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Comb Establishment of fungus-growing termites species Macrotermitinae

(Isoptera: Termitidae) with Termitomyces cylindricus (Basidiomycota:
Agaricales) basidiospores

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Comb Establishment of fungus-growing termites

species Macrotermitinae (Isoptera: Termitidae)
with Termitomyces cylindricus (Basidiomycota:
Agaricales) basidiospores

Khoirul Anwar, Lisdar Idwan Sudirman & Dodi Nandika

To cite this article: Khoirul Anwar, Lisdar Idwan Sudirman & Dodi Nandika (2020): Comb
Establishment of fungus-growing termites species Macrotermitinae (Isoptera: Termitidae) with
Termitomyces�cylindricus (Basidiomycota: Agaricales) basidiospores, Oriental Insects, DOI:
10.1080/00305316.2020.1762775

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 ORIENTAL INSECTS
https://doi.org/10.1080/00305316.2020.1762775

Comb Establishment of fungus-growing termites species

Macrotermitinae (Isoptera: Termitidae) with Termitomyces
cylindricus (Basidiomycota: Agaricales) basidiospores
a
Khoirul Anwar , Lisdar Idwan Sudirmana and Dodi Nandikab
a
Department of Biology, IPB University, Bogor, Indonesia; bDepartment of Forest Product Technology,
IPB University, Bogor, Indonesia

ABSTRACT ARTICLE HISTORY

Combs have a significant role as substrates for Termitomyces in Received 29 July 2019
providing nutrients for Macrotermitinae colonies. However, the Accepted 27 April 2020
process of comb establishment was poorly known. The study KEYWORDS
was to observe the process of fungal comb formation of fungus- Fungal combs; fungus-
growing termites in the laboratory. Each pair of alates from growing termites;
three species, i.e Microtermes insperatus Kemner, Macrotermes Microtermes insperatus;
gilvus Hagen, and Odontotermes bogoriensis Kemner were Macrotermes gilvus;
reared and introduced to Termitomyces sp. basidiospores, mean- Odontotermes bogoriesis;
while another pairs that were not introduced to the basidios- Termitomyces cylindricus
pores were acted as the control. M. insperatus introduced with
and without basidiospore established 100 % of developed
combs and 33.33 % of developed combs respectively.
M. insperatus that succeed in making developed comb had
a relatively similar pattern in growth of colony members
between those introduced and not introduced with basidios-
pores, however, they were in contrast to M. insperatus that did
not succeed in making developed combs. M. gilvus and
O. bogoriensis that were or were not introduced with basidios-
pore established 0 % of the developed comb. The combs were
observed for the first time in the 15th week of the rearing period.
The fungus in developed combs of M. insperatus introduced to
basidiospores and the culture of Termitomyces sp. were in one
clade with Termitomyces cylindricus.

Introduction
Fungus combs or fungus gardens are complex convoluted structures culti-
vated by termite species of the sub-family Macrotermitinae (Isoptera:
Termitidae), a predominantly African group. Abundant clusters of nodules,
small (about 1 mm) white spherules of Termitomyces species and other fungi
grow on these combs (Arshad 1987). Termitomyces is a wild tropical macro-
fungi with only 30 species in Asia and Africa (Otieno 1968; Bels and
Pataragetvit 1982; Van Der Westhuizen and Eicker 1991; Makonde et al.
2013) and all of them are edible and have a very good taste and texture

