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\begin{document}

\title{Assessment of gut microbiota associated with oak tasar silkworm,
Antheraea proylei J. (Lepidoptera: Saturniidae)}



\author[1]{Yumnam Devi}%
\author[2]{Deepak   Lourembam}%
\author[3]{Tourangbam  Shantibala}%
\author[4]{Sinam Subharani}%
\author[5]{Rahul Modak}%
\author[1]{Yallappa Rajashekar}%
\affil[1]{Institute of Bioresources and Sustainable Development}%
\affil[2]{Regional Institute of Medical Sciences}%
\affil[3]{Central Agricultural University}%
\affil[4]{Regional Sericultural Research Station}%
\affil[5]{Kalinga Institute of Industrial Technology}%


\vspace{-1em}



  
  \date{June 10, 2024}


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\selectlanguage{english}
\begin{abstract}
Antheraea proylei J, is an economically important silkworm of North
Eastern region of India reared for the production of the tasar silk. The
silkworm is often exposed to various microbial diseases caused by
bacteria and viruses. The disease causes significant damage to larvae
and elicit pupal mortality, thus posing a serious threat to the linked
economic activities. The gut microbiome of silkworms play an important
role, in nutrient acquisition and immunity. In this study, we have
reported molecular characterization and histopathological assessment of
gut associated bacteria of healthy and diseased tasar silkworms. As
compared to healthy silkworms, diseased infected silk glands shows loss
of turbidity, secretory layer not distinguishable to tunica propria and
lumen distorted. Both secretory and absorptive cells were found to be
hypertrophied. Body fat becomes vacuolated and soft when compared to the
healthy silkworms. Bacterial profile of healthy and diseased silkworm
respectively was identified by 16S rRNA gene sequencing and analysis.
Bacillus toyonensis and Bacillus thuringiensis were commonly found in
healthy larvae whereas Bacillus aryabhattai and Bacillus megaterium were
found in diseased larvae. The family Bacillus of phylum Firmicutes was
dominant in both healthy and diseased silkworms. To the best of our
knowledge, this is the first attempt to study A. proylei midgut
microbiota from a biodiversity hotspot in North Eastern India. The
present study might be helpful in disease prognosis and further
comprehensive analysis on midgut microflora may lead towards the
development of effective strategies for management of these economic
silkworms.%
\end{abstract}%



\sloppy


\textbf{INTRODUCTION}

The North eastern region of India is home to a number of wild
sericigenous insects and is centre of wild silk culture including muga,
eri, oak tasar, and mulberry silk (Lokeshwari \& Shantibala, 2010).
Tasar silk industry has lot of socio-cultural and traditional linkages
in India since immemorial and plays a vital role on rural economy and
hence indicates its impact on the county's economy simultaneously with
agriculture(Reddy, 2010).The oak tasar silkworm, \emph{Antheraea
proylei} J, is an economically important silkworm of Manipur reared for
the production of tasar silk. It is interbred between the male Indian
species of \emph{A. proylei} with the female of Chinese species
of\emph{A. pernyii} G(Jolly, 1969).The yield and quality of tasar silk
cocoons are depended on the climatic conditions, silkworm health and
nutrient absorption. Physiology and pathology of the silkworm's
digestion, absorption, nutrient utilization and diseases emergence of
the silkworm was closely related to microbiota of the larvae's midgut.
Till date, there are no silkworm races at present, which are deemed as
totally resistant to diseases or pests. At the end of the larval stage,
silk productions are aided by silk glands whose infection affects silk
production, resulting in substantial economic losses to the farmers and
industries(Brancalh\selectlanguage{ngerman}ão, Torquato, \& Fernandez, 2009).As disease infected
silkworms fail to spin cocoons, analysis of cytological damages in the
silk gland is essential. Some histopathological studies showed the
susceptibility of silk gland cells to BmNPV (Brancalhão et al., 2009;
Khurad, Mahulikar, Rathod, \& Rai, 2005; Rahman \& Gopinathan, 2004).
Hence knowledge of the microfloral changes of the gut in diseased
conditions will help in understanding the health and nutrition of the
silkworm(Yuan, Lan, Yang, Xiao, \& Zhou, 2006)and give us ideas of
management to improve the diseased condition during infection as such.

