WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

Shreenivasulu et al. World Journal of Pharmacy and Pharmaceutical Sciences

SJIF Impact Factor 6.647

Volume 6, Issue 7, XXX-XXX Research Article ISSN 2278 – 4357

A COMPARATIVE STUDY ON CYTOCOMPATIBILITY OF WEAVER

ANT NEST FIBRE MATS

Kamma Shreenivasulu*, Penumarthi Satya Madhuri and Sagi Himaja

K L University, Green Fields, Vaddeswaram, Guntur District, Andhra Pradesh, India.

ABSTRACT
Article Received on
09 May 2017, Weaver ants are renowned for their nest construction strategies, of
Revised on 30 May l 2017, which adhering in-place leaves stands as an exceptional natural
Accepted on 20 June 2017,
DOI: 10.20959/wjpps20177-9570 episode. This deposition appears as a mat to naked eye integrally
having a fibre network. The mat is expected to be a polymeric
glycoprotein from certain biochemical preliminary confirmations.
*Corresponding Author
Kamma Shreenivasulu
Literature suggests a destined antimicrobial activity for glycoproteins,
K L University, Green antibacterial, in particular. A comparative study has hence been taken
Fields, Vaddeswaram, up on the behaviour of gram-positive (Bacillus subtilis and Clostridium
Guntur District, Andhra
thermocellum) and gram-negative (Escherichia coli and Pseudomonas
Pradesh, India.
aeruginosa) bacteria associated with its presence in culture media.
[email protected],
[email protected], Availability of fibre mat samples from weaver ant (Oecophylla
smaragdina) nests found on three different trees (Citofortunella
microcarpa, Couroupita guianensis and Artocarpus heterophyllus) fostered comparative
assessment of bacterial behavior as consequences of corresponding influence. Enzymatically
digested mat enhanced the growth of all the bacterial strains as expected owing to the
presence of cellulose initially. Its effect on mammalian cell lines has also been studied to
levitate skeptical issues on employing it in medical applications. Suitable statistical methods
are employed to present the results predicting growth inhibition and enhancement in discrete
cases.

KEYWORDS: Fibre mat, Weaver ants, Biopolymer, Cytocompatibility.

INTRODUCTION
Weaver ants are exclusively appreciated in natural science for their nest construction
strategies where worker ants build each of the nests by weaving together the in-place leaves
with larval silk. These territorially eusocial organisms belong to the family, Formicidae and

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are obligatorily arboreal.[1] Their colonies spread extensively comprising of more than a
hundred nests spanning across trees in that vicinity and contain more than half a million
workers. Weaver ants prey on tiny insects and supplement their diet with carbohydrate-rich
honeydew excreted by Hemiptera, like most of the other ant species do. Apart from their
admirable social lifestyle and appreciable teamwork, they deserve an escalated ovation for
their nest architecture. Oecophylla smaragdina is a monogyn species that can be spotted
usually around forest trees.[2]

Weaver ants build their own nests miraculously, around 6 cm long. The principle material
used is just a set of leaves. The first stage is to bring all the leaves together so that they can be
joined. Weaver ants cling to each other and form a living chain to do this. Then worker ants
carry a larva each which actually is as its unborn sister, sheltered in a previously built nest of
this kind. It is taken to the exact spot where the leaves are to be stuck together. Upon being
squeezed gently by mandibles of the worker ant, it immediately starts to produce a sticky
thread. Such act is continued until these glue guns ultimately seal the planar spaces between
the leaves kept in position. Worker ants operate from superficial and posterior directions of
the leaves. Each larval form can produce an approximately definite amount of fibre after
which it loses its potential to develop. Those which sacrifice their further stages of life for the
good of their colony are guarded in the new nest by worker ants till their last breathe. Finally,
the structure that then emerges is one of the rarest possible marvels in engineering owing to
its technology of weaving, strength and ease of use.[3-4]

