Author's Accepted Manuscript

Isolation and characterization of enzyme-

producing bacteria of the silkworm larval
gut in bioregenerative life support system
Xue Liang, Yuming Fu, Hong Liu

www.elsevier.com/locate/actaastro

PII: S0094-5765(15)00280-5
DOI: http://dx.doi.org/10.1016/j.actaastro.2015.07.010
Reference: AA5502

To appear in: Acta Astronautica

Received date: 11 June 2015

Revised date: 4 July 2015
Accepted date: 6 July 2015

Cite this article as: Xue Liang, Yuming Fu, Hong Liu, Isolation and
characterization of enzyme-producing bacteria of the silkworm larval gut in
bioregenerative life support system, Acta Astronautica, http://dx.doi.org/10.1016/j.
actaastro.2015.07.010

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Title:

Isolation and characterization of enzyme-producing bacteria of the

silkworm larval gut in bioregenerative life support system

Authors
Xue Liang1,2,3, Yuming Fu1,2,3, Hong Liu1,2,3*

Affiliations
1 School of Biological Science and Medical Engineering, Beihang University
2Institute of Environmental Biology and Life Support Technology,Beihang
University
3International Joint Research Center of Aerospace Biotechnology&Medical
Engineering, Beihang University

Running title:
enzyme-producing bacteria in gut of the silkworm

* Corresponding author: Hong Liu

Institute of Environmental Biology and Life Support Technology, School of
Biological Science and Medical Engineering,
Beihang University, Beijing 100191, China
Tel /Fax: 86-10-82339837 E-mail: LH64@buaa.edu.cn

Xue Liang and Yuming Fu contributed equally to this study


Abstract:
Silkworm (Bombyx mori L.) larvae were used as an ideal animal protein source for
astronauts in bioregenerative life support system (BLSS). Here, we compared the
differences in bacterial communities of the silkworm larval gut and cellulase
-producing and amylase-producing bacteria between the BLSS rearing way (BRW)
and the traditional rearing way (TRW) through Illumina Miseq sequencing,
culture-dependent approach, 16S rDNA and ITS sequencing, phylogenetic analysis to
find the role of gut bacteria in food digestion. The analysis of Miseq showed that the
gut microbiota in the BRW was significantly changed than that in the TRW. Results
revealed that the isolates can produce cellulose-degrading and starch-degrading
enzymes of gut bacteria of silkworm in the BRW decreased compared with that of the
TRW, but the number of isolates both secrete cellulase and amylase are equal. The
isolates can produce both enzymes in the TRW were Alternaria sp. Preussia sp and
Coprinellus radians. Meanwhile, in the BRW we found Enterococcus, Erwinia and
Pantoea can produce cellulase and amylase. We could use the dominant populations
to make probiotic products for nutrient absorption and disease prevention in BLSS to
improve gut microecology, as well as the yield and quality of animal protein.

Key words: silkworm, BLSS, cellulase-producing bacteria, amylase-producing

bacteria, probiotics


Introduction
The insect gut is inhabited by a wide diversity of microorganisms as a result of
its constituting intestinal microbial ecosystem. The gut microbiota is involved in the
host’s digestion, nutrition, development, resistance to pathogens invasion. The
composition and structure of microbial are dynamic, which can be varied with
changing nutrient availability, physiological environments, and the proximity to other
organisms [1, 2]. Loss of microorganisms often results in abnormal development and
reduces survival of the insect host [3, 4]. Based on the theory of microecology, insect
rely on gut microbes provide a variety of digestive enzymes, to complete its food
digestion, nutrient absorption and metabolism [5]. However, few have discussed the
possibility that microorganisms may produce some of the digestive enzymes to
provide essential nutrients or assist in important metabolism function related to host
food ingestion [1].
Mulberry silkworm (Bombyx mori L.) is an important economical insect whose
importance is reflected not only by its silk production but also by its valuable
nutritional composition. The idea of rearing mulberry silkworm larvae to provide
animal protein for crew in bioregenerative life support system (BLSS) required by
long-term missions to the moon and Mars is widely accepted. This is because
silkworm has many positive merits such as high protein content, reasonable nutrient
compositions and ample contents, a short lifespan, easy breeding method, small
growth room, and little odor and wastewater produced [6]. Due to limitation of space
and resource, mulberry silkworm rearing method in BLSS was different from
traditional rearing method which only uses mulberry leaves. In BLSS, mulberry
silkworms of the first three instars (from the 1st day to 16th day) were fed with
mulberry leaves and for those of the last two instars (from the 17th day to 25th day)
were fed with lettuce leaves [7]. In this rearing way, the yield and the growth rate of
silkworm larvae reared using this method were a little lower than that feeding on
mulberry leaves purely. Prior study has provided evidence that gut microbiota of
silkworms fed on lettuce leaves was relatively simple. The appearance of profitless
bacteria in the gut of silkworm under the BLSS rearing way might break down the

