First record of gregarine protists (Apicomplexa: Sporozoa) in Asian fungus-growing termite Macrotermes barneyi (Blattaria: Termitidae)

Abstract

Macrotermes barneyi , widely distributed in southern China, is the major fungus-growing termite in the subfamily Macrotermitinae. It has no flagellated protists in the guts. Here, we report occurrence of gregarine, a protozoan parasite in the digestive tract of M. barneyi . The general morphology and ultrastructure of the gregarine gamonts and syzygies by light micrograph and scanning electron micrograph are presented. SSU rDNA sequence analysis showed that the termite gregarine has the highest identity (90.10%) to that of Gregarina blattarum from cockroaches. Phylogenetic analysis based on the SSU rDNA sequences from diverse insect eugregarines indicated that the gregarine from M. barneyi is phylogenetically close to G. blattarus, L. erratica and G. tropica from Gregarinidae and Leidyanidae families, and may represent a novel species. This study expands our knowledge about the diversity of terrestrial eugregarines parasitizing in termites.

Full text

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
Scientic Reports | (2021) 11:989 | 
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First record of gregarine protists
(Apicomplexa: Sporozoa)
in Asian fungus‑growing termite
Macrotermes barneyi (Blattaria:
Termitidae)
Shuo Zhang1,2, Zijia Lin1,2, Qihong Huang1, Yulong Shen1* & Jinfeng Ni1*
Macrotermes barneyi, widely distributed in southern China, is the major fungus‑growing termite in
the subfamily Macrotermitinae. It has no agellated protists in the guts. Here, we report occurrence
of gregarine, a protozoan parasite in the digestive tract of M. barneyi. The general morphology and
ultrastructure of the gregarine gamonts and syzygies by light micrograph and scanning electron
micrograph are presented. SSU rDNA sequence analysis showed that the termite gregarine has the
highest identity (90.10%) to that of Gregarina blattarum from cockroaches. Phylogenetic analysis
based on the SSU rDNA sequences from diverse insect eugregarines indicated that the gregarine from
M. barneyi is phylogenetically close to G. blattarus, L. erratica and G. tropica from Gregarinidae and
Leidyanidae families, and may represent a novel species. This study expands our knowledge about the
diversity of terrestrial eugregarines parasitizing in termites.
Apicomplexan represents a diverse group of unicellular eukaryotes which parasitize the body cavities or the cells
of animal. is group include some species pathogenic to human and animals, for example, Plasmodium (haemos-
poridians, causes malaria), and Eimeria and Isospora (coccodians, cause coccidiosis)1,2. Gregarine apicomplexans,
closely related to these parasites, are protists that widely inhabit the digestive tracts, fat bodies, Malpighian tubules
and reproductive organs of invertebrates from marine, freshwater and terrestrial environments1,3. e gregarines
are considered to be the earliest diverged within the phylum Apicomplexa, and have several characteristics such
as extracellular stage, a monoxenous life-cycle and a prevailing presence in marine hosts. erefore, the study on
gregarines is important for the understanding of the phylogenesis of apicomplexans1,2. ese protozoan parasites
may also good materials for studies on parasite-host coevolution and biological control of insects3,4. However,
the gregarines have only been poorly explored compared to those much better studied vertebrate pathogens5,6.
e gregarines are traditionally divided into Archigregarinorida, Eugregarinorida, and Neogregarinorida
based on habitat, morphology and host1, and sometimes two groups: Archigregarinorida and Eugregarinorida
(with Neogregarinorida incorporating into the Eugregarinorida) are presented7. Archigregarines are the ances-
tral group that exist only in marine environment, and Eugregarines are widely found in marine, freshwater and
terrestrial environments8–10.
