Joseph Woodring

Control of the release of digestive enzymes in the larvae of the fall armyworm, Spodoptera frugiperda

There is a basal level of enzyme activity for trypsin, aminopeptidase, amylase, and lipase in the gut of unfed larval (L6) Spodoptera frugiperda. Trypsin activity does not decrease with non-feeding, possibly because of the low protein levels in plants along with high amino acid requirements for growth and storage (for later reproduction in adults). Therefore, trypsin must always be present so that only a minimal protein loss via egestion occurs. Larvae, however, adjust amylase activity to carbohydrate ingestion, and indeed amylase activity is five-fold higher in fed larvae compared to unfed larvae. Gut lipase activity is low, typical of insects with a high carbohydrate diet. A flat-sheet preparation of the ventriculus was used to measure the release of enzymes in response to specific nutrients and known brain/gut hormones in S. frugiperda. Sugars greatly increase (>300%) amylase release, but starch has no effect. Proteins and amino acids have little or no effect on trypsin or aminopeptidase release. The control of enzyme release in response to food is likely mediated through neurohormones. Indeed, an allatostatin (Spofr-AS A5) inhibits amylase and trypsin, and allatotropin (Manse- AT) stimulates amylase and trypsin release. Spofr-AS A5 also inhibits ileum myoactivity and Manse-AT stimulates myoactivity. The epithelial secretion rate of amylase and trypsin was about 20% of the amount of enzyme present in the ventricular lumen, which, considering the efficient counter-current recycling of enzymes, suggests that the secretion rate is adequate to replace egested enzymes.

Control of the release of digestive enzymes in the caeca of the cricket Gryllus bimaculatus

Abstract In Gryllus bimaculatus, more digestive enzymes (amylase, trypsin, aminopeptidase) are secreted in the caecum of fed crickets than in unfed crickets, but the enzymes are released continuously at a basal rate in unfed animals. The rate of synthesis of the enzymes appears to parallel their rate of release. Digestive enzymes are released in response to a specific ratio of nutrients, although a high nutrient component in the food does not necessarily induce a high digestive enzyme release for that component. Rinsed flat‐sheet preparations of the caecum are incubated with specific nutrients (carbohydrates and proteins) and various concentrations of a neuropeptide (type‐A allatostatin), which affects generally the basal rates of secretion. Both maltose and glucose increase the release of amylase in vitro, but starch produces an inhibition of amylase release at lower concentrations. Bovine serum albumin (BSA), peptone and a mixture of amino acids have almost no effect on the release of aminopeptidase or carboxypeptidase, and only low concentrations of peptone increase trypsin release. High concentrations of both BSA and peptone strongly inhibit trypsin activity, perhaps by excess substrate binding to the trypsin active site. The allatostatin Grybi‐AST 5 elevates the release of amylase in vitro, but not of trypsin or aminopeptidase, in 2‐day‐old fed females. In the caeca from 1‐day‐old unfed crickets, both amylase and the trypsin release are stimulated in the presence of AST 5. The paracrine AST 5 is probably released from the gut endocrine cells and binds to the enzyme‐producing caecal cells.

Floral Reward, Advertisement and Attractiveness to Honey Bees in Dioecious Salix caprea

In dioecious, zoophilous plants potential pollinators have to be attracted to both sexes and switch between individuals of both sexes for pollination to occur. It often has been suggested that males and females require different numbers of visits for maximum reproductive success because male fertility is more likely limited by access to mates, whereas female fertility is rather limited by resource availability. According to sexual selection theory, males therefore should invest more in pollinator attraction (advertisement, reward) than females. However, our knowledge on the sex specific investment in floral rewards and advertisement, and its effects on pollinator behaviour is limited. Here, we use an approach that includes chemical, spectrophotometric, and behavioural studies i) to elucidate differences in floral nectar reward and advertisement (visual, olfactory cues) in dioecious sallow, Salix caprea, ii) to determine the relative importance of visual and olfactory floral cues in attracting honey bee pollinators, and iii) to test for differential attractiveness of female and male inflorescence cues to honey bees. Nectar amount and sugar concentration are comparable, but sugar composition varies between the sexes. Olfactory sallow cues are more attractive to honey bees than visual cues; however, a combination of both cues elicits the strongest behavioural responses in bees. Male flowers are due to the yellow pollen more colourful and emit a higher amount of scent than females. Honey bees prefer the visual but not the olfactory display of males over those of females. In all, the data of our multifaceted study are consistent with the sexual selection theory and provide novel insights on how the model organism honey bee uses visual and olfactory floral cues for locating host plants.

