Leishmania donovani is considered an anthroponosis; however, Leishmania-infected animals have been increasingly reported in L. donovani foci, and the role of these animals as reservoirs for human L. donovani infection remains unclear.Human visceral leishmaniasis caused by L. donovani foci in northwestern Ethiopia. Domestic animals were screened for Leishmania DNA and for anti-L. donovani IgG. Serum anti-sand fly saliva antibodies were used as a marker of exposure to the vector sand fly, Phlebotomus orientalis.We conducted a study of domestic animals  in three Leishmania DNA, with positive animals identified among all species studied. Sequencing indicated that the animals were infected with parasites of the L. donovani complex but could not distinguish between L. infantum and L. donovani. A total of 18.9 % of the animals were seropositive for anti-L. donovani IgG, and 23.1 % of the animals were seropositive for anti-P. orientalis saliva IgG, with the highest seroprevalence observed in dogs and sheep. A positive correlation was found between anti-P. orientalis saliva and anti-L. donovani IgGs in cows, goats, and sheep.Of 546 animals tested, 32 (5.9 %) were positive for L. donovani complex DNA in the blood of domestic animals, the reported seroprevalence to the L. donovani antigen, and the widespread exposure to sand fly saliva among domestic animals indicate that they are frequently exposed to Leishmania infection and are likely to participate in the epidemiology of Leishmania infection, either as potential blood sources for sand flies or possibly as parasite hosts.The detection of The online version of this article (doi:10.1186/s13071-015-0976-1) contains supplementary material, which is available to authorized users. Leishmania (Kinetoplastida: Trypanosomatidae), is a neglected tropical and subtropical disease endemic to 98 countries worldwide. In East Africa, life-threatening human visceral leishmaniasis (VL) is caused by Leishmania donovani and primarily affects the poor due to the lack of preventive measures and reduced access to health care facilities [Leishmaniasis, a protozoan disease that is transmitted by sand flies (Diptera: Phlebotominae) and caused by parasites of the genus cilities .L. donovani is believed to be an anthroponosis. However, in Latin America and the Mediterranean Basin, the closely related species L. infantum causes a zoonosis for which canids are the main reservoirs [Leishmania-infected animals have been previously reported in L. donovani foci, including wild and domestic animals [L. donovani VL remains unclear and requires further examination.The optimal strategy for controlling this disease depends on understanding the epidemiology of VL, including its local transmission cycles. Leishmaniasis caused by servoirs . Control animals –5. HowevLeishmania infections in domestic animals in three VL foci in northwestern Ethiopia. Domestic animals were screened for Leishmania DNA and anti-L. donovani IgG in their peripheral blood to detect infection and exposure to Leishmania, respectively. Additionally, anti-sand fly saliva antibodies were used as a marker of exposure [Phlebotomus orientalis, the suspected vector of L. donovani in northwestern Ethiopia [Our study focused on the detection of exposure  to PhlebEthiopia , 8. The Animal blood and serum samples were collected in Addis Zemen, Humera, and Sheraro, three localities in northwestern Ethiopia endemic to human VL. In the Humera district (Tigray region), several outbreaks of VL have been recorded since 1970. Addis Zemen (Amhara region) and Sheraro (Tigray region) are sustained VL foci characterized by a local transmission cycle supported by migrant agricultural laborers returning from Humera [Animal surveys were conducted during two field studies. In October 2010, 266 samples were collected in Addis Zemen and Sheraro, and in November 2010, an additional 280 samples were obtained in Humera , under approval no. 3.10/3398/04. Consent was obtained from the owners of the domestic animals for the collection of blood samples by a veterinarian. International animal experimentation guidelines were followed.Leishmania spp. infection via kDNA real-time PCR as previously described [Leishmania internal transcribed spacer 1 (ITS1) real-time PCR and high-resolution melt analysis (ITS1-HRM PCR) [L. infantum (MCAN/IL/2002/Skoshi), L. tropica (MHOM/IL/2005/ LRC-L1239), and L. major (MHOM/TM/1973/5ASKH) promastigotes. The negative controls included blood samples obtained from five Israeli dogs that had tested negative for Leishmania by PCR. All positive PCR products were submitted for DNA sequencing to the Center for Genomic Technologies at the Hebrew University of Jerusalem. The derived DNA sequences were compared with sequences in GenBank using the NCBI BLAST program (www.ncbi.nlm.nih.gov/BLAST). The percentage of positive animals for each species was calculated based on positive kDNA PCR results followed by sequencing. Samples were considered positive for Leishmania only if their kDNA sequence demonstrated the closest BLAST match to Leishmania and was at least 80 % identical. A species was considered to be identified only when its ITS1 sequence shared 99 to 100 % identity with an existing GenBank sequence.DNA was extracted from whole blood using the guanidine thiocyanate technique . DNA wasescribed , 11. SamHRM PCR) . SamplesHRM PCR) . All samL. infantum and L. donovani infections [L. donovani complex were further evaluated using conventional PCR to determine the species. Two independent PCR assays were carried out to amplify fragments of the Leishmania cysteine protease B (CPB) gene [As ITS1-HRM PCR does not discriminate between fections , samplesPB) gene , 15. FurPB) gene . The samwww.ebi.ac.uk/tools/msa/kalign/) and BioEdit softwares. Only well-defined ITS sequences that were unambiguously assigned to the species L. donovani or L. infantum were downloaded from the GenBank database and used in the analysis  were coated with L. donovani promastigotes  in 20 mM carbonate-bicarbonate buffer (pH 9.25) overnight at 4 °C and incubated with 6 % blocking solution for 60 min at 37 °C. Serum samples were diluted in 2 % blocking solution and incubated in duplicate for 60 min at 37 °C. Thereafter, peroxidase-conjugated secondary antibodies were added, followed by 45 min of incubation at 37 °C. For details on the blocking solutions, sample dilutions, and conjugates employed in these assays, see Additional file An ELISA was used to measure specific anti-L. donovani served as positive controls. Negative serum samples were obtained from healthy cattle (n = 33), horses , goats (n = 21), and sheep (n = 32) from the Czech Republic, which is a sand fly- and Leishmania-free country. Canine-negative (n = 15) and canine-positive (n = 2) control sera were obtained during a previous study [Leishmania or from Leishmania-positive dogs, respectively.Hyperimmune sera from laboratory-bred mice experimentally infected with us study  from labP. orientalis, anti-saliva IgG antibodies were measured via ELISA. The same protocol applied for anti-Leishmania donovani IgG was used, with the following modifications: wells were coated with a salivary gland homogenate , and seP. orientalis salivary gland homogenate with IgG antibodies against the saliva of other sand fly species, sera from mice and dogs that were experimentally exposed to a single sand fly species were used. Canine sera positive for anti-P. perniciosus and anti-L. longipalpis IgG antibodies were available from previous experiments in laboratory-bred beagles exposed solely to P. perniciosus [L. longipalpis [L. infantum. The ELISA protocol described in Additional file P. orientalis (Ethiopia), P. papatasi (Turkey), P. duboscqi , P. arabicus (Israel), or Sergentomyia schwetzi (Ethiopia). The experiments were approved by the Committee on the Ethics of Animal Experiments of Charles University in Prague (Permit Number: 24773/2008-10001) and were performed under a Certificate of Competency (Registration Number: CZU 934/05), in accordance with an Examination Order approved by the Central Commission for Animal Welfare of the Czech Republic.To assess the possible cross-reactivity of niciosus  and L. lgipalpis , respectFor seroprevalence, cut-off values were calculated by the addition of three standard deviations to the mean optical density (OD) of the control sera. The differences in antibody levels between localities were analyzed using the nonparametric Wilcoxon Rank-Sum Test for Differences in Medians. Spearman’s rank correlation matrix was used to assess the correlation between the variables. Statistical analyses were performed using NCSS 6.0.21 software, and the p-value was set at 0.05.Leishmania DNA detected via PCR was 5.9 % (32/546)  were found in Humera, with the highest prevalence observed in cows (18.8 %). At the other localities, only one donkey in Sheraro and one dog in Addis Zemen were found to be positive for Leishmania  shares 100 % identity with sequences from both L. infantum and L. donovani with 100 % coverage, and its phylogeny did not permit discrimination between these two closely related species   Table . Across 18.9 % 10/546   higher anti-als Fig. . Geograpity Fig. .P. orientalis saliva antibodies we used sera from dogs and mice that had been experimentally exposed to a single sand fly species. In dogs, the reactivity of anti-P. perniciosus and anti-Lutzomyia longipalpis sera against P. orientalis salivary gland homogenate (SGH) was similar to that for sera from non-exposed dogs , goats , and sheep  L. donovani has not yet been identified. However, Phlebotomus orientalis is the most probable vector given that it has been found to be infected with L. donovani in nearby Sudanese foci [Leishmania species has been demonstrated experimentally [P. orientalis was assessed using anti-sand fly saliva antibodies as a marker [P. orientalis [L. donovani. Feeding preferences, together with other ecological constraints such as the localization of vector breeding sites [Leishmania infection [In northwestern Ethiopia, the sand fly vector species of ese foci  and its mentally . Exposura marker . Anti-saientalis , thus meng sites  or vectonfection  may helpLeishmania