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Functional and Ecological Effects of Isoform Variation in Insect Flight Muscle

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Summary

Nearly all of the known structural molecules in insect flight muscles exist as multiple isoforms. Both post-transcriptional and post-translational mechanisms are responsible for this variability. Among these mechanisms, alternative splicing is noteworthy for the ability to create a large number of combinatorial arrangements of alternative exons. For example there are over 1K possible distinct combinations of the characterized isoforms of troponin-T and projectin, which are just two of the many alternatively spliced proteins in insect muscle. The potential number of combinatorial possibilities for larger suites of insect muscle proteins is exponentially larger, i.e., numbers that far exceed the total number of coding genes in the insect genome. Thus, isoform variation is a potent source of variation in insect flight muscle and other tissue types, and the control of alternative splicing and other mechanisms that generate protein isoforms is a likely target of natural selection. Presently we know very little about the realized extent of this potential to generate protein variation, and no studies have yet examined constraints such as coordinate regulation of isoform expression of multiple protein species within insect flight muscles.

Functional studies of naturally occurring isoform variation, including the effects of phosphorylation of myosin light chain and alternative splicing of troponin-T, have revealed quantitative effects on muscle contractility and flight performance. In the case of troponin-T in a dragonfly, the isoform mixture affects the calcium sensitivity of muscle activation, power output of intact flight muscle, and wingbeat kinematics. High muscle power output is positively related to success in territoriality and mating, but increases the energetic cost of flight. Thus, alternative splicing of troponin-T in this species appears to be an important mechanism for adjusting between energetically expensive high-performance flight or less costly low-performance flight. Dragonflies show a strong positive relationship between fat reserves and muscle power output, which suggests that there is a signaling pathway that allows an alternative splicing mechanism to adjust muscle energy expenditure rates in accordance with levels of energy reserves. Infection of dragonflies by protozoan gut parasites is associated with a disappearance of the ability to match muscle power output to energy reserves, i.e., it appears that parasites disrupt this signaling pathway. This example shows that isoform variation in insect flight muscle has interesting effects not only on muscle contractile physiology, but is involved with many aspects of whole organism physiology and ecology. The taxonomic and mechanistic diversity within insect flight muscles provides a rich source of material for future studies that seek to understand the functional, ecological and evolutionary context of molecular diversity.

Introduction

This chapter presents an overview and catalog of isoform diversity within insect flight muscles, and summarizes studies that have examined the quantitative effect of naturally occurring isoform variation on muscle contraction and flight performance. Isoforms are defined here as molecules that exist as multiple types because of modifications during or after transcription from a single gene. Small variations in the composition of a protein in insect flight muscle can affect both ultrastructure and mechanics, sometimes independently.1 Thus, achieving a full understanding of the structure and function of insect flight muscles requires knowledge about proteins that exist as multiple isoforms.

An overview of isoform variation also provides an important general perspective because it addresses a central question raised by genome sequencing: how could complex multicellular organisms evolve with as little as a 2-3 fold increase in the number of coding genes compared to unicellular eukaryotes? Protein isoform diversity has been proposed as one of the key mechanisms that has allowed a huge increase in organismal complexity with only a modest inflation in gene number.2-4 By surveying the array of molecular variants known to exist within one tissue type, along with the known functional effects and the as yet poorly documented combinatorial possibilities, we can begin to appreciate the capacity of a relative handful of protein-coding genes to give rise to an exponentially greater array of functionally variable protein combinations.

The final part of this chapter extends this theme by exploring in a more focused manner the biology of isoform diversity in one protein in the flight muscles of dragonflies. By combining mechanistic and ecological perspectives, this work exemplifies the emerging sub discipline of functional ecological and evolutionary genomics.5 Also, because it is based on a species other than Drosophila melanogaster, it provides additional taxonomic breadth and an indication of the diversity of flight muscle physiology within insects, the most taxonomically diverse group of organisms.

