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

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.. In vi

hyperglycemic hormone cDNA from Litopenaeus schmitti Functional analysis by double-stranded RNA interference technique Juana M Lugo1, Yuliet Morera2, Tania Rodrı´guez2, Alberto Huberman3, Laida Ramos2 and

Mario P Estrada1

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], digesreproduc-tion [10], osmoregulareproduc-tion [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 OctoOcto-ber 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-mittiis 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, suggestcorrespond-ing 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.

(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 limosusdifferent 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 schmittiCHH 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

CHHgene 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

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.

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 micropu(Boophi-lus) 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.

(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

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.

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.).

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)

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 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

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)

on ice The reaction mixture was brought to a volume of

20 lL with 1· Moloney murine leukemia virus (M-MLV)

diluted to a final volume of 100 lL The PCR was carried out using 20 lL of 5· diluted RT-mixture, the appropriate PCR buffer to a final concentration of 1· (100 mm Tris ⁄ HCl,

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:

5 min Amplification was completed with an additional

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

DNA labeling system (Amersham) High stringency (0.1·

and membranes were exposed to X-ray (CP-G, Agfa,

Gavaert, Belgium) films for 5 days

dsRNA synthesis

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

precipitated with 2-propanol and dissolved in RNase-free

water Single-stranded RNAs were allowed to anneal by

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 lg of L schmitti CHH

dsRNA treated group), 20 lg of unrelated CHH dsRNA

solu-tion (placebo group) The injecsolu-tions 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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