Table Of ContentPROC. ENTOMOL. SOC. WASH.
95(2), 1993, pp. 131-138
A METHOD OF GENITALIA PREPARATION AND DRY
PRESERVATION FOR COLEOPTERA
Paul E. Skelley
Department ofEntomology and Nematology, Building 970. Hull Road, University of
Florida, Gainesville, Florida 3261 1-0620, U.S.A.
—A
Abstract. technique to evert, inflate, and dry preserve internal sacs ofmale genitalia
is discussed. In addition to pigmented structures, many internal sacs observed have an
asymmetrical form and microscopic structures on the white-fleshy parts. This technique
is compared to more widely used methods ofgenitalia preservation and study.
Key Words: Genitalia, SEM, preparation, internal sac
Male genitalia and associated structures Someresearcherscombinethetwo methods
(collectively called terminalia) are utilized by dissecting the IS's, cutting them down
for taxonomic and systematic studies in one side and mounting them flattened out
most insect taxa. The male internal sacs are on a microscope slide. With this variation
often ignored in studies of taxa where the all three-dimensional information is lost.
sclerotized parameres are distinct among Internal sacs can be studied retracted
species. Internal sacs can yield additional (folded and held within the dark sclerotized
informationintaxawheretheparameresare genitalicstructures)oreverted(extendedand
similar among species (e.g. Chandra 1991). swollen as duringcopulation). Occasionally
Most methods ofinternal sac (IS) (or en- a specimen is killed with thegenitalia evert-
dophallus)preparationand preservationare ed, but this is not a common occurrence.
variationsoftwothemes: 1)relaxandmount Techniques to evert the IS's are few and are
on microscope slides, or 2) relax and store usually delicate procedures.
with glycerin in microvials. Microscope Retracted IS's are studied with transmit-
slides are mostly used for small genitalia ted light in various small-sized taxa where
that are studied at high magnifications with everting the tissue is difficult or impossible
acompoundmicroscope(transmitted light). (as illustrated in Gordon and Cartwright
With this method there are moderate dis- 1980, 1988). Larger insects IS's are often
tortions due to the weight ofthe coverslip. everted by pushing and pulling the relaxed
Some researchers omit the slides altogether tissues with hook-tipped pins or forceps
and preserve the genitalia on a paper point (Sharp and Muir 1912, Sharp 1918, How-
inadropofmountingmedium(Angus 1969: den 1982, d'Hotmanand Scholtz 1990). Af-
2). terall ofthe work to preparegenitalia, most
Larger genitalia are stored in microvials attention is given only to the pigmented
under glycerin. The glycerin preserves the structures on the IS's.
soft tissues and prevents them from drying D'Hotman and Scholtz (1990) everted,
out. This method allows study with the dis- compared, and illustrated the genitalia of
secting microscope (reflected light) and many scarab beetles showing asymmetrical
avoids distortions due to slide mounting. IS's (e.g.. Figs. 1, 2). Thompson (1988) de-
132 PROCEEDINGS OFTHE ENTOMOLOGICALSOCIETY OFWASHINGTON
veloped a glycerin-inflating technique to tergent water solution, 1 ml in a 4 ml vial
studythefleshyIS'sofLeptostethusweevils. was suflicient. The fresh tissues retained
In both of these studies the IS's were ob- much of their membrane integrity and
served and stored in glycerin. Thompson's swelled under osmotic pressure. Genitalia
inflation technique worked well, but the in- were then placed between the thumb and
flated IS's could be studied only when at- forefinger holding the glands and muscula-
tached to the apparatus and under a dis- ture. Withaslow,gentlerollingmotion(like
secting scope. Once the genitalia are squeezingtooth-pasteoutofatubefrom the
removed from the apparatus, they presum- bottom to top) the tissues were forced up
ably deflate. intothemedianlobecausingtheIStoemerge
The intent ofthis study was to develop a from the apex. A similar technique for
method where IS's could be easily everted, everting genitalia has been done with live
preserveddrywithoutcollapseintheirthree- camel crickets (Orthoptera: Gryllacrididae:
dimensional form, and studied under a Ceuthophilus) by T. J. Cohn and the late T.
scanning electron microscope (SEM). H. Hubbell (unpublished) and is also men-
tioned by Sharp and Muir (1912: 483).
Materials and Methods
Hook-tipped pins or forceps were often
Thistechnique involvedtwo majorsteps: helpful in this process.
