SYSTEMATICS

Investigation of Gut-Associated Bacteria in Tenebrio molitor

(Coleoptera: Tenebrionidae) Larvae Using Culture-Dependent
and DGGE Methods
YAO WANG AND YALIN ZHANG1
Key Laboratory of Plant Protection Resources and Pest Management of the Ministry of Education,
Northwest A&F University, Yangling 712100, Shaanxi, China.

Ann. Entomol. Soc. Am. 1–9 (2015); DOI: 10.1093/aesa/sav079

ABSTRACT In this article, the composition and distribution of bacteria associated with the gut of Tene-
brio molitor (L.) larvae were investigated using both culture-dependent and culture-independent dena-
turing gradient gel electrophoresis (DGGE) methods. This work compares bacterial species associated
with four different parts of T. molitor larvae gut: foregut, anterior midgut, posterior midgut, and hindgut.
Five genera, Weissella, Lactococcus, Rahnella, Cronobacter, and Enterococcus, were isolated using nutri-
ent agar. All of these strains were present in the posterior midgut and hindgut. The strains with milk-clot-
ting activity in selective casein-plates assay were sequenced and identified as species of genera Weissella
and Lactococcus, and those with proteolytic activity as Rahnella and Cronobacter, implying that they may
be involved in protein utilization. But none of these strains showed cellulolytic activity. In DGGE experi-
ment, 19 isolated bands belonging to nine taxa (Spiroplasma, Lactococcus, Lactobacillus, Bacillus, an
uncultured Bacillaceae, Clostridium, Enterobacter, Pantoea, and an uncultured Clostridium) were ex-
tracted and identified from DGGE gels. These species could be assigned to three phyla Tenericutes, Fir-
micutes, and Proteobacteria. According to the DGGE analysis, the bacterial communities of the four gut
regions exhibited some differences, with the hindgut showing the highest bands abundance and diversity.
The genus Spiroplasma, which is generally regarded as pathogen or male-killing bacteria in insects, had a
high abundance in the gut environment, their potential role is worthy of a further study.

