Skip to main content
NCBI home page
Integrative and Comparative Biology logoLink to Integrative and Comparative Biology
. 2010 Aug 16;50(5):818–828. doi: 10.1093/icb/icq105

Allocation of Nutrients to Somatic Tissues in Young Ovariectomized Grasshoppers

Evan T Judd *,1, John D Hatle *,1,2, Michelle D Drewry *, Frank J Wessels †, Daniel A Hahn †
PMCID: PMC3140271  PMID: 21558244

Abstract

The disposable soma hypothesis predicts that when reproduction is reduced, life span is increased because more nutrients are invested in the soma, increasing somatic repair. Rigorously testing the hypothesis requires tracking nutrients from ingestion to allocation to the soma or to reproduction. Fruit flies on life-extending dietary restriction increase allocation to the soma “relative” to reproduction, suggesting that allocation of nutrients can be associated with extension of life span. Here, we use stable isotopes to track ingested nutrients in ovariectomized grasshoppers during the first oviposition cycle. Previous work has shown that ovariectomy extends life span, but investment of protein in reproduction is not reduced until after the first clutch of eggs is laid. Because ovariectomy does not affect investment in reproduction at this age, the disposable soma hypothesis would predict that ovariectomy should also not affect investment in somatic tissues. We developed grasshopper diets with distinct signatures of 13C and 15N, but that produced equivalent reproductive outputs. These diets are, therefore, appropriate for the reciprocal switches in diet needed for tracking ingested nutrients. Incorporation of stable isotopes into eggs showed that grasshoppers are income breeders, especially for carbon. Allocation to the fat body of nitrogen ingested as adults was slightly increased by ovariectomy; this was our only result that was not consistent with the disposable soma hypothesis. In contrast, ovariectomy did not affect allocation of nitrogen to femoral muscles. Further, allocation of carbon to the fat body or femoral muscles did not appear to be affected by ovariectomy. Total anti-oxidant activities in the hemolymph and femoral muscles were not affected by ovariectomy. These experiments showed that allocation of nutrients was altered little by ovariectomy in young grasshoppers. Additional studies on older individuals are needed to further test the disposable soma hypothesis.

Introduction

Reduced reproduction extends life span in many animal species (Partridge et al. 2005). The disposable soma hypothesis suggests that when environmental conditions are poor it is beneficial to delay reproduction and increase life span. This can allow the individual to outlast the poor conditions, and then when good environmental conditions return, full reproduction can resume (Kirkwood 1987, 2002). Trade-offs in allocation of nutrients between reproduction and somatic maintenance are thought to underlie this longevity (Kirkwood 1987, 2002; Stearns 1989; Tatar 2001; Boggs 2009). The disposable soma hypothesis suggests that reducing reproduction allows more ingested nutrients to be allocated to the somatic tissues. This enhanced allocation permits increased somatic repair at the cellular level, such as anti-oxidant activities that slow somatic damage, and ultimately increases life span.

Substantial indirect evidence using dietary restriction to extend life span supports the disposable soma hypothesis (Partridge et al. 2005). For example, flies on dietary restriction have reduced reproduction and an extended life span (Bauer et al. 2009; Grandison et al. 2009). Dietary restriction is also associated with reduced accumulation of oxidative damage (Wachsman 1996). Life span in insects can be increased by over-expression of anti-oxidants (Sun and Tower 1999; Sun et al. 2002; but see Orr et al. 2003) or by protein chaperons (Tatar et al. 1997), although similar manipulations may not extend lifespan in mice (e.g., Jang et al. 2009; but see Schriner et al. 2005). Nonetheless, dietary restriction may cause increased cellular maintenance in response to nutritional stress, rather than as a shift in allocation from reproduction toward somatic maintenance. Thoroughly testing the disposable soma hypothesis requires examination of allocation of nutrients in long-lived animals with directly reduced reproduction.

Several previous studies have interpreted the coincidence of reduced reproduction and extended life span as consistent with a shift in storage of nutrients (Hatle et al. 2008), but direct tracking of nutrients is needed to verify the hypothesis. Min et al. (2006) and O’Brien et al. (2008) have shown that fruit flies on dietary restriction that extends life span have greater allocation of ingested nutrients to the soma relative to the allocation to reproduction. However, absolute allocation to the soma is actually reduced in flies on dietary restriction in comparison to flies on full diets (O’Brien et al. 2008). These studies provide critical evidence that extension of life span by dietary restriction can involve a change in allocation of nutrients. However, dietary restriction indirectly reduces reproduction. Here, we seek to use a similar approach to test nutrient allocation upon direct reduction of reproduction via ovariectomy.

Stable isotopes are useful for long-term tracking of nutrients in individual animals because the isotopes do not decay, are nontoxic, and occur naturally in the environment (including in animals’ diets). This avoids many of the potential pitfalls of radioisotopes, which have historically been used to trace nutrients (Gannes et al. 1997; O’Brien et al. 2000, 2008; Martinez del Rio and Wolf 2005; Karasov and Martinez del Rio 2007). Hence, we used stable isotopes to track the allocation of nutrients to reproduction and to multiple somatic tissues of animals after ovariectomy. Diets with distinct stable isotope signatures are typically offered in reciprocal switches in diet (e.g., one group is started on a low-13C diet and switched to a high-13C diet, whereas the second group is started on the high-13C diet and switched to the low-13C diet). This approach helps control for differences in composition between the diets, which may result in different rates of development between the two groups. A goal of this project was to develop two diets that have distinct signatures of stable isotopes yet allow similar rates of reproductive development, thereby minimizing problems due to different developmental rates on different diets. Such diets are ideal for studies of allocation of nutrients.