Snowdrop lectin (Galanthus nivalis agglutinin) in aphid honeydew negatively affects survival of a honeydew-consuming parasitoid.

1u2002Insecticidal proteins can be excreted in the honeydew when sap‐sucking insects feed on insect‐resistant transgenic plants. Honeydew can be an important source of carbohydrates, thus potentially exposing a broad range of honeydew‐feeding insects to transgene products.

Activity, release and flow of digestive enzymes in the cricket, Gryllus bimaculatus

Abstract In the cricket Gryllus bimaculatus, proventricular pressure forces a nutrient fluid from the ground food‐brei through a sieve at the base of the caecae, and the combination of secreted enzymes and water causes a rapid inflation of the caecae on the first day after imaginal ecdysis. The ceacal region of the midgut is the primary site for the secretion of digestive enzymes. Proteases and amylase flow from the caecae into the mostly empty crop on day 1, and carbohydrate and protein digestion starts as soon as food is present (6 h). Thereafter, much of the amylase activity (but not protease) in the crop is synthesized and released by the crop tissues themselves. Regurgitating proteases and amylases from the caecae into the crop after day 1 is most likely accomplished by temporarily halting proventricular peristalsis and allowing the caecal muscles to contract, forcing caecal contents, including enzymes, forward. The total activity of digestive enzymes in the caecae is virtually identical in 2‐day‐old fed and unfed females, indicating little or no secretagogue (prandial) regulation of enzyme secretion. Most of the digestive enzymes in the ventricular endoperitrophic space may originate from the mucus dragged from the caecae. Lipase activity is low in all gut regions in both starved and fed females. Head ligation or injection of trypsin modulating oostatic factor, allatostatin A or B fails to indicate any involvement of nerves or hormones in the release of digestive enzymes in the caecae. Gryllus bimaculatus appears to secrete digestive enzymes continuously, and a considerable loss of enzymes may occur at certain times through egestion.

Oligosaccharide synthesis regulates gut osmolality in the ant‐attended aphid Metopeurum fuscoviride but not in the unattended aphid Macrosiphoniella tanacetaria

Abstract:u2002 An artificial phloem sap (APS) for Metopeurum fuscoviride and Macrosiphoniella tanacetaria, based on analysis of their host plant, Tanacetum vulgare, phloem sap, contained 308u2003μg/μl (900u2003μmol/μl) sucrose (and no other sugars) and a mixture of 14 amino acids with a total concentration of 82.5u2003nmol/μl. There was no significant difference in the total amino acid concentration of the honeydew of adult M. fuscoviride fed on the host plant and aphids fed on APS. Incubation of isolated guts in APS indicated no role of gut bacteria or gut enzymes on the amino acid pattern in the gut. The sugar composition of the honeydew of the ant‐attended M. fuscoviride indicated a rapid digestion of sucrose into glucose and fructose, and the simultaneous synthesis of considerable amounts of melezitose and some trehalose. The sugar composition of the honeydew of the unattended M. tanacetaria in contrast showed only traces of trehalose and melezitose, but up to 20% erlose in plant‐fed aphids. Incubation of isolated guts of M. fuscoviride in APS demonstrated a steady high rate of melezitose synthesis by gut enzymes over an 8‐h period. Incubation of isolated guts of M. tanacetaria on the other hand demonstrated only a moderate rate of erlose synthesis and no detectable melezitose or trehalose. Melezitose in the aphid M. fuscoviride is a signal sugar for ants (ecological function), indicating the presence of abundant sugar rich honeydew [ Woodring et al. (2004)Physiol. Entomol., vol. 29, pp. 311–319]. It was estimated that melezitose reduces the gut osmolality of M. fuscoviride to approximately 25–35% of what it would be without the synthetases (physiological function). M. tanacetaria on the other hand produces very little honeydew, is not attended by ants, and thus there is little need to synthesize large amounts of oligosaccharides to attract ants or for osmoregulation.

REGULATION OF AMYLASE, CELLULASE AND CHITINASE SECRETION IN THE DIGESTIVE TRACT OF THE TWO‐SPOTTED FIELD CRICKET, Gryllus bimaculatus

The secretion of amylase and cellulase in Gryllus bimaculatus is determined by increased food intake, whereby shortly after molting food consumption increases. About half of the standing amylase concentration (activity) in the endothelial cells can be secreted within 30 min. The peak of amylase and cellulase secretion that occurs in the photophase is related to the feeding peak in the previous scotophase. The secretion of chitinase on the other hand is primarily controlled by the molting cycle. Only amylase secretion was affected by calcium in the incubation medium, suggesting an apocrine release mechanism. Refeeding experiments (after 5 days without food) suggest that the release of amylase in response to a nutrient in the lumen (glucose) is not due to simple stimulation of exocytosis, but rather a stimulation of synthesis.

