New Horizons in Insect Science Towards Sustainable Pest Management

190 K. M. Ponnuvel et al.

homology and a total of 29 genes were identi-
fied from the diapause and non- diapause SSH
libraries of which 17 were non-diapause specif-
ic and 12 were diapause specific (Tables 2 , 3 ).
The subtractive genes identified were classified
into six functional groups (regulatory, food uti-
lization, stress response, metabolic, ribosomal,
and transposable elements) (Table 4 ). Based on
the above classification, specific primers were
designed for 11 nonredundant genes (Table 1 )
and validated through qPCR.

Confirmation by QPCR Analysis

The upregulation and downregulation of the
11 putative genes identified from the diapause
and non-diapause subtracted libraries were con-
firmed through qPCR. In most cases, qPCR
analysis confirmed the upregulation or down-
regulation of the cDNAs that were identified
through SSH. However, in one case, the qPCR
results showed a different pattern where expres-
sion was observed in both diapause and non-dia-
pause conditions.

Diapause Upregulated Genes

The following genes were upregulated during

diapause: one regulatory gene (40S ribosomal

protein S19), two stress responsive genes (heat

shock cognate 71 kDa protein and ubiquitin

c-terminal hydrolase), two metabolic genes (sim-

ilar to chitin metabolic process and chitinase do-

main containing protein), six ribosomal protein

genes (Bm ribosomal protein L41, 60S ribosom-

al protein L8,L18,L27a, L13, ribosomal protein

L14), one transposable gene element (negative

regulation of transcription), and two genes with

unknown function (D-37 and D-57) (Fig. 1 ).

Non-diapause Upregulated Genes

The following genes were upregulated during

non-diapause: 11 regulatory genes (pseudouridine

synthase, remodelling, and splicing factor 1, 40S

ribosomal protein S14 and S5, RNA binding pro-

tein, translationally controlled tumor protein ho-

molog, eukaryotic translation elongation factor,

nucleosome assembly protein, nascent protein,

B. mori profilin protein, Bm acyl-coenzyme A

dehydrogenase), one food utilization gene (taste

receptor type 2 member 117), two stress response

Table 1 Primers used in real-time qPCR for confirmation of differentially expressed genes
Gene Forward primer Reverse primer
Propanediol utilization
protein

5′TGTCTACCATCGTGCCAAAG3′ 5′CATGCATTCGTCAAACGAGA3′

Ubiquitin family protein 5′GCCATCTTCAAGCTGTTTGC3′ 5′GAGGTGGCATGCAGATCTTT3′
Translationally controlled
tumor protein homolog

5′ATATTCCATCATGGCAACCA3′ 5′GTAGCAAAATTGGAAGAGAAGG3′

Heat shock cognate 71 kDa
protein

5′CCATACGCTCGATCTCTTCC3′ 5′GTGAGCGTGCTATGACCAAA3′

60S ribosomal protein L3 5′CAGCCTCCACGATCTCTTTC3′ 5′TCGTCATCGTGGTAAGGTCA3′
40S ribosomal protein S14 5′AATGCGGCCAATCTTCATAC3′ 5′GGCACAGGATGTAGCAGAGA3′
Ubiquitin c-terminal
hydrolase

5′CGGAGCTATTTCAGAGCACA3′ 5′TGGAGCTGTTGTGAAATTCG3′

Protein coding gene 5′TGATCTAGCAGTAGAGGACCAA3′ 5′GGTCATGAACTAGAGTCCACAGG3′
Chitinase A precursor 5′TGGCAGAGGTCAACTCGTAA3′ 5′CTTCGATGGTGTCGACATTG3′
Negative regulation of
transcription

5′GCGATAAGAAGGCCACAGTT3′ 5′ACATACAGGCTTCCCGATTT3′

Eukaryotic translation
elongation factor

5′ACGTTGTAGGGCTTGCTCTG3′ 5′TGAAGGCCTACCTACCTGTCA3′