Nature's Versatile Engine

The diversity of flying insects necessitates a highly adaptable flight motor. Flight-capable insects range in body mass over about five orders of magnitude ( < 1mg to ca. 80 g), with wide variation in wingbeat frequency and contractile mechanics. Certain tiny wasps and flies have wingbeat frequencies of 500-1000 Hz, whereas large butterflies and moths use wingbeat frequencies as low as 10-20 Hz. Carpenter bees6,7 are active at midday in hot deserts with muscle temperatures up to 48°C, whereas small winter-active geometrid moths8 can fly with muscle temperatures as low as -3°C. These large differences in contraction frequency and operating temperature have resulted in the evolution of considerable diversity in the ultrastructure and contractile physiology of insect flight muscles.9-12

Insect flight muscles can also undergo phenotypic variation over time within an individual during adult maturation and/or in response to environmental variation. An extreme example is that of bark beetles (Ips pini; Scolytidae), which undergo degeneration of their flight muscles during mating and brood rearing within trees, but can within a few days regenerate their flight muscles and regain the ability to fly.13 Other insects undergo fairly predictable changes in flight muscle size and ultrastructure during the course of adult maturation. The flight muscles of Tsetse flies are not fully mature until after they have consumed a number of blood meals,14 undergoing a total increase in mass of about 75%. Nearly all taxa of dragonflies (Odonata) undergo substantial growth of their flight muscles during adult maturation,15 with dragonflies of the genus Libellula showing as much as a doubling or tripling of muscle size.16

There are important molecular changes during adult maturation of insect flight muscles,17-19 and presently there is fairly little knowledge of how molecular changes are associated with variation in muscle size and usage (but see ref. 20). The extreme taxonomic and functional diversity of insects, along with the ecological importance of variation in flight performance versus energetic costs (a major tradeoff for any flying animal) suggests that this is a rich field for further exploration,21 especially for biologists seeking to relate molecular mechanisms underlying phenotypic plasticity with quantitative functional assays in an ecological context. The remainder of this chapter presents an overview of progress that has been made to date along these lines.

The Underlying Genetics: An Under Inflated Genome and a Hyper Inflated Transcriptome and Proteome

As argued above, the taxonomic and ecological radiation of insects has required tremendous versatility and adaptability of their flight motor, including the ability of individual insects to make adjustments in their muscle size and contractility. As will be shown below, phenotypic adjustments in muscle protein composition generally involve changes in transcripts and proteins rather than changes in expression within gene families. This realization is part of a general paradigm shift away from the “one gene - one protein” model, which has for many decades been a core element of the central dogma of molecular biology. Movement away from this paradigm has been gaining considerable momentum during the last few years in not just muscle physiology, but all aspects of biology. A primary driver has been the revelation that gene number is not hugely different between simple and complex organisms. The first complete eukaryotic genome sequence was for yeast, which revealed a total of 6K coding genes. At that time, estimates of gene number in more complex eukaryotes varied widely, with 100K being the most widely accepted projection for the number of coding genes in humans.22 A common underlying assumption was that evolution of more complex and versatile creatures must have involved a great proliferation of gene number. We now know that complex multicellular eukaryotes have only about 14-30K genes, which is but a 2-5 fold enumeration of the genome size of yeast. This is a rather startling finding, for it reveals that features such as multiple tissue types, nervous systems, behavior, complex life histories, and the ability to make quantitative phenotypic adjustments in the functionality of tissues such as flight muscles, evolved with only a modest inflation of gene number. As this realization has come about, it has also become clear that complex eukaryotes rely heavily on mechanisms that create protein diversity at the transcriptional, post-transcriptional, and post-translational stages. For example, it is presently estimated that at least 40% of human genes are alternatively spliced,23 whereas there are less than a dozen known alternatively spliced genes in yeast.

Isoform diversity appears to be especially common in nerve24,25 and muscle,26 where the ability to vary the molecular composition of ion channels and contractile filaments allows for fine-tuning of the kinetics of electrical or mechanical outputs and therefore specialization of function. A muscle containing a certain protein isoform may have mechanical properties quite different from a neighboring muscle containing a different isoform. Accordingly, it is common in insects to find one particular isoform expressed only in flight muscle and a different isoform in leg muscle. There are also examples in which a single insect flight muscle contains different protein isoforms or isoform mixtures over time or in different ecological settings. Thus, both the functional specificity between muscles, and the ability to adjust to ontogenetic or ecological conditions within muscles appears to be based in large part on the ability to vary protein isoform composition.