The intestinal tract microflora plays an important role in the health of
the host by maintaining a normal ecological balance, regulating
absorption, digestion, and assimilation (Jeon et al., 2011). Gut
microflora are required in pheromone production, pesticide degradation
and survivability, vitamin synthesis, and immunity against
pathogens(Reeson, Jankovic, Kasper, Rogers, \& Austin, 2003).In
addition, these bacteria also resist and compete with the invading
microbes and their propagation and strengthen the immune system (Tokuda,
Watanabe, Matsumoto, \& Noda, 1997). Microbial pathogens infect all
animal species leading to disease and death. But their immune defence
system helps in protection and survival(Miyashita, Kizaki, Kawasaki,
Sekimizu, \& Kaito, 2014). Insects especially their larval forms are
more susceptible to pathogenic bacterial diseases and their virulence
factors than vertebrates which further leads to alteration in host
defense mechanism (Waterfield, Daborn, \& Ffrench\selectlanguage{english}-Constant,
2004).Culture studies in laboratory conditions of insect gut bacterial
communities do not reflect the entire microbial types and strains
(Gilliam, 1997). Culture dependent methods screen only a few predominant
bacteria genera and are unable to detect bacterial genera with low
abundance. The 16S rRNA gene is often used as a marker for identifying
the diversity of bacterial species in insect gut microbiota (Reeson et
al., 2003). It is of notable fact that gut microbiota is involved in
regulating the growth, development and environmental adaptation in the
host.

However, information on the gut bacterial communities of many insects,
including \emph{A. proylei} is limited. The molecular information
regarding this economically important silk moth remains severely limited
till date and only a handful of DNA sequences are available (Shantibala,
Devi, Lokeshwari, Anju, \& Luikham, 2018; Yang et al., 2019).
Additionally, very little is known about the effects of pathogens, its
nutrient utilization and disease emergence of silkworm in its gut
microbiota. Therefore research of the gut microbiota is of great
importance .Little is known about the bacteria inhabiting the gut of
this silk moth and even basic information on the insect's microbial
symbionts is lacking. To date, there is no report on profiling of gut
microflora in silkworm. Hence, in this present study, we aimed to
compare the intestinal microflora of \emph{A. proylei} of normal and
Tiger band disease infected fifth instar larva reared under the same
conditions using 16S rRNA\selectlanguage{english}-based sequencing method. Furthermore, we also
assess the histopathological changes in the midgut and silk glands after
infection to assess the tissue damages. Therefore, our study might
provide insights for improvement and management of disease as a step
towards conservation of wild seri biodiversity for ecological balance
and for sustainable economic viability.

\textbf{MATERIALS AND METHODS}

\textbf{Sample Collection}

Healthy and infected fifth instar larvae of \emph{A. proylei} were
collected from Regional Sericulture Research Station, Mantripukhri,
Manipur during summer season of 2019. The details of the location are
furnished in Table 1. Healthy tasar silkworms and disease silkworms
showing the symptoms of Tiger band disease were collected from the above
mentioned area (Fig. 1 a \& b). The infected larvae suffering from Tiger
band disease were recognized from their black band pattern similar to
tiger stripes appearing on the larva (Shantibala, Fraser, et al., 2018).
Proper sterilized equipment such as forceps, scissors and hand gloves
were used while collecting the insect sample. The collected samples were
stored in an ice cold storage box. Samples were brought from the field
and surface sterilization was done with 70\% ethanol by submersion and
rinsing three times using sterile distilled water prior to dissection.
The gut tissue of larvae and midgut was dissected out in a sterile
environment using dissection scissors. The collected intestinal contents
will be immediately stored at 50\% glycerol at -80\selectlanguage{english}0C for future use
(Broderick, Raffa, Goodman, \& Handelsman, 2004). Three larvae from each
location were taken for gut dissection. Individual larvae were used for
the isolation of gut bacteria. After isolation of gut bacteria the
larvae were transferred to laboratory conditions maintained at 26 \selectlanguage{ngerman}±1º C
and 70 \% RH at Animal Resources Division Laboratory, Institute of
Bioresources and Sustainable Development, Takyelpat, Manipur, India.