Eugregarines (Eugregarinorida) are recurrent guests in insects, which have been reported in order Blat-
tariam, including cockroaches11 and termites12,13 and other orders such as Coleoptera, Diptera, Odonata and
Orthoptera11,14–16. In earlier studies, most of the descriptions were based on morphological traits such as the
shape and size of gregarines, which may underestimate the actual diversity. us molecular data mainly of 18S
rDNA sequences have recently been included to aid taxonomical descriptions17–19.
Termites (Blattaria: Termitidae) are eusocial insects and generally divided into lower termites and higher
termites based on the presence or absence of agellated protists in the hindgut of termites20,21. e subfamily
Macrotermitinae of higher termites, cultivating basidiomycete fungus (Termitomyces spp.) in their nests, are
known as fungus-growing termites22. Macrotermes barneyi is one of the major fungus-growing termites, widely
distributed in southern China23. So far, no gregarine has been reported in Asian termites, as far as we know. In
OPEN
State Key Laboratory of Microbial Technology, Microbial Technology Institute, Shandong University,
 These authors contributed equally: Shuo Zhang and Zijia Lin. *email:
yulgshen@sdu.edu.cn; jinfgni@sdu.edu.cn
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this study, we report a novel occurrence of gregarines in the digestive tract of M. barneyi, the morphology at the
common life-cycle stages and the rst SSU rDNA sequence from termite gregarines.
Results
Presence of gregarines in the gut of M. barneyi. e gregarines were rstly found in the midgut of
termite worker including both minor and major workers of M. barneyi. e abundance of gregarines was higher
in the minor worker termites, ranging from dozens to hundreds. In one paran section of the intestinal part
of the termite worker (Fig.1), four complete cross sections of the gregarine were shown (Fig.1a, arrow). e
nucleus (arrowhead) and nucleolus (double arrowhead) were clearly visible in one cross section (Fig.1b). None
of gregarine was observed in soldier termites. e percentage of termite worker with infected gregarines was
21.45% (Table1).
Morphology of the gregarines. Most of gregarines observed in the midgut were gamonts (Fig.2) and
syzygies (Fig.3a,b). e gregarines were in brownish yellow or brownish black color. e gamonts contained a
protomerite (pm) and deutomerite (dm) that can be dierentiated by size and the presence of the nucleus. e
gamonts were divided by an always present septum (Fig.2, arrow). e front part pm was in at ellipsoid, with
the lengths ranging from 26.8 to 65.1μm, and the widths ranging from 36.7 to 86.4μm. e dm was long ellip-
soid with a length from 182.1 to 377.2μm and widths from 55.8 to 172.2μm, respectively (Table2). e nucleus
(N) was located in the upper middle portion of the posterior segment (Fig.2b).
e syzygies consisted of two or three gamont connected caudofrontally (Fig.3a–c). e majority of syzygies
were composed of two cells (Fig.3a,b). e rst gamont of the syzygies is known as primite (Pr) and the remain-
ing cells are called satellites (Sa). e top of pm was presented in dierent shapes: a smooth anterior tip (Fig.3a)
and a papillary protrusion (Fig.3b). e SEM structure between pm and dm in Pr was shown in Fig.3d. e high
magnication of the apical part of the pm (Fig.3e), the connecting section between Pr and Sa (Fig.3f) and the
end recessed area of Sa (arrow, Fig.3g) were also shown. e longitudinal surface of the observed gamonts and
syzygies were shown in Fig.3h. From the split of the surface, ball-like structures (about 1.2–2μm in diameter)
were observed (Fig.3i). e length and width of two-cell formed syzygies ranged from 322.2 to 945.4μm and
130.9 to 311.1μm, respectively. e three-cell formed syzygies was about 236.8μm in length and 69.9μm in
width, and the size was signicantly smaller than that of two-cell formed syzygies. Gametocysts were found in
Figure1. Transversal histological section of the midgut (MG) of Macrotermes barneyi stained by HE. (a) Four
complete section of gregarines (arrowed) shown in the midgut of the infected host. (b) Higher magnication of
a showing the nucleus (arrowhead) and nucleolus (double arrowhead). Scale bars (a) 100μm; (b) 50μm.