In our experimental system, we directly reduce reproduction by ovariectomizing lubber grasshoppers (Romalea microptera). Ovariectomy increases median survival from ∼165 days in sham-operated females to over 205 days in ovariectomized females, consistent with the predicted trade-off between reproduction and longevity (Hatle et al. 2008).

Ovariectomized grasshoppers retain the ability to allocate nutrients to reproduction. In other common models for aging, such as those based on flies and mice, ovarian hormones are required for reproductive development (Hadley 1996; Chapman 1998). In contrast, for grasshoppers, the main gonadotropin (viz. juvenile hormone) is produced in the corpora allata, near the brain (Fronstin and Hatle 2008). No ovarian hormone is needed for vitellogenesis. Because of this, ovariectomized grasshoppers produce the egg yolk-precursor protein, namely vitellogenin in the hemolymph. They begin to allocate protein to reproduction, but have no ovary to sequester the vitellogenin. About the time of first oviposition in intact grasshoppers, ovariectomized females have very high levels of vitellogenin in the hemolymph. Indeed, the total amount of vitellogenin at this time is similar to the amount of vitellin in the eggs of the first clutch laid by intact females (Hatle et al. 2008). Further, protein is the limiting nutrient for reproduction (Chapman 1998; Hatle et al. 2006a). Therefore, at the age when intact females are laying their first clutch, ovariectomized females have levels of hemolymph proteins that suggest they are allocating these limiting nutrients fully to reproduction (Hatle et al. 2003). This has important implications for predictions on allocation of nutrients that are based on the disposable soma hypothesis.

Consistent with the disposable soma hypothesis, we predict that the allocation of protein to somatic tissues at the time of first oviposition will not differ between ovariectomized females and sham-operated controls, because both are investing protein to reproduction similarly at this age. Within each individual, we expect that levels of incorporation of stable isotopes will vary among somatic tissues. Specifically, we predict that the highly metabolically active fat body will incorporate nutrients ingested by adults faster than the skeletal muscle will incorporate nutrients ingested by adults (after Hobson and Clark 1992; Wolf and Martinez del Rio 2000 for birds; and Miller et al. 2008 for mice). Finally, the disposable soma hypothesis predicts that increased allocation of nutrients to the soma in long-lived phenotypes will allow increased cellular maintenance in these same tissues. Total anti-oxidant activity is one of many metrics of cellular maintenance, so we expect that the activities of anti-oxidants will mirror allocations of nutrients across tissues.

Materials and methods

Experimental animals

Lubber grasshoppers (Romalea microptera) were collected in Miami, FL, USA and shipped to Jacksonville, FL, as juveniles. Juveniles were reared in ventilated cages en masse under heat lamps on a 14L:10D photoperiod, and fed Romaine lettuce and oats ad libitum, with occasional chopped green onions.

Labeled diets

Stable isotopes are substantially less abundant than their natural counterparts, but their concentration can vary naturally in biological systems. Stable isotope concentration is typically reported as a ratio of the less abundant isotope to the more abundant one compared to a standard, commonly known as delta notation (expressed in ‰) (Equation 1). In the case of carbon, animal and plant tissue is always more depleted in 13C than is the standard, thereby resulting in a negative number in delta notation; the less negative the number, the more 13C the tissue contains (Martinez del Rio and Wolf 2005).

graphic file with name icq105m1.jpg (1)

C4 plants have more 13C (δ13C approximately −13‰) than do C3 plants (δ13C approximately −27‰) (Cerling and Harris 1999). In this experiment, we took advantage of this naturally occurring variation in isotope concentrations to create diets labeled with high and low levels of 13C. One dietary protein source was Publix® brand dog food, likely from corn-fed cattle that should present a C4 plant profile and high levels of 13C. The second dietary source of protein was WolfKing® dog food, from grass-fed cattle reared on primarily C3 plants (Oregon free-range) for a low-13C diet. The 13C levels of the diets were clearly different between the Publix® diet and the WolfKing® diet, and the diets also differed in content of 15N (Fig. 1). The Publix®-based artificial diet is hereafter called the high-13C diet, while the WolfKing®-based diet is hereafter called the low-13C diet.

Fig. 1.

Fig. 1

Open in a new tab

Isotopic signatures of foods fed to female grasshoppers from their molt to the adult stage to their first oviposition (or the same period for ovariectomized females). Each individual was offered 0.5 g of lettuce and ad libitum artificial diet daily. Filled symbols each indicate the isotopic signature of one of the components of the diets, namely the lettuce, high-13C (Publix®) food, and low-13C (WolfKing®) food. Open square and triangle are the actual cumulative isotopic signatures of diet ingested by sham-operated females during their first oviposition cycle, as calculated by dry masses of artificial diet and lettuce consumed. Isotopic signatures of ingested diet for ovariectomized females are not shown but are within 1% of that for sham-operated females.

Regimens of diet and surgery

Upon their molt to the adult stage, females were isolated into ventilated 500 cm3 plastic containers and serially assigned to surgery groups, either ovariectomy (=OVX) or sham-operated. Surgeries were performed according to the methods of Hatle et al. (2003) within two days of their molt to adulthood. Females were cold anesthetized for ∼2 h before surgeries. The entire ovary, along with some trachea and fat body, was removed in OVX individuals. In sham-operated individuals the incision was made and some trachea and fat body were removed. Within each surgery group, individuals were then assigned to either an ad libitum high-13C artificial diet or an ad libitum low-13C artificial diet. Preliminary experiments showed that these artificial diets alone were insufficient for oocyte growth. We added 0.5 g Romaine lettuce daily to the ad libitum offering of artificial diet, and this supported the production of eggs (Hatle et al. 2006a). Artificial diets were prepared according to Yang and Joern (1994), with the protein source changed from horse charge to ground dog food. Ratios of protein source to cellulose were adjusted to generate diets with 5% protein by mass. The WolfKing® dog food was noticeably leaner, whereas the protein contents of the two foods were similar; hence, the low-13C diet had less fat. Individuals almost always consumed the entire 0.5 g of lettuce but rarely finished the entire offering of artificial diet. To quantify ingestion, individuals were provided with a known quantity of diet daily and uneaten artificial diet was collected, dried at 37°C overnight, and the dry mass determined (Hatle et al. 2006b).

