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Diversity of insect intestinal microflora

Article  in  Folia Microbiologica · May 2008

DOI: 10.1007/s12223-008-0032-z · Source: PubMed

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Folia Microbiol. 53 (3), 229–233 (2008) http://www.biomed.cas.cz/mbu/folia/

Diversity of Insect Intestinal Microflora

J. MRÁZEKa, L. ŠTROSOVÁa, K. FLIEGEROVÁa, T. KOTTb, J. KOPEČNÝa
aInstitute of Animal Physiology and Genetics, v.v.i., Academy of Sciences of the Czech Republic, 142 20 Prague, Czechia

mrazek@iapg.cas.cz
bInstitute of Animal Science, v.v.i., 104 00 Prague 10-Uhříněves, Czechia

Received 5 November 2007

Revised version 17 January 2008

ABSTRACT. The influence of geographic location, season, age, and part of the digestive tract on bacterial
diversity was evaluated on intestinal microflora of honeybees, wasps, and cockroaches using DGGE ana-
lysis. PCR-DGGE analyses with universal bacterial primers targeting 200-bp region of the 16S rDNA gene
afforded the profile of complex bacterial DNA; specific primers were used to determine the profile of bifido-
bacteria whose concentration in digestive tract was determined by real-time PCR. Selected PCR products
were identified by sequencing. The microflora of the bees exhibited little variations among the hives from
distant locations. Their bifidobacterial population formed 2.8–8.4 % of total bacteria and was very homo-
geneous. The total gut microflora of wasps was also homogeneous, only two samples being affected by the
season or the location; on the other hand, wasp bifidobacterial population was very heterogeneous. Cockroa-
ches showed the highest variations in microflora composition, the age and diet being the ultimate factors;
bifidobacteria counts also varied among tested individuals (0.1–34.1 % of total bacteria). Our results suggest
that nutrition habits are the strongest factor affecting the insect microflora, giving higher variations to omni-
vorous species.

Abbreviations
DGGE denaturing gradient gel electrophoresis T-RFLP terminal restriction fragment length polymorphisms
SSCP single-strand conformation polymorphism

Invertebrates host numerous microorganisms with interactions ranging from symbiosis to patho-
genesis. The gut of insects may represent a large source of as yet unexplored microbial diversity. These bac-
teria utilize a wide range of organic polymers and can be involved in methanogenesis and nitrogen fixation
(Nardi et al. 2002). The gut microflora also plays an important role in pheromone production, pesticide de-
gradation, vitamin synthesis and pathogen prevention (Reeson et al. 2003). Bacterial communities in insects’
intestine have been studied mainly by cultivation-dependent techniques (Gilliam 1997); however, these me-
thods do not reflect entire microbial communities.
A great attention is given to bee species. In honeybees, molecular methods (high-fidelity PCR of
16S rRNA, T-RFLP analysis, SSCP analysis) that were applied revealed several uncultivated bacterial spe-
cies and unidentified bifidobacteria (Jeyaprakash et al. 2003; Mohr and Tebe 2006; Babendreier et al. 2007).
Also the diversity observed was much higher than in studies that had used cultivation-dependent methods.
Less attention has been paid to the ecology and overall picture of gut bacteria in other insect spe-
cies. Cockroaches are investigated as transmitters of pathogenic bacteria and carriers of multiple-antibiotic-
resistance strains (Pai et al. 2005; Elgderi et al. 2006); microflora of their digestive tract has also been stu-
died (Gijzen and Barughare 1992; Zurek and Keddie 1996, 1998). New approach to the study of bacterial
diversity of the gut of American cockroaches using 16S rDNA cloned directly from gut contents suggested
a novel Flavobacterium species (Zurek 1998); Dugas et al. (2001) confirmed this finding by classical micro-
biological methods.
Even less information is available about gastrointestinal microbes of wasps and hornets. While hor-
nets have not been studied at all from this point of view, in wasp gut several bacterial species including
Rickettsiella, Bacillus, Lactococcus, and Lactobacillus were identified by denaturing gradient gel electro-
phoretic analysis (Reeson et al. 2003).
This study targets the gut microbial diversity of bees, wasps and cockroaches with special attention
given to bifidobacterial species. Using real-time PCR and DGGE analysis we attempt to evaluate the effect
of season, location and age on intestinal bacterial communities of selected insects.
230 J. MRÁZEK et al. Vol. 53

