Romalea Research Articles

Herbivore-induced changes in plant carbon allocation: assessment of below-ground C fluxes using carbon-14.

Effects of above-ground herbivory on short-term plant carbon allocation were studied using maize (Zea mays) and a generalist lubber grasshopper (Romalea guttata). We hypothesized that above-ground herbivory stimulates current net carbon assimilate allocation to below-ground components, such as roots, root exudation and root and soil respiration. Maize plants 24 days old were grazed (c. 25-50% leaf area removed) by caging grasshoppers around individual plants and 18 h later pulse-labelled with14CO2. During the next 8 h,14C assimilates were traced to shoots, roots, root plus soil respiration, root exudates, rhizosphere soil, and bulk soil using carbon-14 techniques. Significant positive relationships were observed between herbivory and carbon allocated to roots, root exudates, and root and soil respiration, and a significant negative relationship between herbivory and carbon allocated to shoots. No relationship was observed between herbivory and14C recovered from soil. While herbivory increased root and soil respiration, the peak time for14CO2 evolved as respiration was not altered, thereby suggesting that herbivory only increases the magnitude of respiration, not patterns of translocation through time. Although there was a trend for lower photosynthetic rates of grazed plants than photosynthetic rates of ungrazed plants, no significant differences were observed among grazed and ungrazed plants. We conclude that above-ground herbivory can increase plant carbon fluxes below ground (roots, root exudates, and rhizosphere respiration), thus increasing resources (e.g., root exudates) available to soil organisms, especially microbial populations.

Control of resting ventilation rate in grasshoppers

We examined the effect of extracellular acid-base status and tracheal gas levels on the ventilation rate of resting Romalea guttata and Schistocerca americana grasshoppers. We manipulated haemolymph pH and [HCO3-] within normal physiological ranges using injections of HCl, NaOH, NaHCO3 and NaCl into the haemocoel. In contrast to terrestrial vertebrates, there was no evidence that extracellular acidification increases ventilation rate in grasshoppers. Elevation of haemolymph bicarbonate levels (by NaHCO3 injection) increased ventilation rate, while depression of haemolymph bicarbonate levels (HCl injection) had no effect. Injection of NaHCO3 also increased tracheal PCO2, suggesting that the effect of the NaHCO3 injection might be mediated by a sensitivity of the ventilatory system to tracheal gases. We tested for effects of tracheal gases on ventilation rate by independently manipulating tracheal PCO2 and PO2 using tracheal perfusions. Ventilation rate was positively correlated with tracheal PCO2 and negatively correlated with tracheal PO2. Increasing tracheal PO2 above normal resting levels or decreasing tracheal PCO2 below normal levels decreased ventilation rate. We conclude that quiescent grasshoppers regulate tracheal PCO2 and PO2 by varying ventilation rate and that both PCO2 and PO2 in the trachea stimulate ventilation in normal, resting grasshoppers.

Acid-base status and spiracular control during discontinuous ventilation in grasshoppers

Many insects ventilate discontinuously when quiescent, exhibiting prolonged periods during which little or no gas exchange occurs. We investigated the consequences of discontinuous ventilation (DV) on haemolymph acid­base status and tested whether spiracular opening during DV is due to changes in internal gas tensions in the western lubber grasshopper Taeniopoda eques. At 15 °C, resting T. eques exhibited interburst periods of about 40 min. During the interburst period, haemolymph PCO2 rose from 1.8 to 2.26 kPa, with minimal acidification of haemolymph. Animals in atmospheres in which PCO2 was 2 kPa or below continued to exhibit DV, while atmospheres in which PCO2 was 2.9 kPa or above caused cessation of DV. These data indicate that accumulation of internal CO2 to threshold levels between 2 and 2.9 kPa induces spiracular opening in grasshoppers. In contrast to the situation in lepidopteran pupae, variation in atmospheric PO2 had no effect on interburst duration. Relative to lepidopteran pupae, the internal PCO2 of grasshoppers during DV is threefold lower, the PCO2 required for triggering spiracular opening is also threefold lower, and the open phase spiracular conductance is at least tenfold higher, demonstrating that considerable diversity exists in these aspects of insect respiratory physiology.

