Parasites of the genus Nosema, Crithidia and Lotmaria in the honeybee and bumblebee populations: a case study in India

1 Федеральный исследовательский центр Институт цитологии и генетики Сибирского отделения Российской академии наук, Новосибирск, Россия 2 Новосибирский национальный исследовательский государственный университет, Новосибирск, Россия 3 Институт биологии, экологии и природных ресурсов, Кемеровский государственный университет, Кемерово, Россия 4 Институт наук об окружающей среде, Ягеллонский университет, Краков, Польша 5 Институт систематики и экологии животных Сибирского отделения Российской академии наук, Новосибирск, Россия

Nosema and Tubulinosema species represent the obligate intracellular spore forming organisms that are related to the Fungi (Han, Weiss, 2017). Two microsporidium species, Nose ma ceranae (Fries et al., 1996) and Nosema apis (Zander, 1909), are known to infect honeybees. Nosema bombi is another parasite belonging to the phylum Microsporidia which is widespread in the bumblebee populations (Fantham, Porter, 1914). Analysis of standard nuclear DNA markers of N. bombi in bumblebee colonies from USA, Russia, China and several European countries revealed new genetic variants of the parasite (Fries et al., 2001;Tay et al., 2005;Szentgyörgyi et al., 2011;Cordes et al., 2012;Vavilova et al., 2015). Three new Nosema variants (A, B, and C) isolated from bumblebees in China were suggested to be genetic variants of N. ceranae Vavilova et al., 2015). All detected genetic variants of Nosema species did not receive the status of separate species.
Another microsporidium species Tubulinosema pampeana (Microsporidia: Tubulinosematidae) was described for the first time in Bombus araratus individuals from Argentine (Plischuk et al., 2015). Currently, there are no cases of T. pampeana infections in other regions.
Thus, investigation of these parasites in host populations from new geographical regions allows to characterize new genetic variants and describe it's specific distribution. Despite the high importance of honeybees and bumblebees for the economy of India no studies of their parasites have been performed so far. In this study the diversity of Nosema, Crithidia, and Lotmaria parasites from honeybees and bumblebees in an unexplored regions of India, states Jammu and Kashmir and Karnataka were analyzed.  Table). 39 bumblebee specimens of B. asiaticus, B. lucorum, B. rufofasciatus, B. simillimus and B. trifasciatus species were collected in Jammu and Kashmir state in May, 2007 (see Fig. 1, Table).

Materials and methods
All samples were obtained by entomological sweep nets, identified to the species level in the field and preserved in 70 % ethanol. Total DNA were extracted from abdomens of the specimens fixed in ethanol using DNeasy Blood and Tissue Kit (QIAGEN) according to the manufacturer's protocol.
Parasite prevalence. The prevalence of microsporidia and trypanosomatid parasites was calculated as percentage of the infected specimens to the total number of honeybee or bumblebee specimens in each sampling sites. Confidence intervals for parasite prevalence were defined using STATISTICA by chi-square test.
Comparative and phylogenetic analyses. Logical search was performed to identify rRNA genes sequences from insects parasites of the phylum Microsporidia and family Trypanosomatidae available from the United States National Center for Biotechnology Information. Nucleotide sequences alignments were performed by ClustalW (Larkin et al., 2007) and improved by MUSCLE algorithm (Edgar, 2004) in Unipro UGENE software (Okonechnikov et al., 2012) (http://ugene. unipro.ru) for each parasite group. Phylogenetic analyses were performed using the Neighbor-Joining (NJ) method in MEGA 6.0 and Maximum Likelihood (ML) method in PhyML 3.0 (Guindon, Gascuel, 2003;Tamura et al., 2013). Statistical support for the NJ and ML trees was evaluated by bootstrapping, 100 replications for the ML method and 1,000 replications for the NJ method (Felsenstein, 1985).

