Abstract
Differences in midgut microbial communities of Aedes aegypti, insect vector of dengue virus, might influence the variation observed in the ability of these mosquitoes to propagate dengue viruses. As a first step toward addressing this hypothesis, comparative analysis of bacterial communities from midgut of mosquitoes having differential ability to transmit viruses was done.16S rDNA library and Real Time PCR approaches were used for microbial community structure analysis and eubacterial quantification respectively. Midgut bacteria of three Aedes aegypti strains, MOYO-R (Refractory strain), MOYO-S (susceptible strain), and MOYO-D (Refractory strain) were studied. We found that there was very less microbial diversity in these mosquitoes indicating a very restricted environment for microbial growth in midgut of Aedes aegypti mosquitoes. Pseudomonas related clones dominated all the libraries of three strains. There were significant differences in microbial communities of these three strains as Pedobacter and Janthinobacterium were identified only in MOYO-R strain libraries. Likewise, Bacillus was detected only in MOYO-S strain and Rahnella found in MOYO-R and MOYO-D strains libraries was absent in MOYO-S libraries. Real Time PCR approach confirmed the presence of Pedobacter bacteria only in MOYO-R strain. Real Time PCR based Eubacterial quantification indicated that MOYO-R strain mosquitoes have more 16S rRNA gene copy numbers in their midgut compare to MOYO-S and MOYO-D strain mosquitoes. Our study finds some potential bacteria which could affect the infectivity of dengue virus because of their characteristic physiological properties. This analysis represents the first report of comparison of microbial communities with respect to refractoriness and susceptibility of Aedes aegypti mosquitoes to dengue viruses and will serve to direct future research toward addressing the role of midgut bacteria of Aedes aegypti mosquitoes in the infectivity of dengue virus.
Differential midgut microbial community structure in aedes aegypti , insect vector of dengue virus might influence the variation observed in ability of these mosquitoes to propagate dengue virus. This hypothesis was tested by comparative analysis of bacterial community structure from midgut of mosquitoes having differential ability to transit viruses.
Introduction:
Dengue viruses cause significant morbidity and mortality worldwide and have become the most important arboviruses affecting humans. They are transmitted to humans through the bites of infective female Aedes aegypti mosquitoes, putting about two fifths of the world's population at risk for dengue disease (3). Global incidence of dengue has grown dramatically in recent decades as at present, dengue is endemic in 112 countries in the world causing around 50-100 million infections and 24,000 deaths annually (4, 5, 6).
The major reasons for this catastrophic situation are A. aegypti proliferation, inadequate vector control programs, insecticide resistance in vector populations, increased urbanization, rapid transportation, unavailability of effective vaccines for virus and the decline in socio-economic conditions in many disease endemic countries. The only way to prevent dengue virus transmission is to combat the disease-carrying mosquitoes; therefore there is an urgent need to develop unique mosquito control strategies like paratransgenesis and development of refractory transgenic mosquitoes.
In mosquito, the first point of contact between pathogen ingested with the blood meal and the mosquito is midgut epithelial surface. In order to establish an infection in the midgut, a pathogen must overcome midgut infection barriers such as digestive enzymes, lectins, antimicrobial peptides, nitric oxide and the prophenoloxidase complex (9, 10). Along with these, naturally occurring midgut bacteria might play a vital role as midgut infection barrier since midgut bacteria were reported to inhibit sporogonic development of Plasmodium (11, 12, 13). Also midgut bacteria were reported to influence parasitic life cycle in other insect vectors like sand flies and tsetse flies [15, 23].
Based on these evidences, we hypothesize that the bacteria present in the midgut of mosquito could have important role as determinant of dengue virus development in mosquito and therefore, contribute to the modulation of mosquito competence. To address this hypothesis we studied differences in midgut microbial communities among three A. aegypti strains having differential abilities to transmit dengue virus: MOYO-D, MOYO-R and MOYO-S. MOYO-D is a strain originating from East Kenya having low vector competence for Plasmodium gallinaceum and dengue virus. MOYO-R and MOYO-S were originally selected from MOYO-D for refractoriness and high susceptibility, respectively, to P. gallinaceum (Thathy et al., 1994), however it is also found that MOYO-R is refractory to dengue virus while MOYO-S is highly susceptible to dengue virus(schneider et al.).(reference which is nt here)
The objectives of the study were to study and compare the microbial communities of MOYO-D, MOYO-S and MOYO-R strains of A. aegypti, which could be attributed refractoriness and susceptibility of these mosquitoes to dengue virus. To accomplish this, bacterial 16S rDNA from these three Aedes aegypti strains was amplified by polymerase chain reaction (PCR), cloned and sequenced. Real Time Taqman PCR approach was used for quantification of total bacteria and Pedobacter bacteria.