DNA in animal peripheral blood and Leishmania seropositivity serve as reliable epidemiological markers for assessing infection. PCR positivity indicates the presence of the parasite [Leishmania is highly probable given that its DNA degrades shortly after parasite death [Leishmania infection [Leishmania-positive animals were found in Humera, indicating dynamic transmission to domestic animals in this well-known active focus. However, many L. donovani-seropositive animals were found in all the three surveyed localities, suggesting that exposure to Leishmania parasites also occurred in the foci of Addis Zemen and Sheraro.The presence of parasite , 26. Altte death . Seroposnfection . The majL. donovani and L. infantum is notoriously difficult [L. donovani complex in East Africa is controversial; strains that were previously split into L. donovani, L. archibaldi or L. infantum have now been classified into one group: L. donovani s.s. [The fact that only one-third of the PCR-positive animals were positive for both kDNA and ITS1-HRM PCR, is not surprising because the ITS1 region has a considerably lower copy number , 12. Dueifficult . Moreoveani s.s. .L. donovani are dogs, which are known to play a key role as reservoir hosts in the transmission cycle of the closely related L. infantum [L. donovani foci [Leishmania seroprevalence out of all the species tested at all study sites, with two PCR-positive dogs identified in Humera and Addis Zemen. As a suspected reservoir species, dogs are also highly attractive to the vector [P. orientalis antibodies among the tested animal species. Most importantly, the same Leishmania strains have been recovered from dogs and VL patients [L. donovani.The most suspected animal reservoirs for infantum , 30. Sevani foci , 31–35, ani foci –39. In tpatients , 19 and patients , 37, 39,Leishmania-positive animals have also been found to be seropositive, indicating these domestic animals  as putative host species in local VL foci. Nevertheless, it is important to mention that neither PCR-positivity nor seropositivity indicates that an animal is able to maintain the parasite for a long period of time. This must be primarily demonstrated by the follow-up of infected animals. Several studies of naturally or experimentally infected non-canine domestic animals have demonstrated their different capabilities to maintain Leishmania infection. Cerqueira et al. [L. chagasi (syn. L. infantum). These donkeys remained seropositive until the end of the study, which lasted 12 months; however, the donkeys were able to overcome the infection and failed to infect the vector [et al. indicated that Leishmania infection in goats can persist for at least seven months [L. donovani infection in sheep is likely time-limited because only one out of six experimentally infected sheep was shown to develop measurable amounts of anti-L. donovani antibodies and the transient presence of amastigotes in sampled tissue in a study that included 244 days of monitoring [L. donovani IgG antibodies among Leishmania-positive sheep (Additional files Almost 38 % of a et al.  experimee vector . A PCR sn months . On the nitoring . Thus, tLeishmania while PCR-negative in the blood, and, on the other hand, that out of 32 PCR-positive animals, 20 animals were seronegative, could be explained by several possible mechanisms. Seropositivity and PCR-negativity might be attributable to infection in hosts that have resolved the infection but retain high titers of specific antibodies [Leishmania antigens, as found in some asymptomatic hosts [The fact that many animals were seropositive for tibodies , 43. Anotibodies . The revtibodies , or due ic hosts .L. donovani is still unclear. The present study is the first to report PCR-positive cattle, donkeys, goats, and sheep in Ethiopia. These animals, especially cattle, serve as sources of blood for L. donovani vectors [The role of other domestic animals as hosts or potential reservoirs for  vectors , 46. Eve vectors , 37, 47. vectors .Leishmania reservoir [In addition to the maintenance of persistent infection, the transmissibility competence, e.g. infectivity for the sand fly vector, is an important prerequisite for any mammal to serve as a eservoir , 49. Theeservoir . ValidatL. donovani is traditionally considered to be an anthroponosis in East Africa. However, the present study revealed widespread exposure to L. donovani and sand fly vector bites among domestic animals. The possible involvement of domestic animals as sources of blood for vector sand flies should therefore be considered in VL control strategies. However, the direct involvement of domestic animals in the transmission cycle of L. donovani warrants further investigation, most importantly by xenodiagnosis to determine their transmissibility competence.In conclusion, leishmaniasis caused by


Plasmodium vivax will be an essential component of any comprehensive malaria elimination program, but major gaps in our understanding of P. vivax biology, including the protein-protein interactions that mediate merozoite invasion of reticulocytes, hinder the search for candidate antigens. Only one ligand-receptor interaction has been identified, that between P. vivax Duffy Binding Protein (PvDBP) and the erythrocyte Duffy Antigen Receptor for Chemokines (DARC), and strain-specific immune responses to PvDBP make it a complex vaccine target. To broaden the repertoire of potential P. vivax merozoite-stage vaccine targets, we exploited a recent breakthrough in expressing full-length ectodomains of Plasmodium proteins in a functionally-active form in mammalian cells and initiated a large-scale study of P. vivax merozoite proteins that are potentially involved in reticulocyte binding and invasion.A vaccine targeting P. vivax proteins that are predicted to localize to the merozoite surface or invasive secretory organelles, some of which show homology to P. falciparum vaccine candidates. Of these, we were able to express 37 full-length protein ectodomains in a mammalian expression system, which has been previously used to express P. falciparum invasion ligands such as PfRH5. To establish whether the expressed proteins were correctly folded, we assessed whether they were recognized by antibodies from Cambodian patients with acute vivax malaria. IgG from these samples showed at least a two-fold change in reactivity over naïve controls in 27 of 34 antigens tested, and the majority showed heat-labile IgG immunoreactivity, suggesting the presence of conformation-sensitive epitopes and native tertiary protein structures. Using a method specifically designed to detect low-affinity, extracellular protein-protein interactions, we confirmed a predicted interaction between P. vivax 6-cysteine proteins P12 and P41, further suggesting that the proteins are natively folded and functional. This screen also identified two novel protein-protein interactions, between P12 and PVX_110945, and between MSP3.10 and MSP7.1, the latter of which was confirmed by surface plasmon resonance.We selected 39 P. vivax ectodomains, established that the majority of them contain tertiary structure, and used them to identify predicted and novel protein-protein interactions. As well as identifying new interactions for further biological studies, this library will be useful in identifying P. vivax proteins with vaccine potential, and studying P. vivax malaria pathogenesis and immunity.We produced a new library of recombinant full-length NCT00663546ClinicalTrials.gov  Plasmodium vivax causes malaria in millions of people each year, primarily in Southeast Asia and Central and South America. P. vivax has a dormant liver stage, which can lead to disease recurrence in infected individuals even in the absence of mosquito transmission. The development of vaccines that target blood-stage P. vivax parasites is therefore likely to be an essential component of any worldwide effort to eradicate malaria. Studying P. vivax is very difficult as this parasite grows poorly in the laboratory and invades only small numbers of young red blood cells in patients. Due to these and other challenges, only a handful of P. vivax proteins have been tested as potential vaccines. To generate more vaccine candidates, we expressed the entire ectodomains of 37 proteins that are predicted to be involved in P. vivax invasion of red blood cells. Antibodies from Cambodian patients with P. vivax malaria recognized heat-sensitive epitopes in the majority of these proteins, suggesting that they are natively folded. We also used the proteins to screen for both predicted and novel protein-protein interactions, confirming that the proteins are functional and further supporting their potential as vaccine candidates. As a new community resource, this P. vivax recombinant protein library will facilitate future studies of P. vivax pathogenesis and immunity, and greatly expands the list of candidate vaccine antigens. Plasmodium vivax causes an estimated 70–391 million cases of malaria each year  and non-reducing conditions, and stained [SimplyBlue SafeStain (Invitrogen)] with an increase in molecular mass upon reduction suggesting the proteins contain disulphide bonds, as expected. No homodimer or oligomers are observed by SDS-PAGE. Samples run under native conditions , support the presence of homodimers for P12 and P41 with bands near 146 kDa. Monomers for these correspond to 60 and 66 kDa respectively. MSP7.1 shows a faint band near the expected monomer size of 69 kDa and a dominant band near 242 kDa, suggesting that this protein forms an oligomer of potentially three or four monomers.Recombinant his-tagged (TIF)Click here for additional data file.S2 FigP. vivax P12 (A) or P. vivax P41 (B) were injected over immobilized biotinylated P. vivax P12 (A) or P. vivax P41 (B) with no interactions observed.Increasing concentrations of (TIF)Click here for additional data file.S3 FigP. vivax P12 were injected over immobilized biotinylated P. vivax PVX_110945 with weak binding observed. None of the concentrations used reached equilibrium, which prevented the calculation of an equilibrium dissociation constant (KD).Increasing concentrations of (TIF)Click here for additional data file.