CONTACT Lisdar Idwan Sudirman lisdar.sudirman@yahoo.co.id

© 2020 Informa UK Limited, trading as Taylor & Francis Group

Published online 14 May 2020

2 K. ANWAR ET AL.

(Tibuhwa 2012). Besides its nutritional contents are very high (Gowda and
Rajagopal 1990; Devi et al. 2014), Termitomyces also have medicinal proper-
ties (Breene 1990; Nabubuya et al. 2010; Hsieh and Ju 2018) even they have
been as a source of economic income during the rainy season at prices quite
high, as in Thailand, Vietnam and some African countries (Tibuhwa 2013;
Anwar et al. 2014). However, until today there are no researcher or commu-
nity has succeeded in cultivating them (Gunawan 1997; Pahlevanlo 2013).
Thus, to be able to cultivate Termitomyces, combs as substrates where the
fungi grow are very important things to learn (Arshad 1987; Pahlevanlo 2013).
Termitomyces cultivation is a challenge because the fungi only grow on
a special substrate called fungal comb made by termites (Gowda and
Rajagopal 1990). Termites cultivating Termitomyces include Macrotermes,
Odontotermes, Microtermes, Canthotermes, Hypotermes, and Protermes
(Bels and Pataragetvit 1982; Tibuhwa 2012).
Some researchers have successfully made combs of Macrotermes michael-
seni and Odontotermes montanus by anchoring or introducing spores from
the fruit body T. microcarpus into the copularium in a glass container
(Sieber 1983), but the comb was successfully reported made by anchoring
Microtermes sp. nr. usambaricus without introducing spores in the con-
tainer (Johnson 1981), therefore it is necessary to know the species of
Termitomyces which are responsible for the comb formation. Regarding
previous reason, this study aims to observe the formation of combs by
some species of termite by adding Termitomyces basidiospores into termite
colonies. The Termitomyces that grew in the comb and culture isolate of
Termitomyces were identified molecularly based on the ITS1-5.8 gene
sequence-ITS2 by using a comb as a sample. Besides, the fungal structures
in the comb and termite population were observed.

Materials and methods

Capturing termites

The capturing of termites was carried out at Bogor Agricultural University

(IPB), Darmaga Campus, Bogor, West Java (6°33ʹ19”S, 106°43ʹ27”E) from
November 2016 to November 2017. The termite nests were established in
a glass bottle (volume 150 ml, long 12 cm, diameter 5 cm, neck diameter
2.5 cm, and glass thickness 2 mm) containing 50 g of dry soil then added
with 5 ml of sterile distilled water. A couple male and female alates were
caught and put into nest bottles and plug with cotton (Fig. 1), then stored in
a cardboard box (30 cm × 21 cm × 21 cm) at room temperature (25°C to 30°
C). As many as five millilitres of sterile distilled water was put into nest
bottles using a syringe every two weeks to maintain moisture. A dry wooden
 ORIENTAL INSECTS 3

Figure 1. Installation of termite rearing.

block of Ficus religiosa measuring 1 cm × 1 cm × 5 cm was inserted into each

bottle when the termite colony was formed.

Termite identification
The captured alates were observed and described morphologically. When
the alates succeed in forming the colony in the nest bottle, the soldiers were
taken to be identified based on the key of identification (Ahmad 1958).

Inoculation of Termitomyces basidiospores and comb establishment

The basidiospore suspension of Termitomyces sp. was made by dipping the
entire portion of the pileus with lamella into sterile distilled water (300 ml) for
one minute. Five millilitres of suspension are added using a syringe to each
nest bottle containing soldiers and workers of colonies. The inoculation of
spores carried out when the termite colonies are two months old or when the
worker termites performed foraging activities, indicated by the formation of
soil channels or tunnels in the bottles. The five nest bottles were inoculated
with basidiospore suspension, while the nest bottle’s control was only injected
with sterile distilled water. Parameters of observation included the number of
counted colonies, the number of combs, the cross-section area of the combs,
the number of nodules, and the number of primordium-like structures. The
cross-section area of the combs was measured by Image Raster Software.
Observations were carried out once a week for ± 1 year (52 weeks).

Isolation and identification of Termitomyces

The Termitomyces fruit bodies were found at IPB Darmaga Campus, Bogor,
West Java Province (6°33ʹ19”S, 106°43ʹ27”E) and were taken randomly and
4 K. ANWAR ET AL.

put in a paper bag then identified macroscopically using identification key

(Pegler and Vanhaecke 1994). One piece of tissue (3 mm) from the inside of
the pseudorhiza was taken aseptically and then was grown on MEA (20 g
Malt Extract Broth, 20 g Difco agar, 0.1 g yeast, 0.05% chloramphenicol),
then it was incubated at room temperature (± 25°C) in a dark condition. The
isolates were then observed using a microscope.