Starting at about age 25 days, females were placed on sand three times each week to test for oviposition (as per Hatle et al. 2001). Sham-operated females were reared until first oviposition and then tissues were sampled (see next section). To determine when an ovariectomized female should be sampled, individuals were grouped based on their date of molt. When half of the sham-operated females in a group laid their first clutch, all remaining individuals (from both the sham-operated and OVX groups) were dissected.

Preparation of samples of eggs, femoral muscles, and fat body

Upon first oviposition, the age of the female was recorded, egg clutches were weighed for wet mass, and the number of eggs was counted. Eggs were then kept at –20°C for later analysis of stable isotopes. The average size of an egg was determined by the wet mass of the entire clutch divided by the number of eggs.

As part of this study, we estimated the proportion of protein in first clutch eggs that came from larval capital versus adult income. This allowed a test of whether lubber grasshoppers were income breeders, which would suggest that allocation to reproduction could have a cost in allocation to the soma. Eggs were homogenized with a glass-glass tissue homogenizer in 5 mL phosphate buffered saline (PBS) and the homogenate centrifuged for 10 min at 3000 rpm. The supernatant was transferred to a 25-mL Teflon centrifuge tube and total protein was isolated from the supernatant by precipitation with acetone. The sample was incubated overnight at −20°C, then pelleted at 14,000×g for 10 min. The protein pellet was dried overnight to ensure that no acetone remained, and then the dried protein was weighed for stable isotope analysis (see below).

Femoral muscles and the entire fat body were collected and stored at −20°C. Femoral muscles were removed by making an incision down the length of the femur and scraping out the muscle tissue with forceps. The femoral muscles were lyophilized, bead homogenized, and then weighed for stable isotope analysis (see below). The fat body was picked out of the abdominal body cavity with forceps.

Newly synthesized fats do not strongly incorporate 13C (Post et al. 2007; Wessels and Hahn 2010) so bulk lipids were extracted from the fat body using a protocol modified from Folch et al. (1957). The lyophilized fat body was homogenized in a glass-glass tissue homogenizer in 10 mL of 2:1 chloroform: methanol solution. The homogenate was transferred to a glass 15-mL centrifuge tube and spun at 2500 rpm for 5 min. The supernatant was removed and stored in a 10 mL glass vial with a Teflon screw-cap. The pellet was re-suspended in 10 mL 2:1 chloroform: methanol solution and the extraction procedure repeated two more times for each sample. The lipid-extracted fat body sample was air dried under a fume hood for at least 1 h. The supernatant was transferred to a 25-mL glass separatory flask and phase partitioned by adding ∼5 mL of 0.1% NaCl in H2O and shaking for 30 s. The phases were allowed to partition and the organic layer was removed. The aqueous layer was dried, combined with the pellets, and the solutes were weighed for stable isotope analysis (see below); this preparation is hereafter referred to as fat body aqueous extract.

Stable isotope analyses

Analysis of δ13C and δ15N for each sample was carried out using mass spectroscopy at the University of Florida Stable Isotope Geochemistry Laboratory, according to the methods of Wessels and Hahn (2010). Between 600 and 800 µg of each sample was weighed into a Costech 5× 9 mm pressed tin capsule (Valencia, CA, USA). Samples were first combusted in a Carlo Erba NA 1500 CNS elemental analyzer. The purified N2 and CO2 gas from the elemental analyzer was carried to a ConFlo II interface and then into a Finnigan-MAT 252 isotope ratio mass spectrometer. l-glutamic acid (NIST USGS40) was used as a standard. Results were reported relative to Vienna Peedee Belemnite for δ13C and relative to air for δ15N.

Measurement of total anti-oxidant activities

For a second group, females were isolated upon their molt to the adult stage and then reared solely on ad libitum lettuce. One-third of these females were dissected upon molting to the adult stage. Of the remaining females, one-half were ovariectomized as above, and the other half were sham-operated. As in the previous experiment, sham-operated females were dissected upon laying the first clutch. When half the sham-operated females had laid, all remaining individuals (including sham-operated and ovariectomized females) were dissected. Upon dissection, fat body, femoral muscles, and hemolymph samples were collected, immediately frozen in liquid N2, and then stored at –20°C.

Later, samples of femoral muscles were lyophilized, homogenized in liquid N2, diluted in PBS to an appropriate protein concentration, and assayed for total anti-oxidant power. Fat-body-tissue samples were also lyophilized, homogenized in liquid N2, diluted in PBS to an appropriate protein concentration, and assayed for total anti-oxidant activities. The fat body samples for ovariectomized females were noticeable larger, and this unfortunately led to a failure to lyophilize the samples properly. The larger samples clogged the pinhole used in drying the samples. Because of this, we have no data on activities of anti-oxidants for samples of fat body from ovariectomized females. Samples of hemolymph were simply diluted with PBS and assayed. Anti-oxidant activities were measured as reduction of trolox (after Re et al. 1999, see also Williams et al. 2008).

Mixing models

When using stable isotopes as resource tracers, it is important to account for the discrimination of the tracer as it is metabolized by the consumer. This discrimination results in differences in the composition of the consumer’s tissue and of the diet (Martinez del Rio and Wolf 2005). To measure δ13C and δ15N discrimination, the discrimination in the individuals reared on lettuce (as both juveniles and adults) was calculated directly and applied to the groups reared on artificial diets as adults (Table 1). The weakness of this method is that isotopic routing from dietary protein to somatic protein is likely (see Discussion section). These discrimination estimates were consistent with values estimated by McCutchan et al. (2003), and therefore we believe they are reasonable.