Cryptococcus neoformans is an opportunistic human pathogenic fungus that causes meningoencephalitis. Due to the increasing global risk of cryptococcosis and the emergence of drug-resistant strains, the development of predictive genetics platforms for the rapid identification of novel genes governing pathogenicity and drug resistance of C. neoformans is imperative. The analysis of functional genomics data and genome-scale mutant libraries may facilitate the genetic dissection of such complex phenotypes but with limited efficiency. Here, we present a genome-scale co-functional network for C. neoformans, CryptoNet, which covers ~81% of the coding genome and provides an efficient intermediary between functional genomics data and reverse-genetics resources for the genetic dissection of C. neoformans phenotypes. CryptoNet is the first genome-scale co-functional network for any fungal pathogen. CryptoNet effectively identified novel genes for pathogenicity and drug resistance using guilt-by-association and context-associated hub algorithms. CryptoNet is also the first genome-scale co-functional network for fungi in the basidiomycota phylum, as Saccharomyces cerevisiae belongs to the ascomycota phylum. CryptoNet may therefore provide insights into pathway evolution between two distinct phyla of the fungal kingdom. The CryptoNet web server (www.inetbio.org/cryptonet) is a public resource that provides an interactive environment of network-assisted predictive genetics for C. neoformans. C. neoformans var. grubii (serotype A) and C. neoformans var. neoformans (serotype D) generally cause fatal meningoencephalitis in immunocompromised patients such as HIV/AIDS patients. In contrast, Cryptococcus gattii  affects immunocompetent individuals3C. neoformans remain elusive. C. neoformans requires a high level of integrity of its complex pathways to successfully infect the cells of a human host. A reconstruction of the pathways of pathogenicity and drug resistance in C. neoformans may provide new insights into antifungal treatments.CC. neoformans genes became available, and was used to identify novel genes relevant to virulenceC. neoformans genome. The construction of mutant strains for the remaining genes and the testing for each virulence-related phenotype would be prohibitively expensive and time-consuming.Systematic tools that accelerate the discovery of new genes for pathogenicity and drug resistance are needed to meet the urgent demand for new anticryptococcal treatments. Gene expression signatures from microarray or RNA-seq experiments have proven useful to investigate pathways that modulate pathogenicity and drug susceptibility67Saccharomyces cerevisiae, using a genome-scale co-functional gene network, YeastNet13C. neoformans, provided that a high quality co-functional gene network becomes available.Neither functional genomics data nor reverse genetics resources alone, therefore, can meet the current demand for efficient genetic dissection. Recently, several studies have suggested the use of gene networks as bridges between these two research resources. Co-functional gene networks have been shown to be effective in gene-to-phenotype mapping1011Candida albicans has been reported21Although a few molecular networks for pathogenic fungi have been reported over the past several years, these networks were either small protein-protein interaction networks151618C. neoformans, CryptoNet (www.inetbio.org/cryptonet), which was constructed by integrating 14 distinct types of large-scale data and covers ~81% of the coding genome. We find that CryptoNet is highly predictive for known C. neoformans virulence genes, and apply it to predict novel genes involved in virulence and drug response. Our results expand our view of the pathways relevant to fungal pathogenicity and drug resistance, which potentially may lead to the development of novel therapeutic targets.In this work, we report a genome-scale co-functional network for C. neoformans gene network are summarized in C. neoformans genes . Log likelihood scores (LLS) based on a Bayesian statistical framework and weighted sum (WS) methods were used to integrate diverse types of data derived from three different species ; these methods were used previously in the construction of the co-functional network for S. cerevisiaeThe network construction and network-assisted predictions for the C. neoformans data: the probability of co-citation in Medline articles (CC), co-expression patterns across microarray samples (CX), the co-occurrence of protein domains (DC), phylogenetic profile similarity (PG), and orthologous gene neighborhoods in bacterial genomes (GN). In addition, orthologous functional associations S. cerevisiaeH. sapiensC. neoformans genes have orthologs in either S. cerevisiae or H. sapiens. A total of 14 networks derived from C. neoformans specific data and orthology-based transfer data were integrated into a single network of C. neoformans genes, CryptoNet, which maps 156,506 co-functional links among 5,649 genes . More details about the network construction are described in the www.inetbio.org/cryptonet/). The CryptoNet web server also provides three network search options for hypothesis generation  ‘Infer functions from network neighbors'; and iii) ‘Find context-associated hub genes'. All predictions made in this study were generated by the network search tools from the CryptoNet web server.Co-functional associations between two genes were inferred from five distinct types of neration : i) ‘FinS. cerevisiae and H. sapiens data. To examine the extent to which genomic information derived from C. neoformans contributes to the quality of the integrated network, we divided the CryptoNet links into three species-associated networks, and then measured the accuracy of each network and their intersections using the percentage of gene pairs that share KEGG annotations  and species-specific links . The observed hierarchy of network accuracy indicates that the integration of co-functional links derived from multiple species generally improves the quality of the C. neoformans gene network.Many co-functional links in CryptoNet were derived from otations . The mosC. neoformans pathogenicity. The assessment of predictive power requires known pathogenicity genes. We collected 73 virulence genes for three different virulence phenotypes from the literature: 28, 23, and 22 genes for capsule formation, melanin production, and thermotolerance, respectively  and CryptoNet. We found significantly reduced AUC scores for the YeastNet-derived network: 0.7598, 0.8201, and 0.8068 for capsule formation, melanin production, and thermotolerance, respectively , CNAG_05563 (HOS2), and CNAG_06086 . Although all four of these mutant strains exhibited increased capsule production compared with the parental CMO18 strain in our study (ssn3Δ and kin1Δ mutant strains were tested but reported not to enhance capsule production in Madhani's studyNRG1) and CNAG_06730 (GSK3), were reported to regulate capsulate production in Madhani's study, but were not testable in our study due to a failure in strain recovery. Another independent study has reported that NRG1 is involved in capsule formationCapsule production assays validated four of the 40 tested strains by either Madhani's study or our study , CNAG_03290, SSN3, and KIN1 showed altered levels of melanin production compared with the CMO18 strain (CDC2801), CNAG_00556 (CCK1), and CNAG_04837 (MLN1).We next validated four of the 36 tested strains for melanin production by either study , CNAG_03409 (SKN7), CNAG_03967 , CNAG_04118 (CTK1), CNAG_04282 (MPK2), and CNAG_03811. Five of the 10 tested strains, CNAG_02675 , PDK1, CNAG_03290, CNAG_05558 (KIN4), and CNAG_06845 , were also reported in Madhani's study to show grow defects at 37°CSNF1) and CCK1 in thermotolerance have been reported in the serotype D JEC21 and the serotype A H99 strain backgrounds, respectively2728CDC2801, CNAG_06697 , and KIN1   exhibited increased virulence at 30°C but not at 37°C  . Among t at 37°C , which r at 37°C . These r at 37°C . Notably at 37°C  and indu at 37°C .KIN1, CNAG_05499  and CNAG_04678 (YPK1), that were predicted by CryptoNet to be involved in virulence factor formation, and tested their in vitro and in vivo phenotypes. First, we constructed two independent kin1Δ mutants in the serotype A H99S strain to confirm the altered virulence factor formations in the kin1Δ mutant on a CMO18 background. CMO18  is an attenuated lab passaged derivative of H99Skin1Δ mutants displayed increased thermosensitivity and enhanced melanin, which is in accordance with the phenotypes observed in the CMO18 kin1Δ mutant  or conserved links. This result clearly demonstrates the importance of species-specific data for the prediction of C. neoformans pathogenicity.In the tests described above, network-assisted prediction achieved discovery rates of 10% (4 of 40), 11.11% (4 of 36), and 27.03% (10 of 37) for capsule formation, melanin production, and thermotolerance, respectively. Madhani's study previously tested 1,093 of the 1,201 deletion strains for the three virulence phenotypes and reported 16, 40, and 104 validated genes for capsule formation, melanin production, and thermotolerance, respectively . Our network-assisted genetic screen, therefore, identified genes for three virulence phenotypes with ~6.8-, ~3-, and ~2.8-fold enrichment over Madhani's screen for capsule formation, melanin production, and thermotolerance genes, respectively . Notably, 12 of the 29 identified genes (~41%) for the C. neoformans genes that change their expression levels during the early adaptation to drug stress may provide clues about the mode of action of antifungal drugs and cellular strategies to overcome drug stress. A majority of signature genes result, however, from indirect effects of target pathway perturbations. The identification of genes that directly contribute to the drug response from gene expression data is hampered, therefore, by confounding signals from indirect effects. Moreover, many cellular processes, including drug resistance, may be regulated by post-transcriptional mechanisms. We therefore require a new search algorithm for drug resistance genes to complement the gene expression-based approach.One major reason that cryptococcosis treatment fails is antifungal drug resistance. The emerging complexity of the drug-mediated cell death process suggests that a group of drug resistance genes collaborate to overcome drug stress. The elucidation of the complete set of drug resistance genes may therefore facilitate the development of more efficacious antifungal treatments. Some C. neoformans genes that exhibited >2-fold up-regulation upon treatment with fluconazoleFind context-associated hub genes' search option on the CryptoNet web server. Using this search option, we found that 94 of the 2,135 hub genes were significantly associated with a resistance to fluconazole treatment . We found that 16 of these 94 candidates genes are known to be involved in the ergosterol pathway , CNAG_05538 (JJJ1), CNAG_00711, and CNAG_00869 (PDR5), exhibited an increased resistance or sensitivity to the azole drugs and amphotericin B