Characterization of the Mamestra configurata (Lepidoptera: Noctuidae) larval midgut protease complement and adaptation to feeding on artificial diet,Brassica species,and protease inhibitor

The midgut protease profiles from 5th instar Mamestra configurata larvae fed various diets (standard artificial diet, low protein diet, low protein diet with soybean trypsin inhibitor [SBTI], or Brassica napus) were characterized by one‐dimensional enzymography in gelatin gels. The gut protease profile of larvae fed B. napus possessed protease activities of molecular masses of approximately 33 and 55 kDa, which were not present in the guts of larvae fed artificial diet. Similarly, larvae fed artificial diet had protease activities of molecular masses of approximately 21, 30, and 100 kDa that were absent in larvae fed B. napus. Protease profiles changed within 12 to 24 h after switching larvae from artificial diet to plant diet and vice versa. The gut protease profiles from larvae fed various other brassicaceous species and lines having different secondary metabolite profiles did not differ despite significant differences in larval growth rates on the different host plants. Genes encoding putative digestive proteolytic enzymes, including four carboxypeptidases, five aminopeptidases, and 48 serine proteases, were identified in cDNA libraries from 4th instar M. configurata midgut tissue. Many of the protease‐encoding genes were expressed at similar levels on all diets; however, three chymoptrypsin‐like genes (McSP23, McSP27, and McSP37) were expressed at much higher levels on standard artificial diet and diet containing SBTI as was the trypsin‐like gene McSP34. The expression of the trypsin‐like gene McSP50 was highest on B. napus. The adaptation of M. configurata digestive biochemistry to different diets is discussed in the context of the flexibility of polyphagous insects to changing diet sources. Published 2010 Wiley Periodicals, Inc.     

Restricted diet breadth of the larvae of Antheraea assamensis and the role of the labrum‐epipharynx and galeal sensilla

The silkworm, Antheraea assamensis Helfer (Lepidoptera: Saturniidae), grows primarily on Persea bombycina and Litsea polyantha. To understand if the restricted diet breadth is due to the specific role of gustatory sensilla of the larvae of A. assamensis, the same fifth instar larvae retaining only labrum‐epipharynx or galeal sensilla were subjected to food choice tests. The foods used were leaves of two host‐plant and two non‐host‐plant species. Mean per cent consumption and per cent of choosing larvae were used as parameters for drawing conclusions. The finding indicated involvement of the labrum‐epipharynx for acceptance and galeal sensilla for rejection of a non‐host‐plant species. Scanning electron microscope studies revealed the presence of two sensilla on the galea, one lateral and one medial sensilla styloconicum and two gustatory sensilla in the epipharynx of A. assamensis. The study revealed the key role of galeal sensilla in the restrictive diet‐breadth of A. assamensis.     

Midgut proteases from Mamestra configurata (Lepidoptera: Noctuidae) larvae: characterization,cDNA cloning,and expressed sequence tag analysis

The activities of digestive protease within the midgut of Mamestra configurata (bertha armyworm) larvae were examined using specific substrates and protease inhibitors. The bulk of the activity was associated with serine proteases comprising trypsin-, chymotrypsin-, and elastase-like enzymes. At least 10-15 serine protease isozymes were detected using one-dimension gelatin gel electrophoresis. Cysteine or aspartic protease activities were not present; however, amino- and carboxypeptidase activities were associated with the midgut extract. Midgut proteases were active in the pH range of 5.0-12.0 with peaks at pH 7.5 and 11.0. In general, the middle region of the midgut exhibited a higher pH (approximately 8.0) than either the posterior or anterior regions (approximately 7.3-7.7). Moulting larvae possessed a neutral gut pH that was 0.5-1.5 units below that of feeding larvae. Degenerate PCR and expressed sequence tag (EST)-based approaches were used to isolate 30 distinct serine protease encoding cDNAs from a midgut-specific cDNA library including 8 putative trypsins, 9 chymotrypsins, 1 elastase, and 12 whose potential activities could not be determined. cDNAs encoding three amino- and two carboxypeptidases were also identified. Larvae feeding upon artificial diet containing 0.2% soybean trypsin inhibitor experienced a significant delay in development.     