balance structure of healthy gut microbial community, resulting in reduced digestive
enzyme activity [8]. It has been reported that many silkworm intestinal bacteria
produce digestive enzymes like amylase and cellulose [9]. Thus, it is potentially
possible that the change of gut flora due to lettuce leaf feeding may contribute to the
decrease of physiological activity and cause death of the silkworm. However, how
diet compositions in BLSS shape gut enzyme-producing bacteria is still unknown.
In this study, the gut bacterial diversity and enzyme-producing bacteria of
silkworm larvae reared with the traditional rearing way (TRW) and BLSS rearing way
(BRW) were investigated using culture dependent, culture-independent and Illumina
Miseq approaches. The changes of cellulase-producing bacteria, amylase-producing
bacteria in silkworm larvae in response to lettuce leaf feeding were revealed. This
study may promote the development of probiotic products of animal protein under
BLSS.
Materials and methods
Silkworm strains and rearing methods
The silkworm eggs B. mori L. 872×871 were bought from Guangtong Silkworm
seed Co. Ltd. (Shandong Province, China). The silkworm eggs were incubated under
a 12-h light/12-h dark cycle in an artificial cultivation box at 25 °C and 80 % of
relative humidity. When 20 % of eggs had little black dots on the surface, they were
shaded with black gobo for about 48h to ensure the larvae hatching out at one time.
The silkworm larvae were reared with mulberry leaves from the first to third instar
and then divided into two groups: the BLSS breeding group reared with stem lettuce
leaves and the conventional breeding group still reared with mulberry leaves at the
beginning of the fourth instar [8].
Isolation of gut bacteria and DNA extraction
When the two groups of silkworms grew to the third day of the fifth instar, ten
individuals of each group were selected and subjected to starvation overnight. Those
silkworms were surface decontaminated by wiping with 70 % ethanol solution and
scorched gently in a flame. The content of gut was taken and placed into sterile
microcentrifuge tubes on ice under aseptic condition. According to the screening

standard of bacteria, tenfold serial dilution was spread and incubated for inoculation.
On each nutrient agar plate, 0.1 mL of intestinal content of two groups were spread
and incubated at 37 °C for 2 days. All samples were repeated three times. The media
used for the isolation of bacteria included nutrient agar, potato dextrose agar, Gause’s
No. 1 agar medium, which were autoclaved at 121 °C for 15 minutes, and pH value
was adjusted to 9.2-9.8 [10]. Colonies of each group were picked out, purified three
times by inoculating on the corresponding agar plates, and further transferred to agar
slants.
Pooled DNA samples of the fifth instar in the BLSS and the conventional
breeding groups were composed of DNA extracted from the selected ten individuals,
respectively. DNA was extracted with a Promega DNA Kit (Promega, USA),
quantified with a BioPhotometer (Eppendorf), and stored at −20 °C until used.
Illumina Miseq sequencing and data analysis
High-throughput sequencing was conducted at Majorbio Co., Ltd (Shanghai, China).
The bacterial 16S rRNA gene was amplified with primers 338F
(ACTCCTACGGGAGGCAGCA) and 806 R (GGACTACHVGGGTWTCTAAT)
targeting the V3-V4 region (about 470bp). The fungal 18S rRNA gene was amplified
with primers ITS1-1737 F (GGA AGT AAA AGT CGT AAC AAGG) and ITS2-2043
R (GCT GCG TTC TTC ATC GAT GC) targeting the ITS1-ITS2 region (about
246bp). The PCR amplification was conducted using specific primers with barcode
and high fidelity TrashStart Fastpfu DNA Polymerase (TransGen Biotech, China).
PCR amplification was performed in a total volume of 20 ȝL containing 4 ȝL
5×FastPfu Buffer, 2ȝL 2.5 mM dNTPs, 0.8 ȝL 5ȝM primers, 0.4 ȝL FastPfu
Polymerase and 10 ng DNA template. The bacterial 16S rRNA gene PCR thermal
cycle profile was as follows: 2 min at 95 ; 28 cycles of 30 s at 95 , 30 s at 61 ,
45 s at 72 ; final 10 min at 72 , and cooling at 10 . The fungal 18S rRNA
gene PCR profile was similar to the bacterial profile except that it had five more
cycles.
The Miseq sequencing was collecting the fluorescence signal to read the sequence of
DNA fragment. All sequences were divided depending on the similarity level and

