PHYSIOLOGY,BIOCHEMISTRY, AND TOXICOLOGY Digestive Enzymes and Stylet Morphology of Deraeocoris nebulosus (Hemiptera: Miridae), a Predacious Plant Bug 1 2 DAVID W. BOYD, JR., ALLEN CARSON COHEN, AND DAVID R. ALVERSON Department of Entomology, Clemson University, Clemson, SC 29634 Ann. Entomol. Soc. Am. 95(3): 395Ð401 (2002) ABSTRACT Mixed-feeding habits, such as zoophytophagy, make the ecological roles of many species of insects, especially hemipterans, difÞcult to assess. To understand the feeding adaptations of the predaciousplant 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, ␣-glucosidase, and pectinase were found in the salivary glands. Low levels of trypsin-like, chymotrypsin-like, elastase-like, and pectinase activity, with high levelsof ␣-amylase and ␣-glucosidase activity, were found in the anterior midgut. The insectÕs right maxillary stylet has two rows of at least six recurved barbs on the inner surface pointing away from the head. Thisplant bug isequipped mainly for zoophagy but hasenzymesthat would allow some degree of phytophagy. KEY WORDS Deraeocoris nebulosus, digestive enzymes, Miridae, predator, stylet morphology, omnivory FEEDING HABITS OF the Heteroptera range from strict A theoretical and practical grasp of the role that phytophagy to strict zoophagy (Schaefer and Panizzi heteropterans play in natural and agricultural systems 2000). Several familiescontain omnivoreswhose requiresa thorough understandingof their feeding mixed-feeding habitshave been termed zoophytopha- habits. Such knowledge, however, is difÞcult to obtain gousor phytozoophagous,depending on the relative by direct means, largely because of the cryptic nature degree of animal versus plant consumption (Alomar of the feeding processandthe amorphousnature of and Wiedenmann 1996). The origin of feeding habits the ingested food. Heteropteran workers lament the among the Heteroptera remainscontroversial(Sweet lack of a thorough understanding of heteropteran 1979, Cobben 1979, Cohen 1990, Schaefer 1997, feeding habits, especially for the Miridae (Wheeler Wheeler 2001). The diverse trophic habits of plant 2001). bugsmake the Miridae ideal for studiesoffeeding A consumerÕs ability to use plant or animal materials strategies, including digestive enzyme composition for food is indicated by the presence of speciÞc di- and mouthpart morphology. gestive enzymes and by mouthpart morphology (Bap- Mirids,aswell asall other heteropterans,employ tist 1941; Adams and McAllan 1956; Strong and Kruit- macerate (or lacerate) and ßush feeding (Miles 1972, wagen 1968; Miles1972; Cohen 1990, 1995, 1996, 1998a, Hori 2000, Wheeler 2001) that incorporatespiercing/ 1998b, 2000; Agustõ´ and Cohen 2000; Hori 2000; Zeng sucking mouthparts and watery saliva from the sali- and Cohen 2000a, 2000b). Digestive enzymes speciÞc vary gland complex. Miridsfeed in a manner that is for zoophagy include proteases (e.g., trypsin, chymo- typical of heteropterans, piercing and cutting tissues trypsin, cathepsin), hyaluronidase, and phospholipase with their stylets while injecting digestive enzymes (Cohen 1998b, 2000). SpeciÞc digestive enzymes for through the salivary canal to liquefy food into a nu- phytophagy include amylase and pectinase (Cohen trient-rich slurry. The food slurry is ingested through 1996). the food canal and passed into the alimentary canal Morphological comparisons of both mandibular and where it is further digested and absorbed (Cohen maxillary styletsrevealdifferencesbetween heterop- 2000). teran phytophagesand zoophages.Cobben (1978) showed that the right maxillary stylets of predacious Thisarticle reportsthe resultsofresearchonly. Mention of a heteropterans(e.g., Nabidae, Anthocoridae) are more proprietary product does not constitue an endorsement or a recom- deeply serrated than those of phytophagous heterop- mendation by the USDA for itsuse. terans(e.g., Tingidae). Cohen (1996) showedthat the 1 Current address: Small Fruit Research Station, USDA-ARS, P.O. Box 287, Poplarville, MS 39470 (e-mail: [email protected]). mandibular styletsofphytophagousand predacious 2 Biological Control and Mass Rearing Research Unit, USDA-ARS, pentatomidsvaried in relation to the direction of the P.O. Box 5367, Mississippi State, MS 39762. barbs; those of phytophagous species point away from 0013-8746/02/0395Ð0401$02.00/0 ᭧ 2002 Entomological Society of America 396 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 95, no. 3 the head, whereasthe barbsof predaciousspecies natant wasplaced in a 1.5-ml centrifuge tube and kept point toward the head. The barbson the mandibular at 4ЊC until use (within 48 h). The anterior midguts of styletsofpredaciousheteropteran families(e.g., the