KEY WORDS Tenebrio molitor, larvae, gut bacteria, DGGE

Animals evolve in a bacterial world and occupy various
ecological niches with the help of microorganisms
(McFall-Ngai et al. 2013). An estimated 10–20% of in-
sect species bear intracellular symbionts, which may
play essential or beneficial roles in host development;
these have been described in aphids and many other
insects (Gil et al. 2004, Douglas 2007). As a relatively
open environment, the intestine is very densely colo-
nized by microbes in most insects (Douglas 2011).
Insects also develop specialized gut structures to har-
bor symbionts, such as crypts and enlarged hindguts
(Kikuchi et al. 2005, Nardi and Bee 2012, Chapman
et al. 2013). It has been suggested that the communi-
ties of insect guts may be shaped by the host’s immune
system, the microhabitat of gut regions created by the
pH and structure, and environment factors such as
diets and environment pressures (Douglas 2011, Engel
and Moran 2013). For some insects with social behav-
iors, such as cockroaches, bees, and termites, there is a
transmission of gut microbiota between individuals and
generations (Engel and Moran 2013).
There is growing evidence that the gut microbiota of
insects play an important role in the adaption of hosts
1
Corresponding author, e-mail: yalinzh@nwsuaf.edu.cn.
C The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America.
V
All rights reserved. For Permissions, please email: journals.permissions@oup.com
in several aspects including digestion, nutrition, de-
fense, reproduction, and host metabolism (Dillon and
Dillon 2004, Engel and Moran 2013). For example,
more than 300 microbes coexisting in the hindgut of
termites aid digestion of cellulose, nitrogen fixation,
and carbohydrate fermentation (short-chain fatty acids),
and the gut microbiota coevolve with termite species
(Warnecke et al. 2007, Hongoh 2011). Insects that dis-
play dietary plasticity (i.e., omnivores) were once be-
lieved to rely less on microbial symbionts, because
these insects are able to self-select nutritionally optimal
diets from their environment (Buchner 1965). With
more and more experimental data, microbial symbionts
are gradually known to play a role in facilitating this
omnivory in a number of cockroaches, crickets, carpen-
ter ants, etc. (Kaufman and Klug 1991, Gijzen and Bar-
ugahare 1992, Feldhaar et al. 2007).
The yellow mealworm is an important economic in-
sect, which is mass produced for animal diets. It is also
commonly used as a model insect for experimentation
(Lord et al. 2012). It is omnivorous and can quickly
adapt to many environments. Researchers in China
have tested the possible ways of feeding yellow meal-
worms in bioregenerative life support systems as a
source of animal protein for humans (Li et al. 2013).
The Tenebrio molitor (L.) larval midgut is special with
a pH gradient ranging from 5.2–5.6 to 7.8–8.2 from
2 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA
anterior to posterior part (Vinokurov et al. 2006, Prab-
hakar et al. 2007), and the hindgut has complex struc-
ture with the function of water and nutrition
reabsorption (Grimstone et al. 1968). We have investi-
gated the bacterial communities associated with the gut
of T. molitor larvae using the culture-dependent and
denaturing gradient gel electrophoresis (DGGE) meth-
ods, because we are interested in variations of the bac-
terial community in different regions of the gut and
interaction between host and gut microbiota.
Materials and Methods
Sources of Insects. T. molitor used in this experi-
ment were reared at the Key Laboratory of Plant Pro-
tection Resources of Northwest A&F University for 5
yr, fed on wheat bran and various vegetables. For con-
sistency, all larvae used for bacteria investigations were
about 0.1–0.15 g per individual, 1–2 mo after hatching,
when they were in an active feeding or “gluttony”
period.
Dissection of Larval Guts. Healthy larvae (active
without wounds) were chosen for the study and under-
went starvation for 12 h. They were then externally
sterilized with 75% ethanol for about 2 min and rinsed
five times with sterilized water. A single insect was then
placed on a disposable sterilized glove, the posterior
and anterior tips of the larvae were removed, and the
gut was pulled from the posterior end using sterilized
