Integrative and Comparative Biology 2005 45(3):511-524; doi:10.1093/icb/45.3.511
The Society for Integrative and Comparative Biology
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 Reproduction1
Anthony J. Zera2,1
1 School of Biological Sciences, University of Nebraska, Lincoln, Nebraska 68588-0118
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SYNOPSIS
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The extent to which modifications in intermediary metabolism
contribute to life history variation and trade-offs is an important
but poorly understood aspect of life history evolution. Artificial
selection was used to produce replicate genetic stocks of the
wing-polymorphic cricket,
Gryllus firmus, that were nearly pure-breeding
for either the flight-capable (LW[f]) morph, which delays ovarian
growth, or the flightless (SW) morph, which exhibits enhanced
early-age fecundity. LW(f) lines accumulated substantially more
triglyceride, the main flight fuel in
Gryllus, compared with
SW-selected lines, and enhanced accumulation of triglyceride
was strongly associated with reduced ovarian growth. Increased
triglyceride accumulation in LW(f) lines resulted from elevated
de novo biosynthesis of fatty acid and two morph-specific trade-offs:
(1) greater proportional utilization of fatty acid for glyceride
biosynthesis
vs. oxidation, and (2) a greater diversion of fatty
acids into triglyceride
vs. phospholipid biosynthesis. Even
though SW lines produced less total lipid and triglyceride,
they produced more phospholipid (important in egg development)
than did LW(f) lines. Differences between LW(f) and SW morphs
in lipid biosynthesis resulted from substantial alterations
in the activities of all studied lipogenic enzymes, a result
that is consistent with expectations of Metabolic Control Theory.
Finally, application of a juvenile hormone analogue to LW(f)
females produced a striking SW phenocopy with respect to all
aspects of lipid metabolism studied. Global alterations of lipid
metabolism, most likely produced by alterations in endocrine
regulation, underlie morph specializations for flight
vs. early-age
fecundity in
G. firmus. Modification of the endocrine control
of intermediary metabolism is likely to be an important mechanism
by which intermediary metabolism evolves and contributes to
life history evolution.
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INTRODUCTION
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The functional causes of life-history evolution have been a
central focus of life-history research for over six decades
(Fisher, 1930
; Pianka, 1981
; Townsend and Calow, 1981
; Ricklefs,
1996
; Rose and Bradley, 1998
; Zera
et al., 1998
; Ketterson and
Nolan, 1999
; Sinervo
et al., 2000
; Zera and Harshman, 2001
;
Ricklefs and Wikelski, 2002
; Zhao and Zera, 2002
). At issue
are the physiological, biochemical, and molecular processes
that have been altered by natural selection to produce modified
life-history traits (
e.g., enhanced early-age reproduction),
and trade-offs between traits (
e.g., enhanced early-age reproduction
coupled with decreased longevity). A number of studies have
identified physiological (
e.g., energetic and endocrine) correlates
of life-history variation and trade-offs within species (Service,
1987
; Djawdan
et al., 1996
; Rose and Bradley, 1998
; Sinervo
et al., 2000
; Ketterson and Nolan, 1999
; Zera and Cisper, 2001
;
Harshman and Hoffmann, 2000
; Zera and Larsen, 2001
; Zera and
Harshman, 2001
). More recently, other studies have begun to
focus on the molecular causes of life history evolution (Stearns
and Magwene, 2003
). However, the biochemical-metabolic underpinnings
of life history variation and trade-offs remain understudied
aspects of life history evolution (Zhao and Zera, 2002
; Zera
and Zhao, 2003
b).
A priori, evolutionary modification of intermediary metabolism is expected to be an important factor in life-history evolution. For example, the evolution of increased egg yolk protein biosynthesis is likely to be a key component of the evolution of increased early age fecundity. Similarly, increased longevity or resistance to stressful conditions (e.g., tolerance to desiccation or starvation) are life history traits that are typically associated with enhanced accumulation of energy reserves, and must have evolved via modifications of carbohydrate and lipid metabolism (Zera and Harshman, 2001). Although modification of intermediary metabolism likely plays a key role in life history evolution, only limited information is available on any aspect of this topic (e.g., specific metabolic pathways, enzymes, or regulatory controls that have been altered in genotypes, populations or species to produce differences in life histories). This is especially the case for genetically-based alterations in intermediary metabolism that underlie life history variation and trade-offs. This paucity of information constitutes a major roadblock to attaining a deep understanding of the mechanisms of life-history evolution (Zera and Harshman, 2001; Zera and Zhao, 2003b).
Artifical selection is a powerful tool to investigate the mechanisms of evolution (Falconer and Mackay, 1996; Lynch and Walsh, 1998; Gibbs, 1999; Harshman and Hoffmann, 2000). Recently, a number of studies have begun to use artificial selection to investigate modifications of intermediary metabolism that underlie various aspects of life history evolution (Harshman and Schmidt, 1998; Harshman et al., 1999; Harshman and Hoffmann, 2000; Zera and Larsen, 2001; Zhao and Zera, 2002; Zera and Zhao, 2003a, b). This review primarily focuses on results obtained in my laboratory during the past five years on variation in aspects of lipid metabolism between genetic stocks of the wing-polymorphic cricket, Gryllus firmus, artificially selected to produce morphs adapted for the flight at the expense of reproduction, and vice versa. The ultimate goal of these studies has been to identify genetically-based alterations in lipid metabolism that contribute to the trade-off between flight and reproduction. To put these studies in context I first provide background information on wing polymorphism, and the relationship between lipid physiology and life history trade-offs.
