GENES CONTROLLING SOCIAL BEHAVIOR IN ANTS

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。具体目标#1：确定火蚁的同源蜜蜂基因预测任务行为。我们之前分离的PCR产物1PXIR与蜜蜂或果蝇的觅食基因（蛋白激酶G， PKG）相似，我们用它设计了一个PCR引物来鉴定火蚁的PKG同源物的一个较长的序列，1PXIR对应于蜜蜂PKG mRNA停止密码子约500个核苷酸的位置。利用api PKG序列的23核苷酸5′引物和27核苷酸3′引物，我们获得了300-400 bp的PCR产物。这些被插入到pGem序列向量中。未获得与PKG相关的DNA序列。我们基于api和果蝇觅食基因而非1PXIR设计了新的PKG PCR引物，并正在进行序列的分离。具体目标2：将大脑区域中已知的社会任务特异性大小变化与基因表达差异联系起来。我们从工人身上提取mRNA，在pBluescript中建立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插入物太小，无法发挥作用。制备了兔火蚁载脂蛋白III抗体，并通过亲和层析纯化。用兔抗载脂蛋白ⅲ为第一抗体，AlexaFluor 594驴抗兔IgG为第二抗体（为避免角质层黄色自身荧光，需要红光发射），对雄性和工头冰冻切片进行免疫细胞化学检测。在雄性和工蜂的角质层下的上皮层以及整个大脑中都发现了表达。将多雌性蜂王特异性疏水配体结合蛋白Gp-9b的编码序列插入pET22表达载体中，在大肠杆菌中表达。所表达的蛋白将用于生成兔抗体。具体目标3：沉默特定基因的表达，以测试社交行为的丧失。一种行为测定法被开发出来，用于后续评估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)]，蚂蚁数波动的表达式，其中A为人工蚁巢特定区域内的蚂蚁数，t为总时间，t为特定时间点，t为可变时间偏移量。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，我们发现tc与一群蚂蚁的平均速度成正比。因此，通过测量数自相关函数，我们可以获得蚁群的集体运动信息。

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.