土壤低浓度PAHs胁迫下赤子爱胜蚓差异表达基因初步筛选

为探索土壤低浓度多环芳烃污染的生态毒性及其对土壤生物的致毒机理，本论文初步研究了菲、芘、荧蒽和苯并[a]芘等四种多环芳烃人工土壤污染在0.1mg.kg-1~10.0mg.kg-1浓度水平对赤子爱胜蚓（Eisenia fetida）产卵量、体重变化、排卵激素annetocin基因和翻译控制肿瘤蛋白（translationally controlled tumor protein, TCTP）转录水平的影响，发现在相同的低浓度水平下，只有苯并[a]芘对蚯蚓annetocin前体基因和TCTP基因的表达有显著影响，故其对生物体的生殖风险和致癌风险可能最大。另一方面，低浓度苯并[a]芘对蚯蚓体重和产茧量并无显著影响，这表明基因表达水平作为污染生态监测指标比宏观观测指标更灵敏。 为进一步研究土壤PAHs污染的生态毒性效应，在上述研究基础上，我们采用SSH-PCR的方法构建了人工土壤1.0mg.kg-1苯并[a]芘胁迫下的蚯蚓与对照组蚯蚓之间的消减cDNA文库，随机挑取上调文库301个克隆及下调文库283个克隆进行测序，与NCBI蛋白数据库比对结果表明，其中有391个克隆与已知的75种蛋白质基因显著匹配（期望值< 10-5），其余克隆匹配不显著（期望值> 10-5）或找不到匹配蛋白。显著匹配的基因序列包括：一相解毒酶细胞色素P450，二相解毒酶谷胱甘肽硫转移酶，蛋白质合成所需的核糖体蛋白亚基，参与新合成肽链折叠的热休克蛋白，线粒体基因组编码的呼吸链复合酶体亚基，过氧化物还原蛋白，铁蛋白，钙结合蛋白，半胱氨酸蛋白酶等。表明低浓度苯并[a]芘胁迫引起蚯蚓的生理变化是非常复杂的，涉及污染物降解与解毒、抗氧化保护、能量代谢、蛋白质合成、金属离子调节与蛋白质降解等过程。 Real-time PCR检测验证消减文库中部分差异基因对不同剂量BaP胁迫响应结果表明，各检验基因序列受1.0 mg∙kg-1 BaP 胁迫影响均与消减结果一致，且影响程度均高于0.1 mg∙kg-1浓度水平的BaP；其中，在0.1 mg∙kg-1 BaP胁迫下，过氧化物还原酶PRDX和类似Cyp2R1的P450基因表达未见明显变化。其余的HSP70、HSP90、rpL10、COXⅡ、SCBP、Ferritin等基因在0.1 mg∙kg-1 BaP胁迫组蚯蚓中均有检测到预期表达变化，说明虽然从消减文库中获得的基因在一定的污染物浓度范围内均表现浓度效应，但各个基因对污染物的响应浓度不尽相同。 Real-time PCR检测消减文库中部分差异基因对不同PAHs胁迫响应结果表明，1.0 mg∙kg-1 浓度水平的荧蒽、菲、芘和苯并[a]芘对差异表达基因的影响不尽相同，主要有以下三种情况：(1)广谱响应型：蚯蚓线粒体编码的亚基COXⅡ、可溶性钙结合蛋白、铁蛋白等基因对荧蒽、菲、芘及苯并[a]芘的胁迫均有相似的响应；(2)随芳烃环数而变化型：热休克蛋白HSP70和过氧化物还原酶PRDX表现出响应程度随胁迫多环芳烃的环数增加而提高的现象；(3)仅在苯并[a]芘中有响应型：核糖体蛋白亚基L10和细胞色素P450（类似Cyp2R1）基因，在1.0 mg∙kg-1浓度条件下，它们仅受BaP诱导表达，而芘、菲和荧蒽却没有显示诱导作用。 上述结果表明，在土壤中的低浓度的多环芳烃污染胁迫对蚯蚓的影响是多方面的，这些影响至少涉及能量代谢、污染物降解与解毒、蛋白质合成与修复、信号转导、细胞凋亡、排卵生殖、个体发育等多方面的生理功能。目前蚯蚓基因组还未有完整测序，本文论述的多个差异表达基因是首次在蚯蚓中发现的，这些新发现的基因序列在为低浓度PAHs的生态毒性机理研究提供依据的同时，也为以蚯蚓为模式生物的土壤污染生物监测提供了备选的生物分子标记。另一方面，由于蚯蚓基因组未完整测序，本研究构建的消减文库中仍不少未知功能基因，其功能与调控有待进一步研究。