Comb and fungi DNA extraction

The total DNA isolate and fungal comb were extracted by using a modified
phenol-chloroform method (Siddiquee et al. 2012). A total of 50 mg of comb
or 2 cm Termitomyces colony diameter was put into a 1.5 ml micro-tube
mixed with 500 µl buffer (1 M Tris HCl [pH 8.5], 1 M NaCl [pH 8.5], 1 M
EDTA [pH 8.0] and 10% sodium dodecyl sulphate) and was incubated in
a water bath for 8 hours at 38°C. Furthermore, 350 µl of phenol buffer and
150 µl of chloroform were added to the tube and homogenised for 10 min-
utes. The resulting suspension was centrifuged at 13,000 × g for 10 minutes
at 4°C, then the supernatant was transferred to a new micro-tube and mixed
with 3 µl RNAse solution with a concentration of 1 µg/µl. This mixture was
then incubated at 38°C in a water bath for 15 minutes, and chloroform (1:1)
was added slowly into the sample and homogenised for 10 minutes. The
mixture was then centrifuged at 13,000 × g for 10 minutes at 4°C. The
supernatant was transferred to a new micro-tube. DNA was precipitated
with 250 µl of iso-propane-2-ol and stored overnight at −20°C. Next, the
tube was centrifuged at 13,000 × g for 10 minutes at 4°C. The pellets were
mixed with 500 µl 70% ethanol and centrifuged at 13,000 × g for 10 minutes
at 4°C then the pellets were dried and mixed with TE (Tris EDTA) pH 8 by
50 µl. DNA is stored at −20 °C for further work. The quality of DNA samples
was examined by electrophoresis gel and NanoDrop 2000 C (Thermo
Scientific, USA). Electrophoresis was performed using 1% agarose gel in
a buffer solution one-time TAE (0.045 M Tris-acetate acid and 1 mM EDTA
[pH 8.2]) and run for 30 minutes at 70 V. DNA ladder was 1kb used as
a comparison. The gel was then stained with ethidium bromide (0.5 µl/ml)
and visualised under UV light at a wavelength of 230 nm, 260 nm, and
280 nm. The presence of DNA was visualised by showing gel bands.

PCR amplification of ITS regions from Termitomyces isolate and

Termitomyces on the comb
The amplification of ITS1 and ITS2 rDNA was performed by using ITS1
universal forward primer (5 ‘TCC GTA GGT GAA CCT GCG G 3ʹ) and
ITS2 reverse primer (5 ‘TCC GCT TAT TCC TGA TAT GC 3ʹ) (Siddiquee
et al. 2012). PCR amplification was carried out in a total volume of 50 μl
 ORIENTAL INSECTS 5

consisting of 50 ng DNA extracts, 10 mM dNTPs, 5 units/μl Taq polymerase

DNA, 10 × buffer PCR(200 mM), and 1 µM ITS1 and ITS2 primer. PCR
reaction was carried out in Cycler-200 Thermal Peltier with the following
parameters: initial denaturation for 5 minutes at 95°C, followed by 36
denaturation cycles at 94°C for one minute, attachment at 55°C for
1 minute, final elongation at 72°C for 3 minutes, and a final extension at
72°C for 10 minutes (Siddiquee et al. 2012).

Electrophoresis
PCR product was run on 1% agarose gel in a buffer solution one-time TAE
(0.045 M Tris-acetate acid and 1 mM EDTA pH 8 and was run for 30 minutes at
70 V with DNA ladder 1kb as a standard size. The gel was then stained with
ethidium bromide (0.5 μl/ml). It was visualised under UV light using UV-Vis
spectrophotometry at a wavelength of 230 nm, 260 nm, and 280 nm and
photographed by using the Alphaimager® 2200 gel documentation system
version 5.5.

Phylogenetic analysis
The homology search of DNA sequences was performed by using the
BLAST method (https://www.ncbi.nlm.nih.gov/). Furthermore, the best
homology search results were aligned with the DNA sequence samples
using MEGA6 software. The alignment results were then continued with
the construction of phylogenetic trees by using the Maximum Likelihood
method with 1,000 test bootstraps. DNA sequences were deposited in
GenBank with an access code.
The GenBank access codes from the sequence reported in this study are
MH651799 (culture isolate), MH687931 (Clone1 of Comb), MH687981
(Clone 2 of Comb), MH687979 (Clone3 of Comb), MH697400 (Clone 4
of Comb), and MH680959 (Clone 5 of Comb).

