Appl. Entomol. Zool.

44 (2): 281291 (2009)

http://odokon.org/

Quenching autofluorescence of insect tissues for in situ detection of

endosymbionts

Ryuichi KOGA,* Tsutomu TSUCHIDA and Takema FUKATSU

Institute for Biological Resources and Functions, National Institute of Advance Industrial Science and Technology (AIST);
Tsukuba, Ibaraki 3058566, Japan
(Received 7 October 2008; Accepted 8 December 2008)

Abstract
Oligonucleotide-probed fluorescent in situ hybridization (FISH) targeting 16S rRNA is a powerful technique for de-
tecting and characterizing bacterial cells in environmental samples without cultivation; however, general application
of the technique to insect endosymbionts has been hindered by the strong autofluorescence frequently observed in in-
sect tissues. Here we describe a protocol that markedly reduces autofluorescence of insect tissues by hydrogen perox-
ide (H2O2) treatment, whereby 16S rRNA of bacterial endosymbionts is kept in a FISH-detectable condition. Among
various histological fixatives, Carnoys solution was superior in that whole insects were successfully fixed and auto-
fluorescence of insect tissues was suppressed in comparison with the widely used formaldehyde-based fixatives.
Treatment with both alcoholic 6% H2O2 solution and aqueous 6% H2O2 solution markedly reduced autofluorescence
of the fixed insect tissues, wherein the former kept 16S rRNA of bacterial endosymbiont in a FISH-detectable condi-
tion while the latter failed to do so. The protocol was applicable to endosymbionts of diverse insects such as aphids,
lice and bat flies. The protocol was applicable not only to fresh insect samples but also to archival insect samples pre-
served in acetone for several years. We propose a general and robust protocol for quenching autofluorescence of insect
tissues for FISH detection of bacterial endosymbionts, which is potentially applicable to endosymbionts of a wider
range of organisms with considerable autofluorescence.

Key words: Fluorescent in situ hybridization; hydrogen peroxide; Carnoys solution; quenching autofluorescence of
insect tissues

sequencing. Most of these endosymbiotic bacteria,

INTRODUCTION
Many insects are closely associated with en-
dosymbiotic bacteria, which are harbored in the gut
lumen, in the cecum connected to the gut cavity, in
gut epithelial cells, in the body cavity, or in spe-
cialized cells called bacteriocytes or mycetocytes
(Buchner, 1965). Some endosymbionts are obligate
associates for their hosts and contribute to host fit-
ness, other endosymbionts are facultative compan-
ions of their hosts and cause context-dependent fit-
ness effects, other endosymbionts are parasitic mi-
crobes that negatively affect host fitness, and the
majority of the others are of unknown nature that
have been recognized solely by means of mi-
croscopy, PCR detection and/or 16S rRNA gene
particularly endocellular bacteria of mutualistic
nature, are difficult to culture outside their host
bodies or cells, probably because they have become
highly adapted to the particular environment inside
the host organism and can no longer survive else-
where (Baumann and Moran, 1997). Molecular
phylogenetic characterization based on PCR ampli-
fication and sequencing of the 16S rRNA gene has
been the principal technique for characterizing
these fastidious bacteria (Boone et al., 2001;
Hugenholtz, 2002). Certainly, the molecular phylo-
genetic approach is a powerful and widely applica-
ble technique, but bacterial characterization solely
based on gene sequence is, needless to say, not sat-
isfactory. Multiple endosymbiotic bacteria often

* To whom correspondence should be addressed at: E-mail: r-koga@aist.go.jp

Present address: Molecular Entomology Laboratory, Advanced Science Institute, Riken, Wako 3510198, Japan.
DOI: 10.1303/aez.2009.281