forceps. The fat body and Malpighian tubules were
carefully cleaned. The gut was carefully separated into
four parts: the foregut (F), the anterior midgut (AM),
the posterior midgut (PM), and the hindgut (H), as
shown in Fig. 1. These four parts were placed in differ-
ent microcentrifuge tubes (1.5 ml) and gently washed
three times with sterilized water. A total of 20 guts
were fully homogenized with sterilized plastic pestles.
Homogenate (50 ml) was used for DNA extraction and
50 ml for bacterial culturing. All work was done in a
laminar flow cabinet.
Culturing of Bacteria. Serial dilutions were pre-
pared to reach a final dilution of 106 for each sample.
The 100 ml dilutions from 103 to 106 were spread on
plates of Luria-Bertani agar and beef extract-peptone
medium, and this culturing was repeated three times.
Plates were incubated in a growth chamber at 25 C.
After 2–4 d of culturing, the bacterial colonies with dif-
ferent morphologies (shape, elevation, surface, size,
opacity, pigmentation, etc.) were chosen for polymerase
chain reaction (PCR) amplification using 16S rRNA
gene primers: 27F and 1492R (Marchesi et al. 1998)
and sequenced for species identification. For each
Fig. 1. The gut structure of T. molitor larvae.
unique putative species, at least two clones were
sequenced.
Genome DNA Extraction. DNA extraction was
performed using a standard method for bacteria with
some modifications (Wilson 2001). Gut homogenate
(50 ml) was resuspended in 500 ml TE buffer, 3 ml of
10% SDS, and 20 ml of 20 mg/ml proteinase K, mixed
and incubated 2 h at 37 C, then added 100 ml of 5M
NaCl, mixed thoroughly, followed by adding 80 ml of
CTAB/NaCl solution, mixed and incubated 20 min at
65 C; then followed by the standard phenol/chloro-
form/isoamyl extraction with isopropanol precipitation.
The DNA sample was resuspended in 100 ml TE buffer
and frozen at -20 C until use.
PCR Amplification and DGGE Analysis. The V3
region of bacterial 16S rRNA gene fragments from
DNA samples were amplified using the bacterial
specific primers: 357F and 518R with a GC-clamp
attached at the 5’-end of primer 357F for DGGE anal-
ysis (Myers et al. 1985). Touchdown PCR was
performed using C1000 Thermal Cycler (Bio-Rad,
USA). The 25 ml reaction mixture contained 0.2 mM
dNTPs, 3 mM MgCl2, 0.8 pM primer, 1.5U of Taq poly-
merase (Takara, Recombinant TaqDNA Polymerase,
Japan), 1 ml temple DNA, and 1 PCR buffer. The
reaction conditions consisted of initial denaturation at
94 C for 5 min, 5 cycles of denaturation (30 s at 94 C),
annealing (30 s at 65 C), and extension (40 s at 72 C),
followed by 25 cycles of denaturation (30 s at 94 C),
annealing (30 s at 55 C), and extension (40 s at 72 C),
then a final 5 min extension at 72 C.
PCR products were separated by DCode mutation
detection system (Bio-Rad, USA) according to manu-
facturer’s instructions. Samples (25 ml of each) were
loaded onto 1-mm-thick (16 cm by 16 cm) 8% polyacry-
lamide gels with a denaturing gradient of 40–58%
(100% denaturant is defined as 7M urea and 40%
deionized formamide). DGGE was performed at 60 C,
50 V for 1 h, followed by 60 C, 90 V for 10 h with 1
TAE buffer. Gels were stained with silver nitrate (San-
guinetti et al. 1994). The gels were photographed by a
digital camera and analyzed by Quantity One (Bio-Rad,
USA). Dominant bands were excised from gels, and
DNA was eluted overnight at 4 C in 80 ml sterile dis-
tilled water. Eluted DNA was then reamplified using
primers 357F and 518R (nonclamped primers) as
described above without the first 5 cycles and purified
from agarose gels for subsequent cloning and sequenc-
ing. The diversity of the microbial community was
examined by the Shannon index of general diversity
(Vallaeys et al. 1997). Principle component analysis
(PCA) was conducted to get the relationship the bacte-
rial community using Canoco 4.5 (ter Braak and
Šmilauer 2002).
Nucleotide Sequencing. All sequencing were con-
ducted at the Shanghai Sangon Biological Engineering
Technology and Service Co., Ltd. in China. Sequences
were blasted in GenBank, and we obtained the closest
match or closest affiliation when the closest match did
not have clear affiliation.
All the sequences were deposited in GenBank. The
16S rRNA gene sequences of cultured bacteria are
2015 WANG AND ZHANG: BACTERIA ASSOCIATED WITH THE GUT OF T. Molitor 3