Grasshopper crop and midgut extract effects on plants: an example of reward feedback.

Acid extracts and a resultant fraction from solid-phase extraction (SPE) of Romalea guttata crop and midgut tissues induce sorghum (Sorghum bicolor var. Rio) coleoptile growth in 24-h incubations an average of 49% above untreated controls. When combined with plant auxin, indole-3-acetic acid (IAA), the SPE fraction shows a synergistic reaction, yielding increases in coleoptile growth that average 295% above untreated controls and 8% above IAA standards. The interaction lowered the point of maximum sensitivity of IAA 3 orders of magnitude, resulting in a new IAA physiological set point at 10(-7) g/ml. This synergism suggests that contents in animal regurgitants making their way into plant tissue during feeding may produce a positive feedback in plant growth and development following herbivory. Such a process, also known as reward feedback, may exert major controls on ecosystem-level relationships in nature.

Interactive effects of humidity and temperature on water loss in the lubber grasshopper Romalea guttata

Water loss rates (WLR) at various combinations of temperature and relative humidity were measured in the lubber grasshopper Romalea guttata. WLR increased at higher humidities when rates were corrected for saturation deficit and with increasing temperature when the saturation deficit was held constant (17 mmHg). The higher rates with rising humidity may reflect an increase in cuticular permeability caused by the grasshopper altering or relaxing barrier mechanisms that operate in dry air.

Ventilatory Patterns and Respiratory Transpiration in Adult Terrestrial Insects

Although respiratory surfaces constitute a potential site for dehydration in terrestrial insects, only afew studies have addressed the problem of respiratory transpiration in a manner that explicitly separates water loss associated with gas exchange from cuticular and other avenues of water loss. The most promising of these studies are recent investigations conducted on adult insects that release CO₂ intermittently (discontinuous ventilation [DV] cycle). Using either gravimetric techniques or electronic sensors to compare water lost during interburst (spiracles closed or fluttering, cuticular transpiration only) and burst (spiracles open, cuticular and respiratory transpiration) phases of the cycle, investigators have shown that respiratory water loss accounts for only a small percentage (1.9%-13%) of the total water loss at low to moderate temperatures (15° -30° C). The relatively low amount of water lost during the burst phase in lubber grasshoppers as well the timing and duration of discontinuous CO₂ release raises serious questions regarding the presumed water-saving function of the DV cycle.

Caffeoyltartronic acid from catnip (Nepeta cataria): A precursor for catechol in lubber grasshopper (Romalea guttata) defensive secretions

Adults of the lubber grasshopper (Romalea guttata) secrete increased amounts of catechol from their defensive glands when fed diets containing only catnip leaves (Nepeta cataria). Model compound bioassays showed that these insects were able to sequester and biomagnify simple phenols, such as catechol and hydroquinone, in their defense gland secretions. Excessive catechol secretions from caffeic acid-fortified diets indicated metabolic pathways exist to perform efficiently more complex biochemical conversions. Reverse-phase HPLC of methanol extracts of catnip revealed only one major caffeoyl-polyphenol as a possible precursor for the observed elevated catechol secretions, when this plant is fed to lubbers. The compound was shown to be caffeoyltartronic acid (CTA). During analysis of CTA by probe-MS or gas chromatography (of its silylated derivative), CTA decomposed by loss of carbon dioxide to form caffeoylglycolic acid (CGA), making identification by these methods ambiguous. Only fast atom bombardment mass spectrometry (FAB-MS, negative mode) gave a true molecular weight. Groundivy (Glecoma hederacea), a relative of catnip, was also shown to contain CTA. The mung bean (Phaseolus radiatus=Vigna radiata), a species totally unrelated to catnip, is the only other reported plant source of CTA. Catnip leaves were found to contain about twice as much CTA as mung bean leaves.