statistical analysis of biological information under 97% similar level of OTU. The
community structure was analyzed statistically at different classification levels and
visual analysis of community structure and phylogeny finally.
Screening of cellulase-producing bacteria, amylase-producing bacteria
Each isolate was inoculated on carboxy methyl cellulose (CMC) agar plate
medium (0.1%CMC- nutrient agar, pH =9.2-9.8) and soluble starch agar plate
medium (0.1%starch- nutrient agar, pH =9.2-9.8) using a sterilized toothpick, and
incubated at 30 °C for 48 hours. [11]
Identification of enzyme-producing bacteria
The enzyme-producing bacteria were identified by16S rDNA sequencing. DNA
was extracted by boiling bacterial cell suspension in sterile distilled water [12]. 16S
rRNA genes were amplified with universal primers of 27F
(5'-GAGTTTGATCCTGGCTCAG-3') and1492R
(5'-CGGTTACCTTGTTACGACTT-3').PCR amplification was performed in a total
volume of 25 ȝL containing 5 ȝL of DNA extract, 1 ȝL of each primer (10 ȝL mL−1),
1 ȝL of deoxyribonucleotide triphosphate (dNTP) mixture (10 mmol mL−1), 3 ȝL of
10× Taq PCR buffer (containingMg2+), and 0.5 ȝL of Taq DNA polymerase (TaKaRa,
China).Cycling conditions were as follows: initial denaturation at 95 °C for five min,
30 cycles of 94 °C for one min, 55 °C for one min, 72 °C for one min, and a final
extension at 72 °C for five min. PCR products were examined by electrophoresis in a
1% agarose gel, and bands were visualized by staining with ethidium bromide. PCR
products were further purified with the QIA quick PCR purification kit (Quiagen,
www.quiagen.com) and cloned into pMD18-T vector followed by sequencing.
Sequence analysis was performed using the BLAST (http://www.ncbi.nlm.nih.gov).
For the fungi, the ITS sequence were amplified with ITS1
(5-TCCGTAGGTGAACCTGCGG-3) and ITS4 (5-TCCTCCGCTTATTGATATGC-3)
(Sangon Biotech). Cycling conditions were as follows:94 4min; 30 cycles of 94
30s; 55 30s; 72 45s, and an end extension of at 72 7min.Identifications were
based on 16S rRNA gene sequence and ITS sequence similarity. Phylogenetic tree of
the sequence analysis was constructed from a matrix of pairwise genetic distances by

the neighbor-joining method (MEGA 5.0). The bootstrap analysis of 1,000 replicates
was performed [13].

Results
Shifts in microbial community structure and composition by Miseq
The total genomic DNA was extracted from the gut of silkworm larvae. The
average length of sequence were 470bp for bacterial community and 300bp for fungal
community by high-throughput sequencing were performed in the three sample types,
namely, 5M, gut content sample from the fifth instar larvae of silkworms in TRW; 5L,
gut content sample from the fifth instar larvae of silkworms in BRW; and 4L, gut
content sample from the fourth instar larvae of silkworms in BRW)
Misq sequencing generated total of 58827 and 45956 reads of 1737F-2043R
(fungi primers) and 338F-806R (bacterial primers) for the TRW and BRW samples.
Low quality reads were filtered using the QIIME's scripts and a total of 48420
(1737F-2043R), 38814(338F-806R) effective reads were obtained after trimming the
adapters, barcodes and primers. After denoising, filtering out chimeras and removing
the archaeal sequences, the fungi libraries of 4L, 5L and 5M contained 14555, 14993
and 18872 effective sequences, respectively. The bacterial libraries of 4L, 5L and 5M
contained 13210, 14949 and 10655 effective sequences, respectively.
Biodiversity of the two group samples was investigated based the analyses of
OTUs. In terms of OTUs number, fungal communities had the richer diversity (46
OTUs), whereas bacterial one displayed considerably less richness (35 OTUs).Those
results were supported by venn communities of fungi and bacteria (Fig.1).
Diversity and composition of microbial communities in TRW and BRW were
diverse at different instars (Fig.2). Each sample profile displayed a unique banding
pattern. We have detected that the sample 4L has more diversity than 5M and 5L in
bacterial group (Fig.2A). It had found 21 genotypes of bacteria and only 7 genotypes
in 5M and 5L.Meanwhile, sample 5M has more diversity in fungi group than 4L and
5L, the genotypes of fungi were 28, 18 and 13, respectively (Fig.2B). This analysis
confirmed that feeding with changed feedstuff at the beginning of the fourth instar

resulted in great change in gut microbiota of silkworm in the BRW.

