Polychromophilus, a haemosporidian parasite of bats, is phylogenetically close to human-pathogenic Plasmodium species, and their study may provide further clues for understanding the evolutionary relationships between vertebrates and malarial parasites. Our aim was to investigate the distribution of Polychromophilus spp. in Eastern Europe and test the importance of host ecology and roost site on haemosporidian parasite infection of bats. We sampled bats and their ectoparasites at eight locations in Romania and Bulgaria. DNA was extracted from blood samples and ectoparasites and tested individually for the presence of DNA of Polychromophilus spp. using a nested PCR targeting a 705 bp fragment of cytB. Two species of Polychromophilus were identified: Po. melanipherus in Miniopterus schreibersii and associated ectoparasites and Po. murinus in rhinolophid and vespertilionid bats (6 species) and their ticks and nycteribiid flies. Only cave-dwelling bat species (and their ectoparasites) showed infections, and we found a strong correlation between infections with Polychromophilus parasites and Nycteribiidae prevalence. We report the high genetic diversity of Polychromophilus spp. in Eastern Europe, suggesting that the simultaneous presence of varied host and vector assemblages enhances bat haemosporidian parasite diversity.Malaria is responsible for major diseases of humans, while associated haemosporidians are important factors in regulating wildlife populations.  Rattus macleari) on Christmas Island [Passer domesticus) in Europe [Plasmodium falciparum, being suggested as a recent pathogen with a chimpanzee origin [Polychromophilus species of bats) are phylogenetically close to human-pathogenic Plasmodium species [Haemosporidians causing malaria are responsible for major diseases of humans , with two species infecting different hosts groups in Europe: Po. melanipherus is a parasite of the long-winged bat (Miniopterus schreibersii), while Po. murinus occurs mainly in vesper bats (Vespertilionidae) and certain rhinolophid species [Polychromophilus infections [Nine genera of Plasmodiidae infect bats worldwide: attiella  and basen Europe . The gen species . Unlike fections .Polychromophilus spp. are known in Europe from Great Britain, Italy, the Netherlands, and Switzerland [Polychromophilus spp. are vector-borne parasites, with arthropod ectoparasites suggested to biologically transfer sporozoites between bats [Polychromophilus spp. [Polychromophilus spp. infections. In this context, our aims were to: (a) investigate the distribution of Polychromophilus spp. in Eastern Europe by sampling a diverse range of bat species, and (b) test whether there are differences in Polychromophilus spp. infection between primarily cave-dwelling (roosting in large underground shelters and commonly parasitized by bat flies) and crevice-roosting bat species . We predict that bat species resident in caves should show higher Polychromophilus spp. infection rates, than crevice-dwelling bat species. We also screened different ectoparasites collected from Polychromophilus-positive bats in order to establish their possible carrier role for haemosporidian parasites.The presence of tzerland ,24,25,26een bats . Bat flilus spp. ,28. ThusPolychromophilus parasite prevalence, we assigned each bat species to one group , and sex for DNA extraction. Ticks and bat flies were tested individually (24) or in pools , with Polychromophilus spp. Genomic DNA was extracted from the blood from filter papers using an Isolate II Genomic DNA kit . The genomic DNA of bat ectoparasites was extracted using the QIAamp DNA Mini Kit  according to the manufacturer’s instructions. All the DNA samples were stored at −20 °C until further analysis.Ectoparasites of bats with cytB gene using previously described primers [TM Red Mix, Bioline, London, UK), 7.5 µL water, 1 µL of each primer (10 pmol/μL) and 3 µL aliquot of isolated DNA in the first round and in the second round instead of DNA 1 µL of PCR product from the first reaction was used. The PCR was performed using the T1000TM thermal cycler  with the following condition: initial denaturation at 94 °C for 5 min, then 25 cycles (for the first reaction) and 35 cycles (for the second reaction) of denaturation at 94 °C for 30 s, annealing at 47.2 °C for 30 s (for both reactions), and extension at 72 °C for 45 s and a final extension at 72 °C for 10 min. For each set of reactions (45 samples) 2 negative controls (distilled water) and one positive control, which was Polychromophilus spp.-positive DNA isolated from bat flies of Common bent-wing bat (Mi. schreibersii) collected from Italy were included.A nested PCR targeting a 705 bp fragment of  primers  was usedTM, Thermo Fisher Scientific, Waltham, MA, USA). PCR products were purified and sequenced . Obtained sequences were manually edited, then aligned and compared to those available in GenBankTM by basic local alignments tool (BLAST) analysis. The MEGA model selection method was applied to choose the appropriate model for phylogenetic analyses. In the phylogenetic analyses, reference sequences with high coverage  were retrieved from GenBank and trimmed to the same length. Phylogenetic analyses were conducted by MEGA version 7.0 using the maximum-likelihood method, Hasegawa–Kishino–Yano (HKY) model according to the selection of the program and 1000 bootstraps. The sequences were deposited in GenBank under the following accession numbers .Amplification products were visualized by electrophoresis on 1.5% agarose gel stained with RedSafe™ 20,000× nucleic acid staining solution , and their molecular weight was assessed by comparison to a molecular marker 100 bp DNA Ladder , belonging to 7 species. In the case of positive samples, prevalence showed wide variations among different species, ranging from 10% (Myotis blythii) to 67.3% (Mi. schreibersii), or even 75% , but also showed an identity of 97.7–98.5% with Po. melanipherus collected from Mi. gleni in Madagascar. Blood samples from rhinolophid and vespertilionid bats all hosted different sequences of Po. murinus, with a sequence identity of 96.3–99.8% to reference sequences from bats  sampled in Switzerland and deposited in GenBankTM , while My. daubentonii was infected by I. vespertilionis . Two bat species were infested with ticks . Bat fleibersii .I. simplex pools (8.9%) and three individuals of I. vespertilionis  were positive for Polychromophilus spp. , and further three consisted of three nymphs, collected from five different Mi. schreibersii individuals. The positive I. vespertilionis pool was made from two larvae. All these ectoparasites originated from bats that tested positive for Polychromophilus spp. However, only 20% of all DNA samples of ticks collected from Polychromophilus-positive bats were PCR-positive. The species identified with sequencing was Po. melanipherus (99.5–100% identity with KJ131274.1) in I. simplex pools, while I. vespertilionis harbored Po. murinus (99.2% identity with HM055588.1). Interestingly, corresponding sequences between ticks and their host individual did not show 100% sequence identity in each of the cases, with just a 94.9% identity between a 3 larva pool and the collecting host  were tested for Plasmodiidae DNA, and six lus spp. . Three oPolychromophilus spp. was detected in 23 fly samples . Five different fly species contained the DNA of haemosporidians, with high prevalence rates recorded in Penicillidia conspicua  and N. pedicularia (n = 3). Both species of Polychromophilus spp. were identified in bat flies. In particular, flies collected from Mi. schreibersii  contained the DNA of Po. melanipherus, while flies collected from vespertilionids  all tested positive for Po. murinus. These are the first records of Polychromophilus spp. identified in nycteribiid flies in Eastern Europe (Romania), with first-ever records of Po. melanipherus in Pe. conspicua and Pe. dufourii, and the first-ever records of Po. murinus in N. vexata and Pe. dufourii. DNA of Po. melanipherus was found in Pe. dufourii, collected from Mi. schreibersii (Canarau Fetii), while individuals of the same dipteran species (collected from My. blythii and My. myotis at two different sites) tested positive for Po. murinus.DNA of ua 8/11, . No infePolychromophilus spp. identified , while four haplotype groups were identified among different sequences of Po. murinus , while highest identity was 100% (700/702 bp). We found no geographical structuring in haplotype diversity. High identity sequences (99.99–100%) were found at geographically distant locations , while single sites held high haplogroup diversity .We found high sequence diversity of both entified . AltogetPolychromophilus spp. was identified only in cave-dwelling bat species ((17) = 0.9406, p < 0.001). We found no statistically significant effect of neither bat sex nor age or bat fly sex on Polychromophilus spp. prevalence. Capture season had no effect on prevalence or haplotype diversity.DNA of  species  and showPolychromophilus spp. DNA in the blood of seven European insectivorous bat species and their tick and bat fly ectoparasites. These observations are the first geographical records of malaria-like bat parasites from Romania (both Po. melanipherus and Po. murinus) and Bulgaria (Po. murinus), thus further expanding the known host and geographic ranges of Polychromophilus spp. These results confirm the wide geographical distribution of both species of Polychromophilus in Europe after they were reported from the central, southern and western parts of the continent [Here we report on the occurrence of ontinent ,26,33,34Po. melanipherus was reported only from the common bent-wing bat (Mi. schreibersii), while Po. murinus had a wider host range. Polychromophilus melanipherus is a fairly common parasite of the genus Miniopterus worldwide, with at least 20 different species of bent-winged bats recorded as hosts in Europe, Africa and Australia  and later recorded in at least six other European bat species   [Polychromophilus spp. tested negative in our study. While several of these species were sampled in small numbers (n = 1–12), in the case of N. noctula, the sample size