Gas Exchange, Ventilatory Patterns, and Water Loss in Two Lubber Grasshoppers: Quantifying Cuticular and Respiratory Transpiration

The respiratory physiology and water relations of two acridid grasshoppers (Romalea guttata and Taeniopoda eques) were examined with a flow-through system that simultaneously measures O₂ consumption, CO₂ excretion, and evaporative water loss. Both species exhibited a cyclic pattern of resting gas exchange that was characterized by intermittent bursts of O₂ uptake and CO₂ emission. Increasing temperature shortened both the burst and especially the interburstperiods. Metabolic rates were strongly affected by temperature, but not hydration state. Dehydration, however, degraded the regularity of the respiratory cycles. Water loss rates (WLRs) for Romalea were significantly higher than for Taeniopoda at each temperature, with rates for hydrated animals (both species) greater than for dehydrated individuals. Respiratory transpiration accounted for only 2.1% to 3.8% of the total water loss in Romalea and 3.8% to 5.3% in Taeniopoda between 15° and 30°C These data raise serious questions regarding the presumed w...

Discontinuous Carbon Dioxide Release in the Eastern Lubber Grasshopper Romalea Guttata and its Effect on Respiratory Transpiration

ABSTRACT Ventilatory patterns were examined in the Eastern lubber grasshopper Romalea guttata and correlated with respiratory transpiration. Discontinuous release of CO2 was only observed in quiescent individuals during their scotophase. Interburst periods (spiracles closed) alternated with bouts of CO2 emission and O2 consumption (burst phase); no true ‘flutter’ phase was observed. Cycle duration decreased with increasing temperature in both hydrated and dehydrated individuals. Metabolic rates for this large, sluggish species are lower than those reported for smaller and/or more active grasshoppers. Water loss rates fell within an expected range of values for arthropods from mesic environments. Respiratory transpiration accounted for only 1.9–3.9% of the total water loss between 15 and 30 °C and for only 7% of the water loss during the burst phase of the cycle. These data indicate that the cyclic release of CO2 in this adult insect does not result in substantial savings of water.

Humidity selection by thermoregulating grasshoppers

1. 1.|To make the most effective use of evaporative cooling, an insect must be in an environment of the lowest humidity. 2. 2.|To test the hypothesis that, at temperatures requiring evaporative cooling, grasshoppers were able to select such a favourable environment, we observed the selection of, and time spent in dry (RH ⩽ 5%) or humid (RH ⩾97%) alternative chambers of a shuttle box at a range of temperatures from 28 to 50°C, in two species, Schistocerca americana and Romalea microptera. 3. 3.|Below 45°C, both species tended to remain in the chamber in which they began the trial; above 48°C, they all selected the arid chamber. 4. 4.|These findings suggest that grasshoppers can select the environment best for maintaining their body temperature below lethal levels but do not necessarily mean that they can actually sense relative humidity.

Predator exaptations and defensive adaptations in evolutionary balance: No defence is perfect

The lubber grasshopper,Romalea guttata, is large, aposematic, and extremely toxic. In feeding trials with 21 bird and lizard species, none were able to consume this chemically defended prey. Predators that attempted to eat lubbers, often gagged, regurgitated, and sometimes died. Loggerhead shrikes,Lanius ludovicianus, regularly impale this toxic prey in peninsular Florida. They, like other bird species, are unable to consume fresh lubbers. However, our tests show that they are able to consume lubbers if the prey are allowed to ‘age’ for 1–2 days. This suggests that lubber toxins degrade following death and that shrike impaling behaviour serves as a preadaptation for overcoming the toxic defences of this large and abundant prey. These results also imply that counter adaptations against chemical defences need not involve major morphological or metabolic specializations, but that simple behavioural traits can enable a predator to utilize toxic prey.

