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.     

A physiological and biochemical model for digestion in the ectoparasitic mite,Psoroptes ovis (Acari: Psoroptidae)

Mites are an important group of arthropod pests affecting crops, animals and humans. Despite this, detailed physiological studies on these organisms remain sparse due largely to their small size. Unifying models are required to draw together the diverse information from studies on different groups and species. This paper describes a model for digestion in the parasitic mite, Psoroptes ovis, the causative agent of psoroptic mange or sheep scab disease. The limited information about this species is supplemented with data from other acarines, especially house dust mites and ticks. We review the range of enzymes and allergens found in mites and consider their possible roles in digestion in mites, generally and in particular, P. ovis. Histological studies, enzyme biochemistry and molecular biology and experimental evidence suggest that P. ovis utilises a digestive system reliant upon acid peptidases functioning in a largely intracellular environment. The actions of the digestive enzymes are supplemented by the involvement of bacteria as potential direct and indirect sources of nutrition. It is possible that some extra-corporeal digestion also takes place. The interaction of bacteria and digestive enzymes on the skin surface of the sheep may be responsible for the excessive pathological reactions evident in clinical sheep scab.     

Performance of a generalist grasshopper on a C<Subscript>3</Subscript> and a C<Subscript>4</Subscript> grass: compensation for the effects of elevated CO<Subscript>2</Subscript> on plant nutritional quality

The increasing CO₂ concentration in Earths atmosphere is expected to cause a greater decline in the nutritional quality of C₃ than C₄ plants. As a compensatory response, herbivorous insects may increase their feeding disproportionately on C₃ plants. These hypotheses were tested by growing the grasses Lolium multiflorum C₃) and Bouteloua curtipendula C₄) at ambient (370 ppm) and elevated (740 ppm) CO₂ levels in open top chambers in the field, and comparing the growth and digestive efficiencies of the generalist grasshopper Melanoplus sanguinipes on each of the four plant × CO₂ treatment combinations. As expected, the nutritional quality of the C₃ grass declined to a greater extent than did that of the C₄ grass at elevated CO₂; protein levels declined in the C₃ grass, while levels of carbohydrates (sugar, fructan and starch) increased. However, M. sanguinipes did not significantly increase its consumption rate to compensate for the lower nutritional quality of the C₃ grass grown under elevated CO₂. Instead, these grasshoppers appear to use post-ingestive mechanisms to maintain their growth rates on the C₃ grass under elevated CO₂. Consumption rates of the C₃ and C₄ grasses were also similar, demonstrating a lack of compensatory feeding on the C₄ grass. We also examined the relative efficiencies of nutrient utilization from a C₃ and C₄ grass by M. sanguinipes to test the basis for the C₄ plant avoidance hypothesis. Contrary to this hypothesis, neither protein nor sugar was digested with a lower efficiency from the C₄ grass than from the C₃ grass. A novel finding of this study is that fructan, a potentially large carbohydrate source in C₃ grasses, is utilized by grasshoppers. Based on the higher nutrient levels in the C₃ grass and the better growth performance of M. sanguinipes on this grass at both CO₂ levels, we conclude that C₃ grasses are likely to remain better host plants than C₄ grasses in future CO₂ conditions.     

Seasonal variations in the levels of polyhydroxysteroids and related glycosides in the digestive tissues of the starfish Patiria (Asterina) pectinifera

Seasonal variations in the levels of polar steroids including polyhydroxylated steroids and related glycosides in digestive organs of the starfish Patiria (=Asterina) pectinifera have been studied. The concentration of polar steroids is related to the annual reproductive cycle of the starfish and periods of active feeding. Two peaks in concentrations of polar steroids in pyloric caeca and stomach were found, the first in winter during reorganization and the second in summer during intensive gametogenesis before spawning. Probable biological functions of polyhydroxysteroids and related glycosides are discussed. The data support the hypothesis these compounds are involved in digestion in the starfish.     

Midgut exopeptidase activities in Aedes aegypti are induced by blood feeding

Midgut extracts from Aedes aegypti females exhibited hydrolytic activities against synthetic substrates for carboxypeptidase A, carboxyopeptidase B and leucine-aminopeptidase. The three activities showed a broad pH optimum, with maximum activities at pH between 6.5 and 8.5. Enzymatic activities were further characterized by testing the effects of a variety of protease inhibitors. Captopril and 1-10-phenantroline inhibited the activities of carboxypeptidases A and B, while leuhistin, amastatin and bestatin inhibited aminopeptidase activity. Exopeptidase activities were induced by a blood meal and the highest activities were found during the peak of trypsin activity, about 20-24 h after feeding. An amino acid meal failed to induce significant increases in any of the three exopeptidase activities. The amounts of exopeptidase activities induced were proportional to the protein concentration of the meal. The addition of soy-trypsin inhibitor to the protein meal blocked the post-feeding induction of exopeptidases. The features of the induction of synthesis of the three exopeptidase activities resembled the induction of synthesis of late trypsin during the second phase of digestion.     

