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Advances in insect preparation: bleaching, clearing and relaxing ants (Hymenoptera: Formicidae)


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Myrmecologists use a variety of methods for clearing (macerating) and relaxing ants. Bleaching, however, has virtually never been applied in morphological studies. Here we describe a combination of a common bleaching treatment of insect cuticle with hydrogen peroxide and a subsequent clearing of adhering soft tissues with either lactic acid or proteolytic enzymes. This technique allows viewing of the internal morphology of ants without dissection. The resulting glassy specimens reveal valuable morphological characters and may be used as three-dimensional morphological maps to guide the dissection of additional specimens. Bleached specimens are thus particularly useful as teaching material. Positive side effects of the treatment are the extension of (a) retracted mouthparts, (b) sting apparatus and (c) armatures of the male genitalia. An underwater preparation procedure with subsequent fan drying is also described for relaxing specimens preserved in absolute ethanol. Based on tests of relaxing methods, a much less harmful but equally effective modification of the carcinogenic Barber's fluid is described for relaxing and cleaning purposes of pinned or card-point mounted specimens.
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Myrmecological News 12 15-21 Vienna, September 2009
Advances in insect preparation: bleaching, clearing and relaxing ants (Hymenoptera:
Myrmecologists use a variety of methods for clearing (macerating) and relaxing ants. Bleaching, however, has virtually
never been applied in morphological studies. Here we describe a combination of a common bleaching treatment of in-
sect cuticle with hydrogen peroxide and a subsequent clearing of adhering soft tissues with either lactic acid or proteo-
lytic enzymes. This technique allows viewing of the internal morphology of ants without dissection. The resulting glassy
specimens reveal valuable morphological characters and may be used as three-dimensional morphological maps to guide
the dissection of additional specimens. Bleached specimens are thus particularly useful as teaching material. Positive side
effects of the treatment are the extension of (a) retracted mouthparts, (b) sting apparatus and (c) armatures of the male
genitalia. An underwater preparation procedure with subsequent fan drying is also described for relaxing specimens pre-
served in absolute ethanol. Based on tests of relaxing methods, a much less harmful but equally effective modification of
the carcinogenic Barber's fluid is described for relaxing and cleaning purposes of pinned or card-point mounted specimens.
Key words: Hydrogen peroxide, proteolytic enzymes, lactic acid, Barber's fluid, ethanol preservation, transparent ants.
Myrmecol. News 12: 15-21 (online 6 October 2008)
ISSN 1994-4136 (print), ISSN 1997-3500 (online)
Received 8 May 2008; revision received 17 July 2008; accepted 23 July 2008
Marcus Stüben (contact author) & Prof. Dr. K.E. Linsenmair, Department of Animal Ecology and Tropical Biology,
Biocenter, University of Würzburg, Am Hubland, D-97074 Würzburg, Germany. E-mail:
The continuing biodiversity crisis urgently demands the
assessment of our planet's biological diversity and this re-
quires the best possible preservation of specimens for cur-
rent and future studies (KING & PORTER 2004). The diverse
nature of invertebrate groups and the various entomolo-
gical disciplines require a broad array of preparatory tech-
niques to assure specimen preservation (MARTIN 1977,
2000, MILLAR & al. 2000). Species-specific diagnostic fea-
tures and other morphological characteristics of insect spe-
cimens often have to be revealed by preparatory treatments
like bleaching, clearing and relaxing. Complex structures
like mouthparts, sting apparatus and male genitalia are
mostly retracted and often polluted with solidified body
fluids, dirt or even glue in the case of card-point mounted
specimens. Legs and antennae frequently conceal other
body parts that are important for identification. Dark col-
ours can complicate the analysis of the degree of cuticle
sclerotisation and prevent viewing of internal structures
without dissection. Thus, fully transparent but intact spe-
cimens are very desirable. Additionally, glassy specimens
can provide three-dimensional morphological maps for
dissection of other similar specimens and are valuable for
teaching material in entomology courses. BAUERMEISTER
(1959) published a time-consuming method to produce
transparent molluscs based on the refraction indices of tis-
sues but this approach seems impractical for daily use with
This study on bleaching ants was inspired by our col-
league, Dr. Timo Moritz (pers. comm.), who transforms
alcohol preserved and formalin fixated fishes into entirely
transparent specimens based on the work of DINGERKUS
& UHLER (1977). We present a simple, effective technique
for routine use based on a combination of hydrogen per-
oxide bleaching with clearing procedures. We describe how
retracted mouthparts, sting apparatus and male genitalia
can be extended in bleached specimens. We refine an un-
derwater preparation procedure with subsequent fan drying
for relaxing and a better handling of ants intended for card-
point mounting. Finally, useful chemical solutions like
Barber's fluid (VALENTINE 1942) for cleaning and relaxing
stiff and brittle specimens in dry collections have either
frequently fallen into oblivion or are unacceptable due to
their toxicity. We offer a modified recipe for Barber's fluid
which is a less harmful version of the carcinogenic original.
We chose the ponerine ant Pachycondyla analis (LA-
TREILLE, 1802) (Hymenoptera: Formicidae: Ponerinae) as
a model organism (see Fig. 1). The heavily sclerotised,
dark-coloured, polymorphic workers range from approxi-
mately 9 mm (minor worker) to 19 mm (major worker) in
total body length. The species has winged males and pupae
with dark brown silky cocoons. All specimens were collect-
ed by the first author approximately 4 km west of the Blue
Nile River, Sudan (12° 45' 34" N, 34° 06' 45" E; 444 m a.s.
l.), July 2004. Immediately after collection all specimens
were killed with and preserved in absolute ethanol (p.A.,
min. 99.8 volume percent ethanol) until used for our exper-
iments. The specimens did not show any indication of
leaching or bleaching due to their long storage in their
preservative. All recipes and details of the chemicals are
given in Box 1. Photographs were taken with the single-
lens reflex digital camera Nikon D70 equipped with a
"1:2.8/105 mm AF Micro Nikkor" macro lens. A single
fluorescent light bulb (E27, 15W) served as light source.