Table 1. e termite origin and infection rates of gregarine in Macrotermes barneyi. # Only gregarines
observed in the midgut were counted.
Termite species Location Caste Termites with dissection
#Termites with
gregarines Infection rates %
Macrotermes barneyi
Zhongshan, Guangdong
Collecting:
113° 27″ E, 22° 49″ N
Feeding:
113° 31″ E, 22° 48″ N
Wor kers 303 65 21.45
Soldiers 29 0 0
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the hindgut of M. barneyi and feces (Fig.4). e spherical gametocysts contained two encysted gamonts with
a transparent outer surface (Fig.4a). e surface ultrastructure of gametocyst by SEM was shown in Fig.4b,c.
e average mean diameter of gametocysts was 135.9μm (Table2).
SSU rDNA sequence and phylogenetic tree analysis. e SSU rDNA sequence (MT126033) was
obtained by sequencing of the PCR fragment amplied from the termite gregarine parasite. It has a length of
1778bp and GC content of 48.73%. e majority of reported SSU rDNA sequences are among 1500–1800bp
except for two short sequences (1200bp) from Gregarina chortiocetes (L31841.1) and G. caledia (L31799.1).
Homology analysis showed that the SSU rDNA sequence of gregarine from termite M. barneyi shares approxi-
mately 89–90% identity to the terrestrial eugregarines from cockroach, beetle and cricket. Among three SSU
rDNA sequences from insect gregarines, the sequence from the termite M. barneyi gregarine (MT126033) has
the highest identity (90.10%) to that of gregarine from cockroach (FJ459741).
A phylogenetic tree was constructed using the SSU rDNA sequences from insect eugregarines (Eugrega-
rinorida). ese gregarines belong to the following families: Gregarinidae, Leidyanidae, Hirmocystidae, Stylo-
cephalidae, Actinocephalidae, Stenophoridae and Enterocystidae. e insect hosts include the Orders Blattaria,
Coleoptera, Dermaptera, Diptera, Odonata, Orthoptera, Odonata and ysanuronb. e individual nodes of the
tree were overall well supported for the analysis, but major clades were not well resolved (Fig.5). e eugregarines
from insects formed several clades, and the termite gregarine (MT126033.1) was positioned within a clade that
included Gregarina blattarus, Leidyana erratica and G. tropica. e crab parasite Hematodinium sp (AF286023.1),
designated as the outgroup, was on separate node.
Discussion
is study describes the occurrence of gregarine protozoa in the intestine of the fungus-growing termite M.
barneyi in China. e location, morphology and ultrastructure of gregarines were shown by histological sec-
tion and LM/SEM. Further, the SSU rDNA sequence from termite gregarines and its molecular phylogenetic
analysis were presented.
ere are very few literatures about gregarine from termites12,13. A gregarine (Apicomplexa: Neogregarinida)
reported in lower termite Coptotermes gestroi, which had a lemon-shaped spores, was suggested to belong to
the family Lipotrophidae. e gregarines cysts from workers of C. gestroi was ovoid in shape and was about
80–150μm × 60–110μm. e size of gametocysts isolated from M. barneyi was 82.3–197.2μm, similar to the
above value. According to the book by Desportes16, gregarine species found from dierent termite hosts, belong
to the following families: Actinocephalidae, Gregarinidae, Hirmocystidae, Kofoidinae and Sphaerocystidae16.
ese gregarines were recorded by line drawing, and were mainly found in termites distributed in Italy, USA
and India. ere were not much morphometric data of gregarines available from these termites. e syzygies of
two gamont-connected was 400μm for gregarine of Pleatospora termitis (the host is Macrotermes estherae), and
the total length of syzygies gamonts of Kofoidina ovata from termite Zootermopsis nevadensis in association of
Figure2. Morphology of gamonts observed in dierent termite individuals by light microscope. e gamonts
were isolated from the midgut of termite workers. In (a,c) the samples were observed under dierential
interference contrast (DIC) light microscope, while in (b) the sample was observed under bright eld
microscope. e cell contains two compartments: protomerite (pm) and deutomerite (dm). e nucleus (N)
was visible in the anterior part of the dm (b), and the septum (arrow) separated the pm and dm. Scale bars (a–c)
100μm.
