glucose levels, but it is also involved in other functions such as growth, molting and reproduction. In the present study we describe the first CHH family gene isolated from the Atlantic Ocean shrimp Litopenaeus schmitti. Sequence analysis of the amplified cDNA fragment revealed a high nucleotide sequence identity with other CHHs. Northern blot analysis showed that the isolated CHH mRNA from L. sch- mitti is present in the eyestalk but not in muscle or stomach. We also inves- tigated the ability of dsRNA to inhibit the CHH function in shrimps in vivo. Injection of CHH dsRNA into the abdominal hemolymh sinuses resulted in undetectable CHH mRNA levels within 24 h and a correspond- ing decrease in hemolymph glucose levels, suggesting that functional gene silencing had occurred. These findings are the first evidence that dsRNA technique is operative in adult shrimps in vivo. Abbreviations CHH, crustacean hyperglycemic hormone; RNAi, RNA interference. FEBS Journal 273 (2006) 5669–5677 ª 2006 The Authors Journal compilation ª 2006 FEBS 5669 (CHH-I and CHH-II) have been reported [5,18]. In the shrimp Metapenaeus ensis more than six CHH-like cDNA have been identified and can be divided into CHH-A and CHH-B groups [19]. In the crabs Can- cer pagurus, Carcinus maenas and Libinia emarginata and in the crayfishes Procambarus clarkii and Orconec- tes limosus different CHH-subtypes have been reported [20–24]. Nevertheless, the Litopenaeus schmitti CHH peptide family has been little characterized and until now the CHH mature peptide was the only one that had been isolated [3]. In this work, we have applied the tech- niques of molecular biology to this important species of industrial exploitation. We have isolated, cloned and characterized the first CHH cDNA from an Atlan- tic Ocean shrimp, L. schmitti. We also investigated the ability of dsRNA to inhibit CHH function in shrimps in vivo. So far, there is little information concerning the use of RNA interference (RNAi) in crustacean species. Recently it has been proved that RNA interference mediated gene silencing is operative in shrimp cells in culture [25], but there is no evidence of its functional ability in the whole shrimp organism. This paper constitutes the first evi- dence that the dsRNA technique is functional in adult shrimps in vivo. Results Isolation and cloning of cDNA encoding L. schmitti CHH To date, cDNA sequences encoding CHH neuropep- tide family members are not known for the Atlantic Ocean shrimp, L. schmitti. We decided to obtain a L. schmitti CHH cDNA fragment by RT-PCR, as this approach was widely used to clone cDNAs of the CHH neuropeptide family [26]. The partial CHH sequence was obtained by using fully degenerated primers, as described in Experimental procedures, cor- responding to the N-terminal and C-terminal regions of the mature peptide. To obtain the CHH cDNA, we used 10 lg of total eyestalk RNA from adult shrimp by RT-PCR assays. The quality of synthesized cDNA was confirmed by PCR amplification of L. schmitti b-actin and b-tubulin partial cDNA. In both cases we obtained the expected size of the amplified fragments. A cDNA of 216 bp (oligonucleotides included) was amplified by PCR using the degenerate oligonucleotides designed for CHH gene amplification. The amplified products were subcloned into pGEM-T Easy vector (Promega) for further DNA sequence determination. DNA sequence analysis confirmed that these cDNAs corresponded to the L. schmitti CHH gene (Fig. 1A). The nucleotide sequence obtained for the CHH cDNA from L. schmitti (without primer nucleotide sequences) was compared to other CHH nucleotide sequences reported in penaeid shrimps by clustalw analysis [27,28]. The highest nucleotide identity (89%) was with Marsupenaeus japonicus CHH (Pej-SGP-II). It possessed more than a 70% identity with other eye- stalk CHHs of penaeid shrimps such as Penaeus mono- don (80%), M. ensis (77%) and Litopenaeus vannamei (73%) (Fig. 1A). The deduced amino acid sequence of the obtained cDNA corresponded to the one reported by