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A Protocol For Digesting Internal Soft Tissues And Mounting Spiders For Scanning Electron Microscopy PDF

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2007 (2008). The Journal ofArachnology 35:538-542 SHORT COMMUNICATION A PROTOCOL FOR DIGESTING INTERNAL SOFT TISSUES AND MOUNTING SPIDERS FOR SCANNING ELECTRON MICROSCOPY Fernando Alvarez-Padilla and Gustavo Hormiga: Department of Biological Sciences, The George Washington University, Washington, DC 20052, USA. E-mail: fap(^gwu.edu ABSTRACT. Wedescribeasimpleprotocolfordigestingtheinternalsofttissuesofspidersusinganenzyme complex knownaspancreatin. Thistechniqueispreferred overdigestionswithcausticagentsbecauseitbetter preservesthecuticlesurface, allowingitsstudybymeansofscanningelectronortransmittedlightmicroscopy. In addition, we describe a technique for mounting spider body parts for scanning electron microscopy using an acryloid polymer. Keywords: Digestion, pancreatin, dissection, SEM Protocols for digesting internal soft tissues have see Holm (1979)), such as spermathecae and acces- been widely used in the entomological and arachno- sory ducts. logical literatureeitherto observe themorphology of Examination of entomological or arachnological internal structures (e.g., Purcell 1909, 1910; Lamy specimensforscanningelectronmicroscope(SEM)is 1902; Machado 1951; Millidge 1984; Wibel et al. commonly done by attaching the specimen (or body 1984; Meyer 1989; Sierwald 1989; Platnick et al. part) directlyonto themountingstuborbyattaching 1999; Ramirez 2000; Townsend et al. 2000; Griswold it to the free end ofa piece ofwire, usually made of et al. 2005) or to estimate the proportion ofskeletal gold or copper. Conductive and non-conductive tissuesininsects(Buxton 1932). Potassiumhydroxide adhesives are available to glue the specimen to the (KOH), sodium hydroxide (NaOH) and sodium mount. We have used non-conductive acryloid glue, hypochlorite (NaClO, bleach) are some of the most whichhasthedesiredcharacteristicofproducingthin commonly used substances in these studies. The threads. Thesethreadsareusedtowrapthe specimen purpose of such digestions is to remove the soft at the free end ofthe copper tape. This modification tissues that surround sclerotized organs for study allows us to securely glue the specimen to the copper with scanning electron microscopy (SEM) or optical tape by a small area maximizing the observable microscopy. Digestions with the above mentioned surface of the specimen (Figs. 2C, D, arrows). In chemical agents can damage the chitinous cuticle principle, anygluethatformsthinelasticthreadsand surface ofstructures such as spermathecae, therefore dries out relatively fast can be used for this purpose, the use ofless aggressive substances, such as trypsin, but an acryloid B-72 solution in acetone has the has been recommended (e.g., Sierwald 1989). advantage thatitsviscositycan beeasilymodified by We describe a protocol for digesting spider changingtheproportion ofacetoneto acryloid inthe internal soft tissues with a mixture of digestive mixture. If the acryloid pellets are diluted with too enzymes usually obtained from pig pancreas known much acetone the solution will not form threads or as pancreatin. Pancreatin contains trypsin, amylases, the threads will break easily. Conversely, too little lipases, ribonucleases, and proteases. Furthermore, acetone will result in an unworkable mix of high this enzyme complex is active at room temperature viscosity. The acryloid B-72 glue is easier to and does not require buffers. These enzymes manipulate than the standard graphite conductive effectively digest the soft tissues without apparent glue used for SEM, and once it is covered with gold damage to the cuticle surface of sclerotized organs during the sputter coating process the glue surface (compare Figs. 1 and 2). We have successfully used becomes conductive although sometimes it may pancreatin to study the female internal genitalic present charging problems. — structures as well as the tracheal system anatomy of Pancreatin protocol. Method is modified after a diversity ofspider species (e.g.. Figs. 3-8; Alvarez- Dingerkus & Uhler (1977). Add sodium tetraborate Padilla & Hormiga unpublished ms.). Furthermore, decahydrate (Borax) to one liter of warm distilled digestion of soft tissues with pancreatin greatly water (60° C) until saturation is reached then cool to facilitates the microscopic observation ofsclerotized room temperature (RT « 20° C). (When the solution structures in clearing media (e.g., methyl salicylate, cools, a layer of borax crystals will form at the 538 ALVAREZ-PADILLA & HORMIGA—PREPARING SPIDERS EOR SEM 539 — Eigures 1^. 