Morphology and Proteome Characterization of the Salivary Glands of the Western Chinch Bug (Hemiptera: Blissidae) Crystal Ramm University of Nebraska-Lincoln
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University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Faculty Publications: Department of Entomology Entomology, Department of 2015 Morphology and Proteome Characterization of the Salivary Glands of the Western Chinch Bug (Hemiptera: Blissidae) Crystal Ramm University of Nebraska-Lincoln Astri Wayadande Oklahoma State University Lisa Baird University of San Diego, CA, [email protected] Renu Nandakumar University of Nebraska-Lincoln Nandakumar Madayiputhiya University of Nebraska-Lincoln See next page for additional authors Follow this and additional works at: https://digitalcommons.unl.edu/entomologyfacpub Part of the Entomology Commons Ramm, Crystal; Wayadande, Astri; Baird, Lisa; Nandakumar, Renu; Madayiputhiya, Nandakumar; Amundsen, Keenan; Donze- Reiner, Teresa; Baxendale, Frederick; Sarath, Gautam; and Heng-Moss, Tiffany, "Morphology and Proteome Characterization of the Salivary Glands of the Western Chinch Bug (Hemiptera: Blissidae)" (2015). Faculty Publications: Department of Entomology. 415. https://digitalcommons.unl.edu/entomologyfacpub/415 This Article is brought to you for free and open access by the Entomology, Department of at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Faculty Publications: Department of Entomology by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Authors Crystal Ramm, Astri Wayadande, Lisa Baird, Renu Nandakumar, Nandakumar Madayiputhiya, Keenan Amundsen, Teresa Donze-Reiner, Frederick Baxendale, Gautam Sarath, and Tiffany Heng-Moss This article is available at DigitalCommons@University of Nebraska - Lincoln: https://digitalcommons.unl.edu/entomologyfacpub/ 415 PLANT RESISTANCE Morphology and Proteome Characterization of the Salivary Glands of the Western Chinch Bug (Hemiptera: Blissidae) CRYSTAL RAMM,1 ASTRI WAYADANDE,2 LISA BAIRD,3 RENU NANDAKUMAR,4 NANDAKUMAR MADAYIPUTHIYA,4 KEENAN AMUNDSEN,5 TERESA DONZE-REINER,1 1 6 1,7 FREDERICK BAXENDALE, GAUTAM SARATH, AND TIFFANY HENG-MOSS J. Econ. Entomol. 108(4): 2055–2064 (2015); DOI: 10.1093/jee/tov149 ABSTRACT The western chinch bug, Blissus occiduus Barber, is a serious pest of buffalograss, Buchloe dactyloides (Nuttall) due to physical and chemical damage caused during the feeding process. Although previous work has investigated the feeding behaviors of chinch bugs in the Blissus complex, no study to date has explored salivary gland morphology and the associated salivary complex of this insect. Whole and sectioned B. occiduus salivary glands were visualized using light and scanning electron microscopy to determine overall structure and cell types of the salivary glands and their individual lobes. Microscopy revealed a pair of trilobed principal glands and a pair of tubular accessory glands of differing cellular types. To link structure with function, the salivary gland proteome was characterized using liquid chro- matography tandem mass spectrometry. The salivary proteome analysis resulted in B. occiduus sequences matching 228 nonhomologous protein sequences of the pea aphid, Acyrthosiphon pisum (Harris), with many specific to the proteins present in the salivary proteome of A. pisum. A number of sequences were assigned the molecular function of hydrolase and oxido-reductase activity, with one specific protein sequence revealing a peroxidase-like function. This is the first study to characterize the salivary proteome of B. occiduus and the first of any species in the family Blissidae. KEY WORDS salivary gland, Hemiptera, chinch bug, salivary proteome Chinch bugs are a common pest known for damaging a phloem tissue (Painter 1928, Backus et al. 2013). variety of turfgrasses and field crops including peren- Chinch bug feeding occurs primarily within vascular nial ryegrass, Kentucky bluegrass, zoysiagrass, fescue, tissues and bulliform cells (Anderson et al. 2006). Asso- and bentgrass; and sorghum, corn, wheat, and barley ciated plant tissue injury is a result of direct stylet (Spike et al. 1994). The western chinch bug, Blissus puncture, withdrawal of plant sap, and clogging of vas- occiduus Barber, is a serious pest of buffalograss, cular transport systems via deposition of salivary sheath Buchloe¨ dactyloides (Nuttall), a low maintenance turf- materials (Painter 1928). Buffalograss damage by grass species with comparative freedom from arthro- B. occiduus is a result of feeding, as sap is withdrawn pods and disease (Baxendale et al. 1999). In addition, from tissues around the crown and stolon areas by the B. occiduus has an extensive host range including turf- insect’s piercing–sucking mouthparts. Feeding results grasses such as green foxtail, Kentucky bluegrass, pe- in an