Ontogeny of tracheal dimensions and gas exchange capacities in the grasshopper, Schistocerca americana

How does body size affect the structure and gas exchange capacities of insect tracheae? Do insects become more oxygen-limited as they grow? We addressed these questions by measuring the dimensions of two transverse tracheae within the abdomen of American locusts of different ages, and evaluating the potential for diffusion or convection to provide adequate gas exchange. The grasshopper abdomen has longitudinal tracheae that run along the midgut, heart, nerve cord, and lateral body wall. Transverse tracheae run from each spiracle to the longitudinal tracheae. Dorsal air sacs attach near each spiracle. In both transverse tracheae studied, diffusive capacities increased more slowly than metabolic rates with age, and calculated oxygen gradients necessary to supply oxygen by diffusion increased exponentially with age. However, surgical studies demonstrated that transport of gas through these transverse tracheae occurred by convection, at least in adults. Convective capacities paralleled metabolic rates with age, and the calculated pressure gradients required to sustain oxygen consumption rates by convection were independent of age. Thus, in growing grasshoppers, tracheal capacities matched tissue oxygen needs. Our morphological and physiological data together suggest that use of convection allows older grasshoppers to overcome potential limitations on size imposed by diffusion through tracheal systems.     

Food aversion learning in the polyphagous grasshopper Schistocerca americana

ABSTRACT. The ability of the acridid Schistocerca americana (Drury) (Orthoptera: Acrididae) to learn to avoid a food when it is associated with an artificial aversive stimulus was examined. The aversive stimulus used was an injection of nicotine hydrogen tartrate at a concentration which usually did not cause knockdown. Insects learned to avoid a food which already had limited sustained acceptability. The learning was not found when the aversive stimulus followed a meal on a fully acceptable food.     

Interactions between two neurons in contact chemosensilla of the grasshopper,Schistocerca americana

Summary The tibial contact chemosensilla of Schistocerca americana contain several neurons, one of which responds to nicotine hydrogen tartrate and certain other compounds. The activity of this cell is sometimes briefly interrupted by the firing of a second cell in the same sensillum which usually only fires a few times in the first 500 ms of a stimulation. The evidence suggests that the nicotine-sensitive cell is directly inhibited by the activity of the second cell. Not all cells in the sensillum produce the same effect.     

The nutritional significance of sterol metabolic constraints in the generalist grasshopper Schistocerca americana

Sterols are essential nutrients for grasshoppers, as well as all other insects, but metabolic constraints can limit which phytosterols support normal growth and development. In the current study, the generalist grasshopper Schistocerca americana was used to address two questions related to grasshopper sterol nutrition: (1) how does sterol quantity influence growth and survival, and (2) how do mixtures of suitable and unsuitable sterols at different concentrations influence growth and survival? Results from the first experiment indicated that this grasshopper species had a minimum sterol requirement of 0.05% dry weight; as sterol quantity increased above this concentration, however, survival and performance were not enhanced. Results from the second experiment revealed two novel aspects of sterol nutrition in grasshoppers: (1) when suitable sterols were limiting, most individuals could not use unsuitable sterols to meet the minimum sterol requirement (i.e. no sparing occurred), and (2) above a certain threshold, unsuitable sterols were deleterious even when suitable sterols were present at a concentration that alone permits normal growth and development. We discuss these physiological findings in terms of how sterol metabolic constraints in grasshoppers might influence foraging.     