Results
Based on morphologically identification results, there were Microtermes insper-
atus Kemner, Macrotermes gilvus Hagen, and Odontotermes bogoriensis
Kemner which were tested to establish the comb. The body length of
M. insperatus alates from the field was 8.5 ± 0.5 mm, M. gilvus alates were
15.5 ± 0.5 mm, and O. bogoriensis alates was of 13.5 ± 0.5 mm. The body length
of M. insperatus soldier was 4.74 ± 0.1 mm, almost similar to M. gilvus
4.86 ± 0.2 mm (minor soldier) and 10 ± 0.2 mm (major soldier),
O. bogoriensis soldier was 6.91 ± 0.2 mm. The alates of O. bogoriensis have
6 K. ANWAR ET AL.

rather dark wings compared to M. insperatus and M. gilvus ones (Arinana et al.
2016). The M. insperatus soldier is smaller than its worker (Arinana et al. 2016).
Termitomyces that were found in the field were identified morphologi-
cally as T. cylindricus. It was found four times around the alates-capturing
locations and it seems always found growing on M. insperatus comb.
Usually, one termite nest was overgrown with one or two fruit bodies of
T. cylindricus and were rarely found in groups. Termitomyces cylindricus
pileus was pale brown, without perforatorium, diameters were 4–7 cm. The
stipes were white, solid, length 5–8 cm with a diameter of less than 1 cm.
Three species of termite established combs successfully however, only
M. insperatus was able to form developed comb (Table 1), while the other
species were a success in forming undeveloped combs. The developed
combs consisted of fungal structures i.e. mycelia, nodules, and primordium-
like structure and new mylospheres or pellets that always added by the
termites to the combs. The undeveloped combs consisted of only mylo-
spheres and no fungal structures.
Firstly, the combs and nodules appeared in the nest of M. insperatus in
15th week (after 14 weeks of absence). The wide of the cross-section area
of the comb was 9.62 mm2 which were continued to increase to become
the developed comb. The biggest area of the developed comb that was
successfully achieved in this study was 314 mm2 at 32nd week (Fig. 2A)
and even more, the number of combs that successfully formed was as
much as two combs of one nest bottle. The number of nodules firstly
emerged was 27 in the 15th week and the highest number of nodules was
156 at 21st week (Fig. 2B). The first number of primordium-like structure
emerged at 19th week were 4 and the highest number was 11 at 32nd
week (Fig. 2C). However, the cross-section area of the undeveloped comb
was 9.62 mm2 (not increased).
The size (length × width) of fungal structures were described below: the
hyphal cell nodule of 2.0–2.5 um × 0.5–0.6 um; conidia of 1.0–1.1 um ×
0.5–0.6 um; and nodule sphaerocyst of 2.0–3.0 um × 1.5–2.0 um (Fig. 3A).
The hyphal cell of primordium-like structure were 2.30–4.25 um × 0.5–0.6
um (Fig. 3C), the hyphal cell of culture isolates of 2.0–4.0 um × 0.5–0.6 um
(Fig. 3B). The hyphal cell length of fruiting body’s pseudorhiza were 2.10–-
5.25 um × 0.5–0.6 um (Fig. 3D), ovoid basidiospore with length of 1.0–1.1
um × 0.71–0.81 um, and the basidia width of 1.45–1.81 um.

Table 1. Three species of experiment termites for making combs.

M. insperatus M. gilvus O. bogoriensis
Treatment U1 U2 U3 U1 U2 U3 U1 U2 U3
Basidiospores suspension + + + ± ± ± ± ± ±
Sterile distilled water + ± ± ± ± ± ± ± ±
Description: (+) developed comb, (±) undeveloped comb, U1-U3 = replication
 ORIENTAL INSECTS 7

A
Wide of cross-section (cm2) 350.00
300.00
250.00
200.00
150.00
100.00
50.00
0.00
1 2 3 4 5 15 19 21 22 23 32 41 46 48 51 52
Termite colony age (week)

B
Number of counted

160
140
120
nodules

100
80
60
40
20
0
1 2 3 4 5 15 19 21 22 23 25 32 33 36 41 44 46 48 51 52
Termite colony age (week)

C
25
primordium-like structure

20
Number of counted

15
10
5
0
1 2 3 4 5 15 19 21 22 23 25 32 33 36 41 44 46 48 51 52
Termite colony age (week)

Figure 2A-C. The wide of fungal comb and fungal structure of M. insperatus which was
successful in forming the developed comb. A, the wide (cross-section) area of comb; B, counted
nodules; C, counted primordium-like structures.