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BACKGROUND ON WING POLYMORPHISM IN GRYLLUS FIRMUS
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Wing polymorphism consists of morphs (discontinuous phenotypes)
within a species that are adapted for flight at the expense
of reproduction and vice versa (
Fig. 1). The polymorphism is
common in many insect groups, most notably the Hemiptera/Homoptera
(waterstriders, planthoppers and aphids), Coleoptera (beetles)
and, Orthoptera (crickets and grasshoppers) (Johnson, 1969
;
Harrison, 1980
; Roff, 1986
; Dingle, 1996
; Zera and Denno, 1997
).
Natural populations of the cricket,
G. firmus, the focus of
this review, contain flight-capable and flightless morphs (Veazy
et al., 1976
). The flight-capable morph (denoted LW[f]; [Zera
et al., 1997
];
Fig. 1) has fully-developed wings, large (functional)
flight muscles, and a large reserve of triglyceride, the main
flight fuel in
Gryllus (Zera
et al., 1999
; Zera and Larsen,
2001
). The alternate, flightless (SW) morph has underdeveloped,
non-functional wings and flight muscles, and reduced whole-body
triglyceride. By the end of the first week of adulthood, the
SW morph has substantially-elevated fecundity (100–400%)
relative to the LW(f) morph (
Fig. 1; Zera and Denno, 1997
; Zera
and Harshman, 2001
; see below). Reproductive differences between
morphs occur in the absence of flight, and thus reflect the
negative impact of flight
capability (production and maintenance
of the flight apparatus), rather than flight itself, on egg
production. Wing polymorphism is the most dramatic example of
the trade-off between flight capability and reproduction, a
life-history trade-off of prime important in insects in general
(Johnson, 1969
; Harrison, 1980
; Roff, 1986
; Dingle, 1996
; Zera
and Denno, 1997
).
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FIG. 1. Flight-capable (LW[f]; denoted as "LW" in this figure) and flightless (SW) female morphs of Gryllus firmus of the same age (day 5 of adulthood). In the left panel, the fore wings have been removed to show variation in the hind wings. The middle and right panels illustrate dissections of morphs showing much larger, functional flight muscles, but much smaller ovaries, in the flight-capable female, and substantially-underdeveloped flight muscles but much larger ovaries in the flightless female. See Figure 1 (Zera, 2004) for a color illustration of this figure
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As is the case for most other wing-polymorphic insects, morph
expression in
G. firmus is influenced by both genetic and environmental
factors (
i.e., it is a genetic polymorphism and an environmental
polyphenism). Wing morph expression is under polygenic control
(heritability =
0.60–0.7; Roff, 1990
), and nearly pure-breeding
lines for the LW(f) or SW morphs have been produced in several
laboratories by artifical selection (Roff, 1990
; Zera and Cisper,
2001
;
Fig. 2). Environmental factors such as density, photoperiod,
temperature, and food quality, also strongly affect morph expression
in
G. firmus and in other insects (Denno
et al., 1985
; Zera
and Tiebel, 1988
; Zera and Denno, 1997
).
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FIG. 2. Response to artificial selection on wing morph (LW[f] or SW) in G. firmus. Solid line is the grand mean frequency of LW(f) females (mean of the three LW[f]-selected line means ± SEM), while the broken line is the grand mean frequency of the SW females (mean of three line means) as a function of generation of selection. Approximately 120 males and 120 females of the same morph type were used as breeders for each generation in each line. Females used in biochemical experiments were raised under slightly lower density which increased the frequency of the LW(f) morph in LW(f)-selected lines (to >90%), while keeping the freq. of the SW morph at >90% in SW-selected lines
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Extensive ecological studies over many decades, indicate that
wing polymorphism is maintained by environmental heterogeneity
(Vepsalainen, 1978
; Denno
et al., 1980
, 1991
; Roff, 1994
; Dingle,
1996
; Zera and Denno, 1997
). Within a patch, the more-fecund
flightless morph has a substantial fitness advantage over the
flight-capable morph. However, the flight-capable morph has
the ability to track resources and mates between patches, as
well as to escape deteriorating patches (Denno
et al., 1980;
Langellato and Denno, 2001
).
The physiological causes of morph specialization for flight capability vs. reproduction have been extensively studied in G. firmus and several congeners (Zera and Denno, 1997; Zera and Harshman, 2001). These studies provide important background context for biochemical investigations of morph specialization. Feeding studies in three species of Gryllus have documented that flight-capable and flightless morphs consume and assimilate equivalent or nearly equivalent amounts of nutrients (Mole and Zera, 1993; Zera et al., 1998; Zera and Brink, 2000; A. J. Zera, unpublished data; discussed in more detail below). Thus, differences between the morphs in energy allocated to the flight capability vs. reproduction must be derived almost exclusively from morph-specific differences in internal nutrient allocation (i.e., an internal trade-off) rather than from morph-specific differences in acquisition of nutrients from the diet. The LW(f) morph allocates a greater amount of its energy budget to the growth and maintenance of the large flight muscles and production of large quantities of triglyceride flight fuel (see below), which appear to constrain ovarian growth. At present, it is unclear whether reduced ovarian growth is due to nutrients or space within the LW(f) morph that are insufficient to accommodate large ovaries, in addition to the large thoracic muscles and large lipid reserves necessary for flight (Zera and Harshman, 2001). Alternatively, the endocrine environment necessary to accumulate large triglyceride reserves and to maintain the large flight muscles may have inherent negative effects on ovarian growth independent of nutrient availability (Zera and Cisper, 2001; Zera et al., 1998; see below).