Molecular coordinated regulation of gene expression during ovarian development in the penaeid shrimp

To understand the molecular events of ovarian development in penaeid shrimp, RNA arbitrarily primed polymerase chain reaction (RAP-PCR) was used to identify differentially expressed genes during ovarian maturation in Metapenaeus ensis. From a screening of 700 clones in a cDNA library of the shrimp ovary by the products of RAP-PCR of different maturation stages, 91 fragments with differentially expressed pattern as revealed by dot-blot hybridization were isolated and sequenced. Forty-two of these fragments show significant sequence similarity to known gene products and the differentially expressed pattern of 10 putative genes were further characterized via Northern hybridization. Putative glyceraldehyde-3-phosphate dehydrogenase and arginine kinase are related to provision of energy for active cellular function in oocyte development. Translationally controlled tumor protein, actin, and keratin are related to the organization of cytoskeleton to accomplish growth and development of oocytes. High mobility group protein DSP1, heat shock protein 70, and nucleoside diphosphate kinase may act as repressors before the onset of ovarian maturation. Peptidyl-prolyl cis-trans isomerase and glutathione peroxidase are related to the stabilization of proteins and oocytes. This study provides new insights on the molecular events in the ovarian development in the shrimp.

Comparison of Gene Expression Profiles of Fenneropenaeus chinensis Challenged with WSSV and Vibrio

Microarray technique was used to analyze the gene expression profiles of shrimp when they were challenged by WSSV and heat-inactivated Vibrio anguillarum, respectively. At 6 h post challenge (HPC), a total of 806 clones showed differential expression profile in WSSV-challenged samples, but not in Vibrio-challenged samples. The genes coding energy metabolism enzyme and structure protein were the most downregulated elements in 6 h post WSSV-challenged (HPC-WSSV) tissues. However, a total of 155 clones showed differential expression in the Vibrio-challenged samples, but not in WSSV-challenged samples. Serine-type endopeptidase and lysosome-related genes were the most upregulated elements in tissues 6 h post Vibrio challenge (HPC-Vibrio). Totally, 188 clones showed differential expression in both 6 and 12 HPC-WSSV and HPC-Vibrio samples. Most of the differentially expressed genes (185/188) were downregulated in the samples of 12 HPC-WSSV, whereas upregulated in the samples at 6 and 12 HPC-Vibrio and 6 HPC-WSSV. The expression profiles of three differentially expressed genes identified in microarray hybridization were analyzed in hemocytes, lymphoid organ, and hepatopancreas of shrimp challenged by WSSV or Vibrio through real-time PCR. The results further confirmed the microarray hybridization results. The data will provide great help for us in understanding the immune mechanism of shrimp responding to WSSV or Vibrio.

Assessment of the immunomodulatory effects of raw/farm milk and probiotic bacteria

The overall aims of this study were to investigate the differences between raw/farm milk and pasteurised milk with respect to potential immune modifying effects following consumption and investigate the bacterial composition of raw milk compared to pasteurised milk. Furthermore, in this thesis, panels of potential probiotic bacteria from the Bifidobacterium and Lactobacillus genera were investigated. The overall bacterial composition of raw milk was compared with pasteurised milk using samples obtained from commercial milk producers around Ireland using next generation sequencing technology (454 pyrosequencing). Here the presence of previously unrecognised and diverse bacterial populations in unpasteurised cow’s milk was identified. Futhermore the bacterial content of pasteurised milk was found to be more diverse than previously thought. The global response of the adenocarcinoma cell line HT-29 to raw milk and pasteurised milk exposures were also characterised using whole genome microarray technology. Over one thousand differentially expressed genes were identified which were found to be involved in a plethora of cellular functions. Interestingly a reduction in immune related activity (e.g. Major histocompatability complex class II signalling and T and B cell proliferation) was identified in cells exposed to pasteurised milk compared with raw milk exposures. Further studies comparing human cell response to raw versus pasteurised milk was performed using peripheral blood mononuclear cells (PBMC) from healthy donors. A reduction in CD14 was identified following raw milk exposures compared with pasteurised milk and the pattern of cytokine production may indicate that gram positive bacteria in the raw milk were contributing to the differences in the cellular response to raw versus pasteurised milk. Panels of potentially probiotic bacteria (comprising of lactobacilli and bifidobacteria) were further assessed for immunomodulatory capabilities using cell culture based models. Gene expression and cytokine production were used to evaluate stimulated and unstimulated (LPS) cellular responses as well as interaction mechanisms