Defensive Secretion Production in Lubber Grasshoppers (Orthoptera: Romaleidae): Influence of Age, Sex, Diet, and Discharge Frequency

The lubber grasshoppers Romalea guttata (Houttuyn) and Taeniopoda eques (Burmeister) discharge a defensive secretion from paired metathoracic spiracles. The amount of stored secretion varies greatly among individuals and is influenced by age, stage, sex, diet, species, body weight, and prior discharge. Later instars possess more secretion than younger instars and females possess more than males. When lubbers molt, all stored secretion is lost; thus freshly molted insects lack exudate. Newly molted adult lubbers increased the amount of stored secretion at an age-related variable rate that peaked on day 4 in T. eques and day 7 in R. guttata. Thereafter, secretion production continued, but at reduced rates. In adults of both species, secretion volume was positively correlated with body weight. In T. eques, discharge of secretion from the spiracles does not influence the rate of filling of the gland reservoir. In R. guttata, a polyphagous diet or a monophagous diet of onion leaves, Allium sp., produced significantly more secretion than diets of wild cucumber, Melothria pendula L., or grapefruit, Citrus paradisi Macfady. leaves. Wild R. guttata adults have more secretion than wild T. eques adults, possibly because of the latter's adaptation to a dry desert environment. This study suggests that numerous factors influence secretion volume and hence defensive efHcacy in lubber grasshoppers. Lubber predators are thus confronted with a variably defended prey, whose defensive capabilities increase over time and with certain diets.

Anatomy, ultrastructure, and functional morphology of the metathoracic tracheal defensive glands of the grasshopper Romalea guttata

In the lubber grasshopper Romalea guttata, the respiratory system produces, stores, and delivers a phenolic defensive secretion. The exudate is secreted by a glandular epithelium surrounding the metathoracic spiracular tracheal trunks. Embedded in the glandular tissue are multiple secretory units, each comprised of a basal secretory cell and an apical duct cell. Secretory cells have numerous mitochondria, a tubular, smooth endoplasmic reticulum, well-developed Golgi bodies, and a microvillilined vesicle thought to transfer secretion to the intracellular cuticular duct of a duct cell. Ducts empty into the metathoracic tracheal lumina where the exudate is stored behind the closed metathoracic spiracle. Tactile stimulation elicits secretion discharge, which begins when all spiracles except the metathoracic pair are closed and the abdomen is compressed. Increased hemostatic and pneumatic pressures drive air and secretion out of the spiracle with an audible hiss. Both metathoracic spiracles discharge simultaneously. The secretion erupts first as a dispersant spray, then as an adherent froth, and finally assumes the form of a slowly evaporating repellent droplet. Discharge force and number vary with eliciting stimuli, volume of stored secretion, and age, disturbance state, and temperature of the insect. Molting grasshoppers are unable to discharge because the stored exudate is lost with the shed cuticle. The advantages and limitations of a tracheal defensive system are discussed.

Storage and release of neuropeptides from the corpus cardiacum of the Eastern lubber grasshopper, Romalea microptera

AbstractIn this study, we describe a method for the separation of AKH/RPCH‐family peptides using isocratic high performance liquid chromatography (HPLC). This improved method is simple, rapid, and sensitive and bypasses many of the difficulties associated with gradient HPLC. We have previously shown that the lubber grasshopper, Romalea, accumulates large quantities of the native peptides Ro I and Ro II (Spring and Gäde, Journal of Experimental Zoology 241:41–50, 1987). In the present study, we used isocratic HPLC to follow the changes in the amount of Ro I and Ro II during development. Overall the amount of neuropeptide found in the corpora cardiaca (CC) increases 34‐fold from larval instar III to late adult, with the greatest quantitative increase (3,000 pmol total in males) occurring during the 6 weeks of adult life. The Ro I: Ro II ratio also changes during development, from 5.4 in larval instar III to 2.0 in late instar V and finally to 4.0 in mature adults. Under physiological conditions these neuropeptides can be released from Romalea CC isolated in vitro, when the CC are depolarized with high‐potassium saline. This release is calcium‐dependent and 10% to 15% of the total peptide stores are mobilized during a 1‐hour treatment with high‐potassium, plus calcium, saline. More than one‐third of the mobilized peptide is released within the first 5 min. In locusts, release of the AKH peptides is reported to be mediated by the neurotransmitter octopamine, acting via the adenylate cyclase pathway. In Romalea, however, we find no evidence for either octopamine or cAMP‐mediated release of neuropeptide.

