Research Interests

Overview . My research focuses on the physiological, biochemical, molecular, and endocrine bases of adaptation. Since graduate school I have been working on two interrelated problems: (1) the evolution of the endocrine regulation of development and reproduction in insects, and (2) the physiological, biochemical, and endocrine bases of life history evolution, especially life-history trade-offs. This research has largely been undertaken in wing-polymorphic crickets of the genus Gryllus, and has resulted in the first detailed syntheses of evolutionary genetics, endocrinology, life history evolution, and metabolic biochemistry. My research has been supported continuously by NSF during the past 15 years, with multiple concurrent grants from the same or different panels during this entire period. The hallmarks of this research are its highly interdisciplinary, integrative nature, and its focus on the details of variation in proximate mechanisms that underlie adaptation.

The most noteworthy aspects of my research are the following : (1) It represents the most detailed investigation to date of physiological mechanisms underlying the endocrine control of complex (multi-trait) polymorphism in natural populations of insects; (2) it constitutes the first deep synthesis of quantitative genetics and endocrinology, giving rise to the subdiscipline that I call evolutionary endocrinology; (3) it represents the first detailed synthesis of intermediary metabolism, evolutionary genetics, and life history evolution and provides the most detailed analysis of mechanisms by which intermediary metabolism is altered to produce a life history trade-off; (4) it represents the first investigations of endocrine variation in the field for any insect.

Evolutionary endocrinology of wing-polymorphism, a complex, multi-trait polymorphism.

 Wing polymorphism consists of a flight-capable morph that has fully developed wings and flight muscles, and a flightless morph with underdeveloped, non-functional wings and flight muscles. Importantly, the flightless morph produces 100-400% more eggs than its flight-capable counterpart during early adulthood, and hence trades-off flight capability with early age fecundity. Wing polymorphism is the most extreme example of the trade-off between dispersal and reproduction, which occurs to some degree in most organisms. The polymorphism is very common in many insects groups (crickets, grasshoppers, aphids, waterstriders, beetles), with fightlessness having evolved many times independently in these groups. Since the 1960s, wing polymorphism has served as one of the premier postulated examples of morphological evolution (wings and flight muscles) that results from evolutionary modification of endocrine regulation. The “classical hypothesis’ states that alterations of the levels of key morphogenetic hormones (juvenile hormone, and/or ecdysone) during juvenile development block full development of wings and flight muscles producing a flightless morph. Because these hormones also are important regulators of adult reproduction, alterations in their titers during adulthood also would account for morph-specific differences in fecundity.

Endocrine regulation of morph development by juvenile hormone esterase. Detailed research in my laboratory over the past two decades has provided the first direct evidence that alterations in hormone levels are associated with and may regulate aspects of morph development in a dispersal-polymorphic insect. More importantly, my research is identifying the specific endocrine mechanisms that produce different hormone levels in alternate morphs. The ultimate goal of my studies is to identify the chain of causality that links altered DNA sequence of genes that encode hormonal regulators to alterations in the phenotypic expression of traits important in flight (wings, flight muscles, production of lipid flight fuels) and reproduction (yolk protein production).

A major finding of my research is a polymorphism in the activity of the regulatory enzyme, juvenile hormone esterase (JHE) is tighly correlated with wing polymorphism. Activity of this enzyme may play a key role in regulating the expression of flight-capable and flightless morphs. JHE degrades juvenile hormone and regulates the titer of this hormone in many insects. Activity of this enzyme is several fold higher in G. firmus that are destined to become the long-winged morph. Numerous pieces of evidence are consistent with the idea that JHE regulates genetically-determined wing morph in Gryllus. For example, JHE activity strongly co-segregates with wing morph in crosses and backcrosses between long-winged and short-winged genetic stocks, is positively correlated with in vivo degradation of juvenile hormone, and is negatively correlated with the in vivo JH titer. Additional studies have documented that other hormones (e.g. ecdysteroids) are also likely involved in the regulation of morph expression. Thus far, JHE activity in wing polymorphic crickets is the endocrine regulator that is most tightly correlated with morph expression for any case of complex polymorphism in insects (phase-, cast-, wing-, or flight-muscle polymorphism). My studies on the endocrine regulation of wing polymorphism constitute the most intensively investigated case of juvenile hormone-mediated morphological polymorphism in natural populations of any insect.