Prevalence of parasite infection in honeybee and bumblebee populations
We studied 80 honeybee and 39 bumblebee specimens collected in two Indian states. Nosema spp. were detected by PCR amplification with primers specific to SSU rRNA sequence. Nosema spp. were discovered in 20 honeybee specimens from all investigated honeybee species and 4 bumblebee specimens of B. trifasciatus. The prevalence of Nosema spp. in honeybee populations was 25 and 24 % in Jammu and Kashmir, and Karnataka states, respectively (Fig. 2, a). In Jammu and Kashmir state the prevalence of Nosema spp. in bumblebee population was 10 %.
The trypanosomatid parasites were identified by PCR amplification with primers specific to 18S rRNA sequence in 10 honeybee and 12 bumblebee specimens. A. cerana, A. dor sata, A. mellifera, B. asiaticus, B. simillimus and B. trifasciatus species were infected. In Jammu and Kashmir state the prevalence of trypanosomatid parasites was 16 and 31 % in honeybee and bumblebee populations, respectively (Fig. 2, b). No infected honeybee specimens were found in Karnataka state.
Co-infection by Nosema spp. and one of the trypanosomatid parasites (Crithidia spp. or L. passim) was detected in 6 honey bee (A. cerana and A. mellifera) and 2 bumblebee (B. trifasciatus) specimens in Jammu and Kashmir state (Fig. 2, c).

Genetic diversity of Nosema spp. in honeybee and bumblebee populations
Comparative analyses of the SSU rRNA sequences of Nosema spp. Totally, we obtained 24 nucleotide sequences of Nosema spp. SSU rRNA gene from honeybees and bumblebees (see Table). The results of the comparative analysis showed that 13 out of 20 sequences from honeybee specimens were identical to N. bombi SSU rRNA sequences (KF002566, HG321391, KF188769, JN872234, and JN872233). N. bombi was described for bumblebees only. The remaining 7 sequences were identical to SSU rRNA of N. ceranae (KF640602, JX205150).
Previously Nosema species were considered to be the host specific parasites. N. apis infected only the European honeybee A. mellifera, while N. ceranae was a specific parasite for the Asian honeybee A. cerana (Smith, 2012). In the recent years N. ceranae was identified in A. mellifera and some bumblebee species in the different parts of the world. Moreover, some stains of N. ceranae are predicted to replace N. apis in populations of A. mellifera honeybees (Chen, Huang, 2010; Bars represent confidence intervals defined by chi square test (p = 0.95) in STATISTICA. The studied parasites were characterized by low hostspec ificity; thus, their prevalence was calculated as percentage of the infected specimens to the total number of honeybee or bumblebee specimens in each sampling location.  -Hernández et al., 2012). In this study a bumblebee parasite N. bombi was identified in honeybees from Jammu and Kashmir and Karnataka states, thus additionally refuting the assumption of the host specificity of Nosema species. All Nosema spp. SSU rRNA sequences from infected bumblebee specimens were almost identical to Nosema D sequences (JN872219-JN872229) . A distinguish is only a single nucleotide substitution. No other Nosema as well as Tubulinosema species were found in bumblebees in this study (see Table).

