Substrate specificity in the control of digestive enzymes in larvae of the black carpet beetle

When starved larvae of the black carpet beetle, Attagenus megatoma, were fed selected diets, increases in proteolytic, trypsin, and chymotrypsin activity were correlated with total midgut protein and not with the amount of food consumed. Although larvae initially consumed more of a starch diet than of 2 diets that contained added protein, total protease activity in these larvae was minimal. Starch-fed larvae and larvae fed a casein-sucrose diet had a consistently higher level of sucrase activity than larvae fed an all-casein diet. These total results support a secretagogue mechanism for control of digestive enzyme synthesis in insects. In addition, the absence of parallel stimulation of different digestive enzymes by a single substrate (starch) indicated nutrient class specificity in the control of inducible midgut enzymes in this species.     

Organization of protein digestion in adult Calosoma calidum (Coleoptera: Carabidae)

Organization of protein digestion was examined in adult male Calosoma calidum (Carabidae) fed either ground-beef or waxmoth larvae (Galleria mellonella). Although trypsin activities in the foregut are consistently higher than those in the midgut, the luminal contents of each region are in equilibrium. Movement of fluids between the midgut and foregut is brought about by muscular contractions of the proventriculus. A 42% fall in trypsin activity of the foregut after feeding on ground-beef is due largely to disgorged enzymes being left on the food. A far higher proportion of disgorged trypsin is recovered when C. calidum feed on waxmoth larvae; beetles ingest about 74% prey protein and yet avoid ingesting digestively refractory solids. The retention of undigested macromolecules in the midgut and foregut lumen was determined using [¹⁴C]inulin labelled waxmoth larvae. Nine per cent and 25% of the radiolabel was passed in the faeces in 2 and 4 days respectively, whilst 59 and 91% of the weight gained at feeding was lost in the same intervals. The role that the peritrophic membrane and pyloric valve play in this process, as well as its implications for enzyme conservation, is discussed.     

Rhythmic contractile movements of the larval midgut of the silkworm, Bombyx mori

The raidgut of intact larvae of Bombyx mori exhibited active contractile movements. Contraction waves were generated rhythmically at several regions of the midgut. The waves passed in both oral and aboral directions from their sites of origin. Midgut movements were depressed during moulting. The midgut continued to move normally when tetrodotoxin was injected into the larval haemocoel at doses sufficient to paralyze somatic muscle. Ligation of larvae paralyzed with tetrodotoxin behind the second or third body segment resulted in the abolition of the contraction waves in the midgut portion anterior to the ligature. A ligature applied behind other body segments did not hamper midgut motor activity irrespective of whether or not an abdominal ganglion had been extirpated. The frequency of contraction and the rate of food transport in the midgut were increased when larvae were administered serotonin or when their body temperature was raised.     

Phenol oxidases from Rhodnius prolixus: Temporal and tissue expression pattern and regulation by ecdysone

Rhodnius prolixus 5th instar nymphs have significant PO enzymatic activity in the anterior midgut, fat body and hemolymph. The tissue with the major amount of PO activity is the anterior midgut while those with higher specific activities are the fat body and hemolymph. In this work the temporal pattern of PO enzymatic activity in different tissues was investigated. In fat body, PO peaks occur at 7, 12 and 16 days after a blood meal. In hemolymph, PO diminishes until day 7, and then recovers by day 14. In the anterior midgut tissue, PO peaks on day 9 and just before ecdysis; a similar pattern was observed in the anterior midgut contents. Some of these activities are dependent on the release of ecdysone, as feeding blood meal containing azadirachtin suppresses them and ecdysone treatment counteracts this effect. These results suggest that during the development of the 5th instar, the insect has natural regulating cycles of basal PO expression and activation, which could be related to the occurrence of natural infections. The differences in temporal patterns of activity and the effects of azadirachtin and ecdysone in each organ suggest that, at least in R. prolixus, different tissues are expressing different PO genes.     