Methods and Results
Bleaching with hydrogen peroxide
Depending on the internal structures which are to be re-
vealed bleaching with hydrogen peroxide (for a view on
soft tissues like muscles and tendons) or bleaching in
combination with clearing (for a view on cuticular struc-
tures) had to be chosen. Based on comparative tests using
hydrogen peroxide with concentrations from 10 to 35%,
the best results were obtained with a 35% concentration.
Solutions stronger than this were increasingly hazardous
because of their strong oxidising properties and should be
Major workers, males and pupae within their cocoons
were taken from the absolute ethanol preservative and in-
dividually submerged in excavated glass blocks contain-
ing the 35% hydrogen peroxide. The blocks were covered
with small glass plates to prevent desiccation and stored
in a bright environment at room temperature. Depending
on the colour and the degree of sclerotisation of the cu-
ticle this treatment required between a few hours and 13
days. The specimens brightened within 24 hours. After 48
hours they were noticeably transparent except for the more
sclerotised parts of the cuticle such as the head and the ali-
trunk (see Fig. 2). In an early stage of bleaching when the
cuticle already was transparent but soft tissues were not
yet macerated to pulp, the internal tissues adjacent to the
inner side of the cuticle could be viewed (e.g., muscles and
tendons within the legs). Finally the tissue pulp spread non-
uniformly within the body.
The petiole and the gaster developed transparency. The
cocoons lightened from dark brown to light brown and the
commonly adhering soil particles detached. The cocoons
were not distended by nascent oxygen due to their poro-
sity. After one to two weeks the cuticle of P. analis wor-
kers and males appeared completely transparent and glassy.
Oxygen bubbles were mainly present in the alitrunk, the
coxae and the gaster. The wings of the males lost part of
their lifelike shape by curling up at their tips. The cocoons
of the pupae entirely dissolved leaving the bare pupae be-
As soon as the cuticle became transparent and offered
an unhindered view on inner structures, the specimens were
taken out of the hydrogen peroxide bath and thoroughly
rinsed with water or absolute ethanol depending on the
next treatment. For subsequent clearing with proteolytic
enzymes or lactic acid, water turned out to be superior but
for preservation alcohol was used. The results of the bleach-
ing procedure depended on the light intensity, the duration
of bleaching and the concentration of hydrogen peroxide.
Bleaching in a bright environment was faster than in the
dark. The longer the immersion and the higher the con-
centration of hydrogen peroxide, the more light-coloured
as the cuticle of the specimens. A side effect of bleach- w
Fig. 1: Pachycondyla analis, intermediate worker in ab-
solute ethanol before bleaching and clearing.
Fig. 2: Pachycondyla analis, intermediate worker after 3
days of bleaching with 35% H2O2.
ing was a decrease in the thickness and stability of the cu-
ticles. Soft-bodied specimens like workers or males freshly
emerged from their pupae tended to be partially disinte-
grated if bleached for too long. Specimens left for three
weeks in hydrogen peroxide became too soft for pinning
or card-pointing.
Bleaching in combination with clearing
Bleaching specimens in a 35% hydrogen peroxide solution
lead to transparent cuticles and to pulped, non-uniformly
distributed internal tissue fluffs. Specimens were subse-
quently cleared by treatment with lactic acid or the pro-
teolytic enzyme pepsin as follows. First, the specimens were
bleached as above just until they reached transparency
for an unhindered view of targeted cuticle regions without
too much thinning of the cuticle. Second, the specimens
were thoroughly washed in water to eliminate remaining
hydrogen peroxide. The samples then were transferred
into excavated glass blocks filled with either lactic acid or
a pepsin-solution (see Box 1). The blocks were covered
with glass plates and stored in a dark place at room tem-
perature for up to 13 days. During this period, it was oc-
casionally necessary to renew the pepsin-solution once or
twice when evaporation occurred. The appearance of floc-
culation inside specimens indicated an incomplete diges-
Box 1: Recipes.
Barber's fluid after VALENTINE (1942), in order of mixture!
Original recipe (carcinogenic) our modified recipe, pH-value: 4
265 parts 95% ethanol 51 ml 100% ethanol
35 parts benzene 7 ml acetone (pure)
95 parts ethyl acetate 19 ml ethyl acetate (pure)
245 parts water (aqua dest.) 51 ml water (aqua dest.)
Lactic acid (IUPAC systematic name: 2-hydroxypropanoic acid)
Pure quality, pH value: 0.8
Pepsin-solution after KLESS (1986), pH value: 1.6
2.5 g powdered pepsin
2.5 ml concentrated hydrochloric acid
100 ml water (aqua dest.)
Trypsin-solution after TAYLOR & VAN DYKE (1985), pH value: 9
2 g powdered trypsin (= pancreatin, pancreatic protease, purified trypsin)
75 ml filtered saturated sodium borate solution (sodium tetraborate,
25 ml water (aqua dest.)
tion. When maceration had entirely cleared all soft tissues,
specimens were taken out of the blocks, rinsed thoroughly
with water and transferred into absolute ethanol or glyce-
rine for preservation. Oxygen bubbles emerging during the
bleaching procedure were eliminated by storing the speci-
mens in absolute ethanol.