Huberman et al. [3]; 72 amino acid residues long and possessing six conserved cysteine residues at the same positions as that of other CHHs of penaeid shrimps (Fig. 1B). Tissue-specific gene expression of L. schmitti CHH gene The size and expression of the CHH mRNA in differ- ent tissues were determined by northern blot analysis of total RNA isolated from eyestalk, muscle and stom- ach. We transferred to a nitrocellulose membrane, as described in Experimental procedures, equal amounts (10 lg) of each total RNA sample. For this assay we used as a probe the cDNA corresponding to L. sch- mitti X-organ CHH peptide. To corroborate the qual- ity of total RNA samples, the same nitrocellulose membrane was hybridized with b-actin probe and its transcript was observed as a defined band in all the total RNA samples tested (data not shown). The expression of the isolated X-organ CHH mRNA was observed in the eyestalk, but it was not detected in the muscle or stomach. The estimated size of the CHH RNA transcript was 1 kb (Fig. 2A). We also determined the CHH tissue expression by RT-PCR assays using the specific CHH primers described in Experimental proce- dures. A DNA band at the expected size was amplified from the eyestalk and stomach, and another weak one from muscle (Fig. 2B). In vivo CHH gene suppression using double-stranded RNA To investigate the ability of dsRNA to disrupt the CHH function in adult shrimps, cDNA corresponding to the CHH mature peptide was used as template for synthesizing dsRNA in vitro as described in Experi- mental procedures. The group injected with 20 lg of CHH dsRNA into the abdominal cavity showed a significant decrease Functional analysis of CHH by RNAi J. M. Lugo et al. 5670 FEBS Journal 273 (2006) 5669–5677 ª 2006 The Authors Journal compilation ª 2006 FEBS A B Fig. 1. CHH sequence analysis comparison. (A) Sequence analysis comparison by CLUSTALW analysis among CHH cDNA reported for Marsupenaeus japonicus (AB035724), Metapenaeus ensis (AF109775), Litopeneaus vannamei (AY434016), Peneaus monodon (AF104930) and CHH nucleotide sequence obtained from Litopeneaus schimitti (without primer nucleotide sequences) (DQ355982). The GenBank acces- sion numbers of the sequences are indicated in parentheses. * indicates identical bases. (B) Comparison of the deduced CHH mature peptide sequence among Penaeus shrimps. The conservative cysteine is in bold and shaded gray. * indicates the conserved amino acids within penaeid species. ‘:’ indicates similar amino acid. The gray shaded boxes indicate the amino acids conserved within the CHH family neuropeptides. AB Fig. 2. Tissue-specific expression pattern of L. schmitti CHH gene. (A) Northern blot ana- lysis using as a probe the cDNA correspond- ing to L. schmitti X-organ CHH peptide. (B) Detection by RT-PCR assay using the speci- fic CHH primers. E, shrimp eyestalk total RNA; S, shrimp stomach total RNA; M, shrimp muscle total RNA from L. schmitti; C, Total RNA from bovine tick (Boophi- lus micropulus) as negative control; MW, Molecular mass marker k HindIII (Heber Bio- tec, S.A.). The arrows denote the size of the L. schmitti CHH transcript and the CHH fragment amplified by PCR. RNA ribosomal subunits 18S and 28S are shown. J. M. Lugo et al. Functional analysis of CHH by RNAi FEBS Journal 273 (2006) 5669–5677 ª 2006 The Authors Journal compilation ª 2006 FEBS 5671 (43%) of the hemolymph glucose concentration 24 h after injections (P<0.05) (Fig. 3). To corroborate the specificity of the dsRNA gene silencing mechanism, we included a group of shrimps that were injected with 20 lg of dsRNA unrelated to CHH mRNA. The unre- lated dsRNA was generated from L. schmitti stomach transcript that encodes a chitinase like-protein. This group did not show a significant diminution of the hemolymph glucose concentration (P > 0.05) (Fig. 3). The unrelated CHH gene silencing was corroborated, 24 h after treatment, by northern blot analysis. We observed a signal at the expected size of 500 bp in the stomach total RNA pool from the control animals that