was relatively large (n = 134) from two different locations. Thus, our results indicate a low probability of infection for this bat species, at least in the investigated geographical territory.he genus , originanoctula) . Here weequinum) ,27,36,37Polychromophilus spp. was recorded in three bat species here, with values exceeding previous European records [Po. melanipherus was 67.3% in Mi. schreibersii, while Po. murinus had a mean prevalence of 56.2% in the case of My. daubentonii and an even higher level (75%) in My. myotis. Moreover, these prevalence rates were recorded at more than one site, thus suggesting a widespread presence of Polychromophilus spp. in these bat species.A high prevalence of infection with  records ,27,36,37Polychromophilus species, with 8 out of the 33 tick individuals/pools testing positive (I. vespertilionis came from Daubenton’s bat (My. daubentonii). However, the six positive pools of I. simplex , their vectorial role cannot be excluded and warrant further research. Interestingly, corresponding sequence pairs  did not show 100% identity in two of the cases recorded in this study , not an unusual property of ixodid ticks [Only 20% of ticks collected from bats in this study were found to contain the DNA of either of the two positive . Both pois study . Either hodology ) or tickid ticks . To estaid ticks .Pe. dufourii) tested positive for both Polychromophilus species. Here we report the first-ever records of Po. melanipherus in Pe. conspicua and Pe. dufourii and the first-ever records of Po. murinus in N. vexata and Pe. dufourii. Nycteribiid bat flies were proposed to be the main vectors of Polychromophilus spp. in bats [Polychromophilus spp. recorded in bat flies themselves [Polychromophilus DNA in cave-dwelling bat species known to regularly host bat flies in high abundance in Romania  had low Polychromophilus DNA prevalence or tested negative for this parasite . Bat spmurinus, ).Po. melanipherus sequences clustered with bat-related Po. melanipherus sequences from Central and Southern Europe  to samples collected from vespertilionid bats in Switzerland  and frodentity, ). Polychsee also .Polychromophilus melanipherus showed high haplotype diversity both in bats and their ectoparasites. Two genetically different haplotypes were identified in the two bat fly species collected from bent-winged bats, while I. simplex ticks also provided different haplotypes. Unfortunately, our method prevented us from evaluating the true diversity of Polychromophilus spp. haplotypes in individuals sampled , as the method deployed (nested PCR) amplified only a single sequence from each sample. The six haplogroups identified clustered into two main groups, including all ectoparasite-derived and bat-derived sequences. While all Po. melanipherus-positive bats and ectoparasites were collected at a single site (Canaraua Fetii), this site hosts a huge maternity cluster of Mi. schreibersii, with up to 8000 individuals in summer [Po. melanipherus, all of which may be encountered at the maternity roost at Canaraua Fetii.n summer ,43, but n summer  and BartPolychromophilus spp. sequences encountered in bats, ticks, and bat flies (see Po. murinus) or (ii) the presence of multiple genetic lineages of Po. melanipherus linked to different migratory subpopulations of bent-winged bats.The high diversity of individual lies see  suggest Pe. dufourii, thus suggesting vector competence for both Polychromophilus species. This dipteran species commonly occurs on a number of cave-dwelling bat species like Mi. schreibersii, My. blythii, My. daubentonii or My. myotis [Po. melanipherus to vesper bats and/or Po. murinus to Mi. schreibersii), too. The fact that we found no sign of such cross-infection in any of the host species may be an indication that there is a specific barrier of infectivity at the host level, an idea already suggested for Po. melanipherus [DNA of both haemosporidian species was identified in a polyxenous bat fly species, . myotis , i.e., b. myotis ,46,47. Tnipherus .Polychromophilus murinus in three new bat species and Polychromophilus spp. in two tick and three bat fly species, thus increasing both host and possible vector species spectra. High genetic diversity is reported for both Polychromophilus species, with diverse genetic variants present even at the same location, suggesting that simultaneous presence of diverse host and vector assemblages may enhance malaria-like parasite diversity, too.Our results expanded the known geographical range of bat-associated Plasmodiidae species occurring in the Western Palearctic, showing wide distribution among bats and their ectoparasites in SE Europe. Here we report the first records of


Staphylococcus aureus is an opportunistic pathogen capable of inducing S. aureus pneumonia (SAP), with some lineages also showing multidrug resistance. Given the high level of antibiotic resistance, much research has been focused on targeting S. aureus virulence factors, including toxins and biofilm-associated proteins, in an attempt to develop effective SAP therapeutics. Despite several promising leads, many hurdles still remain for S. aureus vaccine research. Here, we review the state-of-the-art SAP therapeutics, highlight their pitfalls, and discuss alternative approaches of potential significance and future perspectives.Pneumonia is an acute pulmonary infection associated with high mortality and an immense financial burden on healthcare systems.  Pneumonia is one of the leading causes of death in developed countries, with up to 2.56 million recorded fatalities in 2017 . PneumonStreptococcus pneumoniae, although other pathogens, both Gram-positive and Gram-negative, might also be involved . . 195]. Tribed in ), were uribed in , where 1ribed in . ASN100 ribed in . This len day 22 .S. aureus toxins and protects from S. aureus disease in multiple mouse and rabbit infection models [A final, interesting, multivalent vaccine currently under development is IBT-VO2 E. This hn models . Followin models .S. aureus blood and epithelial cell infections) and in a murine dermonecrosis model [In an interesting approach, Wolfmeier et al. used sphingomyelin liposomes F, eitheris model . SphingoBoth liposome types reduced the extent of murine dermonecrosis . It is nS. aureus virulence factors, no suitable vaccine is yet available. mAbs neutralizing single factors have been developed successfully, but many of the conducted in vitro studies did not reach clinical potential  [S. aureus infections, including endocarditis and septic shock, did not show any adverse reactions. More interestingly, eight patients survived and showed clinical improvement after 14 days of treatment [Another promising treatment option involves the bacteriophage cocktail AB-SA01 that contains three myoviruses related to occus φK . The pros (VISA) . Moreoves (VISA) . In an ereatment . FurtherBesides using the phage itself for treatment, other studies explored a possible use of phage-derived lysines. A 2015 study investigating the efficacy of LysGH15 showed that treatment with the lysine achieved 80% survival in mice with SAP. The survival rate further increased to 100% when LysGH15 was combined with apigenin, a naturally occurring flavonoid . In a seBenefits of phage therapy are the little to no adverse host reactions, generally low frequency of spontaneous resistance against the bacteriophage, and their ability to target and kill multidrug resistant clones . HoweverS. aureus vaccine candidates  in Escherichia coli as fusions to a lipoprotein leader sequence. The lipoproteins were compartmentalized in OMVs and elicited high antigen-specific Ab titers in immunized mice. Moreover, the penta-valent OMV-based S. aureus vaccine protected mice from challenge with the S. aureus Newman strain in a sepsis model [S. aureus vaccine.Another interesting approach exploits bacterial outer membrane vesicles (OMVs) as a vehicle for delivering foreign antigens into the host, with the aim of eliciting protective immune responses. For instance, Irene et al. expressed five highly conserved is model . This suS. aureus virulence factors, thus improving our understanding of their mode of action and their role in SAP pathogenesis (S. aureus (In conclusion, much effort has been invested in exploring ogenesis . Classic. aureus . Finally. aureus . SEVs an


Plant genetic resources (PGR) are the foundation of agriculture as well as food and nutritional security.The ICAR-NBPGR is the nodal institution at national level for management of PGR in India under the umbrellaof Indian Council of Agricultural Research (ICAR), New Delhi. India being one of the gene-rich countries faces aunique challenge of protecting its natural heritage while evolving mutually beneficial strategies for germplasmexchange with other countries. The Bureaus activities include PGR exploration, collection, exchange, characterization,evaluation, conservation and documentation. It also has the responsibility to carry out quarantine ofall imported PGR including transgenics meant for research purposes. The multifarious activities are carried outfrom ICAR-NBPGR headquarters and its 10 regional stations located in different agro-climatic zones of India. Ithas linkages with international organizations of the Consultative Group on International Agricultural Research(CGIAR) and national crop-based institutes to accomplish its mandated activities. NBPGR collects and acquiresgermplasm from various sources, conserves it in the Genebank, characterizes and evaluates it for different traitsand provides ready material for breeders to develop varieties for farmers. ICAR-NBPGR encompasses the NationalGenebank Network and at present, the National Genebank conserves more than 0.40 million accessions. NBPGRworks in service-mode for effective utilization of PGR in crop improvement programmes which depends mainlyon its systematic characterization and evaluation, and identification of potentially useful germplasm. NBPGR isresponsible for identifying trait-specific pre-adapted climate resilient genotypes, promising material with diseaseresistance and quality traits which the breeders use for various crop improvement