Schinus terebinthifolius Leaf Extract Causes Midgut Damage,Interfering with Survival and Development of Aedes aegypti Larvae

In this study, a leaf extract from Schinus terebinthifolius was evaluated for effects on survival, development, and midgut of A. aegypti fourth instar larvae (L4), as well as for toxic effect on Artemia salina. Leaf extract was obtained using 0.15 M NaCl and evaluated for phytochemical composition and lectin activity. Early L4 larvae were incubated with the extract (0.3–1.35%, w/v) for 8 days, in presence or absence of food. Polymeric proanthocyanidins, hydrolysable tannins, heterosid and aglycone flavonoids, cinnamic acid derivatives, traces of steroids, and lectin activity were detected in the extract, which killed the larvae at an LC₅₀ of 0.62% (unfed larvae) and 1.03% (fed larvae). Further, the larvae incubated with the extract reacted by eliminating the gut content. No larvae reached the pupal stage in treatments at concentrations between 0.5% and 1.35%, while in the control (fed larvae), 61.7% of individuals emerged as adults. The extract (1.0%) promoted intense disorganization of larval midgut epithelium, including deformation and hypertrophy of cells, disruption of microvilli, and vacuolization of cytoplasms, affecting digestive, enteroendocrine, regenerative, and proliferating cells. In addition, cells with fragmented DNA were observed. Separation of extract components by solid phase extraction revealed that cinnamic acid derivatives and flavonoids are involved in larvicidal effect of the extract, being the first most efficient in a short time after larvae treatment. The lectin present in the extract was isolated, but did not show deleterious effects on larvae. The extract and cinnamic acid derivatives were toxic to A. salina nauplii, while the flavonoids showed low toxicity. S. terebinthifolius leaf extract caused damage to the midgut of A. aegypti larvae, interfering with survival and development. The larvicidal effect of the extract can be attributed to cinnamic acid derivatives and flavonoids. The data obtained using A. salina indicates that caution should be used when employing this extract as a larvicidal agent.     

Sn-protoporphyrin inhibits both heme degradation and hemozoin formation in Rhodnius prolixus midgut

Hematophagy is a feeding habit that involves the ingestion of huge amounts of heme. The hematophagous hemipteran Rhodnius prolixus evolved many genetic resources to protect cells against heme toxicity. The primary barrier against the deleterious effects of heme is the aggregation of heme into hemozoin in the midgut lumen. Hemozoin formation is followed by the enzymatic degradation of heme by means of a unique pathway whose end product is dicysteinyl-biliverdin IX-γ (Rhodnius prolixus biliverdin, RpBv). These mechanisms are complemented by a heme-binding protein (RHBP) in the hemolymph that attenuates the pro-oxidant effects of heme. In this work, we show that when insects are fed with blood enriched with a heme analog, Sn-protoporphyrin (SnPP-IX), both hemozoin synthesis and RpBv production are inhibited in a dose-dependent manner. These effects are accompanied by increased oxidative damage to the midgut epithelium and inhibition of oviposition, indicating that hemozoin formation and heme degradation are protective mechanisms that work together and contributed to the adaptation of this insect to successfully feed on vertebrate blood.     

Activity cycles and the control of four digestive proteinases in the posterior midgut of Rhodnius prolixus Stål (Hemiptera: Reduviidae)

Proteinase levels in the posterior midgut of fifth-instar and adult Rhodnius prolixus follow a cyclic pattern after ingestion of the bloodmeal. In the fifth instar, cathepsin B showed two peaks: the first occurred 6 days after ingestion and the second at the time of ecdysis. Cathepsin D, cathepsin B and lysosomal carboxypeptidase B reached maximal levels 6 days after ingestion. At this time the highest levels of these proteinases were found in mated females, the lowest in males and intermediate levels in virgin females. Maximal levels of aminopeptidase occurred later than catheptic enzymes, and the decline, after maximal levels were achieved, was much more gradual.Catheptic-proteinase levels within the posterior midgut in fifth-instar larvae and adults correlated positively with the amount of protein contained in this gut region. This indicates that production of these proteinases is controlled by a secretagogue mechanism. Aminopeptidase levels were controlled in a different manner. The mated state or sex of adults altered the proteinase levels by changing the amount of protein that was passed into the digestive midgut from the crop.     