Enumeration of the gut microflora

Using the isolation procedure described above, total of 23 isolates were
successfully collected from the gut of silkworm larvae under the TRW and BRW
group. There were 14 strains isolated from the TRW (9 strains from NA, 4 strains
from PDA medium, 3 strains from Gause’s No. 1 agar medium). The rest were from
the BRW (5 strains from NA medium, 1 strain from PDA medium, 3 strains from
Gause’s No. 1 agar medium). Those results showed a similar phenomenon to those
observed by Miseq.

Screening of cellulase-producing and amylase-producing bacteria

According to screening the isolates on CMC- nutrient agar, after incubating at
30 °C for 48 hours, the colonies with degradation capacity were identified using the
Congo red overlay method [14]. For the Congo red method, plates were flooded with
0.1% aqueous Congo red for 10 min and then washed with 5% NaCl solution for 50
min. Cellulose production was observed as a clear zone of hydrolysis around bacterial
colony. Six and three isolates of cellulolytic bacteria were obtained from TRW and
BRW group, respectively. For the isolates of starch- nutrient agar, the iodine method
starch plates were flooded with iodine solution resulting in dark blue plates with
uncoloured zones where the starch had been degraded. The isolates could product
amylase of the BRW and TRW group were11 and 6, respectively.
As identified above, we found three isolates named M1 M4 M11 were
cellulolytic and amylolytic bacteria in the TRW group(Fig.3).Three isolates were all
screened from PDA plate. Meanwhile, L1 L5 L9 were the cellulolytic and amylolytic
bacteria in the BRW group( Fig.4).

Phylogenetic analysis of isolates on cellulose-producing and amylase-producing

bacteria
To further characterize the cellulolytic and amylolytic bacteria, molecular

approach was used. The results showed that the 16S rRNA genes of three isolates had
about 1500 bp DNA fragments by PCR amplification in the BRW group. To clarify
the phylogenetic position of these enzyme-producing bacteria, a phylogenetic tree was
constructed based on 16S rRNA gene sequence homology. The results showed strains
L1 L5 L9 had the highest homology with Enterococcus, Erwinia and Pantoea,
respectively. Meanwhile, three isolates in the TRW group of ITS sequence homology
were related to Alternaria sp. Preussia sp and Coprinellus radians. Phylogenetic tree
for the members of these genera within two types of samples were represented in Fig.
5 and Fig.6.
Discussion
We compared the number and diversity of silkworm larval gut microbiota in
response to two different rearing ways (the TRW and BRW) by high-throughput
sequencing and classical culture techniques. A prior study demonstrated that most of
the cultivable bacteria from the silkworm larvae digestive tract were able to produce
digestive enzymes to utilize plant polysaccharides such as cellulose, starch, xylan, and
pectin[15]. Thus, our results showed diversity decrease and imbalance of gut
microbiota of silkworms in the BRW, indicating that alteration in the gut flora by
feeding lettuce was attributed to digestive enzyme activity and physiological activity
reduction of silkworms.
Based on microecology theory, insects lack a complete enzyme system and thus
need gut microorganisms to provide different kinds of enzymes for food digestion,
nutrient absorption, and biological metabolism [5]. It is supported that the change of
gut flora due to lettuce leaf feeding lead to the decrease of physiological activity of
the silkworms and the appearance of profitless bacteria in the gut of silkworm under
the BRW might break down the balance structure of healthy gut microbial community,
resulting in reduced digestive enzyme activity [8].
In order to show how the enzyme-producing bacteria of silkworm larvae changes
under different rearing methods, we examined diet effect on the composition of gut
bacteria of the silkworm fifth instar by screening the bacteria from different agar
media. Silkworms fed on mulberry leaves from the first instar to fifth instar had