Table 1.

Discrimination factors (permil) used for first clutch tissues

Tissue 13C 15N
Sham-operated Eggs 2.39 0.87
Femoral muscles 2.73 0.52
Fat body aqueous extract 1.91 0.50
Ovariectomized Eggs ND   ND
Femoral muscles 2.67 −0.23
Fat body aqueous extract 1.60 1.29
Open in a new tab

Some females were fed lettuce throughout their juvenile period, then retained on lettuce from their molt to the adult stage to their first oviposition. Discrimination factors were determined by comparing the stable isotope levels of tissues after the first clutch to stable isotope levels of lettuce. These discrimination factors were then applied to females fed the high-13C and low-13C artificial diets during the first oviposition cycle. ND indicates estimates for eggs of ovariectomized females were not determined, because they cannot lay eggs.

We quantified the incorporation of 13C and 15N into eggs and somatic tissue using a two-source linear-mixing model (Equation 2);

graphic file with name icq105m2.jpg (2)

where δ13C or δ15NTISSUE is the consumer tissue of interest, and P represents the proportion of the tissue derived from the protein-rich artificial diet. The term δ13C or δ15NARTIFICIAL DIET is the isotopic composition of the artificial diet and dI represents the discrimination of the artificial diet by the consumer, δ13C or δ15NLETTUCE refers to the isotopic composition of the lettuce component of the diet, and dC represents discrimination of the lettuce component by the consumer. Mixing model estimates for an individual that suggested >100% incorporation were adjusted to a value of 100%.

Statistics

Reproductive outputs on the high-13C diet plus lettuce in comparison to the low-13C diet plus lettuce were analyzed with a one-way MANOVA, with age at first oviposition, number of eggs, and estimated size of an egg as dependent variables. Tests of income or capital breeding were conducted using t-tests, comparing contributions of juveniles’ diet (capital) and adults’ diet (income). Effects of surgery and tissue on allocation were tested with a two-way MANOVA. Similarly, effects of age/surgery and tissue on anti-oxidant activities were tested with a two-way MANOVA. All analyses were conducted with SAS PROC GLM (SAS 1989), except that t-tests were run in Microsoft Excel.

Results

Reproductive parameters of females on artificial diets

The high-13C diet plus lettuce and the low-13C diet plus lettuce did not differ in any reproductive tactic measured (Fig. 2). One-way MANOVA revealed that the age at first oviposition (F1,49 = 0.33; P = 0.569), the number of eggs (F1,49 = 0.82; P = 0.370), and the average wet mass of an egg (F1,48 = 2.12; P = 0.152) were not affected by diet. We conclude that while the two diets differ in exact composition, they result in similar reproductive outputs.

Fig. 2.

Fig. 2

Open in a new tab

Reproductive output for their first oviposition by lubber grasshoppers on two artificial diets, one with low-13C levels and a second with high-13C levels. Both artificial diets were prepared to contain 5% protein by weight. Artificial diets were offered ad libitum, and 0.5 g of Romaine lettuce was also offered daily. Although the diets had different compositions (the high-13C diet had noticeably higher fat), none of the reproductive tactics differed significantly.

Allocation to eggs and somatic tissues

Nitrogen-15 in egg protein from first clutch eggs was derived slightly but significantly more from food ingested since adult molt than from food ingested before adult molt (t-test; P < 0.0001; Fig. 3). Carbon-13 in egg protein from first clutch eggs was derived entirely from the diet ingested from adult molt to first oviposition (P < 0.0001); the estimates are actually >100% derived from carbon ingested as adults, which is likely due to imperfect estimation of 13C discrimination. Considering the 15N and 13C data together, we classify lubber grasshoppers as income breeders.

Fig. 3.

Fig. 3

Open in a new tab

Estimates of allocation of nutrients to first-clutch eggs in lubber grasshoppers. The y-axis label refers to the percentage of nitrogen or carbon in the eggs that was ingested before molt to the adult stage (for “juvenile diet”) or after molt to the adult stage (for “clutch1 diet”). About half the nitrogen came from the juveniles’ diet, while the other half came from the adults’ diet. In contrast, all the carbon in eggs came from nutrients ingested by adults.

The effects of ovariectomy on allocation of nutrients ingested as adults varied slightly between femoral muscles and fat body, when 15N and 13C were considered together (two-way MANOVA; Pillai's Trace F2,22 = 4.78; P = 0.019; Fig. 4). This mild affect was driven by a significant effect of tissue (i.e., femoral muscles versus fat body; P = 0.015) with only a marginal effect of surgery (i.e. ovariectomized versus sham-operated females; P = 0.052). However, neither 15N (P = 0.091) nor 13C (P = 0.112) individually showed significant interactions of surgery and tissue, so the effect is not due entirely to one isotope. That said, the significant result was predominantly due to nitrogen and not carbon, because when each was tested for main effects individually there was a significant difference for 15N (Ptissue = 0.025; Psurgery = 0.027) but not for 13C (Ptissue = 0.312; Psurgery = 0.904). Taken together, the surgery and tissue interaction was largely due to ovariectomized females incorporating more 15N into the fat body than did sham-operated females, but there was no effect of ovariectomy on 15N incorporation into femoral muscles (Fig. 4).

Fig. 4.