Comparing differences in midgut microbial communities between these strains provides a foundation for future work to determine the potential role of midgut microbial communities in mosquito competence. Any variations in midgut bacteria identified in this pilot study will be analyzed further in subsequent experiments to correlate gut microbiota differences and variations in dengue virus infectivity. However, the major goal of this study was the comparison of microbial communities with respect to refractoriness and susceptibility of Aedes aegypti mosquitoes to dengue viruses
Mat n meth
Mosquito rearing
Three Aedes aegypti strains (MOYO-R, MOYO-S and MOYO-D) used in this study were reared in different environmental chambers at 26°C, 84% relative humidity, under a 16-hr light/8-hr dark cycle with a 1-hr crepuscular period at the beginning and the end of each light cycle. Adults were maintained on a 5% sucrose solution ad libitum. Female mosquitoes were blood-fed on anesthetized rats
DNA isolation
Adult female mosquitoes were surface-sterilized in 70% ethanol. The midguts of adult mosquitoes were carefully dissected under sterile conditions and the contents were suspended in 0.85% NaCl. These midgut contents were used for DNA extractions as previously described (Severson, 1997). DNA was checked on 0.8% agarose (USB, USA) gel and quantified using NanoDrop (Thermo Scientific, USA).
Midgut isolation details
Clone library construction
In this study, two sets of universal bacterial primers were used for construction of 16S rDNA library to capture maximum microbial diversity and to remove any PCR bias caused by primer set (REF). In one case 16S rDNA was amplified from extracted mosquito midgut DNA using universal bacterial Primers 530F and 1490R as described previously (Weisburg et al. 1991, Wani et al. 2006) while in the second case, universal bacterial Primers 357F and 1492R were used for PCR amplification (ref) . In both the cases the number of PCR cycles used was reduced to 25 so as to minimize PCR bias (Suzuki et al.1996)
The PCR products were purified using a PCR purification kit (Qiagen, USA). After purification, these were ligated into the TOPO pCR®2.1 vector (Invitrogen, USA) and then transformed into One Shot® TOP10 chemically competent E. coli cells (Invitrogen, USA) following the manufacturer's instructions. PCR screening of clones was performed and positive clones were sequenced using M13 vector primers. Sequencing was performed on an ABI PRISM 3730 DNA Analyzer (Applied BioSystems, USA) using the ABI Big-Dye version 3.1 sequencing kit (Applied BioSystems, USA) as per the manufacturer's instructions.
To ensure minimum PCR bias and capture maximum microbial diversity, 16S rDNA was amplified by using two sets of universal primers.
Nucleotide sequence accession Number
Sequences from this study were deposited in GenBank under Accession numbers …………..
Sequence assembly and phylogenetic analysis
Sequence assembly and editing was done by using ChromasPro (ref). For preliminary identifications, the 16S rRNA gene sequences were analyzed in BLASTn (http://www.ncbi.nlm.nih.gov/BLAST/) and the Ribosomal Database Project II (RDP II) (http://rdp.cme.msu.edu) using the RDP query program (Maidak et al. 1999). The presence of chimeric sequences was checked with Pintail 1.01 module of Mallard 1.02 program (Ashelford et al. 2006) and predicted chimeras were further analyzed by using Bellerophon (Huber et al. 2004). Putative chimeric fragments were excluded from further analysis. Multiple sequence alignment of all the sequences in each 16S rDNA library was performed using ClustalX 1.83 (Thompson et al. 1997) and aligned sequences were edited manually using DAMBE (Xia & Xie 2001) to get unambiguous sequence alignment. Distance matrix of every sequence with the rest of the library was determined using DNAdist module of Phylip 3.64 (ref).