Clearing with either lactic acid or proteolytic enzymes
(pepsin) initially produced similar results. The remaining
tissue fluffs were completely digested and a fully trans-
parent, glassy ant was obtained (see Fig. 3). In a few cases,
the use of pepsin created poor specimens when they were
transferred too quickly into absolute ethanol. The degrada-
tion products of incompletely digested soft tissues (formerly
soluble in water) precipitated throughout the entire speci-
men. The appearance changed immediately from transpa-
rent to translucent. In general, lactic acid and pepsin did
not weaken the cuticle in contrast to long immersion in
35% hydrogen peroxide solution. Enzymatic clearing alone
also proved to be an efficient means of extracting large
series of ant pupae out of their cocoons without damag-
ing their fragile structures. A reverse chronological order
of treatments, i.e., clearing with subsequent bleaching,
proved suboptimal as this led to specimens being translu-
cent rather than really transparent.
Fig. 3: Pachycondyla analis, major worker after 14 days
in 35% H2O2 and subsequent clearing with lactic acid.
cimens and their appendages were relaxed and ready to be
Retracted structures were extended as a side effect of
bleaching. This effect resulted from the combined effects
of soaking, macerating and pushing, with the latter due to
an increasing internal pressure of the nascent oxygen. Struc-
tures like mandibles protruded during the procedure or
were pulled out easily. The sting apparatus became mov-
able and the male genitalia and entire gaster stretched out
of most of the specimens. The boundary of sternites and
tergites with the intersegmental membranes were exposed
and this allowed precise dissections free from the risk of
Bleaching for extending retracted mouthparts,
sting apparatus and male genitalia
The specimens were treated as described in the previous
two sections but the duration of the treatment was reduced
to 3 to 5 days. The hydrogen peroxide bath was halted when
either the retracted appendages protrude or the organs could
be extracted easily with a forceps or a hooked insect pin. In
about one third of the P. analis specimens, the mouth-
parts were extended. With the remaining specimens, closed
mandibles could usually be opened without force and with-
ut breaking the mandibular muscles. Generally the spe-
Underwater preparation and card-point mounting
of specimens preserved in absolute ethanol
For pinning or card-mounting ants that had been preserved
in absolute ethanol the specimens were transferred into o
Petri dishes containing a layer of paraffin wax, about five
millimetres thick and totally submerged in distilled water.
After a few minutes of soaking the specimens were care-
fully turned so that the ventral parts face downward. With-
out piercing the specimens, lifelike postures of the legs,
antennae, wings and the head were fixed with insect pins
on the wax layer. Care had also to be taken when mani-
pulating the joints of the funiculus or the tarsi to avoid pos-
sible breakage. The hind parts of the wings usually needed
special support from additional pins to prevent folding up
during subsequent treatments. Occasionally, specimens of
the same size, colour, and colony reacted differently.
As soon as the specimens were temporarily fixed, the
water within the dishes was emptied and the surface of the
ants was rinsed with absolute ethanol for a few seconds.
The alcohol was poured out and the position of append-
ages, especially of the wings, was checked and rearranged
if necessary. Blow-drying with a hair drier or a computer
fan was then used until the pubescence, pilosity and, par-
ticularly, the wings were dried without sticking together
thus providing a natural appearance. When the wings folded
up the procedure of moistening, smoothing and drying had
to be repeated. After blow-drying the surface of the ants,
the whole specimens were left to dry naturally in air until
their appendages became stiff. Then all insect pins were
removed with care and the specimens were mounted on
card points in the conventional manner (PIECHOCKI & HÄN-
DEL 1996). When large specimens required direct pinning
this was done before the drying procedure. The method
permitted rearrangement of most of the appendages (legs,
head, antennae and gaster) except for the wings, the pos-
ture of which could not be changed due to the position of
thoracic plates at the time of death.
A modified Barber's fluid for cleaning and relaxing
of mounted or dry preserved specimens
Barber's fluid (VALENTINE 1942) is used mainly for clean-
ing and relaxing specimens or their appendages. We mod-
ified the original recipe by substituting acetone for the car-
cinogenic benzene (see Box 1). The ingredients were mixed
at room temperature in the order alcohol, acetone, ethyl ace-
tate and finally distilled water. When the liquids did not
mix entirely, we followed the advice of OEHLKE (1967) to
warm up the fluid using a water bath.
Generally the Barber's fluid was locally applied with a
pair of tweezers or a fine hair brush, but we also sub-
merged specimens in the fluid. A few drops sufficed for
cleaning card-point mounted specimens polluted with non-
water-soluble glue. Within seconds the glue dissolved and
the affected body parts could be cleaned. In the same way
we used the fluid to remove glued specimens from their
card-points. Without exception the glues were successfully
removed, even old glue of unknown composition. For re-
laxing stiff extremities of dried ants, the fluid was also
effective. Legs and coxae of card-point mounted specimens
that were brushed with the fluid became movable in less
than a minute. Femora and tibiae were adjustable as well.
Structures like mandibles sometimes needed repeated brush-
ing or even partial submerging in the fluid for similar re-
sults. Whenever appendages resisted the slight force of the
forceps while testing, the fluid was repeatedly applied to
enable further penetration into specimens. Immersion in the
fluid also proved effective for relaxing whole specimens
formerly preserved in alcohol. Alcohol killed and preserved
material was dried under a lamp and slightly moistened
with the fluid as done by VALENTINE (1942).