The size of DNA sequences of Termitomyces isolate and comb were around
700 bp. Eight ribbons from DNA PCR were obtained by electrophoresis and five
of them (no. 2, 4, 5, 6, & 7) succeed to be sequenced after performing cloning
(Fig. 4). The PCR product of comb was not pure, so it was needed to be cloned to
the pTA2 Vector plasmid using Kit Toyobo TArget Clone-Plus-(Toyobo). The
cloning was carried out by PT. Genetika Science Indonesia. The PCR product
8 K. ANWAR ET AL.

c A B
c c
s

s s

C D

Figure 3A-D. Microscopic structures of Termitomyces cylindricus. A, nodules composed of

conidia (c) and sphaerocyst (s); B, culture isolated from fruiting body; C, primordium-like
structure; D, pseudorhiza.

Figure 4. PCR Products from the comb. Numbers 2, 4, 5, 6, and 7 of the DNA ladder were chosen
to be cloned into a plasmid. M: marker.
 ORIENTAL INSECTS 9

Figure 5. Phylogenetic three designed by the Maximum Likelihood analysis of ITS1 and ITS2
gene sequence to show the position of Termitomyces cylindricus isolate and five clones of fungal
comb among the genus Termitomyces.

was then purified using the Zymoclean Gel DNA Recovery Kit (Zymo
Research). The plasmids were then transformed into bacteria Escherichia coli
Zymo 5α using Kit Mix and Go Competent CellsTM (Zymo Research). PCR was
performed to transformed colonies by using T3 primer and T7 promoters with
Neo KOD FX Kit (Toyobo) 6. Plasmids from transformed colonies were then
isolated by using ZR Plasmid MiniPrep (Zymo Research). The five DNA clones
were similar to T. cylindricus based on the BLAST method.
The phylogenetic analysis of the ITS1-5.8 S-ITS2 genes from the comb
and Termitomyces isolates indicated that both were located in one clade with
T. cylindricus from GenBank and were clustered together sister clade with
Termitomyces symbiont of Microtermes sp (Fig. 5).

Discussion
Combs is a special structure made by fungus-growing termite in their nest.
The structure is composed of many organic materials derived from wood,
10 K. ANWAR ET AL.

litter, or other organic materials that contain lignocellulose (Hesse 2015).

Combs plays an important role in the survival of Macrotermitinae termites
(Harit et al. 2016). Comb in the field-collected from an active mound of
Macrotermes michaelseni consisted of 40% carbohydrates, 10% protein, 30%
of aliphatic compounds, and 20% of aromatic and phenolic compounds.
Besides, the fungal comb is also rich with glucose, arabinose, and xylose
(Arshad 1987). Wood, a lignocellulose-rich material, is a very important
component in the preparation of combs. The type of wood used in this
study is Ficus religiosa wood (Angiospermae) which was found in many
locations around the termite nest and was known as lower-middle-class timber
plants that are susceptible to termites attack. Termites generally eat high
cellulose and low lignin materials as lower quality wood (Nandika et al.
2015). In addition to wood, there are no reports of comb-making experiments
by using litters or grass only but termites eat other materials such as litters and
grass (Nobre and Aanen 2012; Harit et al. 2016). Conifer wood had been
reported to be used as termite feeding, but there is no comb because it contains
resin that inhibits some termite to eat the wood (Nobre and Aanen 2012).

