Protein storage and reproduction increase in grasshoppers on a diet matched to the amino acids of egg yolk protein.

The diets of animals are essential to support development, and protein is key. Accumulation of stored nutrients can support developmental events such as molting and initiation of reproduction. Agricultural studies have addressed how dietary protein quality affects growth, but few studies have addressed the effects of dietary protein quality on developmental transitions. Studies on how dietary quality may affect protein storage and development are possible in arthropods, which store proteins in the hemolymph. We hypothesized that diets with a composition of amino acids that matches the precursor of egg yolk protein (vitellogenin, Vg) will be high quality and support both egg production and accumulation of storage proteins. Grasshoppers were fed one of two isonitrogenous solutions of amino acids daily: Vg-balanced (matched to Vg) or Unbalanced (same total moles of amino acids, but not matched to egg yolk). We measured reproduction and storage protein levels in serial hemolymph samples from individuals. The Vg-balanced group had greater reproduction and greater cumulative levels of storage proteins than did the Unbalanced group. This occurred even though amino acids fed to the Vg-balanced group were not a better match to storage protein than were the amino acids fed to the Unbalanced group. Further, oviposition timing was best explained by a combination of diet, age at the maximum level of storage protein hexamerin-270 and accumulation of hexamerin-90. Our study tightens the link between storage proteins and commitment to reproduction, and shows that dietary protein quality is vital for protein storage and reproduction.

Protein Stores Regulate When Reproductive Displays Begin in the Male Caribbean Fruit Fly.

Many animals exhibit reproductive behavior that requires expenditure of valuable nutrients. In males of many species, competitive energetically demanding displays and the development of sexual ornaments require prior accumulation of nutrient stores. Males must coordinate nutrient stores with ornament development and reproductive displays or they risk depleting their resources mid-development or mid-display, reducing their chance of mating. Males may use nutrient stores to regulate their reproductive behavior. Amino acid reserves may be important for reproduction, but the roles of amino acid stores in initiating maturation and reproductive behavior are less studied than fat stores. Insects store amino acids as hexamerin storage proteins. Many fly species use a specific hexamerin, larval serum protein 2 (LSP-2), as both a juvenile storage medium and to store protein consumed after adult eclosion. Protein stored as LSP-2 has previously been suggested to regulate reproduction in females, but no role has been proposed for LSP-2 in regulating male maturation. We use males of the Caribbean fruit fly, Anastrepha suspensa, a species with nutrient-intensive male sexual displays to test whether LSP-2 stores regulate male reproductive displays. We fed adult A. suspensa males a diet with or without protein, then assayed these males for lsp-2 transcript abundance via qRT-PCR, LSP-2 protein abundance via Western blot, and reproductive display behavior via observation. We found that adult males with ad libitum dietary protein had greater lsp-2 transcript and protein abundance, earlier sexual display behavior, and were more likely to exhibit sexual display behavior than protein-deprived adult males. We show that lsp-2 knockdown via RNAi decreases the proportion of males exhibiting reproductive displays, particularly early in the onset of reproductive behavior. Our results suggest circulating LSP-2 protein stores regulate reproductive behavior in A. suspensa males, consistent with protein stores modulating reproduction in males with expensive reproductive strategies. Our results are consistent with hexamerin storage proteins performing dual roles of protein storage and protein signaling. Our work also has substantial practical applications because tephritid flies are a pest group and the timing and expression of male reproductive displays in this group are important for control efforts using the sterile insect technique.

Life-extending dietary restriction and ovariectomy each increase leucine oxidation and alter leucine allocation in grasshoppers.