Creation of multiple protein isoforms from a single gene occurs in insect flight muscles by a variety of mechanisms, including alternative start codons (e.g., PAR domain protein 1),27 alternative splicing (e.g., troponin-T),28,29 and post-translational modification of proteins by phosphorylation (e.g., flightin).19 Table 1 shows a catalog of proteins that are known to exist as multiple isoforms within insect flight muscles, or have an isoform expressed in flight muscle that is different from isoforms expressed in other muscles. Known functional effects are noted. This list includes components of the thick filaments (myosin heavy chain and myosin light chain), thin filaments (tropomyosin and all of the troponins), structural proteins that affect stiffness, elasticity, and filament anchoring (kettin, projectin, alpha actinin), metabolic enzymes (GPDH), regulatory enzymes (Mlck and Pdp 1), and ion channels (Slowpoke, BSC1). Actin and Troponin C are included in Table 1 for the sake of completeness, although these proteins have multiple isoforms that are encoded by a gene family rather than the one-gene, many-protein pattern of expression that is the operational definition of isoform variation used in this chapter. Altogether, Table 1 includes almost all of the myofibrillar proteins known to exist in Drosophila indirect flight muscle.1

Table 1. Catalog of insect flight muscle proteins that exist as multiple isoforms.

Table 1

Catalog of insect flight muscle proteins that exist as multiple isoforms.

Functional Effects of Isoform Variation

Some of the proteins listed in Table 1 have an isoform that is expressed only in flight muscle. Experiments in which genetic manipulations have caused a nonflight muscle isoform to be expressed in the flight muscle of a Drosophila null mutant have shown effects ranging from no readily observable phenotypic change (tropomyosin30) to flightlessness (GPDH31) or intermediate effects (MHC32). This mixed bag of results prevents the general conclusion that flight muscle specific isoform expression is essential for proper function, although the majority of cases seem to indicate that this is true.

The Importance of Quantitative Measures

Functional variation caused by isoform switching can be subtle and detectable only by quantitative rather than qualitative methods. For example, restoration of flight ability in Drosophila by replacement of IFM-specific tropomyosin with another tropomyosin30 is a qualitative measure that does not address how the level of flight ability might be affected. The following example illustrates how quantitative changes may be detectable only by using sophisticated measurement techniques.

In Drosophila flight muscles, myosin light chain kinase and other phosphorylases appear to become active during the first few hours following adult emergence, since only dephosphorylated MLC is present in late pupae and phosphorylated MLC accumulates in the hours following adult emergence.17,33 MLC phosphorylation increases the ATPase activity of purified D. melanogaster myosin.18,33 These observations, along with the similar time course of MLC phosphorylation and flight acquisition in newly emerged adults, suggest that MLC phosphorylation upregulates muscle contractility.

Genetic manipulations have been used to characterize the in vivo functional effects of variability in MLC phosphorylation.34,35 In these experiments, flightless heterozygotes of homozygous-lethal MLC null mutants were rescued to normal muscle ultrastructure and flight ability by P-element transformation with the wild-type allele. Site-directed mutagenesis was subsequently used to create cDNA constructs in which 2 serine residues, the sites of MLC2 phosphorylation by myosin light chain kinase (MLCK), were replaced by unphosphorylable alanines. These constructs were transposed into MLC2 null mutants, resulting in lines of flies in which the only full-length, functional copy of MLC2 lacked either one or both of the sites that can be phosphorylated by MLCK. The resulting flies were examined for flight muscle ultrastructure, skinned fiber mechanical characteristics, aerodynamic power output and metabolic power input during tethered flight.34,35 Muscles from the flies transformed with MLC2 lacking one or both MLCK phosphorylation sites showed no apparent changes in myofibrillar ultrastructure during rest, maximal activation, or rigor, nor did they show significantly altered calcium sensitivity, cross-bridge kinetics, or maximum steady-state isometric tension. Mutant muscles did show mechanical features indicative of a reduced recruitment of force-producing cross-bridges during stretch activation. Mechanical power output of mutant lines during tethered flight was reduced by 19-28% compared to wild type transformants, along with a similar decrease in metabolic power input, with no change in efficiency. Mutant flies could generally produce sufficient vertical net aerodynamic force to support their body weight, but significantly less than the 1.35 force/weight ratio produced by wild-type rescued and unmanipulated control flies.