programmes. The system hascontributed immensely towards safeguarding the indigenous and introducing useful exotic PGR for enhancingthe agricultural production. Presently, our focus is on characterization of ex situ conserved germplasm anddetailed evaluation of prioritized crops for enhanced utilization; assessment of impact of on-farm conservationpractices on genetic diversity; genome-wide association mapping for identification of novel genes and alleles forenhanced utilization of PGR; identification and deployment of germplasm/landraces using climate analog data;validation of trait-specific introduced germplasm for enhanced utilization.Key words: plant genetic resources; gene banks; wild relatives; biotic and abiotic stresses; marker-assistedselection. Plant genetic resources (PGR) are one of essential componentsof agro-biodiversity and defined as the genetic material ofplants having value as a resource for present and future generations.PGR hold the key to the very foundation of agricultureas well as food and nutritional security for the world. Agriculturalbiodiversity, agri- or agro-biodiversity (a subset ofbiodiversity) is defined as ‘all crops and livestocks, their wildrelatives, and all interacting species of pollinators, symbionts,pests, parasites, predators and competitors’ . Indian subcontinent has a rich and varied heritage ofbiodiversity, encompassing a wide spectrum of habitats fromtropical rainforests to alpine vegetation and from temperateforests to coastal wetlands. It is one of the eight centres oforigin  and is one of the 12 mega gene centresof the world. It possesses 11.9 % of world flora, and about33 % of the country’s recorded flora are endemic to the regionand are concentrated mainly in the North-East, Western Ghats,North West Himalayas and the Andaman and Nicobar islands.Of the 49,219 higher plant species, 5,725 are endemic andbelong to 141 genera under 47 families . Of these3,500 are found in the Himalayas and adjoining regions and1,600 in the Western Ghats alone . The conceptof biodiversity hotspots was originated by Dr. Norman Myersin two articles in “The Environmentalist” (1988), revisedafter thorough analysis by Myers and others in “Hotspots:Earth’s biologically Richest and Most Endangered TerrestrialEcoregions”. The hotspots idea was also promoted by RussellMittermeier in the popular book “Hotspots revisited”. Aroundthe world, 34 biodiversity hotspots exists as of today. Thesesites support nearly 60 % of the world’s plant, bird, mammal,reptile, and amphibian species, with a very high shareof endemic species .From centuries, tribal or traditional farming communitieshave continuously adapted and shapened the dimensions ofrich genetic material available with them. These resources ortraditional varieties or landrace populations often bear specifictraits – early or late maturing, adaptability to a particular soiltype, uses and usually have local names which has enabledthem to survive so long under various biotic and abioticstresses in the centers of diversity along with wild progenitorsof crop plants, wild and weedy relatives. Besides theseresources, potential domesticates  are involved in the PGR spectrum; they are those wildspecies, which are not yet domesticated but are extensivelyused. Some of them grow widely, though genetically andculturally in a near wild state. With richness of plant geneticresources, the tribal regions have therefore been identified as“Hot Spots” of agri-biodiversty. However, they are differentfrom the biodiversity hot-spots  which include all entire endemic biodiversity as apriority for defining hot-spots of a region.During the process of crop evolution crops originated inits centre of origin have changed from the wild progenitors inmorphological, physiological and agronomic traits to newertypes through selection for desired traits. In this whole process,agriculture , cultivation  and domestication have shapened them for appropriatePGR. PGRs are exchanged and searched continuously forspecific traits to improve crops in terms of yield and nutritionalvalue, and their interdependence plays a very important rolein international collection and exchange of germplasm. Everynation is concerned with acquisition of diverse and superiorgermplasm for conservation and utilization.Over the years, India has developed sound and scientificmanagement regimes for ex situ conservation and access to itsgenetic resources . Groups of institutions,scientific societies, non-governmental organizations are addressing the task with ICAR-NBPGR, New Delhi, as thenodal agency for its coordination. It aims at efficient managementof plant genetic resources by providing convenience ofaccess to the various crop improvement programmes. It alsoencompasses the National Genebank Network. At present, thegenebank holds more than 4.4 lakhs accessions. The CryobankFacility in the National Genebank has accessions of variedgermplasm of orthodox, intermediate and recalcitrant seedspecies and also of pollen samples. The in vitro genebankconserves various priority crops which are maintained undershort- to medium-term storage periods. These include tuberousand bulbous crops, tropical fruits species, spices and industrialcrops, medicinal and aromatic plants species.Centre of origin and diversity of crop plants are conceptsrelating to the patterns of distribution and build-up in regionsand habitats, consequent to use and domestication by manin areas representing independent agricultural systems andseparated by major geographical barriers. Various terms havebeen used to designate these ‘geobotanical’ diversity patternsin crop plants viz. primary and secondary centres of diversity,gene centres, cradles/subcradles of agriculture, megacentres,regions , non-centres, microcentres, cradles of angiosperm diversity. However, within centres of diversity, geobotanicalpatterns of variation, add a practical connotation tothe use of these concepts in PGR. The significant point, asstated above is that centres of diversity are the consequenceof continuing processes of evolution and domestication, andhence subject to change. The concept of ‘ecological passports’in different sites where different crops show parallelism incharacters by Vavilov, form the basis for understanding thediversity within the crop species, and in relation to the cropgenepool .PGR conservationThe National Bureau of Plant Genetic Resources (ICARNBPGR)was established by the Indian Council of AgriculturalResearch (ICAR) in 1976 with its headquarters at New Delhi.The chronology of events leading to the present day ICARNBPGRdates back to 1905 when Botany Division was establishedunder the then Imperial Agricultural Research Institute.ICAR-NBPGR has been given the mandate to act as a nodalinstitute at the national level for acquisition and managementof indigenous and exotic PGR for agriculture, and to carryout related research and human resources development forsustainable growth of agriculture. The Bureau is also vestedwith the authority to issue Import Permit and PhytosanitaryCertificate and conduct quarantine checks in seed materialand vegetative propagules introduced from abroad or exported for research purposes.Besides having a 40 ha experimental farm at Issapur village(about 45 km west of Pusa Campus), the Bureau has a strongnational network comprising Regional Stations/Base Centersand ICAR Institutes/SAUs that provide access to representativeagro-ecological situations in the country.The major components of the National Genebank includethe seed genebank, field genebank, cryo-bank and the in vitrogenebank. The focus of seed genebank of NGB is long termconservation of orthodox seeds. The basic parameters that areassessed for conservation are:Uniqueness of the accession. Redundancy is a major issuein all genebanks and best efforts are made to avoidduplication of accessions.Seed quality. The global genebank standards recommendsa minimum of 2000 seeds in self-pollinated crops and4000 seeds in cross pollinated crops. In wild germplasmaccessions, the minimum number has been relaxed to500 seeds.Seed viability. A minimum viability of 85 % is essentialfor seed samples. However in those cases where the IndianMinimum Seed Certification Standards have approved alower level of standard germination, the viability requirementsare accordingly modified in gene bank also.Seed health. Pest free conservation is a priority at NGB.There is a strong collaboration between the Division ofGermplasm Conservation and Division of Plant Quarantine,wherein all accessions are tested for any form of pestinfestation prior to their processing.Availability of passport information. The utilization of theconserved accessions can be facilitated only if all relevantpassport information is available in the database. Hence,only those accessions having basic information on parameterslike biological status, collection details if acquiredthrough exploration, pedigree details if it’s a breeding line/genetic stock or cultivar and any other unique trait if applicable,is accepted for long term conservation.The qualified accessions are then subjected to drying, whichis the most crucial step in genebank processing. A walk-indryingchamber functioning at 15 % RH and 15 °C is usedfor the drying purpose. For species with hard seed coats andwhich require longer drying duration are shifted to batchdryers, after preliminary drying in chamber. The standardmoisture testing method used in gene banks is the hot-airoven method and the procedure recommended for each cropby the International Seed Testing Association is duly followed.Once the desired moisture is achieved (as mentioned below),the seeds are packed in aluminum foil packets. They havethe advantage that they can be resealed and also occupy lessspace than other containers.Conservation of genetic resources is carried out throughtwo types of collections:Collection of seed samples for long term conservation,which is known as base collection. Base collections aremaintainedat –18 to –20 °C, to ensure seed viability formaximum possible time period. The moisture content ofseed to be stored as base collections should be between3 and 7 % depending on the species.Collection of