\textbf{Isolation and culture of the intestinal bacteria}

Guts of healthy and diseased \emph{A. proylei} larvae were homogenized
in 0.86\% NaCl solution (Broderick et al., 2004).The stock solution was
prepared by taking 1 ml of the suspension and was mixed with 9.0 ml
saline. Using the serial dilution method seven dilutions were prepared.
1ml of each dilution was added to a separate plate in triplicates. Then
15 ml of nutrient agar was added to each medium and incubated for 24
hours at 37°C. The dominant colonies were identified, purified three
times by successive inoculation and culture on the corresponding agar
plates, and further transferred to agar slants (Huang \& Xin, 1999).

\textbf{Histopathological Investigation}

For histopathological investigation healthy and diseases infected fourth
instar larvae were collected from Regional Tasar Research Institute,
Manipur, India. Different organs like silk glands and gut were removed
from normal and nucleopolyhedrovirus (NPV) infected silkworms. The
removed tissues were preserved in 10\% formaldehyde solution. The
tissues were again fixed in Bouin's fluid. The water molecules were
removed with the help of alcohol and embedded in paraffin wax for
sectioning. 5-7\selectlanguage{greek}µ\selectlanguage{english}m thick tissues were stained with haematoxylin and
eosin. Structural examination and histopathological changes were
identified by visualization through Leica DM 3000 LED and photographed
with Leica DFC450 C.

\textbf{DNA extraction, amplification, purification and sequencing}

Total genomic DNA was extracted from the intestinal contents using Gsure
Bacterial Genomic DNA isolation kit. The quality of the extracted DNA
was assessed by electrophoresis in 1\% (w/v) agarose gel. The
concentration of the extracted DNAs was determined using a Nanodrop
(Thermo fisher Scientific) and then normalized to 200 ng/\selectlanguage{greek}μ\selectlanguage{english}l. Universal
16S rRNA genes were amplified by PCR in a volume of 25\selectlanguage{greek}μ\selectlanguage{english}l containing 200
ng DNA, 5X Phusion HF buffer, 10mM each dNTP, 2.5 U of Phusion DNA
Polymerase, 0.5\selectlanguage{greek}μ\selectlanguage{english}M of forward and reverse primers as listed before
(Weisburg, Barns, Pelletier, \& Lane, 1991).

The PCR amplification was carried out with the following process:
94\textsuperscript{0}C for 5 min; 35 cycles at 94 \textsuperscript{0}C
for 1 min, 50 \textsuperscript{0}C for 30 sec, 72 \textsuperscript{0}C
for 2 min and a final extension at 72 \textsuperscript{0}C for 10 min.
The amplified PCR products were analysed by electrophoresis on a 1\%
agarose gel and visualized under Gel Documentation System (Bio-Rad). The
PCR product was further purified using Gene JET purification column and
sequenced at sequencing was done using BigDye\selectlanguage{ngerman}® terminator kit following
manufacturer's instructions (Applied Biosystems Inc. ABI, Foster City,
CA) and products analysed on an ABI 3500xL Genetic Analyser platform (at
Eurofins Pvt Ltd, Bangalore, India). Amplicons were sequenced from both
directions using forward and reverse primers.

The purified PCR products were sequenced and aligned with the
corresponding sequences in the GenBank database by using Blast search
algorithm (Altschul et al., 1997).These sequences were deposited in the
GenBank.

\textbf{Sequence analysis}

Online similarity searches were performed using the BLAST algorithm of
GenBank. Sequence alignment was carried out with the CLUSTALW program by
MEGA7 software(S. Kumar, Stecher, \& Tamura, 2016). The phylogenetic
analyses based on the 16S rDNA gene for proteolytic bacteria were
further used to establish relationships among them. The partial 16S rDNA
sequences obtained for were utilized in the search of reference
nucleotide sequence available in NCBI GenBank database using BlastN
algorithm to retrieve the similar sequences(Altschul et al., 1997).
Phylogenetic tree and evolutionary relationships of bacterial isolates
was constructed by Neighbor-Joining method (Saitou \& Nei, 1987) with
Kimura 2-parameter corrections (Kimura, 1980). To calculate the support
for each clade, bootstrap analysis were performed with 1000 replications
(Felsenstein, 1985).