Reduced reproduction and dietary restriction each extend lifespan in many animal models, but possible contributions of nutrient oxidation and allocation are largely unknown. Ovariectomy and eating 70% of ad libitum-feeding each extend lifespan in lubber grasshoppers. When feeding levels between the two groups are matched, ovariectomy increases fat and protein storage while dietary restriction reduces fat storage. Because of these disparities in nutrient investment, metabolism may differ between these two life-extending treatments. Therefore, we examined the allocation and organismal oxidation of one representative of each macronutrient class: leucine, oleic acid, and glucose. Ovariectomy and dietary restriction each increased oxidation of dietary leucine. Dietary leucine may play a special role in aging because amino acids stimulate cellular growth. Speeding oxidation of leucine may attenuate cellular growth. Allocation of leucine to muscle was the clearest difference between ovariectomy and dietary restriction in this study. Ovariectomy reduced allocation of leucine to femur muscle, whereas dietary restriction increased allocation of leucine to femur muscle. This allocation likely corresponds to muscle maintenance for locomotion, suggesting dietary restriction increases support for locomotion, perhaps to search for food. Last, ovariectomy decreased oxidation of dietary oleic acid and glucose, perhaps to save them for storage, but the site of storage is unclear. This study suggests that the oxidation of branched-chain amino acids may be an underappreciated mechanism underlying lifespan extension.

Life-extending Dietary Restriction Reduces Oxidative Damage of Proteins in Grasshoppers but Does Not Alter Allocation of Ingested Nitrogen to Somatic Tissues.

Dietary restriction (DR) extends life span and reduces reproduction in most animals. The disposable soma hypothesis suggests that this longevity is the result of reduced investment in reproduction and increased nutrient allocation to the soma, permitting an increase in cellular maintenance. To investigate the role of nutrient allocation upon life-extending DR, tissue-specific nitrogen allocation was tracked in grasshoppers (Romalea microptera) upon a full or restricted (60% of full) diet. In addition, carbonyl (oxidized protein) assays addressed tissue maintenance. To develop a labeled diet on which grasshoppers could thrive, hydroponically grown Romaine lettuce was enriched with 15N. This allowed quantification of nitrogen allocation upon a normal or restricted diet. There was a 50% decrease in reproductive investment upon DR. At the same time, relative allocation of 15N to the ovary did not change. Most important, relative allocation was similar between restricted and full diet grasshoppers for somatic tissues (ie, mandibular and femur muscle, dried hemolymph, gut, and fat body). Carbonyl assays of muscles, hemolymph, and gut revealed an overall reduction in protein oxidation upon DR. These data suggest that DR does not alter nutrient allocation but does reduce protein oxidation, an observation that is inconsistent with the basic predictions of the disposable soma hypothesis.

Vitellogenin-RNAi and ovariectomy each increase lifespan, increase protein storage, and decrease feeding, but are not additive in grasshoppers.

Reduced reproduction has been shown to increase lifespan in many animals, yet the mechanisms behind this trade-off are unclear. We addressed this question by combining two distinct, direct means of life-extension via reduced reproduction, to test whether they were additive. In the lubber grasshopper, Romalea microptera, ovariectomized (OVX) individuals had a ~20% increase in lifespan and a doubling of storage relative to controls (Sham operated). Similarly, young female grasshoppers treated with RNAi against vitellogenin (the precursor to egg yolk protein) had increased fat body mass and halted ovarian growth. In this study, we compared VgRNAi to two control groups that do not reduce reproduction, namely buffer injection (Buffer) and injection with RNAi against a hexameric storage protein (Hex90RNAi). Each injection treatment was tested with and without ovariectomy. Hence, we tested feeding, storage, and lifespans in six groups: OVX and Buffer, OVX and Hex90RNAi, OVX and VgRNAi, Sham and Buffer, Sham and Hex90RNAi, and Sham and VgRNAi. Ovariectomized grasshoppers and VgRNAi grasshoppers each had similar reductions in feeding (~40%), increases in protein storage in the hemolymph (150-300%), and extensions in lifespan (13-21%). Ovariectomized grasshoppers had higher vitellogenin protein levels than did VgRNAi grasshoppers. Last but not least, when ovariectomy and VgRNAi were applied together, there was no greater effect on feeding, protein storage, or longevity. Hence, feeding regulation, and protein storage in insects, may be conserved components of life-extension via reduced reproduction.