were injected with saline. No other signal was detected in the total RNAs corresponding to the unrelated dsRNA treated animals (Fig. 4). The CHH gene silencing was also corroborated by northern blot analysis and by semiquantitative RT-PCR. The shrimps were killed 24 h after the injec- tions, and the eyestalks were removed to extract total RNA. We transferred to a nitrocellulose membrane, as described in Experimental procedures, equal amounts (20 lg) of each total RNA sample. The northern blot analysis using the amplified CHH cDNA as a probe showed a signal at the expected size of 1 kb in the eyestalk total RNA from the saline trea- ted group. In the samples corresponding to the CHH dsRNA treated shrimp no signal was observed. (Fig. 5A). In the same nitrocellulose membrane, the CHH transcript levels were compared against b-actin probe and the b-actin transcript was observed as a defined band in the all eyestalk total RNA sample tes- ted (Fig. 5B). Similar results were observed in the semiquantita- tive RT-PCR assays, which showed a defined DNA fragment of 216 bp, corresponding to CHH cDNA, in the saline treated group only (Fig. 6A). A DNA band corresponding to b-actin gene was amplified from all eyestalk total RNA samples tested (Fig. 6B). Discussion In this study, we amplified by RT-PCR and character- ized the first cDNA encoding for the CHH mature peptide from an Atlantic Ocean shrimp, L. schmitti. Sequence analysis of the CHH cDNA obtained showed 0 4 8 12 16 20 Saline Unrelated dsRNA CHH dsRNA Glucose (mg/dL) 0 h 24 h Fig. 3. Effects of injection of dsRNA in the profile of the hemo- lymph glucose concentration 24 h after treatments. A group of six shrimps each were injected into the abdominal cavity with 1· NaCl ⁄ P i (saline), 20 lg of unrelated dsRNA or 20 lg dsRNA CHH in 1· NaCl ⁄ P i . The glucose concentration determination was per- formed in triplicate. Error bars represent standard deviations. Statis- tical significance *P<0.05. Fig. 4. Unrelated CHH gene silencing detection by northern blot analysis 24 h after the treatments. Lane 1, stomach total RNA pool from the saline treated group; lanes 2–4, stomach total RNA sam- ples of three animals in the unrelated dsRNA treated group. The arrow denotes the size of the signal obtained only in stomach total RNA pool from the control animals. A B Fig. 5. CHH gene silencing detection by northern blot analysis 24 h after the treatments. (A) Hybridization with L. schmitti CHH DNA probe. (B) Hybridization of the same nitrocellulose membrane with L. schmitti b-actin DNA probe to compare the expression levels of the CHH and b-actin transcripts. Lane 1, eyestalk total RNA pool from the saline treated group; lanes 2–4, eyestalk total RNA sam- ples of three animals in the CHH dsRNA treated group. Functional analysis of CHH by RNAi J. M. Lugo et al. 5672 FEBS Journal 273 (2006) 5669–5677 ª 2006 The Authors Journal compilation ª 2006 FEBS that it shared more than 70% sequence identity with the CHH from other penaeid species. In addition to an identical number of amino acid residues (72), 13 of these were completely positionally conserved with all other members of the CHH family [18]. We also demonstrated by northern blot assay that the X-organ CHH transcript is present in the eyestalk but not in muscle or stomach. This is in agreement with previous finding that have described the eyestalk X-organ–sinus gland complex as the principal source for the CHH family of neuropeptides [1]. This result also agrees with reports of the eyestalk as the only tissue that produces the (translated) X-organ CHH, excepting pericardial organs, which produce a transla- ted splice variant [14]. At present, there are few studies describing the structure or function of endocrine cells in the digestive system of decapod crustaceans [16]. We decided to examine in this research the CHH gene expression in the stomach, including the fore gut site. Recently, CHH was reported in the endocrine cells of the fore gut and hind gut of Carcinus immediately prior to and during