available under accession numbers KM088087–
KM088094. The sequences of different DGGE bands
are available under accession numbers KM088072–
KM088086 and LM643863–LM643866.
Separation of Functional Bacteria. Congo red
agar (10 g/liter carboxymethyl cellulose, 5 g/liter pep-
tone, 0.5 g/liter yeast extract, 1.5 g/liter KH2PO4, 0.2 g/
liter MgSO4, 5 g/liter NaCl, 20 g/liter agar, 0.05 g/liter
congo red) was used for the isolation of cellulolytic
microorganisms. Two kinds of casein-plate (CASN: 5 g/
liter casein, 0.5 g/liter beef extract, 1 g/liter yeast
extract, 1 g/liter NaH2PO4, 0.5 g/liter KH2PO4, 20 g/
liter agar; and CASG: 5 g/liter casein, 1 g/liter glucose,
1 g/liter yeast extract, 1 g/liter NaH2PO4, 0.5 g/liter
KH2PO4, 20 g/liter agar) were used to isolate proteo-
lytic bacteria and chymosin-producing bacteria. Plates
were incubated in a growth chamber at 25 C for 3–5 d.
The cellulolytic and proteolytic activities of microorgan-
isms were detected by a clear zone around the colonies,
and the curdy precipitate around the colonies was an
indicator of chymosin-producing bacteria.
Results
Cultured Bacterium From T. molitor Larvae
Gut. An average of 0.29 6 0.16  105, 1.28 6 0.30 
105, 1.38 6 0.27  106, and 6.64 6 1.20  106 CFU (col-
ony forming unit) per gut were enumerated in the fore-
gut, anterior midgut, posterior midgut, and hindgut,
respectively. Eight bacterial species were isolated in
total, as listed in Table 1. According to their 16 s rDNA
sequences, they belonged to the phyla Firmicutes and
Proteobacteria, including five genera: Weissella, Lacto-
coccus, Rahnella, Cronobacter, and Enterococcus. Lac-
tococcus was the most abundant genera and accounted
for 40–50% of all colonies. All bacterial strains were
present in the posterior midgut and hindgut, with Rah-
nella, Enterococcus, and Cronobacter absent in the
foregut and the latter two absent in the anterior midgut
as well. We did not detect any bacterial strains which
showed cellulolytic activity. Milk-clotting activity was
identified in strains of Weissella and Lactococcus,
which accounted for the major part of the cultured
bacteria. Strains of Rahnella and Cronobacter showed
proteolytic activity.
The DGGE Analysis of Bacteria Communities
in Different Gut Regions of T. molitor Larvae.
A total of 20 isolated bands were extracted from the
DGGE gel. All bands were bacterial genes except band
17, which was a contamination from the host gene
(BLAST analysis showed these were coleopteran 18S
rRNA genes; all further analyses excluded this band).
Among these bands (Fig. 2A and Table 2), band 1,
band 2, band 3, band 4, band 10, and band 11 closely
matched an uncultured bacterium (DQ163951), with
98–100% sequence identity. This bacterium was associ-
ated with T. molitor in research that simply mentioned
this sequence information as a control (Dunn and
Stabb 2005). These sequences were affiliated with Spi-
roplasma (94–95% sequence identity). Band 7 and
band 8 closely matched an uncultured bacterium
(FN823944) associated with Artemia sp. (crustacean
Tkavc et al. 2011) with 96% sequence identity. The
closest affiliation (87%) was an uncultured Clostridium
(KP106087). Band 9 closely matched Bacillus sp.
(EF589780) with 98% sequence identity, and this band
was only detected in the hindgut. Band 13 and band
14, which were again only in the hindgut, were closely
related to Clostridium cellulovorans (CP002160) with
98% sequence identity. Band 19 and band 20, which
were detected in posterior midgut and hindgut, closely
matched Enterobacter sp. (JQ917113) with 99%
sequence identity. The composition and distribution of
bacteria in the T. molitor larvae gut were clearly
observed in the DGGE profile (Fig. 2A).
In terms of the DGGE profile, the Shannon index of
general diversity of each region were F ¼ 0.7453,
AM ¼ 0.6230, PM ¼ 0.7507, and H ¼ 0.9002, respec-
tively. The bacteria species were in three phyla: Teneri-
cutes, Firmicutes, and Proteobacteria, with Firmicutes

