Insects as symbionts

Kukci (Insecta) su najbrojnija i široko rasprostranjena skupina organizama na Zemlji. Žive u različitim oblicima simbioza, a najznačajniji su kao ektoparaziti ljudi, domaćih životinja i kućnih ljubimaca. Svojim domaćinima nanose ozljede, uzrokuju bolesti i prenose patogene. Kao vektori patogena, oblikovali su tijek ljudske povijesti.Insects are the most numerous and widely distributed group of organisms on Earth. They live in different forms of symbiosis, and are most significant as ectoparasites of humans, domestic animals and pets. They inflict host injuries, cause diseases and transmit pathogens. As pathogenic vectors, they shaped the course of human history

Insects as symbionts

Kukci (Insecta) su najbrojnija i široko rasprostranjena skupina organizama na Zemlji. Žive u različitim oblicima simbioza, a najznačajniji su kao ektoparaziti ljudi, domaćih životinja i kućnih ljubimaca. Svojim domaćinima nanose ozljede, uzrokuju bolesti i prenose patogene. Kao vektori patogena, oblikovali su tijek ljudske povijesti.Insects are the most numerous and widely distributed group of organisms on Earth. They live in different forms of symbiosis, and are most significant as ectoparasites of humans, domestic animals and pets. They inflict host injuries, cause diseases and transmit pathogens. As pathogenic vectors, they shaped the course of human history

Psyllid vectors of the apple proliferation phytoplasma in Croatia

Lisne buhe [Hemiptera: Psylloidae, Psyllidae] poznati su vektori fitoplazma voćaka. Istraživanja praćenja vektora fitoplazma voćaka provedena su u razdoblju od 2005. do 2007. godine u glavnim područjima uzgoja voćaka u Republici Hrvatskoj. Praćenje dinamike populacije i skupljanje lisnih buha obavljeno je entomološkom mrežom. Skupljeni vektori determinirani su s pomoću binokulara i ključeva za determinaciju vrsta. U Hrvatskoj su na jabuci nađene i identificirane vrste Cacopsylla melanoneura Forster, Cacopsylla picta Forster (sinonim C. costalis Flor) i Cacopsylla crataegi Schrank. U voćnjacima u kojima su nađene te vrste nisu registrirane izravne štete. Lisne buhe C. picta i C. melanoneura poznati su prirodni vektori fitoplazme Candidatus Phytoplasma mali čija je nazočnost utvrđena u voćnjacima jabuke od Istre do istočne Slavonije. Njihova prisutnost određena je u 12 od ukupno 14 nadziranih županija. Za trogodišnjeg istraživanja dinamike populacije vektora u nasadima jabuke određena je njihova pojava u niskim populacijama. Na brojnost populacije lisnih buha u velikoj mjeri utječe režim kemijske zaštite.Psyllids [Hemiptera: Psylloidae, Psyllidae] are known vectors of fruit tree phytoplasmas. Research of phytoplasma vectors of fruit tree and grapevine was conducted in the period from 2005 till 2007 in the main fruit and grapevine growing regions of Croatia. Monitoring population dynamics and collection of psyllids was carried out in orchards by using entomological net. Determining the presence of vectors was carried out in 39 localities of 14 counties. Two known vectors of the apple proliferation phytoplasma (AP) from genus Cacopsylla were found on apple, vectors Cacopsylla melanoneura Forster and C. picta Forster. The presence of C. crategus Forster, also a possible vector of PD phytoplasma, was identified on apple. In Croatia, vectors of fruit tree phytoplasmas are widespread in all main fruit growing regions from Istria to eastern Croatia. During three years of research and monitoring of vectors population dynamics in orchards, low-number populations were present. The regime of chemical protection greatly affects the population density of psyllids