Resistance to Plant Invasion? A Native Specialist Herbivore Shows Preference for and Higher Fitness on an Introduced Host

The response of native herbivores to the introduction of a new plant to the community has important implications for plant invasion. Under the Enemy Release Hypothesis introduced species become invasive because of reduced enemy control in the new range, while under the New Association Hypothesis introduced species lack effective defenses against native enemies because they do not share an evolutionary history. I tested the response of a native South-American specialist herbivore Utetheisa ornatrix (Lepidoptera: Arctiidae) to a native (Crotalaria incana) and an introduced host (Crotalaria pallida) (Fabaceae: Papilionoideae). I compared seed predation rates between the two hosts in the field, and I tested preference and performance traits with common garden experiments. Utetheisa ornatrix caused much higher seed predation rates on the introduced host than on the native host. Females also preferred to oviposit on the introduced over the native host. Additionally, larvae feeding on the introduced host had higher fitness (higher pupal weight) than larvae feeding on the native host. I discuss how the response of this specialist herbivore to this introduced host plant contradicts the predictions of the Enemy Release Hypothesis and support the New Association Hypothesis. This study shows that the New Association Hypothesis can also be true for specialist herbivores.     

Archives of insect biochemistry and physiology guide for authors

Several carbohydrases and glycosidases from the alimentary cancal and/or salivary glands of feeding larvae of mayetiola destructor have been identified. Pectinase activity was identified in the midgut and may be present in the salivary glands. No endocellulase activity was found in larvae; however, hemicellulase activity was detected in extract of larvae. Amylase activity was present in midguts from feeding larvae and at a low level in extract of salivary glands. Amylases detected in the midgut showed mobilities during polyacrylamide gel electrophoresis similar to the two major amylases in tissues of the insect's host plant. The possibility exists that Hessian fly larvae utilize amylases obtained from their host plant in the digestion of starch. The major glycosidases detected in the midgut lumen of larve were: α-D-glucosidase and α-D-and β-D-galactosidase. The role of these enzymes in the feeding process of Hessian fly larvae is discussed as well as their potential role in feeding damage to wheat.     

Identification and characterization of midgut proteases in <Emphasis Type="Italic">Achaea janata</Emphasis> and their implications

Insect midgut proteases are excellent targets for insecticidal agents such as Bacillus thuringiensis Cry toxins and protease inhibitors. The midgut proteases of Achaea janata have been characterized and Casein zymograms indicated at least five distinct activities corresponding to approx 17, 20, 29 and 80, and 90 kDa. Using a combination of synthetic substrates and specific inhibitors in casein zymograms, photometric assays and activity blots, three trypsin-like and one elastase-like serine proteases were identified but no chymotrypsin-like activity. Various proteinase inhibitors displayed differential inhibitory effects towards the midgut proteases.     

Larval food affects oviposition preference,female fecundity and offspring survival in Yponomeuta evonymellus

1. Yponomeuta evonymellus is a monophagous moth that feeds on Prunus padus which is native to Europe. In recent years, larval feeding and egg clusters have also been observed on non‐native Prunus serotina plants; however, survival of larvae on this new host is very low. 2. The objective of the present study was to determine how the feeding of larvae on each of the two host plants impacts oviposition, offspring survival and fecundity in Y. evonymellus. Our hypothesis was that, under controlled conditions, females will lay eggs on the host on which they fed as larvae. We also hypothesised that the lower survival of young larvae feeding on P. serotina was due to the smaller buds and leaves present in this species, relative to those of P. padus. 3. A dual‐choice experiment conducted under laboratory conditions demonstrated that females preferentially chose to oviposit on the plant species on which they fed as larvae. In the experiment, potential fecundity and offspring survival were significantly higher on P. padus than on P. serotina. The reduced performance of Y. evonymellus on P. serotina was correlated with a smaller bud mass and volume, lower leaf mass and surface area, and difficulty in constructing a protective tent against unfavourable weather conditions. 4. In summary, the identity of the host plant species during larval feeding determines adult oviposition preference for that host species. The survival of larvae on P. serotina growing in the nature is low, but for phenology‐related reasons.     

In vitro and in vivo application of RNA interference for targeting genes involved in peritrophic matrix synthesis in a lepidopteran system

Abstract The midgut of most insects is lined with a semipermeable acellular tube, the peritrophic matrix (PM), composed of chitin and proteins. Although various genes encoding PM proteins have been characterized, our understanding of their roles in PM structure and function is very limited. One promising approach for obtaining functional information is RNA interference, which has been used to reduce the levels of specific mRNAs using double‐stranded RNAs administered to larvae by either injection or feeding. Although this method is well documented in dipterans and coleopterans, reports of its success in lepidopterans are varied. In the current study, the silencing midgut genes encoding PM proteins (insect intestinal mucin 1, insect intestinal mucin 4, PM protein 1) and the chitin biosynthetic or modifying enzymes (chitin synthase‐B and chitin deacetylase 1) in a noctuid lepidopteran, Mamestra configurata, was examined in vitro and in vivo. In vitro studies in primary midgut epithelial cell preparations revealed an acute and rapid silencing (by 24 h) for the gene encoding chitin deacetylase 1 and a slower rate of silencing (by 72 h) for the gene encoding PM protein 1. Genes encoding insect intestinal mucins were slightly silenced by 72 h, whereas no silencing was detected for the gene encoding chitin synthase‐B. In vivo experiments focused on chitin deacetylase 1, as the gene was silenced to the greatest extent in vitro. Continuous feeding of neonates and fourth instar larvae with double‐stranded RNA resulted in silencing of chitin deacetylase 1 by 24 and 36 h, respectively. Feeding a single dose to neonates also resulted in silencing by 24 h. The current study demonstrates that genes encoding PM proteins can be silenced and outlines conditions for RNA interference by per os feeding in lepidopterans.     