Bleaching with hydrogen peroxide
During bleaching, hydrogen peroxide disintegrates into
water molecules and free oxygen radicals through auto-
lysis, with the latter causing the bleaching of the colour
pigments of the insect cuticle. With a 35% hydrogen per-
oxide solution we produced a transparent cuticle and of-
fered a unique view onto lightened internal soft tissues pre-
viously hidden by cuticle. Since the bleaching solution is
water-based, rinsing with water will not distort specimens.
We focused our experiments on a heavily sclerotised spe-
cies. If weakly sclerotised species are to be preserved in
alcohol specimens should be treated stepwise with baths of
gradually increasing alcohol concentrations as published
by PIECHOCKI & HÄNDEL (1996). With bleaching we adopt
a less time-consuming method than the procedure based
on the refraction indices of tissues suggested by BAUER-
MEISTER (1959). Bleaching is a widespread method of se-
lectively eliminating pigmentation of body structures for
different purposes, such as subsequent staining. It has been
used for the bones of vertebrates like fish (TAYLOR 1967),
and for invertebrates like molluscs (PIECHOCKI & HÄN-
DEL 1996). It has also been used for insects (BODE 1975,
SCHAUFF 1986, UPTON 1994, MILLAR & al. 2000).
Bleaching in combination with clearing
Glassy specimens consisting solely of cuticle can be achiev-
ed by a combination of bleaching the cuticle and totally
clearing the inner soft tissues. Prolonged immersion in the
hydrogen peroxide solution leads to maceration but in con-
trast to proteolytic enzymes and lactic acid weakens the
cuticle's strength and structure. To avoid too much thin-
ning, bleaching should be stopped as soon as the cuticle
becomes sufficiently transparent to view underlying struc-
tures. Subsequent clearing of the remaining tissue is best
achieved using the proteolytic pepsin devised by KLESS
(1986). However, earlier clearing experiments for male gen-
italia preparations of Pachycondyla analis revealed that a
trypsin solution (TAYLOR & VAN DYKE 1985) worked as
well, if not better (M. Stüben, unpubl.). M. Maruyama (pers.
comm., Kyushu University Museum, Japan) uses protease
K (a broad-spectrum serine protease) both for DNA extrac-
tion and genitalia cleaning in order to finally embed slide-
mounted male genitalia in Euparal. The advantage of us-
ing proteolytic enzymes is that membranes possessing chit-
inous structures are left intact unlike those treated with the
more customary potassium hydroxide (KOH) solution (KA-
NAAR 1990). Maruyama (pers. comm.) also uses KOH on
occasion and confirmed that genitalia of small ant spe-
cies are sometimes crushed or modified, if not carefully
cleared at low temperature and low concentrations of KOH.
EGUCHI (2002) presented a technique for clearing ant male
genitalia with a consecutive treatment of KOH, lacto-
phenol, aceto-salicylate and finally carbo-xylol in prepara-
tion of mounting genitalia parts on a slide glass with Ca-
nada balsam.
In entomology, clearing by dissection or maceration is
used when the soft inner tissues hinder the study of the
hard chitinous parts and membranous cuticle. Maceration
is achieved most commonly by the use of caustic solu-
tions, such as potassium hydroxide or sodium hydroxide
solution, lactic acid (MILLAR & al. 2000) or by proteolytic
enzymes (PIECHOCKI & HÄNDEL 1996). These approaches
allow morphological studies of mouthparts, sting appara-
tus or the male genitalia armature (HERING 1931, CLAU-
SEN 1938, GURNEY & al. 1964, WEISE 1970, KANAAR 1990,
CUMMING 1992, EGUCHI 2002, EGUCHI & BUI 2007). Relax-
ation of stiff specimens is another application especially
when other relaxing methods fail (KLESS 1986, MENZEL
& MOHRIG 1991). SCHAUFF (1986) suggests bleaching in-
sect specimens that remain too dark after maceration.
The production of whole, fully transparent insect speci-
mens enables in situ functional and morphological studies of
mouthparts, sting apparatus and male genitalia of ants, and
probably insects in general, prior to a dissection or in paral-
lel with dissections of other specimens from a serial sample.
Thus the investigator can become familiar with the struc-
tures which will help in any further anatomical preparation.
Bleaching for extending retracted mouthparts,
sting apparatus and male genitalia
One of the benefits of using the bleaching solution was
that mouthparts, sting apparatus, and male genitalia be-
came extended. Nascent oxygen generated inside the cavi-
ties of the exoskeleton increased the inner pressure. In spe-
cimens with intact chitinous membranes, this pressure dis-
tends or at least loosens previously retracted structures.
The watery solution together with the nascent oxygen
bubbles also cleanses those structures from solidified body
fluids and dirt. Thus our method is superior to the conven-
tional use of relaxing water chambers that do not benefit
from the oxygen pressure. Producing an internal pressure
is an alternative method when structures resist relaxing
treatments with steam or with our modified Barber's fluid
and when clearing the specimen with caustic solutions
or a proteolytic enzyme is not practicable.
MCRAE (1987) reported excellent results with a method
of soaking adult flies briefly in diethyl-ether, and then re-
moving them to let the vapour pressure extend the pro-
boscis. We tested MCRAE's method on the mandibles of
absolute ethanol preserved and air-dried Pachycondyla ana-
lis specimens but it did not work due to the heavily sclero-
tised exoskeleton and strong mandibular muscles (M. Stü-
ben, unpubl.).