Molecular-genetic studies of JHE variation . Recent studies on JHE in Gryllus have expanded to include investigations of the molecular mechanisms responsible for differences in JHE activity that appear to regulate alternate morph development. In collaboration with Drs. John Oakeshott, CSIRO Entomology, Canberra, Australia, and Lawrence Harshman, University of Nebraska, I have recently cloned the JHE gene. Probes derived from this sequence are being used to test the hypothesis that JHE activity differences between morphs result from differential transcription of the JHE gene. An important long-term goal of this project is to identify the regulatory elements that cause differences in JHE activity between long-winged and short-winged stocks of G. firmus.

 Artificial selection on JHE activity in non-polymorphic insects . We have expanded our studies of the microevolution of endocrine regulation in wing-polymorphic crickets to studies of the microevolution of endocrine regulation in insects, in general. To do this we conducted artificial selection on hemolymph JHE activity in a wing monomorphic species of Gryllus. Our main goals were to estimate levels of genetic variation in JHE, to test for genetic correlations between JHE and other endocrine regulators during the same and different stages of the life cycle, and to document phenotypic effects of JHE activity variation. To my knowledge, this is the first, and still the only, study in which an endocrine regulator has been directly subjected to artificial selection. Results of this extensive study (involving 20,000 individually-reared crickets) have many important implications for the microevolution of endocrine regulation. Just to give one example, bidirectional selection on JHE activity in juveniles resulted in a 10-fold difference in enzyme activity between lines during the juvenile stage but no difference in activity between lines during the adult stage (i.e. no significant genetic correlation exists between JHE activity in adult and juvenile stages). This indicates that the endocrine regulation of juvenile development can evolve independently of the endocrine regulation of adult reproduction.

Endocrine regulation of flight capability and reproduction in adult morphs: Implications for the hormonal control of complex polymorphism and life history evolution. The endocrine control of the trade-off between flight capability and reproduction in adults has been another topic of great interest to insect physiologists and evolutionary biologists for over four decades. This topic is not only important with respect to the hormonal control of complex polymorphism, it is also important in the context of the physiological basis of life history evolution. For many decades, evolutionary biologists have been interested in identifying physiological processes responsible for life history trade-offs (negative genetic correlations between life history traits), which occur commonly in organisms. Because hormones regulate many important organismal traits, they are prime candidates as causal factors in life history trade-offs. Yet the hormonal basis of life history trade-offs is only recently beginning to be studied in any detail; my investigations of the endocrine regulation of the trade-off between reproduction and dispersal in Gryllus are spearheading such studies and are an important contributor to the new subdiscipline of evolutionary endocrinology. As is the case with the endocrine regulation of morph development, my research has been the first to directly document, in detail, alterations in endocrine regulatory mechanisms that underlie the flight-reproduction trade-off.

Recent direct quantification of juvenile hormone levels in adult, flight-capable and flightless morphs of G. firmus, have documented an important and unexpected finding: a large-amplitude, morph-specific circadian rhythm for the blood level of this hormone. The JH titer rises and falls 10-50-fold during a four-six hour period in the flight-capable morph during each day of early adulthood, but is temporally constant in the flightless morph. Subsequent studies have verified this morph-specific pattern in field populations of a variety of Gryllus species. This finding contrasts sharply with the widely held “classical” endocrine model of wing polymorphism which proposes that the expression of alternate morph characteristics is simply caused by a JH titer that is above or below some threshold. Importantly, the “classical” model, which has attained the status of dogma, was based on indirect studies of hormone levels in dispersing and flightless morphs. My research demonstrates the importance of undertaking direct, detailed studies of endocrine traits to adequately test hypotheses of endocrine variation.

