192 K. M. Ponnuvel et al.
genes (ubiquitin family protein, polyubiquitin 4),
two metabolic genes (propanediol utilization pro-
tein, chitinase A precursor), and one ribosomal
gene (ribosomal protein L23Ae) (Fig. 2 ).
Genes Unchanged in Diapauses
One ribosomal gene, 60S ribosomal protein L13,
had its expression almost similar in both diapause
and non-diapause conditions (Fig. 1 ).
The results presented herein provide a few
initial clues about the molecular events that
characterize the egg diapause B. mori. Twenty-
nine genes have been identified by SSH whose
expression patterns were confirmed by qPCR.
Twelve genes were upregulated during dia-
pause, 17 during non-diapause, two genes were
with unknown function, and one gene remained
unchanged during diapause and non-diapause.
The above 32 genes were characterized into six
functional groups. The qPCR analysis confirmed
the expression of 11 genes selected for analysis.
Regulatory Genes
Identification of genes regulating the molecular
mechanism may prove useful in understanding
how B. mori can survive in a prolonged inactive
state. A few ribosomal proteins have functions in
regulating cell growth and death in addition to
their roles in translation (Naora and Naora 1999 ;
Horino et al. 1998 ). The appearance of ribosom-
al proteins in the SSH libraries suggests a pos-
sible contribution of these proteins in regulating
the embryonic diapause of B. mori. In an earlier
study on adult diapause of Culex pipiens, it was
observed that two ribosomal proteins have been
downregulated before diapause termination (Ro-
bich et al. 2007 ). Several studies have document-
ed the presence of diapause-associated proteins
from the fat body and haemolymph of diapausing
insects (Brown and Chippendale 1978 ; Brown
1980 ; Osir et al. 1989 ; Salama and Miller 1992 ;
Palli et al. 1998 ; Levenbook 1985). All these have
proven to be storage proteins that are synthesized
before the onset of diapause and are then utilized
when development resumes at the termination
of diapause. In this study, three 40S ribosomal
proteins involved in regulatory role were found,
one from diapause and two from non-diapause.
The qPCR analysis of 40S ribosomal protein S14
showed an upregulation during non-diapause
supporting the earlier reported data.
When diapause is terminated, one would ex-
pect to see a major shift in the patterns of gene
expression. The insect rapidly increases its
metabolic rate and promptly initiates develop-
ment. Thus, one would predict that the genes
involved in the mechanisms that suppress devel-
opment would be switched off and new sets of
genes involved in initiating development would
be switched on (Denlinger 2000 ). From this per-
spective, 11 regulatory genes have been identi-
fied during the non-diapause period.
Pseudouridine synthases catalyze the isomeri-
zation of uridine to pseudouridine (Psi) in a vari-
ety of RNA molecules (Ramamurthy et al. 1999 ).
Depletion of the minifly (mfl) encoded pseudouri-
dine synthase gene of Drosophila causes severe
reduction in size by decreasing both the number
and size of wing cells suggesting that a compo-
nent of the pseudouridine synthase loss of func-
tion phenotype causes defects in notch signalling
(Tortoriello et al. 2010 ). Through qPCR analysis,Clone ID Size (bp) Putative identity Percent identity E-value Matched EST clone ID
D–53 216 60S ribosomal protein L13 99 2e–53 wdV30635
2D–20 133 PREDICTED: similar to ubiqui-
tin c-terminal hydrolase
98 8e–16 FWDP02_T7_O15D–36 140 Chitinase domain-containing
protein 1
96 4e–67 XP_001869617.1Table 3 (continued)