Psyllid vectors of the apple proliferation phytoplasma in Croatia

Lisne buhe [Hemiptera: Psylloidae, Psyllidae] poznati su vektori fitoplazma voćaka. Istraživanja praćenja vektora fitoplazma voćaka provedena su u razdoblju od 2005. do 2007. godine u glavnim područjima uzgoja voćaka u Republici Hrvatskoj. Praćenje dinamike populacije i skupljanje lisnih buha obavljeno je entomološkom mrežom. Skupljeni vektori determinirani su s pomoću binokulara i ključeva za determinaciju vrsta. U Hrvatskoj su na jabuci nađene i identificirane vrste Cacopsylla melanoneura Forster, Cacopsylla picta Forster (sinonim C. costalis Flor) i Cacopsylla crataegi Schrank. U voćnjacima u kojima su nađene te vrste nisu registrirane izravne štete. Lisne buhe C. picta i C. melanoneura poznati su prirodni vektori fitoplazme Candidatus Phytoplasma mali čija je nazočnost utvrđena u voćnjacima jabuke od Istre do istočne Slavonije. Njihova prisutnost određena je u 12 od ukupno 14 nadziranih županija. Za trogodišnjeg istraživanja dinamike populacije vektora u nasadima jabuke određena je njihova pojava u niskim populacijama. Na brojnost populacije lisnih buha u velikoj mjeri utječe režim kemijske zaštite.Psyllids [Hemiptera: Psylloidae, Psyllidae] are known vectors of fruit tree phytoplasmas. Research of phytoplasma vectors of fruit tree and grapevine was conducted in the period from 2005 till 2007 in the main fruit and grapevine growing regions of Croatia. Monitoring population dynamics and collection of psyllids was carried out in orchards by using entomological net. Determining the presence of vectors was carried out in 39 localities of 14 counties. Two known vectors of the apple proliferation phytoplasma (AP) from genus Cacopsylla were found on apple, vectors Cacopsylla melanoneura Forster and C. picta Forster. The presence of C. crategus Forster, also a possible vector of PD phytoplasma, was identified on apple. In Croatia, vectors of fruit tree phytoplasmas are widespread in all main fruit growing regions from Istria to eastern Croatia. During three years of research and monitoring of vectors population dynamics in orchards, low-number populations were present. The regime of chemical protection greatly affects the population density of psyllids

Pathogenic bacteria with highly reduced genomes

Fitoplazme su bakterije koje nemaju staničnu stjenku i zbog toga pripadaju razredu Mollicutes. Ovi patogeni su intracelularni paraziti biljnog floema, a prenose se kukcima koji im služe i kao domaćini. Dok kod kukaca najčešće ne izazivaju nikakve smetnje, učinak fitoplazmi na biljne vrste je često poguban. Najčešći simptomi su filodija, virescencija, rast u obliku vještičje metle, crvenjenje i žućenje listova, nekroza floema. Poznavanje biologije ovih specifičnih organizama je dosta ograničeno zbog toga što se ne mogu kultivirati na hranjivim podlogama. No u posljednje vrijeme počelo se s potpunim sekvenciranjem genoma fitoplazmi, što je dovelo do brojnih novih spoznaja. Imaju iznimno reducirane genome (680 – 1600 kb). Nedostaju im mnogi esencijalni geni u različitim metaboličkim putovima, a najznačajniji nedostatak su geni za sve podjedinice ATP-sintaze, kao i geni za uzimanje i iskorištavanje šećera. To nam ukazuje da fitoplazme aktivno unose metabolite domaćina. No mnogi od tih mehanizama unosa i putova iskorištavanja domaćinskih metabolita nisu poznati kao i onaj o uzimanju ATP-a. Usprkos tome svemu fitoplazme vrlo uspješno preživljavaju i manipuliraju domaćinima iz dva različita carstva. Smatra se da su se tako dobro prilagodile na takav način života zbog plastičnosti genoma. Fitoplazme su kompenzirale svoja ograničenja horizontalnim transferom gena, rearanžmanima DNA i rekombinacijama između kromosoma i PMUs (Potential Mobile Units). Upravo ti PMUs sadrže gene koji su važni za preživljavanje i prilagodbu na različite domaćine. Za sada je najbolji način kojim možemo doći do novih spoznaja o fitoplazmama daljnje sekvenciranje fitoplazmatskih genoma i njihove komparativne analize.Phytoplasmas are bacteria without cell wall belonging to the class Mollicutes. These pathogens are intracellular parasites of plant phloem, transmitted by insects which also serve as their hosts. Phytoplasmas do not cause any disturbance to most insect hosts but their infections of plants is often fatal to this host type. The most common phytplasmoses symptoms in plants are phyllody, virescence, witches broom, reddening or yellowing of leaves, necrosis of the phloem. Knowledge of the biology of these specific organisms is quite limited because they cannot be cultivated in culture medium. Neverheless, in recent time be the complete phytoplasma genome sequencing led to many new insights. Phytoplasmas have an extremely reduced genome (680 to 1600 kb). They lack many essential genes in different metabolic pathways, and most significantly, they can live with the lack of genes for ATP synthase subunits, as well as genes for sugar uptake and usage. It suggests that phytoplasmas effivciently uptake metabolites from the host. Hovewer, many of these uptake mechanisms are including the one for ATP. In spite of everything, phytoplasmas successfully survive and manipulate the hosts from two different kingdoms. It is believed that they have been so well adapted to that way of life because of their genome plasticity. Phytoplasmas compensated their genomic limitations by horizontal gene transfer, rearrangements of DNA and recombination between chromosomes and Potential Mobile Units (PMUs). PMUs contain genes that are important for survival and adaptation to different hosts. For the time being, one of the best ways to gain more knowledge on phytoplasmas is sequencing of additional phytoplasma genomes and comparative genome analysis