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LIPIDS AND LIFE HISTORIES IN INSECTS: CORRELATIONS BETWEEN LIPID RESERVES AND LIFE HISTORY TRAITS
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Lipid is a heterogeneous class of molecules which plays a variety
of important biological roles (Downer, 1985
; Beenakkers
et al.,
1985
). In insects, triglyceride and phospholipid are the two
most abundant lipid classes and together comprise more than
90% of total lipid (Beenakkers, 1985
; Grapes
et al., 1989
).
Triglyceride is the most abundant energy storage molecule, while
phospholipid, the second most abundant lipid class, is the primary
component of biological membranes. In life history studies,
lipid content has most often been investigated in the context
of somatic (non-reproductive) energy stores (Zera and Harshman,
2001
). However, eggs also contain a high content of both triglyceride
and phospholipid and lipid biosynthesis increases during egg
production (Beenakkers
et al., 1985
; Grapes
et al., 1989
; Lipsitz
and McFairlane, 1970
). Total lipid can be easily extracted by
organic solvents and quantified, even in small insects like
Drosophila. Thus, many life history studies in insects (most
studies of
D. melanogaster; Zera and Harshman, 2001
) have measured
whole-body, total lipid (or "neutral" lipid =
primarily non-structural
lipid energy reserves) to estimate calories devoted to somatic
energy reserves. This method only gives a crude estimate of
somatic lipid energy stores because a significant proportion
of total extracted lipid may be due to structural lipid (
e.g., phospholipid). Furthermore, whole-body total or "neutral" lipid
may contain a significant amount of non-somatic lipid (
e.g., from the ovaries), and, depending upon the specific extraction
procedure, a variety of carbohydrates and amino acids (Christie,
1982
). Other studies have documented differences between life-history
phenotypes with respect to specific lipid classes, such as triglyceride
(Nwanze
et al., 1976
; Gunn and Gatehouse, 1993
; Zera and Larsen,
2001
), sometimes measured in specific organs (see below), which
provide a more accurate picture of the functional relationship
between various lipids and life history traits.
Several important associations have been identified between total lipid, or triglyceride, and specific life-history traits in insects. For example, total lipid/triglyceride is typically more abundant in individuals adapted to live longer, to withstand starvation or stress, or in preparation for energy demanding activities such as diapause, or flight (reviewed in Downer, 1985; Dingle, 1996; Zera and Harshman, 2001). Increased lipid/ triglyceride reserves for these demanding activities can accumulate during the juvenile stage (e.g., Nwanze et al., 1976; Gunn and Gatehouse, 1993; Chippendale et al., 1996), and may be associated with slower juvenile growth rate (Chippendale et al., 1996). Alternatively, these reserves may accumulate during adulthood and are often associated with reduced early-age fecundity (Djawdan et al., 1998; Zera and Larsen, 2001; Zera and Harshman, 2001). The most extensive study of lipid classes in the context of a life history trade-off has been reported by Zera and Larsen (2001), and is discussed in detail below.
Consistent correlations between lipid levels and specific life history traits, discussed above for insects, also occur in animals in general (Zera and Harshman, 2001), and strongly suggest that alterations in lipid metabolism are a common aspect of life history evolution. However, until a few years ago, information on specific modifications of lipid metabolism that underlie life history adaptations has been restricted to a few enzymological characterizations of selected lines (Harshman and Schmidt, 1998; Harshman et al., 1999). Essentially no information has been available on variation in in vivo processes such as rates of lipid biosynthesis or oxidation that underlie life history adaptations and trade-offs. Furthermore, interactions among pathways are an important aspect of intermediary metabolism (e.g., pathways of protein and lipid metabolism are linked via the ability of amino acids to be converted into either protein or fatty-acid; Downer, 1985). Yet no information is available on the extent to which modification of pathway interactions contributes to life history adaptation. Finally, flux through pathways of intermediary metabolism is tightly regulated by hormones (Granner and Pilkis, 1996; Sul and Wang, 1998). Yet we are almost completely ignorant of alterations in the endocrine control of metabolism that have given rise to life history adaptations. In the past few years, detailed biochemical studies of lipid metabolism have been undertaken in artificially-selected lines of the wing-polymorphic cricket, Gryllus firmus, that have investigated each of the topics mentioned above. This work is the focus of the rest of the present review.
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DIFFERENCES IN LIPID METABOLISM BETWEEN LIFE-HISTORY MORPHS OF G. FIRMUS
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Artificial selection
To investigate the biochemical-genetic basis of life history
variation and trade-offs, lines of
G. firmus that produce a
high frequency of either the flight-capable (LW[f]) morph or
the flightless (SW) morph were obtained by artificial selection
(
Fig. 2). Details of artificial selection can be found in Zera
and Larson (2001)
and Zera and Cisper (2001)
. Briefly, selection
was conducted in three temporally-separated blocks. Each block
contained a line selected for the LW(f) morph, a line selected
for the SW morph, and a control line. Control lines are not
discussed here. All lines were initiated within a few weeks
from a single base population, which, in turn, had been founded
from
G. firmus collected in Gainesville, Florida (Zera and Cisper,
2001
). During selection, crickets were raised at 28°C, under
a 16 light:8 dark cycle, and were fed the standard (high-nutrient)
diet (see Zera and Larsen, 2001
). Crickets from generations
15–20 were used in biochemical characterizations and were
raised under slightly lower density than those in the standard
artifical selection experiment. This further reduced the frequency
of SW females in the LW(f)-selected lines (to <10%), while
retaining a high freq of SW females in SW-selected lines (>90%).