Fig. 4

Open in a new tab

Estimates of allocation of nutrient to aqueous extracts of fat body and femoral muscles in lubber grasshoppers, from their molt to the adult stage to their first oviposition. The y-axis label refers to the percentage of nitrogen or carbon in the fat body or femoral muscle that was ingested since molt to the adult stage. Ovariectomy slightly increased allocation of nitrogen to the fat body, but not the femoral muscles. For both the femoral muscles and the aqueous fraction of the fat body, all carbon came from materials ingested by adults.

Anti-oxidant activities

Hemolymph, femoral muscles, and fat body (not lipid extracted) were tested for total anti-oxidant activities at three age/surgery combinations: immediately after adult molt, after the first oviposition in sham-operated females, and in ovariectomized females that were matched by age with sham-operated females. There was a significant interaction between age/surgery and tissue for anti-oxidant activities (two-way MANOVA; Pillai's Trace F4,36 = 6.88; P = 0.0004; Fig. 5). Among tissues, activities were highest in the hemolymph immediately after the molt (Tukey's Studentized Range Test; all pairwise comparisons P < 0.05; Fig. 5; note different scales for y-axes), while femoral muscles did not differ across age/surgery combinations (Tukey's; P > 0.05). Whether anti-oxidant activities of fat body tissues were affected by surgery is unclear because of the degraded samples.

Fig. 5.

Fig. 5

Open in a new tab

Total anti-oxidant activities in tissues of female, lettuce-fed grasshoppers. “Molt” refers to females dissected the day of molting to the adult stage. “Sham” refers to females sham operated after molting to an adult, then reared through the first oviposition cycle and dissected after laying. “OVX” refers to females ovariectomized after molting to an adult and then dissected at the same age as sham-operated females. ND indicates that no data were collected for fat body of ovariectomized females. Anti-oxidant activities were measured as the ability of tissue homogenates to reduce ABTS, using trolox standards.

Discussion

No differences in reproductive tactics between the two artificial diets

The high-13C and low-13C diets used in this study have slightly different nutritional compositions, but produced equivalent reproductive outputs in lubber grasshoppers. The compositions of the ground dog foods differed, most notably because the high-13C diet (made from corn-fed cattle) was higher in fat. Despite this, when artificial diets were prepared so that each contained 5% protein, grasshoppers had similar timing in the oviposition of clutches, and similar sizes of clutches and eggs on the two diets. Given that reproduction in grasshoppers is limited by protein (Hatle et al. 2006a), similar reproductive tactics on diets with the same protein content is expected. Timing and size of clutches have previously been shown to explain most of the variation in reproductive tactics in this species (Moehrlin and Juliano 1998; Hatle et al. 2002).

Lubber grasshoppers are income breeders (especially for carbon)

Our data show that lubber grasshoppers are income breeders for the first clutch. Lubber grasshoppers do not become vitellogenic until ∼10 days into adulthood, and they require substantial feeding thereafter to produce a clutch of eggs (Sundberg et al. 2001; Hatle et al. 2001, 2004, 2006a; Juliano et al. 2004); these findings have been interpreted as probable income breeding. However, definitively demonstrating a breeding strategy requires tracking nutrients from ingestion to eggs. We show here that all the carbon and half of the nitrogen in the first clutch of eggs comes from foods ingested as adults.

Allocation of nutrients to somatic tissues in response to ovariectomy

At the time of sample collection, ovariectomized females were likely allocating fully to reproduction (Hatle et al. 2008). From this, we hypothesized that allocation to somatic tissues by females of this age would be unaffected by ovariectomy. Our results for nitrogen were mostly consistent with this prediction. There were no effects of ovariectomy on allocation of nitrogen to femoral muscles. In contrast, ovariectomy slightly increased allocation of nitrogen to the fat body. However, the statistical significance of the result is not clear cut. We hypothesize that allocation of nitrogen generally follows the predictions of the disposable soma hypothesis, but greater resolution over a longer time scale is required to fully resolve this question.

Levels of carbon incorporation reached an average of ∼100% both in ovariectomized and sham-operated females. This makes it unclear whether the two groups incorporated carbon at the same rate. Sampling incorporation over a finer time scale is needed to test this hypothesis. Nonetheless, we can rigorously conclude that, during early adulthood, both tissues fully incorporate carbon ingested as adults.

We hypothesized that the fat body would turn over nutrients faster than would the femoral muscles. Previous studies have shown that turnover in liver is faster than turnover in muscle in vertebrates (Hobson and Clark 1992; Wolf and Martinez del Rio 2000; Miller et al. 2008). The aqueous portion of the fat body is representative of the main organ of intermediary metabolism in insects, active in the breakdown and synthesis of carbohydrates, proteins, and lipids (Chapman 1998). In contrast, femoral muscles can store carbohydrates, but otherwise are an outlay for locomotion. Hence, nutrients were expected to be allocated to the fat body more than to the femur muscle. This was true for nitrogen, but the size of the effect was small. Lubber grasshoppers double their somatic body masses in the time from their molt to the adult stage to first oviposition, and then body masses plateau (Hatle et al. 2006b, 2008). It may be that this rapid somatic growth subsumes differences in turnover rates during the first oviposition cycle. Indeed, preliminary results suggest that during the second oviposition cycle, turnover of carbon and nitrogen in the fat body is at least double that in the femoral muscles in sham-operated females (E.T. Judd and J.D. Hatle, unpublished data).

In addition, stress from surgery might have influenced our results. No unmanipulated control was included in the study. It may be that the stress of surgery influenced allocation of nutrients in the grasshoppers. Few differences in allocation of nutrients were observed between ovariectomized and sham-operated females in this study. It may be that the effects of the ovary were obscured by stress of the surgery. However, ovariectomy extends life span by ∼30% over that of sham-operated controls in this system (Hatle et al. 2008), suggesting that the effect of ovariectomy persists despite any additional stress caused by surgery.