INSECT VECTORS OF PLANT DISEASES

Kukci iz reda Hemiptera i Thysanoptera vektori su brojnih patogenih virusa, bakterija, gljivica, fitoplazmi te spiroplazmi. Karakterizira ih brza reprodukcija te širok spektar biljaka domaćina na kojima čine direktne štete hranjenjem ili indirektne prijenosom patogenih organizama. Današnja istraživanja kukaca vektora biljnih bolesti usmjerena su na razumijevanje mehanizama kojima različiti patogeni utječu na vektore zbog unapređenja vlastitog prijenosa na prigodna domaćina, ali i na praćenje pojava novih vektora te identifikaciju uzročnika biljnih bolesti koje prenose. Lisne uši (Aphididae), štitasti moljci (Aleyroidae) i tripsi (Thripidae) vektori su mnogobrojnih biljnih virusa. Cikade vektori virusa pripadaju podporodicama Agallinae i Deltocephalinae dok su mnogobrojne ostale vrste unutar porodice Cicadellidae, Aphrophoridae i Cercopidae prijenosnici brojnih fitoplazmi i bakterija, uključujući i karantenske patogene poput bakterije Xylella fastidiosa. Istraživanja kukaca vektora uključuju proučavanja opsežnih interakcija unutar patosustava, istraživanja genskih predispozicija vektora za prijenos specifičnog patogena, sinergijske odnose virusa i endosimbiotskih bakterija u tijelu vektora, procjenjuju dinamične učinke patogena na biljke domaćine tijekom razvoja bolesti te molekularne mehanizme kojima patogeni direktno utječu na ponašanje i navike vektora. U ovom radu prikazane su biološke predispozicije različitih vektora za prijenos patogena, istaknuti su odnosi i mehanizmi utjecaja patogena na vektore te načini njihova prijenosa.Insects from the Hemiptera and Thysanoptera order are vectors of numerous pathogenic viruses, bacteria, fungi, phytoplasmas and spiroplasmas. They are characterized by fast reproduction and a broad plant-host spectre which they damage either directly by feeding or indirectly through transmittance of pathogenic organisms. Modern-day research of insect vectors is focused on investigating the mechanisms underlying the pathogen’s influence over its vector. It also aims to discover the molecular and metabolic components in plants, pathogens and their vectors which facilitate pathogen transmission and disease spread. Aphids (Aphididae), whiteflies (Aleyroidae) and thrips (Thripidae) are most important vectors of numerous plant viruses. Leafhoppers belonging only to Agallinae and Deltocephalinae subfamilies transmit viruses whereas numerous species within Cicadellidae, Aphrophoridae and Cercopidae family, transmit phytoplasmas, bacteria as well as quarantine pathogens such as the bacteria Xylella fastidiosa. All pathogens transmitted by insect vectors use complex strategies to enhance effectiveness of their transmission and spread. Most pathogens show high specificity towards their insect vectors since certain anatomical and genetic predispositions must be appeased in order to achieve successful transmission. Pathogens also devised intriguing ways to manipulate vector behaviour and change its feeding and reproductive habits. In this review, we present biological predispositions of different vector-insects for pathogen transmission, highlight mechanisms in plant-vector-pathogen interactions and point out distinct ways of pathogen transmission