Operational taxonomic unit (OTU) determination and community analysis
The resulting distance matrix was used as input for DOTUR 1.53 program (Schloss & Handelsman 2005). Operation taxonomic units (OTUs) were determined with a Similarity cut-off of 97%. The sequences representing each Operational Taxonomic Unit were compared with NCBI database using BLASTn, and the closest matches to bacterial strains were obtained. Phylogenetic trees were constructed by the Neighbor Joining method using Kimura 2 parameter distances in MEGA 4.0 software (Tamura et al. 2007). Chao1 richness estimates was calculated and rarefaction curves were constructed by DOTUR to assess microbial diversity richness
Real Time PCR Assay for Eubacterial quantification
16S rRNA gene copy numbers in midguts of three mosquito strains were determined by Quantitative PCR. In Real-Time PCR study, each reaction was carried out in duplicate by using TaqMan Universal PCR master mix (Applied Bioscience) in (machine name). 16S rDNA based Eubacterial quantification was done by using 331F & 797R Primers (numbering based on Escherichia coli 16S rRNA gene) and (6-FAM)-5´-CGTATTACCGCGGCTGCTGGCAC-3´-(TAMRA) probe, in the same way as described by Nadkarni et. al.. (Ref). Purified Pseudomonas aeruginosa DNA was used in 1:10 dilution series (10pg-100ng) for making standard. Mass of the Pseudomonas aeruginosa genome was calculated according to the following formula:
M= [n] [1.096 e-21 g/bp], where n=genome size.
The CT values (i.e., the cycle no. in which exponential amplification of PCR products crosses threshold) were determined on the basis of the fluorescence signals at the mean baseline during the early cycles of amplification.
PCR efficiency was calculated by using following equation:
Efficiency =10(-1/slope) -1
Real Time PCR based quantification of Pedobacter
The designed probe and primers set were based on regions of identity within 16S rDNA region of Pedobacter sp and designed in the Primer Express Software provided by Applied Biosystems. This software gave some best fit suggestions for the probe and primer set which were checked in NCBI and RDP database for their specificity to Pedobacter genus. The final chosen set included the probe 5'FAM- TAMRA the forward primer, 5'--3' (Tm) , the reverse primer.
Real Time PCR reactions were performed in ABI 7300 (machine name) and each reaction was carried out in duplicate. Primer and probe concentrations were optimized for the ratio of :nM, which were used in the further study. Each PCR reaction was performed in a volume of 25 ul with using Taqman universal PCR master mix supplied by Applied Biosystem. The reaction conditions were 50 °C for 2 min, 95 °C for 10 min and 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Data were analyzed in Sequence Detection Software version 1.6.3 (Applied Biosystems). Purified Pedobacter sp DNA was used for generating standard and PCR efficiency and mass of genome was calculated as described above.
Results
16S rDNA library analysis
16S rDNA libraries representing the midgut bacterial diversity associated with MOYO-R, MOYO-S and MOYO-D strains of A. aegypti were established with two different sets of primers. Comparative analysis of 16S rDNA libraries constructed with universal eubacterial primers 530F and 1490R showed significant difference between midgut microbiota of three strains (table-1). In MOYO-R strain, 196 clones were sequenced resulting in 135 high quality sequences. Out of these, 8 were found to be chimeras and therefore were excluded from further analysis. The phylogenetic analysis of remaining 127 sequences revealed that there were 4 OTUs found in this library. Sequences related to genus Psuedomonas (72% of total clones examined) dominated the library and showed % similarity with Psuedomonas sp. (Genus Pseudomonas dominated the library since 72% of total clones examined were affiliated to Pseudomonas sp.) About 9% of sequences were related to genus Rahnella of enterobacteriece family while 17% clones were associated to uncultured bacteria group. There was a single clone which showed % similarity to genus Pedobacter, a Heparinase secreting bacterium of …… family.
In case of MOYO-S strain a total of 150 good quality sequences were obtained from sequencing of 196 positive clones and 145 sequences were chosen for final analysis after exclusion of 5 chimera sequences. (In MOYO-S strain library, sequencing of 196 clones yielded 150 good quality sequences. Out of these 145 were chosen for final analysis after exclusion of 5 chimeras) In this library 3 OTUs were found and Pseudomonas related clones dominated the library with 66% occurrence. About 33% of total sequences were of uncultured bacteria while one clone showed % identity to Bacillus sp. interestingly there were no bacteria found of enterobactereceae family.