compared with the wild-type strain , the deletion of which increases sensitivity to both amphotericin B and fluconazoleWe tested 11 candidate genes for fluconazole resistance that have available mutant strains in the Madhani collection see . We alsoC. neoformans genes are not yet annotated into pathways, we employed the Gene Ontology biological process (GOBP) annotations for the S. cerevisiae orthologs. We found that four GOBP terms relevant to drug response or ergosterol biosynthesis were significantly enriched: sterol biosynthetic process (GO:0016126), response to drug (GO:0042493), transmembrane transport (GO:0055085), and cell wall organization and biogenesis (GO:0071554) . CryptoNet reveals a modular organization of the pathways to which the drug resistance genes are highly connected . Previous studies have shown that mitochondrial dysfunction related to energy generation is required for azole susceptibility in C. albicansC. glabrata strainC. neoformans.CryptoNet can also provide insights into the pathways that underlie drug resistance. The validated drug resistance genes may be connected to other genes in relevant pathways. We therefore measured the enrichment of pathway annotations related to drug resistance among their network neighbors. Given that the majority of the onnected . Notablyors' see  from theors' see . The topC. neoformans. Network-assisted gene prioritization requires two technical components. First is the need to construct a highly accurate and comprehensive gene network for the target species. We have demonstrated that heterogeneous genomics data can be effectively integrated into a single gene network for C. neoformans. CryptoNet is distinct from the previously constructed fungal networks from several clinical and evolutionary perspectives. CryptoNet is the first genome-scale co-functional network for a fungal pathogen and covers ~81% of the coding genome. In our study, we demonstrated the power of CryptoNet in the study of pathogenicity and drug resistance, which are major challenges in the development of medicine for infectious diseases. CryptoNet is also the first genome-scale co-functional network for a fungal species in the basidiomycota phylum, as S. cerevisiae belongs to the ascomycota phylum. Given the evolutionary distance between C. neoformans and S. cerevisiae, the orthology-based network from YeastNet makes a limited contribution to the C. neoformans gene network  and S. cerevisiae GO annotations, which are described in the The templates for the genome sequence and functional annotations employed in this study are Network benchmarking and data integration using Bayesian data integration were performed as previously describedKIN1), CNAG_04678 (YPK1), or CNAG_05499 (ScSHO1)] were deleted in the H99S serotype A strain. For more details, see the The selected genes [CNAG_01938 . Y.B. supervised the experimental analysis. I.L. supervised the modeling and bioinformatics analysis. H.K., K.J., Y.B., J.H. and I.L. edited the manuscript.H.K., K.J., Y.B. and I.L. conceived the project and wrote the manuscript. H.K. constructed and analyzed the network model. K.J. performed the experiments with the assistance of T.K. and S.M. Y.-L.C. performed the animal studies. J.H. supervised the animal studies. J.S., J.E.S. and S.H. contributed to the data analysis pipeline developments. G.J. constructed the coding genome for Supplementary Materials and Methods


We used differential evolution Markov-chain Monte Carlo (MCMC) to estimate the parameters of a large-scale spiking neural circuit simulation to fit the slice data [disinhibiting the circuit. 5 ms later, the excitatory neurons near the center receive input from excitatory neurons near the middle (i.e. recurrent activity), suggesting that cause for the super-linearity.The superior colliculus (SC) is a midbrain region with visually responsive neurons in the superficial layers and eye movement controlling neurons in the deeper layers. Recently,  performeC Figure . The expC Figure , a+b. Weice data . The modice data . However


Nezara viridula , considered a major pest, was present in 23% of all faecal samples collected. Results also showed that resource partitioning in this system is low, as most bird species shared large proportion of their diets by feeding primarily on lepidopteran, dipteran and arachnids. Dietary composition differed between some species, most likely because of differences in foraging behaviour. Overall, this study reached a level of taxonomic resolution never achieved before in the studied species, thus contributing to a significant improvement in the avian ecological knowledge. Our results showed that bird communities prey upon economically important pests in macadamia orchards. This study set a precedent by exploring avian pest-reduction services using next-generation sequencing, which could contribute to the conservation of avian communities and their natural habitats in agricultural systems.Worldwide, avian communities inhabiting agro-ecosystems are threatened as a consequence of agricultural intensification. Unravelling their ecological role is essential to focus conservation efforts. Dietary analysis can elucidate bird-insect interactions and expose avian pest-reduction services, thus supporting avian conservation. In this study, we used next-generation sequencing to analyse the dietary arthropod contents of 11 sympatric bird species foraging in macadamia orchards in eastern Australia. Across all species and based on arthropod DNA sequence similarities ≥98% with records in the Barcode of Life Database, 257 operational taxonomy units were assigned to 8 orders, 40 families, 90 genera and 89 species. These taxa included 15 insect pests, 5 of which were macadamia pests. Among the latter group,  Birds are one of the most studied groups of animals on the planet because of their importance as bioindicators, providers of ecosystem services and high visibility , 2. NatiMany studies across several crop systems have highlighted the positive contribution of birds to agriculture by reducing insect pest numbers , 5. AlthTraditionally, dietary studies have been conducted using techniques such as the observation of arthropod fragments in faecal samples e.g. , 12). Ot. Ot12]).The development of next-generation sequencing (NGS) has allowed the simultaneous sequencing of millions of amplified DNA templates , represeAmblypelta lutescens lutescens  and Nezara viridula  are considered major macadamia pests [In Australia, the value that insectivorous birds may have in agriculture has not been extensively researched . Most ofia pests . The mosia pests . Alternaia pests . Thus, aIn this study, we used NGS to explore pest-reduction services of birds foraging in macadamia orchards adjacent to riparian habitats in Bundaberg, Australia. Our aims were to: 1) elucidate whether the NGS techniques and protocols, using the Illumina Miseq platform, can be used to study the diet of birds, 2) describe whether bird species in macadamia orchards are feeding on insect pests, and 3) understand resource utilization in the avian community through within- and between-species dietary comparisons.Eucalyptus tereticornis (forest red gum), a number of Melaleuca (tea tree) species, often including M. viminalis (weeping bottlebrush) and M. bracteata (black tea tree), Casuarina cunninghamiana (creek oak), and Waterhousea floribunda (weeping myrtle). The region has a characteristic sub-tropical climate, with hot summers and mild winters, and annual average maximum temperatures of 26.7°C  [We carried out this study in the Bundaberg region in eastern Australia. Sample collection was conducted in a macadamia orchard , located approximately 25 km south of Bundaberg. This orchard was adjacent to a strip of riparian fringing forest, a situation common to most orchards in the region. Riparian habitats in this region are generally composed of a number of tree species, including 59–2015) .Birds were captured at the study site during 4 weeks in November of 2014. The study was timed to coincide with period when the population of major macadamia pests is highest, corresponding to the early nut development stage in the crop. Six mist-nets (approximately 3 × 9 m) were set each day and attended continuously from sunrise till mid-day and from late afternoon till sunset. To maximize the capture probability, mist-nets were evenly distributed throughout the orchard, parallel to the macadamia rows, and their positions were changed every week. Each trapped individual was carefully removed from the mist-net, identified, and then placed individually in a sterile cloth bag for 30 minutes. After this time, the bird was released, and faecal material in the cloth bag collected using a new, sterilized plastic spoon for each faecal sample. Cloth bags were used only once before soaking in hypochlorite solution for 24 hours, washing thoroughly and sterilising for the next use. Each sample was transferred to a 5 ml plastic tube filled with 95% ethanol and immediately placed in a refrigerated container in order to preserve DNA integrity . At the Animal ethics approvals were obtained from the Animal Ethics Committee of Central Queensland University (project no. A14/05-310) and the Department of Environment and Heritage Protection of the Government of Queensland (permit no. WISP14680814).A. lutescens lutescens and sweep-net samplings were performed over the macadamia tree canopies to capture N. viridula.Parallel to the bird trapping, we used two sampling methods to monitor the presence and collect individuals from two of the major macadamia insect pests. Pheromone traps were used to capture Prior to weighing the faecal samples, seeds, fruits and feathers were removed. This cleaning process was performed on one sample at a time to avoid DNA degradation due to an increase in temperature . Wet weiet al. [A. lutescens lutescens and N. viridula, using DNeasy Blood and Tissue Kit , using legs from individuals of each species trapped during the insect pest surveys. Negative controls were included in both DNA extractions to ensure no cross-contamination occurred. Afterwards, DNA was quantified using a full-spectrum spectrophotometer  and then stored at -20°C until PCR analyses.DNA was extracted from faecal samples using QIAamp DNA Stool Mini Kit  following the manufacturer’s instructions, with the modifications proposed by Zeale et al. . AdditioA. lutescens lutescens and N. viridula. Insect pest PCRs were run in 25 μl reactions. Each PCR reaction contained 2 ng/μl of DNA template, 12.5 μ KAPA Hotstart polymerase Readymix (Kapa Biosystems), 1.25 μl of 5% DMSO, 0.75 μl forward primer, and 0.75 μl reverse primer. PCR cycling was performed by initial denaturation at 95° C for 3 minutes, followed by 35 cycles of 98° C for 20 s, 50° C for 30 s, and 72° C for 30 s, followed by a final extension of 72° C for 7min. PCR efficiency was visualised using gel electrophoresis.Arthropod-specific primers ZBJ-ArtF1c (Forward: AGATATTGGAACWTTATATTTTATTTTTGG) and ZBJ-ArtR2c (Reverse: WACTAATCAATTWCCAAATCCTCC)  were usePCR reactions of faecal samples contained 1 to 7 μl of DNA template (0.42 ng/μl–4.2 ng/μl) from the final extraction elutions