FAUNISTICAL AND ECOLOGICAL CAHARACTERISTICS OF HORSE FLIES (TABANIDAE) OF CETINA RIVER

Na području uz rijeku Cetinu u razdoblju od svibnja do kraja rujna 2018. godine obavljena su istraživanja faunističko-ekoloških značajki obada (Tabanidae) na 12 različitih lokaliteta. Sakupljene su 662 jedinke koje su svrstane u 21 vrstu, 6 rodova i 2 podporodice. Pregledom literaturnih podataka i obada u zbirkama kukaca Odjela za biologiju utvđeno je ukupno 35 vrsta obada za istraživano područje. Za područje rijeke Cetine utvrđene su tri nove vrste obada: Hybomitra ciureai, Tabanus lunatus i Philipomyia aprica. Najveći broj obada uzorkovan je na lokalitetu Donje Maovice, a najmanji u Trilju. Najbrojnije vrste obada na istraživanom području su: Tabanus bromius, Tabanus glaucopis i Tabanus tergestinus. Najveću sličnosti faune obada pokazuju lokaliteti Glavice i Naklice, a najmanju lokaliteti Donje Maovice i Trilj (12,5%)On the area of Cetina river, from May till the end of September 2018, studies of faunistical and ecological characteristics of horse flies (Tabanidae) were carried out in twelve localities. 662 specimens of horse flies were collected and classified into 21 species, 6 genera and 2 subfamilies. Reviewing the literature data and horse flies from insect collections of Department of Biology 35 species were determined for the study area. Three new species of horse flies were determined for the area of Cetina river: Hybomitra ciureai, Tabanus lunatus and Philipomyia aprica. The largest number of horse flies was collected in locality of Donje Maovice, while the smallest number of specimens was collected in Trilj. The most common species for the study area were: Tabanus bromius, Tabanus glaucopis and Tabanus tergestinus. The highest faunistical similarity was shown between localities Glavice and Naklice (80%), while between localities Donje Maovice and Trilj the lowest (12,5%)

Pathogenic bacteria with highly reduced genomes

Fitoplazme su bakterije koje nemaju staničnu stjenku i zbog toga pripadaju razredu Mollicutes. Ovi patogeni su intracelularni paraziti biljnog floema, a prenose se kukcima koji im služe i kao domaćini. Dok kod kukaca najčešće ne izazivaju nikakve smetnje, učinak fitoplazmi na biljne vrste je često poguban. Najčešći simptomi su filodija, virescencija, rast u obliku vještičje metle, crvenjenje i žućenje listova, nekroza floema. Poznavanje biologije ovih specifičnih organizama je dosta ograničeno zbog toga što se ne mogu kultivirati na hranjivim podlogama. No u posljednje vrijeme počelo se s potpunim sekvenciranjem genoma fitoplazmi, što je dovelo do brojnih novih spoznaja. Imaju iznimno reducirane genome (680 – 1600 kb). Nedostaju im mnogi esencijalni geni u različitim metaboličkim putovima, a najznačajniji nedostatak su geni za sve podjedinice ATP-sintaze, kao i geni za uzimanje i iskorištavanje šećera. To nam ukazuje da fitoplazme aktivno unose metabolite domaćina. No mnogi od tih mehanizama unosa i putova iskorištavanja domaćinskih metabolita nisu poznati kao i onaj o uzimanju ATP-a. Usprkos tome svemu fitoplazme vrlo uspješno preživljavaju i manipuliraju domaćinima iz dva različita carstva. Smatra se da su se tako dobro prilagodile na takav način života zbog plastičnosti genoma. Fitoplazme su kompenzirale svoja ograničenja horizontalnim transferom gena, rearanžmanima DNA i rekombinacijama između kromosoma i PMUs (Potential Mobile Units). Upravo ti PMUs sadrže gene koji su važni za preživljavanje i prilagodbu na različite domaćine. Za sada je najbolji način kojim možemo doći do novih spoznaja o fitoplazmama daljnje sekvenciranje fitoplazmatskih genoma i njihove komparativne analize.Phytoplasmas are bacteria without cell wall belonging to the class Mollicutes. These pathogens are intracellular parasites of plant phloem, transmitted by insects which also serve as their hosts. Phytoplasmas do not cause any disturbance to most insect hosts but their infections of plants is often fatal to this host type. The most common phytplasmoses symptoms in plants are phyllody, virescence, witches broom, reddening or yellowing of leaves, necrosis of the phloem. Knowledge of the biology of these specific organisms is quite limited because they cannot be cultivated in culture medium. Neverheless, in recent time be the complete phytoplasma genome sequencing led to many new insights. Phytoplasmas have an extremely reduced genome (680 to 1600 kb). They lack many essential genes in different metabolic pathways, and most significantly, they can live with the lack of genes for ATP synthase subunits, as well as genes for sugar uptake and usage. It suggests that phytoplasmas effivciently uptake metabolites from the host. Hovewer, many of these uptake mechanisms are including the one for ATP. In spite of everything, phytoplasmas successfully survive and manipulate the hosts from two different kingdoms. It is believed that they have been so well adapted to that way of life because of their genome plasticity. Phytoplasmas compensated their genomic limitations by horizontal gene transfer, rearrangements of DNA and recombination between chromosomes and Potential Mobile Units (PMUs). PMUs contain genes that are important for survival and adaptation to different hosts. For the time being, one of the best ways to gain more knowledge on phytoplasmas is sequencing of additional phytoplasma genomes and comparative genome analysis