Enzymatic Response of the Eucalypt Defoliator Thyrinteina arnobia (Stoll) (Lepidoptera: Geometridae) to a Bis-Benzamidine Proteinase Inhibitor

Ingestion of proteinase inhibitors leads to hyperproduction of digestive proteinases, limiting the bioavailability of essential amino acids for protein synthesis, which affects insect growth and development. However, the effects of proteinase inhibitors on digestive enzymes can lead to an adaptive response by the insect. In here, we assessed the biochemical response of midgut proteinases from the eucalypt defoliator Thyrinteina arnobia (Stoll) to different concentrations of berenil, a bis-benzamidine proteinase inhibitor, on eucalyptus. Eucalyptus leaves were immersed in berenil solutions at different concentrations and fed to larvae of T. arnobia. Mortality was assessed daily. The proteolytic activity in the midgut of T. arnobia was assessed after feeding on plants sprayed with aqueous solutions of berenil, fed to fifth instars of T. arnobia for 48?h before midgut removal for enzymatic assays. Larvae of T. arnobia were able to overcome the effects of the lowest berenil concentrations by increasing their trypsin-like activity, but not as berenil concentration increased, despite the fact that the highest berenil concentration resulted in overproduction of trypsin-like proteinases. Berenil also prevented the increase of the cysteine proteinases activity in response to trypsin inhibition.     

Evaluation of the Adenanthera pavonina seed proteinase inhibitor (ApTI) as a bioinsecticidal tool with potential for the control of Diatraea saccharalis

Diatraea saccharalis, is a major sugarcane pest, causing damage to the stalks of sugarcane plants. In this study, a trypsin inhibitor (ApTI) was purified from Adenanthera pavonina seeds and was tested for its insect growth regulatory effect. ApTI showed a dose-dependent effect on average larval weight and survival. 0.1% ApTI produced approximately 67% and 50% decreases in weight and survival larval, respectively. The results from dietary utilization experiments with D. saccharalis larvae showed a reduction in the efficiency of conversion of ingested food and digested food, and an increase in approximate digestibility and metabolic cost. The level of trypsin was significantly decreased (ca. 55%) in the midgut of larvae reared on a diet containing 0.05% ApTI and the trypsin activity in ApTI-fed larvae demonstrated sensitivity to ApTI. The action of ApTI on the development of D. saccharalis larvae shows that this protein may have great toxic potential.     

Phenotypic changes and the fate of digestive enzymes during induction of amber disease in larvae of the New Zealand grass grub (Costelytra zealandica)

Amber disease of the New Zealand grass grub Costelytra zealandica (Coleoptera: Scarabaeidae) is caused by ingestion of pADAP plasmid carrying isolates of Serratia entomophila or Serratia proteamaculans (Enterobacteriaceae) and causes infected larvae to cease feeding and clear their midgut to a pale amber colour where midgut serine protease activities are virtually eliminated. Using bacterial strains and mutants expressing combinations of the anti-feeding (afp) and gut clearance (sep) gene clusters from pADAP, we manipulated the disease phenotype and demonstrated directly the relationship between gene clusters, phenotype and loss of enzyme activity. Treatment with afp-expressing strains caused cessation of feeding without gut clearance where midgut protease activity was maintained at levels similar to that of healthy larvae. Treatment with strains expressing sep-genes caused gut clearance followed by a virtual elimination of trypsin and chymotrypsin titre in the midgut indicating both the loss of pre-existing enzyme from the lumen and a failure to replenish enzyme levels in this region by secretion from the epithelium. Monitoring of enzymatic activity through the alimentary tract during expression of disease showed that loss of serine protease activity in the midgut was matched by a surge of protease activity in the hindgut and frass pellets, indicating a flushing and elimination of the midgut contents. The blocking of enzyme secretion through amber disease appears to be selective as leucine aminopeptidase and α-amylase were still detected in the midgut of diseased larvae.     

