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Chrysomela tremula is a polyvoltine oligophagous leaf beetle responsible for massive attacks on poplar trees. This beetle is an important model for understanding mechanisms of resistance to Bacillus thuringiensis (Bt) insecticidal toxins, because a resistant C. tremula strain has been found that can survive and reproduce on transgenic poplar trees expressing high levels of the Cry3Aa Bt toxin. Resistance to Cry3Aa in this strain is recessive and is controlled by a single autosomal locus. We used a larval midgut transcriptome for C. tremula to search for candidate resistance genes. We discovered a mutation in an ABC protein, member of the B subfamily homologous to P-glycoprotein, which is genetically linked to Cry3Aa resistance in C. tremula. Cultured insect cells heterologously expressing this ABC protein swell and lyse when incubated with Cry3Aa toxin. In light of previous findings in Lepidoptera implicating A subfamily ABC proteins as receptors for Cry2A toxins and C subfamily proteins as receptors for Cry1A and Cry1C toxins, this result suggests that ABC proteins may be targets of insecticidal three-domain Bt toxins in Coleoptera as well.

Insect herbivory on forest canopies strongly affects ecosystem nutrient cycling and availability in a variety of ways, e.g. by changing the quantity, quality and timing of nutrient input to forest soils. A qualitative method for measurements of ingestion, egestion and exuviation rates of the insect Chrysomela tremulae on leaves of the hybrid Populus tremula x tremuloides were tested in this study with the aim to detect differences in relative nutrient cycling efficiencies. The assimilation efficiency (AD), efficiency of conversion of digested food (ECD) and efficiency of conversion of ingested food (ECI) increased from 1st, through 2nd and 3rd instar larvae with generally higher efficiencies for nitrogen than carbon. Effects on nutrient limitations for the insect were also tested by increasing the C:N ratio of insect diet. A carbohydrate solution was painted onto leaves which resulted in a significant increase in C:N (p

International audience; Globally, the estimated total area planted with transgenic plants producing Bacillus thuringiensis (Bt) toxins was 12 million hectares in 2001. The risk of target pests becoming resistant to these toxins has led to the implementation of resistance-management strategies. The efficiency and sustainability of these strategies, including the high-dose plus refuge strategy currently recommended for North American maize, depend on the initial frequency of resistance alleles. In this study, we estimated the initial frequencies of alleles conferring resistance to transgenic Bt poplars producing Cry3A in a natural population of the poplar pest Chrysomela tremulae (Coleoptera: Chrysomelidae). We used the F2 screen method developed for detecting resistance alleles in natural pest populations. At least three parents of the 270 lines tested were heterozygous for a major Bt resistance allele. We estimated mean resistance-allele frequency for the period 1999–2001 at 0.0037 (95% confidence interval = 0.000 45–0.0080) with a detection probability of 90%. These results demonstrate that (i) the F2 screen method can be used to detect major alleles conferring resistance to Bt-producing plants in insects and (ii) the initial frequency of alleles conferring resistance to Bt toxin can be close to the highest theoretical values that are expected prior to the use of Bt plants if considering fitness costs and typical mutation rates.

International audience

Genetic modifications of trees may provide many benefits, e. g. increase production, and mitigate climate change and herbivore impacts on forests. However, genetic modifications sometimes result in unintended effects on innate traits involved in plant-herbivore interactions. The importance of intentional changes in plant defence relative to unintentional changes and the natural variation among clones used in forestry has not been evaluated. By a combination of biochemical measurements and bioassays we investigated if insect feeding on GM aspens is more affected by intentional (induction Bt toxins) than of unintentional, non-target changes or clonal differences in innate plant defence. We used two hybrid wildtype clones (Populus tremula x P. tremuloides and Populus tremula x P. alba) of aspen that have been genetically modified for 1) insect resistance (two Bt lines) or 2) reduced lignin properties (two lines COMT and CAD), respectively. Our measurements of biochemical properties suggest that unintended changes by GM modifications (occurring due to events in the transformation process) in innate plant defence (phenolic compounds) were generally smaller but fundamentally different than differences seen among different wildtype clones (e. g. quantitative and qualitative, respectively). However, neither clonal differences between the two wildtype clones nor unintended changes in phytochemistry influenced consumption by the leaf beetle (Phratora vitellinae). By contrast, Bt induction had a strong direct intended effect as well as a post experiment effect on leaf beetle consumption. The latter suggested lasting reduction of beetle fitness following Bt exposure that is likely due to intestinal damage suffered by the initial Bt exposure. We conclude that Bt induction clearly have intended effects on a target species. Furthermore, the effect of unintended changes in innate plant defence traits, when they occur, are context dependent and have in comparison to Bt induction probably less pronounced effect on targeted herbivores.