The identity and physiological actions of an adipokinetic hormone in Acheta domesticus

An octapeptide which mobilizes lipid and inhibits the incorporation of [ 3H]leucine into haemolymph proteins in both Acheta domesticus and Locusta migratoria has been purified from the corpora cardiaca of Acheta. The primary sequence of this Acheta adipokinetic hormone, determined by pulsed liquid-phase Edman sequencing after enzymatic removal of the N-terminal pyroglutamate is: (pGlu)-Val-Asn-Phe-Ser-Thr-Gly-Trp-NH 2. This sequence is identical to that of an octapeptide isolated previously from Gryllus bimaculatus and Romalea microptera. Synthetic material with the assigned structure is biologically active and is chromatographically identical to the natural peptide. In both crickets and locusts, the sensitivity of [ 3H]leucine incorporation to inhibition by Acheta adipokinetic hormone (EC 50 = 5 × 10 −11 M) is two orders of magnitude greater than that of the adipokinetic response (EC 50 = 3 × 10 −9 M). Acheta adipokinetic hormone elicits a full adipokinetic response in both Locusta and in Acheta.

Structure-function studies on neurohormone D: activity of naturally-occurring hormone analogues

The relative potencies of 11 naturally-occurring peptides of the adipokinetic hormone/red pigment-concentrating hormone family (AKH/RPCH-family) have been assessed with respect to increase in heart rate in adult, female American cockroaches, Periplaneta americana, in in vitro and in vivo bioassays. In addition, analogues that lacked the N-terminal pyroglutamate residue or had a free threonine acid at the C-terminus were also investigated. In both bioassays the N- or C-terminal-modified analogues give no or little response suggesting that blocked termini are essential for receptor-binding. In both bioassays the naturally-occurring peptide from the cockroach corpus cardiacum Pea-CAH-I (neurohormone D) is more potent than the second endogenous peptide, Pea-CAH-II. On the basis of this result and previous data it is proposed that neurohormone D is the only physiologically important "true" cardioactive peptide. The dose-response curves of the other peptides indicate that in octapeptides, amino acid residues at positions 2, 6, and 7 are important for receptor-recognition, and that decapeptides are not as effective as octapeptides (exception: the peptide Rom-CC-I isolated from the grasshopper Romalea microptera).

A generalist herbivore in a specialist mode Metabolic, sequestrative, and defensive consequences

Adults of a generalist herbivore, the lubber grasshopper,Romalea guttata, can be converted to functional specialists by feeding them exclusively on catnip,Nepeta cataria. No obvious adverse effects on adult development resulted from this enforced monophagy. Notwithstanding the fact thatR. guttata has had no coevolutionary relationship with this Eurasian mint, it readily sequesters compounds that are identical to or derived from the terpenoid lactones that are characteristic ofN. cataria. R. guttata appears to both biomagnify minor allelochemicals and to sequester metabolites of theNepeta terpenes in its paired defensive glands. The levels of autogenously produced phenolics are not affected by feeding onN. cataria and the defensive secretions of catnip-fed grasshoppers are more repellent to ants than those of wild-fed acridids. Metabolites of theN. cataria monoterpenes are sequestered in the defensive glands when catnip is added to the natural diet ofR. guttata. The ability of a generalist,R. guttata, to facilely bioaccumulate a potpourri of foreign allelochemicals when feeding in a specialist mode is analyzed in terms of its biochemical, physiological, and functional significance. Sequestration is examined as a response to the enteric effronteries represented by the phytochemicals that can be characteristic of the "overload" in a monophagous diet.