Anti-oxidant activities were greatest in the hemolymph and immediately after molt

Our prediction was that anti-oxidant activities would mirror differences in allocation of nutrients. We are confident that our methods were sufficiently sensitive to detect differences because anti-oxidant capacities in hemolymph collected the day of molt to the adult stage was detectably higher than in all other samples measured. The lack of data for fat body from ovariectomized females, the sole tissue to which allocation was altered by surgery, limits our ability to conclude whether anti-oxidant activities mirror nutrient allocation, as was predicted. Although anti-oxidant capacities did not appear to differ due to ovariectomy at ∼50 days, Hatle et al. (2008) previously observed that changes in levels of hemolymph proteins associated with longer life span occurred after the completion of the first oviposition cycle in intact females (i.e., after 50 days). Therefore, the disposable soma hypothesis predicts a shift in allocation to somatic maintenance, and also greater anti-oxidant capacities, in older females.

Discrimination estimates and mixing models

Our estimates suggest that >100% of some tissues were made from carbon ingested as adults. This is impossible, and it suggests that our discrimination estimates are imperfect. Discrimination was estimated by calculating the isotopic differences between lettuce and each tissue from lettuce-fed grasshoppers after their first clutch. This difference was then added to the isotopic signature of tissues from grasshoppers on an artificial diet, to estimate the possible value for tissue samples from grasshoppers fed artificial diet plus lettuce during the first oviposition cycle. Caut et al. (2008) concluded that empirical estimates of discrimination are better than using estimates from the literature. Hence, our estimates are imperfect but likely give the best approximation given our experimental design.

The inaccuracy in our estimates could also be due to the mixing models employed. We chose linear mixing models, but more complex models that incorporate the concentration of macronutrients in the various diets may yield different results (Martinez del Rio and Wolf 2005). However, both artificial diets contained 5% of the limiting nutrient (viz. protein), so adjusting for concentration dependence seems unlikely to improve our estimates. In contrast, isotopic routing might have a strong effect on our estimates. Isotopic routing refers to the fact that dietary amino acids tend to be sent directly to the consumer’s tissues as amino acids, instead of being broken down and then reassembled randomly by the consumer, as linear models assume (Martinez del Rio and Wolf 2005, Martinez del Rio et al. 2009). Because, we fed grasshoppers a high-protein artificial diet and low-protein lettuce, it is possible that the amino acids in the artificial diets were preferentially allocated to the grasshoppers’ tissues for growth and egg production.

Allocation of 13C relative to 15N

For first-clutch eggs, femoral muscles, and fat body, we observed greater assimilation of carbon ingested as an adult than of nitrogen ingested as an adult (t-tests; all P << 0.0001). There are several potential explanations for this result. First, these results could be an artifact of our discrimination estimates or mixing models. A second possible explanation is that young females are actually storing nitrogen ingested as an adult at a higher rate, probably in hexameric storage proteins in the hemolymph. Three hexameric storage proteins make up ∼80% of the total non-vitellogenin protein in the hemolymph of lubber grasshoppers, and their levels change through time in a pattern that is consistent with preparation for reproductive development (Hathaway et al. 2009). The dynamics of isotopic nitrogen metabolism are unclear (Karasov and Martinez del Rio 2007). In the present experiment, females were fed a high-protein artificial diet ad libitum, whereas on more natural plant diets, protein is the limiting nutrient (Chapman 1998; Hatle et al. 2006a). Perhaps grasshoppers with abundant dietary nitrogen reserve as much of it as possible for future clutches, when resources may be less abundant. This hypothesis could be tested by quantitatively comparing allocation of nitrogen in young females fed an artificial diet with 5% protein content plus 0.5 g lettuce to those fed artificial diet with 1% protein content plus 0.5 g lettuce (after Hatle et al. 2006a).

This study lays the groundwork for further experiments on nutrient allocation in long-lived ovariectomized grasshoppers. We have confirmed that we have diets that are functionally similar, and we can track ingested nutrients to multiple tissues and simultaneously measure anti-oxidant activities in those tissues. To our knowledge, this is the first report of concurrent nutrient tracking and quantification of cellular maintenance in a long-lived phenotype, and we have done it in multiple tissues. Measuring nutrient allocation and cellular maintenance in old ovariectomized females will allow us to further test the physiological predictions of the disposable soma hypothesis beyond the first clutch, the point in the lifecycle when mechanisms underlying extension of life span in ovariectomized females are likely to diverge from those of intact females.

Funding

The National Institute on Aging (R15 AG028512-01 to J.D.H.); the National Science Foundation (IOS-641505 to D.A.H.); the United States Department of Agriculture (TSTARC-09051246 to D.A.H.).

Acknowledgments

We thank Jason Curtis of the University of Florida Stable Isotope Geochemistry Laboratory for measuring 13C and 15N levels. We thank Katharine Wright for assisting with the development of the diets and experimental design, Siobhan O’Brien and Cathy Paterson for help with the development of the anti-oxidant assay, Sean Wells and Stephen Melnyk for help with testing females for oviposition, and two anonymous reviewers and the editor for their helpful suggestions.