Table 1. The composition and distribution of cultured bacteria from different gut regions of T. molitor larvae

Strains GenBank Closest match in GenBank % Identity Distribution of strains

accession to closest
no. match F AM PM H

BCH KM088089 KJ095645; Bacteria; Firmicutes; Bacilli; Lactobacillales; 100 þ þ þþþ þþþ
Leuconostocaceae; Weissella
GD KM088090 AB911503; Bacteria; Firmicutes; Bacilli; Lactobacillales; 100 þ þ þþþ þþþ
Leuconostocaceae; Weissella
ZKNR KM088091 EU600924; Bacteria; Firmicutes; Bacilli; Lactobacillales; 100 þ þ þþ þþ
Leuconostocaceae; Weissella
SCH KM088088 HF562963; Bacteria; Firmicutes; Bacilli; Lactobacillales; 99 þþ þþ þþþþ þþþþ
Streptococcaceae; Lactococcus, Lactococcus garvieae
BYZZ KM088087 NR_102968; Bacteria; Firmicutes; Bacilli; Lactobacillals; 99 þþ þþ þþþþ þþþþ
Streptococcaceae; Lactococcus, Lactococcus garvieae
PRO KM088093 GU299866; Bacteria; Proteobacteria; Gammaproteobac- 100 / þ þþ þþ
teria; Enterobacteriales; Enterobacteriaceae; Rahnella
BYP KM088092 JX307659; Bacteria; Proteobacteria; Gammaproteobacte- 97 / / þþ þþ
ria; Enterobacteriales; Enterobacteriaceae;
Cronobacter
LBTR KM088094 NR_113935; Bacteria; Firmicutes; Bacilli; Lactobacillales; 99 / / þ þ
Enterococcaceae; Enterococcus
þ, Present, the number of þ represent the abundance; /, absent.
4 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA

Fig. 2. (A) DGGE profile of 16S rRNA genes from the bacterial communities of the four parts of T. molitor larvae guts;
(B) The bacterial community variations in the hindgut of T. molitor larvae. H in (B) is same as in (A), H1 is the hindgut bacteria
community of larvae feeding on sterilized food. The rest are random individuals in the laboratory population.

dominant in all gut regions, accounting for 79.47% (F),
85.03% (AM), 51.18% (PM), and 49.28% (H) of species
respectively. Based on column chart shown in Fig. 3, it
was clear that bacterial diversity and abundance were
different among the four gut regions. We identified the
highest diversity and abundance of bacterial species in
the hindgut (n ¼ 9 and 41.83% of the entire commun-
ity), followed by the posterior midgut (n ¼ 7 and
29.03% of the entire community), then the foregut
(n ¼ 6 species and 17.63% of the entire community),
and the anterior midgut (n ¼ 5 and 11.52% of the
whole community). It was obvious that Spiroplasma
accounted for a large percentage of bacteria in the pos-
terior midgut and hindgut, the proportions of which
were 38.84% and 30.74%, respectively. The proportion
of Spiroplasma was also the major difference between
2015 WANG AND ZHANG: BACTERIA ASSOCIATED WITH THE GUT OF T. Molitor 5

Table 2. The bacteria from DGGE bands

Bands GenBank Closest affiliation in GenBank % Identity to closest

accession affiliation
no.

Band 1 KM088072 NR_121702 Bacteria

Band 2 KM088073 Tenericutes; Mollicutes;
Band 3 KM088074 Entomoplasmatales; 94–95%
Band 4 KM088075 Spiroplasmataceae;
Band 10 KM088079 Spiroplasma
Band 11 KM088080
Band 5 KM088076 KF148720 Bacteria; Firmicutes;
Band 6 KM088077 Bacilli; Lactobacillales; 99–100%
Streptococcaceae; Lactococcus
Lactococcus garvieae
Band 7 LM643863 KP106087. An uncultured 87%
Band 8 LM643864 Clostridium
Band 9 KM088078 EF589780, Bacteria; Firmicutes; 98%
Bacilli; Bacillales; Bacillaceae;
Bacillus
Band 12 KM088081 AB852173. Bacteria; Firmicute; 99%
Bacilli; Lactobacillales;
Lactobacillaceae; Lactobacillus
Band 13 LM643865 CP002160. Bacteria; Firmicutes; 98%
Band 14 LM643866 Clostridia; Clostridiales;
Clostridiaceae; Clostridium
Band 15 KM088082 EF663420. Bacteria; Firmicutes; 98–99%
Bamd 16 KM088083 Bacilli; Bacillales; An uncultured
Bacillaceae
Band 18 KM088084 AY935243; Bacteria; 99%
Proteobacteria;
Gammaproteobacteria;
Enterobacteriales;
Enterobacteriaceae; Pantoea
Band 19 KM088085 JQ917113, Bacteria; 99%
Band 20 KM088086 Proteobacteria;
Gammaproteobacteria;
Enterobacteriales;
Enterobacteriaceae;
Enterobacter