\textbf{RESULTS}

\textbf{Histopathological Observations}

\textbf{Silk glands}

The three layers of silk glands such astunica propria, glandular layer
and tunica intima were visible in normal silkworm. The secretory cells
have rich secretory granules with a wider layer compared to tunica
propria. Tunica propria is narrow and the lumen consists of silk mass.
Glandular zone has a large number of haemocytes, whose nuclei are
branched. (Fig. 2 a). In disease infected worm, the silk gland shows
loss of tunica propria integrity and secretory layer is not easily
distinguishable from tunica propria. Lumen is seen distorted with
vacuolation in the silk mass. The silk mass loss compactness and becomes
less dense (Fig. 2b).

\textbf{Midgut}

Mid gut in the larvae is mainly involved in digestion and absorption.
Histologically, a stratum of enteric epithelium, the outer ends of whose
cells rest upon a basement membrane lines the midgut. The latter is
followed by an inner layer of circular muscles and an outer layer of
longitudinal muscles. The midgut cell are of two types, columnar cells
and secretory or goblet cells. In addition regenerative cells replacing
the destroyed epithelial cells during moulting are also present. As
healthy larvae mid gut are rich in digestive activity, so the secretory
cells are seen normal and the absorptive cells are normal indicative of
good transport of absorbed material. Regenerative cells and their nuclei
are prominent. The basement membrane and musculature are normal (Fig.
3a).

However in the diseased silkworm, the epithelial layer of the midgut
lacked continuity. After infection, the hypertrophy was much more
pronounced and large vacuoles were formed in epithelial cells of\emph{A.
proylei} midgut. Both secretory and absorptive cells were hypertrophied.
Absorptive cells are fully loaded indicating loss of its absorption. The
inter-cellular spaces widened to a great extent and epithelial tissues
detached from the basement membrane. Also bacterial populations were
seen with dark masses inside lumen indicative of its infections
corroding the epithelial layer (Fig. 3b).

\textbf{Phylogenetic Affiliation of 16S rRNA}

We analysed the 16S rRNA sequences for the phylogenetic affiliation of
both unidentified and identified strains by neighbour-joining analysis
with the optimal criteria set for distance in MEGA 6. 16S rRNA sequences
were compared with sequence data deposited in GenBank using the BLAST
search program. Comparative analysis with GenBank sequences revealed
that most of the intestinal bacteria were showing 99\% similarity and a
few were showing 98\% similarity to their closest relatives retrieved
from the Gene bank database. The 16SrRNA sequences of gut bacterial
isolates generated from our study were submitted into GenBank vide
accession nos.MT416410.1 to MT416415.1 and the details are presented in
Table 2.

Bacterial isolates obtained from the midguts of \emph{A. proylei} were
screened based on their colony characteristics and identified on the
basis of 16S rRNA gene sequences. Based on 16S rRNA gene sequence
analysis, tasar silkworm, \emph{A. proylei} showed Firmicutes as the
dominant group forming a major clade with Bacillus as the dominant genus
with ten different species.

Bacillaceae was found to be abundantly represented in the gut of both
healthy and diseased A. \emph{proylei} (Table 3). Bacillus was found to
be the predominant genera in our study from cultivable gut bacterial
isolates of \emph{A. proylei} of North Eastern India. The predominant
Bacillus genus was represented by species such as \emph{Bacillus
toyonensis, Bacillus thuringiensis, Bacillus pacificus, Bacillus
mobilis} and \emph{Bacillus mycoides} in healthy worms. The diseased
group on the other hand are represented by species such as\emph{Bacillus
megaterium, Bacillus aryabhattai, Bacillus zanthoxyli, Bacillus flexus}
and \emph{Bacillus simplex.}

Phylogenetic analysis revealed that gut microflora diversity of diseased
and normal tasar silkworms is almost similar with only 6 \% differences
between the groups. The evolutionary distances were computed using the
Maximum Composite Likelihood method(Tamura, Nei, \& Kumar, 2004). A
phylogenetic tree was constructed (Fig. 4) using the 16S rRNA sequences
by neighbour-joining method 1000 bootstraps, to better understand the
evolutionary relationship of the intestinal bacteria.

In the diseased silkworms, \emph{B. aryabhattai} seems to be the most
abundant genera found in all the diseased worms. In healthy silkworms,
microflora is affiliated with the species \emph{B. toyonensis}
and\emph{B. thuringiensis} , showing that these species share high
sequence similarity. This showed more than 99\% identity with the
sequences deposited in the GenBank database.