Underwater preparation and card-point mounting
of absolute ethanol preserved specimens
Sampling and storing ants in alcohol has a long tradition
in museum collections. For many years, following the ad-
vice of VIEHMEYER (1918), concentrations of about 70 to
75% were used to prevent the hardening of ant specimens
to keep them in a workable condition for mounting. KING
& PORTER (2004) confirmed the common experience that
specimens killed and stored in 70% and 85% ethanol are
generally flexible and rubbery, with their legs and antennae
readily manipulated without damage, but the appendages
tend to return to their original position and this hinders
mounting. Additionally, specimens stored in 70% alcohol
for six months were noticeably swollen, typically with dis-
tended pleural and intersegmental membranes on the ab-
domen. The difference between these suboptimally preserv-
ed specimens and well preserved ants is self-evident.
The demands of preparatory techniques have changed.
Many collectors are conscious that species which are sam-
pled and preserved have to stand the test of time and have
to ensure multi-purpose studies, because there might never
be a second chance of re-sampling due to environmental
changes and dramatic biodiversity decline. Molecular tech-
niques have increasingly become an integral component
of systematic and ecological work on ants. KING & POR-
TER (2004) recommend killing and storing ants in 95 -
100% ethanol because DNA is best preserved at this type
and concentration of alcohol. They further recommend that
adult ants should be killed and stored in 95% ethanol be-
cause, in addition to ensuring the preservation of DNA,
this will produce specimens that are easier to mount com-
pared to overly brittle specimens resulting from higher con-
centrations or rubbery specimens from lower concentra-
Despite KING & PORTER's (2004) recommendation, the
recent emphasis on molecular techniques has led some myr-
mecologists to use only absolute ethanol. The unfortunate
result of this is that specimens become too hard or brittle
for proper curation without additional treatment. Without
further treatments (e.g., with relaxing fluids or relaxing
chambers) these specimens have limited value for taxo-
nomic identification. A solution is to use short-term water
soaking to speed up relaxation (KING & PORTER 2004). This
method is effective in softening specimens from absolute
ethanol because the alcohol works as a carrier both inside
and on the surface of the specimen (PIECHOCKI & HÄN-
DEL 1996). Following water-softening, specimens can be
rinsed with absolute ethanol and blow-dried to prevent the
pilosity and pubescence from sticking together.
Our procedure should be accessible and easy to use for
amateur and professional entomologists because the com-
ponents are standard in insect preparation techniques. As
a comparison, OEHLKE (1967) treated ichneumonid wasps
preserved in 70% ethanol for seconds to minutes with 96%
ethanol before drying under a heat lamp to prevent pubes-
cence from sticking together. Blow-drying accelerates this
process and gives the most lifelike posture of the mounted
or card-point mounted specimens without affecting pubes-
cence and pilosity. This outcome is especially useful for
producing specimens for reference collections and entomo-
logical exhibitions, where samples should be of the highest
With care taken to avoid shrinking or swelling of the
samples, our method can be easily adapted to other con-
centrations of alcohol. For colour sensitive insect groups
we recommend the technique of MCRAE (1987), using a
sequence of baths in 80% ethanol, absolute ethanol and
finally diethylether to produce mounted specimens, and
modifications compiled by PIECHOCKI & HÄNDEL (1996).
Sampling and storing ants in Scheerpeltz' fluid (SCHEER-
PELTZ 1927, 1936) is not recommended for DNA preser-
A modified Barber's fluid for cleaning and relaxing
of mounted or dry preserved specimens
Conventionally dried specimens are most frequently treat-
ed with steam in relaxing boxes to bring them back to a
workable condition (MARTIN 1977). Using steam with al-
cohol material, however, is not very efficient. It usually
can be applied only to the whole specimen and affects in-
sect wings and pubescence. Most other relaxing methods
are no longer in use, despite the continuing need for proper-
ly curated specimens. For example, in many ecological
studies where passive capture methods (e.g., pitfall trap,
light trap, Malaise, fogging) yield huge amounts of insect
material, insects are often collected and preserved through
direct collection into low-concentration ethanol.
Similarly, large samples of ants are collected and pre-
served in 60 to 70% ethanol (SEIFERT 1996) or 70 to 80%
ethanol because this allows anatomical examinations or
pinning of specimens to be done more or less straight from
the preservative (PIECHOCKI & HÄNDEL 1996). For that rea-
son, according to B. Seifert (pers. comm.), some collec-
tors prefer Scheerpeltz' fluid (SCHEERPELTZ 1927, 1936).
VALENTINE (1942) was the first to publish the recipe
of Barber's fluid which is a very effective and versatile re-
laxing agent. Its applications include cleaning specimens
from old greasy glue, adjustments of extremities during
pinning and corrections of specimens in dry collections.
The last is increasing in importance because digitising type
material has become a common practice (COP-CBD 2006).
If specimens are not properly positioned, even technically
excellent photographs in exemplary databases like that to
be found at can be of very little value for
comparative taxonomic work. Producing high-quality im-
ages is costly and labour-intensive. Thus, non-invasive re-
laxing methods that allow proper adjustment of appen-
dages to expose taxonomically important morphological
features, are particularly valuable. The original Barber's
fluid contained benzene and this is classified by the World
Health Organization as a human carcinogen (FISHBEIN &
O'NEILL 1988).
Our modified recipe uses less harmful chemicals but
still provides excellent relaxing results. As with the original
Barber's fluid (J. Händel, pers. comm.), our specimens be-
came plastic almost instantly and old, greasy specimens
were "rejuvenated". PECK (1974) who used the original
Barber's fluid as preservative for leiodid beetles (Coleo-
ptera, Leiodidae) reported that it did not harden the tissues
of the beetles as alcohol does. We would expect similar
results for our alternative recipe.
