Department of Cellular Biology, University of Georgia, 724 Biological Science Building, 30602, Athens, GA, USA.
Parasit Vectors. 2014 Jan 15;7:24. doi: 10.1186/1756-3305-7-24.
Published data revealed that two of the 243 structural cuticular proteins of Anopheles gambiae, CPLCG3 and CPLCG4, are implicated in insecticide resistance and a third, CPF3, has far higher transcript levels in M than in S incipient species. We studied the distribution of transcripts for these three genes in the tissues of An. gambiae and the location of the proteins in the cuticle itself to gain information about how these cuticular proteins contribute to their important roles. Our data are consistent with CPLCG3/4 contributing to a thicker cuticle thus slowing penetration of insecticides and CPF3 possibly having a role in the greater desiccation tolerance of the M form.
Using RT-qPCR, we established the temporal expression of the genes and by in situ hybridization we revealed the main tissues where their mRNAs are found. Electron microscopy immunolocalization, using secondary antibodies labeled with colloidal gold, allowed us to localize these proteins within different regions of the cuticle.
The temporal expression of these genes overlaps, albeit with higher levels of transcripts from CPF3 in pharate adults and both CPLCG3 and CPLCG4 are higher in animals immediately after adult eclosion. The main location of mRNAs for all three genes is in appendages and genitalia. In contrast, the location of their proteins within the cuticle is completely different. CPF3 is found exclusively in exocuticle and CPLCG3/4 is restricted to the endocuticle. The other CPF gene expressed at the same times, CPF4, in addition to appendages, has message in pharate adult sclerites.
The temporal and spatial differences in transcript abundance and protein localization help to account for An. gambiae devoting about 2% of its protein coding genes to structural cuticular proteins. The location of CPLCG3/4 in the endocuticle may contribute to the thickness of the cuticle, one of the recently appreciated components of insecticide resistance, while the location of CPF3 might be related to the greater desiccation resistance of the M form.
已发表的数据表明，冈比亚按蚊 243 种结构表皮蛋白中的两种，CPLCG3 和 CPLCG4，与杀虫剂抗性有关，而第三种 CPF3 在 M 初期物种中的转录水平远高于 S 期。我们研究了这三种基因在冈比亚按蚊组织中的转录本分布情况，以及这些蛋白在表皮本身中的位置，以获取有关这些表皮蛋白如何发挥其重要作用的信息。我们的数据表明，CPLCG3/4 可能有助于形成更厚的表皮，从而减缓杀虫剂的渗透，而 CPF3 可能在 M 型更高的耐旱性中发挥作用。
使用 RT-qPCR，我们确定了这些基因的时间表达，通过原位杂交，我们揭示了它们的 mRNA 存在的主要组织。使用胶体金标记的二级抗体的电子显微镜免疫定位，使我们能够在表皮的不同区域定位这些蛋白。
这些基因的时间表达重叠，尽管在成虫前体中 CPF3 的转录本水平更高，而且在成虫刚羽化后，CPLCG3 和 CPLCG4 的水平更高。所有三个基因的 mRNA 的主要位置都在附肢和生殖器。相比之下，它们在表皮内的蛋白定位完全不同。CPF3 仅存在于外骨片，而 CPLCG3/4 则局限于内骨片。同时表达的另一个 CPF 基因 CPF4，除了附肢之外，在成虫前体骨片也有 mRNA。
转录本丰度和蛋白定位的时空差异有助于解释冈比亚按蚊将其大约 2%的蛋白质编码基因用于结构表皮蛋白。CPLCG3/4 在内骨片中的位置可能有助于表皮的厚度，这是最近发现的杀虫剂抗性的一个组成部分，而 CPF3 的位置可能与 M 型更高的耐旱性有关。