same 25 or 10 insects were treated as were the Reduviidae) are more numerousthan the barbson the salivary glands. Protein concentrations of all enzyme mandibular styletsofphytophagousheteropteran samples were determined by bicinchoninic acid pro- families (e.g., Lygaeidae, sensu lato) (Cohen 1990). tein assay (Pierce, Rockford, IL), using bovine serum Deraeocoris nebulosus (Uhler) isa predator of plant- albumin as the standard. Three samples from 25 or 10 feeding arthropods including aphids, mites, scale in- insects were used for each tissue. sects, and whiteßies (Gillette 1908, Smith 1923, ␣-Amylase Assay. Amylase activity in the salivary Wheeler et al. 1975, Jonesand Snodgrass1998).Phy- glandsand anterior midgut wasdetermined with a tophagy isunknown in D. nebulosus, but other species diagnostic kit (No. 577Ð3, Sigma, St. Louis, MO) fol- in thispredatory genusfeed facultatively on plants lowing modiÞcationsof Zeng and Cohen (2000a, (McMullen and Jong 1967, RazaÞmahatratra 1981, 2000b, 2000c). The substrate was 4,6 ethylidene (G7)- Wheeler 2001). One of us(D.W.B.) hasobserved D. p-nitrophenyl (G1)-␣, D-maltoheptaside. Enzyme ex- nebulosus with its stylets inserted into sweet potato tracts(10 ␮l) were added to wellsin an ELISA plate. leavesand cabbage, but whether thisbehavior in- The substrate was equilibrated to 37ЊC for 10 min, and volvesthe uptake of nutrientsor justwater isnot 200 ␮l wasadded to each well. The plate wasshaken known. for Þve sand incubated at 37 ЊC for 30 min. Absorbance The objectivesof thisstudywere to determine the wasread at 405 nm in a plate reader (SPECTRA MAX presence of certain digestive enzymes in the salivary Plus, Molecular Devices Corporation, Sunnyvale, gland complex and anterior midgut of D. nebulosus and CA). Absorbance is directly related to amylase activ- to analyze its stylet morphology. We also evaluated ity. Authentic ␣-amylase from barley malt (Sigma thispredaciousmiridÕsability to obtain nutrientsfrom A-2771) wasusedasa positivecontrol (1 mg/ml, 1 animalsand plants. U/mg solid), and buffer and substrate only were used asa negative control. The relative amylaseactivity was calculated asabsorbanceunitsper milligram of pro- Materials and Methods tein. The assay was performed three times for each Insects. A colony was established with D. nebulosus sample. collected from oaks(e.g., Quercus alba L., Q. stellata ␣-Glucosidase Assay. ␣-Glucosidase activity was Wang, and Q. falcata Michaux) in Greenville County tested from both salivary gland and anterior midgut and PickensCounty, SC, during the summerof 1999 extracts, following Agustõ´ and Cohen (2000), by add- and wasmaintained at 25 Ϯ 2ЊC, 50 Ϯ 10% RH, and a ing 100 ␮l of extract to 100 ␮l of 10-mM solution of photoperiod of 14:10 (L:D) h. Deraeocoris nebulosus p-nitrophenyl ␣-d-glucopyranoside (Sigma, N-1377) wasreared on eggsof Ephestia kuehniella Zeller (Lep- in an ELISA plate and incubating at 37ЊC for 1 h. The ␮ idoptera: Pyralidae) (BeneÞcial Insectary, Redding reaction wasstoppedby adding 100 l of 15% Na2CO3. CA, USA) at the Cherry Farm Insectaries, Clemson Invertase from bakers yeast (Sigma I-4504) was used University, Clemson, SC, USA. Voucher specimens as a positive control (1 mg/ml, 400 U/mg solid), and were placed in the Clemson University Arthropod buffer and substrate only were used as a negative Collection. control. Absorbance was read at 405 nm in a plate Sample Preparation. Enzyme samples were pre- reader, and the relative ␣-glucosidase activity was pared by the method of Cohen (1993) with modiÞ- calculated asabsorbanceunitsper milligram of pro- cation. Only adult female insects were used in these tein. The assay was replicated three times. tests. The mirids were starved for 24 h before dissec- General Protease Assay. General protease activity tions to standardize the insects and to allow an accu- was determined using the EnzChek ßuorescence pro- mulation of digestive enzymes. The insects were tease assay kit (E-6638, Molecular Probes, Eugene, placed at Ϫ20ЊC for 4 min and then dissected in ice- OR) following modiÞcationsof Zeng and Cohen cold phosphate buffer saline (pH 7.4) under a dis- (2000a). Extract samples were diluted by adding 10 ␮l secting microscope. The salivary gland complex, in- of extract to 90 ␮l of the reaction buffer in an ELISA cluding all lobes, accessory glands, and tubules, was plate well, and then 100 ␮l of substrate solution buffer exposed by holding the abdomen with Þne forceps and (casein derivatives heavily labeled with the pH-insen- pulling the head and prothorax away from the abdo- sitive BODIPY FL-dye) was added. The assay plate men with another pair of Þne forceps. The anterior wasincubated