Phytosterol structure and its impact on feeding behaviour in the generalist grasshopper Schistocerca americana

Abstract.Sixth-stadium nymphs of the grasshopper Schistocerca americana (Drury) (Orthoptera: Acrididae) were observed in a series of experiments designed to measure feeding behaviour in response to suitable and unsuitable phytosterols. In the first experiment, grasshoppers were presented with artificial diet that contained either sitosterol, a suitable phytosterol, or a spinach lipid extract which contained only unsuitable sterols as well as other spinach lipids. The diet with the spinach lipid extract, but not the sitosterol diet, evoked a deterrent response. To determine if the spinach sterols were responsible for the deterrent response, a second experiment was performed where the spinach lipid extract was separated into three lipid classes, including desmethyl sterols, dimethyl sterols and the remaining spinach lipids. Grasshoppers presented with artificial diet containing the desmethyl sterols (the end-product sterols in spinach) exhibited deterrent responses. Finally, feeding behaviour to a suite of different sterols, including cholesterol (suitable), stigmasterol (unsuitable), and lathosterol (unsuitable), was observed; these sterols were selected because they show variation in the position of double bonds. Grasshoppers presented with diets containing unsuitable sterols again exhibited deterrent responses. Overall, the deterrent effect was strongest when sterols with a double bond at position 22 were in the diet.     

Tarsal chemoreception in the polyphagous grasshopper Schistocerca americana: behavioural assays, sensilla distributions and electrophysiology

ABSTRACT Behavioural and electrophysiological responses of Schistocerca americana (Drury) (Orthoptera: Acrididae) to chemical stimulation of the tarsi were investigated. Using restrained insects, differences in leg-waving behaviour were observed following stimulation by sucrose and nicotine hydrogen tartrate (NHT), compared to control stimulations by water. Furthermore, free-walking insects were able to detect NHT on leaf surfaces, resulting in leg-raising to avoid tarsal contact.
SEM studies showed the presence of numerous peg chemoreceptor sensilla on the ventral surface of the tarsus. Tip recordings from such pegs showed activity from up to three chemosensitive neurones, plus a mechanoreceptor neurone. Stimulation by NaCl and KC1 elicited similar responses from two or three neurones in all sensilla tested, with increased firing rates at higher concentrations. Sucrose caused an increase in firing rate in few sensilla. In such cases several neurones were stimulated, and there was no evidence of a specific neurone sensitive to sucrose. In contrast, NHT elicited rapid firing in a single neurone, which was not sensitive to NaCl. Stimulation by NHT also inhibited the activity of the NaCl-sensitive neurones.
Possible mechanisms for chemical discrimination in S. americana tarsi are compared with those previously proposed for grasshopper mouthpart sensilla, and the significance of a NHT-sensitive neurone in tarsal sensilla is discussed.     

Phytosterol unsuitability as a factor mediating food aversion learning in the grasshopper Schistocerca americana

Abstract Sixth stadium nymphs of the grasshopper Schistocerca americana (Drury) (Orthoptera: Acrididae) were presented with spinach with or without added cholesterol or β-sitosterol, and the lengths of the first four meals were monitored. Spinach alone evoked a typical aversion learning pattern in which successive meals were of decreasing duration; however, when cholesterol or β-sitosterol was added the spinach remained acceptable. Nymphs fed spinach for 24h preferred glass-fibre discs with added cholesterol in a choice test, but nymphs fed wheat (an acceptable food) did not prefer cholesterol-treated or control discs. Nymphs pretreated with spinach did not distinguish between discs treated with stigmasterol (an unsuitable phytosterol for grasshoppers) and control discs. In no-choice tests, nymphs pretreated with spinach also fed significantly longer on discs with added 10% cholesterol or 1%β-sitosterol, compared to meals on discs with stigmasterol or sucrose controls. Detection of appropriate sterols is likely to involve a post-ingestive feedback mechanism, as significantly longer meals on discs treated with only sucrose resulted when spinach-fed nymphs, were force-fed gelatin capsules packed with β-sitosterol or cholesterol, compared with stigmasterol. Contact chemoreception is unlikely to play a role in this behaviour, as discs (lacking sucrose) treated with cholesterol, β-sitosterol, stigmasterol, or a chloroform control, all required similar numbers of contacts before feeding was initiated.     