Termitomyces cylindricus and termite-growing fungi

Termitomyces cylindricus is found in West Java but the previous report (Salma
and Rifai 2016) stated that in West Java only T. clypeatus, T. eurrhizus,
T. striatus, and T. microcarpus, were found while T. cylindricus was not
reported. On separate results based investigation in the field T. cylindricus is
always found associated with Microtermes in Pasuruan and Bogor (Anwar
et al. not been published) but in China, it was reported that it associated with
Macrotermes.
In this study, T. cylindricus spores were chosen in introducing in the
termite colonies because T. cylindricus was found together with tested alates
in the field. We considered that the termites will succeed to establish comb
with T. cylindricus, but based on the results only M. insperatus succeed to
develop comb however M. gilvus and O. bogoriensis were not able to devel-
oped comb. There was no report about the association between M. insperatus
and T. cylindricus, but T. cylindricus was ever found associated with
Macrotermes sp. in China (Pegler and Vanhaecke 1994). It was reported
that M. gilvus was found to live in association with T. globulus, T eurhizus,
T. albiceps, and T. cartilaginous. Odontotermes bogoriensis was found to be
associated with T. eurhizus.
Some species of Termitomyces, such as T. eurhizus and T. medius, are
reported as symbionts of several species of more than two genera of ter-
mites, respectively. Therefore, Termitomyces was reported that it had low
symbiont specificity and high host specificity (Aanen et al. 2007). Based on
the three termite species studied, only M. insperatus managed to form
 ORIENTAL INSECTS 11

a comb with spore inoculation T. cylindricus while two other species failed
to form a comb. Maybe both colonies need spores from other fungi species
Termitomyces to form combs. It needs to be investigated more that is the
T. cylindricus only grew by Microtermes insperatus or M. insperatus able to
grow other species of Termitomyces.
In this study, M. gilvus and O. bogoriensis died at the age of 5–6 months
because they did not succeed to develop comb. The workers died firstly then
followed by the queen. The death of termite colonies was possibly caused by
fungal attacks on the colony, nematode worms, drought, and the failure of
colonies to form mushroom gardens (Harit et al. 2016).

Comb making process and the fungal structure

In this study, the comb was successfully formed by anchoring the alates of
M. insperatus Kemner, which were introduced with spore suspension
T. cylindricus. The comb was formed after 15 weeks of captivity with the
largest area was 314 mm2, which was at 32 week. Johnson reports that
Microtermes sp. nr. usambaricus succeeded to form the new comb in
10 weeks after introducing pieces of old fungal comb pieces to glass contain-
ers containing only termite colonies. The process of making combs on
termites has been reported by (Johnson 1981; Sieber 1983).
The largest comb area that was successfully formed was 15 mm2 for
23 weeks of captivity and formed a nodule structure (Fig. 6). Sieber also
reports that termites Macrotermes michaelseni and Odontotermes montanus
have successfully formed combs and nodules by introducing traces of spores
T. microcarpus on filter paper into glass containers containing termite
colonies. (Johnson et al. 1981) also reported Macrotermes bellicosus
(Smeanthman), Microtermes sp. A, Microtermes sp. B, and Microtermes sp.
D succeed to form a comb by anchoring the larva only, without introducing
spores and/or combs from the outside. It turned out that the intestinal part
of the termite carried spores after being dissected.
The comb structures were composed of groups of pellets or milosphere.
According to (Johnson 1981; Bels and Pataragetvit 1982; Sieber 1983) the
comb formation process is as follows. Pellets were first formed utilising
lignocellulosic material in the form of wood chips digested in part by worker
termites, then added liquid from the digestive system to form small dots. The
pellets were then arranged to form a comb (Fig. 6). Furthermore, when the
candidate is overgrown with fungal mycelium Termitomyces, then the worker
termites continuously add the pellets to the formed comb so that the size of
the comb increases (developed comb). This is consistent with the results of
this study that there is a comb pellet that has a black and white colour (Fig. 7).
Black pellets are new pellets that still contain high lignin while white pellets are
old pellets that contain low lignin (Nandika et al. 2015). Worker termites
12 K. ANWAR ET AL.

Figure 6. The undeveloped comb (left) and the developed comb (right). Developed comb by
M. insperatus consisting of nodules, primordium-like structures, and mylosphere.

Figure 7. The developed comb was composed of black and white pellets (mylosphere). Black
pellets indicated as new pellets while white pellets as old pellets. One or two white pellets were
seen between black pellets that indicated termites inoculated new pellets at old pellets.