M. Stüben was supported by grants from the German Re-
search Foundation (DFG-GRK 200/5) and the University
of Würzburg. We express our sincere thanks to Prof. Ali
Sharaf El-Din, Sudanese Institute for Natural Sciences
(SIFNS). We are grateful to our Sudanese colleagues at the
University of Khartoum and the Faculty of Agriculture in
Abu Naama (University of Sinnar). We thank very much
Brian Taylor for proofreading and Joachim Händel for ac-
cess on hardly accessible articles. We appreciate the use-
ful comments made by Timo Moritz and the anonymous re-
viewers. Their personal comments on this project greatly
improved this manuscript.
Myrmekologen verwenden eine Reihe von Methoden zum
mazerierenden Aufhellen und Aufweichen von Ameisen,
während das Bleichen für morphologische Studien prak-
tisch unbekannt ist. Wir beschreiben eine Kombination des
gebräuchlichen Bleichens von Insektencuticula mit Was-
serstoffperoxid mit der Mazeration anhaftender, weicher
Gewebe durch Milchsäure oder proteolytische Enzyme.
Diese Technik erlaubt einen Einblick in die innere Morpho-
logie der Ameisen ohne Präparation. Die resultierenden
gläsernen Ameisen offenbaren wertvolle morphologische
Merkmale und können als dreidimensionale morphologi-
sche Karte für eine Sektion weiterer Exemplare fungieren
oder als Lehrmaterial dienen. Positive Nebeneffekte des
Bleichens sind die Erleichterung des Lösens (a) zurück-
gezogener Mundwerkzeuge, (b) des weiblichen Stachelap-
parates und (c) der männlichen Genitalarmatur. Eine Me-
thode zur Unterwasserpräparation mit anschließender Lüf-
tertrocknung wird als Präparationsmethode für Ameisen aus
absolutem Ethanol beschrieben. Basierend auf Tests von
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... Organic matter Bacteria Mould/Fungi [33,34] A pictorial archive of specimens before and after treatments was created using a Keyence VHX-S90BE digital microscope, equipped with Keyence VH-Z250R and VH-Z20R lens and VHX-2000 Ver. software (Keyence, Osaka, Japan). ...
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Diptera puparia may represent both in forensic and archaeo-funerary contexts the majority of the entomological evidence useful to reconstruct the peri and post-mortem events. Puparia identification is quite difficult due to the lack of identification keys and descriptions. In addition, external substances accumulated during the puparia permanence in the environment make the visualization of the few diagnostic characters difficult, resulting in a wrong identification. Six different techniques based on physical and chemical treatments have been tested for the removal of external substances from puparia to make identification at species level feasible. Furthermore, the effects of these methods on successful molecular analyses have also been tested as molecular identification is becoming an important tool to complement morphological identifications. The results of this study indicate that cleaning via warm water/soap, the sonication and treatment with a sodium hydroxide solution are the best methods to achieve a good quality of the samples.
... In addition, it has long been known that the prolonged illumination of insect cuticle with intense light, in the presence of oxygen, leads to irreversible bleaching of melanin by its oxidative degradation (Sarna & Swartz 2006), and that other agents, such as hydroquinone, can also be used for this purpose (Arndt & Fitzpatrick 1965). Clearing protocols that allows the investigation of soft tissue as well as the cuticle, making both, cuticle structures and organs have also been reported (Stüben and Linsenmair 2009;Smolla et al. 2014). Combining these methods with advanced QUBIC could enable better whole-mount clearing of large beetles. ...
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Internal tissues of multicellular organisms cannot directly be seen because they contain pigments. For this reason, whole‐body clearing methods have been developed and applied to mammals such as mice. Insects such as beetles, however, cannot be cleared by the mammalian method because of pigments such as melanin in their exoskeletons. In this study, we tried to develop a whole‐body clearing method for large beetles. We first bleached the exoskeleton using a hydrogen peroxide treatment, and applied advanced Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis (CUBIC) reagents to make the internal tissues transparent. The combined method of hydrogen peroxide and advanced CUBIC allowed us to successfully undertake whole‐body clearing of large beetles.
... The high fidelity of CLSM-derived images as compared to those collected by conventional microscopy allows to visualise very thin or small body parts of insects (Klaus and Schawaroch 2006;Michels 2007). Thicker layers can hardly be visualised without resorting to the use of clearing agents to bleach tissues such as hydrogen peroxide (Stüben and Linsenmair 2008), methyl salicylate, lactic acid (Michels 2007) on Murray's clearing solution (1 part benzyl alcohol + 1 part benzyl benzoate) (Zucker 2006). ...
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There are several harmful and yield decreasing arthropod pests, which live within plant tissues, causing almost unnoticeable damage, e.g. Ostrinia nubilalis Hbn., Cydia pomonella L., Acanthoscelides obtectus Say. Their ecological and biological features are rather known. The process leading to the damage is difficult to trace by means of conventional imaging techniques. In this review, optical techniques—X-ray, computer tomography, magnetic resonance imaging, confocal laser scanning microscopy, infrared thermography, near-infrared spectroscopy and luminescence spectroscopy—are described. Main results can contribute to the understanding of the covert pest life processes from the plant protection perspective. The use of these imaging technologies has greatly improved and facilitated the detailed investigation of injured plants. The results provided additional data on biological and ecological information as to the hidden lifestyles of covertly developing insects. Therefore, it can greatly contribute to the realisation of integrated pest management criteria in practical plant protection.