Vitellogenin RNAi halts ovarian growth and diverts reproductive proteins and lipids in young grasshoppers.

Reduced reproduction extends lifespan of females in many animals. To test the effects of reproduction on storage of macronutrients, we block reproductive output in the lubber grasshopper by injecting RNAi against the precursor to egg-yolk protein, vitellogenin, in early adulthood. Controls were injected with either buffer or RNAi against the major storage protein in the hemolymph, hexamerin-90. Vitellogenin RNAi greatly reduced both levels of mRNA for vitellogenin and ovarian growth, in comparison to both controls. Fat body mass was increased upon vitellogenin RNAi, but concentrations of the three hexameric storage proteins from the hemolymph were not. Surprisingly, hemolymph vitellogenin levels were increased upon vitellogenin RNAi. Total reproductive protein (hemolymph vitellogenin plus ovarian vitellin) was unchanged by vitellogenin RNAi, as reproductive protein was diverted to the hemolymph. Similarly, the increased lipid storage upon vitellogenin RNAi was largely attributable to the reduction in lipid in the ovary, due to decreased ovarian growth. A BLAST search revealed that the 515 bp sequence of vitellogenin used for RNAi had three 11 bp regions identical to the vitellogenin receptor of the cockroach Leucophaea maderae. This suggests that our treatment, in addition to reducing levels of vitellogenin transcript, may have also blocked transport of vitellogenin from the hemolymph to the ovary. This would be consistent with halted ovarian growth simultaneous with high levels of vitellogenin in the hemolymph. Nonetheless, the accumulation of vitellogenin, instead of hexameric storage proteins, is inconsistent with a simple model of the trade-off between reproduction and storage. This was observed in young females; future studies will address whether investment of proteins may shift to the soma as individuals age. Overall, our results suggest that blockage of reproduction in young grasshoppers redirects lipids to storage and reproductive proteins to the hemolymph.

Life-extending ovariectomy in grasshoppers increases somatic storage, but dietary restriction with an equivalent feeding rate does not.

Reduced diet or reduced reproduction each extends lifespan in many animals. It is often thought that reduced reproduction and reduced diet may act through the same mechanisms. In grasshoppers, ovariectomy extends lifespan and reduces feeding to a level similar to that used for life extension by dietary restriction, further suggesting mechanistic overlap. Here, we measure the feeding rate of ovariectomized grasshoppers and, by manipulating feeding levels, create a sham-operated & dietary restricted group with matched daily feeding. Both groups show ~25% increased survivorship near the median age of mortality for fully fed and reproductive controls. Ovariectomy results in a doubling of fat body mass and hemolymph volume in comparison to both a feeding-matched dietary restriction group and a sham-operated & fully fed control, which do not differ from each other. Total anti-oxidant activity in the hemolymph and the skeletal muscle was unchanged upon ovariectomy or dietary restriction, so it does not appear to be a major factor in lifespan extension. Next, we measured mitochondrial counts using qPCR to determine mitochondrial cytochrome-b concentrations relative to nuclear (genomic) beta-actin. Mitochondrial counts in the ovariectomized group were lower than sham-operated and fully fed controls but not than the dietary restriction group. Last, in the fat body, transcript levels of hexamerin-90 (a hemolymph storage protein) were affected by neither ovariectomy nor dietary restriction. Hence, ovariectomy resulted in large magnitude increases in organismal storage. The matched-fed dietary restricted group differed from the ovariectomized group only in organismal storage, and not in any of the cellular parameters measured here. This study suggests that longevity via ovariectomy has distinct physiological mechanisms from longevity via dietary restriction in grasshoppers that are independent of daily feeding rate, particularly for protein and fat storage.

Divergent egg physiologies in two closely related grasshopper species: Taeniopoda eques versus Romalea microptera (Orthoptera: Romaleidae).