References

  1. Bauer JH, Morris SNS, Chang C, Flatt T, Wood JG, Helfand SL. dSir2 and Dmp53 interact to mediate aspects of CR-dependant life span extension in D. melanogaster. Aging. 2009;1:38–48. doi: 10.18632/aging.100001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Boggs CL. Understanding insect life histories and senescence through a resource allocation lens. Funct Ecol. 2009;23:27–37. [Google Scholar]
  3. Caut S, Angulo E, Courchamp C. Caution on isotopic model use for analyses of consumer diet. Can J Zool. 2008;86:438–45. [Google Scholar]
  4. Chapman R. The insects: structure and function. 4th. Cambridge, UK: University Press; 1998. [Google Scholar]
  5. Cerling TE, Harris JM. Carbon isotope fractionation between diet and bioapatite in ungulate mammals and implications for ecological and paleoecological studies. Oecologia. 1999;120:347–63. doi: 10.1007/s004420050868. [DOI] [PubMed] [Google Scholar]
  6. Folch J, Lees M, Stanley GHS. A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem. 1957;226:497–509. [PubMed] [Google Scholar]
  7. Fronstin RB, Hatle JD. A cumulative feeding threshold require for vitellogenesis can be obviated with juvenile hormone in lubber grasshoppers. J Exp Bio. 2008;211:79–85. doi: 10.1242/jeb.009530. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gannes LZ, O’Brien DM, Martinez del Rio C. Stable isotopes in animal ecology: assumptions, caveats, and a call for more laboratory experiments. Ecology. 1997;78:1271–6. [Google Scholar]
  9. Grandison RC, Piper MDW, Partridge L. Amino-acid imbalance explains extension of lifespan by dietary restriction in Drosophila. Nature. 2009;462:1061–4. doi: 10.1038/nature08619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hadley MC. Endocrinology. 4th. Upper Saddle River, NJ, USA: Prentice-Hall, Inc; 1996. [Google Scholar]
  11. Hathaway M, Hatle JD, Li S, Ding X, Barry T, Hong F, Wood D, Borst DW. Characterization of hexamerin protein and their mRNAs in the adult lubber grasshopper: the effects of nutrition and juvenile hormone on their levels. Comp Biochem Physiol A. 2009;154:323–32. doi: 10.1016/j.cbpa.2009.06.018. [DOI] [PubMed] [Google Scholar]
  12. Hatle JD, Juliano SA, Borst DW. Hemolymph ecdysteroids do not affect vitellogenesis in the lubber grasshopper. Arch Insect Biochem Physiol. 2003;52:45–57. doi: 10.1002/arch.10067. [DOI] [PubMed] [Google Scholar]
  13. Hatle JD, Waskey T, Jr, Juliano SA. Plasticity of grasshopper vitellogenin production in response to diet is primarily a result of changes in fat body mass. J Comp Physiol B. 2006a;176:27–34. doi: 10.1007/s00360-005-0028-9. [DOI] [PubMed] [Google Scholar]
  14. Hatle JD, Andrews AL, Crowley MC, Juliano SA. Interpopulation variation in developmental titers of vitellogenin, but not storage protein, in lubber grasshoppers. Physiol Biochem Zool. 2004;77:631–40. doi: 10.1086/420946. [DOI] [PubMed] [Google Scholar]
  15. Hatle JD, Borst DW, Eskew ME, Juliano SA. Maximum titers of vitellogenin and total hemolymph protein occur during the canalized phase of grasshopper egg production. Physiol Biochem Zool. 2001;74:885–93. doi: 10.1086/324475. [DOI] [PubMed] [Google Scholar]
  16. Hatle JD, Crowley MC, Andrews AL, Juliano SA. Geographic variation of reproductive tactics in lubber grasshoppers. Oecologia. 2002;132:517–23. doi: 10.1007/s00442-002-0994-5. [DOI] [PubMed] [Google Scholar]
  17. Hatle JD, Paterson CS, Jawaid I, Lentz C, Wells SM, Fronstin RB. Protein accumulation underlying lifespan extension via ovariectomy in grasshoppers is consistent with the disposable soma hypothesis but is not due to dietary restriction. Exp Gerontol. 2008;43:900–908. doi: 10.1016/j.exger.2008.08.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hatle JD, Wells SM, Fuller LE, Allen IC, Gordy LJ, Melnyk S, Quattrochi J. Calorie restriction and late-onset calorie restriction extend lifespan but do not alter protein storage in female grasshoppers. Mech Age Dev. 2006b;27:883–91. doi: 10.1016/j.mad.2006.09.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hobson KA, Clark RG. Assessing avian diets using stable isotopes I: turnover of 13C in tissues. Condor. 1992;94:181–8. [Google Scholar]
  20. Jang YC, et al. Overexpression of Mn superoxide dismutase does not increase life span in mice. J Gerontol A Biol Sci Med Sci. 2009;64:1114–25. doi: 10.1093/gerona/glp100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Juliano SA, Olson JR, Murrell EG, Hatle JD. Plasticity and canalization of insect reproduction: testing alternative models of life history transitions. Ecology. 2004;85:2986–96. [Google Scholar]
  22. Karasov WH, Martínez del Rio C. Physiological ecology: how animals process energy, nutrients, and toxins. Princeton NJ, USA: Princeton University Press; 2007. [Google Scholar]
  23. Kirkwood TBL. Immortality of the germ-line versus the disposability of the soma. In: Finch CE, Schneider EL, editors. Handbook of the biology of aging. 2nd. New York: Van Nostrand Reinhold; 1987. [Google Scholar]
  24. Kirkwood TBL. Evolution of ageing. Mech Age Dev. 2002;123:737–74. doi: 10.1016/s0047-6374(01)00419-5. [DOI] [PubMed] [Google Scholar]
  25. Martinez del Rio C, Wolf BO. Mass-balance models for animal isotopic ecology. In: Starck JM, Wang T, editors. Physiological and ecological adaptations to feeding in vertebrates. Enfield, NH, USA: Science Publishers; 2005. [Google Scholar]