The fibre mat is predominantly found to consist partially digested cellulose along with
considerable traces of protein.[5] It is however obvious that glycoproteins sanction the
properties to stretch and stick.[6-7] It has been attempted here to check the antibacterial
activity of the fibre mat separated from nests woven by Oecophylla smaragdina colonies on
the branches of three different trees.[8-9] Gram positive Bacillus subtilis, gram positive
Clostridium thermocellum, gram negative Escherichia coli and gram negative Pseudomonas
aeruginosa were considered for assessing their growth in the presence of fibre mat separated
from weaver ant nests found on Citofortunella microcarpa, Couroupita guianensis
(Cannonball tree) and Artocarpus heterophyllus (Jack tree). Interesting results paved a path to
behavioural analysis of bacterial growth in the presence of enzymatically digested fibre mat
and presenting it as a media component. Cytotoxic study against mammalian cell lines was
carried out to check for its use in medical applications.[10-12]

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MATERIALS AND METHODS

Weaver ant nests spotted on the branches of the above mentioned trees were collected with
care so as not to miss the chance of visualizing their internal architecture. Fibre mat had been
separated under sterile conditions from each of them collected after evacuating their
residents. Fibre mat separated from the weaver ant nest formed on the branches of
Citrofortunella microcarpa was named Sample 1, that from Couroupita guianensis
(Cannonball tree) as Sample 2 and Artocarpus heterophyllus (Jack tree), Sample 3. Before
actually proceeding with the experimentation designed for behavioural study of gram positive
and gram negative bacteria, all the three fibre mat samples were subjected to certain
preliminary confirmatory tests to ensure the potential presence of cellulose and protein
content, after which each of them had been quantified. The quantification of glucose units
released post enzymatic digestion using dinitrosalicylic acid (DNS colorimetric assay) can be
relied on to estimate the amount of cellulose present. Protein content has been quantified by
Bradford method (Quick Start Bradford Protein Assay 1-800-4 BIORAD).

The above mentioned bacterial strains were incubated overnight with pre-sterilized pieces of
fibre mat after being cut uniformly into circular pieces of 5mm diameter, along with
respective positive controls at 370C and 150 rpm in orbital shaker. After the destined
incubation period, their absorbance had been recorded spectrophotometrically. The same had
also been done with enzymatically digested mat (1% in culture broth) (HIMEDIA-
Cellulase“onozuka R-10”). To further predict the influence of protein content present in the
test samples, each enzymatically digested mat was pre-treated with Proteinase K and added to
the broth inoculated with each bacterial strain.

The henceforth obtained distribution-free data set was preliminarily analysed based on two-
sided sign test results designed for matched pairs, fairly considering the probability of
negative occurrences, if the overall trend predicted inhibition or that of positive occurrences,
if the overall trend predicted an enhancement in bacterial growth, as threshold. Each trial data
set obtained was keenly evaluated statistically so as to predict certain reliable deductions.

The effects of each silk fibre mat sample on mammalian cell lines were investigated by
incubating with Jurkat cells. Jurkat cells were thawed in a water bath at 37°C after removing
from liquid nitrogen storage unit. Approximately 3x105 cells were added to 5 ml of
mammalian cell growth medium and incubated at 37°C in the presence of 5% CO2. They
were then left to grow for a couple of days, during which the pH of the culture was recorded

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daily. After incubating for the stipulated time period, cell proliferation had been evaluated by
employing MTT Reduction Assay. This procedure was performed exclusively for those trial
cases which gave an appreciable antibacterial activity result.

RESULTS
Physical observation and biochemical composition of samples
Physical observation of all the three fibre mat samples served a blind basis, in terms of their
opacity level, to hypothesize trends in trial data set. Sample 1 appeared extremely translucent,
almost opaque, though not completely and hence cannot be considered so. Sample 2
appeared fairly translucent and loosely hanging comparable to spider silk, unlike Sample 1
which resembled cellulose paper. Sample 3 appeared convincingly translucent and bound
compared to that of sample 2, though not as organized in structure as Sample 1. Preliminary
confirmations revealed the potential presence of cellulose and protein. Sample 1 was found to
contain 0.56μg/μl cellulose, sample 2 0.43μg/μl and sample 3 0.35μg/μl. Bradford protein
assay elucidated the protein content in sample 1 to be 0.79μg/μl, in sample 2 to be 0.53μg/μl
and in sample 3 to be 0.47μg/μl.