We compared egg survivorship and egg development time at different soil moistures for two closely related grasshopper species from divergent habitats: marsh-inhabiting Romalea microptera (Beauvois) versus desert-inhabiting Taeniopoda eques (Burmeister). These two species can interbreed and produce viable offspring. In nature, both species have a similar 8-9 mo subterranean egg stage, but their soil environments differ dramatically in water content. We predicted that the eggs of the two species would exhibit differential survivorship and development times under different moisture levels. Our laboratory results show that the eggs of both species survived a wide range of soil moistures (≈ 0.5 to 90%), maintained for 3 mo. However, the eggs of the marsh grasshopper, R. microptera, better tolerated the highest soil moistures (95 and 100%), whereas the eggs of the desert species, T. eques, better tolerated the lowest soil moistures (0.0 and 0.1%). Sixty-five percent of marsh-inhabiting R. microptera eggs, but no desert T. eques eggs, survived 3 mo submersion under water. In contrast, 49% of desert T. eques eggs, but only 3.5% of R. microptera eggs, survived after being laid into oven-dried sand and then maintained with no additional water until hatch. In the laboratory at 26 °C, the two species differed significantly in the mean length of the oviposition-to-hatch interval: 176 d for R. microptera versus 237 d for T. eques. These divergent traits presumably benefit these insects in their divergent habitats. Our results suggest the evolution of physiological divergence that is consistent with adaptations to local environments.

Ovariectomy in grasshoppers increases somatic storage, but proportional allocation of ingested nutrients to somatic tissues is unchanged.

Reduced reproduction increases storage and extends lifespan in several animal species. The disposable soma hypothesis suggests this life extension occurs by shifting allocation of ingested nutrients from reproduction to the soma. A great deal of circumstantial evidence supports this hypothesis, but no direct tracking of nutrients has been performed in animals that are long-lived because of direct reduction in reproduction. Here, we use the stable isotopes to track carbon and nitrogen from ingestion to somatic organs in long-lived, ovariectomized grasshoppers. Three estimates of somatic storage (viz., quantity of hemolymph storage proteins, amount of femur muscle carbohydrates, and size of the fat body) all doubled upon ovariectomy. In stark contrast, ovariectomy did not increase the proportion of these tissues that were made from recently ingested foods. In other words, the physiology underlying relative allocation to these somatic tissues was not affected by ovariectomy. Thus, at the level of whole tissue storage, these results are consistent with a trade-off between reproduction and longevity. In contrast, our stable isotope data are inconsistent with the prediction that enhanced storage in ovariectomized females results from a physiological shift in allocation of ingested nutrients.

Does it pay to delay? Flesh flies show adaptive plasticity in reproductive timing.

Life-history plasticity is widespread among organisms. However, an important question is whether it is adaptive. Most models for plasticity in life-history timing predict that animals, once they have reached the minimal nutritional threshold under poor conditions, will accelerate development or time to reproduction. Adaptive delays in reproduction are not common, especially in short-lived species. Examples of adaptive reproductive delays exist in mammalian populations experiencing strong interspecific (e.g., predation) and intraspecific (e.g., infanticide) competition. But are there other environmental factors that may trigger an adaptive delay in reproductive timing? We show that the short-lived flesh fly Sarcophaga crassipalpis will delay reproduction under nutrient-poor conditions, even though it has already met the minimal nutritional threshold for reproduction. We test whether this delay strategy is an adaptive response allowing the scavenger time to locate more resources by experimentally providing supplemental protein pulses (early, mid and late) throughout the reproductive delay period. Flies receiving additional protein produced more and larger eggs, demonstrating a benefit of the delay. In addition, by tracking the allocation of carbon from the pulses using stable isotopes, we show that flies receiving earlier pulses incorporated more carbon into eggs and somatic tissue than those given a later pulse. These results indicate that the reproductive delay in S. crassipalpis is consistent with adaptive post-threshold plasticity, a nutritionally linked reproductive strategy that has not been reported previously in an invertebrate species.