MATERIAL AND METHODS

Insect collection. Members of four insect species were tested: honey bees (Apis mellifera), wasps
(Vespula vulgaris), hornet (Vespa crabro) and cockroaches (Nauphoeta cinerea oliv).
Adult honeybee workers (n = 19) were taken directly from the hives from five different locations of
Czechia (Máslovice, Havlíčkův Brod, Rosice u Brna, Přerov and Vsetín) in May 2006; each location was at
least 50 km away from another one, average altitude was 295 ± 80 m a.s.l. Wasps (n = 15) and hornets were
collected in two different towns of Central Bohemia and Moravia (Prague – location PG and Olomouc – OL)
in the autumn 2006 and spring 2007. Cockroaches (n = 12) were laboratory - bred (experimental cockroach-
keeping of Institute of Entomology, Acad. Sci. Czech Rep., České Budějovice) on bread and vegetables; the
body length ranged from 10 to 26 mm. The same DGGE analysis as used for bees and wasps was applied to
their intestinal tract to detect the influence of body size (correlating with age) on the diversity of their gut
microflora.
Processing and DNA isolation. The insects were killed by decapitation. The whole intestine was
carefully removed from the clipped insects with sterile forceps. Intestinal sections of cockroaches (crop,
anterior midgut, posterior midgut and hindgut) were separated with a sterile scalpel. The digestive tract con-
tents were used immediately for bacterial DNA isolation by PowerSoilTM DNA Kit (MoBio Laboratories,
USA) according to the manufacturer’s protocol.
Primers and PCR program for DGGE. Amplification of total bacterial community DNA was done
by targeting 16S rRNA gene sequences with universal bacterial primers 338GC (5´-CGC CCG CCG CGC CCC
GCG CCC GGC CCG CCG CCG CCG CCG CAC TCC TAC GGG AGG CAG CAG-3´) and RP534 (5´-ATT ACC
GCG GCT GCT GG-3´). PCR assay was performed with the following amplification program: denaturation
(3 min 94 °C), 35 cycles (1 min 94 °C, 30 s 55 °C, 1 min 72 °C), and final elongation (10 min 72 °C)
(Muyzer et al. 1993). For bifidobacteria detection the nested PCR was done using specific primers Lm26F
(5´-GAT TCT GGC TCA GGA TGA ACG-3´) and Lm3R (5´-CGG GTG CTI CCC ACT TTC ATG-3´) with
denaturation (5 min 94 °C), 3 amplification cycles (45 s 94 °C, 2 min 55 °C, 1 min 72 °C), followed by
another 30 amplification cycles (20 s 94 °C, 1 min 55 °C, 1 min 72 °C) and elongation step (7 min 72 °C)
(Kaufmann et al. 1997). PCR reaction was performed using ReadyMixTM Taq PCR Reaction Mix (Sigma-
Aldrich, Germany), PCR products were purified with QIAquick PCR purification kit (Qiagen, Germany)
according to the manufacturer’s instructions. The presence of PCR product was checked by electrophoresis
in a 0.8 % agarose gel stained with ethidium bromide and viewed by UV transilluminator (Sambrook et al.
1989). Subsequently, a second PCR (with primers 338GC and RP534) was performed, using the amplicons
from the first PCR as a template DNA (Temmerman et al. 2003).
DGGE analysis. PCR products were separated and analyzed by DGGE on DCodeTM Universal
Mutation Detection System (BioRad, USA) on 9 % polyacrylamide gel with 35–60 % denaturing gradient of
7 mol/L urea and 40 % formamide (V/V) for total bacteria and 45–65 % denaturing gradient for bifido-
bacteria. The electrophoresis was carried out in 1× TAE (40 mmol/L Tris, 20 mmol/L acetic acid, 1 mmol/L
EDTA) buffer at 55 V for 19 h and 60 °C (Fischer and Lerman, 1983). The gels were stained in SYBR®
Green I dye (10 ppm) for 25 min and visualized by UV light on Vilber Lourmat System (France). The stan-
dard ladder consisting of the following organisms was used: Bacteroides uniformis AR20, Lachnospira multi-
para ATCC 19204, Ruminococcus albus SY3, Pseudobutyrivibrio ruminis JK 618, Treponema 704, Rumino-
coccus flavefaciens 627, Faecalibacterium praustnitzii A2165, Butyrivibrio fibrisolvens ATCC 19171,
Clostridium proteolasticum X2, Escherichia coli JM 109, Megasphera elsdenii AW 106 (total bacteria stan-
dard) and Bifidobacterium bifidum ATCC 29521, B. breve ATCC 15700, B. longum ATCC 15707, B. in-
fantis ATCC 17930, B. catenulatum CCM 4989 and B. adolescentis CCM 4987 (bifidobacteria standard).
Identification of bands frequently retrieved. Bands of interest were cut from the documented stained
polyacrylamide gel with a sterile scalpel blade. DNA was eluted by the addition of 100 μL of sterile distilled
H2O and centrifuged (167 Hz, 10 min). One μL of this solution was used for amplification with primers
FP341 (CCT ACG GGA GGC AGC AG) and RP534 under PCR-DGGE program (Muyzer et al. 1993). The re-
sulting PCR products were cleaned with QIAquick PCR purification kit (Qiagen) and amplified again with
primer FP324 using ABI PRISM® BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems,
USA) on PCR thermocycler T-personel Combi (Biometra, Germany). This PCR product was (after ethanol
precipitation) resuspended in formamide HiDi (high-deionized) and analyzed on 3100 Avant Genetic Analy-
zer (Applied Biosystems). The resulting sequences were compared with the GenBank database using the
BLASTn algorithm (Maidak et al. 1994).
Real-time PCR analysis. The quantification of bacterial DNA was done with MX3005P QPCR Sys-
tem (Stratagene, USA). The qPCR 2× SYBR Master Mix (Top-Bio, Czechia) was used for PCR amplificat-
ion of total bacteria with universal primers 27fp and 515r (Kobayashi et al. 2000) and bifidobacteria with
2008 INSECT INTESTINAL MICROFLORA 231

