Publications

2009
Machado HE, Pollen AA, Hofmann HA, Renn SCP. Interspecific profiling of gene expression informed by comparative genomic hybridization: A review and a novel approach in African cichlid fishes. Integrative and Comparative Biology. 2009;49 (6) :644 - 659.Abstract

Modern genomic approaches have facilitated great progress in our understanding of the molecular and genetic underpinnings of ecological and evolutionary processes. Analysis of gene expression through heterologous hybridization in particular has enabled genome-scale studies in many ecologically and evolutionarily interesting species. However, these studies have been hampered by the difficulty of comparing-on a common array platform-gene-expression profiles across species due to sequence divergence altering the dynamics of hybridization. All too often, comparisons of expression profiles across species were limited to contrasting lists of gene or even of just functional categories. Here we review these issues and propose a novel solution. Exploiting the diverse cichlid lineages of East Africa as our model-system, we then present results from an experimental case study that compares the neural gene-expression profiles of males and females of two species that differ in mating system. Using a single microarray platform that contains genes from one species, Astatotilapia burtoni, we conducted a total of 16 direct comparisons for neural gene-expression level between individual males and females from a pair of sister species, the polygynous Enantiopus melanogenys and the monogamous Xenotilapia flavipinnis. Next, we conducted a meta-analysis with previously published data from two different intra-specific expression studies to determine whether sex-specific neural gene expression is more closely associated with behavioral phenotype than it is with gonadal sex. Our results indicate that the gene expression profiles are species-specific to a large extent, as relatively few genes show conserved expression patterns associated with either sex. Finally, we describe how competitive genomic DNA hybridizations between the two focal species allow us to assess the degree to which divergence of sequences biases the results. We propose a masking technique that correlates interspecific expression ratios obtained with cDNA with hybridization ratios obtained with genomic DNA for the same set of species and determines threshold sequence divergence to reduce false positives. Our approach should be applicable to a wide range of interesting questions related to the evolution and ecology of gene expression.

2009.machado.icb_.pdf
2008
Salzburger W, Renn SCP, Steinke D, Braasch I, Hofmann HA, Meyer A. Annotation of expressed sequence tags for the East African cichlid fish Astatotilapia burtoni and evolutionary analyses of cichlid ORFs. BMC genomics [Internet]. 2008;9 :96 - 96. Publisher's VersionAbstract

BACKGROUND: The cichlid fishes in general, and the exceptionally diverse East African haplochromine cichlids in particular, are famous examples of adaptive radiation and explosive speciation. Here we report the collection and annotation of more than 12,000 expressed sequence tags (ESTs) generated from three different cDNA libraries obtained from the East African haplochromine cichlid species Astatotilapia burtoni and Metriaclima zebra.\n\nRESULTS: We first annotated more than 12,000 newly generated cichlid ESTs using the Gene Ontology classification system. For evolutionary analyses, we combined these ESTs with all available sequence data for haplochromine cichlids, which resulted in a total of more than 45,000 ESTs. The ESTs represent a broad range of molecular functions and biological processes. We compared the haplochromine ESTs to sequence data from those available for other fish model systems such as pufferfish (Takifugu rubripes and Tetraodon nigroviridis), trout, and zebrafish. We characterized genes that show a faster or slower rate of base substitutions in haplochromine cichlids compared to other fish species, as this is indicative of a relaxed or reinforced selection regime. Four of these genes showed the signature of positive selection as revealed by calculating Ka/Ks ratios.\n\nCONCLUSION: About 22% of the surveyed ESTs were found to have cichlid specific rate differences suggesting that these genes might play a role in lineage specific characteristics of cichlids. We also conclude that the four genes with a Ka/Ks ratio greater than one appear as good candidate genes for further work on the genetic basis of evolutionary success of haplochromine cichlid fishes.