... The specimens studied were pinned dry or preserved in ethanol. Dry specimens were relaxed with Barber's fluid modified from Stuben and Linsenmair (2009), and the left proleg was removed, cleaned manually with forceps and a fine brush with isopropyl alcohol, kept submerged in contact lens solution Renu ® for 24 h, and then agitated in an ultrasonic bath (5.400 kHz) with warm water and detergent solution for 3 min or less for small structures. ...
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Assassin bugs (Hemiptera, Heteroptera, Reduviidae) have diverse and complex morphological and behavioral adaptations for prey capture. Several of these morphological adaptations occur on the proleg. The prolegs of Emesinae are typically raptorial and they are used for grooming, grasping and hunting prey. Several morphological characters that define Emesinae as a group are found on the proleg, such as the anterior opening of the acetabula, the elongation of the procoxa, and the lateral (campaniform) sensilla on the protibia. Metapterini comprises 28 genera and approximately 280 described species, and are characterized by a conspicuous basal process of the anteroventral series of the profemur, and highly modified pretarsal structures. In this study, structures of the proleg are documented for 13 genera of Metapterini, using stereomicroscopy and scanning electron microscopy (SEM). Detailed descriptions and digital macrophotographs are provided for most of the genera for the first time, and from this morphological documentation 38 phylogenetic characters are coded, presented as a data matrix, and analyzed cladistically, and their potential usefulness for resolving relationships among Metapterini is discussed.
... We therefore tested different reagents for their effectiveness, which were already proposed for cleaning insects for the SEM (e.g.,Beutel, Friedrich, Yang, & Ge, 2014). However, we did not used hydrogen peroxide, because this substance can damage the chitinous cuticle surface structures (Stueben and Linsenmair, 2009).An incubator is not mandatory, as the specimens can incubate at room temperature. ...
The objective of the present study was to compare cleaning methods for delicate insect specimens for investigations with scanning electron microscopy (SEM). As typical specimens we used aquatic larvae of mosquitoes, springtails, larvae of mayflies and caterpillars because they are very fragile and large parts of their body consist of soft tissue. Additionally their cuticle is very often covered with dirt, soil particles or other materials. Cleaning with ultrasonic sound, as the most common cleaning method used for SEM, will destroy fragile insects. Therefore we tested different procedures to remove the dirt particles. In a first approach we compared cleaning with Potassium hydroxide (KOH), Proteinase K, and Triton X in aquatic larvae of flies, which were available in numbers and kept under the same conditions. As our results showed that the treatment with KOH gives the best results we treated in a second approach springtails, larvae of mayflies and caterpillars only with KOH. The springtails and caterpillars were largely free of particles after treatment with KOH; however, the larvae of mayflies were still covered with remnants of diatoms and precipitates of calcium carbonate of the algae. KOH dissolves organic impurities, on the other hand silicon dioxide and lime crusts are not solved. With this limitation, treatment with KOH is a simple technique for routine use as cleaning method for fragile insect specimens for SEM.
... A combination of toluidine blue, borax and pyronin G is widely used as staining agents for these sections [9]. In CLSM and LFSM bleaching with various agents [42][43][44][45] is required for non-transparent samples. Additionally, the fluorescence of certain tissues can be increased or altered with different staining techniques or by combining bleaching with standard whole-mount immunocytochemistry (e.g. ...
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Chitin is the major scaffolding component of the insect cuticle. Ultrastructural analyses revealed that chitin adopts a quasi-crystalline structure building sheets of parallel running microfibrils. These sheets called laminae are stacked either helicoidally or with a preferred orientation of the microfibrils. Precise control of chitin synthesis is mandatory to ensure the correct chitin assembly and in turn proper function of cuticular structures. Thus, evaluation of chitin-metabolism deficient phenotypes is a key to our understanding of the function of the proteins and enzymes involved in cuticle architecture and more generally in cuticle biology in insects. Usually, these phenotypes have been assessed using electron microscopy, which is time-consuming and labor intensive. This stresses the need for rapid and straightforward histological methods to visualize chitin at the whole tissue level. Here, we propose a simple method of chitin staining using the common polysaccharide marker Fluorescent brightener 28 (FB28) in whole-mount Drosophila melanogaster. To overcome the physical barrier of FB28 penetration into the cuticle, staining is performed at 65°C without affecting intactness. We quantify FB28 fluorescence in three functionally different cuticular structures namely wings, dorsal abdomens and forelegs by fluorescence microscopy. We find that, as expected, cuticle pigmentation may interfere with FB28 staining. Down-regulation of critical genes involved in chitin metabolism, including those coding for chitin synthase or chitinases, show that FB28 fluorescence reflects chitin content in these organs. We think that this simple method could be easily applied to a large variety of intact insects.
Although scanning electron microscopy (SEM) has been broadly used for the examination of fixed whole insects or their hard exoskeleton‐derived structures, including model organisms such as Drosophila, the routine use of SEM to evaluate vulnerable soft internal organs and tissues was often hampered by their fragile nature and frequent surface contamination. Here, we describe a simple four‐step protocol that allows for the reliable and reproducible preparation of the larval and prepupal salivary glands (SGs) of Drosophila for SEM devoid of any surface contamination. The steps are to: first, proteolytically digest the adhering fat body; second, use detergent washes to remove contaminating coarse tissue fragments, including sticky remnants of the fat body; third, use nonionic emulsifying polysorbate emulsifiers to remove fine contaminants from the SGs surface; and fourth, use aminopolycarboxylate‐based chelating agents to detach sessile hemocytes. Short but repeated rinses in 100 μL of a saline‐based buffer between steps ensure efficient removal of remnants removed by each treatment. After these steps, the SGs are fixed in glutaraldehyde, postfixed in osmium tetroxide, dehydrated, critically point‐dried, mounted on aluminum stubs, sputter coated with gold–palladium alloy and examined in the SEM.