Generally, \emph{Bacillus} species are widely used as biopesticides and
biofertilizers in agricultural practice (Pérez-García, Romero, \& De
Vicente, 2011), as potential probiotics for both animals and humans
(Cutting, 2011; Elshaghabee, Rokana, Gulhane, Sharma, \& Panwar, 2017).
Hence our results also might help in potential application
of\emph{Bacillus} species against a variety of pathogenic gut
microflora.

\textbf{DISCUSSION}

Many Lepidopteran insects including silkworms are described as
beneficial insects in the past two millennia. Gut microflora of most of
the insects comprises a wide variety ranging from obligate endosymbionts
to facultative bacteria and others (Dillon \& Dillon, 2004).
Comprehensive analysis of bacterial diversity within the gut of insects
from different geographical regions will enable us to better understand
their ecological roles and their interactions with the insect host. Very
little information is available on the gut bacterial diversity of tasar
silkworm, \emph{A. proylei} which is exclusively present in the world in
North Eastern regions of India. Culture-dependent 16s rRNA gene sequence
based approaches in this study was used to assess the cultivable gut
bacterial communities associated with these tasar silkworm populations
in Manipur, North Eastern state of India.

Our analysis shows that the tasar silkworm harboured unique bacterial
flora in its gut with Firmicutes group as the major one and Bacillus
being the predominant genera constituting about 100 per cent of total
gut bacterial isolates from \emph{A. proylei.} Earlier studies have also
documented the presence of Pseudomonas and Bacillus species as the
dominant bacterial communities in the gut of gypsy moth, \emph{L.
dispar} (Lepidoptera)(Broderick et al., 2004). These results were in
agreement with that of \emph{Bombyx mandarina} and \emph{Bombyx mori}gut
bacterial diversity where phylum Firmicutes was found to be the
predominant bacteria and Enterococcus to the predominant bacterial genus
through 16S rRNA gene sequencing(D. Kumar et al., 2019). Bacillus was
also the most dominant bacterial genus in an earlier study, accounting
for 18\% of the total gut community in 21 different insect species (Yun
et al., 2014).

Gut morphology was early being described as a potential phylogenetically
informative character in a study on Passalids (Bess
beetles)(Reyes-Castillo, 1970). A limited diversity was revealed in 16S
rRNA gene analysis of gut bacteria of \emph{P. xylostella} larvae
(Indiragandhi et al., 2007). The phylogenetic analysis of gut bacterial
isolates from tasar silkworm indicates that tasar silkworm has limited
gut bacterial diversity.

We also investigated the histopathological importance of healthy and
diseased silkworm larvae to understand in-depth pathogenic effects a
disease imposes on the cells, tissues and organs. As infected silkworms
fail to spin cocoons analysis of cytological damages in the silk gland
is very essential. The digestive system in silkworm larvae needs much
attention as oral entry of pathogenic microbes are quite common. Apart
from dual function of digestive and absorptive, the midgut region also
provides a barrier to invading parasites too. Here we report for the
first time the histopathological effects in \emph{A. proylei} silkworm.
Our results show that silk glands that contains tissue of infected
larvae were ruptured and deformed along with the formation of lump cells
compared with healthy larvae. In disease infected worm, the silk mass
shows a notable destruction and vacuolation of the fat body cells and
the fat tissues became soft and compactness as compared to those of the
healthy insect. Also the IV instar larvae with disease stop feeding and
colour change appears in the integument. The abnormal swelling, cell
hypertrophy and other cytotoxic effects are the result of loss of
silkworm ability to maintain homeostasis.

Histopathological changes in midgut by bacterial or viral infections
have been documented in other silkworms (Choudhury et al., 2004;
Jurat-Fuentes \& Jackson, 2012; Mohanta et al., 2015; Ponnuvel et al.,
2003). The midgut portion of nuclear poly hydrosis infected silkworm
larvae showed hyper trophism of nuclei. Hypertrophic nuclei were swollen
compared to normal nuclei. We observed notable hypertrophy, hyperplasia
and multi-layered epithelial cells of the midgut.

Also, phylogenetic tree analysis of the gut bacterial 16S rRNA gene
sequences in our study (Fig. 4) showed Firmicutes as the dominant family
forming a major clade with Bacillus as the dominant genus with ten
different species.