International audience; The insect midgut is the primary target site for Bt-derived insecticides and Bt alternatives. However, despite extensive recent study, the precise role and nature of different Bt receptors remains a subject of considerable debate. This problem is fuelled by a lack of understanding of the genes expressed in the insect midgut and their physiological roles. The poplar leaf beetle, Chrysomela tremulae, is an important model for understanding the mode of action of, and resistance to, coleopteran-specific Bt toxins and currently shows the only known naturally occurring case of resistance to Cry3A toxins. Moreover it belongs to the same family as the corn rootworm, Diabrotica virgifera, an economically important beetle pest and target of hybrid corn expressing Cry3 toxins. Pyrosequencing is a fast and efficient way of defining the transcriptome of specific insect tissues such as the larval midgut. Here we use 454 based pyrosequencing to sample the larval midgut transcriptome of C. tremulae. We identify candidate genes of putative Bt receptors including transcripts encoding cadherin-like proteins, aminopeptidase N and alkaline phosphatase. We also describe a wealth of new transcripts predicting rapidly evolving gene families involved in plant tissue digestion, which have no homologs in the genome of the stored product pest the Red Flour beetle, Tribolium castaneum.

In this study, the effects of various hosts on adults of Chrysomela populi L. and C.tremulae F. (Col.:Chrysomelidae), important poplar pests in Central Anatolia Region, were investigated. The studies were carried on four hosts (Populus nigra L., P. x euramericana (Dode) Guiner, P. alba L. and Salix babylonica L.) at 24±1°C and 65±5 % R.H. and 18 h photoperiod. Average total number of eggs laid by each female was found to change between 32.90-989.18 in C.populi, and between 571.80-1320.10 in C.tremulae. The life longevity was found to change between average 45.00-54.18 and 2.67-106.70 days in the females, 48.30-71.70 and 91.96-123.68 days in the males in C.populi and C.tremulae, respectively. Fertility tables of females of the two species were made for different hosts, and biological parameters were determined. Studies showed that P. x euramericana was sensitive and P.alba was resistant for both species if only the poplar species were taken in hand., Bu çalışmada Orta Anadolu bölgesinde kavakların önemli zararlıları olan Chrysomela populi L. ve C.tremulae F.(Col.:Chrysomelidae) erginlerine değişik konukçu bitkilerin etkileri araştırılmıştır. Çalışmalar Populus nigra L., P. x euramericana (Dode) Guiner, P.alba L. ve Salix babylonica L. olmak üzere dört konukçu bitkide, 24±1°C sıcaklık, %65±5 orantılı nem ve 18 saat gün uzunluğu koşullarında yürütülmüştür. C.populi'de bir dişinin bıraktığı toplam yumurta sayısı konukçu bitkiye göre ortalama 32.90-989.18, C.tremulae'da ise 571.80-1320.10 adet arasında değişmiştir. Dişi ömrünün C.populi ve C.tremulae'da sırasıyla ortalama 45.00-54.18 ve 2.67-106.70, erkek ömrünün ise aynı sırayla 48.30-71.70 ve 91.96-123.68 gün arasında değiştiği belirlenmiştir. Her iki türün dişilerinde değişik konukçu bitkilere göre doğurganlık tabloları yapılarak biyolojik parametreler bulunmuştur. Çalışmalar sonunda sadece kavak türleri ele alındığında, P. x euramericana'nın her iki tür için duyarlı, P.alba'nın ise dayanıklı olduğu belirlenmiştir.