seed samples for immediate use, termed asactive collection. Active collection are maintained in conditionsthat ensure at least 65 % viability for 10–20 years.The moisture content of seeds to be stored as active collectionsshould be between 3 and 8 % for seeds havingpoor storability and between 7 and 11 % for seeds havinggood storability, depending on the temperature used forstorage.These collections are conserved in different types of storagefacilities. Depending on the duration of storage, three basictypes of storages are recognized:Short term storage. The period for short term storage ofseeds is from one year upto 18 months. It requires a cool and dry atmosphere (20–22 °C and 45–50 % RH) wherethe seeds can be conveniently stored for one to two yearswithout much loss in their viability.Medium term storage. The active collection, which aregenerally larger than those meant for base collection areconserved in the medium term storage. The accessions arefor regular distribution and therefore the time period forstorage is not more than 5 yrs. The active collections arestored at temperatures ranging from 0–10 °C and relativehumidity of 20–30 %.Long term storage. This is the storage facility for basecollection, where the seeds that meet all the above mentionedcriteria for NGB conservation are maintained at–18 to –20 °C.The ex situ genebank at NBPGR comprises 12 long termmodules holding the base collection. The active collections aredistributed in 22 medium term modules maintained at 4 °C forstoring germplasm at active sites. The genebank is currentlybeing upgraded with new infrastructure and all efforts arebeing made to bring the NGB to global standards.Crop wild relativesCrop wild relatives (CWR) are wild taxa closely related to cropplants, including wild progenitors and/or wild forms of crops.Maxted et al. (2006) defined a CWR as a wild plant taxon thathas an indirect use derived from its close genetic relationshipto a crop. The closer the species related, the more the possibility/practicality to get their traits incorporated. They form animportant source of useful traits such as agronomic, quality,biotic and abiotic stresses, which are identified as criticalcomponent for food security and environmental sustainabilityin the 21st century. CWRs are often associated with disturbedhabitats and neither these habitats are offered adequate protectionby ecosystem conservation agencies nor their diversity properly conserved ex situ. CWR diversity,like that for many species, is at a declining stage; which is associatedwith the loss of genetic diversity . This necessitates the need to establish CWR inventorieswhich is also an indispensable tool for exploration, surveys andcollection of CWR. Therefore, the need for novel genes fordeveloping climate resilient varieties, increasing pressure onwild species populations and habitats and the present meagreex situ collections, all accentuate the importance of locatingand collecting germplasm of wild relatives.Arora and Nayar (1984) reported the occurrence of over320 wild relatives of crops  inIndia. The NHCP of ICAR-NBPGR serves as a nodal point forconfirming the botanical identity of crop wild relative’s taxa.With the identification of diversity-rich spots, availability oflocation details of intended taxa, India is moving forward inthe systematic collecting of CWR from diverse habitats forconservation and sustainable use. Only one third of shortlistedtaxa have been assembled by ICAR-NBPGR; among themmore than half the taxa with <10 accessions. Analysis of gapsin collection in a scientific manner  through a mission-mode approachis currently being employed the way. In addition, detailed studies on habitat ecology, floral biology and breeding system,crossability (with crop), seed dormancy and storage behaviourof species would enable their meaningful conservation andsustainable utilization. Crossability studies aids in realizationof gene-pool concept in crops, and knowing the closer relatives(even from different genera). Ensuring correct taxonomic identity,safe conservation and supply of germplasm to crop-basedinstitutes would strengthen the pre-breeding/base-broadening/gene-pyramiding activities through designing suitable longterm multi-parental breeding programmes. All these indicatethe need for trained expertise in classical subjects like taxonomyand cytogenetics, with long-term commitment. Also itis imperative to undertake studies on assessing the gene flowbetween wild and cultivated taxa in the wake of concerns of biosafety. Alltaxonomic related species may not have an equal potential asa gene donor to crops . Prioritization ofCWRs for management preferably on genetic relationship isimportant for optimization of resources. Economic importanceof the crop, crossability relationship, threat and rarity of thetaxa and habitat, conservation status in the genebank are theother criteria for prioritization.Conservation of niche-specific taxa needs attention as theyare often rare and endemic. Predicted extinction of speciesis more likely to affect RET taxa. Various steps involved inthe effective management of CWRs such as development ofan inventory, prioritization of CWRs taxa and habitats, ecogeographicand genetic analysis of CWRs, threat analysisand genetic erosion assessment of individual CWRs taxa,gap analysis and fixing conservation targets, development ofex situ/in situ strategies, leading to conservation and finallyutilization and sustainable availability for crop improvement are all important in the Indian contextalso. Constituting specialized group in the country devoted tothese aspects of CWRs may be a feasible option.The above studies would facilitate a national-level mappingof CWR distribution after incorporating additional informationfrom eco-geographic studies, which will help in theidentification of CWR hotspots, which can be matched withexisting protected area network in the country, thereby areasand taxa demanding conservation can be identified . Strong networking among all the stakeholdersworking on characterization, evaluation and conservation isthe need of the hour, as it is difficult for a single institute tocollect, conserve and evaluate all the target species due topaucity of land, resources and expertise.PGR characterization and evaluationThe utilization of PGR in crop improvement programs restson identification of promising accessions. The collected orintroduced germplasm is characterized and evaluated to assessits potential, by recording data on agronomic traits such asyield, quality, and tolerance to biotic and abiotic stresses. Thegermplasm is also evaluated for new traits using moleculartools to identify the genes to develop new varieties as perrequirement of the farmers. Approximately 10,000 accessionsare characterized/evaluated every year at ICAR-NBPGR andits regional stations. Till date, more than 2.35 lakhs accessionsof different agri-horticultural crops have been characterizedand evaluated and passport data is available.Core sets have been developed to facilitate the enhancedutilization of germplasm. Genetic diversity in large collectionhas been determined using morphological and DNA fingerprintingmarkers. Mega programme on characterization andevaluation under the National Initiative for Climate ResilientAgriculture (NICRA) executed in collaboration with SAUs for21,822 accessions of wheat and 18,775 accessions of chickpea.Generation, validation and utilization of genomic resources isone of the major objective of ICAR-NBPGR. These resourcesare utilized for value addition to the plant germplasm resourcesharboured in the genebank and for generating molecular profilesvarieties of agri-horticultural crops. The advent of nextgeneration sequencing with improved chemistries and lowerinput costs have resulted in high throughput data that couldbe mined for generating SSR and SNP markers.Application of genomic toolsfor PGR utilization and pre-breedingOne of the major objectives of ICAR-NBPGR is to supplygermplasm, collected indigenously or from exotic sources, tothe breeders and other researchers in the country. These germplasmaccessions have helped to develop improved varietiesin various national programmes. Till date more than 5 lakhsamples were supplied for utilization to various stakeholdersfor use in crop improvement programmes. Ever-increasingsignificance of conservation and utilization of PGR on onehand and advancements in computer technology for digitizationand management of data on the other have catapultedPGR Informatics into limelight.Genomics has provided various technologies includingsequencingand re-sequencing platforms, availability of genomesequences as references, high-throughput genotypingplatforms, SNP arrays, genome editing tools, etc. These technologiesare shortly described here.Genome sequencing. The inexpensive sequencing andresequencing technologies are the major driving forces behindincreased number of assembled plant genomes of differentcrops including wild relatives. A single reference genomedoes not represent the total diversity within a species, hence,resequencing of cultivars, landraces and wild accessions is requiredto harness the total genetic variation and to identify thesuperior alleles for the target traits. Genome information availabilityhas generated many next-generation sequencing-basedplatforms for allele mining and candidate genes identification.Next generation sequencing and whole-genome resequencingis required for discovery, validation, and assessment of diagnosticmarkers in different crops and it provides genome-widemarkers. The draft genome sequences are now available in anumber of crops through different genome sequencing consortiafor rice International Rice Genome Sequencing Project(IRGSP 2005), pigeonpea , chickpea, wheat International Wheat GenomeSequencing Consortium (IWGSC 2018), etc.The genome sequencing using NGS has resulted in largecollections of functional markers which enhance gene assistedbreeding, reducing the possibility of losing the desirable traitvariation due to recombination. Sequencing and resequencingof populations developed in crossing programs or of naturalpopulation (germplasm) along with high-throughput