molting, which is responsible for water uptake at this time, thus establishing a physiologically relevant role for a brain ⁄ gut peptide in an arthropod [7,16]. We performed RT-PCR assays with specific CHH primers and observed a DNA fragment at the CHH expected size in stomach tissue. This finding suggests that there may be low level differential expression of CHH in the stomach tissues, which might be molt-stage dependent. The weak DNA band amplified from muscle tissue suggests a similar CHH mRNA expression pattern to the one observed in stomach. In order to analyze the efficiency of gene silencing by direct injection of dsRNA into adult shrimps, we synthesized dsRNA corresponding to CHH mature peptide from L. schmitti. We observed that the group injected with CHH dsRNA showed, 24 h after injec- tion, a significant decrease of the hemolymph glucose concentration compared with the saline treated group (P<0.05). This result was corroborated by northern blot analysis of the eyestalks total RNA sample from the CHH dsRNA treated shrimps and by semiquanti- tative RT-PCR assays. We observed a signal corres- ponding to the CHH transcript in the eyestalks total RNA from the saline treated group. In the CHH dsRNA treated group we did not detect any signal. Similarly we observed the amplification by RT-PCR of a defined band of 260 bp in the saline treated group only. These results suggest the possible complete deg- radation of the CHH transcript because of dsRNA gene silencing mechanism. RNA interference is the phenomenon in which long dsRNA is able to silence cognate gene expression, thereby providing an opportunity to investigate the corresponding protein’s function [29]. In the present study a dramatic CHH knockdown was observed. A complete silencing of the CHH transcript could be achieved in 24 h. Numerous factors could influence the efficacy of interference RNA in vivo, for example, the length of target mRNA, the length and concentration of dsRNA, the region of homology between the dsRNA and the target, as well as other lesser know mechanisms [29]. In recent investigations it was dem- onstrated that the length and dose of dsRNA deter- mine the potency of gene suppression in the shrimp cells in culture, obtaining the best results when larger dsRNA length and higher dosage were used [25]. Similar results to ours were obtained by Dzitoyeva and coworkers by intra-abdominal dsRNA injection B A Fig. 6. CHH gene silencing detection 24 h after the treatments by semiquantitative RT-PCR assays. (A) PCR reaction with L. schmitti CHH specific primer. (B) PCR reaction with Oreochromis mossam- bicus b-actin specific primer. Lane 1, RT-PCR negative control (without template); lanes 2–5, eyestalk cDNA samples from four animals in the CHH dsRNA treated group; lane 6, eyestalk cDNA from the saline treated group; MW1, molecular mass marker 100 bp DNA ladder (Promega); MW2, k HindIII (Heber Biotec S.A.). J. M. Lugo et al. Functional analysis of CHH by RNAi FEBS Journal 273 (2006) 5669–5677 ª 2006 The Authors Journal compilation ª 2006 FEBS 5673 in adult Drosophila that express lacZ transgene in the central nervous system [30]. They observed that the injection of lacZ dsRNA into naive adult wildtype flies completely removed the endogenous intestinal b-galactosidase activity when assayed 72 h after injection. They also observed that higher dosage of dsRNA (0.16–0.32 lg) was effective in abolishing the enteric X-gal staining 24 h after the injection, whereas a lower concentration (0.1 lg) was fully effective after 48 h [30]. Others authors observed that unfed adult female ticks (Amblyomma americanum) injected with cystatin dsRNA and then allowed to partially feed on a rabbit showed approximately 80% decrease in cystatin transcript level when compared to mock-injected ticks or ticks injected with unrelated dsRNA. They suggest that the complete silencing of the gene transcript could not be achieved due to dilu- tion of dsRNA in the feeding stage of the female tick [31]. On the other hand, Acosta and coworkers obtained