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Quantitative Gene Expression of C-Type Lectin in Litopenaeus vannamei

Collected from Shrimp Farms with White Spot Syndrome Virus Disease Outbreak

Article  in  Philippine Agricultural Scientist · March 2014

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PHILIPP AGRIC SCIENTIST ISSN 0031-7454
Vol. 97 No. 1, 92–95
March 2014

Quantitative Gene Expression of C-Type Lectin in Litopenaeus

vannamei Collected from Shrimp Farms with White Spot Syndrome
Virus Disease Outbreak
Yasser C. Cabansag1,2, Jonathan V. Lazaro1 and Apolinario V. Yambot2,*
1
College of Veterinary Science and Medicine, Central Luzon State University, Science City of Muñoz, Nueva Ecija
3120, Philippines
2
Molecular Biology and Biotechnology Laboratory, College of Fisheries, Central Luzon State University, Science City
of Muñoz, Nueva Ecija 3120, Philippines
*
Author for correspondence; e-mail: polyambot@yahoo.com; Mobile: +639088854861
Portion of the undergraduate thesis of YCC under a research project funded by the Philippine Council for Agriculture,
Aquatic and Natural Resources Research and Development (PCAARD), Department of Science and Technology
(DOST), Philippines.

White spot syndrome virus (WSSV) is one of the most devastating diseases affecting the Pacific
white shrimp (Litopenaeus vannamei) worldwide. Because shrimps rely on their innate immunity to
recognize and eliminate different pathogens, the gene expression of an antimicrobial peptide C-type
lectin in L. vannamei collected from shrimp farms with WSSV outbreaks was quantified using real-
time quantitative polymerase chain reaction (RT-qPCR). The study compared the gene expression of
C-type lectin in WSSV-infected and uninfected L. vannamei.
WSSV infection in shrimp from different farms was confirmed using nested polymerase chain
reaction (PCR). A total of 20 WSSV-infected and 20 uninfected L. vannamei samples were subjected
to quantification of C-type lectin gene expression. A significant increase in C-type lectin gene
expression was manifested in WSSV-infected shrimp compared with the gene expression of
uninfected L. vannamei (p<0.05). This increase indicates that the up-regulation of C-type lectin has a
major role as an immune gene against WSSV infection in shrimp farm.

Key Words: C-type lectin, gene expression, Litopenaeus vannamei, Pacific white shrimp, real-time quantitative
polymerase chain reaction, White spot syndrome virus

Abbreviations: PCR – polymerase chain reaction, RT-qPCR – real-time quantitative polymerase chain reaction,
WSSV – White spot syndrome virus

INTRODUCTION the White spot syndrome virus (WSSV) (Lightner and

Litopenaeus vannamei, commonly known as Pacific
white shrimp, is the most widely cultured shrimp species
in the world (Flegel 2006; Wang et al. 2007). Since
shrimp as an invertebrate only possesses innate
immunity, pathogen recognition proteins (PRRs) like
lectins are very important components of the shrimp’s
defense against different pathogens. Calcium-dependent
or C-type lectin I is one kind of lectin in L. vannamei that
can only be expressed in the hepatopancreas (Ma et al.
2007). It is a carbohydrate-binding protein that plays an
important role in shrimp defense against bacterial and
viral infection such as White spot syndrome virus (Ma et
al. 2007; Zhao and Wang 2008).
One of the major problems in culturing penaeid
shrimp is its susceptibility to bacterial and viral diseases,
thereby causing significant losses to the shrimp industry
(Zhao et al. 2009). One of the most devastating viral
diseases affecting the shrimp industry around the world is
Redman 1998). High mortality due to WSSV infection
emerges as the key constraint to the sustained production
of cultured shrimp. Therefore, the study of immune
defense and genes that control the immune competence
of the shrimp during disease outbreak becomes important
(Wang et al. 2007).
In this study, gene expression of C-type lectin I from
WSSV-infected samples collected directly from
commercial farms raising Pacific white shrimp was
determined using real-time quantitative polymerase chain
reaction (RT-qPCR). The gene expression of the C-type
lectin I in WSSV-infected and uninfected shrimp samples
was compared using relative quantification. Since C-type
lectin I is an antimicrobial protein, a part of the shrimp’s
innate immunity, we hypothesized that penaeid shrimp
infected with WSSV has an up-regulated gene expression
of C-type lectin I gene unlike WSSV-uninfected penaeid
shrimp.