phenotypinghelps in identification and linking of variations in gene sequences to their phenotypes. Kim et al. (2016) reported thewhole-genome resequencing of the 137 rice mini core collection,potentially representing 25,604 rice germplasms inthe Korean genebank of the Rural Development Administration(RDA) based on the Nipponbare reference genome, andresequencing data yielded more than 15 million SNPs and1.3 million INDELs. Further study of this rice mini core withphylogenetic and population analysis using 2,046,529 highqualitySNPs successfully assigned rice accessions to therelevant rice subgroups, suggesting that the SNPs capture evolutionarysignatures present in rice subpopulations. Similarly,a population structure analysis of 300 rapeseed accessions was carried outbased on the 201,817 SNPs obtained from sequencing, dividedaccessions in nine subpopulations .However, hierarchical clustering and principal componentanalysis showed intermingle of spring type accessions withsemi-winter types pointing out towards frequent hybridizationbetween spring and semi-winter ecotypes in China.Sequence-based markers associated with rare elite allelesfacilitate positional cloning and prebreeding. In case of PGRincluding landraces and wild relatives, screening of collectionto be used for genomic analysis can be done based on passportdata  in combinationwith evaluation data. Sequencing based approaches provideopportunity to identify novel variations for a large number ofgenes through genotype-phenotype associations. Resequencingof large number of genotypes helps in determining processof origin, domestication, population structure and identifieslines with deleterious mutations in the genomes that can beeliminated to minimize the genetic load in the crop speciesas observed in case of maize . NGS technologiestogether with precise phenotyping have been usedfor identification of marker trait associations in several crops,for example, rare wheat haplotypes effective against abiotic orbiotic stresses were developed through introgression of usefuland novel stress and quality traits’ alleles to lines derivedfrom crosses of exotics with CIMMYT’s best elite germplasmunder CIMMYT’s Seeds of Discovery (SeeD) initiative . Singh et al. (2018) used next-generationsequencing, together with multi-environment phenotyping tostudy the contribution of exotic genomes to 984 three-waycrossderived(exotic/elite1//elite2) pre-breeding lines (PBLs)for accelerating grain yield gains using exotic wheat geneticresources.Molecular markers and genetic maps. Recent developmentsin genome sequencing and or resequencing has resultedin development of large number of molecular markers indifferent crops. Availability of molecular markers linked tospecific traits enhances pre-breeding efficiency and effectivenessthrough marker assisted selection (MAS). Molecularmarkers that are linked to the genes of a desired trait knownas diagnostic markers can be indirectly used for selection oftarget traits . A major earlier success forcrop breeding using genomic markers was the marker-assistedintrogression of the ethylene response factor, known as Submergence1A (Sub1A) gene, for submergence tolerance intohigh-yielding commercial rice varieties which acts by limitingshoot elongation during the inundation period . Riar et al. (2012) used polymorphic D-genomespecificSSR markers for analysing the cosegregation of the5DS anchored markers with the rust resistance in an F2 population, and mapped theleaf rust resistance gene  on the short arm of wheat chromosome 5D.Vikal et al. (2014) used SSR markers for pyramiding of candidategenes for xa8, the resistance gene against Bacterial blightdisease in elite rice varieties. Ellur et al. (2016) incorporated anovel Bacterial blight resistance gene Xa38 in variety PB1121from donor parent PR114-Xa38 using a modified markerassistedbackcross breeding (MABB) scheme.Genomics has provided powerful approaches to understandinteraction between many genes and complex signalling pathwaysin case of polygenic traits like resistance to abiotic andbiotic stresses. In rice breeding, high-density genome mapsare being effectively used in background selection integratedwith foreground selection of bacterial blight resistance (xa13and Xa21 genes), amylose content (waxy gene) and fertilityrestorer gene in order to identify superior lines with maximumrecovery of Basmati rice genome along with the quality traitsand minimum non-targeted genomic introgressions of the donorchromosomes . Quantitativetrait loci (QTL) analysis of the genome linked to quantitativephenotypic traits, has yielded climate goverened QTL indiverse crop species . Rodrigues et al.(2017) determined protein content and genetic divergenceof twenty-nine soybean genotypes using 39 microsatellitemarkers from QTL regions of the trait grain protein contentfor plant breeding purposes. The pairs of genotypes withgreater genetic distances and protein contents were selected toproduce populations with higher means and genetic variancesand greater gains with selection.Genome wide association studies (GWAS) could overcomeseveral constraints of conventional linkage mappingand provide a powerful complementary strategy for dissectingcomplex traits. GWAS make use of past recombinations indiverse association panels to identify genes linked to phenotypictraits at higher resolution than QTL analysis. GWAS hasbecome a powerful tool for QTL mapping in plants because abroad range of genetic resources may be accessed for markertrait association without any limitation on marker availability.Different approaches used for GWAS include:SNP marker arrays or SNP chips approach. Discovery andtagging of new genes using GWAS or QTL analysis havenow become much easier. The availability of high-densitySNP marker arrays has opened a way for cost effectiveGWAS using natural populations. Wang et al. (2017) developeda high-throughput NJAU 355K SoySNP array andconducted GWAS in 367 soybean accessions (including105 wild and 262 cultivated) across multiple environmentsand reported a strong linkage disequilibrium region onchromosome 20 significantly correlated with seed weight.Zhao et al. (2019) carried out meta-analysis GWAS using775 tomato accessions (including wild accessions) and2,316,117 SNPs from three GWAS panels and discovered305 significant associations for the contents of sugars,acids, amino acids, and flavor related volatiles.Genotyping by sequencing (GBS) approach. As the costof sequencing is continuously declining, GBS also known as next generation genotyping method, is becoming morecommon for discovering novel plant SNPs and used themfor GWAS studies . Kim et al. (2016)reported the whole-genome resequencing of 137 rice minicore collection and conducted genome wide associationstudies on four agriculturally important traits including‘grain pericarp colour’, ‘amylose content’, ‘protein content’,and ‘panicle number’ and identify some novel alleles.Similarly, Arora et al. (2017) genetically characterized177 A. tauschii accessions using GBS to study the variationfor grain size using genome-wide association study.Genomic selection. Genomics assisted breeding approachknown as genomic selection (GS) is a better approach whichsimultaneously uses large genotypic data (genome wide) (exceedingphenotypic data), phenotypic data and modelling usingstatistical tools to predict the genomic estimated breedingvalues (GEBVs) for each individual . In genomic selection,a statistical modelis generated using a representative population of the breedingpopulation known as training population. This model issubsequently used to calculate the allelic effects of all markerloci, i. e. genomic assisted breeding values without havingphenotypic data and these values can be used for preselectionof trait-specific genotypes . Xu etal. (2012) and Spindel et al. (2016) highlighted that couplingof genome wide data with genomic selection offered greatspecificity and predictability which can be used to accelerateprebreeding. Using GS, complex traits can be improved rapidlythrough generation of reliable phenotypes by shorteningthe selection cycle. GS application in pasture grass Loliumperenne resulted in four-year reduction in the breeding cycle. In genomic selections, genomic estimatedand true breeding values were found to be closely correlated,even for polygenic traits with low heritability . GS can facilitate selection of complex traits, e. g., grainyield  and tolerance to abiotic andbiotic stress.In genomic selection, genetic diversity specific to the populationor family (species) of interest is captured throughmarkers developed through GBS which minimized the ascertainmentbias. GS is superior in respect of fixing all the geneticvariation and to select individuals with higher GenomicBreeding Value (GEBV) without any phenotyping. In case ofpolyploid species, polysomic inheritance and possibility ofdouble reduction requires specific consideration while usingfor genomic selection.Genome editing. Recent advancements in genomics havealso made feasible the editing of genomes and their use incrop improvement programs. Pre-breeding involves genetictransformation through recombination and genome editing(GE) tools provide an alternative. To replace conventionalgenetic engineering, a number of genome editing technologieshave been developed during last two decades includingantisense, RNA interference (RNAi), virus-induced gene silencing(VIGS), oligonucleotide directed mutagenesis (ODM),zinc finger nuclease (ZFN), transcription activator-like effectsnucleases (TALENs), and clustered regularly interspacedshort palindromic repeats/Cas9 (CRISPR/Cas9) . These genome editing technologies can acceleratepre-breeding programs through beneficial knockout mutations, e. g. identification of genes for disease resistance or suppressingof unwanted traits linked with desired traits in wild species,as products of these technologies are not considered agenetically modified organism (GMO) .Most of these genome editing tools except RNAi, act by inserting,removing or replacing specific regions of genome withthe help of specific nucleases known as “molecular scissors”.GE approaches can be used to modify