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2–14 gamonts was 636 μm16. e size of gregarines from dierent termite species are variable. In this study we
have presented the detailed morphometric data of common life stages (gamont, syzygy and gametocyst), which
would be useful for further characterization of gregarines.
Figure3. Light micrograph (LM) and scanning electron micrographs (SEM) showing the morphology and
surface ultrastructure of the syzygies from the midguts of M. barneyi. (a,b) LM of syzygies. e syzygy contains
two parts: primite (Pr) and Satellite (Sa). e nucleus (N) is visible in the middle of each individuals. e top
structures in Pr (double arrowhead) and the connection (arrowhead) between the Pr and Sa were dierent
between (a,b). (c) LM of a syzygy with three parts dyed by Meilan dye solution. (d–g) SEM showing the partial
structure of syzygy; (d) the connecting part between protomerite (pm) and deutomerite (dm) in Pr.; (e) showing
a recessed area (arrow) in the center of the top of the pm in (a); (f) higher magnication of the connecting part
between Pr and Sa; (g) SEM of the posterior end of the Sa. (h,i) SEM showing the surface structure of syzygy;
the ball-like structure was observed from the ruptured surface in (h). Scale bars (a,b) 100μm; (c) 50μm; (d–g)
10μm; (h,i) 2.5μm.
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Termites are known as eusocial insects, which include the reproductive, worker and soldier termites. e
gregarine was initially found in the worker of M. barneyi, then the presence of gregarine in the digestive tract
of king, queen and soldier termites were examined, and no gregarine was found in these castes of M. barneyi.
Similar phenomenon was reported in other termite workers13 and wasps workers24. Workers and soldiers from
eld colonies of lower termite C. gestroi were checked for the presence of gregarine, the gregarine cyst was found
only in worker termites13.e gregarines seem to be specic to workers, the reason might be related to the roles of
termites. e worker termites mainly collect foods and transfer them to the nest, which increase their exposure
to the parasite oocysts. An alternative explanation may be that gregarine infection is related to host’s natural
microbiota. e forage termite workers have a more diverse microbiome than other termite castes1,25, which
may make workers more suitable to harbor gregarines. Of course, further studies on host–microbe interactions
should be explored.
It is noted that infection rate (frequency of the infected individuals), here in M. barneyi, was about 21%,
which is higher than that reported in termites C. gestroi (0.6–3.3%)13 and Odontotermes sp (5%)12. e rate of
gregarine infection varied with dierent host species, and even changes with dierent seasons in same species,
which could reach the highest 35% in workers of Polybia species, neotropical swarm-founding wasps24. By now,
the eect of gregarines on the host has not been claried, no eects or the negative eects ranging from high
mortalities to negligible eects have been reported in all cases1,26–29. is seems to depend on the quantity and
gregarine species as well as environment of the host’s habitat1.
e invertebrate hosts are usually infected with gregarines by swallowing mature oocysts, which are released
into the environment through feces and the gregarines are oen found in the digestive tract and body cavity of
the insect11. In the current study, three life-cycle stages of gregarines such as gamont, syzygy and gametocyst
have been obviously observed in midgut, hindgut and/or fece of M. barneyi, and other stages of the gregarine
have not been found. It was dicult to maintain fungus-growing termite M. barneyi alive in the laboratory for
a long time. We noted that gregarine has the papilla like structure at the top of the cell (Figs.2c, 3b), suggesting
that they are probably trophozoites. However, more morphological data are needed to give an accurate taxonomic
identication of the gregarine. e majority of gregarines observed are two-cell type of syzygies and we observed
Table 2. Morphometric data of gregarines from this study. Max maximum, Min minimum, SD standard
deviation.