Thermal constraints for range expansion of the invasive green mussel, Perna viridis, in the southeastern United States.

Cold temperatures are thought to be among the most important determining factors of geographic distribution for tropical and sub-tropical marine invertebrates. The Asian green mussel, Perna viridis, has been introduced into coastal waters of Florida where its current distribution is hypothesized to be limited by low temperatures during winter. Lethal and sub-lethal effects (heat shock protein/Hsp70 expression) of cold water and air temperatures were analyzed in two size classes of P. viridis from Florida in an effort to determine the effects of current and forecasted temperatures on the potential for range expansion. Mussels were exposed to water temperatures of 14, 10, 7 and 3°C for up to 30 days, or to air temperatures of 14, 7, 0 and -10°C for periods of 2 hr. Mortality was significantly increased at all water and air temperatures ≤14°C. No differences in mortality rates were observed between small (15-45 mm) and large (75-105 mm) size classes except after exposure to 7°C air, in which small mussels had higher mortality. Significant increases in Hsp70 expression were observed after a 2-hour exposure to 10°C water, but Hsp70 expression was not significantly increased at any temperatures in which mortality was not also significant. The temperature threshold for survival in this population appears to be between 10 and 14°C, suggesting that under current conditions P. viridis may already be at the northern edge of its potential range in the United States. If water temperatures increase with global climate change, northerly flowing currents may permit range expansion as temperatures allow.

Allocation of nutrients to somatic tissues in young ovariectomized grasshoppers.

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.

Protein accumulation underlying lifespan extension via ovariectomy in grasshoppers is consistent with the disposable soma hypothesis but is not due to dietary restriction.

Reduced reproduction extends lifespan in many experimental animals, but the mechanism by which this occurs is unclear. The disposable soma hypothesis suggests that when reproduction is reduced, more nutrients are allocated to the soma and lifespan is extended. Alternatively, the reproductive tissues or the process of reproduction may have a direct (i.e., non-nutritional) negative effect on lifespan. We used ovariectomized grasshoppers to examine the effects of reduced reproduction throughout the lifespan at the physiological level. We focused on protein, the limiting nutrient for egg production. Ovariectomized females lived significantly longer than sham females. Because both groups ingested similar amounts, the effect was independent of dietary restriction. Despite this, ovariectomized females gained less body mass than sham females. Ovariectomized grasshoppers produced the egg yolk-precursor protein vitellogenin. At the time sham females laid their first clutch, cumulative reproductive protein was similar in ovariectomized and sham females. By advanced ages, however, ovariectomized females had produced about five-fold less cumulative reproductive protein than sham females. In contrast, old ovariectomized females had at least two-fold more hemolymph storage protein. These results are consistent with ovariectomy extending lifespan in part via enhanced protein allocation to storage at the expense of reproduction.

Life history plasticity after attaining a dietary threshold for reproduction is associated with protein storage in flesh flies.