depending on the DNA concentration of each sample. PCR reactions and cycling conditions were the same as described above. PCRs were run twice to test amplification reproducibility.The sequencing was performed using Illumina MiSeq platform following the guide for metagenomics sequencing library . SequencBioinformatic analysis was performed in Qiime (v.1.8) . Only seWe assigned OTU taxonomy against an arthropod taxa using the online DNA barcoding workbench Biodiversity of Life Database (BOLD) version 3 , which iWe calculated accumulation curves using the number of OTUs in each faecal sample per bird species, by randomizing the samples 9999 times. We further examined dietary richness using the differences in the number of OTUs between species. Additionally, we checked for differences in the frequency of orders present per bird species, based on the average frequency of each arthropod order per faecal sample.Throughout this study, Permutational analysis of variance (PERMANOVA) was used. PERMANOVA is a statistical test that uses resemblance-based methods for univariate or multivariate analyses . This roAccumulation curves, PERMANOVA, PERMDISP, and PCO, were calculated with PRIMER software (v.6.0) with the PERMANOVA+ add-on (v.1.0.6) .Meliphaga lewinii) , Eastern Yellow Robin  (13), Little Shrike-Thrush (Colluricincla megarhyncha) (12), White-throated Treecreeper (Cormobates leucophaea) (7), Fairy Gerygone  (6), Varied Triller  (4), Red-browed Finch  (3), White-throated Honeyeater  (2), White-browed Scrubwren  (2), Brown Honeyeater (Lichmera indistincta) (1), Fan-tailed Cuckoo (Cacomantis flabelliformis) (1), Grey Shrike-thrush (Colluricincla harmonica) (1), and Silvereye  (1). Only species represented with n≥4 were included in the analysis.We captured 82 individuals from 13 bird species: Lewin's Honeyeater  and represented 31.74% of all the sequences. These 9 OTUs belonged to three lepidopteran families; Oecophoridae (19.91% of sequences), Saturniidae (11.04%), and Bombycidae (0.80%). One hundred and forty six OTUs occurred infrequently . After all OTUs were blasted against BOLD database based on ≥98% similarity, 257 OTUs  were assigned to an arthropod taxon, while no taxonomic match was found for 222 OTUs  . The mosAll assigned OTUs were taxonomically divided into 8 orders and 40 families . LepidopHyposada hydrocampata, Atholosticta oxypeuces, and Phloeocetis sp. ANIC5) (Seventy out of 90 genera we found (77.78%) belonged to the order Lepidoptera. Three lepidopteran species were present in the diet of all bird species (. ANIC5) .N. viridula, Nysius vinitor , Olene mendosa , Candalides absimilis , and Isotenes miserana  (Hysteroneura setariae (rusty plum aphid) [Piezodorus oceanicus (redbanded shield bug) [Orgyia australis (red tussock moth) [Cleora repetita (grey looper) and Ectropis spp. (loopers) [Agrotis munda (brown cutworm) [Chrysodeixis argentifera (tobacco looper) [Cryptoblabes adoceta (sorghum head caterpillar) [Adoxophyes fasciculana (bell moth) [Dudua aprobola (tortrix moth) [Among the arthropod taxa found in faecal samples, we found 5 pests of macadamia: itborer) . Furtherm aphid) , and oneeld bug) , and 8 lck moth) , 2 from loopers) , 56, 2 fcutworm)  and Chry looper) , 1 from rpillar) , and 2 fll moth)  and Duduix moth)   .C. leucophaea to a maximum of 159 in G. palpebrosa . Results showed non-significant differences in the number of OTUs between species .The number of OTUs ranged from a minimum of 118 in lpebrosa  . However, PERMDISP results were also significant , showing dispersion in the data. No significant differences were found in the frequency of any other order . Five ar species .E. australis and those of G. palpebrosa (t = 1.379 P = 0.037), M. lewinii , C. megarhyncha , and C. leucophaea , and also between the diet composition of C. megarhyncha and those of G. palpebrosa , L. leucomela , and C. leucophaea . When presence/absence data were used, PERMANOVA results were similar to those obtained when using the number of sequences per OTU, showing significant differences in dietary composition between bird species (Pseudo-F = 1.622 P = 0.003). Only one pairwise test comparison  produced a non-significant result, in contrast with the other method. Significant differences were found between the dietary composition of E. australis and those of M. lewinii , C. megarhyncha , and C. leucophaea , and also between that of C. megarhyncha and those of G. palpebrosa , L. leucomela , and C. leucophaea . PERMDISP results confirmed that differences in dietary composition between bird species obtained with PERMANOVA were due to multivariate location, and not to multivariate dispersion . Between-species differences showed in PERMANOVA analyses were visualized using PCO ordination plots, where the first two axes explained a total variation of 27.6% on both log-transformed and presence-absence datasets . Pairwise tests showed significant differences between the diet composition of datasets .This is the first study using NGS and bioinformatics analyses to explore the diet of birds and associated ecosystem services by sequencing the arthropod DNA present in their faeces. Using the MiSeq platform of Illumina, we successfully sequenced DNA in 62 faecal samples from 11 bird species foraging in macadamia agro-ecosystems. Our results revealed that avian communities deliver pest-reduction services in this system. This study showed differences in dietary composition, but most of the bird species had wide insectivorous diets and shared a large proportion of prey items, indicating a low resource partitioning in these systems.N. vinitor, O. mendosa, C. absimilis, and I. miserana), and 1 ‘major’ N. viridula. The later species, commonly known as the green vegetable bug, is a threat to macadamia production in Australia [N. viridula was present in 23% of bird faecal samples. Specifically, it was present in 32% and 25% of all faecal samples from two of the most commonly trapped birds, M. lewinii and E. australis, respectively. In general, its presence was recorded in 6 out of 11 bird species. Nezara viridula seasonally alternates between crop and non-crop plant species [M. lewinii and E. australis makes these two species the most important ones from a pest-management point of view. Unfortunately, even though A. lutescens lutescens presence was recorded during insect pest surveys, its DNA could not be amplified with the arthropod-specific primers used in this study, thus species-specific primers need to be developed in future studies in order to understand whether this pest is consumed by avian communities.We report the presence of 5 macadamia-specific insect pests. Four of these are considered ‘minor’ . In species , and preet al. [Although at present it is not possible to quantify the number of individuals of any eaten insect pest using NGS , our reset al.  showed net al. , 68) sugH. setariae) [P. oceanicus, O. australis, C. argentifera, C. adoceta) [D. aprobola) [Ectropis spp., C. repetita) [Agrotis spp.) [Additionally, our results indicate the presence of 10 insect pests from other crops in the diet of the studied birds. Some of these pests can damage economically important crops present in the study area such as sugarcane (etariae) , summer adoceta) , 58, 59,probola) , avocadoepetita) , 56, andis spp.) . These rSpecies accumulation curves indicated that, most likely due to low sample sizes, we were not able to fully describe the entire insectivorous diet of any of the bird species. We found no significant differences in the number of OTUs between bird species. The frequency of individual arthropod orders found in faecal samples did not show differences between bird species, suggesting that the majority of avian species foraging in riparian woodland-macadamia interfaces shared food resources, by feeding primarily on lepidopteran, dipteran and arachnids. Several records of birds in riparian habitats show that the tree canopy is the most used habitat stratum , supportE. australis differed from most species, as visually shown by the cluster formed by its samples in the PCO plots. These differences are likely due to the foraging behaviour of E. australis, considered mostly a ground-forager, as opposed to the other species, which are mostly arboreal [C. megarhyncha showed a different dietary composition compared to those of L. leucomela, E. australis, G. palpebrosa and C. leucophaea. Previous studies in northern Australia found that C. megarhyncha showed higher foraging habitat diversity than a number of other bird species [C. megarhyncha showed higher foraging plasticity across vegetation types than other bird species, including E. australis, and C. leucophaea, whose numbers were biased towards particular habitats [C. megarhyncha has a high foraging plasticity, which is in accordance with the differences in diet composition with other species found in this study. Although L. leucomela was, together with C. megarhyncha, characteristic of the same riparian zone class in northern Australia [C. megarhyncha [C. leucophaea dietary composition differed from those of C. megarhyncha and E. australis. These three species are insectivorous, but a number of studies have reported the presence of a majority of ants in the diet of C. leucophaea . Ou. OuE. au species , tend to species , such as species .While NGS offers a powerful tool to analyse the diet of wildlife, it also presents a number of limitations, such as the impossibility of determining whether any arthropod’s DNA detected is a result of primary or secondary predation . It is aAn additional limitation is the number of arthropod sequences characterised for the COI region in BOLD or any other online database, which constrains the reconstruction of the diet of the studied birds. Nevertheless, these databases are growing at an incredible speed  and NGS-G. palpebrosa, L. leucomela, and C. megarhyncha.The data from the study clearly demonstrate the applicability of NGS analyses to explore the diet of birds and infer ecosystem functions. The diet composition comparisons based on NGS data were consistent with known foraging behaviours of the captured avian species. Additionally, the species-specific diet description at order and family levels using NGS is consistent with previous records based on traditional methods , 49–51, The dietary analysis of the studied bird species is in accordance with the research previously published about their foraging behaviour. Because of their rich vegetation structure, remnant riparian habitats provide a wide range of foraging strata that maximise the bird guilds inhabiting them . Guild-rOverall, our study showed that NGS using the MiSeq Illumina platform and subsequent bioinformatics analyses offer a solid, relatively fast and recently increasingly affordable way to explore the diet of mixed-guild avian communities, delivering a wide scope and detailed resolution on the prey taxonomic level, which offers an opportunity to better understand their diet. Our results highlight the benefits that conserving birds foraging in orchards can bring to the macadamia industry and stress the need to protect the riparian habitats in this region to maintain avian richness.