Presence of a single abundant storage hexamerin in both larvae and adults of the grasshopper, Schistocerca americana

We identified a single hexameric storage protein in the grasshopper, Schistocerca americana, and monitored its abundance through the last larval instar and up until reproductive competence in adults of both sexes. This storage hexamerin, termed Schistocerca americana Persistent Storage Protein (saPSP) was the most abundant soluble protein in both larvae and adults. In both sexes, saPSP abundance started out low at the onset of the last larval instar and accumulated during feeding, peaking just prior to molting. Adults of both sexes contained significant amounts of saPSP after eclosion. In adult males, saPSP content dropped continuously after eclosion and was lowest once individuals reached reproductive maturity. In contrast, adult females depleted saPSP reserves during the first days of adulthood, but subsequently accumulated significant saPSP stores. In adult females, saPSP stores peaked just prior to the completion of egg provisioning. Given the overall patterns of abundance, saPSP has functions in both larvae and adults. In addition, the observed pattern of storage hexamerin accumulation differs from patterns of accumulation in the other known grasshoppers, Locusta migratoria and Romalea microptera, suggesting that significant functional diversity has evolved in storage hexamerins among the grasshoppers.     

Direct visualization of hemolymph flow in the heart of a grasshopper (Schistocerca americana)

Background  

Hemolymph flow patterns in opaque insects have never been directly visualized due to the lack of an appropriate imaging technique. The required spatial and temporal resolutions, together with the lack of contrast between the hemolymph and the surrounding soft tissue, are major challenges. Previously, indirect techniques have been used to infer insect heart motion and hemolymph flow, but such methods fail to reveal fine-scale kinematics of heartbeat and details of intra-heart flow patterns.     

Ultrastructure of the replication of the grasshopper crystalline-array virus in Schistocerca americana compared with other picornaviruses

The replication of the grasshopper crystalline-array virus (CAV), a picornavirus, was studied in thoracic muscles and pericardial cells. Two types of intracellular vesicles were evident that apparently functioned in viral synthesis. Those with smooth membranes were predominant in perinuclear regions and appeared to originate as protrusions from the outer nuclear membrane; those with particle-associated membranes were evident throughout the cytoplasm and appeared to be derived from existing membranous structures such as Golgi bodies and endoplasmic reticulum. In general, the replication of the grasshopper-CAV appears similar to the replication of picornaviruses in vertebrate systems.     

Ontogeny of identified cells from the median domain in the embryonic brain of the grasshopper Schistocerca gregaria

In this paper, we propose an ontogeny for previously identified cells from the median domain in the midline of the embryonic brain of the grasshopper Schistocerca gregaria. The so-called lateral cells (LCs) are characteristically located laterally within the median domain at its border with the protocerebral hemispheres. The LC occurs singly and can be identified in the early embryo on the basis of their expression of the cell surface lipocalin Lazarillo. Using immunocytochemical, dye injection, electron microscopical and histological methods, we show that these LC are neurons and derive as postmitotic cells directly from the epithelium of the median domain. Further, they and the other identified cells of the median domain such as the protocerebral commissure pioneers (PCP), co-express the Mes-3 antigen, consistent with a derivation from the mesectodermal germ layer of the embryo. Subsequent to axogenesis, electron microscopy reveals that these Mes-3-expressing LC fasciculate with the co-expressing PCPs within the developing protocerebral commissure. We present a model for the origin of all these cells based on histological data and bromodeoxyuridine incorporation. The model suggests a delamination of cells from the mesectoderm followed by a migration to their ultimate sites within the median domain.     

Developmental expression and molecular characterization of two gap junction channel proteins expressed during embryogenesis in the grasshopper Schistocerca americana