This finding has several important implications . First, to my knowledge, it is the first example of a naturally-occurring, genetic polymorphism for a circadian rhythm for a hormone titer . This finding opens up a whole new area of research on the microevolution of functionally-important circadian rhythms in natural populations. The large amplitude of the cycle, and its tight genetic association with the flight-capable morph, makes this a powerful experimental model for microevolutionary and functional studies of circadian rhythms.

Second, this result indicates a novel endocrine mechanism regulating the expression of morph-specific traits and driving the evolution of flightlessness in wing-polymorphic crickets (Discussed in Zhao and Zera, 2004).

Third, studies of the morph-specific circadian rhythm for the JH titer has resulted in the first studies of endocrine variation in natural populations of insects . Endocrine studies in field populations of vertebrates are common. Surprisingly, no endocrine studies have been conducted in field populations of insects prior to my current investigations of morph-specific circadian rhythms in Gryllus species. Field endocrinology of the flight-reproduction trade-off will be an important ongoing aspect of my research for the foreseeable future.

The metabolic-biochemical basis of life history trade-offs ; microevolution of intermediary (lipid) metabolism

The energetic basis of life history trade-offs has been a central topic in evolutionary biology for over six decades. However, this aspect of life history evolution is still poorly understood. For the past 10 years I have been using wing polymorphism as a model to investigate this topic. The most general finding derived from a variety of studies is that flight-capable females allocate a significant proportion of their energy budget to maintenance metabolism of the large flight muscles, and to accumulation of large quantities of triglyceride flight fuel. This, in turn, constrains egg production and is an important physiological cause of the reduced fecundity of flight-capable vs. flightless females. These studies are the first to directly identify physiological costs of flight capability, a subject of speculation for many decades, and are the most detailed investigations to date of the energetic causes of a genetically-based life history trade-off.

Ongoing research is breaking new ground by identifying specific alterations of intermediary metabolism that underlie increased accumulation of lipid (flight fuel) in the flight-capable morph. Using radiotracers, we have documented large-magnitude, genetically-based alterations in flux through pathways of fatty acid and triglyceride biosynthesis that account for the increased accumulation of triglyceride flight fuel in the flight-capable morph. This work is now being cited in basic textbooks on evolution as a classic example of a biochemically-based allocation trade-off that underlies a life history trade-off (e.g. Evolutionary Analysis by Freeman and Harron, 3 rd ed., 2004; pp 458-459). We have further documented that morph-specific differences in flux through pathways of lipid biosynthesis result from large-scale changes in the activities of enzymes that comprise these pathways. Finally, preliminary data suggest that that these large-scale alterations in lipid metabolism may be primarily caused by morph-specific alterations in the endocrine control of metabolism. This important result suggests that alterations in intermediary metabolism that underlie a key life history trade-off in G. firmus may be primarily due to alterations in regulation, rather than due to limited availability of internal resources, the most widely held explanation for life history trade-offs. These studies are beginning to open the black box of intermediary metabolism as it relates to life history trade-offs, and have resulted in the first detailed synthesis of evolutionary genetics, metabolic biochemistry, and endocrinology. A recently funded NSF grant expands above-mentioned research in new direction. We are currently investigating the the molecular/enzymatic mechanisms (gene transcription; post-translational modification) responsible for the elevated activities of lipogenic enzymes that cause the enhanced biosynthesis of triglyceride flight fuel in the flight-capable morph.

Recent Publications

• Zera, A. J. and Z. Zhao. 2006. Intermediary metabolism and life history trade-offs: Diffeential metabolism of amino acids underlies the dispersal-reproduction trade-off in a wing-polymorphic cricket. American Naturalist (revised manuscript in review).
• Zera, A.J. 2006. Wing polymorphism in Gryllus: Energetic, endocrine, and biochemical bases of morph specializations for flight vs. reproduction. In: Insects and Phenotypic Plasticity (T. N. Ananthakrishnan and D. W. Whitman, eds.) (In Press).
  