Recombinant Expression, Purification, and Characterization of Three Isoenzymes of Aspartate Aminotransferase fromArabidopsis thaliana

Five different genes encoding isoenzymes of aspartate aminotransferase (AAT) have been identified in the plantArabidopsis thaliana.cDNA sequences encoding three of these AAT isoenzymes,asp1(mitochondrial),asp2(cytosolic), andasp5(plastid), were manipulated into bacterial expression vectors and the recombinant proteins expressed were purified from liquid culture using conventional methods. Yields of the purified isoenzymes varied from 11.5 mg/g wet wt cells (AAT5) to 0.95 mg/g wet wt cells (AAT2), an improvement of more than 1000-fold over typical yields of native isoenzymes obtained from plant tissues of other species. Analysis of the recombinant proteins on denaturing PAGE gels indicated subunitM_rs of between 44 and 45 K. Kinetic parameters (K_mandk_cat) obtained for all four substrates (aspartate, α-ketoglutarate, glutamate, and oxaloacetate) were consistent with values obtained for native AAT isoenzymes from other plant species. Further characterization of the purified recombinant enzymes alongside native enzymes fromA. thalianaleaf tissue on AAT activity gels confirmed the identity ofasp1andasp2as the mitochondrial and cytosolic AAT genes but indicated thatasp5may encode an amyloplastic rather than the chloroplastic enzyme.     

Maintenance of narrow diet breadth in the monarch butterfly caterpillar: response to various plant species and chemicals

In order to better understand the maintenance of a fairly narrow diet breadth in monarch butterfly larvae, Danaus plexippus L. (Lepidoptera: Nymphalidae: Danainae), we measured feeding preference and survival on host and non-host plant species, and sensitivity to host and non-host plant chemicals. For the plant species tested, a hierarchy of feeding preferences was observed; only plants from the Asclepiadaceae were more or equally preferred to Asclepias curassavica, the common control. The feeding preferences among plant species within the Asclepiadaceae are similar to published mean cardenolide concentrations. However, since cardenolide data were not collected from individual plants tested, definitive conclusions regarding cardenolide concentrations and plant acceptability cannot be made. Although several non-Asclepiadaceae were eaten in small quantities, all were less preferred to A. curassavica. Additionally, these non-Asclepiadaceae do not support continued feeding, development, and survival of first and fifth-instar larvae. Preference for a host versus a non-host (A. curassavica versus Vinca rosea) increased for A. curassavica reared larvae as compared to diet-reared larvae suggesting plasticity in larval food preferences. Furthermore, host species were significantly preferred over non-host plant species in bioassays using a host plant or sucrose as a common control. Larval responses to pure chemicals were examined in order to determine if host and non-host chemicals stimulate or deter feeding in monarch larvae. We found that larvae were stimulated to feed by some ubiquitous plant chemicals, such as sucrose, inositol, and rutin. In contrast, several non-host plant chemicals deterred feeding: caffeine, apocynin, gossypol, tomatine, atropine, quercitrin, and sinigrin. Additionally the cardenolides digitoxin and ouabain, which are not in milkweed plants, were neutral in their influence on feeding. Another non-milkweed cardenolide, cymarin, significantly deterred feeding. Extracts of A. curassavica leaves were tested in bioassays to determine which components of the leaf stimulate feeding. Both an ethanol extract of whole leaves and a hexane leaf-surface extract are phagostimulatory, suggesting the involvement of both polar and non-polar plant compounds. These data suggest that the host range of D. plexippus larvae is maintained by both feeding stimulatory and deterrent chemicals in host and non-host plants.     

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.     

