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

2007·4.9 MB·English

by  Fernando Alvarez-Padilla

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
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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 '
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