Studies were conducted exclusively on females, both because
of the labor intensive nature of the biochemical characterizations
(performed on two-three days of adulthood, on nine lines, fed
three diets), and because we were primarily interested in the
biochemical basis of the trade-off between flight capability
and early-age fecundity. The few (5–10%) SW females produced
in the LW(f)-selected lines, or LW(f) females produced in the
SW-selected lines were not characterized. In addition, by day
5 of adulthood, the last day on which crickets were compared
for lipid characteristics, about 10% or less LW females had
histolyzed their flight muscles (flight muscle histolysis is
common in crickets and increases with age; see Zera
et al.,
1997
). These flightless females with long wings (designated
LW[h]) are phenotypically very similar to SW females with respect
to reproductive, physiological, and biochemical traits and are
not considered here (
e.g., see Zera
et al., 1997
; 1999
; Zera
and Larsen, 2001
; Zera and Cisper, 2001
; Zhao and Zera, 2001
).
Biochemical characterizations were performed on stocks fed a
variety of diets (standard, low-nutrient, and high carbohydrate;
see Zera and Larsen, 2001
). However, data presented here were
either pooled across the various diets or were obtained on the
standard diet alone. Genetically-based differences in aspects
of lipid metabolism were ascertained using paired
t-tests, which
compared the difference in the means of a trait between LW(f)
and SW lines of a particular block, averaged across blocks (see
Zera and Larsen, 2001
; Zhao and Zera, 2002
and
Table 1; also
see Rose
et al., 1996
). In essence, the paired
t-test measures
the consistency of differences between LW(f) and SW-selected
lines across blocks. In some cases (
e.g., Zera and Zhao, 2003
b),
LW(f) and SW lines were crossed and backcrossed to document
co-segregation between various traits. See Zera and Cisper (2001)
and Zera and Larsen (2001)
for additional details of the selection
experiment and genetic analyses.
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TABLE 1. Morph-specific trade-off in the proportional biosynthesis of triglyceride vs. phospholipid in G. firmus.
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Differences in lipid levels between flight-capable (LW[f]) and flightless (SW) morphs
Triglyceride and phospholipids were measured in genetic stocks
of
G. firmus nearly pure-breeding for LW(f) (flight-capable)
or SW (flightless) morphs (data for one block on the standard
diet are presented in
Fig. 3; see Zera and Larsen [2001]
for
data on all blocks and diets). The LW(f) morph exhibited a genetically-based,
greater accumulation of triglyceride than the SW morph on each
of several diets that differed in total calories, carbohydrate,
or lipid content. Enhanced accumulation of triglyceride in the
LW(f) morph occurred during the first week of adulthood, precisely
when ovarian growth was substantially reduced relative to the
SW morph (
Fig. 3). Furthermore, enhanced accumulation of triglyceride
was associated with decreased accumulation of phospholipid (Zera
and Larsen, 2001
). Thus, in
G. firmus, there is a strong trade-off
between triglyceride accumulation and ovarian growth, or phospholipid
accumulation (Zera and Larsen, 2001
). These morph-specific differences
make functional sense because triglyceride is the main flight
fuel in
Gryllus (Zera
et al., 1999
), while phospholipid is an
important component of vitellogenin (yolk protein) (Beenakkers
et al., 1985
). In addition to these whole-organism differences
in triglyceride and phospholipid, the LW(f) morph allocates
a disproportionately greater amount of whole-organism triglyceride
and phospholipid to somatic (non-ovarian) tissues, while the
SW morph allocates a disproportionately greater amount to the
ovaries (A. J. Zera, unpublished data). As mentioned previously,
lipid is important for reproduction as well as for flight, and
eggs have high triglyceride and phospholipid contents (Beenakkers
et al., 1985
). The substantially greater amount of total lipid
and triglyceride found in the dispersing morph, of
G. firmus and other dispersal-polymorphic species (Nwanze
et al., 1976
;
Gunn and Gatehouse, 1993
), indicates that lipid requirements
are greater for dispersal than for reproduction during early
adulthood.
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FIG. 3. Relationships among whole-body triglyceride mass (top, left), ovarian mass (bottom, left), rate of triglyceride biosynthesis (top, right), and rate of fatty acid oxidation (bottom, right), during the first week of adulthood in LW(f) and SW adult G. firmus. Data are from LW(f) and SW lines of one representative block (Block 3) of the selection experiment (data from Zera and Larsen, 2001; Zhao and Zera, 2002; Zera and Zhao, 2003a). Differences of similar magnitude were observed between LW(f) and SW-selected lines of the other two blocks (see above references and text). Whole-body triglyceride masses were adjusted to whole-body, fat-free dry mass by ANCOVA. "Y" axes of triglyceride biosynthesis and fatty acid oxidation graphs represent amounts of radioactivity (DPM) incorporated into triglyceride or CO2 during a standard period of time (see text, Zhao and Zera [2002], and Zera and Zhao [2003a] for experimental details)
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Lipid acquisition from the diet does not differ between LW(f) and SW morphs
The substantial differences in lipid levels between LW(f) and
SW-selected lines of
G. firmus (Zera and Larsen, 2001
) could
result from morph-specific differences in any of several aspects
of lipid metabolism (
e.g., biosynthesis or oxidation). Alternatively,
increased accumulation of lipid in the LW(f) morph during the
first week of adulthood could simply result from greater nutrient
intake (
i.e., increased consumption and/or assimilation), rather
than morph-specific differences in internal metabolism. Surprisingly,
most recent physiological-energetic studies life-history trade-offs
have not quantified or controlled for potential differences
in nutrient input between life history phenotypes (
e.g., nearly
all studies of
Drosophila; Zera and Harshman, 2001
).