Hydrolysis of solubilized fibroin and silk proteins in the midgut of the pharate adult of Bombyx mori

Midgut protease in the pharate adult hydrolysed native silk proteins and solubilized fibroin by ethylenediamine cupric hydroxide or lithium bromide. By agar gel electrophoresis one to three protease bands moving toward the anode were detected, and the number of bands and the electrophoretic mobility were different among the various strains. Optimal activity of the enzyme was at about pH 8·3. The protease activity was found to decrease in higher concentrations of the substrates. One peak of protease activity was seen in Sepharose 6B chromatography, and the elution pattern and peak position of the enzyme were very similar to those of protease activity with casein. In DEAE-cellulose chromatography, the peak of activity for casein overlapped but did not coincide with a broad peak of protease hydrolysing native silk proteins. The results obtained support the assumption that the midgut protease in the pharate adult is one of the sources of the cocoon-digesting enzyme.     

A chymotrypsin-like serine protease cDNA involved in food protein digestion in the common cutworm, Spodoptera litura: Cloning, characterization, developmental and induced expression patterns, and localization

A full-length cDNA (Slctlp2) encoding a chymotrypsin-like serine protease was cloned from Spodoptera litura. This cDNA encoded a putative serine protease with a predicted molecular mass of 30.6 kDa, which contained a serine protease catalytic motif GDSGGPL. Temporal and spatial expression of Slctlp2 mRNA and protein detected by Northern blotting, RT-PCR, qPCR and Western blotting analyses revealed that both Slctlp2 mRNA and protein were mainly present in the foregut and midgut of the 5th and 6th instar larvae during the feeding stages. In situ hybridization and immunohistochemistry confirmed that both Slctlp2 mRNA and protein were predominately present in the midgut. Expression of the gene was not induced by bacterial infection. Juvenile hormone III induced the gene expression, while 20-hydroxyecdysone had no impact on the expression. The expression of Slctlp2 mRNA and protein was down-regulated by starvation but up-regulated by re-feeding. The SlCTLP2 protein was detected in the lumen residues of the anterior, middle and posterior midgut and feces of the feeding 6th instar larvae, suggesting that it was secreted from the epithelium into the lumen of the gut. The results suggest that this Slctlp2 gene may be involved in digestive process of food proteins during the feeding stages of the larval development.     

Protein digestion in larvae of the red oak borer Enaphalodes rufulus

Abstract In the Ozark Mountains of the U.S.A., the red oak borer Enaphalodes rufulus contributes to the destruction of red oaks. To understand nutrient digestion in E. rufulus larvae, digestive proteinases are compared in both larvae fed heartwood phloem and those transferred to artificial diet. The pH of gut extracts is approximately 6.3 in the midgut and foregut and decreases to 5.5 in the hindgut region. The hydrolysis of casein by midgut extracts from E. rufulus larvae fed either artificial diet or phloem from tree sections increases in buffers greater than pH 6.19, with maximum hydrolysis being observed at pH 10.1. Casein zymogram analysis reveals two major proteinase activities in larval midgut extracts of diet‐fed larvae, with molecular masses of approximately 25 and 40–60 kDa, whereas phloem‐fed larvae have proteinase activities corresponding to 40, 45, 60, 80 and >100 kDa. Substrate analysis indicates at least one major trypsin‐like activity in both gut extracts with a molecular mass of >100 kDa, but two chymotrypsin‐like activities of approximately 25 and >200 kDa are found only in diet‐fed larvae. Inhibitors of serine proteinases are most effective in reducing the general proteolytic activity of midgut extracts from larvae fed either food source. The data indicate that serine proteinase inhibitors have the potential to reduce E. rufulus larval damage to oaks. In particular, transgenic technologies incoporating trypsin inhibitors may be effective in reducing protein digestion in phloem‐feeding larvae.     

Ultrastructural changes in the midguts of Hessian fly larvae feeding on resistant wheat

The focus of the present study was to compare ultrastructure in the midguts of larvae of the Hessian fly, Mayetiola destructor (Say), under different feeding regimens. Larvae were either fed on Hessian fly-resistant or -susceptible wheat, and each group was compared to starved larvae. Within 3 h of larval Hessian fly feeding on resistant wheat, midgut microvilli were disrupted, and after 6 h, microvilli were absent. The disruption in microvilli in larvae feeding on resistant wheat were similar to those reported for midgut microvilli of European corn borer, Ostrinia nubilasis (Hubner), larvae fed a diet containing wheat germ agglutinin. Results from the present ultrastructural study, coupled with previous studies documenting expression of genes encoding lectin and lectin-like proteins is rapidly up-regulated in resistant wheat to larval Hessian fly, are indications that the midgut is a target of plant resistance compounds. In addition, the midgut of the larval Hessian fly is apparently unique among other dipterans in that no peritrophic membrane was observed. Ultrastructural changes in the midgut are discussed from the prospective of their potential affects on the gut physiology of Hessian fly larvae and the mechanism of antibiosis in the resistance of wheat to Hessian fly attack.     