Bacillus cereus strains isolated from food products and food processing environments and two laboratory strains, B. cereus ATCC 10987 and B. cereus ATCC 14579. Subsequently, genome sequences of these strains were analysed together with 11 additional B. cereus reference genomes to provide an overview of the different types of carbohydrate transporters and utilization systems found in B. cereus strains. The combined application of API tests, defined growth media experiments and comparative genomics enabled us to link the carbohydrate utilisation capacity of 22 B. cereus strains with their genome content and in some cases to the panC phylogenetic grouping. A core set of carbohydrates including glucose, fructose, maltose, trehalose, N-acetyl-glucosamine, and ribose could be used by all strains, whereas utilisation of other carbohydrates like xylose, galactose, and lactose, and typical host-derived carbohydrates such as fucose, mannose, N-acetyl-galactosamine and inositol is limited to a subset of strains. Finally, the roles of selected carbohydrate transporters and utilisation systems in specific niches such as soil, foods and the human host are discussed.We characterised carbohydrate utilisation of 20 newly sequenced  Metabolic activity and growth of bacteria requires energy that can be acquired during carbohydrate catabolism. Carbohydrates can be present in nature in complex forms such as polysaccharides including starch, cellulose or glycogen, but also as mono- and disaccharides exemplified by glucose and sucrose. Efficient use of available carbohydrates by microorganisms is determined by the different types of carbohydrate transporters and utilization systems encoded by chromosome- and plasmid-located genes and gene clusters ,2. In adEscherichia coli  ,34 B. ceAPI 50CH  test was used in combination with API 50CHB/E medium  according to manufacturer’s instruction. Shortly, single colonies were used to inoculate API 50CHB/E medium to a turbidity equivalent of 2 McFarland. Test strips were filled with inoculated medium, incubated at 30°C, and checked for media colour change after 24 and 48 h. For each strain, three independent repetitions were performed.595 was measured every 10 min and read outs were used to score for positive or negative growth. Chemically defined Y1 medium [Ten ml of BHI was inoculated with a single colony and incubated overnight at 30°C with aeration at 200 rpm. One ml of overnight grown culture was pelleted in a table top centrifuge , washed with Peptone Physiological Salt buffer  and resuspended in 1 ml of PFZ. 96-well plates were filled with 20 μl of 10x diluted washed culture and 180 μl of test media. Plates were then transferred into a plate reader  for incubation at approximately 200 rpm at 30°C. OD1 medium  was sligB. cereus strains were included in the comparative analysis. Next to the 20 newly sequenced food-spoilage B. cereus strains [B. cereus genomes including B. cereus ATCC 14579 and B. cereus ATCC 10987 were obtained from the NCBI-Genbank database (http://www.ncbi.nlm.nih.gov/genome/genomes/157) and compared to each other and to the genome of reference strain Bacillus subtilis 168  in the genomes were determined using OrthoMCL . A databB. subtilis 168 listed in the Subtiwiki database (highly curated) [B. cereus OG table. Additional genes and systems were found with keyword searches and genome context analysis. Subsequently, selected genes/proteins were manually curated by comparison against sequence databases , family/domain databases (using e.g. InterPro [The carbohydrate utilization systems of curated)  were iniInterPro ), enzymeInterPro ) and patInterPro ). Gene cInterPro . DetailsB. cereus strains and B. subtilis 168, additional circular reference genomes of 18 strains from the B. cereus sensu lato group were included, i.e. 6 B. anthracis strains, 11 B. thuringiensis strains and 1 B. weinenstephanensis strain. On the basis of an OrthoMCL analysis of all these genomes, all OGs were selected with a single orthologous gene in each of the different genomes. The protein sequences of these core OGs were aligned using Muscle [For the construction of a phylogenetic tree, next to the genomes of 33 g Muscle  and aligg Muscle .B. cereus sensu lato group strains to the phylogenetic groups (I to VII) proposed by Guinebretière et al. [panC gene sequences from the OG group and implementing in the online tool (https://www.tools.symprevius.org/Bcereus/english.php).Affiliation of 51 e et al.  was perfB. cereus genomes were found to contain 1775 core ortholog groups (OGs), i.e., orthologous genes that occur in all genomes only in one copy. A phylogenetic tree was built based on differences in all encoded core proteins derived from 33 B. cereus genomes and an additional 18 reference genomes from the B. cereus sensu lato group and B. subtilis 168  were used to search for ortholog systems in the B. cereus OG table, and additional cassettes were identified by keyword searches and manual annotation. The identified cassettes are shown schematically in B. cereus ATCC 14579 genome, but since the corresponding genes are functional based on the experimental data, the non-interrupted fragments of the pseudogenes may still be functional. The various B. cereus carbohydrate utilization systems are described below.The B. cereus strains grow on glucose , trehalose (trePAR), N-acetyl-glucosamine (nagABR) and N-acetyl-muramic acid  (All  murPQR)  Tables.  murPQR) .B. cereus strains grow on maltose, a disaccharide , and a maltose operon transcriptional regulator MalR , a ribokinase RbsK and a ribose operon transcriptional regulator RbsR is present in all B. cereus as well as in all considered B. cereus sensu lato group genomes and B. subtilis 168. All B. cereus strains grow on ribose  is another candidate for the import of cellobiose (a beta-glucoside), but the bglP gene was found only in B. cereus strains B4077, B4083, B4088, B4117, E33L and FRI_35, which also does not agree with the API growth results on cellobiose. Thus further work is required to elucidate the roles of these clusters in carbohydrate transport and metabolism.llobiose . This coB. cereus strains  not included in our study) and this was indeed the only strain growing on D-xylose . The corresponding 4 genes are also present in B. subtilis 168 (but not in a single cassette) and are known to be responsible for growth on sucrose [B. cereus strains and their ability to grow on sucrose (13 of 14 strains)  or animal  origin into food products. In general, raw materials and final food products are considered nutritious commodities. However, some groups of products may offer limited carbohydrate supplies, for example staple foods contain mainly starch while dairy products contain mainly lactose.glgPADCB present in all B. cereus genomes, however 8 of the B. cereus strains are not able to utilise glycogen and starch  which was proposed to support growth on starch [amyS gene (OG_4318) in the B. cereus strains correlates exactly with growth on starch or glycogen, indicating that this is the sole gene responsible for degradation of these polymeric substrates , suggesting that these strains will also not grow on starch/glycogen. The encoded protein AmyS of 513 residues is most likely secreted, since it has a signal peptide and a typical signal peptidase cleavage site (AYA|D). The extracellular degradation of starch or glycogen will lead to release of glucose units which can be accumulated by the maltose and glucose uptake systems and subsequently metabolised as described above.Starch is a polymer of glucose units, and consists of the linear-chain amylose and the branched-chain amylopectin. An analogue of starch, glycogen, has a similar structure to amylopectin, but more extensively branched and compact than starch. There is a glycogen biosynthesis gene cassette d starch . The genn starch . Alpha-abstrates . MoreoveB. cereus is commonly associated with dairy environments, and therefore we tested for utilisation of lactose, the main carbohydrate present in milk [lacGEFRABCD in their genomes (B. cereus strains of this study (data not shown). A BLASTP analysis to the NCBI database shows presence and 100% protein sequence identity of this lactose cassette in B. cereus strains m1293, VD102, VD140, NVH 0075–95, and MHI 86  and in genomes of B. cereus strains 03BB102, F837_76, Q1 and in B. subtilis 168. The cassette encodes a mannose-specific PTS transporter ManP, a mannose 6P isomerase ManA and a BglB family transcriptional antiterminator ManR , F837_76 and FRI_35. None of the strains grew on inositol in the API test, but 11 strains were found to grow in the aerobic growth experiment in defined medium . Therefore there must be an additional cassette for inositol utilization.A 9-gene cassette d medium  includinTable B inB. cereus strains B4079, ATCC 10987, E33L, and FRI_35 which does not correlate well with observed growth on N-acetyl-galactosamine. Nevertheless none of the strains grow on tagatose in static conditions (Table B inB. cereus strains and two reference genomes (Q1 and NC7401)  , but it B. cereus [spsA gene was specifically expressed during oral infection of Galleria mellonella [B. cereus genomes, and all but three  experimentally validated strains were observed to grow on glucose-6-phosphate  is present in all B.cereus genomes, and next to the gntP, gntK and gntZ genes it contains genes encoding 6-phosphogluconolactonase Pgl, glucose-6-phosphate 1-dehydrogenase, transketolase, and transaldolase  phospho-beta-D-glucosidases, i.e. bglA (BSU40110), bglC/yckE (BSU03410), and bglH (BSU39260) [B. cereus genomes there is no equivalent of bglA of B. subtilis. The bglC gene (OG_4907) of B. subtilis 168 has one ortholog in 11 B. cereus strains, but it is not part of a sugar utilisation cassette, while a second ortholog of bglC is found only in strains B4082 and AH820 as part of a 7-gene cassette for cellobiose