GENES CONTROLLING SOCIAL BEHAVIOR IN ANTS

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。具体目标#1：确定预测任务行为的蜜蜂基因的火蚁同源物。我们先前分离到的与蜜蜂或果蝇觅食基因(蛋白激酶G，PKG)相似的PCR产物1PXIR被用来设计一个PCR引物来鉴定PKG的火蚁同源物的更长序列。1PXIR对应于蜜蜂PKG mRNA终止密码子的约500个核苷酸的位置。根据Apis PKG序列设计了23个核苷酸的5‘端和27个核苷酸的3’端引物，扩增出300-400个碱基的扩增产物。将其插入到pGEM序列载体中。未获得与PKG对应的DNA序列。我们已经设计了新的基于蜜蜂和果蝇觅食基因的PKG的聚合酶链式反应引物，而不是1PXIR，序列的分离正在进行中。具体目标2：将已知的社会任务特定脑区大小变化与基因表达差异联系起来。我们从工作人员中提取mRNA，以开发pBluescrip中的cDNA文库，用于后续的差异显示实验。获得了含有插入物的菌落，但我们通过测序抽查的那些插入物太小而没有用。利用亲和层析技术制备并纯化了火蚁载脂蛋白III兔抗体。用兔抗载脂蛋白III为第一抗体，AlexaFluor 594驴抗兔Ig G为第二抗体(为避免角质层黄色自发荧光)，对雄性和工人头部冰冻切片进行免疫细胞化学检测。在男性和工人的角质层下面的上皮层，以及整个大脑中都发现了这种表达。将多炔女王特异性疏水配体结合蛋白GP-9b的编码序列插入pET22表达载体，并在大肠杆菌中表达。表达的蛋白将用于产生兔抗体。具体目标#3：抑制特定基因的表达，以测试社交行为的丧失。开发了一种行为测试，用于随后评估RNAi实验的效果。我们测量了自相关函数，c(T)=(1/T)？[A(T)A(t+t)]，其中A是人工蚁穴中定义区域内的蚂蚁数量，T是总时间，t是特定时间点，t是可变时间偏移量。我们发现，c(T)以指数函数的形式衰减，相关时间为tc。通过对随机运动的建模，我们证明了TC与一群蚂蚁的平均速度成正比。因此，通过测量数字自相关函数，我们可以获得关于一群蚂蚁的集体运动的信息。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Specific aim #1: Identify fire ant homologs of bee genes predictive of task behavior. Our previously isolated PCR product, 1PXIR, which is similar to the honey bee or Drosophila foraging gene (protein kinase G, PKG) was used to design a PCR primer to identify a longer sequence of the fire ant homolog of PKG. 1PXIR corresponds to a position that is about 500 nucleotides from the stop codon of honey bee PKG mRNA. Using a 23 nucleotide 5' primer and a 27 nucleotide 3' primer based on the Apis PKG sequence, we obtained a range of 300-400 bp PCR products. These were inserted into a pGem sequence vector. No DNA sequences corresponding to PKG were obtained. We have designed new PCR primers for PKG based the Apis and Drosophila foraging gene rather than 1PXIR, and the isolation of the sequence is in progress. Specific aim #2: Correlate known social task-specific size changes in brain regions with differences in gene expression.We extracted mRNA from workers to develop a cDNA library in pBluescript for subsequent differential display experiments. Colonies containing inserts were obtained, but those inserts which we spot-checked by sequencing were too small to be useful. A rabbit antibody to fire ant apolipophorin III was generated and purified by affinity chromatography. Frozen sections of male and worker heads were examined by immunocytochemistry, using rabbit anti-apolipophorin III as the first antibody and AlexaFluor 594 donkey anti-rabbit IgG as the second antibody (red emission is necessary to avoid cuticle yellow autofluorescence). Expression was found in epithelial layers underlying the cuticle in both males and workers, and also throughout the brain. The coding sequence for the polygyne queen-specific hydrophobic ligand-binding protein Gp-9b was inserted into a pET22 expression vector and expressed in E. coli. The expressed protein will be used to generate a rabbit antibody. Specific aim #3: Silence expression of specific genes to test for loss of social behavior. A behavior assay was developed for subsequent use in evalulating effects of RNAi experiments. We measured the autocorrelation function, c(t) = (1/T) ? [A(t) A(t + t)], of ant number fluctuations, where A is the number of ants in a defined area of an artificial nest, T is the total time, t is a particular time point, and t is a variable time offset. We found that c(t) decays as an exponential function, with a correlation time tc. Through modeling of random motion, we showed that tc is directly proportional to the average velocity of the ants in a group. Thus, by measuring the number autocorrelation function, we can obtain information about the collective motion of a group of ants.