It appears that the histopathological effects with respect to the Tiger
band disease are mainly localized in the silk glands, midgut, and
muscles surrounding the alimentary canal and body fat. The most common
pathological changes observed include hypertrophy, vacuolation
ultimately leading to insect death. Molecular phylogenetic analysis
further reveals that Firmicutes and Bacillus being the predominant clade
and genera in both healthy and diseased silkworms. Detailed
characterization and further investigation from different geographical
locations in the North eastern region of India would advance our
understanding of diversity and composition of gut bacteria of oak tasar
silkworms and its disease management.

\textbf{CONCLUSION}

To the best of our knowledge, this is the first attempt towards a
genetic and histopathological characterization of the midgut microbiota
of \emph{A. proylei} found in Manipur, North East India. A more
comprehensive knowledge about midgut bacteria may lead to better
understanding the direct or indirect involvement of microbiota in the
immune response, nutrition and reproduction of these important silkworm.
This present study not only offers insight into the process of gradual
degradation \emph{A. proylei} larvae midgut, but also elucidates the
extent of damage that occurs therein, after the infection which also
affects the silk glands. The probiotic candidature of role of the
potential gut microflora will also be a challenge ahead. Further our
findings might advance our understanding in the field of disease
resistance traits and silk worm \emph{A. proylei} management.

\textbf{ACKNOWLEDGEMENTS}

The authors are gratefully acknowledged the help and support rendered by
Director, Institute of Bioresources and Sustainable Development (IBSD),
Manipur, Dept. of Pathology, RIMS and Regional Sericultural Research
Station, Imphal for the conduct of this study. While the first author
acknowledges~Institute of Bioresources and Sustainable Development,
Manipur India for financial assistance through IBSD-Ph.D. programme.

\textbf{Conflict of interest:} The authors have no conflict of interest
to declare.

\textbf{AUTHORS CONTRIBUTION}

Conceived and designed the experiments: YRD, DSL, TS, YR. Sample
collection and performed the experiments: YRD, SS. Analysed results and
data: DSL YRD. Wrote Manuscript DSL, YRD, YR. YR supervised the study.

\textbf{DATA AVAILABILITY STATEMENT}

DNA sequences used in study have been released in NCBI with GenBank
Accession numbers MT416410.1 -- MT416415.1.

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\textbf{Figure Legends}

\textbf{Figure 1:} Oak tasar silkworm larvae, \emph{Antheraea proylei} J
(a) healthy and (b) disease larvae suffering from Tiger band disease.

\textbf{Figure 2 :} Microscopic examination of silk gland of normal
silkworm and diseased at 10x magnification. (a\textbf{)} Silk gland of
normal silkworm showing intact tunica propria (TP) and rich nuclei
showing horseshoe shaped nuclei (N) (Bar=100\selectlanguage{greek}µ\selectlanguage{english}m) (b)The diseased silk
gland showing loss of tunica propria (TP) integrity. At advance stage of
infection, larvae shows small spherical nuclei (N) with appearance of
vacuoles (V) (Bar=100\selectlanguage{greek}µ\selectlanguage{english}m).

\textbf{Figure 3:} (a) Cross section of midgut of healthy fourth instar
larvae of \emph{A. proylei} showing columnar epithelial cells (CEC)and
compact darkly stained nucleus (N) (Bar= 200\selectlanguage{greek}µ\selectlanguage{english}m) (b) Cross section of
midgut of TBD fourth instar larvae of \emph{A. proylei} showing hyper
tropheid columnar epithelial cells (CEC) and vacuoles (V) in cytoplasm
(Bar= 200\selectlanguage{greek}µ\selectlanguage{english}m).

\textbf{Figure 4: Phylogenetic tree of gut bacteria isolated from gut
of\emph{Antheraea proylei}}

The evolutionary history was inferred using the Neighbor-Joining method.
The optimal tree with the sum of branch length = 0.66091549 is shown.
The percentage of replicate trees in which the associated taxa clustered
together in the bootstrap test (1000 replicates) are shown above the
branches. The tree is drawn to scale, with branch lengths in the same
units as those of the evolutionary distances used to infer the
phylogenetic tree. The evolutionary distances were computed using the
Maximum Composite Likelihood method and are in the units of the number
of base substitutions per site. Evolutionary analyses were conducted in
MEGA7. Diseased silkworm (red circles) and healthy silkworms (square)
are represented in analysis.\selectlanguage{english}
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