Allen Carson Cohen

Digestive Enzymes and Stylet Morphology of Deraeocoris nebulosus (Hemiptera: Miridae), a Predacious Plant Bug

Abstract Mixed-feeding habits, such as zoophytophagy, make the ecological roles of many species of insects, especially hemipterans, difficult to assess. To understand the feeding adaptations of the predacious plant bug Deraeocoris nebulosus (Uhler), the digestive enzymes from the salivary glands and anterior midgut were analyzed, and the mouthpart stylets were investigated with scanning electron microscopy. Evidence of trypsin-like enzyme, α-glucosidase, and pectinase were found in the salivary glands. Low levels of trypsin-like, chymotrypsin-like, elastase-like, and pectinase activity, with high levels of α-amylase and α-glucosidase activity, were found in the anterior midgut. The insect’s right maxillary stylet has two rows of at least six recurved barbs on the inner surface pointing away from the head. This plant bug is equipped mainly for zoophagy but has enzymes that would allow some degree of phytophagy.

Partial characterization of α-amylase in the salivary glands of Lygus hesperus and L. lineolaris

Abstract The α-amylases in the salivary glands of Lygus hesperus Knight and L. lineolaris (Palisot de Beauvois) were isolated and purified by ion exchange chromatography, and by isoelectric focusing, respectively. The α-amylase from L. hesperus had an isoelectric point (p I ) of 6.25, and a pH optimum of 6.5. The specific activity of α-amylases in the salivary glands of L. hesperus was 1.2 U/mg/ml. The α- amylase from L. lineolaris had a p I of 6.54, and a pH optimum of 6.5. The specific activity of α-amylase from L. lineolaris was 1.7 U/mg/ml. The activity of α-amylase in both species was significantly inhibited by α-amylase inhibitor from wheat and also by EDTA and SDS. Sodium chloride enhanced α-amylase activity for both species. The enzyme characteristics and relative activities are discussed in the context of differences phytophagous versus zoophagous habits in these two congeneric species.

New oligidic production diet for Lygus hesperus Knight and L. lineolaris (Palisot de Beauvois).

A new oligidic (undefined) diet for rearing Lygus hesperus Knight (Heteroptera: Miridae) and L. lineolaris (Palisot de Beauvois) is described. The diet (referred to as Nl diet) is a semisolid slurry that accommodates the solid-to-liquid feeding habits of Lygus spp. The Nl diet consists of an “entomophage component” (cooked, whole chicken eggs, chicken egg yolks, sugar, and yeast) combined with plant components (soy bean flour, wheat germ, lima bean meal, and soy lecithin). Biological fitness estimates for L. hesperus indicated that mean biomass production per cage, adult wet and dry weights, survival to the adult stage, and egg production were significantly greater for the Nl diet than for the existing standard, Debolt (1982) diet. The ingredients in the Nl diet cost about ⅛ those in the Debolt diet, and preparation requires less than ½ of the labor. The cost of diet for production per 1000 eggs was approximately

Comparison of α-amylase and protease activities of a zoophytophagous and two phytozoophagous Heteroptera ☆

0.004 compared to

Partial characterization of trypsin-like protease and molecular cloning of a trypsin-like precursor cDNA in salivary glands of Lygus lineolaris.

0.04 for an equal number of eggs from Debolt diet. Recent work, started a...

Molecular cloning and partial characterization of a trypsin-like protein in salivary glands of Lygus hesperus (Hemiptera: Miridae)

To better understand the nature of facultative phytophagy in the zoophytophagous Geocoris punctipes (Say), and facultative zoophagy in phytozoophagous Lygus hesperus (Knight) and Lygus lineolaris (Palisot de Beauvois), we compared the activities of both the starch digesting enzyme alpha-amylase and of general proteases in these species. The alpha-amylases and proteases were demonstrated in L. hesperus, L. lineolaris and G. punctipes. The presence of alpha-amylase in the salivary gland complexes of G. punctipes indicates a disposition of this species toward utilization of nutrients that can be derived only from plants, either directly from ingestion of plant macromolecules or from second-hand ingestion of plant material from the digestive system of their prey. The alpha-amylase activity in G. punctipes was much less than those of phytozoophagous L. hesperus and L. lineolaris. The relative importance of amylolytic activity and proteolytic activity is also discussed.

Induction of Elastase in a Zoophytophagous Heteropteran, Lygus hesperus (Hemiptera: Miridae)

Based on substrate specificity, an alkaline pH optimum, sensitivity to selected proteinase inhibitors, and molecular analysis, we provide evidence for the presence of a trypsin-like serine proteinase in the salivary gland complex (SGC) of the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois) (Heteroptera: Miridae). The predominant activity in extracts of the SGC against N(2)-benzoyl-L-arginine-p-nitroanilide (L-BApNA) was at pH 10, but a minor peak of activity also occurred at pH 5. The major BApNAase activity focused at 10.4 during preparative isoelectric focusing and was eluted with an apparent molecular weight of 23,000 from a calibrated gel filtration column. The BApNAase fraction gave a single major band when analyzed on a casein zymogram. The activity was completely suppressed by the serine protease inhibitors, phenylmethylsulfonyl fluoride (PMSF) and lima bean trypsin inhibitor. A cDNA coding for a trypsin-like protein in the salivary glands of L. lineolaris was cloned and sequenced. The 971bp cDNA contained an 873-nucleotide open reading frame encoding a 291-amino acid trypsin precursor. The encoded protein included amino acid sequence motifs that are conserved with four homologous serine proteases from other insects. Typical features of the putative trypsin-like protein from L. lineolaris included the serine protease active site (His(89), Asp(139), Ser(229)), conserved cysteine residues for disulfide bridges, the residues (Asp(223), Gly(252), Gly(262)) that determine trypsin specificity, and both zymogen signal and activation peptides. Cloning and sequencing of a trypsin-like precursor cDNA provided additional direct evidence for trypsin like enzymes in the salivary glands of L. lineolaris.