genes with definedquantitative trait nucleotides (QTNs) that cause a sizeable phenotypiceffect. Further, GE tools can be used for broadeningthe allele pool through generating targeted variations usefulfor genomic selection . Using GE tools, desired traits can be physicallylinked to ensure their co-segregation known as “trait stacking”. For example, ZFN-assisted gene targetinghelped insertion of heritably insert herbicide-resistant genes(SuRA/SuRB and PAT) in the Z. mays genome . Zhang et al. (2016) recently used CRISPR/Cas9system for production of homozygous transgene free wheatmutants. Jenko et al. (2015) showed that GE and GS, bothcan be combined and referred as the promotion of allelesby genome editing (PAGE) which also has a great potentialfor pre-breeding. Recently, Gupta (2019) reviewed the latestmodification of CRISPR/Cas9 system, a base editing technologyapplicable to DNA as well as RNA, has revolutionizedGE and demonstrated in several crops including rice, maize,wheat, etc. will be highly useful for base broadening to beused for genomic selection and relating phenotypes to genesthrough mutant development, particularly in primitive landracesand wild species and will provide a new direction topre-breeding programmes.PGR InformaticsPGR Informatics is the management  and analyses  of diverse information . PGR Informatics has assumedsignificance because of the following factors: (i) increasedawareness about PGRFA, (ii) various international agreements coming into force, (iii) availability ofinformation in text, images, maps, videos, etc., (iv) technologiesto record, link and archive such diverse types of information,(v) growing power  of computers andinternet to facilitate access and retrieval. Fundamental meritof an organized digital information system is that it providesfair and just opportunity for all to access. On-line portals, as aconsequence of PGR Informatics, enable non-exclusive accessto PGR information to a large number of users involved inoverlapping research areas on PGR management. Typicallyinformation is collected on details of multitude of passportdata including taxonomy, biogeography, and ethnobotany ofthe germplasm acquisitions (domestic collections and exoticintroductions), their seed health, multiplication for supplyand conservation, regeneration, experimental data on characterizationand evaluation leading to utilization. In additionto field data, it also includes biochemical and genomic dataas well as publications. Once the information is digitized andstored, computer technologies allow management and analysis irrespective of the scale and types of data leading to bettervisualization and predictions.The need for countries to develop, maintain and exchangeinformation “from all publicly available sources, relevantto conservation and sustainable use of biological diversity”including “results of technical, scientific and socio-economicresearch” has been recognized in the Convention on BiologicalDiversity , and the Global Plan ofAction . Information ofthis nature is imperative for planning and implementing activities;sustainable use and sharing of benefits accrued fromits use. Global assessment indicates that many of the world’sPGR are insufficiently and poorly documented. The passportinformation and characterization and evaluation data ongenebank accessions conserved in genebanks are either lackingor poorly recorded or scattered at different places, suchas passport data sheets, reports of collection and explorationmissions, crop catalogues, published articles, etc. In addition,there exist informal or non-coded knowledge held by traditionalfarmers and indigenous people. To use this informationefficiently and effectively, valuable information needs to becollected, collated, maintained and exchanged with the helpof PGR Informatics.Important PGR Informatics applications developed andmaintained at NBPGR are:pgrportal.nbpgr.ernet.inPGR Portal exchange.nbpgr.ernet.inexchange.nbpgr.ernet.inImport Permit and EC Data Search genebank.nbpgr.ernet.ingenebank.nbpgr.ernet.inGenebank Dashboard PGR Map pgrinformatics.nbpgr.ernet.in/pgrmap 5pgrinformatics.nbpgr.ernet.in/nhcpNational Herbarium of Crop Plants pgrinformatics.nbpgr.ernet.in/cwrBiosystematics Portal pgrinformatics.nbpgr.ernet.in/pgrclimPGR Climate http://pgrinformatics.nbpgr.ernet.in/ip-pgr/PGR and IPRs Recent advances in PGR Informatics in IndiaNBPGR has been striving to establish PGR information set upsince 2002 . Development of mobileapps in PGR Informatics facilitates enhanced access to PGRinformation which in turn could lead to enhanced utilization.NBPGR has developed two mobile apps “Genebank” and“PGR Map”. Both the apps are first of their kind for anygenebank in the world. The apps have been developed for bothAndroid and iOS. No other ICAR app is available for iPhone.Licenses were purchased and the apps have been hosted onGoogle Play and App Store.Genebank app provides a dashboard view of indigenouscollections (state-wise), exotic collections (country-wise),addition of accessions to genebank, etc. The app also helpsgenerate routine genebank reports. The app uses databases liveon the backend and hence always gives updated information.PGR Map app offers three benefits: “What’s around me”helps user to obtain quickly the accessions that have beencollected and conserved in the genebank from a particularlocation in India where the user is located at the moment;“Search the map” helps user to list the accessions that havebeen collected and conserved in the genebank from anyselected location in India; “Search for species” helps user tomap the collection sites of a crop species.PerspectivesAll countries are interdependent for their PGR requirementsand cannot acquire and conserve resources to satisfy all theirneeds. There is a need to collaborate at local, regional andinternational levels for the acquisition and conservation of thegermplasm. It is also imperative to obey the quarantine andbiosafety rules for the safe movement of germplasm. Prioritycollection trips are required to identify areas which are notsufficiently covered so far and a repeated visits are required tobe made in areas that showed diversity in the past. New toolsof geographical information system (GIS) and remote sensingneed to be deployed to supplement the existing ground datain PGR programmes to exploit agro-biodiversity particularlyin difficult/inaccessible areas.There is a need to adopt complementary conservation strategiesinvolving both in situ and ex situ approaches. For in situconservation due attention is required to be given to geneticallyrich hotspots including tribal belts and to strengthen andexpand the network of germplasm conservation by includingall the stakeholders, including the communities. Characterizationand evaluation are essential to promote the utilizationof materials. These tasks require substantial inputs and a decentralizedevaluation network. There is a need to modify thedescriptors for evaluation accordingly, and make the search forthe desired characteristics in the database as quick and efficientas possible. The core collection concept is more structured andefficient approach to identify limited sets of diverse germplasmand utilise the same more effectively. Pre-breeding is needed toincorporate new kinds of pest resistance, to bring in new levelsof productivity and stability of performance, and to providequality traits for food and feed products. Awareness generationof the people at various levels  about the value of PGR wealth,its protection and conservation is essential. In addition theinterface among different stakeholders is likely to bring outnew useful PGR management alternatives.The authors declare no conflict of interest.Archak S., Agrawal R.C. PGR informatics at the National Bureau ofPlant Genetic Resources: status, challenges and future. 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Whether priority effects may influence competition in Tribolium remains to be tested. The present study examined the importance of priority effects in competitions between two common species of flour beetle (Coleoptera: Tenebrionidae): Tribolium castaneum and T. confusum. To investigate whether priority effects confer competitive advantages to Tribolium beetles, relative introduction times of T. castaneum and T. confusum to competitive arenas were manipulated, and adult populations were measured for seven months. Four important patterns were noted: (1) Tribolium species given two-weeks priority access to experimental arenas attained larger populations than their late-arriving competitor, (2) when founding adults were introduced simultaneously, T. castaneum was competitively dominant, (3) T. castaneum benefited more from priority arrival than T. confusum, and (4) available bran resources largely predicted population decline in adult beetles toward the end of the experiment. These results suggest competitive outcome in Tribolium is not always predicted by species’ identity, and that performance could instead be determined by the timing of species’ arrivals and available resources.Flour beetles are a classic model system for studying competitive dynamics between species occupying the same ecological niche. Competitive performance is often interpreted in terms of biological species traits such as fecundity, resource use, and predation. However, many studies only measure competitive ability when species enter an environment simultaneously, and thus do not consider how the relative timing of species’ arrival may determine competitive outcome ( Tribolium flour beetles (Coleoptera: Tenebrionidae) are excellent model organisms for testing hypotheses in genetics and population ecology [T. castaneum (Herbst) and T. confusum  could cause the elimination of one of the two species. Despite some level of unpredictability in outcome [Tribolium could be influenced by adjusting initial environmental conditions, such as humidity and temperature. Park’s experiments suggested competitive performance is not intrinsic to a species, and that the outcome of interspecific competition depends upon the initial conditions of an ecological space. ecology –3, espec ecology –6, was f outcome , Park [4 outcome  revealedi.e., “priority effects”), competitive performance appears to be determined by both the relative order of arrival and the amount of time between the arrivals of the interacting species [Tribolium beetles in competition research, priority effects in this system have not been measured.More recent investigations of animal competition emphasize the importance of historical contingency in predicting competitive performance and community assembly ,9. Speci species –17. Prio species , ectomyc species , and ins species –22. HoweTribolium have strongly supported the role of competitive interactions in structuring ecological communities [Tribolium species may be influenced by behavioral and ecological characteristics such as reproductive interference [T. confusum and T. castaneum under identical environmental conditions. The following predictions were made: (1) if priority effects determine interspecific competition more so than species identity, reversing the order of species arrival should reverse the outcome; and (2) when competitors arrive simultaneously, competitive outcome will be determined by how species characteristics  are expressed within the experimental environment. This study contributes to a growing body of research that emphasizes the role of historical contingency in animal competition and community assembly.Experiments involving munities . This rerference , intragurference  as well rference ,5,26. HoTribolium castaneum (Herbst) and T. confusum  are widely used in population ecology research due to their ease of culture in laboratory conditions and their economic importance as common grain pests [T. castaneum and T. confusum are more genetically related to each other than to any other member of the Tribolium genus [in pests . Consequum genus ,29. The um genus . Both spum genus .Tribolium appears to be highly sensitive to relative humidity (R.H.) and temperature, though optimal living conditions differ slightly between species [i.e., egg to adult), and may lay 2–16 eggs per day [T. castaneum is fastest in warm and humid environments (35–37.5 ºC and >70% R.H.), whereas T. confusum performs best in slightly cooler conditions (32.5 ºC and 70% R.H) [Tribolium competition studies [Fecundity and generation time in  species ,5. Both  per day . In favo per day , though  per day . Develop70% R.H) . It is i studies .Tribolium castaneum and Tribolium confusum using 200 stock adults purchased from Ward’s Science, Rochester NY. Seven months before trials, beetles were kept in a special bran medium which had been conditioned to 26 ± 1°C and 55 ± 5% R.H. All beetles were kept in medium consisting of eight parts bran, four parts flour, four parts brewer’s yeast, and one part Fluker’s Cricket supplement .Colonies were established for T. castaneum and T. confusum, beetles were assigned to three treatments in which relative introduction times were manipulated for each species, and two single-species populations as controls. Each treatment consisted of ten competitive arenas (5 x 5.5 x 3 cm Tupperware enclosures) containing an initial five g of medium (see above for proportions). In single-species populations, 20 adult beetles  were introduced to ten arenas and allowed to colonize the experimental medium without any interspecies interactions. In priority treatments, species arrival was manipulated by allowing a prior population of ten adults of either species to remain isolated until introducing ten adults of the second species two weeks later. In CF Priority, T. confusum was introduced the experimental medium first, whereas in CS Priority, T. castaneum was given early arrival. Immediate competition of Tribolium was also examined, when ten adults of both species were added to the arenas at the same time . Thus, once trials began, all 50 arenas contained an initial combined total of 800 adult beetles, and 200 beetles added two weeks later. All beetles were selected at random from the stock colonies, without preference for age or sex, and maintained at the environmental conditions described above throughout the experiment. Every 30 days, , live adult beetles were sifted from the medium, weighed, counted, and identified to species. Adult cadavers were also counted and identified when possible. Live and dead adults and larvae were then returned to the arenas along with the medium. Populations were measured seven times during the experimental period . Population measurements ceased when adult T. castaneum populations collapsed in all treatments during the final months of the study.To test the effect of priority establishment on competition between Tribolium species was also estimated by measuring the mass of the residual medium, which also contained unidentified eggs and larvae. Substrate was returned to the arena after each census. Fresh bran substrate was not added at any time during the experiment.Finally, monthly resource use between post-hoc tests were performed to assess significant differences between treatment means. Tribolium species were considered to be competitively successful if their average adult population was significantly larger than its competitor species during the experimental period.A generalized linear model  was used to measure the effects of treatment, beetle species, and their interactions on the adult beetle populations. If fixed-effects were significant overall, post hoc Tukey HSD test was used to examine differences in average beetle survivorship between each treatment. If net substrate loss significantly affected survivorship, a simple linear-regression was used to assess the relationship between substrate levels and beetle survivorship.Adult populations appeared to collapse in treatment groups between month six and seven the experiment. To examine whether declining substrate levels impacted survivorship in beetle populations, a three-factor ANOVA was used to examine the effect of treatment, beetle species, net substrate loss, and their three-way interactions on the proportion of adults in month seven relative to month six . Prior to the analysis, values of survivorship were transformed using a log(x+1) transformation. To determine relevant factors in the model, non-significant effects were eliminated in a stepwise manner using a Bonferroni-adjusted threshold for significance (α = 0.0071). If treatment significantly affected survivorship, a Tribolium species and month (1–7) were treated as fixed-effects of substrate mass (g). Each population  was fitted as a random effect. All statistical tests were performed in JMP Pro [Finally, a repeated measures linear mixed-model was used to estimate bran consumption by beetles during single-species trials.  JMP Pro . Means aT. castaneum; 19.0 ± 1.5 in T. confusum). An overall significant effect of treatment  and beetle species  on adult Tribolium populations was supported by the generalized linear model. An interaction between beetle species and treatment was also supported by the analysis . Thus, both T. castaneum and T. confusum populations were not equally affected by treatments . Over the seven month experimental period, T. confusum single-species populations contained an average of 46.8 ± 2.5 adults, whereas T. castaneum had only 28.1 ± 2.5 adults .When 12, 67 = 16.2, p<0.0001, 4, 67 = 5.36, p = 0.0008), and three-way interactions between treatment, beetle species, and net substrate loss  and when beetle were added to arenas at the same time (0.20 ± 0.069). Survivorship was highest in CF Priority and CF Solo . Survivorship in CF Solo was significantly higher than CS Solo (p<0.0001), CS Priority (p = 0.0004), and when beetles were added simultaneously to arenas (p = 0.0158). Adult survivorship was also significantly higher in CF Priority, relative to CS Solo (p<0.0016) and CS Priority (p<0.0342). In all other treatment comparisons, beetle survivorship was approximately even between species . When comparing beetle survivorship between species, net substrate loss explained 51% of the variation in T. castaneum survivorship , according to a mixed model analysis. However, substrate disappeared more rapidly in T. castaneum populations than T. confusum , despite T. castaneum attaining smaller average populations  in similar experimental conditions is typically 30 days [Tribolium are known to cannibalize eggs [T. castaneum, populations also appeared to collapse after month six, which was correlated with reduced quantities of the experimental medium. Adult populations therefore may have been impacted by the combined effects of food limitation and cannibalism between life stages. However, unlike T. castaneum, single-species populations of T. confusum did not collapse. It is difficult to predict whether T. confusum in single-species trials would have persisted beyond the seven month experimental period. 