Mechanisms of CD8+ T Cell Mediated Virus Inhibition in HIV-1 Virus Controllers

CD8+ T cells are associated with long term control of virus replication to low or undetectable levels in a population of HIV+ therapy-naïve individuals known as virus controllers (VCs; <5000 RNA copies/ml and CD4+ lymphocyte counts >400 cells/µl). These subjects' ability to control viremia in the absence of therapy makes them the gold standard for the type of CD8+ T-cell response that should be induced with a vaccine. Studying the regulation of CD8+ T cells responses in these VCs provides the opportunity to discover mechanisms of durable control of HIV-1. Previous research has shown that the CD8+ T cell population in VCs is heterogeneous in its ability to inhibit virus replication and distinct T cells are responsible for virus inhibition. Further defining both the functional properties and regulation of the specific features of the select CD8+ T cells responsible for potent control of viremia the in VCs would enable better evaluation of T cell-directed vaccine strategies and may inform the design of new therapies.

Here we discuss the progress made in elucidating the features and regulation of CD8+ T cell response in virus controllers. We first detail the development of assays to quantify CD8+ T cells' ability to inhibit virus replication. This includes the use of a multi-clade HIV-1 panel which can subsequently be used as a tool for evaluation of T cell directed vaccines. We used these assays to evaluate the CD8+ response among cohorts of HIV-1 seronegative, HIV-1 acutely infected, and HIV-1 chronically infected (both VC and chronic viremic) patients. Contact and soluble CD8+ T cell virus inhibition assays (VIAs) are able to distinguish these patient groups based on the presence and magnitude of the responses. When employed in conjunction with peptide stimulation, the soluble assay reveals peptide stimulation induces CD8+ T cell responses with a prevalence of Gag p24 and Nef specificity among the virus controllers tested. Given this prevalence, we aimed to determine the gene expression profile of Gag p24-, Nef-, and unstimulated CD8+ T cells. RNA was isolated from CD8+ T-cells from two virus controllers with strong virus inhibition and one seronegative donor after a 5.5 hour stimulation period then analyzed using the Illumina Human BeadChip platform (Duke Center for Human Genome Variation). Analysis revealed that 565 (242 Nef and 323 Gag) genes were differentially expressed in CD8+ T-cells that were able to inhibit virus replication compared to those that could not. We compared the differentially expressed genes to published data sets from other CD8+ T-cell effector function experiments focusing our analysis on the most recurring genes with immunological, gene regulatory, apoptotic or unknown functions. The most commonly identified gene in these studies was TNFRSF9. Using PCR in a larger cohort of virus controllers we confirmed the up-regulation of TNFRSF9 in Gag p24 and Nef-specific CD8+ T cell mediated virus inhibition. We also observed increase in the mRNA encoding antiviral cytokines macrophage inflammatory proteins (MIP-1α, MIP-1αP, MIP-1β), interferon gamma (IFN-γ), granulocyte-macrophage colony-stimulating factor (GM-CSF), and recently identified lymphotactin (XCL1).

Our previous work suggests the CD8+ T-cell response to HIV-1 can be regulated at the level of gene regulation. Because RNA abundance is modulated by transcription of new mRNAs and decay of new and existing RNA we aimed to evaluate the net rate of transcription and mRNA decay for the cytokines we identified as differentially regulated. To estimate rate of mRNA synthesis and decay, we stimulated isolated CD8+ T-cells with Gag p24 and Nef peptides adding 4-thiouridine (4SU) during the final hour of stimulation, allowing for separation of RNA made during the final hour of stimulation. Subsequent PCR of RNA isolated from these cells, allowed us to determine how much mRNA was made for our genes of interest during the final hour which we used to calculate rate of transcription. To assess if stimulation caused a change in RNA stability, we calculated the decay rates of these mRNA over time. In Gag p24 and Nef stimulated T cells , the abundance of the mRNA of many of the cytokines examined was dependent on changes in both transcription and mRNA decay with evidence for potential differences in the regulation of mRNA between Nef and Gag specific CD8+ T cells. The results were highly reproducible in that in one subject that was measured in three independent experiments the results were concordant.