92 The Philippine Agricultural Scientist Vol. 97 No. 1 (March 2014)

Gene Expression of Lectin in Infected Shrimp Yasser C. Cabansag et al.

MATERIALS AND METHODS Table 1. Nucleotide sequences of primer pairs used in

the study.
Primer Sequence (5’-3’) Products Reference
A total of 94 Pacific white shrimps were collected from Name (bp)
farms with reported mortalities in the provinces of WSSVF1 5’-GAA ACT ATT GAA 211 Natividad et
Zambales (n=60, harvest size at 30 g), Bulacan (n=17, AAG GCT TTG CCT C-3 al. (2008)
harvest size at 30 g) and Batangas (n=10, PL 18 and n=7, WSSVR1 5’-GTT CCT TAT TTA
30 g) from 2011 to 2012. Surviving and moribund CTA CTA CGG CAA- 3’
WSSVF2 5’- ATG AGA TTG AGT 161
samples were caught for analysis in the laboratory. TTG AGA GAT GCA-3’
Hemolymph (0.1 mL) from each harvestable size WSSVR2 5’- AAC CAA ACA ATC
shrimp was drawn from the dorsal region under the first ATC AGC GAC ACC-3’
abdominal segment using 1-mL sterile syringe; muscle LvCTL1-F 5’-TCT GTC GCC AGT Zhao et al.
tissues of PL 18 were collected. The collected tissues GTT CGT TC-3’ (2009)
LvCTL1-R 5’-CGT ATT CCC ACT
were transferred to a microcentrifuge tube and stored ATT AGG CTC-3’
immediately in liquid nitrogen tank for further processing β-actin F 5’CCA CGA GAC CAC 142 Wang et al.
and WSSV detection was conducted using 2-step nested CTA CAA C-3’ (2007)
PCR. β-actin R 5’-AGC GAG GGC AGT
Genomic DNAs from each hemolymph and muscle GAT TTC-3’
tissue (PL 18) of Pacific white shrimps were extracted
using Wizard Genomic DNA Purification Kit (Promega, Relative gene expression was calculated using the
Madison, WI, USA) following the manufacturer’s 2-∆∆CT method as proposed by Livak and Schmittgen
instruction. The concentration and quality of the (2001). Comparison between infected and uninfected
extracted DNA were measured at A260nm/A280nm groups was conducted using Student’s t test (p< 0.05).
(NanoDrop 2000c Spectrophotometer, Thermo Scientific,
USA) to ensure the amount and purity of the extracted
DNA. One-step and 2-step nested PCR were conducted RESULTS AND DISCUSSION
using gene specific primers for WSSV (Table 1).
Thermal profiles for 1-step and 2-step nested PCR All 17 shrimps from Bulacan were negative for WSSV in
were as follows: initial denaturation at 95 °C for 1 min, the 1-step and 2-step nested PCR. Only one sample
followed by 25 cycles of denaturation at 95 °C for 30 s, (1.7%) from Zambales was WSSV positive in the 1-step
annealing at 60 °C for 1 min and extension at 72 °C for 1 PCR and none from Batangas. However, after 2-step
min and one cycle of final extension at 72 °C for 5 min nested PCR, 18 (30%) samples from Zambales were
WSSV positive and 17 (100%) samples from Batangas
(Tapay et al. 1999). PCR products from 1-step PCR were
were WSSV positive (Table 2). Gel electrophoresis after
used as template of 2-step nested PCR. PCR products 2-step nested PCR confirmed the expected amplicon with
were then subjected to electrophoresis. a length of 161 bp.
Relative gene expression of C-type lectin I gene was
C-type Lectin I Gene Expression Quantification calculated using β-actin as internal control. Results
A total of 20 WSSV-infected and 20 uninfected L. showed a significant 9.2-fold increase in gene expression
vannamei were randomly selected from the samples from 20 WSSV-infected L. vannamei and only a 1.7-fold
weighing 30 g (± 2 g) and subjected to quantification of increase in gene expression from 20 uninfected L.
C-type lectin gene expression. Hepatopancreas was vannamei (p< 0.05) relative to the control (Fig. 1).
collected from each shrimp sample transferred into WSSV is a highly infectious disease affecting
microcentrifuge tubes and stored immediately in liquid cultivated shrimps. Interestingly, during the sample
nitrogen tank until RNA extraction. Total RNA from collection, no “white spots” as characteristic gross sign of
WSSV-infected and uninfected groups was obtained from WSSV infection was observed but after the 2-step nested
each hepatopancreas using SV Total RNA Isolation PCR, results showed most samples were WSSV positive.
System (Promega/Madison, WI, USA) following the Detection of WSSV from Batangas and Bulacan samples
manufacturer’s instruction. The concentration and quality only after 2-step nested PCR (except for one sample from
of the RNA was measured at A260nm/A280nm (NanoDrop Zambales which was 1-step positive) suggests a low level
2000c Spectrophotometer, ThermoScientific, USA). First infection which is supported by the study of Thakur et al.
strand cDNA was reverse-transcribed using 1 µg total (2002) and corroborated by Tsai et al. (1999). With this,
RNA as template by iScript™ cDNa Synthesis Kit (Bio- early detection of WSSV through molecular approach is
Rad Laboratories, Inc., CA, USA). deemed important to avoid massive mortality and future
For quantification of C-type lectin gene, gene economic loss.
specific primers for C-type lectin I were used (Table 1). Shrimp immune system depends entirely on the
Quantification was done using LightCycler®480 Real- innate immunity and the defense mechanisms are
Time PCR with the following thermal profiles: pre- initiated by cell surface host proteins that include C-type
incubation at 95 °C for 5 min, followed by 45 cycles of lectin. Several kinds of lectin in shrimp are involved in
denaturation at 95 °C for 10 s, annealing at 60 °C for 15 s innate immune responses such as promotion of
and extension at 72 °C for 25 s. β-actin gene was used as phagocytosis, nodule formation and encapsulation (Gross
internal control (Wang et al. 2007). et al. 2001; Mistry et al. 2001; Jack and Turner 2003;