This example shows quite clearly that subtle but important functional effects can result from changes as small as the addition of one or two phosphates on a single protein.

Alternative Splicing and the Generation of Combinatorial Complexity

Phenomena such as tissue-type specificity of isoform expression and functional effects of protein phosphorylation are reasonably familiar to most biologists. What is considerably less familiar, and completely missing from undergraduate textbooks in cellular and molecular biology, is the fact that a single tissue can express myriad forms of alternatively spliced transcripts from a single gene.

Figure 1 shows the splicing patterns of two genes that each encode a diversity of transcripts and proteins within insect muscle. The troponin T gene (TnT) in dragonflies encodes seven distinct transcripts,28 including all but one of the eight possible combinations of a cassette of three alternatively spliced exons. The relative abundance of these transcripts within an individual dragonfly matches, at least qualitatively, the relative abundance of different TnT protein variants from flight muscle on 2-d gels,29 which indicates that these transcripts are translated and the protein is incorporated into muscle. A more elaborate example is the projectin gene in Drosophila (for more detail, see chapter by Ayme-Southgate and Southgate), which has been shown to encode at least 16 distinct transcripts36 (and probably many more, since the basic pattern shown in Figure 1B allows at least 144 combinations of the 13 alternatively spliced exons). These projectin transcripts were charaterized from total adult RNA, so it remains to be determined which isoforms are expressed in flight muscle. Suppose for the sake of illustration that these two genes undergo independently regulated alternative splicing in a single insect flight muscle. In that case, there could be 7 × 144 = 1,008 combinations of distinct proteins, a total that approaches 10% of the number of coding genes in an insect genome. Expansion of these combinatorial possibilities by inclusion of other alternatively spliced genes in muscle, or the plethora of post-translational modifications such as phosphorylation19 would quickly expand this number of protein combinations to very large numbers.

Figure 1. Gene structure and alternative splicing pattern of two insect muscle genes.

Figure 1

Gene structure and alternative splicing pattern of two insect muscle genes. Panel A shows the 5' end of the troponin-T gene from the dragonfly Libellula pulchella.,, Alternative exons are white, constitutive exons are black, and exons within the 5' UTR (more...)

Although there is presently very little knowledge of the extent to which organisms actually use this potential to generate different protein combinations, and no studies have yet examined constraints such as coordinate regulation of isoform expression of multiple protein species in insect flight muscles, it is clear that the capacity for generating different combinations of proteins is extremely high. It is also interesting to note that natural selection might commonly affect loci that control alternative splicing, thereby causing shifts in the relative abundance of isoforms of alternatively spliced proteins. Such a response to selection could result in large changes in function despite little or no genetic change at the loci of the relevant structural proteins or enzymes. A rough example of this is the recent finding that the chromosomal locations of quantitative trait loci significantly associated with the activity of a number of glycolytic enzymes in Drosophila (glycogen synthase, hexokinase, phosphoglucomutase, trehalase) is different from the chromosomal locations of those enzymes.37 There is no indication as yet that this particular example involves isoform variation, but it serves to illustrate the point that genetic variation underlying functional differences does not necessarily reside in genes that encode the proteins that carry out a particular function.

Functional Consequences of Naturally Occurring Isoform Variation

Most of the work on functional effects of insect flight muscle isoforms has focused on genetic manipulations that cause expression of non-IFM isoforms in flight muscles,30-32,38-42 or mutations that cause a failure to express the wild type IFM isoform or isoform mixture.43 Such studies are excellent tools for understanding the molecular basis of muscle development and contraction, but they reveal little about naturally occurring variation because they create phenotypes that do not exist in nature. There has been relatively little work aimed at determining the functional consequences of naturally occurring variation in IFM isoform content. The example of MLC2 phosphorylation17,18,33-35 discussed above comes close to doing this, but does not squarely hit the mark because variation in MLC2 phosphorylation is only known to occur during early adult maturation, so that flight-capable wild type flies are presumably invariant in their MLC2 phosphorylation state. The only studies that have specifically addressed the functional effects of naturally occurring variation in flight muscle isoforms examine alternative splicing of troponin-T in dragonfly flight muscles.28,29 Here I present an overview of that work and place it in an ecological context that broadens the ability to appreciate functional significance.