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Comparative and phylogenetic analyses of SSU rRNA, ITS2 and partial LSU rRNA sequences from Nosema spp. To expand information about Nosema D we obtained SSU rRNA, ITS2, and partial LSU rRNA sequences for four B. trifasciatus specimens. Sequences of Vairimorpha spp., N. bombi, N. ceranae, N. apis, and Nosema sp. from Pieris rapae from GenBank, as well as the obtained sequences were used for phylogenetic analysis. Sequences of several Tubulinosema species were taken as an outgroup. Phylogenetic tree built by the NJ method is presented in Fig. 3.
The phylogenetic tree was divided on outgroup and three clusters (see Fig. 3). The outgroup was presented by sequences of T. pampeana, which were described as parasite of B. araratus from South America and two parasites of Drosophila spp.
(T. ratisbonensis and T. kingi). The first cluster (I) consists of sequences of N. bombi WS2 and N. bombi WS3 that were previously described in populations of bumblebee from West Siberia (Vavilova et al., 2015). The second cluster (II) includes two clades. The sequences of N. bombi previously identified from the Europe, USA and West Siberia formed the first clade (Tay et al., 2005;Sokolova et al., 2010;Szentgyörgyi et al., 2011). The second clade consists of the newly identified Nosema D sequences. The third cluster (III) is also split into two clades. Sequence of N. apis, obtained from A. mellifera apiary specimens in New Zealand (Gatehouse, Malone, 1998), is in the first clade of the third cluster. The second clade of this cluster consists of sequences of several Vairimorpha spp. from Bombyx mori and Manayunkia speciosa (Liu et al., 2012;Malakauskas et al., 2015); sequences of N. ceranae from Taiwan honeybees (Huang at al., 2007); and sequence of unspecified Nosema from Pieris rapae . Thus, the analysis of complete SSU rRNA, ITS2 and partial LSU rRNA gene sequences confirmed that Nosema D is a genetic variant of N. bombi and distributed in the bumblebee populations at least in China and India.

Genetic diversity of Crithidia spp. and Lotmaria passim in honeybee and bumblebee populations
Comparative analyses of the 18S rRNA sequences of Cri thidia spp. and Lotmaria passim. We obtained 10 and 12 nuc leotide sequences of 18S rRNA gene of trypanosomatid parasites in honeybees and bumblebees, respectively  Fig. 3. Neighborjoining (NJ) phylogenetic tree was based on SSU rRNA, ITS2, and the partial LSU rRNA nucleotide sequences of several N. bombi genetic variants, Nosema spp. and Vairimorpha spp. from various insects and annelids available from GenBank. Tubulinosema spp. were used as outgroup.
Statistical support was evaluated by bootstrapping (1 000 replications). The nodes with bootstrap values over 50 % are indicated.    Table). The results of comparative analysis showed that nine of infected bumblebee specimens refer to C. expoeki (KM980187) and three others refer to C. bombi (FN546181,KM980184,KM980185). The distinguish between C. bombi and C. expoeki sequences amounted five nucleotide substitutions (Fig. 4).