1)eversion and potential inflation ofthe IS, Genitalia were again placed in the deter-
and 2) drying the genitalia. gent water solution and the "squeezing"
Dry museum specimens were relaxed in process repeated until the genitalia re-
a weak solution ofdetergent water(approx- mained inflated. The time required in the
imately 1 partdetergent: 9 partswater), and detergent solution varied dramatically
the genitalia dissected. The IS's were evert- among specimens. Larger genitalia often
edmanuallywithhook-tippedpinsandjew- tookseveraldaysinthesolutionandseveral
elers forceps as in Sharp and Muir (1912: "squeezings."Smallgenitaliaoftenrequired
483-484). These specimens were dried for only one "squeezing" and a few hours in
study, but inflating attempts failed and the the detergent.
tissues remained folded and wrinkled. In- Delicate IS's could be fixed or hardened
flation with a syringe (Hardwick 1950) did before drying. Various chemical fixatives
not improve the results. like osmium tetroxide, hexamethyldisali-
Inflated IS preparations were made from zane (Nation 1983), or formaldehyde may
freshlykilled specimens, thefresherthe bet- be used. See Glauert (1980) for discussions
ter. Rates oftissue hardening varied greatly ofvarious fixation techniques which can be
among specimens depending on size, used. I did not employ any ofthese for this
strength of tissues, and method of killing. study.
Cyanide or ethyl acetate produced the best The specimens, once everted and/or in-
results. Alcohol submersion worked ade- flated, are ready to bedried with thecritical
quately but appeared to kill the insects in a point dryer. Other drying techniques (i.e.
tense stateandrapidlyhardenedthetissues. freeze drying) may be useful, but they were
Few good inflations were made from old not usedhere. To bedriedinacriticalpoint
alcohol-killed and preserved specimens. dryer, the specimens needed to be dehy-
After the insect died, the genitalia was dratedthroughalcoholbathsinto 100%eth-
removed and placed in weak detergent wa- anol. I raised the alcohol percentage in the
tersolution(1 partdetergent: 9partswater). vials by slowly adding 70% isopropanol; a
Carewastakentoremovegenitaliawith the fewdropsatfirst,thendoublingthevolume.
associated glands and musculature intact. When near 70%, I poured off"the liquid and
The genitalia were then covered with de- added straight 70% alcohol. From there a
VOLUME NUMBER
95, 2 133
normal dehydration series was used. I left
the specimen in each liquid change from 1
to several hoursallowingampletime forthe
specimen to come to an equilibrium with
the solution before changing it.
Once dehydrated and in the third change
of100%ethanol, genitaliawerecritical point
driedwithaTousimis, Samdri®-780A. The
genitalia were mounted on a paper point
and pinned under the rest ofthe specimen
for study with a dissecting microscope or
coated and studied under a scanning elec-
tron microscope (SEM). Specimens illus-
tratedherewerecoatedwithgold-palladium
in a Denton Vacuum DESK II sputtercoat-
er and photographed with a Hitachi S-570
SEM. Specimens studied are deposited in
Figs. 1-2. Canthon pilulanus (Linnaeus) (Scara- the Florida State Collection ofArthropods,
baeiaae)genitaliawithevertedinternalsac,dorsalview. Gainesville, Florida.
1, Line = 0.18 mm. 2, Line = 1.83 mm.
Fig. 3. AtaeniussaramariCartwright(Scarabaeidae)genitaliawith evertedinternal sac, lateral view. Line =
0.23 mm.
)
134 PROCEEDINGS OFTHE ENTOMOLOGICALSOCIETY OF WASHINGTON
Results
The original workwas done on Phylloph-
aga (Scarabaeidae) while helpingto prepare
the SEM genitalia illustrations in Woodruff
andBeck(1989). Thetechniqueproveduse-
fulonvariousotherfamiliesandscarabgen-
era, a few ofwhich are illustrated.
In liquid IS's are clear, except for the ob-
vious pigmented structures. When they are
critical point dried, soft tissues turn an
opaquewhiteandanyinternalstructuresare
obscured. This white tissue contrasts with
Figs. 4-5. Platytomus longidus(Cartwright) (Scar-
= pigmentedstructuresandcanbestudiedun-
abaeidae) genitalia with everted internal sac. Line
0.20 mm. 4, Dorsal view. 5, Lateral view. der a dissecting microscope at lower mag-
nifications(20-100X). Specimenscoated for
study at higher magnifications (100-1000x
with the SEM lost this contrast. Examina-
tionwiththeSEMrevealedvaryingamounts
Fig. 6. AphodiusbadipesMelsheimer(Scarabaeidae)genitaliawith everted internal sac, lateral view. Line =
0.60 mm.
VOLUME NUMBER
95, 2 135
PROCEEDINGS OFTHE ENTOMOLOGICALSOCIETY OFWASHINGTON
136
Fig. 9. Epicauta heterodera Horn (Meloidae) genitalia with everted internal sac, lateral view. Line =
0.23 mm.
Microscope slide mounting is the most
widely available technique to study speci-
mens at high magnifications. Genitalia can
beadequatelypreparedfromdriedmuseum
specimensandinternal features studiedwith
transmitted light. There are drawbacks to
this technique. Slide mounted genitalia are
kept separate from the pinned specimen,
and the association between them is easily
lost. Clarity with transmitted light at high
magnifications is adequate but the fleshy
microsculpture is difficult to discern. Once
set in mounting medium, the specimen be-
comes two dimensional and can be viewed
only from the top and bottom. Being three
dimensional, there are distortions that oc-
cur as they are flattened.