• (Zera, A. J. 2005. Intermediary metabolism and life history trade-offs: Lipid metabolism in lines of the wing-polymorphic cricket, Gryllus firmus, selected for flight capability vs. early-age reproduction. From the symposium: Artificial selection as a tool to investigate the biochemical-genetic basis of life history trade offs. (Invited Review) Integrative and Comparative Biology. 45:511-524.
  
• (Zhao, Z. and A. J. Zera. 2004. A morph-dependent daily cycle in JH biosynthesis underlies the morph-dependent daily cycle in the JH titer in a wing-polymorphic cricket. Journal of Insect Physiology. 50:965-973.
  
• Zhao, Z. and A. J. Zera. 2004. The hemolymph JH titer exhibits a large-amplitude, morph-dependent, diurnal cycle in the wing-polymorphic cricket, Gryllus firmus. Journal of Insect Physiology. 50:93-102.
  
• Zera, A. J. and Z. Zhao. 2004. Effect of a juvenile hormone analogue on lipid metabolism in a wing-polymorphic cricket: Implications for the biochemical basis of the trade-off between reproduction and dispersal. Biochemical and Physiological Zoology. 77:255-266.
• Zera, A. J. and Z. Zhao. 2003. Morph-dependent fatty-acid oxidation in a wing-polymorphic cricket: Implications for morph specialization for dispersal vs. reproduction J. Insect Physiology 49:933-943.
  
• Zera, A. J. 2003. The endocrine regulation of wing polymorphism: State of the art, recent surprises, and future directions. (Invited Review) Integrative and Comparative Biology . 43:607-616.
• Zera, A. J. and Zhao, Z. 2003. Life history evolution and the microevolution of intermediary metabolism: Activities of lipid-metabolizing enzymes in life-history morphs of a wing-dimorphic cricket. Evolution 57:586-596.
• Zhao, Z. and Zera, A. J. 2002. Differential lipid biosynthesis underlies a trade-off between reproduction and flight-capability in a wing-polymorphic cricket. Proc. Natl. Acad. Sci. USA. 99:16829-16834.
• Zera, A. J., T. Sanger, J. Hanes and L. G. Harshman. 2002. Purification and characterization of juvenile hormone esterase from Gryllus assimilis Archives of Insect Biochemistry and Physiology 49:41-55.
• Zhao, Z., A. J. Zera. 2001 Enzymological and radiotracer studies of lipid metabolism in the flight-capable and flightless morphs of the wing polymorphic cricket, Gryllus firmus. Journal of Insect Physiology 47:1337-1347.
• Zera, A. J, and A. Larsen. 2001. The metabolic basis of life history variation: Genetic and phenotypic differences in lipid reserves among life history morphs of the wing-polymorphic cricket, Gryllus firmus Journal of Insect Physiology 47:1147-1160.
• Zera, A. J. and L. G. Harshman. 2001. Physiology of life history trade-offs in animals Annual Review of Ecology and Systematics 32:95-106.
• Zera, A. J. and G. L. Cisper. 2001 Genetic and diurnal variation in the juvenile hormone titer in a wing-polymorphic cricket: Implications for the evolution of life histories and dispersal. Physiol. Biochem. Zool. 74:293-306.
• Zera, A. J. and J. Bottsford. 2001 The endocrine-genetic basis of life history variation: Relationship between the ecdysteroid titer and morph-specific reproduction in the wing polymorphic cricket, Gryllus firmus. Evolution 55:538-549.

Current Active External Grants

NSF-ECOLOGICAL AND EVOLUTIONARY PHYSIOLOGY. Molecular and biochemical causes of trade-offs in lipid biosynthesis that underlie a life history trade-off (7/05-6/09; $400,000).

NSF-ECOLOGICAL AND EVOLUTIONARY PHYSIOLOGY . Physiological and molecular causes of genetic variation/covariation in endocrine regulation. ($360,000; 1/1/03-12/31/06)

NSF-ECOLOGICAL AND EVOLUTIONARY PHYSIOLOGY . Morph-dependent cyclic JH titer in a wing-polymorphic cricket: Adaptive significance and underlying causes ($297,000; 6/1/02-5/31/06).