Coupling diet quality and Bowman-Birk and Kunitz-type soybean proteinase inhibitor effectiveness to Diatraea saccharalis development and mortality

We used bioassays to investigate the effect of Bowman‐Birk and Kunitz‐type soybean proteinase inhibitors on two artificial diets (diets 1 and 2) which are commonly used to feed laboratory colonies of larvae of the moth Diatraea saccharalis, monitoring food intake and utilization, and larval development and mortality. Diet 1 was less nutritious, with a low protein content and reduced mineral and essential amino acid (e.g., cysteine, lysine, and methionine) content, while diet 2 was richer and more complete. When proteinase inhibitors were incorporated into the artificial diets, the effects on larval development were significantly greater for those larvae fed diet 1, with the chronic ingestion of proteinase inhibitors reducing the level of trypsin‐like activity in the midgut of larvae fed this diet. Larvae fed diet 2 also showed a reduced level of tryptic activity in the midgut, but this was less marked than for diet 1. These results indicate that despite their inhibitory effect on midgut enzymes, the effectiveness of proteinase inhibitors is directly dependent on the quality of the diet. The different effects seen on insect biology when proteinase inhibitors are added to rich or poor diets suggests that the role of anti‐nutritional proteins in the control of insects might not be adequately addressed by bioassays based on the incorporation of inhibitors into artificial diets.     

The effects of three metabolic inhibitors on digestive proteinase production in Rhodnius prolixus Stål (Hemiptera: Reduviidae)

Mated female Rhodnius prolixus were fed citrated rabbit blood containing cordycepin, cycloheximide and 5-fluorouracil. The effect of these compounds on cathepsin B, cathepsin D and aminopeptidase levels in the posterior midgut was determined. Cycloheximide and 5-fluorouracil decreased the levels of all three proteinases in a dose-dependent manner. Cordycepin decreased the levels of cathepsin B and D but did not alter the aminopeptidase levels in a similar manner. These results indicate that after ingestion of a blood meal, production of the extracellular catheptic enzymes is dependent on both RNA and protein synthesis while production of the intracellular aminopeptidase depends on protein synthesis alone. The effects of these compounds on proteinase activity cycles also supports this conclusion.     

Ultrastructural localisation of cathepsin B in the midgut of Rhodnius prolixus Stål ( Hemiptera : Reduviidae) during blood digestion

The artificial substrate N-benzoyl-dl-arginine-β-naphthylamine, was used to localise cathepsin B in midgut cells of the haematophagous insect, Rhodnius prolixus Stål (Hemiptera : Reduviidae), during blood digestion. Cathepsin B was localised primarily in the lysosomes of cells from all 3 midgut regions and in Golgi vesicles of the digestive intestinal regions, but not in association with any other cellular structures. The timing of localisation correlated with previously described cycles of endoproteinase activity and with known ultrastructural modifications to the midgut cells. Secretory vesicles, which originated from the Golgi complexes, were present only in the intestinal regions, and in the anterior intestine, they showed a strong positive correlation (r = 0.939, P = 0.01) with post-feeding cathepsin B activity. Cathepsin B plays a major role in primary extracellular digestion of blood proteins, and is active in the midgut lumen and lysosomes rather than in association with the microvilli.     