  26. Martinez del Rio C, Wolf N, Carleton S, Gannes LZ. Isotopic ecology ten years after a call for more laboratory experiments. Biol Rev. 2009;84:91–111. doi: 10.1111/j.1469-185X.2008.00064.x. [DOI] [PubMed] [Google Scholar]
  27. McCutchan JH, Lewis WM, Jr, Kendall C, McGrath CC. Variation in trophic shift for stable isotope ratios of carbon, nitrogen, and sulfur. Oikos. 2003;102:378–90. [Google Scholar]
  28. Miller JF, Millar JS, Longstaffe FJ. Carbon- and nitrogen-isotope tissue—diet discrimination and turnover rates in deer mice, Peromyscus maniculatus. Can J Zool. 2008;86:685–91. [Google Scholar]
  29. Min K-J, Hogan MF, Tatar M, O’Brien DM. Resource allocation to reproduction and soma in Drosophila: a stable isotope analysis of carbon from dietary sugar. J Insect Physiol. 2006;52:763–70. doi: 10.1016/j.jinsphys.2006.04.004. [DOI] [PubMed] [Google Scholar]
  30. Moehrlin GS, Juliano SA. Plasticity of insect reproduction: testing models of flexible and fixed development in response to different growth rates. Oecologia. 1998;115:492–500. doi: 10.1007/s004420050546. [DOI] [PubMed] [Google Scholar]
  31. O’Brien DM, Schrag DP, Martinez del Rio C. Allocation to reproduction in a hawkmoth: a quantitative analysis using stable carbon isotopes. Ecology. 2000;81:2822–31. [Google Scholar]
  32. O’Brien DM, Min K-J, Larsen TL, Tatar M. Use of stable isotopes to examine how dietary restriction extends Drosophila lifespan. Current Biol. 2008;18:R155–6. doi: 10.1016/j.cub.2008.01.021. [DOI] [PubMed] [Google Scholar]
  33. Orr WC, Mockett RJ, Benes JJ, Sohal RS. Effects of overexpression of copper-zinc and manganese superoxide dismutases, catalase, and thioredoxin reductase genes on longevity in Drosophila melanogaster. J Biol Chem. 2003;278:26418–22. doi: 10.1074/jbc.M303095200. [DOI] [PubMed] [Google Scholar]
  34. Partridge L, Gems D, Withers DJ. Sex and death: what is the connection? Cell. 2005;120:461–72. doi: 10.1016/j.cell.2005.01.026. [DOI] [PubMed] [Google Scholar]
  35. Post D, Layman C, Arrington D, Takimoto G, Quattrochi J, Montaña C. Getting to the fat of the matter: models, methods and assumptions for dealing with lipids in stable isotope analyses. Oecologia. 2007;152:179–89. doi: 10.1007/s00442-006-0630-x. [DOI] [PubMed] [Google Scholar]
  36. Re R, Pelligrini N, Proteggente A, Pannala A, Yang M, Rice-Evans C. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Rad Biol Med. 1999;26:1231–7. doi: 10.1016/s0891-5849(98)00315-3. [DOI] [PubMed] [Google Scholar]
  37. SAS Institute. SAS/STAT User’s Guide. Vol. 2. Cary, NC, USA: SAS Institute; 1989. [Google Scholar]
  38. Schriner SE, et al. Extension of murine life span by overexpression of catalase targeted to mitochondria. Science. 2005;308:1909–11. doi: 10.1126/science.1106653. [DOI] [PubMed] [Google Scholar]
  39. Stearns SC. Trade-offs in life-history evolution. Funct Ecol. 1989;3:259–68. [Google Scholar]
  40. Sun J, Tower J. FLP recombinase-mediated induction of Cu-Zn superoxide dismutase transgene expression can extend life span of adult Drosophila melanogaster flies. Mol Cell Biol. 1999;19:216–28. doi: 10.1128/mcb.19.1.216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Sun J, Folk D, Bradley TJ, Tower J. Induced overexpression of mitochondrial Mn-superoxide dismutase extends the life span of adult Drosophila melanogaster. Genetics161. 2002:661–72. doi: 10.1093/genetics/161.2.661. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Sundberg SV, Luong-Skovmand MH, Whitman DW. Morphology and development of oocyte and follicle resorption bodies in the lubber grasshopper, Romalea microptera (Beauvois) J Orthop Res. 2001;10:39–51. [Google Scholar]
  43. Tatar M. Senescence. In: Fox CW, Roff DA, Fairbairn DJ, editors. Evolutionary ecology: concepts and case studies. Oxford: Oxford University Press; 2001. [Google Scholar]
  44. Tatar M, Khazaeli AA, Curtsinger JW. Chaperoning extended life. Nature. 1997;390:30. doi: 10.1038/36237. [DOI] [PubMed] [Google Scholar]
  45. Wachsman JT. The beneficial effects of dietary restriction: reduced oxidative damage and enhanced apoptosis. Mutation Res. 1996;350:25–34. doi: 10.1016/0027-5107(95)00087-9. [DOI] [PubMed] [Google Scholar]
  46. Wessels FJ, Hahn DA. Carbon 13 discrimination during lipid biosynthesis varies with dietary concentration of stable isotopes: implications for stable isotope analyses. Funct Ecol. 2010 Available online April 2010, doi: 10.1111/j.1365-2435.2010.01716.x. [Google Scholar]
  47. Williams JB, Roberts SP, Elekonich MM. Age and natural metabolically-intensive behavior affect oxidative stress and antioxidant mechanisms. Exp Geron. 2008;43:538–49. doi: 10.1016/j.exger.2008.02.001. [DOI] [PubMed] [Google Scholar]
  48. Wolf BO, Martinez del Rio C. Use of saguaro fruit by White-winged doves: isotopic evidence of a tight ecological association. Oecologia. 2000;124:536–43. doi: 10.1007/s004420000406. [DOI] [PubMed] [Google Scholar]
  49. Yang Y, Joern A. Compensatory feeding in response to variable food quality by Melanoplus differentialis. Physiol Entomol. 1994;19:75–82. [Google Scholar]

Articles from Integrative and Comparative Biology are provided here courtesy of Oxford University Press

RESOURCES