Characters Mean (μm) SD (μm) Min (μm) Max (μm) Numbers
Gamonts
Protomerite length 42.8 11.7 26.8 65.1 10
Protomerite width 55.3 15.6 36.7 86.4 10
Deuteromerite length 321.4 53.8 182.1 377.2 10
Deuteromerite width 120.4 34.0 55.8 172.2 10
Syzygies
Prime length 348.7 78.0 202.8 563.6 20
Satellite length 325.6 84.9 119.4 472.7 20
Total length 674.3 149.4 322.2 945.4 20
Prime width 112.2 28.7 53.2 171.8 20
Satellite width 105.4 24.3 59.9 165.7 20
Total width 217.6 45.3 130.9 311.1 20
Gametocysts
Diameter 135.9 31.3 82.3 197.2 10
Figure4. Dierential interference contrast (DIC) light micrograph and scanning electron micrographs (SEM)
showing the general morphology of the gametocyst from the termite hindgut. (a) DIC light micrograph of
gametocyst showing initial stage of gametocyst formation in which individual gamonts associate (arrow) to
form a spherical structure. (b) SEM of gametocyst showing the surface ultrastructure. (c) A mechanically
ruptured gametocyst. Scale bars (a) 200μm; (b) 50μm; (c) 100μm.
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the three-cell type of syzygies once. Multiple or bi-association syzygies was reported in Hirmocystidae family
such as type species Kofoidina ovata16, and it was an aseptate species found in the gut of Z. nevadensis, while
the gregarine observed in M. barneyi is septate gregarine. e current data suggested that this gregarine found
in M. barneyi might be a novel species. e species name has not been determined due to incomplete morpho-
logical structure and limited molecular sequence data available from termite gregarines. Although the major
clade in phylogenetic analysis is not well resolved, overall tree topology is generally consistent to the reported
analysis7,14,17, which indicates that the gregarine from M. barneyi is phylogenetically close to G. blattarus, L.
erratica and G. tropica from families of Gregarinidae and Leidyanidae, and belongs to Gregarinidae group I14.
In summary, we report the presence of the gregarine in a fungus-growing termite M. barneyi for the rst
time. e current study expands the understanding of distribution and diversity of terrestrial eugregarines in
L31841.1 Gregarina chortiocetes
L31799.1 Gregarina caledia
FJ459741.1 Gregarina blattarum
FJ459752.1 Leidyana erratica
MT126033.1 Gregarine MbGr from Macrotermes barneyi
FJ459749.1 Gregarina tropica
KC890798.1 Apicomplexa sp. 1 KCW-2013
FJ459737.1 Amoebogregarina nigra
MK181531.1 Amoebogregarina sp. JMD-2019a
FJ459746.1 Gregarina kingi
FJ459740.1 Gregarina basiconstrictonea
FJ459744.1 Gregarina cuneata
KY697695.1 Enterocystis dorypterygis
FJ459757.1 Protomagalhaensia granulosae
FJ459758.1 Protomagalhaensia wolfi
FJ459751.1 Gregarina cubensis
FJ459753.1 Leidyana haasi
FJ459754.1 Leidyana migrator
FJ459743.1 Gregarina coronata
FJ459745.1 Gregarina diabrotica
FJ481523.1 Apicomplexa sp. Phaedon brassicae/Daegwallyeong
FJ459742.1 Gregarina cloptoni
KU664396.1 Gregarina sp. isolate GSPS-1
FJ459747.1 Gregarina niphandrodes
FJ459748.1 Gregarina polymorpha
FJ459759.1 Pyxinia crystalligera
FJ459760.1 Stenophora robusta
FJ459750.1 Hoplorhynchus acanthatholius
FJ459756.1 Prismatospora evansi
FJ459755.1Paraschneideria metamorphosa
FJ459739.1 Geneiorhynchus manifestus
FJ459738.1 Colepismatophila watsonae
FJ459762.1 Xiphocephalus ellisi
FJ459761.1 Stylocephalus giganteus
FJ459763.1 Xiphocephalus triplogemmatus
AF286023.1 Hematodinium sp. MF-2000
100
99
100
83
100
100
100
97
72
69
85
100
100
97
99
99
99
98
90
80
0.05
Insects
Blattaria
Coleoptera
Diplopoda
Odonata
Thysanuron
Coleoptera
Blattaria
Coleoptera
Orthoptera
Orthoptera
Crab
Coleoptera
Decapoda
Figure5. Molecular phylogenetic analysis using SSU rDNA sequences from termite gregarine (black marked)
and other eugregarines from various insects. e order of insect hosts was listed on the right of the tree. e
tree was constructed by Maximum Likelihood method, and evolutionary analyses were conducted in MEGA7.