281
282 R. KOGA et al.
coexist in the same host insects, from which sev-
eral different 16S rRNA gene sequences are
obtained (cf. Fukatsu and Nikoh, 1998, 2000;
Tsuchida et al., 2002; Moran et al., 2005). Local-
ization of these co-infecting endosymbionts may
reflect their biological functions and host-symbiont
and symbiont-symbiont interactions, but gene se-
quences alone provide little insight into such bio-
logical aspects of insect-microbe endosymbiosis.
To integrate cytological information with molec-
ular data, therefore, oligonucleotide-probed in situ
hybridization targeting 16S rRNA has been an
essential component for bacterial characterization
(Amann and Fuchs, 2008). Using this technique,
even if the bacteria of interest in environmental or
insect samples cannot be cultured, each of 16S
rRNA sequences obtained from the samples can be
linked to each of the bacterial cells in the samples
under a microscope. In particular, 16S rRNA-tar-
geted fluorescent in situ hybridization (FISH) has
been widely used to analyze bacteria in environ-
mental samples, because of such merits as (i) easy
detection and visualization with synthetic oligonu-
cleotide probes under a fluorescent microscope, (ii)
high sensitivity and resolution due to fluorescent
detection, (iii) convenient simultaneous visualiza-
tion of multiple bacterial species with probes la-
beled with different fluorochromes, and (iv) easy
and rapid optical sectioning and image analysis
using a laser confocal microscope; however, insect
tissues often exhibit significant autofluorescence,
which has limited the range of FISH applications
to insect materials (Fukatsu et al., 1998; Thimm
and Tebbe, 2003).
To circumvent the autofluorescence problem, in
situ hybridization with enzymatic detection sys-
tems has been attempted (cf. Fukatsu and Nikoh,
1998, 2000; Fukatsu et al., 1998, 2000; Fukatsu,
2001; Wilkinson et al., 2003), but the approach has
disadvantages such as a tedious experimental pro-
cedure, lower spatial resolution of signals, and dif-
ficulty of simultaneous detection of multiple en-
dosymbionts in the same samples. Another ap-
proach is to quench the autofluorescence of insect
tissues, thereby eliminating the background noise
for FISH detection. Recently, in a series of trials,
we have developed an experimental protocol that
markedly reduces the autofluorescence of insect
tissues by H2O2 treatment while 16S rRNA of bac-
terial endosymbionts is kept in a FISH-detectable
condition (Koga et al., 2003, 2007; Moran et al.,
2005; Sakurai et al., 2005; Tsuchida et al., 2005;
Sasaki-Fukatsu et al., 2006; Fukatsu et al., 2007;
Kono et al., 2008). In these studies, however, the
technical aspects were briefly described in the
method section only. We have performed a series of
systematic examinations to optimize the protocol
toward better quenching of autofluorescence, im-
proved fixation medium and procedure, better
archival preservation technique for insect materials
before analysis, and improved procedure for sam-
ple processing, most of which have not yet been
published.
Here we describe the details of the optimization
process to quench the autofluorescence of insect
tissues for FISH detection of bacterial endosym-
bionts, which we expect to be extremely useful for
researchers working on symbiotic/parasitic bacteria
associated with diverse insects and other organ-
isms.
MATERIALS AND METHODS
Aphids. A laboratory strain of the pea aphid
Acyrthosiphon pisum Harris (Homoptera: Aphidi-
dae), designated IS, which harbors the secondary
endosymbiont Serratia symbiotica in addition to
the primary endosymbiont Buchnera aphidicola
(Fukatsu et al., 2000), was reared on seedlings of
the broad bean Vicia faba L. (Fabales: Fabaceae) at
20C under a long-day regimen of 16 h light and
8 h dark. A laboratory-maintained colony of the
social aphid Colophina arma Aoki (Homoptera:
Pemphigidae), which harbors the primary endo-
symbiont B. aphidicola and an unnamed secondary
endosymbiont (Fukatsu and Ishikawa, 1993; Ijichi
et al., 2005), was reared on potted plants of Clema-