besides adding pellets are also seen adding yellowish stool liquid to the comb.
Intestinal termites contain digestive enzymes that help degrade organic matter
in combs (Nobre and Aanen 2012). Besides, (Um et al. 2013) reported that in
 ORIENTAL INSECTS 13

the digestive tract termites grow fungi found bacteria that produce antifungal
compounds of Bacillaceae. These inhibit the growth of Pseudoxylaria sp.,
Umbelopsis sp., Fusarium sp., Trichoderma sp., and Coriolopsis sp. but does
not inhibit the growth of Termitomyces sp (Um et al. 2013). This situation
results in a monoculture condition in the comb, so that only Termitomyces
successfully grows in a comb (Qian et al. 2011).
The area of the comb was continuously increasing until reached 314 mm2
in the 32nd week (Fig. 2A). It was caused by the worker termite activity by
adding new pellets to the comb. However, the area of the comb decreased after
the 32nd week because the termites possibly eat the comb and stop adding new
pellets. It is possibly caused by the availability of raw materials. It was
supported by the observation results of the hollow wood condition although
it looked intact from the outside. The other report said that termite made and
ate the comb to fulfill their nutritional intake (Rouland-Lefèvre et al. 2006).

Nodules and primordium-like structure in combs

Nodules on M. insperatus comb were first seen in the 15th week (after 14 weeks
of absence), but the other report said that they were first seen in the 10th week
(after 9 weeks of absence) on Microtermes sp. nr. usambaricus comb (Johnson
1981). The nodules, known as anamorph (asexual phase) of Termitomyces,
were composed of short-sized cell hyphae collection, conidia, and sphaerocyst
(Fig. 3) (De Fine Licht et al. 2006). The primordium-like structure had a long
hypha with septa. It is called a primordium-like structure because its hyphae
were more similar to pseudorhiza hyphae of the fruiting body compared to the
culture and nodule hyphae. The conidia and sphaerocyst were found in
Termitomyces culture and nodule but not in primordium-like structure and
pseudorhiza (Botha and Eicker 1991). It was occurred a changing from sexual
morph (Termitomyces sp.) to asexual morph (Termitosphaera sp.) at the
culture media. It happened in the fungi such as Aspergillus sp. (asexual
morph) that changes to Eurotium sp. (sexual morph) due to the environ-
mental factors as the medium of isolation (Han et al. 2003).
Nodules and primordium-like structure had not been found in the new
black pellets of the comb but the old white pellets of combs. They were
formed after black pellets changes into white pellets which contain low
lignin (Nandika et al. 2015). The degradation of lignin was caused by the
enzyme of Termitomyces and the termite activity (Nobre and Aanen 2012).
Only nodules and primordium-like structures Termitomyces were grown in
the comb but no greenish colony of Xylaria. It means the condition of the
combs was probably monoculture (Shinzato et al. 2005, 2007; Pahlevanlo
2013). Qian also said the same thing in the field that the comb would change
to polyculture with Trichoderma, Xylaria, and other fungi when the comb
was abandoned by termites (Qian et al. 2011). According to Bels and
14 K. ANWAR ET AL.

Pataragetvit (1982) the fruiting bodies in nature usually were formed when
the humidity is high and the level of CO2 decreases due to the reduction of
individual members of termite colonies in the nest. For this reason, the same
research using a larger container and setting the CO2 level needs to be done
to get the fruiting body.

Phylogenetic analysis of fungi that grow in a comb

Phylogenetic analysis confirmed that the DNA sequence of fungi from the
comb and the isolate from the fruiting body was Termitomyces. They were
close-relation and one cluster with T. cylindricus.

Conclusions
The comb establishment is very essential for fungus-growing termite for
survival. Microtermes insperatus, with or without basidiospore suspension,
could establish the developed comb although M. insperatus with basidios-
pore suspension is more successful than with sterile water. Macrotermes
gilvus and O. bogoriensis with or without basidiospore suspension unable
to established developed comb. Termitomyces cylindricus basidiospores
may have a role to help Macrotermitinae species to established comb but
we still need to investigate more, especially about the role of basidiospore
presence of Termitomyces species in a Termite species for comb establish-
ment. Besides, the specificity of the symbiosis between species of
Termitomyces and termite is still unclear, so it is needed to be further
investigated.

Acknowledgments
The authors wish to express their appreciation to Lembaga Pengelola Dana Pendidikan
(LPDP) of the Ministry of Finance of the Republic of Indonesia for helping in financing
during the research. The authors also wish to thank Sepriyadi Rihi for molecular
assistance.

Disclosure statement
No potential conflict of interest was reported by the author(s).

ORCID
Khoirul Anwar http://orcid.org/0000-0003-3414-438X
 ORIENTAL INSECTS 15

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