2008.salzburger.bmc_.pdf
Pollen AA, Hofmann HA. Beyond neuroanatomy: Novel approaches to studying brain evolution. Brain, Behavior and Evolution. 2008;72 (2) :145 - 158.Abstract

The study of the evolution of brain structure and function, although fascinating, has been contentious, largely due to the correlative nature of neuroanatomical comparisons and the often ill-defined categorizations of habitat and behavior. We outline four conceptual approaches that will help the field of brain evolution emerge from a historical focus on descriptive comparative neuroanatomy. First, reliable, efficient and unbiased behavioral assays must be developed to characterize relevant cross-species differences in addition to focused studies of neuroanatomy. Second, developmental and physiological processes underlying neuroanatomical and behavioral differences can be analyzed using the comparative approach. Third, genome-wide comparisons including genome-wide linkage mapping, transcriptional profiling, and direct sequence comparisons, can be applied to identify the genetic basis for phenotypic differences. Finally, signatures of selection in DNA sequence can provide clues about adaptive genetic changes that affect the nervous system. These four approaches, which all depend on well-resolved phylogenies, will build on detailed neuroanatomical studies to provide a richer understanding of mechanistic and selective factors underlying brain evolution.

2008.pollen.bbe_.pdf
Duftner N, Larkins-Ford J, Legendre M, Hofmann HA. Efficacy of RNA amplification is dependent on sequence characteristics: Implications for gene expression profiling using a cDNA microarray. Genomics. 2008;91 (1) :108 - 117.Abstract

Minute tissue samples or single cells increasingly provide the starting material for gene expression profiling, which often requires RNA amplification. Although much effort has been put into optimizing amplification protocols, the relative abundance of RNA templates in the amplified product is frequently biased. We applied a T7 polymerase-based technique to amplify RNA from two tissues of a cichlid fish and compared expression levels of unamplified and amplified RNA on a cDNA microarray. Amplification bias was generally minor and comprised features that were lost (1.3%) or gained (2.5%) through amplification and features that were scored as regulated before but unregulated after amplification (4.2%) or vice versa (19.5%). We examined 10 sequence-specific properties and found that GC content, folding energy, hairpin length and number, and lengths of poly(A) and poly(T) stretches significantly affected RNA amplification. We conclude that, if RNA amplification is used in gene expression studies, preceding experiments controlling for amplification bias should be performed. ?? 2007 Elsevier Inc. All rights reserved.

2008.duftner.genomics.pdf
Greenwood AK, Wark AR, Fernald RD, Hofmann HA. Expression of arginine vasotocin in distinct preoptic regions is associated with dominant and subordinate behaviour in an African cichlid fish. Proceedings of the Royal Society of London B: Biological Sciences [Internet]. 2008;275 (1649) :2393 - 2402. Publisher's VersionAbstract

Neuropeptides have widespread modulatory effects on behaviour and physiology and are associated with phenotypic transitions in a variety of animals. Arginine vasotocin (AVT) is implicated in mediating alternative male phenotypes in teleost fish, but the direction of the association differs among species, with either higher or lower AVT related to more territorial behaviour in different fishes. To clarify the complex relationship between AVT and alternative phenotype, we evaluated AVT expression in an African cichlid in which social status is associated with divergent behaviour and physiology. We compared AVT mRNA expression between territorial and non-territorial (NT) males in both whole brains and microdissected anterior preoptic areas using transcription profiling, and in individual preoptic nuclei using in situ hybridization. These complementary methods revealed that in the posterior preoptic area (gigantocellular nucleus), territorial males exhibit higher levels of AVT expression than NT males. Conversely, in the anterior preoptic area (parvocellular nucleus), AVT expression is lower in territorial males than NT males. We further correlated AVT expression with behavioural and physiological characteristics of social status to gain insight into the divergent functions of individual AVT nuclei. Overall, our findings highlight a complex association between AVT and social behaviour.

2008.greenwood.procrsocb.pdf
Renn SCP, Aubin-Horth N, Hofmann HA. Fish and chips: functional genomics of social plasticity in an African cichlid fish. Journal of Experimental Biology [Internet]. 2008;211 (18) :3041 - 3056. Publisher's VersionAbstract

... Arginine vasotocin {(AVT;} represented by multiple clones on the array), the non-mammalian homolog of arginine vasopressin {(AVP} ... In teleost fish , {AVT} is known to play a role in male mating tactics [peacock blenny {(Grober} et al., 2002; Carneiro et al., 2003); midshipman ...