anterior midgut and posterior midgut. The insects feed-
ing on sterilized wheat bran harbored more Spiro-
plasma, which might indicate its uniqueness. A further
analysis of PCA (Fig. 4, the distance between the sym-
bols in the diagram approximates the dissimilarity of
their species composition, measured by their Euclidean
distance) showed that the bacterial community of the
four regions exhibited some differences. The foregut
and anterior midgut had similar species composition,
and the posterior midgut bacterial community was
more similar with that of the hindgut. We also made a
comparison between the hindgut bacterial communities
of individuals, revealing some differences between indi-
viduals under similar feeding conditions based on the
band diversity and abundance and PCA analysis
(Figs. 2B and 4).
Discussion
A total of 14 groups were identified from the gut of
the T. molitor larvae using both culture-dependent and
DGGE methods. The DGGE method detected more
species than the culture-dependent method, but among
those cultured bacteria with low abundance, Weissella,
Rahnella, Cronobacter, and Enterococcus were not
detected using the DGGE method. Among the cultura-
ble bacteria detected, only the highly abundant
Lactococcus was detected by the DGGE method. This
suggests that the DGGE method may underestimate the
abundance of some species to some degree, which may
be caused by the DNA extraction and PCR amplification
procedure (Shi et al. 2010), or the sensitivity of the
DGGE gel itself may influence the result. While rela-
tively few bacterial species can be cultured, DGGE can-
not detect some culturable strains that are low in
abundance. Here, we get a better overall representation
of species by using both methods. Bands 8 and 9 had a
low identity with known species, indicating that there
are some novel species; besides, the information of V3
region (16S rRNA) is really limited (we failed to con-
struct a reliable phylogenetic tree), so a comprehensive
study of the bacterial community is necessary.
Intestinal bacterial communities of insects have been
widely studied, and their diversity and abundance
exhibit huge variations. The number of major gut bac-
terial species vary from one to hundreds (Fukatsu and
Hosokawa 2002, Nakajima et al. 2005). Our results
showed that the gut bacterial community of T. molitor
was very simple. This corresponds with the research of
insect intestines from other insects such as honeybee
larvae (Babendreier et al. 2007, Vojvodic et al. 2013),
aphids (Haynes et al. 2003), ant lions (Dunn and Stabb
2005), and gypsy moth larvae (Broderick et al. 2004).
The low complexity of T. molitor larvae gut
6 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA

Fig. 3. The composition and distribution of bacteria from the DGGE gel. The amount of each isolated part was evaluated
by the relative density of the DGGE bands. (A) Both the total abundance and diversity of the four gut regions. (B) The
composition and relative abundance of the bacteria in different gut regions.