Interactions between Bacillus thuringiensis subsp. kurstaki HD-1 and midgut bacteria in larvae of gypsy moth and spruce budworm

We examined interaction between Bacillus thuringiensis subsp. kurstaki HD-1 (Foray 48B) and larval midgut bacteria in two lepidopteran hosts, Lymantria dispar and Choristoneura fumiferana. The pathogen multiplied in either moribund (C. fumiferana) or dead (L. dispar) larvae, regardless of the presence of midgut bacteria. Inoculation of L. dispar resulted in a pronounced proliferation of enteric bacteria, which did not contribute to larval death because B. thuringiensis was able to kill larvae in absence of midgut bacteria. Sterile, aureomycin- or ampicillin-treated larvae were killed in a dose-dependent manner but there was no mortality among larvae treated with the antibiotic cocktail used by [Broderick et al., 2006] and [Broderick et al., 2009]. These results do not support an obligate role of midgut bacteria in insecticidal activity of HD-1. The outcome of experiments on the role of midgut bacteria may be more dependent on which bacterial species are dominant at the time of experimentation than on host species per se. The L. dispar cohorts used in our study had a microflora, that was dominated by Enterococcus and Staphylococcus and lacked Enterobacter. Another factor that can confound experimental results is the disk-feeding method for inoculation, which biases mortality estimates towards the least susceptible portion of the test population.     

Allatotropin Modulates Myostimulatory and Cardioacceleratory Activities in Rhodnius prolixus (Stal).

Haematophagous insects can ingest large quantities of blood in a single meal and eliminate high volumes of urine in the next few hours. This rise in diuresis is possible because the excretory activity of the Malpighian tubules is facilitated by an increase in haemolymph circulation as a result of intensification of aorta contractions combined with an increase of the anterior midgut peristaltic waves. It has been previously described that haemolymph circulation during post-prandial diuresis is stimulated by the synergistic activity of allatotropin (AT) and serotonin in the kissing bug Triatoma infestans; resulting in an increase in aorta contractions. In the same species, AT stimulates anterior midgut and rectum muscle contractions to mix urine and feces and facilitate the voiding of the rectum. Furthermore, levels of AT in midgut and Malpighian tubules increased in the afternoon when insects are getting ready for nocturnal feeding. In the present study we describe the synergistic effect of AT and serotonin increasing the frequency of contractions of the aorta in Rhodnius prolixus. The basal frequency of contractions of the aorta in the afternoon is higher that the observed during the morning, suggesting the existence of a daily rhythmic activity. The AT receptor is expressed in the rectum, midgut and dorsal vessel, three critical organs involved in post-prandial diuresis. All together these findings provide evidence that AT plays a role as a myoregulatory and cardioacceleratory peptide in R. prolixus.     

Midgut clearance and digestive enzyme levels in larvae of Attagenus megatoma following removal from food

Total protein and amino n levels in midguts of larvae of the black carpet beetle, Attagenus megatoma declined dramatically within 24 hr following removal from both the standard Purina^® diet and nutrient-treated wool. However, the secretion of proteolytic enzymes and sucrase (E.C.3.2.1.26) continued even after midgut clearance. The gradual decline in midgut digestive enzymal level observed was attributed to a reduced rate of secretion and to excretion of enzyme with the voided faeces. Although Purina-fed larvae had a 5-fold higher level of sucrase activity than the wool-fed larvae, proteolytic activity was higher in the wool-fed larvae. These results are in accord with a secretagogue mechanism as well as nutrient class specificity for the control of digestive enzymes in this species.     

The salivary and crop apyrase activity of Rhodnius prolixus

A calcium dependent apyrase activity (ATP→AMP + 2Pi) has been characterized in the salivary secretion of Rhodnius prolixus. High levels of this activity were found in the crop of all stages of larvae and the adults after a single blood or saline meal. The activity persisted for several days but was totally absent in the crop insects from which the salivary glands had been removed. The use of this activity as a saliva marker shows that the insect salivates during the whole meal and most of the saliva is ingested with the food. The physiological role of this activity is discussed. A simple method for saliva collection and a technique for the surgical ablation of the salivary glands in adult insects are described.