30 days . Adult Tize eggs ,41, whicT. castaneum attained higher population sizes than T. confusum. Successful competition of T. castaneum during immediate competition which could be explained by this species’ enhanced reproduction in relatively warm and moist environments [T. castaneum over T. confusum  [T. confusum in single-species populations were higher than T. castaneum, which is unexpected if T. castaneum development was unevenly favored by the experimental climate.When beetles were introduced simultaneously to arenas, ronments . All pha70% R.H) . HoweverT. confusum received priority establishment, T. confusum attained larger populations than T. castaneum, but performed worse relative to single-species trials. Thus, interspecies interactions appeared to reduce population growth in T. confusum, despite successful competition overall. Similarly, T. castaneum was competitively dominant when given priority access to arenas, and average population size was nearly the same as immediate competition and single-species trials. Thus, priority arrival benefited T. castaneum performance more than T. confusum. Given the present experimental conditions, T. castaneum receives a larger interspecific competitive advantage from priority than T. castaneum. Other studies investigating competition between insect herbivores, have similarly described how priority unevenly improves species’ performance [T. castaneum, which allowed this species to benefit more from priority than T. confusum. However, because this study was conducted under the same temperature and humidity levels throughout, it is uncertain whether T. confusum would have equally benefited from priority had experimental conditions been more favorable. Future study could determine whether the importance of priority arrival in competition is constrained by how much the environment already favors the early arriving species.Competitive outcome for both species was strongly predicted by priority arrival. When formance ,43. In [formance  a nativeT. confusum and T. castaneum obtain from the two-week period are unclear. Previous research investigating Tribolium behavior suggests egg and larval predation is the primary source of interspecies interactions [T. castaneum females more than T. confusum [T. castaneum populations in immediate and priority competition did not differ from their single-species controls. Thus, colonization priority may allow Tribolium to produce enough eggs so as to minimize predation costs from the larval and adult stages of a late-arriving species.Precisely what advantages ractions –46. Egg confusum ,48, whicTribolium populations are known to release toxic metabolites as waste products into the surrounding medium which can disrupt reproductive success of other competitors [T. castaneum appears to be more negatively impacted by conditioned medium than T. confusum [Tribolium populations may have become nutrient limited. If true, Tribolium benefits from early-arrival by preempting high quality resources, which is a primary mechanism underlying priority effects in plant and animal communities [The effect of priority may be enhanced when early arriving species impact their environment in a manner that limits the success of late-arriving species . For inspetitors ,50. Chemconfusum . In addimunities .Callosobruchus chinensis reduced the egg production of C. maculatus females much more than when C. maculatus attempted copulation with C. chinensis females [Tribolium, female fecundity in T. castaneum becomes more inhibited as the density of adult T. confusum males increases [A final, alternative explanation may involve asymmetrical reproductive interference arising between the two competitors, which in some cases can drive species exclusion more often than resource competition . For ins females . This asncreases , and thiT. castaneum populations most strongly impacted. In single-species populations, total bran mass in T. castaneum trials declined more rapidly than T. confusum trials, despite T. castaneum attaining smaller monthly populations.Adult beetle populations appeared to be limited by available bran substrate, with T. confusum populations appeared to collapse only in immediate competition trials, and when T. castaneum adults were introduced two weeks prior. T. confusum survivorship was near 100% during single-species trials and when given priority arrival, which suggests competitive interactions, in addition to limited food, prevented long-term establishment. In contrast, T. castaneum populations unexpectedly collapsed in all treatments except when T. confusum received priority.In all trials, adult survivorship between month six and seven differed across treatments. T. castaneum was more negatively affected by limited resources than T. confusum. For both species, adult survivorship was highest when less substrate was consumed. Substrate consumption also increased with population size, which explains why survivorship was highest for both species in arenas with smaller populations, such as when T. confusum received priority. In competition studies where food is not renewed, coexistence appears to be stable for much longer than in environments where resources are continuously resupplied [Tribolium, competitive pressures may reduce population growth in both species, thereby reducing the rate of resource decline [Adult survivorship in both species was strongly predicted by total substrate loss, however supplied ,56. In T decline . While tTribolium has been documented by previous competition studies and is typically attributed to excessive cannibalism [Tribolium population growth in high densities [T. castaneum may be exacerbated by nutritionally deficient medium, which can lead to the “near-suicide” of populations [T. castaneum could be compensating for nutrients missing from the substrate environment by increasing predation on egg and immature stages. In the present study, the experimental medium contained brewers’ yeast, which can provide the nutrition homogenous grain mixtures may lack [Tribolium collapse. Additionally, given that reduced survival was observed in Tribolium adults, increased egg or larval cannibalism cannot fully explain the population collapse. These patterns instead suggest that Tribolium populations are sensitive to limited resources, and may have exceeded the carrying capacity within the experimental environment.Population collapse in nibalism ,26. Cannensities ,59. Howeensities . Additioulations . In suchmay lack ,61. Howe


Caenorhabditis elegans, communicate environmental and developmental information with conspecifics through a class of small-molecule pheromones termed ascarosides . Nematodes share ascaroside signaling pathways , but are also capable of eavesdropping on chemical signals of predatory species . Ascarosides signal vast arrays of information, either individually or as blends, based on concentration, sex, physiological state, and other ascarosides sensed . For instance, octopamine-succinylated ascaroside #9 (osas#9) is able to signal starvation conditions in the absence of other ascarosides .Nematodes, such as the model organism C. elegans (Cel) is an androdioecious species, with the majority of the natural population comprised of self-fertilizing hermaphrodites, and a small proportion (<0.2%) being male . There are two other similarly androdioecious species in the genus, C. briggsae (Cbr) and C. tropicalis (Ctr). All three species evolved their hermaphroditism separately and uniquely . Of the male-attracting ascarosides secreted by C. elegans , ascr#8 is the most potent . Since ascr#8 is a male attractant in this hermaphroditic species, we asked if other hermaphroditic species retained the ability to attract males using this cue. Males from the gonochoristic  sister species to C. briggsae and C. tropicalis – C. nigoni (Cni) and C. wallacei (Cwa), respectively – were also assayed for their ability to respond to ascr#8. The closest relative of C. elegans, the gonochoristic C. inopinata , which has been recently characterized , was also tested, along with the Japonica Group gonochoristic species C. japonica (Cja) and C. afra (Caf).et al., 2016). Dwell times were transformed using a Base 2 Exponentiation  to generate only non-zero data in order to calculate fold-changes. The Logbase2 of the fold-changes was then calculated to normalize the data. All data sets were first checked for normality using a D’Agostino & Pearson normality test before comparisons were performed. Species in which both vehicle and ascr#8 dwell times were normally distributed were compared using a paired t-test, while those in which one or both data sets were not normally distributed were compared using a Wilcoxon matched-pairs signed rank test (GraphPad Prism 7.03). Transformed values were compared between related species via an unpaired t-test.Dwell times were analyzed as previously described using a Spot Retention Assay . The other well studied hermaphroditic model species, C. briggsae, responded significantly, although not robustly as C. elegans, spending approximately 40 seconds in ascr#8 compared to C. elegans’ dwell time of nearly 150 seconds. . Surprisingly, the sister-species of C. briggsae, C. nigoni, responded in a more robust manner . Similarly, while C. tropicalis exhibited no response to ascr#8 – its dwell time in ascr#8 being no different than that of the vehicle control  – C. wallacei spent significantly more time in ascr#8 than the vehicle , and therefore exhibited significantly more attraction than C. tropicalis . These data suggest that females of gonochoristic species have retained their ability to attract males via ascr#8 signaling, while in androdioecious species, this communicatory mechanism is lost, potentially due to a lack of selective evolutionary pressure. However, neither Japonica Group member responded to ascr#8, nor did the closest C. elegans relative, C. inopinata . This lack of attraction to ascr#8 may be due to the highly specialized insect-commensal life-cycle of C. inopinata. This species may have lost the necessary olfactory receptors to sense ascr#8 within its reduced genome , in a manner similar to Trichinella .C.briggsae and C. nigoni both inherited mechanisms to sense ascr#8. However, given the recent evolution of C. briggsae  , the reduced response compared to C. nigoni indicates less selective pressure to retain ascr#8 sensing mechanisms. Similarly, C. tropicalis likely evolved the ability to self-fertilize even before the nigoni-briggsae split , leading to its inability to respond attractively to ascr#8. Together, these data suggest that ascr#8 functions as a C. elegans species-specific male attractant, with other species having lost the necessary mechanisms to sense and respond to this chemical. As different neurons and receptors may play roles in the ascr#8-mediated dauer development pathways, the comprehensive ability to sense this pheromone in the species tested is yet to be determined.Given that hermaphroditism evolved multiple times during nematode evolution, it is plausible that C. elegans strain CB1489 (him-8(e1489)) was obtained from Maureen Barr at Rutgers University. RE980 (C. briggsae him-8(v188)) and RE1017 ) were generously provided by Ronald Ellis at Rowan University. The C. wallacei (JU1873), C. nigoni (JU1422), C. afra (JU1286), and C. japonica (DR5081) wild isolates were obtained from the Caenorhabditis Genetics Center. C. inopinata (NK74SC) was generously provided by the Forestry and Forest Products Research Institute in Japan.The