Ecological and population genetics Vavilov Journal of Genetics and Breeding
All ten 18S rRNA gene sequences were identical and they could belong to either C. mellificae or L. passim parasites. Sequences of C. mellificae/L. passim differ in 4 and 3 nuc leotide substitutions from C. bombi and C. expoeki, respectively (see Fig. 4). Using the primers for 18S rRNA specific to L. passim and to C. mellificae (Arismendi et al., 2016) on the next step, we proved that all the obtained sequences belonged to L. passim (KJ713378, KM980188, KT252553, KX953206).
Summarizing the data about microsporidian and trypanosomatid parasites in honeybee and bumblebee population from India, we identified that two A. cerana and one A. mellifera specimens were co-infected by N. ceranae and L. passim; three specimens of A. mellifera were infected by both N. bombi and L. passim. Co-infection by Nosema D and C. expoeki in bumblebee populations was established in two B. trifasciatus specimens. No cases of Nosema D and C. bombi co-infection were found in this study.
Co-infection by N. ceranae and L. passim was also previously established in honeybee samples from Switzerland (Tritschler et al., 2017). Infection of both N. ceranae and C. mellificae parasites was described for honeybees from Belgian apiaries (Ravoet et al., 2013) (see Fig. 4). Nevertheless, sequences of C. mellificae 18S rRNA, identified by Ravoet et al. (2013), were identical for C. mellificae and L. passim. Thus, these data should be clarified. Gallot-Lavallée et al. (2016) investigated co-infection by Nosema spp. and Crithidia spp. in the bumblebee populations from Mexico and established the cases of shared parasite infection (Fig. 5). However, species of Nosema and Crithidia genera found in infected bumblebee samples were not specified. Our data about co-infection of honeybee and bumblebee specimens by microsporidia and trypanosomatid parasites coincide with previously described studies. For the first time N. bombi/L. passim and N. bombi/C. expoeki co-infection were detected.
Geographic distribution of Nosema spp., Crithidia spp. and L. passim in honeybee and bumblebee populations The results of this study supplement the knowledge of the distribution of microsporidia parasites among the honeybee and bumblebee populations all over the world (see Fig. 5).
Apian parasites of genus Nosema (N. apis and N. ceranae) are wildly distributed in honeybee populations. Joint presence of these parasites was described in numerous studies (Table S1 in the Supplementary material) 1 . Nevertheless, there are cases in the several countries such as Indonesia, Israel, Kenya and Zimbabwe of honeybee infections by N. apis only (see Table S1). Presence of N. ceranae only was established in the honeybee's population from countries of Latin America (except Brazil), several European countries, Iran, Mongolia, Saudi Arabia and Vietnam (see Table S1). In this study, we discovered that honeybee populations were infected by N. ceranae. There were no cases of N. apis presence. Presence of N. bombi parasite in honeybee specimens was detected for the first time (see Fig. 5).
N. bombi is widespread in the natural and commercial bumblebee populations of North and South America, Eurasia and New Zealand (Gallot-Lavallée et al., 2016;Brown, 2017). Several N. bombi genetic variants (WS1, WS2 and WS3) were described in Siberian bumblebee populations (Vavilova et al., 2015). Four new Nosema variants (A, B, C and D) were isolated from bumblebees in China . The microsporidian parasite, T. pampeana, was described in bumblebee populations from Argentina (Plischuk et al., 2015). In the recent decades the cases of bumblebees infection by apian parasites N. ceranae (Argentina, China, Colombia, Mexico, UK and Uruguay), N. apis (Mexico and UK) and other Nosema species (Chile, China and Mexico) have been described (Gallot-Lavallée et al., 2016;Brown, 2017). We established the presence of Nosema D in bumblebee population from Jammu and Kashmir state (India). Nosema D was previously described by ) (see Fig. 5).
Presence of two trypanosomatid parasites, C. mellificae and L. passim, was indicated in honeybee populations globally. C. mellificae was found in honeybee specimens from Australia, Belgium, USA and Spain (Table S2). L. passim infections were described for honeybees from Belgium, Japan, Serbia and Switzerland (see Table S2). In this study distribution of L. passim in Indian honeybee populations were established (see Fig. 5). There were no cases of honeybee infection by C. mellificae.
The cases of trypanosomatid infections were determined in commercial and native populations of bumblebees on the territories of North and South America and Eurasia. C. bombi is the most common trypanosomatid parasite that infects bumblebees from Argentina, Belgium, Chile, Germany, Italy, Russia, Switzerland and UK (Table S3). The second species C. expoeki is presented in Mexican and Swiss bumblebee populations (Schmid-Hempel, Tognazzo, 2010;Gallot-Lavallée et al., 2016). Bumblebee infection by C. mexicana was indicated in Mexico (Gallot-Lavallée et al., 2016). Both C. bombi and C. expoeki are distributed among bumblebee populations from India (see Fig. 5).
Thus, in this study the prevalence of Nosema, Crithidia and Lotmaria parasites in honeybee and bumblebee populations of Jammu and Kashmir and Karnataka states were identified. In addition, co-infection by Microsporidia and Trypanosomatidae parasites was identified in several honeybee and bumblebee specimens from Jammu and Kashmir state. Honeybee and bumblebee specimens from India studied were infected by several microsporidian parasites (N. bombi, N. ceranae and Nosema D). Trypanosomatid parasites of C. bombi, C. expoeki and L. passim species were detected in honeybee and bumblebee populations. Moreover, for the first time N. bombi infection was detected in the honeybee population. Thus, further investigations are required to determine distribution of microsporidia and trypanosomatid parasites among the honeybee and bumblebee populations all over the World.