Host suitability and diet mixing influence activities of detoxification enzymes in adult Japanese beetles

Induction of cytochrome P450, glutathione S transferase (GST), and carboxylesterase (CoE) activity was measured in guts of the scarab Popillia japonica Newman, after consumption of single or mixed plant diets of previously ranked preferred (rose, Virginia creeper, crape myrtle and sassafras) or non-preferred hosts (boxelder, riverbirch and red oak). The goal of this study was to quantify activities of P450, GST and CoE enzymes in the midgut of adult P. japonica using multiple substrates in response to host plant suitability (preferred host vs non-preferred hosts), and single and mixed diets. Non-preferred hosts were only sparingly fed upon, and as a group induced higher activities of P450, GST and CoE than did preferred hosts. However, enzyme activities for some individual plant species were similar across categories of host suitability. Similarly, beetles tended to have greater enzyme activities after feeding on a mixture of plants compared to a single plant type, but mixing per se does not seem as important as the species represented in the mix. Induction of detoxification enzymes on non-preferred hosts, or when switching between hosts, may explain, in part, the perceived feeding preferences of this polyphagous insect. The potential consequences of induced enzyme activities on the ecology of adult Japanese beetles are discussed.     

Life history and host range of Sauris nr. purpurotincta,an unsuitable biological control agent for Chinese tallowtree

Foreign surveys in China discovered a defoliating insect species feeding on the leaves of Chinese tallowtree (Triadica sebifera), an invasive weed of the southeastern U.S.A. The life history of this species, Sauris nr. purpurotincta (Lepidoptera: Geometridae), was examined and larval no-choice and adult multiple-choice host range tests were conducted in quarantine to evaluate their suitability for biological control of Chinese tallowtree. The results indicated that the larvae have five instars and require approximately 22 days to complete development to the adult stage. Host range tests indicated that the larvae could not feed and complete development on most species tested. However, 40% of the larvae survived when fed leaves of Hippomane mancinella, a state-listed endangered species in Florida, and all larvae survived when fed Morella cerifera, a common native species of the southeastern U.S.A. Multiple-choice oviposition tests indicated eggs were laid on leaves of both a south Florida native plant Gymnanthes lucida and Chinese tallowtree. Considering this broad host range, this species will not be considered further for biological control of Chinese tallowtree in the U.S.A.     

Laboratory host range testing of Lilioceris sp. near impressa (Coleoptera: Chrysomelidae) – a potential biological control agent of air potato,Dioscorea bulbifera (Dioscoreaceae)

Air potato, Dioscorea bulbifera, is an invasive, herbaceous, climbing vine, which dominates invaded native vegetation in Florida. The fortuitous discovery of Lilioceris sp. near impressa defoliating D. bulbifera vines and feeding on the bulbils (aerial tubers) in the Katmandu Valley of Nepal initiated a project to assess the potential of this leaf beetle for biological control of air potato in Florida. Quarantine host specificity tests were conducted on 41 plant species in 24 families and 13 orders, with 26 species outside of the Dioscoreaceae and 15 species within the Dioscoreaceae. Adults test fed (nibbled) on 4/12 of tested Dioscorea species, but no larval feeding or development occurred on any plant other than the target, D. bulbifera. The larvae feed gregariously and quickly skeletonize offered leaves of air potato. Air potato bulbils that received any feeding damage to the primary meristematic region did not sprout. The ability of the beetle larvae and adults to feed on the bulbils is important because in Florida, the plant rarely flowers or produces fruit, so these aerial tubers are the primary means of persistence and spread. The adults can live for several months without food. This extremely specialized herbivore from part of the weed's native range appears to have great promise as a biological control of air potato.     

Control of protease secretion in the intestine of fifth instar larvae of Rhodnius prolixus.

Groups of four Rhodnius prolixus larvae in the 5th-instar received no food or were given once only through a special feeder, similar weights of food solutions. These were either protein solutions (whole human blood, its plasma or erythrocytes, egg albumin, bovine serum albumin, haemoglobin) or non-protein solutions (saline, casein hydrolysate, dextran, maltose). Following feeding, the protein content and caseinolytic activity of the larval midgut homogenates, determined between the 3rd and 14th days, were practically the same in starved larvae and in larvae receiving non-protein solutions. In the protein-fed larvae the protein content and the specific protease activity increased. These findings exclude neurosecretory control and indicate that ingested protein stimulate the proteolytic activity of R. prolixus midgut.