Similarly Pseudomonas related clones were also prevalent in MOYO-D strain library with 77% occurrence out of 109 final sequences. (Similarly out of 109 final sequences analyzed from MOYO-D strain library, sequences affiliated to Pseudomonas were the most dominant with 77% occurrence.) Two clones showed homology to bacteria yet to be cultured while about 20% sequences were related to genus Rahnella.
To capture maximum bacterial diversity, all the three 16S rDNA libraries associated with the three strains were repeated with a different primer set (357F/1492R), however it showed almost the same distribution of bacteria in the libraries as Pseudomonas related clones were again predominant in all the three libraries (table-2). The only significant feature obtained was the presence of Janthinobacterium related clones in MOYO-R strain library which could not be detected with first primer set while genus Pedobacter and Bacillus related sequences were not obtained with this second primer set.
Phyogenetic trees were constructed to show phylogenetic affiliation of all the members detected in the midgut of each strain. Based on dendrogram, clones in MOYO-R strain mosquitoes, midgut bacteria belong to Bacteriodetes, gammaproteobacteria and betaproteobacteria groups. In case of MOYO-S strain betaproteobacteria and gammaproteobacteria classes of phylum proteobacteria were present along with firmicutes. In MOYO-D strain mosquitoes, bacteria affiliating with class gammaproteobacteria were only present.
Real Time PCR based Eubacterial quantification
Mass of the Pseudomonas aeruginosa genome for generating DNA standard was calculated as 6.9 fg. DNA standard for 16S rRNA gene quantification showed a slope of -3.15 which corresponds to about 107% PCR efficiency. Real-Time PCR based Quantification of total bacteria showed a significant difference in copy no. of 16S rRNA gene from three mosquito strains (table). The copy numbers of 16S rRNA gene ranged from 106 to 109 copies per ml and were maximum in midgut of MOYO-R strain followed by MOYO-D and MOYO-S strains respectively.
Determination of Pedobacter 16S rRNA gene copy no.
Real Time PCR based quantification of Pedobacter bacteria showed that these bacteria are present only in the midgut of MOYO-R strain as the amplification was in the range of no template control in case of MOYO-S and MOYO-D strains. We found that no of Pedobacter bacteria were …. in MOYO-R strain contributing ….. percent of total bacteria.
Discussion:
Today, most control efforts for emergence of epidemic dengue have focused on A. aegypti, the primary vector of dengue virus which has facilitated the emergence of epidemic dengue in urban centers around the world. Traditional methods used for disease prevention and control have largely been unsuccessful due to failure of vaccine development and organized mosquito control programmes coupled with rapid emergence of insecticide resistance in vector populations. So keeping this in view the elucidation of factors affecting susceptibility or resistance to virus infection in vector mosquitoes could help in exploring potential novel disease prevention strategies.
Previous efforts to explore the factors affecting DENV susceptibility in Aedes aegypti strains have been inconsistent (Bennette et al., 2002; Gorrochotegui-escalante et al., 2005). In addition to strong genetic components, substantial influence of environmental factors on vector competence for DENV was noted (Bosio et al., 1998). Previously the differential ability of A. aegypti strains MOYO-R, MOYO-S and MOYO-D to transmit pathogens was co-related with genetic factors (ref), also Schneider et al., (2007) confirmed that although significant genetic variation exists among these strains in terms of adult body size and dengue vector competence, there was not considerable evidence of interaction between the phenotypic characteristic and dengue virus dissemination (ref).
Recently, the role of natural microbiota was investigated toward the determination of the mosquito's succeptibility to various pathogens and hence their vectoral capacity. Indeed it was found that pathogen development was significantly more influenced by the mosquito's basal level immunity rather that induction of immune response upon parasite infection (Frolet et al., 2007). Previously Xi et al reported that naturally occurring midgut bacteria of A. aegypti mosquitoes can modulate dengue virus infection by activating Toll immune pathway but could not find a direct bacteria-virus interaction and raised the possibility that mosquito midgut bacteria can hinder virus interaction with the midgut epithelium (ref). However as every bacterium has its unique niche in midgut environment this interaction may vary bacterium to bacterium. In another possible mechanism, midgut bacteria may form a physical barrier which block the virus access to the epithelium; this is common mechanism by which the vertebrate microbiota protect against pathogenic bacterial infection.(Pamer., 2007).