Feeding, nutrient flow, and digestive enzyme release in the giant milkweed bug, Oncopeltus fasciatus

Abstract The giant milkweed bug, Oncopeltus fasciatus, is an intermittent feeder, spending most of its time in compact groups away from the food, with feeding bouts lasting 30–60 min. They macerate a small area of the seed with their stylets, secreting large amounts of saliva (which suspends and emulsifies the seed tissue), and quickly suck the emulsion into the gut. Therefore, little digestive activity occurs in the seed tissue. Freshly ingested food in the first gut region (V1) consists of 50% water, 46% lipid, 5% protein, and 1% soluble carbohydrates. The junction of the midgut and hindgut lacks a lumen in the larvae stages, and a 3 μL large oil drop (> 85% triacylglycerides) accumulates in the third gut region (V3). Approximately 24 h after the imaginal moult, this large volume of oil and faeces is defecated, and thereafter an oily faeces is defecated several times daily. It is postulated that the delayed egestion in larvae protects them against suffocation due to self‐soiling with oily faeces. Serine proteases are absent in the gut and, instead, a cystein proteinase, inhibited by E64, is present (gut pH 6.0). High lipase activity was found throughout the gut, but only limited amylase activities. Basal levels of all three enzymes occurred throughout the gut, but at lower levels in starved bugs. Salivary gland extracts contain a cysteine proteinase and a lipase, but no amylase.

Environmental control of trypsin secretion in the midgut of the two-spotted field cricket, Gryllus bimaculatus

The two most important environmental factors controlling the release of trypsin in Gryllus bimaculatus are temperature and food consumption. Food consumption is in turn controlled by food availability (quantity), food quality (contained nutrients, inhibitors), developmental stage, age, sex and the daily light-dark cycle. The secretion of trypsin was higher at an acclimation temperature (AT) of 22°C than at 32°C, although the weight of caecal tissue and body weight were lower. The trypsin secretion at both experimental temperatures (25°C and 35°C) was almost 2 times greater in crickets maintained at 22°C AT since egg hatch than those maintained only since the last larval stage, but not at 32°C AT. Acclimation became increasingly rotational with increased exposure time at different rearing temperatures. The more food consumed the higher the trypsin secretion. Secretion was highest on day 3 in adult females and day 2 in males, corresponding to the day of maximal food consumption. Secretion was less than 20% in starved or cellulose fed females compared to those fed a control diet. Food reached the caeca in starved crickets within 30min and induced an increased trypsin secretion. Crickets started feeding at the onset of darkness, and trypsin secretion was significantly elevated near the end of the scotophase. The in vivo response to 0.4% soybean trypsin inhibitor (SBTI) fed throughout the last larval stage resulted in reduced growth and a 50% decrease in trypsin secretion in 2day old adult females. An adaptation to the reduction of trypsin secretion occurred when G. bimaculatus was fed 0.1% and 0.2% SBTI, but not when fed with 0.4%.

Exogenous and endogenous protease inhibitors in the gut of the fall armyworm larvae, Spodoptera frugiperda.

A dose-dependent inhibition of endogenous trypsin and aminopeptidase occurs in the lumen of Spodoptera frugiperda after feeding L6 larvae exogenous inhibitors soybean trypsin inhibitor (SBTI), tosyl-L-lysine chloromethyl ketone-HCl (TLCK), or bestatin, respectively, for 3 days. TLCK inhibits trypsin in tissue extracts and in secretions more strongly than SBTI. The aminopeptidase released into the lumen (containing the peritrophic membrane) is strongly inhibited by bestatin, but the membrane-bound enzyme is not. A bound enzyme may be more resistant to an inhibitor than unbound. A cross-class elevation of aminopeptidase activity occurs in response to ingested trypsin inhibitor, but there was no cross-class effect of aminopeptidase inhibitor (bestatin) on trypsin activity. An endogenous trypsin and aminopeptidase inhibitor is present in the lumen and ventricular cells. The strength of the endogenous trypsin inhibition seems to be in the same range as that resulting from ingestion of the exogenous inhibitor SBTI. In some insect species, considerable trypsin secretion occurs in unfed as well as in fed animals, and endogenous protease inhibitors might function to protect the ventricular epithelium by inactivation of trypsin when less food is available.