Trials
A total of 20 trials were carried out to examine the behaviour of bacteria in the presence of
each fibre mat pieces collected from weaver ant nests found on three different trees. Similarly
20 each with enzymatically digested fibre mat and 10 each post Proteinase K treatment. The
analysis being designed to be in terms of bacterial growth deprecated the need for a negative
control to be taken into consideration. Broth inoculated at the same time as that done for each
test run served as positive control. After 24 hours of incubation at above mentioned
conditions, absorbance at 660 nm recorded for all the trial runs has been presented
graphically below.

Bacterial behavior in the presence of sterilized fibre mat samples

Figure I depicts the behaviour of the above considered micro-organisms in the presence of
sterilized pieces of fibre mat sample 1, assessed spectrophotometrically in each trial.
Likewise, Figure II corresponds to incubation with sample 2 and Figure III with sample 3. A
significant proportion of trials, 17 out of 20 with C. thermocellum, and 14 with B. subtilis
showed growth inhibition while the results obtained with E. coli (9/20) and P. aeruginosa
(12/20) were moderately significant when incubated in the presence of sample 1. A
significant proportion of trials, 18 out of 20 with C. thermocellum, 17 with B. Subtilis and 17

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with E. coli showed growth inhibition while 14 with P. aeruginosa can be considered
moderately significant, when incubated in the presence of sample 2. The results recorded
upon incubation with sample 3 unfolded a significant proportion of trial positives, 17 out of
20 for E. Coli , 15 for C. thermocellum and 15 for P. aeruginosa, while 14 out of 20 for B.
Subtilis which is convincingly significant. The mean absorbance of positive control and the
test i.e., in the presence of raw fibre mat were calculated from the trial data set and are
tabulated below along with standard deviation and the probabilities of significant
observations (Table I).

Table I. Statistical parameters calculated from absorbance data recorded after

stipulated incubation of each bacterial culture with pieces of each sterilized fibre mat
sample 1, 2 and 3 (20 trials).
Mean Mean Standard Significance of
Trial set absorbance of absorbance of deviation of Inhibition in
positive control the test inhibition % terms of Probability
C. thermocellum 0.4555 0.4095 11.1478 0.85
B. subtilis 0.5155 0.4300 27.3202 0.70
Sample 1
E. coli 0.4755 0.4780 6.7501 0.45
P. aeruginosa 0.4945 0.4775 11.8591 0.60
C. thermocellum 0.5155 0.4350 10.2840 0.90
B. subtilis 0.4755 0.4215 8.7059 0.85
Sample 2
E. coli 0.4945 0.4455 6.5015 0.85
P. aeruginosa 0.4555 0.4200 10.5213 0.70
C. thermocellum 0.4755 0.4575 5.1824 0.75
B. subtilis 0.4945 0.4765 6.1642 0.70
Sample 3
E. coli 0.4555 0.4345 4.3281 0.85
P. aeruginosa 0.5155 0.4960 6.4565 0.75

Figure I: Growth inhibition elucidated from absorbance data recorded

spectrophotometrically after stipulated incubation of each bacterial culture with pieces
of sterilized fibre mat sample 1 (20 trials).

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Figure II: Growth inhibition elucidated from absorbance data recorded

spectrophotometrically after stipulated incubation of each bacterial culture with pieces
of sterilized fibre mat sample 2 (20 trials).

Figure III: Growth inhibition elucidated from absorbance data recorded

spectrophotometrically after stipulated incubation of each bacterial culture with pieces
of sterilized fibre mat sample 3 (20 trials).