Gap junctions are membrane channels that directly connect the cytoplasm of neighboring cells, allowing the exchange of ions and small molecules. Two analogous families of proteins, the connexins and innexins, are the channel-forming molecules in vertebrates and invertebrates, respectively. In order to study the role of gap junctions in the embryonic development of the nervous system, we searched for innexins in the grasshopper Schistocerca americana. Here we present the molecular cloning and sequence analysis of two novel innexins, G-Inx(1) and G-Inx(2), expressed during grasshopper embryonic development. The analysis of G-Inx(1) and G-Inx(2) proteins suggests they bear four transmembrane domains, which show strong conservation in members of the innexin family. The study of the phylogenetic relationships between members of the innexin family and the new grasshopper proteins suggests that G-Inx(1) is orthologous to the Drosophila 1(1)-ogre. However, G-Inx(2) seems to be a member of a new group of insect innexins. We used in situ hybridization with the G-Inx(1) and G-Inx(2) cDNA clones, and two polyclonal sera raised against different regions of G-Inx(1) to study the mRNA and protein expression patterns and the subcellular localization of the grasshopper innexins. G-Inx(1) is primarily expressed in the embryonic nervous system, in neural precursors and glial cells. In addition, a restricted stripe of epithelial cells in the developing limb, involved in the guidance of sensory growth cones, expresses G-Inx(1). G-Inx(2) expression is more widespread in the grasshopper embryo, but a restricted expression is found in a subset of neural precursors. The generally different but partially overlapping expression patterns of G-Inx(1) and G-Inx(2) supports the combinatorial character of gap junction formation in invertebrates, an essential property to generate specificity in this form of cell-cell communication.     

Larval nutrition affects lipid storage and growth, but not protein or carbohydrate storage in newly eclosed adults of the grasshopper Schistocerca americana

Nitrogen availability from dietary protein can have profound effects on the physiology and evolutionary ecology of insect herbivores. While many studies consider the effects of nutrition on consumption and gross body composition of protein and other important nutrients, few consider partitioning to storage for future use. I used chemically defined artificial diets to quantitatively manipulate the amount of dietary carbohydrates and proteins available to growing larvae of the grasshopper Schistocerca americana to determine how larval nutrient availability affects growth and all three classes of stored nutrients (proteins, lipids, and carbohydrates) carried over from larval feeding into adulthood. Larvae on poor diets increased consumption, but could not compensate for diet quality, eclosing small and containing no significant nutrient stores at adulthood. Individuals fed intermediate to high nutrient content diets as larvae were significantly larger and contained a significantly greater proportion of lipid stores at adult eclosion, but not protein or carbohydrate stores than individuals fed low nutrient content diets. This suggests that larvally derived lipid stores may be more important to adult fitness than carbohydrate or protein stores. This result is contrary to previous studies performed on the role of larval nutrition and allocation to protein stores, and this difference is likely due to variation in the relative availability of protein in adult diets across species.     

Arsenic hyperaccumulation in the Chinese brake fern (Pteris vittata) deters grasshopper (Schistocerca americana) herbivory

Brake fern, Pteris vittata, not only tolerates arsenic but also hyperaccumulates it in the frond. The hypothesis that arsenic hyperaccumulation in this fern could function as a defense against insect herbivory was tested. Fronds from control and arsenic-treated ferns were presented to nymphs of the grasshopper Schistocerca americana. Feeding damage was recorded by visual observation and quantification of the fresh weight of frond left uneaten and number of fecal pellets produced over a 2-d period. Grasshopper weight was determined before and after 5 d of feeding. Grasshoppers consumed significantly greater amounts of the frond tissue, produced more fecal pellets and had increased body weight on control plants compared with grasshoppers fed arsenic-treated ferns. Very little or none of the arsenic-treated ferns were consumed indicating feeding deterrence. In a feeding deterrent experiment with lettuce, sodium arsenite at 1.0 mm deterred grasshoppers from feeding whereas 0.1 mm did not. In a choice experiment, grasshoppers preferred to feed on lettuce dipped in water compared with lettuce dipped in 1.0 mm sodium arsenite. Our results show that arsenic hyperaccumulation in brake fern is an elemental defense against grasshopper herbivory.     