2008.renn_.jeb_.pdf
Hofmann HA, Shumway CA. Preface. Brain, Behavior and Evolution. 2008;72 (2) :89 - 90. 2008.hofmann.bbe_.pdf
Cummings ME, Larkins-Ford J, Reilly CRL, Wong RY, Ramsey M, Hofmann HA. Sexual and social stimuli elicit rapid and contrasting genomic responses. Proceedings of the Royal Society of London, Series B. 2008;275 (1633) :393 - 402.Abstract

Sensory physiology has been shown to influence female mate choice, yet little is known about the mechanisms within the brain that regulate this critical behaviour. Here we examine preference behaviour of 58 female swordtails, Xiphophorus nigrensis, in four different social environments (attractive and unattractive males, females only, non-attractive males only and asocial conditions) followed by neural gene expression profiling. We used a brain-specific cDNA microarray to identify patterns of genomic response and candidate genes, followed by quantitative PCR (qPCR) examination of gene expression with variation in behaviour. Our microarray results revealed patterns of genomic response differing more between classes of social stimuli than between presence versus absence of stimuli. We identified suites of genes showing diametrically opposed patterns of expression: genes that are turned 'on' while females interact with attractive males are turned 'off' when interacting with other females, and vice versa. Our qPCR results identified significant predictive relationships between five candidate genes and specific mate choice behaviours (preference and receptivity) across females exposed to males, with no significant patterns identified in female or asocial conditions or with overall locomotor activity. The identification of stimulus- and behaviour-specific responses opens an exciting window into the molecular pathways associated with social behaviour and mechanisms that underlie sexual selection.

2008.cummings.procrsocb.pdf
2007
Pollen AA, Dobberfuhl AP, Scace J, Igulu MM, Renn SCP, Shumway CA, Hofmann HA. Environmental complexity and social organization sculpt the brain in Lake Tanganyikan cichlid fish. Brain, Behavior and Evolution. 2007;70 (1) :21 - 39.Abstract

Complex brains and behaviors have occurred repeatedly within vertebrate classes throughout evolution. What adaptive pressures drive such changes? Both environmental and social features have been implicated in the expansion of select brain structures, particularly the telencephalon. East African cichlid fishes provide a superb opportunity to analyze the social and ecological correlates of neural phenotypes and their evolution. As a result of rapid, recent, and repeated radiations, there are hundreds of closely-related species available for study, with an astonishing diversity in habitat preferences and social behaviors. In this study, we present quantitative ecological, social, and neuroanatomical data for closely-related species from the (monophyletic) Ectodini clade of Lake Tanganyikan cichlid fish. The species differed either in habitat preference or social organization. After accounting for phylogeny with independent contrasts, we find that environmental and social factors differentially affect the brain, with environmental factors showing a broader effect on a range of brain structures compared to social factors. Five out of seven of the brain measures show a relationship with habitat measures. Brain size and cerebellar size are positively correlated with species number (which is correlated with habitat complexity); the medulla and olfactory bulb are negatively correlated with habitat measures. The telencephalon shows a trend toward a positive correlation with rock size. In contrast, only two brain structures, the telencephalon and hypothalamus, are correlated with social factors. Telencephalic size is larger in monogamous species compared to polygamous species, as well as with increased numbers of individuals; monogamy is also associated with smaller hypothalamic size. Our results suggest that selection or drift can act independently on different brain regions as the species diverge into different habitats and social systems. Copyright © 2007 S. Karger AG.

2007.pollen.bbe_.pdf
Aubin-Horth N, Desjardins JK, Martei YM, Balshine S, Hofmann HA. Masculinized dominant females in a cooperatively breeding species. Molecular Ecology. 2007;16 (7) :1349 - 1358.Abstract

The molecular mechanisms underlying complex social behaviours such as dominance are largely unknown. Studying the cooperatively breeding African cichlid Neolamprologus pulcher, we show that dominant females were similar to dominant males in dominance behaviour, high testosterone levels and brain arginine vasotocin expression (a neuropeptide involved in vertebrate territorial, reproductive and social behaviours) compared to subordinate helpers, but had lower levels of 11-ketotestosterone than males. Furthermore, brain gene expression profiles of dominant females were most similar to those of the males (independent of social rank). Dominant breeder females are masculinized at the molecular and hormonal level while being at the same time reproductively competent, suggesting a modular organization of molecular and endocrine functions, allowing for sex-specific regulation.