specific primers Lm26f and Lm3r as previously. Temperature program was the same as for DGGE described
above. Dilutions of purified genomic DNA of control strains B. uniformis and B. bifidum were used to con-
struct calibration curves and to calculate the numbers of bifidobacteria as a percentage of total bacterial count.

RESULTS AND DISCUSSION

Honeybees. The profile of complex bacterial DNA was detected by using the PCR-DGGE analyses
with universal bacterial primers targeting 200-bp region of the 16S rDNA gene; similarly, specific primers
were used to detect the profile of bifidobacteria. DGGE band patterns of total bacterial DNA (Fig. 1 left)
showed some variation in the upper part of the gel, while bands in the lower part were quite similar. Even if

Fig. 1. DGGE band pattern of honeybee microflora from different hives. Left: total bacteria, right: bifidobacteria; each line 1–19 repre-
sents a profile obtained from a single worker. 1–3 (location Máslovice), 4–7 (location Havlíčřkův Brod), 8–11 (location Rosice u Brna),
12–15 (location Přerov), 16–19 (location Vsetín); lines S are the standards. Bands: A – uncultured β-proteobacterium (97 %), B, G
and H – uncultured γ-proteobacterium (97 %), C and D – uncultured Serratia sp. (97 %), E – Lactobacillus apis (89 %), F – uncultured
Firmicutes bacterium (99 %), I – Bifidobacterium indicum (89 %), J – Bifidobacterium minimum (97 %); for further details see the text.

differences could be found for individual bees, generally, the location of hives did not have a strong in-
fluence on bacterial composition. This low diversity can be explained by uniform diet of workers that con-
sists exclusively of pollen, nectar and diluted honey. Bacterial population could be partly stabilized by
apidecins, proline-rich antibacterial peptides active against Gram-negative bacteria (Otvos 2002). Sequenced
bands (Fig. 1 left) were compared with the GenBank database; similarity of 99 % with uncultured species of
Serratia (Jeyaprakash et al. 2003), Firmicute and γ-proteobacterium (Bebendreier et al. 2007) found in
Japanese and South African honeybee intestines was shown for bands D, F and G, respectively. The bifido-
bacterial profile of studied bees was also very homogeneous (Fig. 1 right). The strong band occurring in all
workers (band I) yielded a sequence corresponding to a strain B. indicum, with only 89 % homology, so this
band could represent a new taxon. Band J had 97 % homology to B. minimum and/or B. indicum. Bifido-
bacterial count expressed as a percentage of total bacterial numbers ranged among tested workers from 2.8
to 8.4 %.
Wasps and hornet. Using the PCR-DGGE analysis the influence of location and season was asses-
sed on wasp intestinal microflora. DGGE profile (Fig. 2 left) showed surprisingly little variations of total
bacterial content of wasps from different locations, mainly the upper part of gel being homogeneous. Only two
samples (lane 14 and 15) from Prague had a considerably different profile. Also differences between wasps
collected in autumn and spring were slight but again samples 14 and 15 collected in spring had different
band composition. For comparison, lane 16 represents DGGE profile of hornet gut that markedly differs from
wasp intestine content. In wasps the sequences of bands of interest were homologous with Pseudomonas
232 J. MRÁZEK et al. Vol. 53