This data suggests that mRNA stability, in addition to transcription, is key in regulating the direct anti-HIV-1 function of antigen-specific memory CD8+ T cells by enabling rapid recall of anti-HIV-1 effector functions, namely the production and increased stability of antiviral cytokines. We have started to uncover the mechanisms employed by CD8+ T cell subsets with antigen-specific anti-HIV-1 activity, in turn, enhancing our ability to inhibit virus replication by informing both cure strategies and HIV-1 vaccine designs that aim to reduce transmission and can aid in blocking HIV-1 acquisition.

Effect of pooling samples on the efficiency of comparative studies using microarrays

Motivation: Many biomedical experiments are carried out by pooling individual biological samples. However, pooling samples can potentially hide biological variance and give false confidence concerning the data significance. In the context of microarray experiments for detecting differentially expressed genes, recent publications have addressed the problem of the efficiency of sample pooling, and some approximate formulas were provided for the power and sample size calculations. It is desirable to have exact formulas for these calculations and have the approximate results checked against the exact ones. We show that the difference between the approximate and the exact results can be large.

Immune activation in Retinal Aging: A Gene Expression Study.

PURPOSE
To investigate changes in gene expression during aging of the retina in the mouse.

METHODS
Total RNA was extracted from the neuroretina of young (3-month-old) and old (20-month-old) mice and processed for microarray analysis. Age-related, differentially expressed genes were assessed by the empiric Bayes shrinkagemoderated t-statistics method. Statistical significance was based on dual criteria of a ratio of change in gene expression >2 and a P < 0.01. Differential expression in 11 selected genes was further verified by real-time PCR. Functional pathways involved in retinal ageing were analyzed by an online software package (DAVID-2008) in differentially expressed gene lists. Age-related changes in differential expression in the identified retinal molecular pathways were further confirmed by immunohistochemical staining of retinal flat mounts and retinal cryosections.

RESULTS
With ageing of the retina, 298 genes were upregulated and 137 genes were downregulated. Functional annotation showed that genes linked to immune responses (Ir genes) and to tissue stress/injury responses (TS/I genes) were most likely to be modified by ageing. The Ir genes affected included those regulating leukocyte activation, chemotaxis, endocytosis, complement activation, phagocytosis, and myeloid cell differentiation, most of which were upregulated, with only a few downregulated. Increased microglial and complement activation in the aging retina was further confirmed by confocal microscopy of retinal tissues. The most strongly upregulated gene was the calcitonin receptor (Calcr; >40-fold in old versus young mice).

CONCLUSIONS
The results suggest that retinal ageing is accompanied by activation of gene sets, which are involved in local inflammatory responses. A modified form of low-grade chronic inflammation (para-inflammation) characterizes these aging changes and involves mainly the innate immune system. The marked upregulation of Calcr in ageing mice most likely reflects this chronic inflammatory/stress response, since calcitonin is a known systemic biomarker of inflammation/sepsis. © Association for Research in Vision and Ophthalmology.

DNA methylation profiling in cell models of diabetic nephropathy

We have previously identified differentially expressed genes in cell models of diabetic nephropathy and renal biopsies. Here we have performed quantitative DNA methylation profiling in cell models of diabetic nephropathy. Over 3,000 CpG units in the promoter regions of 192 candidate genes were assessed in unstimulated human mesangial cells (HMCs) and proximal tubular epithelial cells (PTCs) compared to HMCs or PTCs exposed to appropriate stimuli. A total of 301 CpG units across 38 genes (similar to 20%) were identified as differentially methylated in unstimulated HMCs versus PTCs. Analysis of amplicon methylation values in unstimulated versus stimulated cell models failed to demonstrate a >20% difference between amplicons. In conclusion, our results demonstrate that specific DNA methylation signatures are present in HMCs and PTCs, and standard protocols for exposure of renal cells to stimuli that alter gene expression may be insufficient to replicate possible alterations in DNA methylation profiles in diabetic nephropathy.