Bacterial behavior in the presence of enzymatically digested fibre mat samples

Figure IV unfolds the behaviour of the above considered micro-organisms when the culture
broth is added with 1% enzymatically (cellulase) digested fibre mat sample 1, assessed
spectrophotometrically in each trial. Likewise, Figure V corresponds to incubation with
sample 2 and Figure VI with sample 3 in the same fashion. Gram positive bacteria exhibited
significant negative inhibition in all the 20 trials with enzymatically digested sample 1 while
E. coli and P. aeruginosa flaunted a moderately significant proportion of 20 trials, 9 and 12

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respectively. All the 20 trial runs carried out for each of the four microbes taken into
consideration in this study have exhibited negative inhibition upon incubation in the presence
of cellulase digested fibre mat sample 2. The same was found to be with sample 3 also.
Further statistical estimations including mean absorbance of positive control and the test i.e.,
in the presence of digested fibre mat were calculated from the trial data set and are tabulated
below along with standard deviation and the probabilities of significant observations (Table
II).

Table II. Statistical parameters calculated from absorbance data recorded after
incubation of each bacterial culture with each of the enzymatically digested fibre mat
sample 1, 2 and 3 (20 trials).
Mean Standard Significance of
Mean Absorbance
Trial set absorbance of deviation of Inhibition in terms
of positive Control
the test inhibition % of Probability
C. thermocellum 0.4945 0.5835 13.9072 Negative
B. subtilis 0.4345 0.5620 17.6786 Negative
Sample 1
E. coli 0.5155 0.6955 13.1838 Negative
P. aeruginosa 0.4755 0.5695 14.4637 Negative
C. thermocellum 0.4755 0.5425 14.8796 Negative
B. subtilis 0.5155 0.5945 6.1936 Negative
Sample 2
E. coli 0.4755 0.5940 12.8636 Negative
P. aeruginosa 0.4755 0.5315 3.6670 Negative
C. thermocellum 0.4945 0.5665 3.8863 Negative
B. subtilis 0.4555 0.5695 14.4476 Negative
Sample 3
E. coli 0.5155 0.6700 13.7804 Negative
P. aeruginosa 0.4945 0.5560 10.1875 Negative

Figure IV: Growth inhibition elucidated from absorbance data recorded

spectrophotometrically after stipulated incubation of each bacterial culture with
enzymatically digested fibre mat sample 1 (20 trials).

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Figure V: Growth inhibition elucidated from absorbance data recorded

spectrophotometrically after stipulated incubation of each bacterial culture with
enzymatically digested fibre mat sample 2 (20 trials).

Figure VI: Growth inhibition elucidated from absorbance data recorded

spectrophotometrically after stipulated incubation of each bacterial culture with
enzymatically digested fibre mat sample 3 (20 trials).

Bacterial behavior in the presence of Proteinase K treated enzymatically digested

fibremat samples
Figure VII reveals the behaviour of gram positive and gram negative microorganisms
employed in this study in the presence of 1% Proteinase K pre-treated enzymatically
(cellulase) digested fibre mat sample 1, assessed spectrophotometrically in each trial (a total
of 10). Likewise, Figure VIII corresponds to incubation with sample 2 and Figure IX with
sample 3, carried out accordingly. All the three samples yielded a highly significant unitary

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proportion of positive trials (negative inhibition to mention in specific, growth enhancement

being its implied sense). The mean absorbance of positive control and the test i.e., in the
presence of Proteinase K treated cellulase digested fibre mat calculated from the trial data set
are tabulated below along with standard deviation, supported by t-test results, to arrive at a
general numerical insight (Table III).

Table III. Statistical parameters calculated from absorbance data recorded after
stipulated incubation of each bacterial culture with each of the Proteinase K pre-treated
cellulase digested fibre mat sample 1, 2 and 3 (10 trials)
t-test
Trial set A B C D
t p
C. thermocellum 0.4780 0.7330 15.5213 -11.969 3.9351E-07 Positively
B. subtilis 0.4120 0.6320 16.5998 -12.749 2.2949E-07 Positively Unitary
Sample 1 E. coli 0.4960 0.7550 17.8358 -16.761 2.1433E-08 Positively
P. aeruginosa 0.5030 0.7450 16.7959 -14.473 7.6887E-08 Positively Unitary
C. thermocellum 0.4780 0.6220 11.4496 -11.290 6.4589E-07 Positively Unitary
B. subtilis 0.4120 0.5200 7.9454 -10.590 1.1078E-06 Positively Unitary
Sample 2
E. coli 0.4960 0.6410 11.2146 -8.7615 5.3149E-06 Positively Unitary
P. aeruginosa 0.5030 0.6550 11.4935 -10.363 1.328E-06 Positively Unitary
C. thermocellum 0.4780 0.6300 13.7091 12.8667 2.1209E-07 Positively Unitary
B. subtilis 0.4120 0.5330 10.6502 17.5258 1.4502E-08 Positively Unitary
Sample 3
E. coli 0.4960 0.6450 14.4541 12.8556 2.1367E-07 Positively Unitary
P. aeruginosa 0.5030 0.6560 14.9574 7.5653 1.7244E-05 Positively Unitary