tis stans Siebold & Zucc (Ranunculales: Ranuncu-
laceae) at 20C under the long-day regimen.
Other insects. The National Institute of Infec-
tious Diseases (NIID) strain of the human body
louse Pediculus humanus L. (Phthiraptera: Pedicul-
idae) was maintained and provided by Minoru Mi-
hara (Sasaki-Fukatsu et al., 2006). The insects
were harvested on 23 March 2005. Samples of the
slender pigeon louse Columbicola columbae L.
(Phthiraptera: Philopteridae) were collected from
the rock pigeon Columba livia L. (Columbiformes:
Columbidae) in Tsukuba, Ibaraki by Takema
Fukatsu on 10 November 2006 (Fukatsu et al.,
 Quenching Autofluorescence of Insect Tissues
2007). Samples of the nycteribiid bat fly Penicil-
lidia jenynsii Westwood (Diptera: Nycteribiidae)
were collected from the Eastern bent-winged bat
Miniopterus fuliginosus Hodgson (Chiroptera: Ves-
pertilionidae) in Sayoh, Hyogo by Nobutaka Urano
on 4 December 2007.
Acetone preservation. Live insects were placed
in 6 ml or 13.5 ml glass vials (Screw Glass Vials
No. 2 or No. 4, Maru-Emu Co. Ltd., Japan) filled
with acetone, which were tightly capped and kept
at room temperature until use (Fukatsu, 1999).
Prior sample processing. To prepare dissected
embryos from fresh aphids, adult insects were dis-
sected in 70% ethanol with fine forceps under a
binocular microscope, and the dissected embryos
were transferred to a new vial filled with fixative.
For whole aphid specimens, live adult insects were
decapitated in a fixative-filled Petri dish, and their
legs, cauda, and cornicles were removed. The in-
sects were then transferred to a new vial filled with
fixative. For acetone-preserved aphid samples, the
insects were placed in 70% ethanol for 1030 min
to soften the tissues, and dissected or decapitated
as described above. For acetone-preserved human
louse and pigeon louse samples, the insects were
decapitated and their cuticle was pricked with a
needle and/or cut with a razor in several places to
facilitate the infiltration of reagents before in situ
hybridization. For acetone-preserved bat fly sam-
ples, their legs were removed and their cuticle was
pricked with a needle and/or cut with a razor in
several places before in situ hybridization.
Fixation. The compositions of the fixatives used
in this study are listed in Table 1. Using aqueous
fixatives (i.e., buffered formalin, buffered glu-
taraldehyde and Bouins solution), fresh aphids
were dissected in a fixative-filled Petri dish, fixed
with fresh fixative in a glass vial overnight at 4C,
and washed and stored in phosphate buffer (pH
7.4) at 4C. Using alcoholic fixatives (i.e., alco-
holic Bouins solution, alcoholic formalin and
Carnoys solution), whole insects were placed in
fixative-filled glass vials and fixed at room temper-
ature. Samples were kept for several days in alco-
holic Bouins solution and alcoholic formalin, and
then washed with 80% ethanol before use. Using
Carnoys solution, the samples were kept overnight
and then washed with 100% ethanol.
Hydrogen peroxide treatment. With reference
to in situ hybridization protocols in previous stud-
283
ies using frogs, mice and chicks (Dent et al., 1989;
Conlon and Herrmann, 1993; Denkers et al.,
2004), we used 6% H2O2 treatment. Aqueous 6%
H2O2 solution was prepared by combining one vol-
ume of 30% H2O2 and four volumes of distilled
water. The fixed insect samples were hydrated
through an ethanol-water series and incubated in
aqueous H2O2 solution until they were decolorized.
The H2O2 solution was exchanged every two or
three days for the first two or three weeks of incu-
bation. The bleached insects were thoroughly
washed with PBSTx (0.8% NaCl, 0.02% KCl,
0.115% Na2HPO4, 0.02% KH2PO4, 0.3% Triton X-
100) and observed by light and epifluorescent mi-
croscopy.
Alcoholic 6% H2O2 solution was prepared by
combining one volume of 30% H2O2 and four vol-
umes of 100% ethanol. The fixed insect samples
were thoroughly washed with 100% ethanol and in-
cubated in alcoholic H2O2 solution until they were