The Pigment-Dispersing Hormone Family: Chemistry, Structure-Activity Relations, and Distribution

This report summarizes recent work on the pigment-dispersing hormone (PDH) family, a set of related neuropeptides common to arthropods. The primary structures are known for the major form of PDH in several crustacean species (Pandalus borealis, Uca pugilator, Cancer magister, Penaeus aztecus, Procambarus clarkii) and for related pigment-dispersing factors from two insects (Acheta domesticus, Romalea microptera). In this peptide family, the amino acid chain length (18 residues), termini (N-terminal Asn, C-terminal Ala-NH2), and at least 50% of the sequence are conserved. Synthetic analogs have been used to analyze the structure-activity relations of PDH, leading to: an evaluation of the role of specific residues; a tentative identification of the message sequence; and the preparation of stable and superpotent analogs including tyrosinated analogs for radioiodination. An enzyme-linked immunosorbant assay (ELISA) has been developed for β-PDH. Antisera raised against α-PDH and β-PDH were used to determine the distribution of PDH. This distribution and other evidence indicate that, besides its role in humoral regulation of the pigmentary system, PDH may serve extra-pigmentary functions. The functions of the PDH-related peptides in insects are unknown.

Reduction in diet breadth results in sequestration of plant chemicals and increases efficacy of chemical defense in a generalist grasshopper

The lubber grasshopper,Romalea guttata, is a generalist feeding on a broad diet of many herbaceous plant species and has a metathoracic defensive secretion normally containing phenolics and quinones synthesized by the insect. When insects were reared on a restricted diet of wild onion, they sequestered sulfur volatiles from the plant into their defensive secretions. These compounds were not detected by gas chromatography-mass spectroscopy in secretions of insects on an artificial diet or a natural, generalist diet of 26 plants that included wild onion as a component, nor were they present in secretions from field-collected insects. Defensive secretions of insects reared on wild onion were significantly more deterrent, by as much as an order of magnitude, to two species of ant predators than secretions from insects on either of the other two diets, despite a reduction in the concentration of autogenous defensive chemicals in secretions of insects on the onion diet. Sequestration of plant chemicals that increased defensive efficacy occurred when diet breadth was reduced. We suggest that this occurs because under conditions of specialization, plant secondary metabolites are more likely to be ingested and bioaccumulated in sufficient concentrations to have biological activity against predators. What we define as casual bioaccumulation of bioactive plant chemicals following dietary specialization may lead to evolution of sequestered defense syndromes in insects, and this process may not necessarily require specific adaptation to or coevolution with a toxic host plant.

Activation of fat body glycogen phosphorylase in the eastern lubber grasshopper (Romalea microptera) by the endogenous neuropeptides Ro I and Ro II

AbstractRomalea corpus cardiacum (CC) contains large quantities of two neuropeptides, named Ro I and Ro II, that are capable of eliciting strong hyperlipemic (locusts) and hypertrehalosemic (cockroaches) responses in test species (Spring and Gäde: Journal of Experimental Zoology 241:41–50, 1987). Although clearly members of the locust adipokinetic hormone/crustacean red pigment‐concentrating hormone (AKH/RPCH) family of peptides (Gäde: Zeitschrift für Naturforschung, Section C 41:315–320, 1986), these neuropeptides have no discernible effect on hemolymph lipids or carbohydrates in Romalea itself, and neither do large doses of synthetic locust AKH I (1,000 pmol) nor stick insect hypertrehalosemic factor II (HTF II: 500 pmol). Injections of its own CC are capable of stimulating Romalea fat body glycogen phosphorylase in a dose‐dependent manner, and this effect can be mimicked by Ro I and Ro II. Neither extracts of pars intercerebralis nor subesophageal ganglion have any significant effect on phosphorylase activation. Using synthetic peptides, we determined dose‐response curves for phosphorylase activation. Ro I is the more effective in stimulating phosphorylase, with a maximum activation of 70%, compared to 50% for Ro II. Ro II is more potent, however, producing a significant activation with 0.1 pmol, compared to 0.5 pmol of Ro I. Although these peptides are powerful activators of endogenous fat body glycogen phosphorylase in Romalea, this function does not explain the enormous quantities of neuropeptide (2,800 pmol Ro I; 650 pmol Ro II) found in the CC. Glycogen phosphorylase is also activated by starvation, and this effect is reversed by feeding. It is not clear at present whether the starvation‐induced activation is hormonally controlled.