Although as discussed earlier, previously lots of attention has been given to screen genetic factors to explain the susceptibility or refractoriness of different mosquito strains to pathogens (ref), still role of naturally occurring mosquito midgut bacteria in this aspect has been ignored. In this study, we are first time attempting to explore the importance of these midgut bacteria in the differential ability of mosquito strains to transmit pathogens.
To find out whether it is population richness or presence/absence of key bacteria that could be correlated to refractoriness or susceptibility of A. aegypti to dengue virus, we used Real Time PCR and 16S rDNA clone library approaches. Our study provides an additional demonstration of the utility of 16S rDNA library analysis and Real Time PCR based Eubacterial quantification for describing differences in complex microbial communities of mosquito midgut. Since it has been anticipated that almost 99% of all microorganisms can not be cultivated by standard methods (Hugenholtz et al. 1998); and available conventional culture techniques limit the isolation and identification of all the microorganisms of mosquito midgut, we, therefore chose culture-independent analysis to assess microbial diversity in Aedes aegypti midgut.
Based on RT PCR analysis?, we found that there are significant differences between midgut bacterial population of MOYO-R, MOYO-S and MOYO-D mosquitoes which could play an important part in refractiveness and sensitivity of these mosquitoes to dengue virus.
The dominant bacteria in the midguts of these three mosquito strains belonged to---- Pseudomonas. Previously Pseudomonas was also reported as dominant from the midgut of Culex mosquitoes (ref). Rahnella, an enterobacteriacae family member was identified in MOYO-R and MOYO-D strains. Rahnella aquatilis was commonly isolated from freshwater (ref) and also has been linked to human diseases in some instances (ref). Previously, it was also reported from gut of Hepialus gonggaensis larvae and red turpentine beetle (ref). The characteristic physiological properties of Rahnella include reducing nitrate to nitrite, producing acid from D-glucose. It can also ferment a lot of? carbohydrates like lactose, maltose, rhamnose, raffinose, salicin and D-gluconic. Therefore, this bacterium may have an important part in insect metabolic system and nutrition, and in turn related to the intestinal microbial ecosystem.
In case of MOYO-R strain, the significant difference was the presence of bacteria Pedobacter and Janthinobacterium. Pedobacter was previously reported to be found in water and soil (Ref), also its association has been reported from scorpion intestine and a plant parasite nematode (ref) and in this study we are first time reporting its presence in insect midgut. It is a heparinase secreting bacterium which degrades heparan sulfate, an important receptor moiety used by many pathogens like ……..(ref) for binding to cell surface.
Among the different factors such as virus load, the virulence of the dengue virus and the immunity and susceptibility of the mosquito host that influence the transmission efficiency of the vector mosquito, susceptibility of vector mosquito to dengue infection is a critical parameter which is determined by interaction between midgut cell membrane receptors and the virus envelope glycoprotein in receptor-mediated endocytosis.
and the immunity and susceptibility (lang change) of the mosquito host
It is known that the transmission efficiency of the vector mosquito is influenced by the virus load, the virulence of the dengue virus and the immunity and susceptibility (lang change) of the mosquito host. Of these factors, susceptibility of vector mosquito to dengue infection is a critical parameter which is determined by interaction between midgut cell membrane receptors and the virus envelope glycoprotein in receptor-mediated endocytosis. (Some receptor ).
Recently (ref) observed similarities between mosquito heparan sulfate and human liver heparan sulfate. They have observed the importance of this moiety for the transmission of Plasmodium in mosquito host and also raised the possibility that other mosquito borne pathogens like dengue virus may utilize mosquito heparan sulfate for their survival in host mosquitoes. It has been also observed that heparin lyase can significantly inhibit the binding of dengue virus and yellow fever virus by degrading the heparin sulfate (ref). Having a potential of degrading heparan sulfate, this bacterium may (may have role to play in determining) play an important part in refractiveness of these mosquitoes to dengue virus and Plasmodium, also may be utilized in developing a novel strategy to control many insect transmitted diseases.