e dinoagellates sequence from crab was used as outgroup. e numbers on the nodes indicate the bootstrap
values of higher than 60%.
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insects, and enriches our knowledge on termite parasites, which will contribute to the further gregarines study
and phylogenetic evolution research.
Materials and methods
Termite collection, feeding and identication. Macrotermes barneyi colony harboring all castes and
fungus combs was collected from Zhongshan, Guangdong Province in southern area of China (113° 27″ E, 22°
48″ N). e colony was carefully wrapped and brought to the laboratory. Aer arriving the lab, the dead termite
and broken combs were removed, and the complete fungus combs with healthy termites were put into a custom-
ized plastic container, which was kept in incubator at 27°and 85% humidity . e termite species was identied
by the morphology of the soldier and mitochondrial COII gene sequencing30.
Termite dissection and isolation of the gregarines. Worker and soldier termites were rinsed in dis-
tilled water, 75% ethanol and distilled water in turn, then the termites were dissected and the whole guts were
removed to the sterilized plates. e guts were separated into foregut, midgut and hindgut under Olympus SZ2-
ILST dissection microscope (Olympus Corporation, Tokyo, Japan). e gregarines were checked in each region
of the digestive tract and were isolated carefully using pipette for observation or stored at − 20° for later DNA
extraction.
Paran section preparation and observation. Aer dissection, the midguts were transferred into 4%
paraformaldehyde PB buer and xed for 24h. e samples were prepared for paran sectioning and stained
with HE (hematoxylin–eosin) according to protocol providing by Wuhan Sevier Biotechnology Co., Ltd. e
obtained slides were observed and photographed with a Nikon Eclipse80i bright-eld microscope (Nikon Cor-
poration, Tokyo, Japan) equipped with a Nikon DS_Ri1 (Nikon Corporation, Tokyo, Japan) camera.
Light microscope observation. Live observations of the gregarines were performed with a Nikon Eclip-
se80i dierential interphase contrast (DIC), and bright eld microscope (Nikon Corporation, Tokyo, Japan)
equipped with a Nikon DS_Ri1 camera for microphotographic records of the cells. Optical micrographs were
arranged using Adobe Photoshop CS5 (Adobe Systems Incorporated, San Jose, CA, USA). e width and length
of each compartment of gregarines were measured at dierent stage, mainly gamonts and syzygies.
Scanning electron microscope observation. For scanning electron microscope (SEM), e gregarines
were xed in 2.5% glutaraldehyde for 3h, aer which the cells were transferred to PBS buer and washed three
times to wash o the xing solution, dehydrated with 10%, 30%, 50%, 70%, and 90% (v/v) ethanol for 15min
each time, and then in anhydrous ethanol for 15min twice. e solution containing the gregarines was dropped
in the center of the slide, and the slide carrying the sample was placed in a Leica EM CPD300 critical point dryer
for drying. e slide was then adhered to the sample stage with conductive tape and gold plated for 240s using
a Cressington 108 ion sputtering device. SEM stubs were observed and photographed under a Quanta 250 FEG
scanning electron microscope (ermo Fisher Scientic, Massachusetts, USA), and the photos were edited with
Adobe Photoshop CS5.