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A complementary description for Neanopidium mexicanum Dajoz, 1975, type species of the genus, is provided based on specimens from the type locality Valle Nacional (Oaxaca, Mexico), including description and illustrations of male and female abdominal terminalia, which is provided for the first time to a Neanopidium species.
Eleven new species of Hlavaciellus Jaloszynski, 2006 are described from Malaysia: H. adelphos, H. angustifrons, H. bifoveatus, H. cirrus, H. clandestinus, H. decoratus, H. extremalis, H. metrios, H. miser, H. peninsularis, and H. smetanai. Diagnostic characters, including head modifications of males and aedeagi are illustrated, and detailed morphology of Hlavaciellus is described for the first time on the basis of disarticulated specimens. A preliminary parsimony-based phylogenetic analysis of most currently known genera of the Cephenniini (with two genera of the Eutheiini as an outgroup) provided a robust support for the previously suggested placement of Hlavaciellus as a sister group taxon to Cephennodes Reitter, 1884. The Cephennomicrus group of genera was placed as a sister group to Cephennodes + Hlavaciellus.
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Collectors use a variety of concentrations and types of alcohols to preserve ant specimens. We evaluated existing literature, experimental evidence, and expert myrmecological advice to determine what kind and concentration of alcohol will result in the best preserved specimens for card-point mounting and DNA extraction. For our experimental evaluation, we killed and stored Solenopsis invicta, Camponotus floridanus, and Dorymyrmex bureni workers in isopropanol and ethanol at four concentrations (70, 85, 95, 100 %) over three time periods (24 h, 1 month, 6 months). We then compared specimen condition and amenability to manipulation for mounting on card points. Specimens stored in either 95% isopropanol or 95 % ethanol for time periods longer than 24 h produced the best specimens for mounting. A literature review revealed that DNA is best preserved in 95 – 100 % ethanol due to the ability of ethanol to more rapidly penetrate cellular membranes and deactivate DNase activity than other primary alcohols. We recommend that general collections of adult ant specimens should be killed and stored in 95 % ethanol. Following this recommendation will result in ant specimens that are easier to mount for museum collections and better preserved for molecular studies. A variety of other killing and preservation techniques relevant to the study of ants are also discussed.
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In the collection of the Institut für Pflanzenschutzforschung Kleinmachnow, Bereich Eberswalde, Abt. Taxonomie der Insekten there are 50 males and 79 females of Sciaridae collected by H. SAUTER in Taiwan. There is described a new method for the preparation of dry mounted specimens on slides. All males and one female of the Taiwan collection were mounted on slides. Three species were new combined: Lycoriella pammela (EDWARDS, 1928), Peniosciara cornuta (LENGERSDORF, 1927) and Bradysia dissimilis (LENGERSDORF, 1927). Redescriptions of Sciara copiosa LENGERSDORF, 1927, Eurysciara rostrata LENGERSDORF, 1927, Bradysia dissimilis (LENGERSDORF, 1927) and Peniosciara cornuta (LENGERSDORF, 1927) are given, Bradysia sauteri is described as new. All taxonomic and faunistic data given by LENGERSDORF (1927) were checked, corrections to these data are given. [in German]
The myrmicine ant genus Parvimyrma is newly established for a single new species found from N. Vietnam. The genus is undoubtedly placed in the Solenopsis genus group, and it is distinguished from the other genera belonging to the genus group by a combination of the following features: posteromedian portion of clypeus narrowly inserted between frontal lobes; masticatory margin of mandible with 5 distinct teeth; antenna 11-segmented, with a 2-segmented club; eye completely absent; promesonotum in profile almost flat or very weakly convex dorsally; metanotal groove relatively shallowly impressed dorsally; propodeum unarmed; propodeal spiracle small, situated a little behind the midlength of the sides of propodeum; metapleural gland large; petiolar peduncle with a small anteroventral process; postpetiole narrowly attached to the anteriormost end of gaster; sting poorly developed.
Sodium hydroxide in the form of household flake lye is a substitute which is more gentle on delicate tissues than potassium hydroxide. Several types of small dishes and cavity plates are convenient containers in which to study genitalic preparations. Suggestions are made for the storage of genitalia in glycerine within microvials, especially the avoidance of too much glycerine and the need to slant the vials to keep the corks dry when they are pinned. Plastic microvials and synthetic rubber stoppers are suggested as substitutes for glass microvials and cork stoppers.
The structure of the organs taking part in oviposition of Thripidae (ovipositor, ectodermal exit system, spermatheca, accessory gland) has been investigated by light- and electron microscopy. Some histochemical tests were also performed. The general structure of the ovipositor corresponds to that known for other pterygote insects, except for a few pecularities, e.g. the absence of third valvulae (or gonapophyses IX). The spermatheca is a thin-walled vesicle consisting of wall cells and single gland cells. It is connected to the vagina by socle cells containing a well-developed system of infoldings of the apical plasma membrane. The spermathecal duct has a valve. The sperm ball in the spermathecal lumen is surrounded by an enveloping structure. Several authors have categorized it as a spermatophore, a problematic interpretation in some respects. The accessory gland is a typical ectodermal insect gland, which probably produces a mucoprotein secretion as a lubrication and/or cementing agent for oviposition. The ultrastructure of the gland cells depends on the maturity stage of the animal. The accessory gland duct is also provided with a valve. A hypothesis concerning the mechanical function of the oviposition apparatus as well as some cytological observations are discussed; and the structure of the Thripid ovipositor is compared with that in other Pterygota.