Molt-related chitin synthase and chitinase genes

of whiteleg shrimp Penaeus vannamei

Jorge Gustavo Rocha-Estrada *, Julio Humberto Córdova-Murueta1, Fernando Luis
1

García-Carreño1, Gloria Yepiz-Plascencia2 and Alma Peregrino-Uriarte2.

1
Biochemistry Lab. Centro de investigaciones Biológicas del Noroeste, (CIBNOR); La Paz, B.C.S., Mexico.
2
Molecular Biology of Aquatic Organisms. Centro de investigaciones en alimentacion y desarrollo (CIAD); Hermosillo, Sonora,
Mexico. Pacific Fisheries and Technologists meeting, San Francisco, CA, USA. Feb, 08.

INTRODUCTION MATERIALS, METHODS AND RESULTS

Crustaceans grow discontinuously by losing Identification of A B C D0-D1 D2-D3
and regeneration of exoskeleton in a cyclic molting stage in shrimps
process known as molt cycle. The whole cycle by the structure of the
is divided into stages A through D3, and it is uropods in an 1
stereoscope (3x zoom).
known to induce biochemical, physiological
and behavioural changes such as gene
expression or feeding, among many others1,
therefore, it is a major event in crustaceans’ Methodology for sample
lifecycle. preparation. RNA DNA free cDNA
Crustacean exoskeleton contains chitin as a RNA
major component. Chitinases (E.C. 3.2.1.14) DNAse I SSRT III
and chitin synthase (E.C. 2.4.1.16) are
responsible for exoskeleton chitin cleavage 2 3 4
and synthesis, respectively2. 1. Sampling 6 organisms in each molt stage.
2.Dissection of midgut gland and cuticular tissue.
A)
B) 3.RNA extracion and analysis in 1% agarose denaturing gel electrophoresis to detect
integrity.
4.Removal of contaminant genomic DNA with DNAse I and cDNA synthesis.

A) Domains of chitinase 3 SEARCHING FOR MOLT-RELATED GENES AND THEIR CHARACTERISTICS

B) Structure and domains
of chitin synthase4.
The study of chitin synthase and chitinase
genes can provide insights about the
coordination of two opposite function proteins
that participate in the molting cycle, a process
of basic (research in physiology) and applied
(aquaculture) relevance.
OBJECTIVES
To identify the molting related chitin synthase
(PvChS) and chitinase (PvChi) genes from
whiteleg shrimp Penaeus vannamei and
quantify the mRNA of these genes during the
molt cycle.
Ch1 Chi2 Chi3 • Three chitinase genes (Chi1, Chi2, Chi3) have been
previously reported for Penaeid shrimps (P. japonicus, P.
monodon). As Chi1 and Chi3 were found in midgut gland
mRNA, the proteins they encode could have digestive
function. Chi2, which is encoded in cuticular tissue, is more
likely to be involved in molting5.
• In Penaeus vannamei, we found these three chitinase genes.
• PvChi2 mRNA (99% simmilar to Penaeus japonicus Chi2)
Chi2 ChS was found in cuticular tissue (1), but not in midgut gland
Ct Mg Ct Mg (2).
• Chitin synthase (ChS) gene or protein has no been reported
for any crustacean
• PvChS mRNA (86% similar to Manduca sexta ChS) was
found in both cuticular tissue (3) and midgut gland (4).
Primers for ChS and Chi2 were tested by PCR using cDNA(c)
or gDNA (g). In both cases, larger products were generated
when using gDNA. Nucleotide sequence of these fragments
that is under process will define their sequence and length.

Conclusion: So far we have identified 3 chitinase isoform genes, one of them being expressed in cuticular tissue,
and with a putative rol in the molting cycle. We have also found one chitin synthase gene. This is the first report of a
crustacean chitin synthase gene or protein. In the future, we aim to evaluate the expression pattern of both genes
during the molt cycle and to obtain full-lenght sequences of Penaeus vannamei ChS and Chi2 cDNA.

AKNOWLEDGMENTS
CONACYT grant 45964 (Penaeus vannamei
transcriptome); partners from BQ lab at La Paz,
B.C.S. and AMB lab at Hermosillo, Son. Special thanks
to M. E. Luna for support.
REFERENCES
1. Chang, ES (1995) Physiological and biochemical changes during the molt cycle in decapod crustaceans: An overview. Journal of Experimental Marine Biology and Ecology 193: 1–14.
2. Bolognesi R, Arakane Y, Muthukrishnan S, Kramer KJ, Terra WR & Ferreira C (2005) Sequences ofcDNAs and expression ofgenes encoding chitin synthase and chitinase in the midgut
of Spodoptera frugiperda. Insect Biochemistry and Molecular Biology 35: 1249-1259.
3. Arakane Y, Zhu Q, Matsumiya M, Muthukrishnan S & Kramer KJ (2003) Properties of catalytic, linker and chitin-binding domains of insect chitinase. Insect Biochemistry and Molecular
Biology 33: 631–648.
4. Merzendorfer, H (2006) Insect chitin synthase: a review. J Comp. Physiol. B 176: 1-15.
5. Watanabe, T & Kono, M (1997) Isolation of a cDNA encoding a chitinase family protein from cuticular tissues of the kuruma prawn Penaeus japonicus. Zoological Science, 14, 65-68.