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:
(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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