We conducted extensive feeding studies in three Gryllus species and documented that, in each case, flight-capable and flightless females consume the same, or nearly the same (within 10%), amount of food and do not differ in assimilation of total nutrients, lipid, carbohydrate, or protein when fed the standard diet (Mole and Zera, 1993; Zera et al., 1998; Zera and Brink, 2000; A. J. Zera, unpublished data). (Note: Significantly-reduced consumption by flightless vs. flight-capable females of G. firmus was originally reported by Mole and Zera [1994]. However, that result was due to abnormal retention of eggs by the more fecund SW females, which compressed their gut and inhibited feeding during the second week of the two-week feeding trial. Subsequent studies, restricted to the first week of adulthood, which is the same period of time during which lipid studies were conducted, demonstrated that mean consumption was only 10% lower in LW(f) vs. SW females [A. J. Zera, unpublished data].) Thus, elevated lipid levels in LW(f) vs. SW females must result exclusively from differential metabolism of internal nutrients rather than from differential acquisition of nutrients from the diet. G. firmus is one of the only insects for which differential acquisition of nutrients can be eliminated as a potential cause of phenotypic differences in energy reserves.
LW(f) and SW morphs differ in rates of lipid biosynthesis and oxidation
In vivo rates of total lipid, triglyceride and phospholipid biosynthesis were compared between LW(f) and SW morphs by quantifying the amount of radiolabelled precursor (14C-acetate or 14C-palmitic acid) incorporated into these lipids (see Zhao and Zera [2002] and the legend of Figure 4 for experimental details). Figure 4 (upper panel) illustrates a schematic diagram of the de novo pathways of triglyceride, and phospholipid biosynthesis. Briefly, fatty acids, most commonly 16–18 carbons in length (Beenakkers et al., 1985; Grapes et al., 1989), are synthesized from acetate (acetyl-CoA). These fatty acids are then combined with glycerol phosphate, and converted into either triglycerides or phospholipid, which comprise greater than 90% of total lipid in Gryllus and most insects (Beenakkers et al., 1985; Grapes et al., 1989; Zera and Larsen, 2001). Thus, biosynthesis of triglycerides and phospholipids consists of two parts: (1) the production of fatty acids that comprise the bulk (>90%) of these molecules, and (2) the partitioning of fatty acids into these two lipid classes.
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FIG. 4. Top panel. Simplified pathway of de novo biosynthesis of triglyceride and phospholipid from acetate (see Downer, 1985). Glycerol-3P = glycerol-3-phosphate. Box under phosphatidate indicates that this compound is composed of two fatty acids (FA) linked to glycerol-3-phosphate. Boxes next to triglyceride and phospholipid indicate that these compounds are produced from phosphatidate by removal of the phosphate group (to produce diglyceride) and subsequent addition of (1) a third fatty acid (triglyceride), or (2) a phosphorylated compound such as phosphocholine (phospholipid). 14C-acetate, next to acetate, and 14C-palmitate, next to fatty acid, indicate that radiotracer studies of lipid biosynthesis were conducted by injection of either radiolabelled actetate or palmitate into whole crickets. Amount of radiolabel incorporated into triglyceride and phospholipid during a given period of time was subsequently quantified. See Zhao and Zera (2001, 2002) for experimental details. Bottom panel. Morph-specific trade-offs in glyceride (triglyceride vs. phospholipid) biosynthesis in G. firmus identified in radiotracer studies of Zhao and Zera (2002). "Y" diagrams illustrate relative flow of 14C through de novo pathway of fatty acid biosynthesis in LW(f) (denoted as "LW" in this figure) and SW females. Total glyceride biosynthesis (=total fatty acid biosynthesis; denoted by width of base of "Y") is greater in LW(f) than in SW females. Total and relative biosynthesis of triglyceride is greater in LW(f) than in SW females, while total and relative biosynthesis of phospholipid is greater in SW than in LW(f) females. Widths of lines, which are only meant to illustrate rank-order and not quantitative differences between morphs, are based on data from Table 1, Figure 5, and additional data from Zhao and Zera (2002)
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On the first day of adulthood, rates of triglyceride or phospholipid
biosynthesis were low and equivalent in LW(f) and SW females
(Zhao and Zera, 2002
), consistent with the low and equivalent
levels of these compounds in LW(f) and SW morphs found on day
0 (
Fig. 3; Zhao and Zera, 2002
). By contrast, on day 5 of adulthood,
we observed a substantially higher rate of triglyceride biosynthesis
and lower rate of phospholipid biosynthesis in LW(f)
vs. SW
selected lines (
Figs. 3 and
5). No interactions were observed
between diet or line type (LW[f]
vs. SW) and data in
Figure 5 are biosynthetic rates pooled across the three diets. These
results are consistent with an increased rate of triglyceride
biosynthesis, and a decreased rate of phospholipid biosynthesis
being important factors that cause the greater accumulation
of triglyceride and lesser accumulation of phospholipid in the
LW(f)
vs. SW morphs (
Fig. 3; Zhao and Zera, 2002
). Finally,
the elevation in triglyceride biosynthesis was much greater
than the decrease in phospholipid biosynthesis in the LW(f)
morph. Thus, biosynthesis of total lipid (=total fatty acid)
was higher in LW(f)
vs. SW females (data not shown; see
Table 1 of Zhao and Zera, 2002
).