decolorized. The H2O2 solution was exchanged
every two or three days for the first two or three
weeks of incubation. The bleached insects were
thoroughly washed with 100% ethanol, and either
subjected to microscopic observation, in situ hy-
bridization, or storage at 20C until use.
Tissue sectioning. The fixed insect samples
were dehydrated and cleared through a water-
ethanol-xylene series, embedded in paraffin, cut
into serial 5 m m tissue sections, and mounted on
silane-coated glass slides. The tissue sections were
then dewaxed through a xylene-ethanol series, and
dried in air prior to in situ hybridization and cyto-
logical staining.
Probes and counter-staining reagents. The
following fluorochrome-labeled oligonucleotide
probes targeting bacterial 16S rRNA were used for
in situ hybridization: Cy5-Apis2a (5-Cy5-CCT
CTT TTG GGT AGA TCC-3) targeting the pri-
mary endosymbiont Buchnera of A. pisum (Koga et
al., 2003); Cy3-PASSisR (5-Cy3-CCC GAC TTT
ATC GCT GGC-3) targeting the secondary en-
dosymbiont Serratia of A. pisum (Koga et al.,
2003); CA-P-Cy5 (5-Cy5-TTC CAG TGT GGC
TGA TCA-3) targeting the primary endosymbiont
Buchnera of C. arma; CA-S-Cy3 (5-Cy3-TTC
CAG TGT GGC TGG CCA-3) targeting the sec-
ondary endosymbiont of C. arma; and Cy3-
EUB338 (5-Cy3-GCT GCC TCC CGT AGG
AGT-3) targeting eubacteria in general (Amann et
284 R. KOGA et al.
al., 1990). For counter-staining of host insect nu-
clei, the following DNA-binding fluorochromes
were added to the hybridization buffer: 0.5 m M
SYTOX green (Invitrogen, Carlsbad, CA) and/or
100 ng/mL of 4,6-diamino-2-phenylindole (DAPI)
(Wako, Osaka, Japan).
In situ hybridization of tissue sections. About
150 m l hybridization buffer (20 mM Tris-HCl [pH
8.0], 0.9 M NaCl, 0.01% sodium dodecyl sulfate,
30% formamide) containing 100 pmol/ml each of
the probes and the counter-staining fluorochrome
was applied to tissue sections on a glass slide and
covered with a coverslip, and incubated in a hu-
midified chamber at room temperature overnight.
After brief washing with phosphate buffer, the tis-
sue sections were mounted in Slowfade antifade
solution (Invitrogen) with a coverslip, and ob-
served under an epifluorescent microscope (Axio-
phot; Carl Zeiss, Oberkochen, Germany).
In situ hybridization of whole mount samples.
The fixed insect samples were hydrated with
PBSTx, pre-incubated with the hybridization buffer
without probe three times, and incubated with the
hybridization buffer containing the probes and the
counter-staining fluorochrome overnight. After
thorough washing with PBSTx, the insect samples
were equilibrated with Slowfade antifade solution,
and observed under the epifluorescent microscope
and/or a laser confocal microscope (Pascal5, Carl
Zeiss).
RESULTS AND DISCUSSION
Color and autofluorescence of aphid tissues
Live aphids usually exhibit vivid coloration: A.
pisum is green (Fig. 1A) while C. arma is reddish
brown (Fig. 1F). Even embryos in ovarioles have
similar colors, yellowish green in A. pisum and
brown in C. arma, wherein larger embryos are
more strongly colored (Fig. 2A and E). These in-
sects emit intense autofluorescence when observed
by fluorescent microscopy (data not shown).
Permeability of fixatives into aphid tissues
We tested the following standard histological fix-
atives: buffered formalin, buffered glutaraldehyde,
Bouins solution, alcoholic Bouins solution, alco-
holic formalin and Carnoys solution (Table 1).
With the aqueous fixatives represented by buffered
formalin, buffered glutaraldehyde and Bouins so-
lution, whole insects were poorly fixed because of
inferior permeability: the insect tissues were often
damaged when processed into paraffin sections,
and the histology of the tissues was highly degen-
erative. Meanwhile, dissected embryos were suc-
cessfully fixed with the aqueous fixatives. With the
alcoholic fixatives represented by alcoholic Bouins
solution, alcoholic formalin and Carnoys solution,
both whole insects and dissected embryos were
successfully fixed and processed into histological
preparations (Table 1). Since dissecting insects for