Morphology of the Alimentary Canal of Chrysoperla rufilabris (Neuroptera: Chrysopidae) Adults in Relation to Microbial Symbionts

Trypsin-like enzymes from the salivary gland complex (SGC) of Lygus hesperus Knight were partially purified by preparative isoelectric focusing (IEF). Enzyme active against Nalpha-benzoyl-L-arginine-p-nitroanilide (BApNA) focused at approximately pH 10 during IEF. This alkaline fraction gave a single activity band when analyzed with casein zymograms. The serine proteinase inhibitors, phenylmethylsulfonyl fluoride (PMSF) and lima bean trypsin inhibitor, completely inhibited or suppressed the caseinolytic activity in the crude salivary gland extract as well as the IEF-purified sample. Chicken egg white trypsin inhibitor also inhibited the IEF-purified sample but was not effective against a major caseinolytic band in the crude salivary gland extract. These data indicated the presence of serine proteinases in the SGC of L. hesperus. Cloning and sequencing of a trypsin-like precursor cDNA provided additional direct evidence for serine proteinases in L. hesperus. The encoded trypsin-like protein included amino acid sequence motifs, which are conserved with five homologous serine proteinases from other insects. Typical features of the putative trypsin-like protein from L. hesperus included residues in the serine proteinase active site (His(89), Asp(139), Ser(229)), conserved cysteine residues for disulfide bridges, residues (Asp(223), Gly(252), Gly(262)) that determine trypsin specificity, and both zymogen signal and activation peptides.

Stylet Bundle Morphology and Trophically Related Enzymes of the Hemlock Woolly Adelgid (Hemiptera: Adelgidae)

Abstract One potential biochemical adaptation to mixed feeding habits, or zoophytophagy, is enzyme induction/enzyme repression. We investigated the potential of the highly polyphagous mirid Lygus hesperus Knight to increase its production of the specialized proteolytic enzyme, elastase (E. C. 3. 4. 31.36). Efforts to induce elastase production were made by feeding L. hesperus on an artificial diet spiked with elastin. Short-term (less than one generation) and long-term (three generations) tests were conducted to determine the effect of acute versus chronic exposure to elastin, a protein that may be present in extra cellular matrix of prey. Elastase activity was much greater in the salivary gland complex (SGC) of individuals fed elastin-containing diet than it was in those fed the control diet. The elevated elastase activity in the elastin treatment group indicated the inducibility of this proteolytic enzyme, especially in the SGC. The results also indicated that the SGC, rather than the gut, is the principal site of elastase production.

Fatty Acid Distributions as Related to Adult Age, Sex and Diet in the Phytophagous Heteropteran, Lygus hesperus (Heteroptera: Miridae)

Abstract A study of the internal morphology of the alimentary canal of Chrysoperla rufilabris (Brumeister) adults in relation to yeast symbionts was conducted using light, scanning, and transmission electron and epifluorescence microscopy. The alimentary canal of field-collected adults possessed a single large (≈300–400 μm in length) diverticulum at the posterior end of the foregut. Although yeast cells (4.0–6.5 μm) were distributed throughout the alimentary canal, large numbers of blastically dividing cells (i.e., yeast) were observed within the diverticulum. The diverticulum interior was highly convoluted and folded transversely and longitudinally, and yeast cells were observed to accumulate within the folds. Large tracheal trunks were attached to the lateral side of the diverticulum, suggesting a high demand for gas exchange within this organ. The diverticulum was lined with cuticle, and the underlying tissues did not contain large amounts of endoplasmic reticulum, mitochondria, or Golgi complex, indicating that minimal absorption occurred within this gut region. This suggested that the high potential for gas exchange in the diverticulum by the tracheal trunks was primarily to support yeast metabolic activity. All size classes (i.e., 0.1, 4.0, and 10.0 μm) of fluorescence particles ingested by newly eclosed adults eventually ended up in the midgut and hindgut regions, indicating that the foregut and/or diverticulum do not possess an absolute mechanism for retaining particles based on size. However, all size classes of the fluorescent particles typically persisted within the diverticulum. The evident confluence between the diverticulum lumen and the gut lumen suggested a free exchange or flow of fluids between these regions. The proventriculus (proximal to the diverticulum) was pronounced and consisted of a series of long “hairs,” short “hairs,” and small spine-like structures projecting into the midgut. Large numbers of yeast cells were observed in association with the proventricular hairs, and these hairs may play a role in the retention of yeast cells. The midgut possessed typical absorptive structures (i.e., microvilli), and large numbers of mitochondria, rough endoplasmic reticulum, and Golgi complex were observed in midgut epithelial cells. Because evidence indicated no or minimal absorption of nutrients within the diverticulum, it was concluded that nutritional factors provided by the yeast must be transferred to the midgut where absorption occurs. Large numbers of yeast cells enclosed within a well-developed peritrophic matrix were observed in the midgut, suggesting that the yeast themselves may serve as a source of nutrients. Whereas the exact mechanism by which yeast contribute to the nutrition of C. rufilabris adults was not determined, morphological evidence obtained in this study supported the hypothesis that chrysopids form a mutualistic symbiosis with yeast and that the esophageal diverticulum was a specialized structure for housing them.