1. Spermatheca of Chrysometa alajuela Levi 1986 (Tetragnathidae) digested with KOH; 2. Spermatheca of C alajuela digested with pancreatin; 3. Glenognathafoxi (McCook 1894) (Tetragnathidae) tracheal system digested with pancreatin (left tracheal trunk); 4. Detail of the left tracheal trunk and tracheoles of G.foxi. 540 THE JOURNAL OF ARACHNOLOGY — Figures 5-8. 5. Spermathecae of Leucauge venusta (Walckenaer 1842) (Tetragnathidae) digested with pancreatin; 6. SpermathecaeofNcmometasp. (Tetragnathidae)digestedwithpancreatin; 7. Leftmalepedipalp ofChrysometaalajuelci (Tetragnathidae), mesal view(arrow indicates acryloid gluethread oncoppertape); 8. Cephalothorax of Tetragnatha versicolor Walckenaer 1842, ventral view (arrow indicates dried acryloid glue thread around coxa IV on tip ofcopper tape). bottom.) Dissolve 1 g of pancreatin 4X USP grade 24 h ofdigestion. Thecleaningprocess must bedone enzyme complex in 70 ml of distilled water, add carefully to avoid damaging the internal sclerotized 30 ml of borax saturated aqueous solution and mix structures. Transfer the sample to distilled waterand well. Filter the solution with cotton and a funnel to remove the partially digested tissue with streams of remove the coagulated proteins. Distribute the water produced by a pipette. Return the sample to solution into several vials and keep them frozen the pancreatin solution until digestion is completed. (—20° C). The solution can be thawed and frozen Transfer the specimen to 75% alcohol (= ethanol) several times, and it will work for 3 da at RT. and wash it with a pipette until the tissue is To enable the pancreatin solution to contact completely removed. If some soft tissues remain internal tissues a wide opening must be made in the undigested, repeat the transfer to distilled water and abdomen (e.g., Platnick et al. 1999); or a large then to the pancreatin solution. Once the soft tissues section ofthe specimenmustberemovedtoallowthe have been digested, transfer the specimens to clean pancreatin solution to enter (e.g., cut a large window 75% alcohol. on the dorsal cuticle oftheabdomen). Forspecimens For inspection with a compound microscope preserved in ethanol, transfer the specimens to a vial transfer the specimens to clove oil, methyl salicylate with distilledwaterforonetotwohours oruntil they or some other clearing substance. Temporary slide- sink; then transfer the samples to the pancreatin mounts for these preparations are described else- solution. where (e.g., Grandjean 1949; Coddington 1983). Specimens < 5 mm'* will take overnight to digest KOH digestions work fine for study with stereo or completely. Smaller samples, such as epigyna, will transmitted light microscopy. However, the cuticle take4 5 h butcan be left overnight too. Samples left surface may have been over digested by the caustic > 16 h can be completely digested and destroyed. process making this type ofspecimen unsuitable for Digestion times for specimens > 5 mm^ will be ~ 1- SEM. For SEM study follow standard protocols for 3 da. Incubating the samples at ~ 37° C will reduce critical point drying and sputter coating of the digestion times. All samples must be cleaned after specimen. ALVAREZ-PADILLA & HORMIGA—PREPARING SPIDERS FOR SEM 541 — SEM mounting protocol. Dissolve Acryloid B-72 LITERATURE CITED (Paraloid B-72) pellets in acetone until they form Buxton, P.A. 1932. The proportion ofskeletal tissue a gluey homogenous mix. The viscosity of the in insects. Biochemical Journal 26:829-832. mixture can be adjusted by adding or evaporating Coddington, J.A. 1983. A temporary slide mount acetone. The mix should be such that it will produce allowing precise manipulation ofsmall structures. athinthreadwhen aneedletouchesits surfaceandis Verhandlungen des Naturwissenschaftlichen Ver- then pulled about one centimeter away. If the eins in Hamburg NP 26:291-292. preparation ofthe acryloid solution produces many Dingerkus, G. & L.D. Uhler. 1977. Enzyme clearing airbubbles(asaconsequenceofstirringthepelletsin ofalcian blue stained whole small vertebrates for acetone), sonicate the vial until all the bubbles have demonstration of cartilage. Stain Technology risen to the surface. Unused acryloid solution can be 52:229-232. stored in a closed vial and its viscosity readjusted Grandjean, F. 1949. Observation etconservation des with acetone when needed. Any glue that forms thin tres petit arthropodes. Bulletin du Museum elastic threads and dries out relatively fast could be National d’Histoire Naturelle Paris 21:363-370. potentially used, but we only have experience with Griswold, C.E., M.J. Ramirez, J.A. Coddington & Acryloid B-72. N.I. Platnick. 