Microvial-stored specimens can be re-
movedandstudied fromall views, andmay
be re-inflated for later study ifneeded, as in
Figs. 10-11. SaprinuslugensErichson(Histeridae)
Leptostethus preparations described by
genitalia with everted internal sac, lateral view. 10,
Line = 0.06 mm. 11, Line = 0.60 mm. Thompson (1988). Specimens stored this
VOLUME 95, NUMBER 2 137
Figs. 12-13. Listronotuscchmodon O'Brien(Curculionidae)genitaliawithevertedinternalsac,lateralview.
12, Line = 0.43 mm. 13, Line = 0.086 mm.
way are generally larger and can be studied association between the specimen and its
with both transmitted and reflected light. genitalia.
Pigmented structures, both internal and ex- Genitaliadried with acritical point dryer
ternal, are obvious and easily studied, can be studied with a dissecting microscope
whereas unpigmented structures are diffi- or a SEM. Being dry, the genitalia can be
cult to discern. Microvials and glycerin are pinned under the specimen with no chance
readily available. Problems can arise with for loss ofthe association. The genitaha are
the extra weight ofthe vial when added to light in weight and take up little space, de-
the insect pin. The bulk can become a nui- creasing potential hazards to other speci-
sance in usurping storage space and aid in mens. Thegenitaliacontainnoliquidwhich
dislodging specimens from the box bot- can soil the specimens, although the body
toms. If too much glycerin is used, it can oils from specimens may soil the genitalia.
seep through the microvial stopperand soil Thelimitationsofthistechniquecan be for-
the label or the specimen. Ifthe specimen midable. The equipment needed is expen-
is large and takes up most ofthe insect pin, sive and many institutions may not have a
the microvial is pinned separately next to critical point dryer available. Dry museum
the specimen. This can lead to a loss ofthe specimens can be used, ifa certain amount
,
138 PROCEEDINGS OFTHE ENTOMOLOGICALSOCIETY OFWASHINGTON
ofwrinkling is acceptable. The best prepa- Chandra, K. 1991. Newgenitalic characters fordis-
rations come from freshly killed material, tinguishingAnomala dorsalis from Anomalafra-
thus, rare or endangered taxa may be un- terna. TheColeopterists' Bulletin45(3): 292-294.
Glauert,A. M. 1980. Fixation,dehydrationandem-
available for study. Critical point drying bedding of biological specimens. Vol. 3, Part I,
turns the tissues opaque, eliminating study 207 pp. InGlauert, A. M., ed.. Practical Methods
by transmitted light, but allows study with on Electron Microscopy. North-Holland Publish-
aSEM. ingCompany, New York.
TheuseofcriticalpointdryersandSEM's Gordeonm,HRe.mDi.spahnedrOe.sLp.ecCiaerstworfiRghhty.ss1e9m8u0s.aTnhdeTWriecshti--
in IS studies have advantages and difficul- orhyssemus (Coleoptera: Scarabaeidae). Smith-
ties depending on the taxa. The exact pro- sonian Contributions to Zoology 317, 29 pp.
ceduresusedforinflating, potentialfixation, . 1988. NorthAmericanrepresentativesofthe
and drying IS's will vary with each taxon. tribe Aegialiini (Coleoptera: Scarabaeidae: Apho-
The method described is not useful for all diinae).SmithsonianContributionstoZoology461
37 pp.
taxa. Becauseofthe taxonomic information Hardwick, D. F. 1950. Preparation ofslide mounts
that can be gained, this method should be of lepidopterous genitalia. The Canadian Ento-
considered in systematic studies. mologist 82: 231-235.
d'Hotman,D.andC. H.Scholtz. 1990. Comparative
morphologyofthemalegenitaliaofderivedgroups
Acknowledgments
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Gainesville,FL,fortheirencouragementand American Entomological Institute 19(6), 180 pp.
help. IthankM. C. ThomasandJ. M. King- Nation,J. L. 1983. Anewmethodusinghexamethyl-
solver, Florida State Collection of Arthro- disalizane forpreparation ofsoft tissues forscan-
pods, Gainesville, FL; H. Cromroy, Uni- ningelectronmicroscopy.StainTechnology58(6):
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versity ofFlorida, Gainesville, FL; and the
Sharp,D. 1918. StudiesinRhynchophora. IV.Apre-
anonymous reviewers for their editorial liminarynote on the malegenitalia. Transactions
comments. ofthe Entomological Society ofLondon 1918(1-
I additionally thank P. M. Choate, Uni- 2): 209-222, pi. IX.
versity of Florida, Gainesville, FL; P. W. Sharp,D.andF.A.G. Muir. 1912. Thecomparative
Kovarik,OhioStateUniversity, Columbus, anatomy ofthe male genital tube in Coleoptera.
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versity,Tallahassee, FL;andR. B. Selander, Thompson, R. T. 1988. A method for inflating the
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