utilization. Finally, B. cereus strains B4077, B4083, B4088, B4117, E33L and FRI_35 carried orthologues of B. subtilis 168 bglPH operon encoding a beta-glucoside-specific PTS system BglP, and an aryl-phospho-beta-D-glucosidase or aryl-6-phospho-beta-glucosidase BglH [bgl operon antiterminator BglG. Taken together, there are 14 B. cereus strains that have 1 or 2 genes encoding an aryl-6-phospho-beta-glucosidase. It is not clear what the transporter is for aryl beta-glucosides in these strains, but it is possibly a more general PTS for beta-glucosides, such as for cellobiose (see above for 3 putative PTS cellobiose utilization cassettes). This occurrence of one or more aryl-6-phospho-beta-glucosidases in 12 B. cereus strains correlates almost perfectly with the growth observed on salicin , consisting of a glucose moiety linked to an aromatic ring. The SU39260) . In the  salicin . The onlglpPFKD genes (BSU09270-BSU09300) of B. subtilis 168, was found to be present in all B. cereus genomes. The cassette encodes a glycerol uptake facilitator protein GlpF, a glycerol kinase GlpK, an aerobic glycerol-3-phosphate dehydrogenase GlpD, and a glycerol uptake operon regulator Glp  strains showed growth . A glyceB. cereus genomes, but not in strains B4082, B4083, B4088, B4147, 03BB102, and F837_76. Moreover, all B. cereus genomes encode a glycerol-3-phosphate permease (OG_511). However this substrate was not experimentally tested.A glycerol-phosphate uptake system, based on an ABC transporter UgpBEAC, was found in most of B. subtilis 168 which did not have ortholog cassettes in any of the B. cereus strains. These include the mannitol cassette mtlAFD , the sorbitol/glucitol cassette gutRBP-fruC (BSU06140-BSU06170), the arabinan/arabinose cassette abnA-araABDLMNPQ-apfA (BSU28720-BSU28810), the L-rhamnose cassette rhaAMBR(EW) (BSU31180-BSU31220), and the oligomannoside cassette gmuBACDREFG (BSU05810-BSU05880). In agreement with this lack of genes, the selected B. cereus strains in our study did not grow on mannitol, sorbitol, arabinose or rhamnose  and may be specific for breakdown of more complex polysaccharides as levan is a fructose polymer (fructan).Orthologs of the levan/fructose-specific PTS system B. cereus with a broad range of carbohydrate transporter and utilisation systems supports their transmission from soil to host. Comparative analysis of genotypic and phenotypic diversity of carbohydrate utilisation among 20 newly sequenced B. cereus food isolates and B. cereus ATCC 14579 and ATCC 10987 suggests an adaptative metabolic capacity to different environmental niches represented by carbohydrates such as cellobiose or xylose found in soil, starch or lactose in food and dairy products, and fucose and glucose-6-phosphate found in intestinal tracts of animal and human hosts. In addition to such strain-specific features, all the strains could utilise compounds typically reported to support growth of B. cereus such as glucose, fructose, maltose, trehalose, N-acetyl-glucosamine, and ribose [Equipment of ubiquitous food-borne human pathogens such as d ribose ,69,70.B. cereus isolates may correspond to the classification of the isolates to four of the phylogenetic groups identified previously by Guinebretière [B. cereus sensu lato group. For example strains in groups VI and II show a broader substrate utilisation capacity compared to strains belonging to group III. Members of the latter group, with exception of strain B4087, did not grow on glycogen, starch or salicin. In addition, the genomes of these strains lack aryl-6-phospho-beta-glucosidases involved in the utilisation of two other aromatic glucosidases, esculin and arbutin. Notably, strain B4087 displayed a distinctive growth pattern and genome potential compared to the other members of group III. Within group IV, a sub-branch encompassing strains isolated from vegetables , showed limited substrate utilisation. Interestingly, two strains (B4079 and ATCC 10987) representing a sub- branch within group III showed the least carbohydrate utilisation capacity, and both strains originate from spoilage incidents, namely from a retorted can with chocolate beverage and spoiled cheese, respectively.The diversity in sugars utilised by bretière  within tB. cereus isolates and identified a significant number of new carbohydrate utilisation cassettes. The collective capacity of B. cereus strains to utilise carbohydrates is visualised in B. cereus genomes (see worksheet PTS systems in Carbohydrate uptake in bacteria is mediated by different transport mechanisms including phosphoenol-pyruvate-dependent phosphotransferase systems (PTS systems), ATP-binding transporters (ABC transporters) and secondary transporters (permeases). We searched for transporters and associated metabolic enzymes in the genomes of newly sequenced B. cereus from soil, via food processing environments and food, to the host, is discussed below.The possible roles of specific carbohydrate transporter and utilisation systems in transmission of nagEABR and murRQP cassettes in all tested isolates perfectly correlates with the observed growth on these substrates.The soil environment is rich in chitin, a constituent of insects exoskeletons and cuticles but also fungal cell walls. Chitin is a 1–4 polymer of N-acetyl-glucosamine and by activity of commonly found chitinases, simple amino sugars can be released and become available for soil bacteria . Amino sB. cereus isolates possess three or four cassettes for cellobiose utilisation, only five B. cereus isolates were found to utilise cellobiose in static conditions, which suggests that activation of cellobiose systems is not achieved in most cases. Conceivably, activation of cellobiose uptake and utilisation systems requires specific conditions that remain to be elucidated. In Listeria monocytogenes presence of β-glucosides  in the environment causes repression of virulence genes via a specific regulatory pathway (involving bvr locus) independent from carbon catabolite repression (CCR) mechanism [B. cereus enterotoxin production has not been reported up to now.Cellobiose is a catabolite of cellulose degradation, and is therefore also present in soil and decaying vegetation. While all the echanism ,72 as weB. cereus ATCC 10987, and this perfectly matched the unique presence of the previously described cassette for xylose utilisation in this strain [Notably, growth on xylose, another common plant-derived carbohydrate was only observed for s strain .B. cereus strains. This is in line with previous observations, that showed limited ability to utilise lactose among B. cereus dairy farm isolates, although prevalence increased along the production chain, reaching 20% lactose-positive strains among B. cereus household milk isolates [B. cereus strains not included in this study  is also bound to proteins in mucins. Prior to uptake and utilisation these mucin-bound sugars can be released by endo-alpha-N-acetylgalactosaminidases which are generally found in the human gut. Notably, 8 of the tested B. cereus strains were shown to utilise glucose-6-phosphate, while genomes of all the strains carry the conserved sugar-phosphate specific spsRKABC cassette [B. cereus sugar phosphate sensing and uptake during growth in the intestinal environment of Galleria mellonella larvae [hlyII, encoding the pore-forming toxin haemolysin II, by activation of HlyIIR in plasmid-curated acrystalliferous B. thuringiensis [Finally, 19 of tested cassette . Sugar pcassette . In parta larvae . Moreovengiensis .B. cereus biofilm formation and virulence remain to be established.Besides the effect of available carbohydrates on the expression of virulence factors, that may facilitate the host infection as well as outcompeting competitive flora, carbohydrates were shown to affect biofilm formation both in gram- positive and gram-negative bacteria –78. FormThe analysis of complete carbohydrate transporter and utilization cassettes allowed for the prediction of substrate utilization. Particularly for carbohydrate substrates used by only small numbers of strains such as xylose, lactose, mannose, sucrose, starch/glycogen, and salicin, there is an excellent correlation with the presence of the corresponding gene cassettes. Discrepancies between observations and predictions are conceivably due to: i. lack of expression of cassettes in tested conditions, e. g. availability of oxygen, as the number of strains utilising gluconate, L-fucose and inositol was higher in defined, aerated media compared to static API tests; ii. miss-identification of cassettes based on incorrect or incomplete prediction of carbohydrate specificity of transporters; and iii. lack of identification of cassettes e.g. presence of several ABC transporters with unknown specificity and proteins annotated only as “membrane proteins” may function as transporters (e.g. permeases). The latter two points of the genome-mining approach are clearly limitations for the identification of novel sugar utilization pathways which have not been described in literature earlier.B. cereus strains as well as the corresponding potential to utilise carbohydrates for growth, reflecting strain diversity and the capacity to occupy different niches including soil, food products as well as intestinal tract of insects and mammals.In conclusion, this study provides extensive information on the genetic potential of S1 Table(PDF)Click here for additional data file.S2 Table(A) PTS system cassettes, (B) ABC transporter cassettes, (C) permease cassettes. * genes with newly predicted function.(PDF)Click here for additional data file.S3 TableThe information in each cell represents: NCBI accession code or contig number (NODE/scaffold) | locus tag | nt coordinates | number of encoded amino acids. Blue shading indicates putative pseudogenes. * genes with newly predicted function.(XLSX)Click here for additional data file.S4 Table(PDF)Click here for additional data file.