Body condition affects the timing and magnitude of life history transitions. Therefore, identifying proximate mechanisms involved in assessing condition is critical to understanding how these mechanisms affect the expression of life history plasticity. Nutrient storage is an important body condition parameter, likely playing roles in both attaining minimum body-condition thresholds for life history transitions and expression of life history traits.We manipulated protein availability for females of the flesh fly Sarcophaga crassipalpis to determine whether reproductive timing and output would remain plastic or become fixed. Liver was provided for 0, 2, 4, or 6 days of adult pre-reproductive development. Significantly, liver was removed after the feeding threshold had been attained and females had committed to producing a clutch.We also identified the major storage proteins and monitored their abundances, because protein stores may serve as an index of body condition and therefore may play an important role in life history transitions and plasticity.Flesh flies showed clear post-threshold plasticity in reproductive timing. Females fed protein for 2 days took ~30% longer to provision their clutch than those fed for 4 or 6 days. Observations of oogenesis showed the 2-day group expressed a different developmental program including slower egg provisioning.Protein availability also affected reproductive output. Females fed protein for 2 days produced ~20% fewer eggs than females fed 4 or 6 days. Six-day treated females provisioned larger eggs than 4-day treated females, followed by 2-day treated females with the smallest eggs.Two storage proteins were identified, LSP-1 and LSP-2. LSP-2 accumulation differed across feeding treatments. The 2- and 4-day treatment groups accumulated LSP-2 stores but depleted them during provisioning of the first clutch, whereas the 6-day group accumulated the greatest quantity of LSP-2 and had substantial LSP-2 stores remaining at the end of the clutch. This pattern of accumulation and depletion suggests that LSP-2 could play roles in both provisioning the current clutch and future clutches, making it a good candidate molecule for affecting reproductive timing and allotment. LSP-1 was not associated with post-threshold plasticity; it was carried over from larval feeding into adulthood and depleted uniformly across all feeding groups.

Calorie restriction and late-onset calorie restriction extend lifespan but do not alter protein storage in female grasshoppers.

Calorie restriction (CR) and late-onset CR enhance longevity in many organisms. Resource allocation theory suggests that longevity is enhanced by increasing somatic storage, at the expense of current reproduction. Phytophagous insects accumulate amino acids as hemolymph storage proteins for major developmental events. We hypothesized that protein storage is involved in life extension from CR. In a longitudinal experiment, we tested whether CR altered protein storage in female grasshoppers. Individuals on CR (60 or 70% of ad libitum) or late-onset CR had at least 60% greater longevity than ad libitum individuals. Age at first oviposition, dry mass of the first clutch, or lifetime fecundity were not affected by CR, but CR did increase the number of clutches produced. Most important, females on life-extending CR and late-onset CR did not differ in the concentration of hemolymph storage of proteins in comparison to ad libitum females. Protein storage changed with time in all groups, demonstrating sufficient sensitivity in our methods. Previous experiments have shown that severe CR ( approximately 30% of ad libitum) can reduce hemolymph storage. Therefore, the reduction in intake needed to extend lifespan is not sufficient to reduce protein storage in the hemolymph. These results do not support the hypothesis that protein storage is involved in life extension from CR.

Plasticity of grasshopper vitellogenin production in response to diet is primarily a result of changes in fat body mass.

Life history plasticity is the developmental production of different phenotypes by similar genotypes in response to different environments. Plasticity is common in early post-embryonic or adult development. Later in the developmental stage, the transition from developmentally plastic to canalized (i.e., inflexible) phases is often associated with the attainment of a threshold level of storage. Thresholds are often described simply as total body mass or cumulative consumption of food. The physiological characteristics of thresholds, such as the contributions of the growth of particular organs or the production rate of proteins, are largely unstudied. To address the physiology underlying a threshold-induced developmental transition, total vitellogenin production in response to diet quality in the lubber grasshopper was studied. For individuals that differed in age or dietary protein, somatic mass, ovarian mass, fat body mass, mass-specific vitellogenin production, vitellogenin titer, and storage protein titer were measured. Age and diet strongly affected these parameters, with ovarian mass and fat body mass contributing most to the differences. During mid vitellogenesis, females were highly plastic in response to changing food quality. Only during late vitellogenesis were females unresponsive to changes in food quality. Fat body mass was a more important component of plasticity than was mass-specific vitellogenin production. Because these two variables together make up total vitellogenin production, the greater contribution of fat body mass than mass-specific vitellogenin production suggests that growth factors may be more important than tissue stimulators in producing developmental changes in total vitellogenin production. To our knowledge, this is the first study to demonstrate that mass gain of an organ is more important to developmental plasticity than is the output of that same organ.

Starvation affects vitellogenin production but not vitellogenin mRNA levels in the lubber grasshopper, Romalea microptera.