Parsing parallel evolution:Ecological divergence and differential gene expression in the adaptive radiations of thick-lipped Midas cichlid fishes from Nicaragua

The study of parallel evolution facilitates the discovery of common rules of diversification. Here, we examine the repeated evolution of thick lips in Midas cichlid fishes (the Amphilophus citrinellus species complex) - from two Great Lakes and two crater lakes in Nicaragua - to assess whether similar changes in ecology, phenotypic trophic traits and gene expression accompany parallel trait evolution. Using next-generation sequencing technology, we characterize transcriptome-wide differential gene expression in the lips of wild-caught sympatric thick- and thin-lipped cichlids from all four instances of repeated thick-lip evolution. Six genes (apolipoprotein D, myelin-associated glycoprotein precursor, four-and-a-half LIM domain protein 2, calpain-9, GTPase IMAP family member 8-like and one hypothetical protein) are significantly underexpressed in the thick-lipped morph across all four lakes. However, other aspects of lips' gene expression in sympatric morphs differ in a lake-specific pattern, including the magnitude of differentially expressed genes (97-510). Generally, fewer genes are differentially expressed among morphs in the younger crater lakes than in those from the older Great Lakes. Body shape, lower pharyngeal jaw size and shape, and stable isotopes (dC and dN) differ between all sympatric morphs, with the greatest differentiation in the Great Lake Nicaragua. Some ecological traits evolve in parallel (those related to foraging ecology; e.g. lip size, body and head shape) but others, somewhat surprisingly, do not (those related to diet and food processing; e.g. jaw size and shape, stable isotopes). Taken together, this case of parallelism among thick- and thin-lipped cichlids shows a mosaic pattern of parallel and nonparallel evolution. © 2012 Blackwell Publishing Ltd.

NuGO contributions to GenePattern

NuGO, the European Nutrigenomics Organization, utilizes 31 powerful computers for, e.g., data storage and analysis. These so-called black boxes (NBXses) are located at the sites of different partners. NuGO decided to use GenePattern as the preferred genomic analysis tool on each NBX. To handle the custom made Affymetrix NuGO arrays, new NuGO modules are added to GenePattern. These NuGO modules execute the latest Bioconductor version ensuring up-to-date annotations and access to the latest scientific developments. The following GenePattern modules are provided by NuGO: NuGOArrayQualityAnalysis for comprehensive quality control, NuGOExpressionFileCreator for import and normalization of data, LimmaAnalysis for identification of differentially expressed genes, TopGoAnalysis for calculation of GO enrichment, and GetResultForGo for retrieval of information on genes associated with specific GO terms. All together, these NuGO modules allow comprehensive, up-to-date, and user friendly analysis of Affymetrix data. A special feature of the NuGO modules is that for analysis they allow the use of either the standard Affymetrix or the MBNI custom CDF-files, which remap probes based on current knowledge. In both cases a .chip-file is created to enable GSEA analysis. The NuGO GenePattern installations are distributed as binary Ubuntu (.deb) packages via the NuGO repository.

Differences in Mucosal Gene Expression in the Colon of Two Inbred Mouse Strains after Colonization with Commensal Gut Bacteria

The host genotype has been proposed to contribute to individually composed bacterial communities in the gut. To provide deeper insight into interactions between gut bacteria and host, we associated germ-free C3H and C57BL/10 mice with intestinal bacteria from a C57BL/10 donor mouse. Analysis of microbiota similarity between the animals with denaturing gradient gel electrophoresis revealed the development of a mouse strain-specific microbiota. Microarray-based gene expression analysis in the colonic mucosa identified 202 genes whose expression differed significantly by a factor of more than 2. Application of bioinformatics tools demonstrated that functional terms including signaling/secretion, lipid degradation/catabolism, guanine nucleotide/guanylate binding and immune response were significantly enriched in differentially expressed genes. We had a closer look at the 56 genes with expression differences of more than 4 and observed a higher expression in C57BL/10 mice of the genes coding for Tlr1 and Ang4 which are involved in the recognition and response to gut bacteria. A higher expression of Pla2g2a was detected in C3H mice. In addition, a number of interferon-inducible genes were higher expressed in C3H than in C57BL/10 mice including Gbp1, Mal, Oasl2, Ifi202b, Rtp4, Ly6g6c, Ifi27l2a, Usp18, Ifit1, Ifi44, and Ly6g indicating that interferons may play an essential role in microbiota regulation. However, genes coding for interferons, their receptors, factors involved in interferon expression regulation or signaling pathways were not differentially expressed between the two mouse strains. Taken together, our study confirms that the host genotype is involved in the establishment of host-specific bacterial communities in the gut. Based on expression differences after colonization with the same bacterial inoculum, we propose that Pla2g2a and interferon-dependent genes may contribute to this phenomenon.