similar results in a vertebrate aquatic organ- ism; they observed that zebrafish embryos micro- injected with myostatin dsRNA showed, 24 h postfertilization, a drastic reduction in the myostatin transcript level [32]. We also suggest that CHH dsRNA injection can specifically suppress CHH gene function in adult shrimps, because injection of unrelated dsRNA did not result in reduction of blood glucose levels, and in addi- tion, off-target reduction in b-actin was not observed after injection of all dsRNA constructs. Our results show for the first time that dsRNA injections into the abdominal body cavity of adult shrimps can be used to trigger RNA interference and to cause the consequent removal of the respective gene product. This could be used as a powerful tool to study gene function in crustaceans. Experimental procedures Animals Adult shrimps of approximately 10 g were provided by the Cultizaza Company (Tunas de Zaza, Cuba), and were kept alive in aerated seawater until used. Water temperature was maintained between 28° and 30 °C and salinity between 3.3% and 3.5%. Oligonucleotide primers Degenerate primers F-LsCHH [5¢-GCIAA(C ⁄ T)TT(C ⁄ T) GA(C ⁄ T)CCI(T ⁄ A)(C ⁄ G)ITG(C ⁄ T)ACIGG-3¢] and R- LsCHH [5¢-IACIGT(T ⁄ C)TGIAC(A ⁄ G)TGIGC(T ⁄ C)TG (A ⁄ G)TA(C ⁄ T)TC-3¢] were designed based on the amino acid sequence of the CHH mature peptide from L. schmitti [3], and inosine (I) was included in the more degenerate sites. The specific primers used in the control PCR were F-act (5¢-ACACTGTGCCCATCTACGAGGG-3¢), R-act (5¢-CGATCCAGACGGAGTATTTACGC-3¢), F-tub (5¢- CCCTTCCCTCGTCTCCAC-3¢) and R-tub (5¢-GCCAGT GTACCAGTGAAGGGA-3¢). These primers were designed based on tilapia (Oreochromis mossambicus) b-actin gene (GenBank accession number AB037865) and prawn (Mac- robrachium rosenbergii) b-tubulin gene [33] sequences, respectively. In the in vitro transcription reaction and in the RT-PCR assays to determine the CHH tissue expression, the CHH specific primers used were F-CHH (5¢-GCGAA CTTTGATCCGTCGTGC-3¢) and R-CHH (5¢-GACGGT CTGGACGTGGGCCT-3¢). Eyestalk dissection A total of 120 eyestalks were collected from L. schmitti immediately after anesthetizing with the methanesulfonate salt of 3-aminobenzoic ethyl ester dissolved in water. The cuticle and non-neuronal tissues were removed; the dissec- ted eyestalks were ground to fine powder in liquid nitrogen by means of mortar and pestle for RNA extraction. RNA isolation Total RNA from different shrimp tissues was extracted using RNAgents Total RNA Isolation System (Promega, Madison, WI, USA) and was quantified by measuring the absorbance at 260 nm. RT-PCR First-strand cDNA was synthesized from eyestalk total RNA. Five microliters of total RNA and 1 lL oligo (dT) (0.5 lgÆlL )1 ) were incubated at 70 °C for 5 min and placed on ice. The reaction mixture was brought to a volume of 20 lL with 1· Moloney murine leukemia virus (M-MLV) Reverse Transcriptase buffer, 1 lm each dNTP, 1 UÆlL )1 rRNasin, and 15 UÆlg )1 of M-MLV RT (Promega), incuba- ted at 42 °C for 30 min and at 95 °C for 5 min, and then diluted to a final volume of 100 lL. The PCR was carried out using 20 lLof5· diluted RT-mixture, the appropriate PCR buffer to a final concentration of 1· (100 mm Tris ⁄ HCl, 500 mm KCl, pH 8.3), 1.5 mm MgCl 2 , 50 pmoles each designed degenerate oligonucleotide from L. schmitti CHH amino acid sequence, and 2.5 units of Taq DNA polymerase (Heber Biotec S.A., Havana, Cuba). The amplification of CHH cDNA was carried out in 30 cycles as follows: denatur- ation 30 s at 95 °C, 1 min annealing at 65 °C and 1 min of extension at 72 °C, after the initial denaturation at 95 °C for 5 min. Amplification was completed with an additional extension step at 72 °C for 5 min. Functional analysis of CHH by RNAi J. M. Lugo et al. 5674 FEBS Journal 273 (2006) 5669–5677 ª 2006 The Authors Journal compilation ª 2006 FEBS The cycle number for the gene expression study in the shrimps treated with CHH dsRNA was determined by a val- idation test in which the PCR was performed as described