fixation in field surveys is too tedious, use of
the aqueous fixatives, including the widely used
buffered formalin, could not be recommended.
Hence, the alcoholic fixatives were further exam-
ined for compatibility with FISH detection of
aphid endosymbionts.
Effect of fixatives on autofluorescence of aphid
tissues
When the insects fixed with alcoholic Bouins
solution were observed by fluorescent microscopy,
the tissues emitted very strong autofluorescence
(data not shown), probably because of picric acid
and formalin contained in the fixative (Fukatsu et
al., 2005). Fixation with alcoholic formalin was
much better, but the tissues still exhibited strong
autofluorescence (Figs. 1C, H and 2B, F). Fixation
with Carnoys solution was better than with alco-
holic formalin; the color and autofluorescence of
the tissues became remarkably weaker, but still
persisted (data not shown). Autofluorescence of the
tissues was generally stronger in the short-wave-
length range (purple to blue) than in the long-
wave-length range (green to red), as has been re-
ported previously (Fukatsu et al., 1998; Thimm and
Tebbe, 2003).
Quenching autofluorescence with hydrogen per-
oxide
We then attempted to decolorize the aphid tis-
sues fixed with Carnoys solution by using hydro-
gen peroxide (H2O2), following the protocols in
previous studies of mice and other animals (Dent et
al., 1989; Conlon and Herrmann, 1993; Denkers et
al., 2004). When treated with aqueous 6% H2O2 so-
lution, the tissues were certainly decolorized, but
were ragged and the nuclei of the tissues exhibited
poor stainability with DAPI and SYTOX green
(data not shown), indicating the degradation of tis-
 Quenching Autofluorescence of Insect Tissues
Fig. 1. Body color and autofluorescence of adult insects
of the pea aphid Acyrthosiphon pisum (AE), and those of 1st
instar nymphs of the wooly social aphid Colophina arma
(FJ). (A and F) Live insects, bright-field observation; (B and
G) insects fixed with alcoholic formalin, bright-field observa-
tion; (C and H) insects fixed with alcoholic formalin, epifluo-
rescent observation; (D and I) insects fixed with Carnoys solu-
tion and treated with alcoholic H2O2 solution, bright-field ob-
servation; (E and J) insects fixed with Carnoys solution and
treated with alcoholic H2O2 solution, epifluorescent observa-
tion. The bright-field and epifluorescent images (B) and (C),
(D) and (E), (G) and (H), and (I) and (J) are taken from the
same specimens, respectively. The epifluorescent images are
generated by merging three monochrome images through three
optical filter sets (FITC, Cy3 and Cy5) with the same exposure
time. In these images, blue, green and red represent the images
through FITC, Cy3 and Cy5 filter sets, respectively.
285
Fig. 2. Body color and autofluorescence of dissected em-
bryos of the pea aphid A. pisum (AD), and those of the wooly
social aphid C. arma (EH). (A and E) Embryos fixed with al-
coholic formalin, bright-field observation; (B and F) embryos
fixed with alcoholic formalin, epifluorescent observation; (C
and G) embryos fixed with Carnoys solution and treated with
alcoholic H2O2 solution, bright-field observation; (D and H)
embryos fixed with Carnoys solution and treated with alco-
holic H2O2 solution, epifluorescent observation. The bright-
field and epifluorescent images (A) and (B), (C) and (D), (E)
and (F), and (G) and (H) are taken from the same specimens,
respectively. The epifluorescent images are generated as in
Fig. 1. Bars show 200 m m.
sues and nucleic acids. Meanwhile, when treated
with alcoholic 6% H2O2 solution, not only the tis-
sues of embryos but also the strongly colored tis-
sues of whole insects were successfully decol-
orized (Figs. 1D, I and 2C, G), and the histology of
the tissues remained in a good condition, exhibit-
ing normal stainability with DAPI and SYTOX
green (data not shown). When bleached tissues
were observed by fluorescent microscopy, the auto-
fluorescence of the tissues was markedly reduced
(Figs. 1E, J and 2D, H).
286 R. KOGA et al.