significantly more bacteria (14 isolates) than those fed on mulberry leaves from the
first instar to third instar and on lettuce during the fourth instar and fifth instar (9
isolates).Among the TRW group, 3 isolates out of 14 can produce cellulose, 11
isolates out of 14 can produce amylase. The proportions of cellulase-producing
bacteria and amylase-producing bacteria in the TRW samples were 21.4% and 78.5%,
respectively. In contrast, cellulose-producing bacteria and amylase-producing bacteria
separately accounted for 33.3% and 66.6 % in the BRW groups. The proportion of
cellulase-producing bacteria in the BRW group was higher by 12% than that in the
TRW group. Otherwise, amylase-producing bacteria occurred in the BRW group with
a lower percentage of 12 %.This difference may relate to the different content of
cellulose and starch. Additionally, lettuce leaves contain fewer sterols than that of
mulberry leaves, sterols are indispensable in growth and development of the silkworm
larvae [16].In the TRW group, three isolates which can both secrete cellulose and
amylase are Alternaria sp. Preussia sp and Coprinellus radians. Alternaria is a
ubiquitous fungal genus associated with a wide variety of substrates including seeds,
plants, agricultural products, animals, soil, and the atmosphere. Moreover, genus
Alternaria was able to use carboxymethylcellulose as substrates. It is reasonable that
the function of Alternaria in the silkworm gut is associated with nutrient and energy
digestion to ensure the normal growth [17]. Preussia sp and Coprinellus radians
associated with the growth and development of the silkworm larva gut whether or not
that remains to be further proved.
A remarkable change of cultivable enzyme-producing bacteria in the BRW
samples due to lettuce leaf feeding. Enterococcus is a predominant bacterium in the
silkworm gut according to Xiang et al. [18]. In the silkworm larvae, Enterococcus is
present at high frequency in the digestive tract, which can lower the gut pH and
inhibit the suppression of Nosema bombycis germination, contributing to resistance to
disease [19, 20]. Erwinia and Pantoea are belonged to the family
Enterobacteriaceae. Studies have showed that Erwinia sp. from fifth instar B. mori
larvae could utilize pectin efficiently which involves in digestion of pectin from
lettuce leaves [21]. Pantoea is found in a wide range of natural environments,


including water, soil, as part of the epi and endophytic flora of various plant hosts,
and in the insect gut. Some strains have proven effective as biological control agents
and plant-growth promoters. As Pantoea is frequently isolated from invertebrate hosts,
the presence of such a peptide may be of interest for the biological control of insect
pests [22].
Only bacteria exist in most studies of insect gut microbial diversity. Our results
showed three isolates of enzyme-producing in the silkworm gut by the rearing method
in TRW group are fungi. However, although culture-based methods can provide a
good indication of gut microbes, they do not necessarily provide comprehensive kinds
on the composition of gut microbial communities because they are limited in scope to
organisms that are amenable to cultivation with particular growth media and
conditions. Moreover, no isolates of fungi have been found in the BRW group
that shown to optimize the structure of silkworm intestinal microbial flora is
necessary.
Based on the data obtained in this study, it can be concluded that the gut of
silkworm larvae is a rich cellulolytic and amylase microorganism resource and that
the isolate of Enterococcus, Erwinia and Pantoea, seem to be a prospective organisms
for further biotechnological exploitation to make probiotics for improving yield and
quality of silkworm larvae reared in BLSS.

Acknowledgements
This research was supported by International Science-Technology Cooperation Progra
m of China (2012DFR30570).

Reference

[1]McKillip J.L., Small C.L., Brown J.L., Brunner J.F., Spence KD. (1997).

Sporogenous midgut bacteria of the leafroller, Pandemis pyrusana (Lepidoptera:


Tortricidae). Environ. Entomol., 26, 1475–1481

[2] Dillon R.J., Vennard C.T., Buckling A., Charnley A.K., 2005. Diversity of locust

gut bacteria protects against pathogen invasion. Ecol. Lett., 8, 1291–1298

[3] Eutick M.L., O’Brien R.W., Slaytor M., 1978.Bacteria from the gut of Australian

termites. Appl. Environ. Microbiol., 35, 823-828.

[4]Fukatsu T, Hosokawa T., 2002. Capsule transmitted gut symbiotic bacterium of

theJapanese common plataspid stinkbug,Megacopta punctatissima. Appl. Environ.

Microbiol., 68, 389-396

[5]Wei X. 1985. Normal flora and health.Shanghai Science Press.

