Elsevier

Journal of Insect Physiology

Volume 52, Issues 11–12, November–December 2006, Pages 1226-1233
Journal of Insect Physiology

Upregulation of two actin genes and redistribution of actin during diapause and cold stress in the northern house mosquito, Culex pipiens

https://doi.org/10.1016/j.jinsphys.2006.09.007Get rights and content

Abstract

Two actin genes cloned from Culex pipiens L. are upregulated during adult diapause. Though actins 1 and 2 were expressed throughout diapause, both genes were most highly expressed early in diapause. These changes in gene expression were accompanied by a conspicuous redistribution of polymerized actin that was most pronounced in the midguts of diapausing mosquitoes that were exposed to low temperature. In nondiapausing mosquitoes reared at 25 °C and in diapausing mosquitoes reared at 18 °C, polymerized actin was clustered at high concentrations at the intersections of the muscle fibers that form the midgut musculature. When adults 7–10 days post-eclosion were exposed to low temperature (−5 °C for 12 h), the polymerized actin was evenly distributed along the muscle fibers in both nondiapausing and diapausing mosquitoes. Exposure of older adults (1 month post-eclosion) to low temperature (−5 °C for 12 h) elicited an even greater distribution of polymerized actin, an effect that was especially pronounced in diapausing mosquitoes. These changes in gene expression and actin distribution suggest a role for actins in enhancing survival of diapausing adults during the low temperatures of winter by fortification of the cytoskeleton.

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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      In addition, the GO terms related to actin filament organization were enriched among the downregulated genes (in response to both supercooling and freezing). Cytoskeletal stabilization and rearrangement has emerged as a recurring theme linked to both insect cold acclimation (Kim et al., 2006; Des Marteaux et al., 2017, 2018a,b), and cold injury repair (Kayukawa and Ishikawa, 2009; Teets et al., 2012), particularly with regards to defense of polymerized actin. KEGG mapping failed to identify any enriched pathway downregulated in response to supercooling.

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    1

    Current address: Harvard School of Public Health, Immunology and Infectious Disease, 665 Huntington Avenue, Boston, MA 02115, USA.

    2

    Current address: BioSciences Research Laboratory, USDA-ARS, 1605 Albrecht Boulevard, Fargo, ND 58105, USA.

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