2005. Atlas ofphylogeneticdatafor Takethecriticallypointdried specimenwithafine entelegyne spiders (Araneae: Araneomorphae: brushorsoftforceps; conventionalforcepswilleasily Entelegynae) with comments on their phylogeny. damage the specimen. Place the specimen (e.g., Proceedings of the California Academy of cephalothorax, pedipalp, leg, etc.) on the free end Sciences 56:1-324. ofthe sticky copper tape (the other end ofthe tape Holm, A. 1979. Ataxonomic study ofEuropean and has been attached to the SEM mount). At this point East African species ofthe genera Pelecopsis and avoid moving the copper tape because this action Trichopterna (Araneae, Linyphiidae), with de- may easily catapult the specimen. To permanently scriptions of a new genus and two new species attach the specimen to the copper tape, collect of Pelecopsis from Kenya. Zoologica Scripta enough glue with the tip of a needle mounted on 8:255-278. a handle (or a thin and straight piece ofmetal wire), Lamy, E. 1902. Les trachees des araignees. Annales touch the base of the mount with the glue-loaded Des Sciences Naturelles - Zoologie et Biologic needle tip and pull the needle until a thin thread of Animale 15:149-280. glue is formed. Wrap the specimen and the copper Machado, A. de B. 1951. Subsidios para o estudo da tape with this glue thread several times by going biologia na lunda Ochyroceratidae (Araneae) de around the area in a circular motion. Once the fAngola. Publgoes Culturais Companhia de Dia- glue has dried, examine the mounted specimen mantes de Angola 8:1-88. with the stereoscope at high magnification. If any Meyer, E.P. 1989. Corrosion cast as a method for dirt is visible on the specimen, try to remove it by investigation of the insect tracheal system. Cell gently blowing air with a thin glass pipette con- Tissue Research 256:1-6. nected to surgical latex tubing or with the help of Millidge, A.F. 1984. The taxonomy ofthe Linyphii- a needle or brush. Carefully bend the tip ofcopper dae, based chiefly on the epigynal and tracheal tape with the mounted specimen to the desired characters (Araneae: Linyphiidae). Bulletin ofthe orientation. Proceed to sputter coating the prepara- British Arachnological Society 6:229-267. tion. Platnick, N.L, C.J. Grismado & M.J. Ramirez. 1999. ACKNOWLEDGMENTS On the genera ofthe spider subfamily Otiothopi- nae (Araneae: Palpimanidae). American Museum Ichthyologists John Burns and Robert Javonillo Novitates 3257:1-25. (The George Washington University) suggested the Purcell, F. 1909. Development and origin of the use of pancreatin for enzymatic digestion of soft respiratory organs in Araneae. Quarterly Journal tissues. Martin J. Ramirez (Museo Argentino de ofMicroscopical Science 54:1-110. Ciencias Naturales, Buenos Aires) first recom- Purcell, F. 1910. The phylogeny of the tracheae in mended the use of copper sticky tape for SEM Araneae. Quarterly Journal of Microscopical mounts. Martin J. Ramirez, Diana Silva, Lara Science 54:519-564. Lopardo, Dimitar Dimitrov, Suresh Benjamin, Ana- Ramirez, M.J. 2000. Respiratory systemmorphology hita Shaya, Vanessa Degrassi, John Burns, and and the phylogeny of Haplogynae spiders (Ara- Robert Javonillo provided extremely useful com- neae: Araneomorphae). Journal of Arachnology mentsonanearlierdraftofthismanuscript. Support 28:149-157. for this work has been provided by a NSF-PEET Sierwald, P. 1989. Morphology and ontogeny of grant to Gustavo Hormiga and Gonzalo Giribet femalecopulatory organsin American Pisauridae, (DEB-0328644), a George Washington University with special reference to homologous features REF grant, and a doctoral scholarship form CON- (Arachnida: Araneae). SmithsonianContributions ACYT (Mexico) to the senior author. to Zoology 484:1-23. THE JOURNAL OF ARACHNOLOGY 542 ' Townsend, V.R., Jr., R.S. Hazelton, B.E. D.L. Baumgartner. 1984. Scanning electron mi- Felgenhauer & J.H. Spring. 2000. New tech- croscopy on antennal sense organs of Nasonia nique for examining invertebrate membranes vitripennis (Hymenoptera: Pteromalidae). Trans- using high resolution scanning electron microsco- actions of the American Microscopical Society py. Microscopy Research and Technique 49: 103:329-340. 209-211. Wibel, R.G., J.D. Cassidy, H.E. Buhse, Jr., M.R. Manuscript received 16 October 2006, revised 18 Cummings, V.P. Bindokas, J. Charlesworth & January2007.

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