fluorescens (band C, 99 %) and Salmonella enterica (band E, 98 %). The one band analyzed from hornet (D)
showed 98 % similarity with Acinetobacter species. In contrast to total bacteria, DGGE profile of Bifido-
bacteria was very heterogeneous with no relation to location or time of wasp collection (Fig. 2 right). The
number of bifidobacterias varied (0.1–37.1 %). Also homology with GenBank data was very low and did not
correspond to bifidobacterial species. No bacteria described earlier for wasps by Reeson et al. (2003) were
found in this study. The relatively homologous profile of total bacterial contents of wasp intestine was also
different from the data of Reesson et al. (2003) who found a great variety and differences of gut bacterial
profiles of wasps from the same nest. This discrepancy can be associated with nutrition habits. Wasps eat
a broad diet and it is assumed that their nutrition is not dependent on microbial processes. Nevertheless, it
seems that wasp intestine could be a source of new bacteria.

Fig. 2. DGGE profile of wasp and hornet microflora. Left: total bacteria, right: bifidobacteria. Lines 1–10 show profile from insects
obtained in autumn 2006, lines 11–15 are from April 2007; line 16 is from hornet; wasps representing lines 1–3 are from location
Olomouc, 4–16 are from location Prague; S – standards. Bands: A – Escherichia coli (92 %), B – uncultured bacterial clone, C – Pseudo-
monas fluorescens (99 %), D – Acinetobacter sp. (98 %), E – Salmonella enterica (98 %), F – Corynebacterium glutamicum (94 %),
G – Bifidobacterium adolescentis (90 %), H – Propionibacterium acnes (90 %), I – Bifidobacterium adolescentis (94 %), J – Clostri-
dium perfringens (87 %), K – Corynebacterium diphtheriae (95 %); for further details see the text.

Cockroaches. A comparison of bacterial DNA profile of different parts of digestive tract was car-
ried out for one cockroach. Very high bacterial variability was typical for the studied bugs (Fig. 3 left) with
no correlation to their body size (age). All tested subjects contained Blattabacterium sp. that is an obligate
mutualistic endosymbiont present in all cockroaches (Dasch et al. 1984) except Nocticola genus (Lo et al.
2007). This bacterium resides within specialized cells of the fat body tissue, so it can be a contaminant. How-
ever, the strong band belonging to this bacterium in our DGGE gel (band B) is noteworthy. Two sequenced
bands were homologous with unknown uncultured bacterial clones (band A and D), and two bands (C and E)
had only low similarity with Lactobacillus species (93 and 94 %). From the comparison of different parts of
cockroach intestinal tract it can be concluded that hindgut has different bacterial profile, while crop, anterior
midgut and posterior midgut are more similar to each other. DGGE bifidobacterial profile of cockroaches is
also heterogeneous. The strongest band (F) had only 91 % homogeneity with B. adolescentis. The numbers
of cockroach bifidobacteria were also dependent on individual samples and represented 0.1–34.1 % of total
bacteria. The high variability found in this report can again be explained by food habits – cockroaches as
omnivores have diverse nutrition sources and their digestion is parallel to human digestive tract.

This project was supported by Grant Agency of the Czech Republic (project no. 303/06/0974), by the Ministry of Agriculture
of the Czech Republic (project no. MZE 0002 701 401) and Institutional Research Program IAPG (no. AV 0Z 5045 0515).
 2008 INSECT INTESTINAL MICROFLORA 233

Fig. 3. DGGE band pattern of cockroache microflora. Left: total bacteria, right: bifidobacteria. Lines 1–4 show different parts of diges-
tive tract of a single bug (1 – crop, 2 – anterior midgut, 3 – posterior midgut, 4 – hindgut), lines 5–16 represent bacterial-band profile
from the entire digestive track of insects with different body size: 5 – 10 mm, 6 – 10, 7 – 15, 8 – 17, 9 – 22, 10 – 22, 11 – 23, 12 – 24,
13 – 25, 14 – 25, 15 – 26, 16 – 26; S – standards. Bands: A and D – uncultured bacterium clone, B – Blatabacterium sp. (97 %),
C – Lactobacillus sp. (93 %), E – Lactobacillus paracolinoides (94 %), F – Bifidobacterium sp. (91 %); for further details see the text.

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