Because the biosynthesis of triglyceride and phospholipid was
measured in each individual, we also could estimate the proportional
biosynthesis of these two key lipid classes via ANCOVA. Results
presented in
Table 1 indicate a genetically based trade-off
in the biosynthesis of these two lipid classes. That is, relative
to SW-selected lines, LW(f) selected lines consistently converted
a
proportionately greater amount of acetate or palmitate into
triglyceride. Thus, two important, genetically-based alterations
in lipid biosynthesis were observed in selected lines of
G. firmus (
Fig. 4, lower panel). First, LW(f) lines exhibited increased
flux through the
de novo pathway of fatty acid biosynthesis
(resulting in the biosynthesis of a greater amount of fatty
acid and hence total lipid). Second, there was a more downstream
trade-off involving the greater diversion of fatty acids into
triglyceride
vs. phospholipid in the LW(f) morph. Even though
the rate of total lipid biosynthesis was lower in the SW morph,
the greater diversion of fatty acid into phospholipid resulted
in a higher rate of phospholipid biosynthesis in this morph,
compared with the LW(f) morph. As mentioned above, phospholipid
is an important component of vitellogenin, and plays an important
role in embryo development (Beenakkers
et al., 1985
). To our
knowledge, this represents the first direct documentation of
genetically-based alterations in the
in vivo flux through pathways
of intermediary metabolism leading to the differential production
of end products central to the specialization of phenotypes
for alternate life histories (Zhao and Zera, 2002
).
In addition to its genetically-based elevation in total lipid and triglyceride biosynthesis, the LW(f) morph also exhibited a genetically-based reduction in the rate of fatty acid oxidation relative to its SW counterpart (Figs. 3 and 6, upper panel). Rate of fatty acid oxidation was measured as the amount of radiolabelled, injected fatty acid (palmitate) or acetate that was converted into CO2 during a standard incubation period (see Zera and Zhao, 2003a for experimental details). On the day of emergence, the rate of fatty-acid oxidation was equivalent in the LW(f) and SW morphs, similar to the situation for lipid biosynthetic rate. However on day 5, the rate of fatty acid oxidation was significantly higher in the SW-selected line relative to the LW(f) line of the same block for each of the three blocks. This was the case for lines fed each of the three diets (see Zera and Zhao, 2003a). As was the case for lipid biosynthesis, no interaction between line type and diet was observed, indicating that differences between the morphs in fatty acid oxidation were roughly equivalent on each of the three diets.
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FIG. 6. Top panel. Rate of fatty acid oxidation in LW(f) and SW-selected lines of G. firmus on day 5 of adulthood raised on the standard (high) diet. Histograms refer to the mean (±SEM) amount of injected 14C-palmitic acid converted into 14C-CO2 during the standard four-hour incubation period. Asterisks above the histograms refer to results of t-tests of LW(f) and SW individuals within a block, while asterisk within the parenthesis refer to results of paired t-test comparing line means across blocks (*** = P < 0.005; * = P < 0.05). Note the consistently greater rate of fatty acid oxidation in SW vs. LW(f) lines. Bottom panel. Genetically-based trade-off between LW(f) and SW lines with respect to the proportional oxidation of fatty acid vs. utilization for glyceride (triglyceride and phospholipid) biosynthesis. Values represent mean percentage of radiolabelled 14C palmitic acid that was oxidized to CO2 vs. oxidized into CO2 plus incorporated into glycerides for a representative pair of LW(f) and SW lines. These mean percentages differ significantly as do values for LW(f) and SW lines from the other two blocks (Results of ANCOVAs: P < 0.005 in each case; see Zera and Zhao, 2003a for statistical analyses)
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A genetically-based trade-off was observed in the proportion
of fatty acid (or acetate) that was oxidized
vs. converted into
glycerides (
i.e., triglyceride and phospholipid) (
Fig. 6, lower
panel; Zera and Zhao, 2003
a). Thus, the elevated triglyceride
reserves in the LW(f) morph resulted from a decreased rate of
fatty acid oxidation, in addition to an elevated rate of glyceride
biosynthesis. By contrast, the elevated rate of fatty-acid oxidation
in the SW morph reduces the availability of precursors for glyceride
biosynthesis, but probably contributes a significant amount
of energy necessary for the enhanced biosynthesis of vitellogenin
(yolk protein) in SW females. Recent studies indicate a complimentary
situation for amino acid metabolism in
G. firmus: LW(f) females
oxidize a greater amount of amino acids than do SW females,
which likely reduces yolk protein production in LW(f) females,
but which contributes energy to drive the enhanced biosynthesis
of fatty acid for subsequent conversion to triglyceride flight
fuel (A. Zera and Z. Zhao, unpublished data).
Morphs differ in the allocation of biosynthesized triglyceride and phospholipid to somatic vs. reproductive organs
In addition to measuring whole-organism biosynthesis and oxidation of triglyceride and phospholipid, we also measured, in a separate experiment, the amounts of newly biosynthesized triglyceride and phospholipid that were allocated to somatic vs. reproductive (ovarian) tissue in selected lines of one block (Fig. 7; Zhao and Zera, 2002). Not only did the LW(f) morph biosynthesize a significantly greater amount of triglyceride than did the SW morph (as in the previous experiment discussed above), it also allocated a disproportionately greater amount of triglyceride to somatic vs. ovarian tissue (Fig. 7). Conversely, the SW morph both biosynthesized more phospholipid and allocated more of this lipid class to ovarian tissue. These results have important implications for energetic studies of life history trade-offs. For example, they illustrate the degree to which precision in the estimate of energy devoted to the soma vs. reproduction can be increased when individual lipid components are measured in individual body compartments. Just to give one example of this point, LW(f) females biosynthesized 60% more whole-body triglyceride compared with SW females (Zhao and Zera, 2002). Assuming that whole-body triglyceride has an exclusively somatic function, a common assumption in insect life history studies (Zera and Harshman, 2001), one would conclude from these data that there is a 60% greater lipid allocation to somatic function in LW(f) vs. SW females. However, this turns out to be a substantial underestimate. Using biosynthesized triglyceride that is actually found in the somatic body compartment (i.e., non-ovarian tissues), a 200% increased allocation to somatic lipid is observed in LW(f) females. The difference between these two estimates is due to the former estimate not taking into account the disproportionately reduced allocation of whole-body triglyceride to the soma in the SW morph. Indeed, the majority of whole-body triglyceride in the SW morph is found in the ovaries (Zhao and Zera, 2002). Estimating allocation of triglyceride to reproductive vs. somatic functions is likely to be even more complex, because the ovarian and fat-body triglyceride pools are probably dynamic and interconvertible to some degree.