Pathogenic bacteria with highly reduced genomes

Fitoplazme su bakterije koje nemaju staničnu stjenku i zbog toga pripadaju razredu Mollicutes. Ovi patogeni su intracelularni paraziti biljnog floema, a prenose se kukcima koji im služe i kao domaćini. Dok kod kukaca najčešće ne izazivaju nikakve smetnje, učinak fitoplazmi na biljne vrste je često poguban. Najčešći simptomi su filodija, virescencija, rast u obliku vještičje metle, crvenjenje i žućenje listova, nekroza floema. Poznavanje biologije ovih specifičnih organizama je dosta ograničeno zbog toga što se ne mogu kultivirati na hranjivim podlogama. No u posljednje vrijeme počelo se s potpunim sekvenciranjem genoma fitoplazmi, što je dovelo do brojnih novih spoznaja. Imaju iznimno reducirane genome (680 – 1600 kb). Nedostaju im mnogi esencijalni geni u različitim metaboličkim putovima, a najznačajniji nedostatak su geni za sve podjedinice ATP-sintaze, kao i geni za uzimanje i iskorištavanje šećera. To nam ukazuje da fitoplazme aktivno unose metabolite domaćina. No mnogi od tih mehanizama unosa i putova iskorištavanja domaćinskih metabolita nisu poznati kao i onaj o uzimanju ATP-a. Usprkos tome svemu fitoplazme vrlo uspješno preživljavaju i manipuliraju domaćinima iz dva različita carstva. Smatra se da su se tako dobro prilagodile na takav način života zbog plastičnosti genoma. Fitoplazme su kompenzirale svoja ograničenja horizontalnim transferom gena, rearanžmanima DNA i rekombinacijama između kromosoma i PMUs (Potential Mobile Units). Upravo ti PMUs sadrže gene koji su važni za preživljavanje i prilagodbu na različite domaćine. Za sada je najbolji način kojim možemo doći do novih spoznaja o fitoplazmama daljnje sekvenciranje fitoplazmatskih genoma i njihove komparativne analize.Phytoplasmas are bacteria without cell wall belonging to the class Mollicutes. These pathogens are intracellular parasites of plant phloem, transmitted by insects which also serve as their hosts. Phytoplasmas do not cause any disturbance to most insect hosts but their infections of plants is often fatal to this host type. The most common phytplasmoses symptoms in plants are phyllody, virescence, witches broom, reddening or yellowing of leaves, necrosis of the phloem. Knowledge of the biology of these specific organisms is quite limited because they cannot be cultivated in culture medium. Neverheless, in recent time be the complete phytoplasma genome sequencing led to many new insights. Phytoplasmas have an extremely reduced genome (680 to 1600 kb). They lack many essential genes in different metabolic pathways, and most significantly, they can live with the lack of genes for ATP synthase subunits, as well as genes for sugar uptake and usage. It suggests that phytoplasmas effivciently uptake metabolites from the host. Hovewer, many of these uptake mechanisms are including the one for ATP. In spite of everything, phytoplasmas successfully survive and manipulate the hosts from two different kingdoms. It is believed that they have been so well adapted to that way of life because of their genome plasticity. Phytoplasmas compensated their genomic limitations by horizontal gene transfer, rearrangements of DNA and recombination between chromosomes and Potential Mobile Units (PMUs). PMUs contain genes that are important for survival and adaptation to different hosts. For the time being, one of the best ways to gain more knowledge on phytoplasmas is sequencing of additional phytoplasma genomes and comparative genome analysis

Multigene characterization of ''Candidatus Phytoplasma asteris' isolate from oilseed rape (Brassica napus ssp. oleifera (DC.) Metzg.)