2007.aubin-horth.me_.pdf
Shumway CA, Hofmann HA, Dobberfuhl AP. Quantifying habitat complexity in aquatic ecosystems. Freshwater Biology. 2007;52 (6) :1065 - 1076.Abstract

Summary 1. Many aquatic studies have attempted to relate biological features, such as species diversity, abundance, brain size and behaviour, to measures of habitat complexity. Previous measures of habitat complexity have ranged from simple, habitat-specific variables, such as the number of twigs in a stream, to quantitative parameters of surface topography, such as rugosity. 2. We present a new video-based technique, called optical intensity, for assaying habitat complexity in aquatic ecosystems. Optical intensity is a visual, quantitative technique modifiable for any scale or for a nested analysis. We field-tested the technique in Lake Tanganyika, Tanzania, on 38 quadrats (5 x 5 m) to determine if three freshwater habitats (sand, rock and intermediate) were quantitatively different. 3. A comparison of the values obtained from optical intensity with a previous measure of surface topography (rugosity) showed that the two corresponded well and revealed clear differences among habitats. Both the new measure and rugosity were positively correlated with species diversity, species richness and abundance. Finally, whether used alone or in combination, both measures had predictive value for fish community parameters. 4. This new measure should prove useful to researchers exploring habitat complexity in both marine and freshwater systems.

2007.shumway.fb_.pdf
Trainor BC, Hofmann HA. Somatostatin and somatostatin receptor gene expression in dominant and subordinate males of an African cichlid fish. Behavioural Brain Research. 2007;179 (2) :314 - 320.Abstract

Somatostatin is a neuropeptide best known for its inhibitory effects on growth hormone secretion and has recently been implicated in the control of social behavior. Several somatostatin receptor subtypes have been identified in vertebrates, but the functional basis for this diversity is still unclear. Here we investigate the expression levels of the somatostatin prepropeptide and two of its receptors, sstR2, and sstR3, in the brains of socially dominant and subordinate Astatotilapia burtoni males using real-time PCR. Dominant males had higher somatostatin prepropeptide and sstR3 expression in hypothalamus compared to subordinate males. Hypothalamic sstR2 expression did not differ. There were no differences in gene expression in the telencephalon. We also observed an interesting difference between dominants and subordinates in the relationship between hypothalamic sstR2 expression and body size. As would be predicted based on the inhibitory effects of somatostatin on somatic growth, sstR2 expression was negatively correlated with body size in dominant males. In contrast sstR2 expression was positively correlated with body size in subordinate males. These results suggest that in A. burtoni social status affects the relationships between somatostatin prepropeptide and receptor gene expression in the hypothalamus and the control of somatic growth. ?? 2007 Elsevier B.V. All rights reserved.

2007.trainor.bbr_.pdf
2006
Hofmann HA. Gonadotropin-releasing hormone signaling in behavioral plasticity. Current Opinion in Neurobiology. 2006;16 (3) :343 - 350.Abstract

Sex and reproduction sculpt brain and behavior throughout life and evolution. In vertebrates, gonadotropin-releasing hormone (GnRH) is essential to these processes. Recent advances have uncovered novel regulatory mechanisms in GnRH signaling, such as the initiation of sexual maturation by kisspeptins. Yet despite our increasing molecular knowledge, we know very little about environmental influences on GnRH signaling and reproductive behavior. Alternative model systems have been crucial for understanding the plasticity of GnRH effects within an organismal context. For instance, GnRH signaling is under the control of seasonal cues in songbirds, whereas social signals regulate GnRH in cichlid fishes, with crucial consequences for reproduction and behavior. Analyzing cellular signaling cascades within an organismic context is essential for an integrative understanding of GnRH function. ?? 2006 Elsevier Ltd. All rights reserved.