Declining acceptability of a food plant for the polyphagous grasshopper Schistocerca americana: the role of food aversion learning

ABSTRACT Successive contacts with a food that was initially acceptable to the acridid Schistocerca americana (Drury) (Orthoptera: Acrididae) were monitored. It was found that acceptability, as measured by meal length, declined with experience until the food, spinach, was completely rejected. Reduced acceptability of spinach was maintained even after feeding for three meals on a fully acceptable and suitable food, a period of 2–3 h. Experiments with spinach-filled capsules placed in the gut suggested that the decline in acceptability was not due to direct feedback from the gut or haemolymph on the sensory or feeding control systems. Food aversion learning is indicated as the mechanism underlying the decline in acceptability.     

The relationship between plant acceptability and suitability for survival and development of the polyphagous grasshopper,Schistocerca americana (Orthoptera: Acrididae)

The feeding behavior of final-instar nymphs ofSchistocerca americana was observed when they first encountered plants that ranged in acceptability from being eaten in large amounts to not being eaten at all. Growth and survival on the same plants through the last stadium were also studied and the results combined as a suitability index to facilitate comparison with the behavior. Although the plants that were eaten most gave the highest suitability index and those that were not eaten permitted no survival, there was no simple relationship between the amounts eaten and the suitability for growth and survival. The possibility that the insects might become habituated to plants that were initially unacceptable was investigated, but no habituation was found over a 3-day period. It is suggested that food intake is largely determined by the presence of deterrent compounds in the less acceptable foods and that nutritional differences between the plants are likely to have been of minor importance. The behavior on some foods suggests that food aversion learning may be involved. It is concluded that the variability of the insects' behavior makes it impossible to predict the suitability of a plant from their immediate behavioral responses. In the field, insects may sometimes reject foods that would be suitable for survival and development, and feed on plants that are nutritionally deficient or even toxic.     

Neural regulation of discontinuous gas exchange in Periplaneta americana

Patterns of gas exchange among terrestrial arthropods are highly variable from continuous to discontinuous with discretely partitioned phases. The underlying initiation and co-ordination of these patterns is relatively poorly understood. Here we present a novel method for the simultaneous measurement of central nervous system (CNS) activity of the metathoracic ganglion and VCO(2) in medium to large sized live terrestrial arthropods. Using Periplaneta americana at four oxygen levels (40%, 21%, 10% and 2% at 25 degrees C; n=6 per treatment), we present minimally invasive visualization of nervous output relative to typical resting discontinuous gas exchange (DGE) data for the first time. DGE was maintained when cockroaches were exposed to hyperoxia or moderate hypoxia, but was lost in severe hypoxia. CNS activity was manifested in three signal types: large CNS output coinciding with peak CO(2) production during a burst, moderate CNS output coinciding with CO(2) sawtoothing and fluttering, and minimal CNS activity during the closed phase of DGE in normoxia. Large and moderate CNS outputs were associated with observed abdominal pumping and congruent CO(2) peaks. At 10% oxygen, VCO(2) was significantly elevated during the inter-burst period in association with almost constant moderate CNS output between the periodic large CNS output. At 2% oxygen, DGE and large CNS output are lost to continuous CO(2) release and largely continuous moderate CNS output. As previously reported for this species, a central pattern generator for ventilation in the metathoracic ganglion is supported and we infer the presence of localized oxygen chemoreceptors based on clear CNS response to a change in oxygen tension.     

Embryonic expression of muscle-specific antigens in the grasshopper Schistocerca gregaria

Monoclonal antibodies (MAbs) are used to investigate molecules that are expressed during embryonic muscle differentiation and that may be involved in muscle pioneer and muscle attachment site formation. MAb F2A5 immunoreactivity appears in all muscle pioneers as soon as they extend processes, and continues in all muscle precursors. MAb 4H1 immunoreactivity is strongly expressed only after mesodermal cells have fused with the muscle pioneers; then it is concentrated at their growth-cone-like ends near developing attachment sites. During later embryonic development, MAb F2A5 and MAb 4H1 immunoreactivity become associated with the myofibrillar network. Biochemical experiments indicate that MAb 4H1 recognises a 47 kDa antigen, and MAb F2A5 recognises an 80 kDa antigen.