Sistemas de informação para DNA microarrays

O projecto de sequenciação do genoma humano veio abrir caminho para o surgimento de novas áreas transdisciplinares de investigação, como a biologia computacional, a bioinformática e a bioestatística. Um dos resultados emergentes desde advento foi a tecnologia de DNA microarrays, que permite o estudo do perfil da expressão de milhares de genes, quando sujeitos a perturbações externas. Apesar de ser uma tecnologia relativamente consolidada, continua a apresentar um conjunto vasto de desafios, nomeadamente do ponto de vista computacional e dos sistemas de informação. São exemplos a optimização dos procedimentos de tratamento de dados bem como o desenvolvimento de metodologias de interpretação semi-automática dos resultados. O principal objectivo deste trabalho consistiu em explorar novas soluções técnicas para agilizar os procedimentos de armazenamento, partilha e análise de dados de experiências de microarrays. Com esta finalidade, realizou-se uma análise de requisitos associados às principais etapas da execução de uma experiência, tendo sido identificados os principais défices, propostas estratégias de melhoramento e apresentadas novas soluções. Ao nível da gestão de dados laboratoriais, é proposto um LIMS (Laboratory Information Management System) que possibilita a gestão de todos os dados gerados e dos procedimentos realizados. Este sistema integra ainda uma solução que permite a partilha de experiências, de forma a promover a participação colaborativa de vários investigadores num mesmo projecto, mesmo usando LIMS distintos. No contexto da análise de dados, é apresentado um modelo que facilita a integração de algoritmos de processamento e de análise de experiências no sistema desenvolvido. Por fim, é proposta uma solução para facilitar a interpretação biológica de um conjunto de genes diferencialmente expressos, através de ferramentas que integram informação existente em diversas bases de dados biomédicas.

Notable differences in gene expression between injured turbot (Scophthalmus maximus) and brill (Scophthalmus rhombus) skin

Dissertação de mestrado, Engenharia Biológica, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2015

Deciphering the molecular role of DJ-1 in the etiology of Parkinson’s and Huntingtons’s disease

Tese de doutoramento, Ciências Biomédicas (Neurociências), Universidade de Lisboa, Faculdade de Medicina, 2014

A simple genetic basis for complex social behaviour mediates widespread gene expression differences.

A remarkable social polymorphism is controlled by a single Mendelian factor in the fire ant Solenopsis invicta. A genomic element marked by the gene Gp-9 determines whether workers tolerate one or many fertile queens in their colony. Gp-9 was recently shown to be part of a supergene with two nonrecombining variants, SB and Sb. SB/SB and SB/Sb queens differ in how they initiate new colonies, and in many physiological traits, for example odour and maturation rate. To understand how a single genetic element can affect all these traits, we used a microarray to compare gene expression patterns between SB/SB and SB/Sb queens of three different age classes: 1-day-old unmated queens, 11-day-old unmated queens and mated, fully reproductive queens collected from mature field colonies. The number of genes that were differentially expressed between SB/SB and SB/Sb queens of the same age class was smallest in 1-day-old queens, maximal in 11-day-old queens and intermediate in reproductive queens. Gene ontology analysis showed that SB/SB queens upregulate reproductive genes faster than SB/Sb queens. For all age classes, genes inside the supergene were overrepresented among the differentially expressed genes. Consistent with the hypothesized greater number of transposons in the Sb supergene, 13 transposon genes were upregulated in SB/Sb queens. Viral genes were also upregulated in SB/Sb mature queens, consistent with the known greater parasite load in colonies headed by SB/Sb queens compared with colonies headed by SB/SB queens. Eighteen differentially expressed genes between reproductive queens were involved in chemical signalling. Our results suggest that many genes in the supergene are involved in regulating social organization and queen phenotypes in fire ants.