Short-term adaptation of digestive processes in the cockle Cerastoderma edule exposed to different food quantity and quality

Feeding and digestive parameters were analysed in cockles Cerastoderma edule fed for 3 days on two foods of different qualities, both foods given in two different concentrations. With low quality food, gut content was found to increase with ingestion rate. Such increased capacity of the gut to allocate food precludes negative effects upon throughput time, and so absorption efficiency remained nearly constant at the two food concentrations. With high quality food, gut content remained at high constant values and consequently enhancement of food ingestion rate with a high food ration leads to a significant reduction in throughput time, resulting in lower absorption efficiencies. Significantly higher levels of amylases and cellulases have been found within the digestive gland of cockles fed high quality diets. Coincidentally, absorption of carbohydrates is increased and absorption of lipids decreased in such diets as compared to low quality diets. Implications of the positive correlation between digestive enzyme activity and food quality are discussed in relation to the role that both digestive investments and endogenous faecal losses play in digestive processes. Results obtained in this study indicate that investments in the form of digestive enzymes are a key factor in the functional response of cockles to short-term variations in the food regime. Accepted: 13 September 1997     

An analysis of chewed food particle size and its relationship to molar structure in the primatesCheirogaleus medius andGalago senegalensis and the insectivoranTupaia glis

The chewed food particle size and shearing capacity of the lower molars of two primate species, the fat-tailed dwarf lemur,Cheirogaleus medius and the bushbabyGalago senegalensis, and an insectivoran, the tree shrew,Tupaia glis, were compared. Differences in the shearing design of the lower molars correlate strongly with the chewed food particle size in these species: the greater the shearing capacity, the smaller the chewed food particles. These three species are of comparable size but differ greatly in diet in the wild.C. medius primarily eats fruit and nectar, whileG. senegalensis andT. glis are largely insect-eaters. The lower molars ofG. senegalensis andT. glis show a much greater shearing capacity than do those ofC. medius. The average length of chewed food particles ofC. medius is significantly larger than that ofG. senegalensis, while that ofT. glis is intermediate between the two primates but is closer to that ofG. senegalensis. Our findings that insect-eating species grind their food more finely than do fruit- and resin-eating species can be correlated with digestibility of foods: finely chewing foods such as fruits which are low in relatively undigestible cell wall components would not greatly improve their digestibility, so a highly efficient food processing apparatus would be less important to the animal's survival. Insect-eaters much more finely chew their foods, implying that there is some constituent of insect bodies difficult to digest, and that grinding increases its digestibility. We suggest that this constituent is chitin.     

Isolation and Characterization of Total DNA from Several Endangered Species and Their Allies

A low pH extraction medium with high salts, which avoids ionization and subsequent oxidation of phenolic compounds during tissue grinding and precipitation of large amounts of materials, were successfully used to obtain total DNA from Cathaya argyrophylla Chun et Kuang, Paeonia suffruticosa var. spontanea, Cimici fuga nanchuanensis Hsiao, Adenophora potaninii (Congeneric species with A. lobophylla) etc. The DNA yields, quality and purity were characterized. These isolated DNA could be used directly for RFLP and RAPD analysis which are useful as molecular genetic markers without sedimentation in cesium chloride gradient or column chromatography. A fast, inexpensive and reliable procedure has been developed for detecting the genetic diversity of endangered plants.     

Digestive enzymes of leaf-cutting ants, Acromyrmex subterraneus (Hymenoptera: Formicidae: Attini): distribution in the gut of adult workers and partial characterization

Enzyme activities associated with the labial glands, midgut and rectum of adult Acromyrmex subterraneus were investigated in order to understand their role in digestion of plant and fungal material. High chitinolytic activity was detected in the labial glands, indicating a possible role in the degradation of fungus ingested by the ants. Chitinolytic activity seen in other compartments of the alimentary canal probably originated in the labial glands. The highest activity detected in the midgut was for alpha-glucosidase, which was considered to be of insect origin due to its association with midgut epithelium and it is probably involved in glucose assimilation from nutrient sources such as maltose and sucrose present in plant material. A large range of enzyme activities were detected in the rectal lumen contents, and as in the midgut the highest values were for alpha-glucosidase activity. The absence of activity associated with the epithelium, in the particulate fraction, indicates that the rectal epithelium does not have a secretory function. The detection of enzymes in the rectal lumen contents, which were not detected in the midgut lumen contents, indicates that the rectum acts as a reservoir, accumulating enzymes. The major digestive enzymes were partially characterized using hydrophobic interaction chromatography, gel filtration and SDS-PAGE. The pH of the adult intestinal tract and flow rate of dye through the tract was investigated. A gradual acidification of the intestinal tract was noted commencing with the crop (pH 6-8.2) and terminating with the rectum (pH 3-5). The flow of dye through the different compartments of the tract showed a rapid fill time for all the gut compartments and a short residence time in the crop. In all other compartments, the dye remained detectable for 10 days or longer.     

Rapid and efficient subcloning of DNA without dephosphorylation or gel electrophoresis

Conventional subcloning into plasmid vectors often involves dephosphorylation, gel electrophoresis, DNA extraction, and purification to isolate the target insert and the cleaved plasmid. This is not only time-consuming but very often problematic. We have developed strategies that can circumvent these steps by mixing digested donor and recipient plasmids together for ligation. These strategies capitalizes on: (1) the ability to enhance the ligation efficiency of desired DNA fragments into the target vector by the generation and removal of small (<50 bp) fragments from nontarget DNA using peripheral restriction sites and spin column technology and (2) the elimination of undesired ligation products after ligation by using the Lac Z gene, differences in antibiotic resistance among plasmid vectors, and unique restriction sites situated in nontarget DNA fragments.