communities may be due to the simple, efficient, and observe some differences in the bacterial communities;
well-organized gut environment (Grimstone et al. 1968, this probably is the result of complex physical and
Vinokurov et al. 2006, Prabhakar et al. 2007). Although chemical conditions that result in regional differences
the overall pattern was simple, we were still able to of the gut environment (Egert et al. 2003, Lemke et al.
2015 WANG AND ZHANG: BACTERIA ASSOCIATED WITH THE GUT OF T. Molitor
Fig. 4. PCA analysis of the DGGE profile. The bands are scored as present or absent (binary method).
2003). It should be noted that the hindgut bacterial
communities of T. molitor larvae were relatively diverse
and abundant, similar to the situation in several other
insects such as termites, crickets, cockroaches, and
some beetles in which the hindgut bacteria aid host
development in various ways (Kaufman and Klug 1991,
Gijzen and Barugahare 1992, Morales-Jiménez et al.
2009, Hongoh 2011). As an omnivorous insect, gut bac-
teria of T. molitor may be important for host adaptation
to different foods. Genta et al. (2006) indicated that gut
microbiota might play subtle roles in the life of the
T. molitor, being involved in the digestion of refractory
food, detoxification of secondary plant compounds, and
modifying the volatile profiles of the insect host. How-
ever, the functions of gut microbiota may be subtle and
only detected in certain situations such as unbalanced
diet and controlled environment (Dillon and Dillon
2004, Lundgren and Lehman 2010, Storelli et al.
2011): for instance, we know that that Pantoea agglom-
erans in the insect gut participates in nitrogen fixation
and the production of antifungal phenols (Bridges
1981, Bridges et al. 1984, Dilion and Charnley 1996,
Vasanthakumar et al. 2006), while Lactobacillus, Clos-
tridium, and Bacillus are sugar-fermenting bacteria in
some Coleopteran insects (Rajagopal 2009). More
research will be needed to determine the role of micro-
biota in T. molitor.
Spiroplasma, normally viewed as a male-killing sym-
biont or pathogen in arthropods (Gregory and Francis
2000, Jung et al. 2014), is widespread in insects. In this
study, several bands with a few sequence differences
belonged to Spiroplasma, demonstrating both abun-
dance and heterogeneity; however, neither sequence
matched any species in the database. Prior research on
the gut bacterial community of T. molitor also showed
that the proportion of Spiroplasma was quite high,
7
above 90% in some samples (Jung et al. 2014). These
authors also demonstrated that after 10 d of feeding
bran and soil, the gut bacteria in T. molitor was domi-
nated by Spiroplasma; here in our study, the yellow
mealworms raised with sterilized wheat bran also har-
bored more Spiroplasma in hindgut. Noting the rela-
tive lack of reports of Spiroplasma inhabiting the insect
gut, Jung et al. (2014) suggested that their prevalence
in the gut might constitute an unusual situation among
insects. We also found that the Spiroplasma sequence
was closely related (97–98%) with those of another
omnivorous tenebrionid, Gonasida inferna (Casey)
(Colman et al. 2012), even though the authors did not
mention the abundance of the genus. We could infer
that Spiroplasma may be heritable and coevolve with
the gut of T. molitor, or they are just more competitive
than other bacterial species. Apart from reproductive
regulation, Spiroplasma can convey increased resist-
ance to nematode infections in Drosophila (Jaenike
et al. 2012) and improve overwintering ability in the
leafhopper Dalbulus maidis (DeLong & Wolcott)
(Ebbert and Nault 1994). The potential role of Spiro-
plasma in T. molitor should be explored further
because they appear harmless to the host (they are
pathogen to some insects such as honey bee) and do
not show male-killing characterization.
The gut bacterial communities can be shaped by cer-
tain diets. A case in point is that several cellulolytic bac-
teria were isolated from the T. molitor larvae gut after
treatment with lignocellulosic waste for 2 wk (Qi et al.
2011). But in our study, we did not detect any cultura-
ble cellulolytic bacteria in an aerobic environment.
This may to some degree reflect the process of coevolu-
tion of insect-microbe interaction. Microbes that have
adopted endosymbiotic life styles not only have evolved
to live in specialized habitats within living organisms,
8 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA
but the living habitats have also evolved to accommo-
date them (Nardi et al. 2006). Since some proteolytic
bacteria and chymosin-producing bacteria were cul-
tured, we made a comparison of midgut protease activ-
ity and chymosin activity between conventionally
reared and antibiotic-treated insects (data not shown).
Chymosin activity was much higher after treatment.
But we do not fully understand this phenomenon. As
with the previous study (Jung et al. 2014), we also
made an attempt to clone nifH and cellulase gene but
failed. More research efforts should be made to clarify
the role of gut bacterial communities in the high effi-
ciency of food utilization and excellent adaptation
capacity of T. molitor.
Acknowledgments
We sincerely thank Prof. John Richard Schrock (Emporia
State University, USA) for reviewing this manuscript.
This research was supported by the Special Fund for the
Public Interest (Agriculture) (200903052) by The Ministry of
Science and Technology and The Ministry of Agriculture
of China, and the “13115” Sci-Tech Innovation Project of
Shaanxi Province (2007ZDKG-14).
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Received 15 September 2014; accepted 14 July 2015.