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

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.

www.nature.com/scientificreports/ the hindgut of M. barneyi and feces (Fig. 4). The spherical gametocysts contained two encysted gamonts with a transparent outer surface (Fig. 4a). The surface ultrastructure of gametocyst by SEM was shown in Fig. 4b,c. The average mean diameter of gametocysts was 135.9 μm (Table 2).
SSU rDNA sequence and phylogenetic tree analysis. The SSU rDNA sequence (MT126033) was obtained by sequencing of the PCR fragment amplified from the termite gregarine parasite. It has a length of 1778 bp and GC content of 48.73%. The 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 approximately 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 (Eugregarinorida). These gregarines belong to the following families: Gregarinidae, Leidyanidae, Hirmocystidae, Stylocephalidae, Actinocephalidae, Stenophoridae and Enterocystidae. The insect hosts include the Orders Blattaria, Coleoptera, Dermaptera, Diptera, Odonata, Orthoptera, Odonata and Thysanuronb. The individual nodes of the tree were overall well supported for the analysis, but major clades were not well resolved (Fig. 5). The 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. The crab parasite Hematodinium sp (AF286023.1), designated as the outgroup, was on separate node.

Discussion
This study describes the occurrence of gregarine protozoa in the intestine of the fungus-growing termite M. barneyi in China. The location, morphology and ultrastructure of gregarines were shown by histological section and LM/SEM. Further, the SSU rDNA sequence from termite gregarines and its molecular phylogenetic analysis were presented.
There are very few literatures about gregarine from termites 12,13 . A gregarine (Apicomplexa: Neogregarinida) reported in lower termite Coptotermes gestroi, which had a lemon-shaped spores, was suggested to belong to the family Lipotrophidae. The gregarines cysts from workers of C. gestroi was ovoid in shape and was about 80-150 μm × 60-110 μm. The size of gametocysts isolated from M. barneyi was 82.3-197.2 μm, similar to the above value. According to the book by Desportes 16 , gregarine species found from different termite hosts, belong to the following families: Actinocephalidae, Gregarinidae, Hirmocystidae, Kofoidinae and Sphaerocystidae 16 . These gregarines were recorded by line drawing, and were mainly found in termites distributed in Italy, USA and India. There were not much morphometric data of gregarines available from these termites. The 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 www.nature.com/scientificreports/ 2-14 gamonts was 636 μm 16 . The size of gregarines from different 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. www.nature.com/scientificreports/ Termites are known as eusocial insects, which include the reproductive, worker and soldier termites. The 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 workers 13 and wasps workers 24 . Workers and soldiers from field colonies of lower termite C. gestroi were checked for the presence of gregarine, the gregarine cyst was found only in worker termites 13 .The gregarines seem to be specific to workers, the reason might be related to the roles of termites. The 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. The forage termite workers have a more diverse microbiome than other termite castes 1,25 , 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 . The rate of gregarine infection varied with different host species, and even changes with different seasons in same species, which could reach the highest 35% in workers of Polybia species, neotropical swarm-founding wasps 24 . By now, the effect of gregarines on the host has not been clarified, no effects or the negative effects ranging from high mortalities to negligible effects have been reported in all cases 1,[26][27][28][29] . This seems to depend on the quantity and gregarine species as well as environment of the host's habitat 1 .
The invertebrate hosts are usually infected with gregarines by swallowing mature oocysts, which are released into the environment through feces and the gregarines are often found in the digestive tract and body cavity of the insect 11 . 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 difficult 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 identification of the gregarine. The majority of gregarines observed are two-cell type of syzygies and we observed  www.nature.com/scientificreports/ the three-cell type of syzygies once. Multiple or bi-association syzygies was reported in Hirmocystidae family such as type species Kofoidina ovata 16 , and it was an aseptate species found in the gut of Z. nevadensis, while the gregarine observed in M. barneyi is septate gregarine. The current data suggested that this gregarine found in M. barneyi might be a novel species. The species name has not been determined due to incomplete morphological 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 analysis 7,14,17 , 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 I 14 .
In summary, we report the presence of the gregarine in a fungus-growing termite M. barneyi for the first time. The current study expands the understanding of distribution and diversity of terrestrial eugregarines in  Figure 5. Molecular phylogenetic analysis using SSU rDNA sequences from termite gregarine (black marked) and other eugregarines from various insects. The order of insect hosts was listed on the right of the tree. The tree was constructed by Maximum Likelihood method, and evolutionary analyses were conducted in MEGA7. The dinoflagellates sequence from crab was used as outgroup. The numbers on the nodes indicate the bootstrap values of higher than 60%. www.nature.com/scientificreports/ 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 identification. 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). The colony was carefully wrapped and brought to the laboratory. After arriving the lab, the dead termite and broken combs were removed, and the complete fungus combs with healthy termites were put into a customized plastic container, which was kept in incubator at 27°and 85% humidity . The termite species was identified by the morphology of the soldier and mitochondrial COII gene sequencing 30 .
Termite dissection and isolation of the gregarines. Worker and soldier termites were rinsed in distilled water, 75% ethanol and distilled water in turn, then the termites were dissected and the whole guts were removed to the sterilized plates. The guts were separated into foregut, midgut and hindgut under Olympus SZ2-ILST dissection microscope (Olympus Corporation, Tokyo, Japan). The 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.
Paraffin section preparation and observation. After Table S1). The alignment was performed on MEGA 7 using MUSCLE algorithm with default parameters 31 . After alignment, the obvious gaps were deleted. The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura-Nei model 32 . The tree with the highest log likelihood (− 9754.60) is shown. The 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 Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. The 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. www.nature.com/scientificreports/