2006.hofmann.con_.pdf
Trainor BC, Hofmann HA. Somatostatin Regulates aggressive behavior in an african cichlid fish. Endocrinology. 2006;147 (11) :5119 - 5125.Abstract

Animals respond to environmental and social change with plasticity in the neural substrates underlying particular behavioral states. In the African cichlid fish Astatotilapia burtoni, social dominance status in males is accompanied by reduced somatic growth rate as well as increased somatostatin neuron size in the preoptic area. Although somatostatin is commonly studied within the context of growth, we show here for the first time that this ancient neuropeptide also plays a role in controlling social behavior. Somatostatin antagonists increased aggressive behavior in a dose-dependent fashion and the potent somatostatin agonist octreotide decreased aggression. We cloned and sequenced the genes encoding two somatostatin receptor subtypes in this species to study transcription in the gonads. When we examined somatostatin receptor gene expression in testes, expression of the somatostatin type 3 receptor was negatively correlated with an aggressive display and androgen levels. However, octreotide treatment did not reduce plasma testosterone or 11-ketotestosterone levels, suggesting that the behavioral effects of somatostatin are not mediated by androgens. These results show that somatostatin has important effects on social behavior. In dominant male A. burtoni, somatostatin may function to contain energetically costly processes such as somatic growth and aggressive behavior.

2006.trainor.endo_.pdf
2005
Aubin-Horth N, Landry CR, Letcher BH, Hofmann HA. Alternative life histories shape brain gene expression profiles in males of the same population. Proceedings of the Royal Society B: Biological Sciences [Internet]. 2005;272 (1573) :1655 - 1662. Publisher's VersionAbstract

Atlantic salmon (Salmo salar) undergo spectacular marine migrations before homing to spawn in natal rivers. However, males that grow fastest early in life can adopt an alternative ‘sneaker’ tactic by maturing earlier at greatly reduced size without leaving freshwater. While the ultimate evolutionary causes have been well studied, virtually nothing is known about the molecular bases of this developmental plasticity. We investigate the nature and extent of coordinated molecular changes that accompany such a fundamental transformation by comparing the brain transcription profiles of wild mature sneaker males to age-matched immature males (future large anadromous males) and immature females. Of the ca. 3000 genes surveyed, 15% are differentially expressed in the brains of the two male types. These genes are involved in a wide range of processes, including growth, reproduction and neural plasticity. Interestingly, despite the potential for wide variation in gene expression profiles among individuals sampled in nature, consistent patterns of gene expression were found for individuals of the same reproductive tactic. Notably, gene expression patterns in immature males were different both from immature females and sneakers, indicating that delayed maturation and sea migration by immature males, the ‘default’ life cycle, may actually result from an active inhibition of development into a sneaker.

2005.aubinhorth.procrsocb.pdf
Aubin-Horth N, Letcher BH, Hofmann HA. Interaction of rearing environment and reproductive tactic on gene expression profiles in Atlantic salmon. Journal of Heredity. 2005;96 (3) :261 - 278.Abstract

Organisms that share the same genotype can develop into divergent phenotypes, depending on environmental conditions. In Atlantic salmon, young males of the same age can be found either as sneakers or immature males that are future anadromous fish. Just as the organism-level phenotype varies between divergent male developmental trajectories, brain gene expression is expected to vary as well. We hypothesized that rearing environment can also have an important effect on gene expression in the brain and possibly interact with the reproductive tactic adopted. We tested this hypothesis by comparing brain gene expression profiles of the two male tactics in fish from the same population that were reared in either a natural stream or under laboratory conditions. We found that expression of certain genes was affected by rearing environment only, while others varied between male reproductive tactics independent of rearing environment. Finally, more than half of all genes that showed variable expression varied between the two male tactics only in one environment. Thus, in these fish, very different molecular pathways can give rise to similar macro-phenotypes depending on rearing environment. This result gives important insights into the molecular underpinnings of developmental plasticity in relationship to the environment.