The Philippine Agricultural Scientist Vol. 97 No. 1 (March 2014) 93

Gene Expression of Lectin in Infected Shrimp
Table 2. Detection of White spot syndrome virus
(WSSV) using 2-step nested polymerase chain
reaction (PCR).
2-step Nested PCR Results
Collection Collected (WSSV Positive)
Sites Samples
1st Step 2nd Step
Zambales 60* 1 (1.7%) 18 (30%)
Batangas 17** 0 17 (100%)
Bulacan 17* 0 0
*
At harvestable size (30 g)
**
Muscle tissue (PL 18) (n=10) and at harvestable size (30 g) (n=7)
Fig. 1. Gene expression of C-type lectin I from White spot
syndrome virus (WSSV)-infected and uninfected
samples. Gene expression of WSSV-infected (n =
20) versus uninfected (n = 20) Litopenaeus
vannamei. Values (bar) show mean ± standard
error of mean (SEM) of WSSV. Means without a
common letter differ (p<0.05, Student’s t-test).
Zhao et al. 2009; Yang et al. 2011; Wang et al. 2013). In
Penaeus monodon, PmAV lectin gene is involved in
virus resistance (Luo et al. 2003) while in
Fenneropenaeus chinensis, FcCLec-1 and FcCLec-2
genes have antibacterial properties (Liu et al. 2007). In L.
vannamei, C-type lectin I or LvCTL1 has both
antibacterial and antiviral properties (Zhao et al. 2009;
Wei et al. 2012). C-type lectin I and FcCLec-1 have two
carbohydrate recognition domains while PmAV and
FcCLec-2 contain a single carbohydrate recognition
domain. The up-regulation of C-type lectin in naturally
WSSV-infected L. vannamei collected in farms in the
present study is supported by Luo et al. (2003), Liu et al.
(2007) and Ma et al. (2007) on similar up-regulation of
lectins such as PmAV, FcCLec-1 and C-type lectin
(LvLT), respectively, in artificially WSSV-infected
shrimps. The quantity of the gene expressed reflects the
increase in the amount of lectin secreted, which indicates
a major role in enhancing the immunity of the shrimp to
resist the infection caused by WSSV. This study is the
first report on the up-regulation of C-type lectin in L.
vannamei cultured in farms with actual outbreaks of
WSSV.
94
Yasser C. Cabansag et al.
CONCLUSION
Up-regulation of C-type lectin in WSSV-infected L.
vannamei indicates that it is functioning as an immune
gene against WSSV infection. This finding can be used
as baseline information in the genetic improvement of
Pacific white shrimp with regard to selection of
molecular markers for disease resistance.
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