Figure VII: Probacterial behaviour observed from absorbance data recorded

spectrophotometrically after stipulated incubation of each bacterial culture with
enzymatically digested, Proteinase K treated fibre mat sample 1 (10 trials).

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Figure VIII: Probacterial behaviour observed from absorbance data recorded

spectrophotometrically after stipulated incubation of each bacterial culture with
enzymatically digested, Proteinase K treated fibre mat sample 2 (10 trials).

Figure IX: Probacterial behaviour observed from absorbance data recorded

spectrophotometrically after stipulated incubation of each bacterial culture with
enzymatically digested, Proteinase K treated fibre mat sample 3 (10 trials).

Cytotoxic evaluation against Jurcat cells

MTT Reduction test revealed that any of the three fibre mat samples weren’t cytotoxic
against Jurcat cells. As evident from the spectrophotometric results tabulated in Table IV,
Jurcat cells could considerably proliferate in the presence of fibre mat.

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Table IV. MTT Reduction Assay Results.

Absorbance of Absorbance of
Absorbance of
Sample Negative control Positive control Cytotoxicity
Trial at 570 nm
at 570 nm at 570 nm
1 0.574 0.658 0.697 Negative
2 0.521 0.596 0.625 Negative
3 0.496 0.583 0.598 Negative

DISCUSSIONS
The fibre mat collected from weaver ant nest contains a protein apart from cellulose, partially
digested (by the larval forms to be in specific). Such mats usually contain antimicrobial
substances to defend its decomposition owing to nest structural durability. Extrapolating this
very obvious feature in correlation with the extent of translucence, cellulose and protein
contents assist in predicting bacterial behaviour accompanied by statistical deductions.

Sterilized fibre mat samples could inhibit bacterial growth to a considerable extent as evident
from sign test results. The significance of inhibition in terms of probability reported in Table
I represent the sign test threshold for each of the micro-organisms. An appreciable proportion
of trial set results indicated inhibition, though the overall level of inhibition cannot evaluate
fibre mat sample to have bactericidal properties, instead it can be called bacteriostatic. Gram
positive bacteria exhibited a higher level of antibacterial behaviour against all the three fibre
mat samples, over gram positive bacteria, which can be accredited to their outer membrane
architecture. However, E. Coli behaviour was observed to be randomly non-classifiable
across the entire dataset. It might be elucidated probably by carrying out more than 20 runs.

Upon enzymatic digestion of the fibre mat known to contain cellulose, the level of bacterial
growth inhibition was significant, though slightly lower than that observed with each fibre
mat as such. An attempt to trace out the influence of protein content on bacterial growth, by
treating each enzymatically digested sample with Proteinase K, yielded satisfactory results
sound enough to support the hypothesis made before starting experimentation. Cellulase
digestion of each mat containing reasonable amount of cellulose, harvested considerable
amount of glucose units which can be utilized by each of the bacterial cultures. The result set
recorded for this attempt revealed the fact of protein content being responsible for
antibacterial activity of weaver ant nest fibre mat. On the other hand, it also unfolded the
probacterial behaviour owing to the presence of glucose.

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Relying on the statistical data presented, Proteinase K pre-treated cellulase digested fibre mat
can be employed as an additional component to growth media. 1% of it in the broth itself
could enhance the growth leading to an intuition that systematic design of culture runs can
achieve even more. Comparing the entire data set obtained in this experimental study
divulged to an understanding that the fibre mat as such, i.e., cellulose associated with fibre
protein content innately has bacteriostatic properties. This is an added feature to the weaver
ant nest, one of the nature’s architectural marvel.

