Upregulation of two actin genes and redistribution of actin during diapause and cold stress in the northern house mosquito, Culex pipiens
Introduction
The cytoskeleton has significant roles in nuclear/cell division, cell signaling, motility and polarity of cells, and cell shape (Amos and Amos, 1991; McIlwain and Hoke, 2005; Ramaekers and Bosman, 2004). Low-temperature alterations of the cytoskeleton have been noted in several species of plants and animals, and these changes appear to be critical for low-temperature survival. For example, actin filaments and microtubules of tobacco cells exposed to 0 °C for a few minutes are depolymerized immediately, and after recovery at 25 °C, the filaments and microtubules are repolymerized (Pokorna et al., 2004). In winter wheat (Triticum aestivum L.), cold acclimation is achieved by disassembly of the microtubules in response to low temperature (4 °C) and the reorganization of the microtubules into a cold-tolerant arrangement (Abdrakhamanova et al., 2003). Cells of homeothermic animals depolymerize most of their microtubules at low temperatures, while poikilotherms, by contrast, frequently assemble microtubules at low temperature and thereby prevent depolymerization (Pucciarelli et al., 1997). For example, both the poikilothermic Antarctic fish, Notothenia coriiceps, (Detrich et al., 1989) and the Antarctic ciliate, Euplotes focardii, (Pucciarelli et al., 1997) undergo microtubule assembly in response to low temperatures. In addition, unique cold-adapted tubulins have been found in some organisms such as the Antarctic ciliate, E. focardii (Pucciarelli and Miceli, 2002).
Little is known about cytoskeletal responses of insects to low temperature. Our interest in potential cytoskeletal changes in insects as a low-temperature adaptation was prompted by the observation that actin is upregulated during adult diapause of the northern house mosquito, Culex pipiens (L.) (Robich et al., 2006). In this paper, we report the full length sequences of that actin and an additional actin, both of which are shown to be diapause upregulated. In addition, we use fluorescent staining and confocal microscopy to note changes in actin distribution and abundance in Cx. pipiens that have entered diapause and/or have been exposed to low temperature.
Section snippets
Insect rearing
Our anautogenous colony of Cx. pipiens (Buckeye strain) was maintained at 25 °C, 75% R.H., with 15L(light):9D(dark) (Nondiapause, 25 °C). Larvae and adults were reared as described by Robich et al. (2006). To induce diapause, the second instar larvae were moved to an environmental room at 18 °C, 75% R.H., with 9L:15D (Diapause, 18 °C). To eliminate temperature as a variable, a third group of mosquitoes was reared at 18 °C, 75% R.H., with 15L:9D (nondiapause, 18 °C). Adults from the diapause 18 °C
Clone identification
The full-length cDNA of actin 1 obtained by RACE is a 1247 bp sequence (GenBank accession number DQ385449) that encodes 391 amino acids. The open reading frame (ORF) of actin 1 is 1156bp, from nucleotides 26 to 1156, with a 25 bp 5′untranslated region and a 91 bp 3′untranslated region including the poly-A tail. The putative polyadenylation signal (AATAAA) was identified at nucleotide positions 1209–1214. A multiple sequence alignment of the deduced actin 1 amino acid sequence with the sequences of
Discussion
Actin is a highly conserved protein in eukaryotic cells. Three main isotypes (α, β, and γ) have been reported (Carlini et al., 2000), and they play important roles in a range of cellular functions including muscle contraction, cell motility, cytoskeletal structure, cell division, intracellular transport, and cell differentiation (Herman, 1993). The actin gene family consists of 8–44 different genes in plants (Reece et al., 1992), but insects have, at most, six actin genes (Fyrberg et al., 1980
Acknowledgments
This work was supported in part by NSF Grant 10B-0416720 and NIH Grant R01-AI058279.
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Current address: Harvard School of Public Health, Immunology and Infectious Disease, 665 Huntington Avenue, Boston, MA 02115, USA.
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Current address: BioSciences Research Laboratory, USDA-ARS, 1605 Albrecht Boulevard, Fargo, ND 58105, USA.