but terminated at different cycle numbers. A kinetic profile of the amount of PCR product generated at different PCR cycles was constructed and the cycle number used was chosen within the exponential region of the amplification curve. This was to ensure that the amount of PCR product reflected the amount of template in the original sample. We used 25 cycles for b -actin and 30 cycles for CHH gene. Cloning of PCR amplified DNA fragment The PCR product was cloned using the pGEM-T Easy vector system I kit (Promega). Both strands of the cloned cDNA were sequenced in an automatic DNA sequencer (Amersham Pharmacia, Buenos Aires, Argentina) using a Thermo Sequenase Premixed cycle Sequencer Kit (Amersham Phar- macia) according to the instructions of the manufacturer. Northern blot analysis Northern blot analysis was used to characterize the expres- sion of the shrimp CHH gene. Ten micrograms of each RNA sample were separated on 1.5% formaldehyde agarose gel, transferred to a Hybond N+ membrane (Amersham, Little Chalfont, UK) by overnight capillary blotting and hybridized in Church and Gilbert hybridiza- tion buffer (7% SDS; 1 mm EDTA; 0.5 m phosphate buf- fer, pH 7.2) containing a L. schmitti CHH specific probe at 65 °C overnight. The L. schmitti CHH cDNA was labeled with [ 32 P]dATP[aP] by random priming with Megaprime TM DNA labeling system (Amersham). High stringency (0.1· NaCl ⁄ Cit and 0.1% SDS at 65 °C) washes were performed and membranes were exposed to X-ray (CP-G, Agfa, Gavaert, Belgium) films for 5 days. dsRNA synthesis Single-stranded RNAs were produced from opposing strands of a 216 bp L. schmitti CHH cDNA clone intro- duced into pGEM-T Easy vector (Promega), by in vitro transcription with the T7 and Sp6 polymerases from Ribo- MAX Large Scale RNA Production Systems (Promega). Prior to in vitro transcription the plasmid DNA was linea- rized with the HincII and NaeI restriction enzymes and purified with QIAGEN gel extraction kit (Qiagen, German- town, MD, USA). Afterward, the reaction mixture was treated with RNase free DNaseI, to remove the DNA tem- plate. Then, the mixture was extracted once with phe- nol ⁄ chloroform and once with chloroform, and RNA was precipitated with 2-propanol and dissolved in RNase-free water. Single-stranded RNAs were allowed to anneal by mixing equal amounts of each strand, heating to 100 °C for 1 min, and cooling gradually to room temperature for 3–4 h. Single-stranded RNAs and the annealed RNA (dsRNA) were checked on denaturing agarose gels. To produce a nontarget dsRNA, which was used to investigate possible general effects of off-target silencing, a stomach transcript from a L. schmitti gene that encodes to a chitinase like-protein, was cloned into the pGEM-T Easy vector (Promega); this construct generated a dsRNA of 492 bp in length. Injection of dsRNA into adult shrimps and CHH biological activity detection Adult shrimps were anesthetized before the injection of dsRNA. Hemolymph (100 lL) was removed before injec- tions for baseline measurement of glucose. Afterward, indi- vidual shrimps were injected with 20 lgofL. schmitti CHH dsRNA reaction mixture in 1· NaCl ⁄ P i (137 mm NaCl, 2.7 mm KCl, 4.3 mm Na 2 HPO 4 7H 2 O, pH 7.3) (CHH dsRNA treated group), 20 lg of unrelated CHH dsRNA reaction mixture in 1· NaCl ⁄ P i or with 1· NaCl ⁄ P i solu- tion (placebo group). The injections were placed into the abdominal body cavity. Twenty-four hours after injections, hemolymph (100 lL) was extracted from each shrimp to measure glucose concentration. A glucose oxidase diagnos- tic kit (Sigma, Atlanta, GA, USA), was used to determine glucose concentrations. All glucose determination was car- ried out in triplicate. Statistical analysis Results were presented as mean ± SD. Statistical signifi- cance was assessed by a one-way analysis of variance fol- lowed by Student’s t-test. Acknowledgements We thank the personnel of the Cultizaza Company, of Tunas de Zaza, Cuba, for their help in providing the shrimps. 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