Table 1. Histological fixatives used in this study

Preservation of Intensity of
histology autofluorescence
Fixative Composition
Whole Dissected Whole Dissected
insects embryos insects embryos

Buffered formalin 10% formalin (3.7% formaldehyde) in Bad Good Strong

phosphate buffer (pH 7.4)
Buffered glutaraldehyde 2.5% glutaraldehyde in phosphate buffer Bad Good Very
(pH 7.4) strong
Bouins solution Picric acid-saturated water : formalin : acetic Bad Good Very
acid15 : 5 : 1 strong
Alcoholic Bouins 53% ethanol, 27% formalin (10% Good Good Very Very
solution formaldehyde), 7% acetic acid, 0.5% picric acid strong strong
Alcoholic formalin Ethanol : formalin3 : 1 Good Good Strong Strong
Carnoys solution Ethanol : chloroform : acetic acid6 : 3 : 1 Good Good Moderate Moderate

FISH detection of bacterial endosymbionts in
aphid tissues bleached with hydrogen peroxide
When the aphid samples fixed with alcoholic
formalin were subjected to whole-mount FISH tar-
geting 16S rRNA of their primary and secondary
endosymbiotic bacteria, hybridization signals were
certainly observed, but strong background signals
due to autofluorescence obscured the true signals.
In the dissected embryos that exhibited relatively
weak autofluorescence, we were able to barely rec-
ognize the localization of the primary and second-
ary endosymbionts in the bacteriome (Fig. 3A, C)
(cf. Fukatsu et al., 2000; Koga et al., 2003). In the
whole insects that were vividly colored and emitted
strong autofluorescence, hybridization signals were
difficult to recognize with certainty due to the
strong background noise (Fig. 3B, D). On the other
hand, when the aphid samples fixed with Carnoys
solution and bleached with alcoholic H2O2 solution
were subjected to the same FISH procedure, hy-
bridization signals were clearly visualized not only
in dissected embryos but also in whole insects (Fig.
3EH).
Successful quenching and FISH with acetone-
preserved archival insect samples
In the above experiments, fresh insects main-
tained in the laboratory were fixed, processed and
subjected to FISH detection of their endosym-
bionts; however, fresh samples are not always
available, particularly when working on diverse
field-collected archival materials. Conventionally,
ethanol has been widely used to preserve biological
samples for DNA analyses (Hillis et al., 1996);
however, we found that acetone is superior to
ethanol to preserve samples for molecular analyses
in that (i) acetone preserves DNA better than
ethanol, particularly when water is contaminated,
and, notably, (ii) acetone also preserves RNA well
(Fukatsu, 1999). Thus far, acetone preservation has
been successfully applied for DNA analyses such
as PCR detection, molecular phylogenetics and
quantitative PCR using insect samples preserved
up to for 8 years (Fukatsu, 1999, 2001; Fukatsu et
al., 2001; Kikuchi and Fukatsu, 2003; Kutsukake et
al., 2004), and also for RNA analyses such as in
situ hybridization, RT-PCR and quantitative RT-
PCR using insect samples preserved up to for 8
years (Fukatsu, 1999, 2001; Kutsukake et al.,
2004). Hence, we expected that, when aphids and
other insect samples preserved in acetone for sev-
eral years were transferred to Carnoys solution,
bleached with alcoholic H2O2 solution and sub-
jected to the FISH procedure, their endosymbiotic
bacteria would be successfully visualized with a
low autofluorescent background.
In order to confirm this expectation, we at-
tempted H2O2 bleaching and the FISH procedure to
detect bacterial endosymbionts of acetone-pre-
served archival insect samples: 22-month-old
human body louse Pediculus humanus, 21-month-
old slender pigeon louse Columbicola columbae,
and 10-month-old nycteriibiid bat fly Penicillidia
jenynsii. It has been described that these insects are
associated with endocellular symbiotic bacteria
(Aschner, 1931; Ries, 1931; Buchner, 1965;
 Quenching Autofluorescence of Insect Tissues
Fig. 3. Laser confocal images of whole mount FISH of
the pea aphid A. pisum and the wooly social aphid C. arma.
(AD) Specimens fixed with alcoholic formalin; (EH) speci-
mens fixed with Carnoys solution and treated with alcoholic
H2O2 solution. (A and E) Mature embryos of A. pisum; (B and
F) ovarioles in adult females of A. pisum; (C and G) mature
embryos of C. arma; (D and H) 1st instar nymphs of C. arma.
Blue, green and red colors represent images through FITC,
Cy3 and Cy5 filter sets, respectively. Without autofluorescence
of the insect tissues (i.e. EH), host insect nuclei, the second-
ary endosymbionts, and the primary endosymbionts Buchnera
are clearly visualized in blue by SYTOX green, in green by
Cy3, and in red by Cy5, respectively. Bars show 200 m m.
Sasaki-Fukatsu et al., 2006; Fukatsu et al., 2007).
The human louse samples were relatively pale, but
contained plenty of black blood meal in the gut
287
(Fig. 4A). After 8 days of H2O2 treatment, the sam-
ples were successfully bleached (Fig. 4B). With lit-
tle autofluorescence, FISH targeting bacterial 16S
rRNA clearly visualized the endosymbiotic bacte-
ria localized in a stomach disc (Fig. 4C) (cf.
Sasaki-Fukatsu et al., 2006). The pigeon louse
samples were brownish (Fig. 4D), but after 7 days
of H2O2 treatment, the samples were almost com-
pletely bleached (Fig. 4E). Although some autofluo-
rescence remained in the cuticle, FISH successfully
detected the symbiotic bacteria localized in ovaries
(Fig. 4F) (cf. Fukatsu et al., 2007). The bat
fly samples were dark brown, with a thick, hard
and colored cuticle (Fig. 4G). After 18 days
of H2O2 treatment, the samples were definitely
bleached (Fig. 4H), but considerable autofluores-
cence remained in the cuticle (data not shown);
however, endosymbiotic bacteria localized in well-
developed bacteriomes were clearly observable by
FISH (Fig. 4I). These results indicate that (i) H2O2
bleaching and the FISH procedure to detect bacte-
rial endosymbionts is generally applicable to a
wide array of endosymbiont-associated insects; (ii)
the procedure is generally applicable to acetone-
preserved archival insect specimens; (iii) even
when insect samples are strongly colored, pro-
longed H2O2 treatment can reduce the color and
autofluorescence; and (iv) even after 3 months of
H2O2 treatment, bacterial 16S rRNA molecules in
insect tissues are preserved in a FISH-detectable
condition (data not shown).
Robust and general procedure for FISH detec-
tion of endosymbionts in archival insect speci-
mens
From all these results, we propose the following
procedure as a robust and general protocol for 16S
rRNA-targeted FISH detection of endosymbionts
in archival insect specimens: (i) Insect samples can
be preserved in acetone before processing, at least
for several years. This step is optional but practi-
cally useful, particularly when working on field-
collected and archival materials. (ii) Acetone-pre-
served samples are fixed in Carnoys solution. This
non-aqueous fixative rapidly infiltrates whole in-
sect tissues through the cuticle, and suppresses the
development of autofluorescence in insect tissues,
unlike conventional formaldehyde-based fixatives.
(iii) The fixed samples are bleached with alcoholic
H2O2 solution. This step markedly reduces the
288 R. KOGA et al.

