Identification and characterization of heat shock 70 genes in Aedes aegypti (Diptera: Culicidae)

Abstract

Heat shock genes are highly evolutionarily conserved and are expressed to varying degrees in all organisms in response to stress. Heat shock 70 (hsp70) genes have been well characterized in a number of organisms, most notably Drosophila melanogaster, but not as yet for any of the major arthropod-borne viral mosquito vectors. To identify hsp70 genes in the yellow fever mosquito, Aedes aegypti (Diptera: Culicidae), basic local alignment searches of the Ae. aegypti genome were performed using D. melanogaster Hsp70 protein sequences as query. Two clusters of six previously unannotated AaHsp70 genes were identified and found to be organized into three pairs of nearly identical open reading frames, which mapped to two genomic scaffolds. Consistent with a designation as heat shock genes, no detectable level of expression of AaHsp70 genes was observed under normal rearing conditions (28 degrees C), with robust expression observed with a heat shock of 37-39 degrees C. Northern analysis showed heat-inducible expression of putative AaHsp70 genes at all life stages and in all tissues tested in a time- and temperature-dependent manner. Monitoring of AaHsp70 gene expression levels in field-caught Ae. aegypti may serve as a general marker for stress. In addition, promoter sequences from AaHsp70 genes may be used to control the expression of transgenes in an inducible manner.

Fig. 1

Genome organization and relationship of AaHsp70 genes. Arrangement of AaHsp70 genes in Ae. aegypti. AaHsp70 genes are indicated by solid arrows. Lines under each gene indicate areas reamplified, cloned, and sequenced in this project. Distances between genes are labeled in base pairs. n/a, not available due to gaps in sequence assembly.

Fig. 2

Alignment of Ae. aegypti, An. gambiae, and D. melanogaster hsp70–predicted proteins. Gene sequences listed in the Materials and Methods section were aligned using MEGA 4.1 and bootstrap analysis was performed using the neighbor-joining method with 1,000 replications. Bootstrap support (>80) is listed on the node of each branch, where applicable. AaHsp70Bb′ (repaired) indicates the predicted protein after removing two deleterious mutations.

Fig. 3

Sequence comparison of AaHsp70 genes. Complete transcripts for six AaHsp70 genes were aligned using ClustalW (Geneious, Biomatters, Aukland, New Zealand) to produce an identity graph. The start of each open reading frame is marked with a green arrow, and the end with a red arrow. Dark marks on lines representing each AaHsp70 gene indicate base changes from the consensus AaHsp70 sequence.

Fig. 4

Expression levels of AaHsp70 genes increase with heat shock temperature. Female Ae. aegypti were heat shocked at temperatures from 35 to 39°C, and total RNA was hybridized to a ³²P-labeled probe common to all AaHsp70 genes. All blots were hybridized simultaneously to ensure equivalent exposure and probe normalization (specific activity = 5.4 × 10⁷ CPM). Control mosquitoes (cnt) were not heat shocked. Ethidium bromide–stained rRNA loading controls are shown below each blot.

Fig. 5

Northern analysis of individual AaHsp70 paralogs. Northern analysis was performed using a ³²P-labeled probe derived from the 3′ untranslated regions of AaHsp70 paralogs. Exposure times were standardized based on specific activity of each probe (7.3 × 10⁷–3.0 × 10⁸ CPM). Mosquitoes were heat shocked at 39°C, and control mosquitoes (cnt) were not heat shocked. Ethidium bromide–stained rRNA loading controls are shown below each blot.

Fig. 6

Tissue and life stage expression of AaHsp70 genes. Expression in specific tissues (A) and life stages (B) was analyzed by Northern analysis using a ³²P-labeled probe designed to hybridize to all AaHsp70 genes. Tissues were dissections from female Ae. aegypti that had been heat shocked at 39°C for 1 h or kept at 28°C as controls (C). Tissues include whole body (WB), head (H), salivary glands (SG), thorax without salivary glands (T), ovaries (O), and midguts (M). RNA from males (¤), third and fourth instar larvae (L), and pupae (P) heat shocked at 39°C for 1 h was used for the life stage study. Ethidium bromide–stained rRNA loading controls are shown below each blot.