Fitoplazme (rod 'Candidatus Phytoplasma') su bakterije bez stanične stijenke koje parazitiraju u floemu biljaka i stanicama kukaca te ih nije moguće uzgajati u čistoj kulturi in vitro. Fitoplazme imaju iznimno reducirane genome (680 - 1600 kb) i nedostaju im mnogi esencijalni geni za različite metaboličke putove. U svom životnom ciklusu obavezno izmjenjuju dva domaćina – biljku i kukca koji je ujedno i vektor. Dok kod kukaca najčešće ne izazivaju nikakve smetnje, njihov učinak na biljke je često vrlo štetan. U biljkama se zadržavaju unutar floema što im osigurava unos potrebnih metabolita za preživljavanje i umnažanje. Bolesti proliferacije uljane repice često se povezuju se s infekcijom unutarstaničnim fitogenim bakterijama roda 'Candidatus Phytoplasma'. Budući da ove bakterije nije moguće uzgojiti u čistoj kulturi in vitro, dokaz prisutnosti bazira se isključivo na molekularnim metodama. Stoga je cilj ovog diplomskog rada bila identifikacija te multigenskaa tipizacija izolata fitoplazme 'Ca. P. asteris' dobivenih iz simptomatičnih uzoraka uljane repice prikupljenih u Zagrebu na eksperimentalnom zemjištu Agronomskog fakulteta. Zarazu fitoplazmatskom vrstom 'Ca. P. asteris' dokazala sam PCR/RFLP analizom gena za 16S rRNA, a filogenetska analiza fitoplazmatskog gena za 16S rDNA iz izolata simptomatične uljane repice potvrdila je rezultate PCR/RFLP klasifikacije te pripadnost vrsti 'Ca. P. asteris' kao i u prethodno identificiranim fitoplazmama na istom domaćinu. Analiza još tri genska lokusa izvan 16S rRNA gena nije samo potvrdila pripadnost podskupini I-B ribosomske skupine Aster Yellows i vrsti 'Ca. P. asteris', nego je po prvi puta pružila i detaljnu molekularnu karakterizaciju nekog izolata fitoplazme iz uljane repice.Phytoplasmas (genus 'Candidatus Phytoplasma') are bacterial pathogens that cause numerous diseases in plants. Because of their small size and lack of a cell wall they are affiliated to the class Mollicutes. Phytoplasmas have extremely reduced genomes (680 - 1600 kb) and lack many essential genes for different metabolic pathways. Life cycle of phytoplasma involves two hosts – plants and insects. Although in insects they usually do not cause negative effects, their effect on plants is often harmful. In the infected plants phytoplasmas retain within phloem which ensures them to get required metabolites for growth and multiplication. Rapeseed proliferation diseases are often associated with infection with the intracellular phytopathogenic bacteria of the genus 'Candidatus Phytoplasma'. Since these bacteria cannot be grown in pure in vitro culture, detection and diagnosis are based mainly on molecular methods.Therefore, the aim of this study was the identification and multigene typing of 'Ca. P. asteris' phytoplasma obtained from symptomatic oilseed rape samples collected in Zagreb on the experimental plot of the Faculty of Agriculture. The infection with the ‘Ca. P. asteris' phytoplasma species was detected by PCR/RFLP gene analysis of 16S rRNA. Phylogenetic analysis of the phytoplasma 16S rDNA gene from isolates of symptomatic oilseed rape confirmed the results of PCR/RFLP classification and affiliation to the 'Ca. P. asteris' species, as in the previously identified phytoplasma isolates from the same host. The analysis of three more genetic loci outside the 16S didn't only confirm belonging to the subgroup I-B of the Aster Yellows group and 'Ca. P. asteris' species, but also provided detailed molecular characterization of an oilseed rape phytoplasma isolate for the first time