initial reddish discoloration of plant tissue, fol- rennial ryegrass, and zoysiagrass and small grains such lowed by patches of yellowing or browning turf, and as corn, sugarcane, wheat, and barley (Ferris 1920, eventually plant death (Baxendale et al. 1999). Bird and Mitchener 1950, Farstad and Staff 1951, Sla- In recent years, increased attention has been given ter 1964, Baxendale et al. 1999, Eickhoff et al. 2004). to insect saliva and its role in plant–insect interactions Chinch bugs are categorized as salivary sheath feeders, (Cherqui and Tjallingii 2000, Francischetti et al. 2007, lying down sheaths of gelling saliva that harden around Cooper et al. 2010, Carolan et al. 2011, DeLay et al. the insect stylets as they advance toward the target 2012, Nicholson et al. 2012, Will et al. 2012, Rao et al. 2013). Previous research has documented salivary gland morphology and the composition of salivary se- 1 Department of Entomology, University of Nebraska, Lincoln, NE cretions in Hemipteran insects (Sogawa 1965, Waya- 68583. dande et al. 1997, Madhusudhan and Miles 1998, Ni 2 Department of Entomology and Plant Pathology, Oklahoma State et al. 2000, Swart and Felgenhauer 2003, Carolan et al. University, Stillwater, OK 74078. 2011). Miles (1987, 1990)andBurd et al. (1998) sug- 3 Department of Biology, University of San Diego, CA 92110. 4 Department of Biochemistry, University of Nebraska, Lincoln, NE gested that associated damage and symptoms caused 68588. by insects with piercing–sucking mouthparts were due 5 Department of Agronomy and Horticulture, University of Ne- to the injection of salivary phytotoxins into plant tissues. braska, Lincoln, NE 68583. Salivary secretion composition is quite diverse and 6 Grain, Forage and Bioenergy Research Unit, USDA-ARS, Lin- coln, NE 68583. consists of many different enzymes and metabolites 7 Corresponding author, e-mail: [email protected]. depending upon the insect and the host plant Published by Oxford University Press on behalf of Entomological Society of America 2015. This work is written by US Government employees and is in the public domain in the US. 2056 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 108, no. 4 (Miles 1968). Hemipteran saliva has been documented microscope slide, covered with a cover slip, and viewed to contain digestive enzymes such as amylase, protease, under a phase contrast microscope (Olympus BX2 invertase, and lipase in the salivary glands of the milk- compound microscope with digital imaging). For histo- weed bug (Bronskill 1958) and reductants, surfactants, logical cross-section analysis, principal and accessory cellulases, polyphenol oxidase, and peroxidase in aphid glands were dissected out of chinch bugs in a chilled watery saliva (Miles 1999). Pectinases in saliva may solution of 2% gluteraldehyde in 0.1 M Sorenson’s help to not only break down the middle lamella of the sodium phosphate buffer (Clark et al. 1981), pH 7.2. plant cell wall but also function in gustatory exploration Glands were transferred into Sorenson’s fixative solu- and overcoming plant resistance (Campbell and Dreyer tion for 2 h. Glands were rinsed in buffer, dehydrated 1985). The oxido-reductases peroxidase and polyphenol with a standard ethanol series, and embedded in JB-4 oxidase may allow these insects to detoxify plant defen- resin. Thick sections (4–6 mm) were cut with a glass sive phytochemicals such as hydrogen peroxide (Ni knife, mounted on glass slides, and stained with tolui- et al. 2000). In addition, gelling sheath saliva may help dine blue. Sections were visualized using an Olympus with stylet support, lubrication, penetration, protection BX-51 light microscope. from bacteria and viruses, and suppression of wound For histological cross sections, confirmation of the signaling (Miles 1968, 1972, 1987, 1999; Cherqui and accessory gland and the separate lobes of the principal Tjallingii 2000). gland was accomplished by separating the trilobed Despite an increasing interest in the role of insect principal gland into the anterior, posterior, and lateral salivary secretions in plant feeding (Wayadande et al. lobes. The individual lobes of the principal gland and 1997, Miles 1999, Cherqui and Tjallingii 2000, Franci- the unilobed accessory gland were then processed fol- schetti et al. 2007, Cooper et al. 2010, Carolan et al. lowing LM methods as previously described, sectioned, 2011, Will et al. 2012, Nicholson et al. 2012, DeLay and observed by LM. et al. 2012, Rao et al. 2013), and the economic impor- SEM of B. occiduus Salivary Glands. B. occiduus tance of the Blissus species (Mize and Wilde 1986, Pot- salivary glands were collected as described above in a ter 1998, Baxendale et al. 1999, Vittum et al. 1999, chilled solution of 2% gluteraldehyde in 0.1 M Soren- Eickhoff et al. 2004), little is known about the chinch son’s sodium