Fig. 4. Body color, H2O2 bleaching and whole mount FISH of the human body louse Pediculus humanus (AC), the slender
pigeon louse Columbicola columbae (DF) and the nycteribiid bat fly Penicillidia jenynsii (GI). (A, D and G) Acetone-preserved
insects; (B, E and H) insects after treatment with alcoholic H2O2 solution; (C and I) epifluorescent microscopic images of FISH de-
tection of bacterial endosymbionts in H2O2-treated insects; (F) laser confocal image of FISH detection of bacterial endosymbionts
in H2O2-treated insect. In panels C, F and I, host insect nuclei and bacterial endosymbionts are visualized in blue by SYTOX green
and in red by Cy3, respectively. Bars show 0.5 mm (A to E), 0.1 mm (F) and 1 mm (G to I).

autofluorescence of insect tissues while 16S rRNA
molecules of endosymbionts are kept in a FISH-
detectable condition. (iv) The bleached samples are
processed into tissue sections, dissected tissues, or
whole-mount specimens, and subjected to oligonu-
cleotide-probed FISH targeting endosymbiont 16S
rRNA. (v) FISH specimens are observed by epifluo-
rescent or laser confocal microscopy. Significant
elimination of background autofluorescence en-
ables simultaneous visualization of multiple en-
dosymbionts with different fluorochromes. We sug-
gest that this technique will provide a powerful tool
for observing endosymbiotic systems in insects,
the most diverse metazoan group in the terrestrial
ecosystem. It is of interest whether this FISH pro-
tocol is also applicable to non-insect arthropods
and other animals with bacterial endosymbionts.
Duration of hydrogen peroxide treatment
Hereafter, we discuss several important factors
that may affect the applicability of H2O2 bleaching
and the FISH procedure on the basis of our rather
preliminary results. The length of treatment that is
either sufficient or optimal for quenching autofluo-
rescence of insect tissues while not damaging FISH
detectability has not been established. Thus far, we
have empirically stopped H2O2 treatment when the
insects looked whitish in color. The dark-colored
pigeon louse and bat fly samples were successfully
bleached after 13 weeks of H2O2 treatment (cf.
Fig. 4). In the case of A. pisum, FISH signals of the
endosymbionts were observed in samples kept in
alcoholic H2O2 solution for 3 months, but were not
detectable in samples kept for 6 months (Koga, un-
published data). Hence, we suggest that the dura-
tion of H2O2 treatment for quenching autofluores-
cence of insect tissues can be up to 3 months.
Condition of endosymbiont 16S rRNA mole-
cules after hydrogen peroxide treatment
At a glance, it may appear surprising that 16S
rRNA molecules of endosymbionts are detectable
 Quenching Autofluorescence of Insect Tissues
by FISH after treatment with H2O2, a strong oxi-
dizing reagent. Actually, DNA and RNA molecules
in insect tissues treated with H2O2 are degraded;
for example, when total nucleic acids were ex-
tracted from fresh or acetone-preserved aphid sam-
ples and run on agarose gels, DNA and rRNA
bands were clearly seen; however, after H2O2 treat-
ment, these bands disappeared while a smear
band of low molecular weight was observed (Koga
and Fukatsu, unpublished data). Nevertheless, 16S
rRNA molecules of the endosymbionts were de-
tectable by oligonucleotide-probed FISH in the
aphid samples (cf. Fig. 3). Probably, the 16S rRNA
molecules are certainly broken down but the ma-
jority of the products are over the size of the
oligonucleotide probes (around 20 bps), which can
hybridize with the probes.
Period of acetone preservation
Thus far, we have successfully extracted and an-
alyzed DNA and RNA molecules by PCR, cloning,
sequencing, quantitative PCR, RT-PCR, quantita-
tive RT-PCR and in situ hybridization from some
insect samples preserved in acetone at room tem-
perature up to for 8 years (Fukatsu, 1999, 2001;
Fukatsu et al., 2001; Kikuchi and Fukatsu, 2003;
Kutsukake et al., 2004). This depends on the condi-
tion of the samples, but we suggest that, if appro-
priately prepared and stored, insect samples pre-
served in acetone for several years can be subjected
to H2O2 bleaching and the FISH procedure to de-