Ericerus pela, represents one of the most dramatic examples of sexual dimorphism in any insect species. In this study, we showed that although E. pela males display complete metamorphosis similar to holometabolous insects, the species forms the sister group to Acyrthosiphon pisum and cluster with hemimetabolous insects. The gene expression profile and Gene Ontology (GO) analyses revealed that the two sexes engaged in distinct developmental programs. In particular, female development appeared to prioritize the expression of genes related to cellular, metabolic, and developmental processes and to anatomical structure formation in nymphs. By contrast, male nymphal development is characterized by the significant down-regulation of genes involved in chitin, the respiratory system, and neurons. The wing and appendage morphogenesis, anatomical and tissue structure morphogenesis programs activated after male nymphal development. Transcription factors  and DNA methyltransferase were also differentially expressed between females and males. These results may indicate the roles that these differentially expressed genes play in regulating sexual dimorphism through orchestrating complex genetic programs. This differential expression was particularly prominent for processes linked to female development and wing development in males.The Chinese white wax scale insect,  Most animal species are sexually dimorphic and display phenotypic differences in morphology, physiology, and/or behavior between females and males of the same species. Exaggerated dimorphic traits are predominantly used in the competition for mates12Ericerus pela (Chavannes) (Hemiptera: Coccidae) is a famous resource insect due to its role in economic production. It has been bred in China for more than a thousand years. The males are the best-known wax producers, and they secrete large amounts of pure white wax during the second-instar nymphal stage. The white wax has historically been used in traditional medicine, printing, and candle production, and its use has since expanded to the food, chemical, pharmaceutical, and cosmetic industriesThe Chinese white wax scale insect Females and males are so morphologically distinct that they can be mistaken for members of different species . The fem456E. pela is associated with substantial developmental changes and results in a distinctive adult form that is capable of flight and can engage in courtship behavior. E. pela males and females are distinctly different at each postembryonic developmental stage, and the sexually dimorphic traits accumulate gradually during the life cycle. Detailed molecular studies of these sexually dimorphic features are still lacking.By contrast, males display more dramatic morphological transitions, reminiscent of the ones seen in holometabolous insects, which we describe here as “holometabolous-like”. After embryonic development, males undergo first and second-instar nymph, prepupal, pupal, and adult stages . The mal456The female neotenous characteristics and male holometabolous-like development raise the possibility that the juvenile hormone (JH) and ecdysone signaling pathways may play important roles in regulating sexually dimorphic processes in scale insects. Hormone-controlled regulatory networks interact with Hox genes and cell signaling proteins to activate tissue-specific and stage-specific responses891011E. pela at different developmental stages using RNA-Seq to provide a global transcriptomic characterization of its dimorphic sexes. By comparing gene expression profiles between females and males, we defined gene sets that build the molecular bases of sexually dimorphic development. These results will improve our understanding of sexual dimorphism in scale insects and other insect species.In this study, we constructed ten libraries of E. pela was obtained by pooling individual eggs, first/second instar nymphs, male pupae, and male and female adults. Illumina sequencing produced a total of 88,153,000 reads covering a total of 7,933,770,000 bp after removal of low-quality sequences (SRA accession number SRA047325.1). These reads were assembled into 1,481,387 contigs ranging from 50 bp to 4,489 bp . These contigs were further assembled into 231,175 scaffolds  using paired end-joining and gap-filling (−5) BLAST hits against the non-redundant (nr) database.One transcriptome of -filling . We finaThere were 13,257, 21,361, and 9,315 unigenes suitable for annotation in the Gene Ontology (GO), KEGG orthology (KO), and Cluster of Orthologous Groups (COG) databases, respectively, based on sequence homology .E. pela, despite the males displaying complete metamorphosis similar to holometabolous insects, formed the sister group to Acyrthosiphon pisum and clustered with hemimetabolous insects. As demonstrated in previous studies1314Pediculus humanus  and formed a sister group to Holometabola.The PhyML (Phylogenetic estimation using Maximum Likelihood) phylogenetic tree  showed tWe constructed ten RNA-Seq libraries from males and females at different developmental stages (GEO accession number GSE52228 and GSE63934), including female first- (FF) and second-instar nymphs (FS) and early- (FA1), middle- (FA2), and late-stage adults (FA3) as well as male first- (MF) and second-instar nymphs (MS), pre-pupae (MPP), pupae (MP), and adults (MA). Each of the ten RNA-Seq libraries generated over 7.0 million clean reads .We identified 1,771 differentially-expressed genes (DEGs) between the first (FF) and second instar (FS), with 829 up-regulated and 942 down-regulated genes in FS . The bioThe number of DEGs during the molt from first instar (MF) to second instar (MS)  was very different from the values in females (FF vs. FS). Male nymphal development was characterized by significant down-regulation of most GO biological process terms related to chitin, the respiratory system, and neurons and up-regulation of muscle-related terms . There wThere were a total of 2,817 DEGs between the FF and MF libraries, with 1,984 up-regulated and 833 down-regulated genes in MF . There wWhen female adults at different stages were compared to male adults (MA), the number of DEGs increased in each comparison. Most DEGs were enriched in molecular function terms related to “catalytic activity” and “hydrolase activity”. Additionally, many DEGs were enriched in “somatic muscle development”, “fatty acid synthase activity”, “DNA or RNA related process”, and many other GO terms. The KEGG pathway annotation yielded similar results, but “insulin signaling pathway” and “steroid hormone biosynthesis” were also found .df = 20, P < 0.01). The discrepancies at a few developmental stages between the qRT-PCR and RNA-Seq results may have been caused by a sensitivity bias between the two methods or by the use of different statistical methods and threshold values in qRT-PCR and RNA-Seq.The gene expression profiles were validated by qRT-PCR. The results were closely consistent with the RNA-Seq results  conveys the JH signal to prevent precocious metamorphosis through the maintenance of high expression of Kruppel homolog 1 (Kr-h1)1617Functional molting hormone receptors consist of the products of two genes, ecdysone receptor (EcR) and ultraspiracle (usp)Blattella germanicaE. pela. It exhibited the highest expression level during the male prepupal stage (MPP) , which regulates wing development, was enhanced by JH in ge (MPP) . Hormone cascade .E. pela.Hox genes, which are related to limb development , wing development , and segmentation formation , are key factors in establishing differential structures91011E. pela and found its expression level increased sharply at the developed adult stage (FA3), in which the embryos have begun development. It also exhibited pronounced expression in male first-instar nymphs (MF) that decreased progressively with development  trees in the Research Institute of Resources Insects. E. pela from each developmental stage were collected for the pooled transcriptome. Thousands of eggs without distinguishing between females and males were collected for the pooled transcriptome. For the first-instar nymphal stage, more than 500 female and 500 male individuals were collected. During this stage, females and males inhabited different sides of the leaves, making it is easy to distinguish females from males. More than 100 female and 100 male second-instar nymphs were collected. For the developed female adults (FA3), approximately 20 individuals were collected. For the other developmental stages, approximately 50–100 individuals of each sex were collected. The pooling approach was used for sample collection39A pooled RNA sample (20 μg) of every developmental stage  was used for cDNA library preparation. The library was constructed using the same techniques as in our previous studyDrosophila melanogaster (Diptera), Anopheles gambiae (Diptera), Bombyx mori (Lepidoptera), Tribolium castaneum (Coleoptera), Apis mellifera (Hymenoptera), and Nasonia vitripennis (Hymenoptera)) were downloaded from the following websites, respectively: ftp.flybase.net(dmel_r5.27); http://www.vectorbase.org/Anopheles_gambiae(AgamP3.6); http://www.silkdb.org/silkdb/(release-2.0); ftp://bioinformatics.ksu.edu/pub/BeetleBase(version3.0); http://hymenopteragenome.org/drupal/sites/hymenopteragenome.org.beebase/(release-2.0); and http://genomes.arc.georgetown.edu/nasonia/nasonia_genome_consortium (OGSv1.2). Sequences from three other hemimetabolous insects (Pediculus humanus (Phthiraptera), Acyrthosiphon pisum (Hemiptera), and Bemisia tabaci (Homoptera)) were downloaded from the following websites, respectively: https://ftp.vectorbase.org/public_data/organism_data/phumanus); http://www.aphidbase.com/ (version 1); and http://www.ncbi.nlm.nih.gov/bioproject/PRJNA50499. Sequences from Daphnia pulex were downloaded from http://wfleabase.org. Alternative splicing was filtered out of the CDS, and the longest sequence was reserved.The CDS and protein sequences of six holometabolous insect species (The TreeFam method41http://code.google.com/p/phyml/) was applied to these sequence sets to build phylogenetic trees under the HKY85 + invgamma model for nucleotide sequences. We used 100 rapid bootstrap replicates to assess branch reliability.The phylogenetic tree was reconstructed based on the identified 382 single-copy gene families that were present in all species. The corresponding amino acid sequences were aligned with the MUSCLE (v3.8.31) program and then trimmed to remove ambiguous sites or missing data. The CDS results were aligned using the protein alignment result as a guide. The CDS alignments for each gene were then concatenated into a super-alignment containing 599,412 positionsThe mRNA of each sample  was enriched separately using oligo (dT) magnetic beads after extracting the total RNA from each sample. The mRNA was fragmented (approximately 200 bp) after the addition of fragmentation buffer. First-strand cDNA was synthesized using random hexamer-primers using the mRNA fragments as templates. Buffer, dNTPs, RNase H, and DNA polymerase I were added to synthesize the second strand. The double-stranded cDNA was then purified using a QiaQuick PCR extraction kit and washed using EB buffer for end repair and the addition of single adenines. Sequencing adaptors were ligated to the fragments, and the required fragments were purified by agarose gel electrophoresis and enriched by PCR amplification. Finally, ten RNA-Seq libraries were constructed and sequenced using Illumina HiSeq™ 2000.The original image data were transformed into sequence data by base calling, which is defined as raw reads. To obtain clean reads, reads with adaptors, reads with more than 10% unknown bases, and low-quality reads were removed prior to data analysis. The clean reads were mapped to reference sequences using SOAPaligner/soap2The sequences were assessed by read quality, statistics of alignment analysis, sequencing saturation analysis, distribution of reads on the reference gene, and distribution of reads on the reference genome. This analysis constitutes the general information of the study.Gene expression levels were calculated by the numbers of reads mapped to the reference sequence for every gene based on the RPKM (reads per kb per million reads) methodP-values in multiple tests. We used stringent criteria  to determine significant differences in gene expressionTo identify differentially expressed genes (DEGs) between two samples, the clean tag frequency in each RNA-Seq library was statistically analyzed according to a strict algorithm developed from the significance of digital gene expression profilesP-value underwent Bonferroni Correction, and the corrected P-value ≤ 0.05 was chosen as a threshold. GO terms fulfilling this condition were defined as significantly enriched GO terms in DEGsThe DEGs were chosen for further analysis of GO and KO enrichment. DEGs were first mapped to GO terms in the database to calculate the number of genes for every term. Then, the GO terms that were significantly enriched in the DEGs relative to the genomic background were found using a hypergeometric test. The formula is as equation (1).Pathway enrichment analysis provides significantly enriched signal transduction pathways, metabolic pathways, and other pathways in DEGs relative to the transcriptome background. The formula used in this analysis is the same as the one used for GO enrichment, with N representing the total number of genes with KEGG annotations (Kyoto Encyclopedia of Genes and Genomes)49−ΔΔCt methodP = 0.01 levelTo validate the results of RNA-Seq analysis, we performed qRT-PCR experiments with nine genes. The concentration of total RNA was determined using the Agilent 2100 Bioanalyzer . One μg of total RNA from each sample was reverse-transcribed separately in a 20 μl reaction system according to the protocol provided with the M-MLV first-strand kit . Then, qRT-PCR was performed using SsoFast™ EvaGreen Supermix . β-actin was used as an internal standard. Three replicates were conducted for each developmental stage. The relative expression levels of each gene were calculated using the 2P.Y. and X.M.C. conceived and designed the experiments. P.Y. wrote the paper. P.Y. and Y.F. analyzed the data. W.W.L. and T.S. performed the qRT-PCR analysis. All authors reviewed the manuscript.Supplementary InformationDataset 1Dataset 2Dataset 3Dataset 4