As shown in Figure 1, the troponin-T gene in the dragonfly Libellula pulchella contains three alternative exons near the 5' end of the coding region. One of the alternative exons contains only three nucleotides that encode a single amino acid (lysine), thus demonstrating that alternative splicing can provide the finest possible level of control over amino acid content of a protein. Interestingly,Drosophila troponin-T also has three alternative exons in the 5' coding region, including a micro-exon that encodes a single lysine residue.44 In Drosophila however, flight muscles contain only one splice variant and the microexon is expressed only in adult hypodermic and visceral muscles. From this it appears that there is wide variation among insects in their patterns of expression of troponin-T splice variants.

We have characterized six distinct transcripts of L. pulchella troponin-T from either cDNA clones or PCR products from flight muscles, along with a seventh cDNA that occurs in leg and body wall muscles but is absent from flight muscles.28,29,45 A PCR fragment that corresponds with the size of the eighth possible combination of the three alternative exons is sometimes detectable as a rare transcript but has not been captured in any of our sequenced subclones.

To quantify the relative abundance of the different splice forms of TnT, we used PCR primers for constitutive regions flanking the cassette of three alternatively spliced exons. One of these primers carried a fluorescent tag, which allowed the PCR product to be fractionated **********> and quantified according to the relative abundance of each fragment size.28,29,45 The primer pair used in this experiment generated TnT transcript fragments that were 243, 246, 258, 261, 267, 270, and 285 nucleotides in length (Table 2). This array of fragment sizes agrees precisely with sizes predicted from sequence data of the seven known splice variants.

Table 2. Fragment sizes (number of nucleotides) and mean relative abundances (% of total TnT transcripts within flight muscles from an individual dragonfly) of the PCR products obtained from amplification of the 5' alternatively spliced region of L. pulchella TnT cDNA.

Table 2

Fragment sizes (number of nucleotides) and mean relative abundances (% of total TnT transcripts within flight muscles from an individual dragonfly) of the PCR products obtained from amplification of the 5' alternatively spliced region of L. pulchella (more...)

To determine how variation in the alternative splicing of TnT affects muscle function, we combined measurements of the relative abundance of TnT splice variants within an individual dragonfly with assays of muscle contractile performance. One important caveat in these experiments is that in order to isolate a sufficient amount of RNA, all of the flight muscles from one half of the thorax were homogenized prior to generating cDNA. Thus, when comparing the relative abundance of different troponin-T transcripts to the contractile performance of a single muscle or to the flight performance of the dragonfly, we made the simplifying assumption that within an individual the major flight muscles are fairly homogeneous in their isoform composition.

At the cellular level, there was a strong correlation between the summed relative abundance of transcripts 261 & 267 and the sensitivity of skinned fibers to activation by calcium (Fig. 2A). There was nearly as strong a correlation between the summed relative abundance of transcripts 261 & 267 and the power output of the intact basalar muscle, which drives the downstroke of the forewing leading edge (Fig. 2B). (Note that these results are consistent with the way alternatively spliced forms of troponin-t affect the calcium sensitivity and other contractile properties of human cardiac muscle).46 From high-speed video recordings of free-flying dragonflies, we showed that there is a significant correlation between the relative abundance of transcripts 261 & 267 and wingstroke amplitude and frequency (Fig. 3A), the main kinematic variables that insects use to adjust aerodynamic force and power output. Finally, dragonflies with greater wingbeat amplitude and frequency were shown to have significantly higher rates of flight metabolism (Fig. 3B).

Figure 2. Relationship between the abundance of two troponin T transcripts and A) the calcium sensitivity of skinned fibers and B) power output of the intact basalar muscle during workloop contractions.

Figure 2

Relationship between the abundance of two troponin T transcripts and A) the calcium sensitivity of skinned fibers and B) power output of the intact basalar muscle during workloop contractions. Data are from ref. . Open symbols are females; closed symbols (more...)

Figure 3. Relationship between the abundance of two troponin t transcripts and A) the product of wingbeat amplitude and frequency during free flight, and B) effect of the product of wingbeat amplitude and frequency on the energetic cost of flight.