tect their endosymbionts. Suggestions for good
preservation include the sample per preservative
ratio and the volatility of the preservative. The
amount of insect samples must not exceed 10% of
the volume of acetone: water contamination tends
to result in degradation of DNA and RNA. Acetone
is more volatile than ethanol, so the containers
must not be plastic, but glass vials that can be
tightly sealed (Fukatsu, 1999).
Prior sample processing
Acetone and Carnoys solution are so permeable
that whole insects can be thrown into the solutions.
Alcoholic H2O2 solution is also permeable. How-
ever, the other aqueous solutions used for hy-
bridization do not readily enter the tissues of whole
insects. Hence, particularly for whole-mount FISH
detection, prior sample processing, such as decapi-
tation, pricking the cuticle with a needle, cutting
289
the cuticle with a razor, and dissection of the tis-
sues of interest, is needed to facilitate the infiltra-
tion of probes and reagents. The trade-off between
histological preservation and the permeability of
reagents should be optimized depending on the
samples and purposes.
Applicable histological preparations
We demonstrated that H2O2 bleaching and the
FISH procedure are applicable to paraffin tissue
sections and whole mount preparations. Thimm
and Tebbe (2003) reported that cryosectioned spec-
imens are useful for in situ hybridization of en-
dosymbionts of microarthropods. Whether our pro-
cedure is applicable to cryosections requires future
examination.
Recommended procedure for FISH detection of
endosymbionts
Finally, we briefly present an overview of the
recommended procedure for FISH detection of en-
dosymbiotic bacteria in archival insect materials.
For details, also refer to Materials and Methods.
Note that, in all of the following steps, no shaking
is needed for incubation; diffusion is sufficient as
shaking may damage the whole mount samples.
1. Preservation
Preserve insect samples in acetone at room tem-
perature, which can be up to for several years.
2. Fixation
The insect samples are fixed with Carnoys solu-
tion at room temperature overnight.
3. Bleaching
After thorough washing with 100% ethanol, the
insect samples are transferred to alcoholic 6%
H2O2 solution and incubated at room temperature
until decolorized. Exchange the solution every two
or three days for the first two or three weeks of the
incubation. The incubation period can be up to for
3 months. After the bleaching step, wash the sam-
ples thoroughly with 100% ethanol. At this step,
samples can be stored at 20C for over a year.
4. Sample processing
After thorough washing in 70% ethanol to re-
move H2O2, the bleached insects are decapitated,
pricked, cut or dissected to facilitate the infiltration
of reagents. For FISH on tissue sections, the insect
samples are embedded in paraffin, cut into serial
sections, mounted on glass slides, dewaxed through
a xylene-ethanol series, and dried. For whole
290 R. KOGA et al.
mount FISH, the insect samples are hydrated by in-
cubating with PBSTx buffer several times. The
samples are then incubated with hybridization
buffer without probe several times.
5. Hybridization
For sectioned samples, hybridization buffer con-
taining fluorochrome-labeled probe and counter-
staining reagent (e.g., DAPI, SYTOX green) is
applied to the tissue sections, covered with a co-
verslip, and incubated at room temperature in a
humidified chamber overnight. For whole mount
samples, pre-hybridization buffer is replaced with
hybridization buffer containing the probe and
counter-staining dye, and the samples are incu-
bated at room temperature overnight.
6. Mounting
The samples are thoroughly washed with
PBSTx, equilibrated with and mounted in Slow-
Fade antifade solution or any equivalent reagent.
7. Observation
Finally, the samples are observed under either an
epifluorescent microscope or a laser confocal mi-
croscope.
ACKNOWLEDGEMENTS
We thank Minoru Mihara and Nobutaka Urano for insect
samples, and Junko Makino for technical assistance. T.T. was
supported by the Research Fellowship of the Japan Society for
the Promotion of Science for Young Scientists.
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