LW(f) and SW morphs differ in specific activities of lipogenic enzymes
In vitro specific activities of a representative group of enzymes
involved in lipid biosynthesis were compared between LW(f) and
SW-selected lines. Activities were measured in fat body, the
most important lipogenic organ in insects (Downer, 1985
). Names
and metabolic roles of enzymes are given in
Figure 8, while
specific activities measured on crickets fed the standard diet
are given in
Figure 9 (see Zera and Zhao, 2003
b for additional
enzymes, assays performed on crickets fed different diets, and
assay conditions). Lipogenic specific activities were measured
to independently assess whether lipid biosynthesis is elevated
in the LW(f) morph, and to identify specific enzymes involved
(Zera and Zhao, 2003
b). Specific activity of
each lipogenic
enzyme studied was substantially elevated genetically in LW(f)
vs. SW lines (
Fig. 8; Zera and Zhao, 2003
b). Thus, the genetic
differences in fatty acid biosynthesis between LW(f) and SW
morphs appear to result from a global alteration of the activities
of enzymes involved in lipid biosynthesis rather than from modifications
of a few "key" enzymes.
In vivo differences in activities of
these enzymes between LW(f) and SW morphs, are even greater
than indicated in
Figure 9. This is because there is about a
40% greater amount of fat body in the LW(f) compared with the
SW morph (on the standard diet), and activities in
Figure 9 were standardized to the same amount of fat body protein (
i.e., they are specific activities). Line-crosses and backcrosses
documented strong co-segregation among activities of each of
these enzymes, wing length, flight muscle mass, and ovarian
mass (Zera and Zhao, 2003
b).
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FIG. 8. Enzymes of the de novo pathway of fatty-acid biosynthesis (Downer, 1985) whose activities were compared between LW(f) and SW morphs. ACL (ATP-citrate lyase) converts citrate, transported outside the mitochondrion, into acetate (=acetyl CoA). Fatty acid synthase (FAS) is a complex enzyme that converts malonyl CoA (produced from acetyl CoA in the preceeding step in the pathway) into a 16 carbon fatty acid through a series of chemical reactions denoted by the dotted line. Reducing equivalents (NADPH) required for fatty acid biosynthesis are produced from NADP by the enzymes glucose-6-phosphate dehydrogenase, isocitrate dehydrogenase, and malate dehydrogenase
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Metabolic differences between LW(f) and SW lines result from differences in endocrine regulation
LW(f) and SW-selected lines differ with respect to a wide variety
of biochemical traits (summarized in
Table 2), as well as various
morphological and reproductive traits (
e.g., size of wings,
flight muscles, ovaries, and fat body; Zera
et al., 1997
; Zera
and Zhao, 2003
b, 2004
). The co-ordinate expression of these
alternate sets of diverse traits in LW(f) and SW-selected lines
likely results from genetic differences in endocrine regulation.
One potential candidate regulator is juvenile hormone (JH),
a hormone of central importance in insects (Nijhout, 1994
; Wyatt
and Davey, 1996
). JH positively affects ovarian growth, and
negatively affects many aspects of flight-capability such as
the size of wings, flight muscle mass, fat body mass, and lipid
accumulation in many insects including
Gryllus (Nijhout, 1994;
Zera
et al., 1998
; Zera, 2004
). Thus, a genetically-specified
difference in the titer of, or tissue sensitivity to, JH could
account for the differential co-expression of the various biochemical,
morphological, and reproductive traits observed in SW and (LW[f])-selected
lines.
Although the hormonal control of intermediary metabolism has
been extensively studied in vertebrates (
e.g., Granner and Pilkis,
1990
; Sul and Wang, 1998
), the endocrine regulation of intermediary
metabolism is much less understood in insects. As a first step
in determining whether differences in the expression of morph-specific
traits between selected lines result from differences in endocrine
regulation, we applied methoprene, a juvenile hormone analogue,
to adult females of one of the LW(f) selected lines (Zera and
Zhao, 2004
). This manipulation produced a remarkable SW phenocopy
with respect to numerous reproductive, anatomical, and biochemical
traits (
Fig. 10). For example, relative to LW(f) controls, LW(f)
females treated with methoprene had larger ovaries, decreased
flight-muscle mass, decreased rate of triglyceride biosynthesis,
decreased specific activities of lipogenic enzymes, increased
rate of fatty acid oxidiation, and increased rate of phospholipid
biosynthesis, all characteristics of the SW morph (
Fig. 10;
Zera and Zhao, 2004
). Remarkably, hormonally-treated LW(f) and
control LW(f) females differed in these traits to the same degree
as did untreated SW and LW(f) females (
Fig. 10; Zera and Zhao,
2004
). These results provide strong evidence that morph-specific
differences in lipid metabolism are caused by variation in endocrine
regulation. However, differences in the
in vivo JH titer between
LW(f) and SW morphs are complex (Zera and Cisper, 2001
; Zhao
and Zera, 2004
), and it is presently unclear whether JH itself
or other hormones regulate differences in lipid metabolism between
the morphs. For example, the blood titer of ecdysteroids, a
group of hormones that regulates numerous traits in insects
(Nijhout, 1994
), also differs substantially between LW(f) and
SW adult
G. firmus (Zera and Bottsford, 2004
; Zhao and Zera,
2004
), and the titer of this hormone may be strongly altered
by topical application of methoprene (Zera, 2004
). Thus differences
in the titer of ecdysteroids (or other as yet unidentified hormones)
may regulate the morph-specific differences in lipid metabolism.