Figure 3

Relationship between the abundance of two troponin t transcripts and A) the product of wingbeat amplitude and frequency during free flight, and B) effect of the product of wingbeat amplitude and frequency on the energetic cost of flight. Each data point (more...)

Male dragonflies engage in vigorous and sometimes highly escalated flight contests to establish and defend territories, and to acquire and defend mates. Thus, it is not surprising to find that both territorial and mating success have a significant positive relationship with muscle power output47 (Fig. 4).

Figure 4. Relationship between muscle power output of male L.

Figure 4

Relationship between muscle power output of male L. pulchella dragonflies and their lifetime territorial (A) and mating (B) success. Data are from ref. .

If having high muscle power output is strongly related to territorial and mating success, what is the purpose and utility of dragonflies being able to vary the contractility and power output of their flight motor? One answer comes from ontogenetic studies, where we have shown that contractility increases steadily during the course of adult maturation.29 L. pulchella dragonflies approximately double in body mass between adult emergence and sexual maturity, and mating and territoriality occur only when dragonflies are fully mature. Thus, it appears that muscle and flight performance are up-regulated only at maturity when intense aerial battles are used by males to establish and defend territories and acquire mates, and by females to evade unwanted copulation attempts by males. Reduced power output by immature adults should reduce their energetic cost of flight, which may be critically important given that approximately two-thirds of newly emerged L. pulchella adults lose body mass and disappear from the population (i.e., they appear to starve).48 Seen in this context, it appears that dragonflies use isoform variation of troponin T to adjust the tradeoff between muscle performance and the energetic cost of flight (Fig. 5).

Figure 5. Alternative splicing of troponin-T allows dragonflies to achieve either low performance, low cost flight that helps conserve energy, or high performance flight that increases territorial and mating success.

Figure 5

Alternative splicing of troponin-T allows dragonflies to achieve either low performance, low cost flight that helps conserve energy, or high performance flight that increases territorial and mating success.

Not all L. pulchella fully upregulate their muscle performance at maturity, as some have a relatively low muscle power output even after they have been mature for a number of weeks (i.e., the low power output data points in (Fig. 4). This brings us back to the question of why this species, even at sexual maturity when flight performance is critical for territorial and mating success, has evolved the ability to down-regulate muscle contractility and flight performance. We have recently obtained what appears to be a good answer to this question, for we have found that nutritional status is an important factor determining the variation in muscle performance among mature males.47 Males that are not infected with gregarine gut parasites (protozoans) show a strong positive correlation between total body lipid content and muscle power output, whereas infected individuals show no such relationship (Fig. 6).

Figure 6. Relationship between total body lipid content and the power output of dragonfly flight muscles.

Figure 6

Relationship between total body lipid content and the power output of dragonfly flight muscles. Healthy dragonflies show a strong positive relationship between fat reserves and muscle power output, but this relationship is absent in individuals infected (more...)

The ability of healthy dragonflies to adjust their muscle contractile performance may allow their maximum energy consumption rate to match the rate at which energy can be mobilized from storage pools. Variation among individuals in levels of stored fat may be caused by short term differences in territorial effort and foraging success (which are affected strongly by weather), but there are also likely to be long-term differences in foraging ability that keep some individuals relatively energy-poor throughout their adult lives.

In L. pulchella dragonflies infected with gregarine gut parasites, the apparent loss of the ability to match muscle contractility to the size of the energy storage pool suggests that gregarines may cause physiological changes that affect signaling pathways and energy homeostasis. Indeed, we have found that dragonflies with gregarine trophozoites in their midgut have chronically activated p38 map kinase in their flight muscles (Schilder & Marden, unpub. data). In vertebrates, this molecule is involved in pathways that control a wide variety of cellular functions and gene expression,49 including insulin signaling, glucose transport, and the function of fat storage cells.50,51 Changes in p38 MAPK signaling are also known to affect certain components of the molecular machinery that controls alternative splicing.52 Although we have only begun to scratch the surface of this intriguing interaction between gut parasites and the ability of dragonflies to adjust their performance and energetic costs, what we have found so far in this single species suggests that there is likely to be a wealth of interesting biology involved in the physiology and ecology of isoform variation in the flight muscles.

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