[6] Yang Y., Tang L., Tong L., Liu H., 2009. Silkworms culture as a source of protein

for humans in space. Adv. Space. Res., 43, 1236–1242

[7] Yu X, Liu H, Tong L., 2008. Feeding scenario of the silkworm (Bombyx mori L.)

in the BLSS. Acta. Astronaut., 63, 1086–1092

[8] Liang X., Fu Y.M, Tong L., Liu H., 2014. Microbial shifts of the silkworm larval

gut in response to lettuce leaf feeding. Appl. Microbiol. Biotechnol., 98, 3769–3776

[9] Kalpana S, Hatha A.A.M., Lakshmanaperumalsamy P .,1994. Gut microflora of

the larva of silkworm, Bombyx mori. Insect. Sci. Appl., 15, 499–502

[10] Huang X.L.1999. Guidance of microbiology experiment. China Higher

Education Press. pp114-116.

[11] Berg B., Hofsten B.V., Pettersson G.1972.Growth and cellulase formation by

Cellvibriofulvus. J. Appl. Bacteriol., 35, 204-21 4

[12] Brousseau R., Saint-Onge A., Préfontaine G., Masson L, et al. 1993. Arbitrary


primer polymerase chain reaction, a powerful method to identify Bacillus

thuringiensis serovars and strains. Appl. Environ. Microbiol., 59,114-119.

[13] Saitou N., Nei M.,1987. The neighbor-joining method: a new method for

reconstructing phylogenetic trees. Mol. Biol. Evol., 4, 406–425

[14] Sneath P.H.A., Nair N.S., Sharpe E.M., Holt J.G., 1984. Bergey’s manual of

systemic bacteriology. 1st edition, William and Wilkins. pp. 408-494.

[15] Anand AA, Vennison SJ, Sankar SG, Prabhu DI, Vasan PT, Raghuraman T,
Geoffrey CJ, Vendan SE (2010) Isolation and characterization of bacteria from the
gut of Bombyx mori that degrade cellulose, xylan, pectin and starch and their impact
on digestion. J Insect Sci 10:1–20

[16]Xue Y.W., 2009. Influence of feeding the Cudrania triloba, Cudrania tricuspidata

(Carr.) Bur. leaves on resistant physiological functions of silkworm, Bombyx mori.

MS thesis, Southwest University, Chongqing,China.

[17] Deng J.X., Cho H.S, Narayan C.P, Lee H.B., et al.2014.A Novel Alternaria

species isolated from Peucedanum japonicum in Korea. Mycobiology, 42, 12-16

[18] Xiang H., Li M., Zhao Y., Zhao L., et al. 2007. Bacterial community in midguts

of the silkworm larvae estimated by PCR\DGGE and 16SrDNA gene library analysis.

Acta. Entomol. Sin., 3, 222–233

[19] Takizawa Y., Iizuka T., 1968. The aerobic bacterial flora in the gut of larvae of

the silkworm Bombyx mori L. (I) The relation between media and the numbers of

living cells. J. Sericultural. Sci. Jpn. 37, 295–305

[20] Lu X, Wang F., 2002. Inhibition of cultured supernatant of Enterococci strains on

germination of Nosema bombycis spores in vitro. Sci Sericulture., 28, 126–128 (In


Chinese)

[21] Feng W, Wang X.Q, Zhou W, Liu G.Y, Wan Y.J. 2011.Isolation and

characterization of lipase-producing bacteria in the intestine of the silkworm, Bombyx

mori, reared on different forage. J. Insect. Sci., 11,135


[22] Maayer P.D., Chan W.Y., Enrico R., Stephanus N.V., et al. 2014 .Analysis of

the Pantoea ananatis pan-genome reveals factors underlying its ability to colonize and

interact with plant, insect and vertebrate hosts. BMC Genomics, 15,404



Figure legend:

Fig.1: Venn of fungal and bacterial communities of BRW and TRW based on Misq
sequencing A) is bacteria B) is fungi
Fig.2: Relative abundance of community proportions at genus level in BRW and
TRW groups, A) is bacteria B) is fungi

Fig.3 Plate showing cellulase and amylase degrading bacteria in TRW group

Fig.4 Plate showing cellulase and amylase degrading bacteria in BRW group

Fig.5: Neighbor-joining tree of ITS sequences depicting the phylogenetic

relationships of cellulolytic and amylolytic bacteria from TRW

Fig.6Neighbor-joining tree of 16S rRNA gene sequences depicting the phylogenetic

relationships of cellulolytic and amylolytic bacteria from BRW


Differences of silkworm gut microbes under BLSS rearing and traditional

rearing ways

cellulase and amylase producing bacteria were separated in both rearing

ways

Enterococcus, Erwinia and Pantoea are cellulase and amylase producing

isolates in BRW

The isolates can be developed into probiotics for improving animal

protein in BLSS


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