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SUMMARY AND SYNTHESIS
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Modifications of intermediary metabolism, that lead to changes
in the production of key life-history components, such as yolk
protein for eggs, triglyceride for somatic maintenance and dispersal,
and energy for growth and metabolism, almost certainly are key
components of life history evolution. A deep understanding of
the mechanisms underlying life-history evolution thus requires
detailed knowledge of these modifications. For example, only
by identifying these alterations in intermediary metabolism
can the chain of causality be traced from variation in DNA sequence
to variation in the phenotypic expression of whole-organism
life histories. Detailed information on variation within and
interactions among metabolic pathways will be required to understand
many central issues in life-history evolution such as the nature
and stability of genetic correlations between life-history traits
and the nature of constraints in life-history evolution (see
O'Brien
et al. [2002]
for a recent excellent example of this
latter point).
Recent studies of lipid metabolism in selected lines of G. firmus, described above, currently represent the most detailed investigations of alterations in intermediary metabolism underlying a naturally-occurring, genetically-based life history trade-off (Table 3; Zhao and Zera, 2002; Zera and Zhao, 2003a, b, 2004). Most notably, recent studies in G. firmus have directly documented that the differential flow of metabolites through specific pathways of intermediary metabolism underlie an internal, resource-based trade-off important for morph specialization. That is, the SW and LW morphs differ genetically in the degree to which fatty acids are (1) oxidized for energy vs. used for glyceride biosynthesis, and (2) partitioned between triglyceride and phospholipid biosynthesis (Zhao and Zera, 2002; Zera and Zhao, 2003a). These trade-offs result in enhanced production of triglyceride flight fuel in the LW(f) morph, and enhanced production of phospholipid and energy in the SW morph. These two functionally-important, biochemical trade-offs are now useful models to investigate the molecular and hormonal regulation of resource-allocation trade-offs. More recent studies have identified additional, functionally-important interactions and trade-offs between amino-acid and lipid metabolism in LW(f) and SW females (A. Zera and Z. Zhao, unpublished data). These studies collectively illustrate the remarkable alterations in intermediary metabolism that underlie the evolution of alternate life histories in dispersing vs. reproductive morphs of G. firmus.
Another important finding of the Gryllus studies is that differential flux through the pathway of lipid biosynthesis in LW(f) and SW morphs appears to result from substantial alterations in the activities of many enzymes of lipogenesis (Figs. 8–9; see Zera and Zhao [2003b] for activities of other enzymes). This finding is similar to results of other recent artifical or laboratory selection studies, in which changes in carbohydrate or lipid metabolism are associated with alteration of activities or mRNA abundance of many pathway enzymes (e.g., Clark et al., 1990; Asante et al., 1991; Ferea et al., 1999). Similarly, changes in pathway flux due to hormonal regulation (e.g., changes in glycolysis and glucoeneogenesis modulated by insulin or glucagon) also appear to result from co-ordinate modulation of many pathway enzymes (e.g., Granner and Pilkis, 1990), termed "multisite modulation" (Fell, 1997). Thus, short-term or evolutionary changes in flux through metabolic pathways, appear to require co-ordinate alteration of many enzymes, a finding that is consistent with theoretical expectations of Metabolic Control Theory (Kascer and Burnes, 1979; Fell, 1997). These observations further suggest that hormones (or other regulators) that co-ordinate the expression of multiple enzymes of a pathway are likely to be particularly important targets of selection on pathway flux (Ferea et al., 1999; Zera and Zhao, 2004).
Thus, another important finding of the Gryllus studies is that alteration of hormonal regulation appears to be an important cause of morph-specific differences in lipid metabolism (Fig. 10; Zera and Zhao, 2004). Although hormones have been implicated as causal factors in life history trade-offs (Ketterson and Nolan, 1999; Sinervo, 2000; Zera and Cisper, 2001; Zera and Harshman, 2001), they have typically not been invoked to explain differential allocation of resources (Zera and Zhao, 2003b). Indeed, some workers have viewed differential resource allocation vs. differential regulation as alternate explanations for life history variation and trade-offs (Rose and Bradley, 1998; Leroi, 2001). This is reminiscent of the previous dicotomization of "structural" vs. "regulatory" changes as being the most important causes of molecular evolution (e.g., Wilson, 1976). An important message of the Gryllus studies is that modification of endocrine regulation may be the primary cause of differential resource allocation, which, in turn, is a key component of the trade-off between early-age reproduction and flight-capability in G. firmus.
Finally, these studies illustrate the power of wing polymorphism in Gryllus as an experimental model to investigate the functional causes of life history variation and trade-offs (Zera and Harshman, 2001; Zera and Zhao, 2003b, 2004). Morph-specific differences in life histories are of sufficient magnitude, and crickets are large enough, to allow analyses of the physiological, biochemical, and endocrine causes of life history variation to be studied in individual organs (e.g., organ-specific enzyme activities). Highly significant genetic differences were documented between morphs of G. firmus in numerous biochemical traits, even though statistical power was low (2 df in paired t-tests; = [number of blocks] – 1). Because of the extensive data on variation
TOP
SYNOPSIS
INTRODUCTION