DNA extraction and sequencing. Approximately 50 gamonts or syzygies were taken and pooled from
M. barneyi for DNA extraction. Aer washing with PBS buer three times, the genomic DNA of gregarines
was extracted following the instruction provided by the TIANamp Mico DNA Kit (Tiangen Biotech Co., Ltd.,
Beijing, China). DNA concentration and quality were detected using a Neno-200 micronucleic acid analyzer
(Allsheng Instruments Co., Ltd, Hangzhou, China). For PCR amplication of the SSU rDNA, two universal
primers: F1 (5′-GCG CTA CCT GGT TGA TCC TGCC-3′) and R1 (5′-GAT CCT TCT GCA GGT TCA CCTAC-3′)
were used2. e total volume of the amplication reaction mixture is 50 μL, which contained 25 μL of 2X M5 Taq
HiFi PCR MIX (Mei5 Biotechnology Co., Ltd., Beijing, China), 1 μL each of upstream and downstream primers
(10μM), 1 μL of template DNA (about 45ng) and ddH2O 22 μL. e PCR was performed in a TaKaRa TP600
thermal cycler (Takara Bio Inc., Dalian, China) using the following procedure: initial denaturation at 94°C for
5min; followed by denaturation at 94°C for 45s, annealing at 55°C for 45s, 72°C for 2min, and nal extension
at 72°C for 10min. e amplied fragment was ligated into pMD19-T vector (Takara) and the recombinant
plasmid was sequenced by Shanghai Shengong Company. e sequence was analyzed using BLAST (http://blast
.ncbi.nlm.nih.gov).
Molecular phylogenetic analysis. SSU rDNA sequence from M. barneyi gregarines (MbGr), deposited
in GenBank under accession number MT126033.1, was aligned with other 35 apicomplexan sequences mainly
from insects (min coverage > 52%, identity > 77.28%), and the dinoagellate Hematodinium sp. (AF286023) was
used as an outgroup (Supplementary TableS1). e alignment was performed on MEGA 7 using MUSCLE algo-
rithm with default parameters31. Aer alignment, the obvious gaps were deleted. e evolutionary history was
inferred by using the Maximum Likelihood method based on the Tamura-Nei model32. e tree with the high-
est log likelihood (− 9754.60) is shown. e percentage of trees in which the associated taxa clustered together
is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying
Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Compos-
ite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. e tree is
drawn to scale, with branch lengths measured in the number of substitutions per site. A total of 36 nucleotide
sequences were used in the analysis. All positions containing gaps and missing data were eliminated.
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General parasitological observations. Both worker and soldier termites were subjected to dissection
and the presence of parasites inside the digestive tract were registered. e number of termites and gregarines
were counted to calculate the general prevalence and intensity. e infection rate was presented as the percentage
of the number of hosts infected at least one parasite cell13.
Received: 15 September 2020; Accepted: 8 December 2020
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Acknowledgements
is study was funded by National Natural Science Foundation of China (31272370 and 31970119). We thank
Dr. Medina-Duran JH for valuable comments and advice. We also thank Professors Weibo Song and Xiaofan
Zhao for suggestions on this study.
Author contributions
S.Z. and Z.L. performed the experiments and contributed to the analysis of data. Y.S. and J.N. conceived the
project. J.N. and Q.H. contributed to the design of the experiments. S.Z., Z.L. and J.N. wrote the paper. All
authors edited the paper.
Competing interests
e authors declare no competing interests.
Additional information
Supplementary Information e online version contains supplementary material available at https ://doi.
org/10.1038/s4159 8-020-79671 -7.
Correspondence and requests for materials should be addressed to Y.S.orJ.N.
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