Mammalian cell lines could proliferate in the presence of each of the fibre mats. This can
hence robustly suggest the usage of weaver ant fibre mat in medical applications like
preparation of tissue grafts, etc., however, it has to be validated further. The results of this
study merely elevated the span of tremendous applications associated with weaver ant nest
fibre mat.

CONCLUSIONS
Serial molecular elimination imposed on each sample revealed that the protein content was
merely responsible for antibacterial activity. In certain stage, it has also uncovered the
probacterial activity of each mat, wherein the cellulosic content, glucose units in particular
post cellulase digestion, enhanced bacterial growth, bearing protein degradation as a primary
pre-requisite. The samples employed in this study did not considerably disturb the growing
pattern of Jurkat cells. These properties of weaver ant nest fibre mat can hence be exploited
in growth media and medical applications.

ACKNOWLEDGEMENTS
The authors are very much thankful to the management of K L University, Vaddeswaram,
Guntur, Andhra Pradesh for providing required facilities to carry out this work.

REFERENCES
1. Himender Bharti. List of Indian ants (Hymenoptera: Formicidae). Halteres, 2011; 3:
79–87.
2. Ross H. Crozier, Philip S. Newey, Ellen A. Schlüns & Simon K.A. Robson. A
masterpiece of evolution – Oecophylla weaver ants (Hymenoptera: Formicidae). Article
in Myrmecological News, May, 2010; 13: 57-71.
3. M. Prajwal, M. A. Sangamesha, and K. Pushpalatha. Ant larvae silk fibres mat. Current
Science, 2015 108: 1544-1547.

www.wjpps.com Vol 6, Issue 7, 2017.

Shreenivasulu et al. World Journal of Pharmacy and Pharmaceutical Sciences

4. Altman GH, Diaz F, Jakuba C, Calabro T, Horan RL, Chen J, Lu H, Richmond J, Kaplan
DL. Silk-based biomaterials. Biomaterials, 2003; 24(3): 401-416.
5. Minoura, N., Tsukada, M. and Nagura, M. Physico-chemical properties of silk fibroin
membrane as a biomaterial. Biomaterials, 1990; 11: 430-434.
6. Tara D. Sutherland, Sarah Weisman, Holly E. Trueman, Alagacone Sriskantha, John W.
H. Trueman,_ and Victoria S. Haritos. Conservation of Essential Design Features in
Coiled Coil Silks. Molecular Biology and Evolution, November, 2007.
7. Arunrat Khamhaengpol, Santi Maensiri, Sineenat Siri. Fibroin Protein Extract from Red
Ant Nests for a Production of Electrospun Nanofibers. Khon Kaen University Research
Journal, 2015; 10: 919-929.
8. Simon Wright and Sara L Goodacre. Evidence for antimicrobial activity associated with
common house spider silk. BMC Research Notes, 2012; 5: 326-331.
9. G.K. Khaira, A. Ganguli and M. Ghosh. Synthesis and evaluation of antibacterial activity
of quaternized biopolymer from Klebsiella terrigena. Journal of Applied Microbiology,
2013; 116: 511-518.
10. Liu, T.L., Miao, J.C., Sheng, W.H., Xie, Y.F., Huang, Q., Shan, Y.B. and Yang, J.C.
Cytocompatibility of regenerated silk fibroin film: a medical biomaterial applicable to
wound healing. JZUS-B, 2010; 11: 10-16.
11. Ronald E. Ungera, Michael Wolfa, Kirsten Petersa, Antonella Mottab, Claudio
Migliaresib, C. James Kirkpatricka. Growth of human cells on a non-woven silk fibroin
net: a potential for use in tissue engineering. Biomaterials, 2004; 25: 1069–1075.
12. Cragg GM, Newman DJ, Snader KM. Natural products in drug discovery and
development. Journal of natural products, 1997; 60(1): 52–60.

www.wjpps.com Vol 6, Issue 7, 2017.