2005.aubin-horth.jh_.pdf
2004
Renn SCP, Aubin-Horth N, Hofmann HA. Biologically meaningful expression profiling across species using heterologous hybridization to a cDNA microarray. BMC genomics [Internet]. 2004;5 (1) :42 - 42. Publisher's VersionAbstract

BACKGROUND: Unravelling the path from genotype to phenotype, as it is influenced by an organism's environment, is one of the central goals in biology. Gene expression profiling by means of microarrays has become very prominent in this endeavour, although resources exist only for relatively few model systems. As genomics has matured into a comparative research program, expression profiling now also provides a powerful tool for non-traditional model systems to elucidate the molecular basis of complex traits.\n\nRESULTS: Here we present a microarray constructed with approximately 4500 features, derived from a brain-specific cDNA library for the African cichlid fish Astatotilapia burtoni (Perciformes). Heterologous hybridization, targeting RNA to an array constructed for a different species, is used for eight different fish species. We quantified the concordance in gene expression profiles across these species (number of genes and fold-changes). Although most robust when target RNA is derived from closely related species (<10 MA divergence time), our results showed consistent profiles for other closely related taxa (approximately 65 MA divergence time) and, to a lesser extent, even very distantly related species (>200 MA divergence time).\n\nCONCLUSION: This strategy overcomes some of the restrictions imposed on model systems that are of importance for evolutionary and ecological studies, but for which only limited sequence information is available. Our work validates the use of expression profiling for functional genomics within a comparative framework and provides a foundation for the molecular and cellular analysis of complex traits in a wide range of organisms.

2004.renn_.bmc_.pdf
2003
Hofmann HA. Functional genomics of neural and behavioral plasticity. Journal of Neurobiology. 2003;54 (1) :272 - 282.Abstract

How does the environment, particularly the social environment, influence brain and behavior and what are the underlying physiologic, molecular, and genetic mechanisms? Adaptations of brain and behavior to changes in the social or physical environment are common in the animal world, either as short-term (i.e., modulatory) or as long-term modifications (e.g., via gene expression changes) in behavioral or physiologic properties. The study of the mechanisms and constraints underlying these dynamic changes requires model systems that offer plastic phenotypes as well as a sufficient level of quantifiable behavioral complexity while being accessible at the physiological and molecular level. In this article, I explore how the new field of functional genomics can contribute to an understanding of the complex relationship between genome and environment that results in highly plastic phenotypes. This approach will lead to the discovery of genes under environmental control and provide the basis for the study of the interrelationship between an individual's gene expression profile and its social phenotype in a given environmental context.

2003.hofmann.jn_.pdf
2001
Hofmann HA, Schildberger K. Assessment of strength and willingness to fight during aggressive encounters in crickets*. Animal Behaviour [Internet]. 2001;62 :337 - 348. Publisher's VersionAbstract

Game theory predicts that at least some of the behaviour patterns displayed during aggressive encounters are used to assess asymmetries in variables that indicate fighting ability and resource value. Game theoretical models such as the sequential assessment game see assessment as the major activity during a fight. However, while these models acknowledge the existence of physical and motivational assessment parameters, there are only a few examples where a mechanism for the assessment of fighting readiness has been shown. In staged encounters between male Mediterranean field crickets, Gryllus bimaculatus, fighting behaviour follows a stereotyped escalation cascade with ritualized displays in the beginning and physical combat towards the end. Despite their larger size, heavier animals lost 30% of the encounters even if weight asymmetry was large. To examine whether the contestants provide assessment cues that might explain this surprising result, we analysed two stereotyped displays in detail (antennal fencing and mandible spreading). The duration of antennal fencing, which is necessary to initiate a fight, was independent of experience and weight asymmetry between the contestants, but was prolonged after shortening the antennae by almost 90%. Fights escalated only when antennal movement frequencies were high in both contestants. In blinded crickets few contests were settled by another ritualized display, mandible spreading, and fights that escalated beyond this stage were significantly shorter than in untreated crickets. We suggest that antennal fencing may be used to assess fighting readiness of the opponent, while mandible spreading may indicate fighting ability. We conclude that high willingness to fight may help crickets to overcome inferior fighting ability.

2001.hofmann.ab_.pdf
Hofmann HA, Fernald RD. What cichlids tell us about the social regulation of brain and behaviour. Journal of aquaculture and aquatic sciences. 2001;9 :17 - 31. 2001.hofmann.jaas_.pdf

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