The interaction of juvenile hormone (JH) and nutrition was studied during the oviposition cycle of the Eastern Lubber grasshopper (Romalea microptera). Starvation of females early or in the middle of the cycle inhibited oocyte growth. Starvation for 4 days also reduced hemolymph levels of JH III and vitellogenesis (Vg) to 25% and 15%, respectively, of the levels in fed animals. Likewise, Vg production by fat body fragments incubated in vitro was reduced to 2% of the levels in fed animals and total protein synthesis was reduced to 25%, suggesting that starvation had a stronger effect on Vg synthesis than on protein synthesis. These effects were reversed when starved animals were fed again. However, fat body levels of Vg-mRNA were similar in fed and starved animals, indicating that starvation did not affect transcript levels. We tested whether the decline in JH levels mediated the other starvation effects by infusing animals with JH III or vehicle for 2 days at the onset of starvation. Infusion of JH elevated JH and Vg-mRNA levels 670% and 103%, respectively, above the levels in vehicle-infused animals. However, Vg production and hemolymph levels of Vg were similar to the levels in vehicle-infused animals. These data suggest that JH alone is insufficient to stimulate Vg production.

Interpopulation variation in developmental titers of vitellogenin, but not storage proteins, in lubber grasshoppers.

We examined simultaneous plastic and latitudinal interpopulation variation in the time course of hemolymph protein titers during egg production in the lubber grasshopper. Our goal was to gain insight into possible evolutionary changes in the physiology underlying reproductive plasticity. We used lubbers from three locations in the United States (Florida [FL], Louisiana [LA], and Georgia [GA]), each offered three daily food rations. Previous genetic analysis indicated that grasshoppers from FL (the low-latitude population) and GA (the high-latitude population) were phylogenetically closer to each other than to LA grasshoppers (the intermediate-latitude population). The ages at maximum titers of vitellogenin (Vg(max)) and three storage proteins that were referred to as major hemolymph proteins (MHP(max)) were used as indices of the progress of oocyte development. Age at Vg(max) was affected significantly both by diet and by population. Perhaps most importantly, age at Vg(max) was less for GA grasshoppers than for FL and LA grasshoppers; this pattern differs from the phylogenetic relationships of the populations. Age at MHP(max) was significantly affected only by diet and not by population. Hence, the regulation of these proteins may differ across populations. Finally, we found no evidence that plasticity of reproductive investment in response to food availability differs across populations (as indicated by nonsignificant interactions of population and feeding environment).

Hemolymph ecdysteroids do not affect vitellogenesis in the lubber grasshopper.

The role of hemolymph ecdysteroids in the reproduction of non-dipteran insects is unclear. We examine the role(s) of hemolymph ecdysteroids during egg production in the lubber grasshopper, Romalea microptera. In all individuals, hemolymph ecdysteroids rose to a sharp peak with similar maxima and then fell to undetectable levels. The time from the adult molt to the maximum ecdysteroid titer (E(max) titer) varied in response to food availability, whereas the time from E(max) titer to oviposition was unrelated to food availability. Because both the timing of egg production and the timing of E(max) responded similarly to environmental changes, ecdysteroids may be involved in egg production. We hypothesized that this role is the stimulation of vitellogenesis. Ovariectomized females had vitellogenin but no ecdysteroids, so ecdysteroids are not necessary for vitellogenin production. In addition, treatment of females with ecdysteroids altered neither Vg titers nor ovarian growth. Ovarian ecdysteriods increased at the same age in development as hemolymph ecdysteroids. In contrast to hemolymph ecdysteroids, ovarian ecdysteroids persisted until oviposition. Despite this, [(3)H]ecdysone injected into the hemolymph was detected later only at very low levels in the ovary, suggesting that hemolymph ecdysteroids are not sequestered by the ovary. In summary, our studies indicate that hemolymph ecdysteroids in adult females of the lubber grasshopper are associated with the timing of egg production, but they neither regulate vitellogenesis nor act as a source of ecdysteroids for the ovary.