Jathinobacterium is (which was) commonly isolated from rivers, lakes and springs (Ref) and it can also (was also reported to cause) cause opportunistic infections (ref). This bacterium has also been reported from midgut of eonymph of saw fly (ref). Janthinobacterium produces a water insoluble pigment violescin, which has been reported to show anti-bacterial, anti-viral, anti-leishmanial and anti-tumoural activity (ref) and this might help in developing immunity of mosquitoes to pathogens. (Here we hypothesize that the presence of these two bactetria) So presence of these two above mentioned bacteria in refractory MOYO-R strain might be co linked with the refractive ness of these mosquitoes towards dengue virus and Plasmodium because of their unique physiological properties.
In MOYO-S strain, the significance was the presence of Bacillus sp. and absence of Enterobacteria. An intermediate display of above two strains was seen in MOYO-D strain with presence of Enterobacteria and absence of Pedobacter, Janthinobacterium and Bacillus sp.
In our study, phylogenetic analysis showed a very low chao1 estimate in all the six clone libraries predicting a very low microbial diversity in Aedes aegypti midgut environment. Rarefaction curve analysis also showed that Clone libraries were completely saturated. Some previous studies also reported a very low midgut bacterial diversity in these mosquitoes (ref) suggesting a very stringent mosquito midgut environment
Why RT PCR?
(For an accurate estimation of total no of bacteria, it was postulated that DNA standard in Real Time PCR study should be constructed with the (DNA from / of the bacteria) using the bacteria which predominate the given habitat. In order to minimize the variations caused by differences in 16S rDNA copy No and effect of generation time of the bacteria during estimation of total no of bacteria by in Real Time PCR study, we also used purified DNA of Pseudomonas aeruginosa, a dominant bacterium in clone library study for generating standard.)
As it has been postulated that DNA standard in Real Time PCR study should be constructed with the (DNA from / of the bacteria) using the bacteria which predominate the given habitat for an accurate estimation of total no of bacteria to minimize the variations caused by difference in 16S rDNA copy no and effect of generation time of the bacteria, so we have taken Pseudomonas aeruginosa purified DNA for generating standard because it was the dominant bacterium in clone library study.
Real Time PCR based Eubacterial quantification showed that no. of 16S rRNA gene copies were more in MOYO-R strain (than) followed by MOYO-D and MOYO-S strains. To our best knowledge, this is the first attempt to quantify the mosquito midgut bacteria using Real Time Taqman PCR based technology. To confirm the clone library results, we have rechecked the presence of Pedobacter bacteria with Real Time Taqman PCR approach and confirmed its presence only in MOYO-R strain. We also confirmed the exact no of these bacteria in mosquito midgut and found that … percent of total bacteria are Pedobacter. This result is contrasting to the results obtained in clone library approach where only … percent clones were affiliated to Pedobacter but as chances of bias are more associated with clone library approach, also as Real Time PCR gives more exact enumeration, we can rely on the results obtained with Real Time PCR based quantification. This is also the first report of quantification of these bacteria with Real Time PCR approach with highly specific Taqman probe and as these bacteria are used for commercial production of heparinase enzyme, this report may prove valuable in this aspect also.
As the condition of mosquito borne diseases is getting worsen, there is an urgent need to explore alternative strategies like genetic modification of mosquitoes and paratransgenesis approach to combat with this drastic situation. Therefore, now a days there is increased attention for the study of endogenous microbiota of mosquito midgut to search for potential bacteria or potential molecule for paratransgenesis studies. In a study it was reported that Asaia bacteria can stably associate with the Anopheles stephensi mosquitoes, therefore can be a suitable candidate for paratransgenesis approach for expressing potential anti parasite molecule (ref). Along with it recent studies on mosquito midgut bacteria also shows increasing attention of the scientific community in this field (ref) and our study will provide one additional step in fight against mosquito borne diseases.
So it can be concluded that our study provided identification of some potential bacteria which might be related with infectivity of dengue virus and therefore might be an important part in refractoriness and susceptibility of mosquitoes to dengue virus. The present findings might contribute to the identification of new targets for anti dengue strategies. Whether the differences in midgut bacterial communities reported here will be maintained between Aedes aegypti strains in other habitats and will eventually be correlated with vector competence, requires further experimentation.
New papers reference
Moyo-s moyo-r
Different use-para,n all with review
Real time insertion everywhere
Discussion gud start
in this paper we report our findings on bacterial richness and diversity associated with three different A. aegypti strains with differential ability to transmit dengue virus.
Tree interpretation
Real time
Moyo s and moyo R
New paper
