Isolation and identification of genes expressed during diapause in horn fly, Haematobia irritans (L.)

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Kalischuk-Tymensen, Lisa
University of Lethbridge. Faculty of Arts and Science
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Lethbridge, Alta. : University of Lethbridge, Faculty of Arts and Science, 2001
There is a discrepancy in the current literature concerning the stage of development in which horn flies arrest during pupal diapause. A study was therefore conducted to describe the morphologies of horn fly pupae and its central nervous system (CNS) throughout nondiapause pupal development and diapause. Morphologies of diapausing pupae and CNS indicated that developmental arrest occured early in pupal development during the interval between head eversion and pupal-adult apolysis. Morphological descriptions are necessary for defining compariable tissues between nondiapausing insects and diapausing insects. These tissues can then be used for molecular differential analysis to determine genes specific to either diapause or nondiapause. One such differential analysis technique, subtractive hybridization, was used to isolate putative diapause up-regulated genes from the horn fly. Seven different cDNAs were closed and partially sequenced. Comparisons of the cDNA sequences with known DNA and protein sequences indicated homology with transferrin, cytochrome oxidase I, Kunitz family serine protease inhibitor, tyrosine hydroxylase (TH), and carboxylensterase. Two cDNAs did not have homology to entries in DNA and protein databases. Northern blot analyses were used to study expression of each gene by probing total RNA extracted from whole pupae throughout nondiapause pupal development and diapause. Expression of TH was also determined in total RNA extracted from CNS tissue of nondiapausing and diapausing pupae. Cytochrome oxidase was equally expressed in nondiapause and diapause destined pupae, and therefore not considered to be a diapause up-regualted gene. Expression patterns differed slightly for each of the remaining clones; however, expression tended to be highest in diapause destined pupae during pupation compared to nondiapausing pupae. These genes and their products are involved in many aspects of insect phsiology including metamorphosis, melanization and sclerotization of the puparium and cellular defense. The possible functions of these genes and products are discussed in the context of the diapause process.
xii, 92 leaves : ill. ; 28 cm.
Dissertations, Academic , Horn fly -- Dormancy , Diapause , Gene expression