International audience; Chrysomela tremulae is one of the most threatening leaf-feeding beetles in young poplar plantations and nurseries in France. Adults and larvae feed on young poplar leaves and destroy the new growth. They cause reductions in biomass or the death of young nursery plants and decrease the biomass production of short rotation coppices. Previous studies have revealed significant differences in responses among poplar species and hybrids, and knowledge on the host plant resistance and its genetic variation is a first step to achieve selection for durable resistance. To determine the genetic basis of resistance to C. tremulae, an interspecific Fl family P. deltoides x P trichocarpa of 330 individuals was used. The P. deltoides parent was resistant and the male parent P. trichocarpa suceptible. The evaluation of resistance was carried both in laboratory and field conditions on respectively 330 and 184 genotypes. The preferences of C. tremulae were determined (1) in the laboratory by evaluation of adults feeding through multiple choice tests on leaf disk (2) in the field, by visual notations of damage in a randomised plantation. It allowed to classify the progenies according to their level of sensivity. The distributions of clonal means observed fit with a polygenic control for the traits. Resistance to C. tremulae showed a higher broad sense heritability in the laboratory tests than in the field evaluation. The genetic maps of the 2 parents : the female parent P. deltoides and the male parent P. trichocarpa realized, using RAPD, RFLP, AFLP and SSR markers were used to understand the genomic structure of resistance. Based on 'interval mapping' we have identified for laboratory test 3 QTL, explaining between 6.7% and 14.5% of clonal differences on P. deltoides and P. trichocarpa linkage groups.

International audience; The ‘high dose–refuge’ (HDR) strategy is commonly recommended and currently used for delaying or preventing pest adaptation to transgenic plants producing Bacillus thuringiensis (Bt) toxins. The efficiency of this strategy depends, among other factors, on the initial frequency of Bt resistance alleles and on the fitness costs associated with these alleles. Two years ago, an allele conferring resistance to Bt poplar was detected in a French population of the poplar pest Chrysomela tremulae F. Although this pest had never been subjected to Bt selection pressure due to human activities, the frequency of this allele was estimated at 0.0037, with a 95% credible (CI) interval of 0.00045–0.0080. We investigated the frequency of this allele in a second sample of C. tremulae collected more than 500km from the site of the initial population. The estimated frequency in this sample was 0.0113 (95% CI 0.0031–0.0247), reinforcing the conclusion that resistance to Bt plants may be present at detectable frequencies in pest populations before selection resulting from pest management by humans. The frequency of the Bt resistance allele over the two samples was 0.0049 (95% CI 0.0020–0.0091). We also followed five laboratory lines in which the frequency of this allele was initially fixed at 0.500. After five generations maintained on non-Bt poplar leaves, the frequency of this allele decreased in all lines, whereas allelic frequencies at a neutral locus were unaffected. Thus, the Bt resistance allele detected in French populations of C. tremulae is probably associated with a fitness cost.

Many strategies have been proposed for delaying the development of insect resistance to Bacillus thuringiensis (Bt). The current paradigm for Bt resistance management is the high dose-refuge strategy. For this strategy to be successful: (i) heterozygotes must be killed in treated areas, (ii) resistant alleles must be rare (frequency10-3), and (iii) there must be a high level of gene flow between populations to ensure random mating. We studied gene flow within and between populations with a view to managing the resistance of Chrysomela tremulae (Coleoptera: Chrysomelidae) to new transgenic, highly toxic poplars expressing a synthetic Bt gene. In this study, we assessed the extent of gene flow in C. tremulae within and between 16 sites in France and Belgium, using allozyme markers. We found a high level of genetic variability in C. tremulae, with a mean of 0.206 +/- 0.16. There were no obvious limitations to gene flow between populations of C. tremulae over large geographical distances (several hundreds of kilometres). Nevertheless, a very low level of genetic differentiation was observed between a site located in the south of France and the sampled sites from the Centre region.

İÜ ZE T Doktora Tezi ORTA ANADOLU BÖLGESİNDE KAVAKLARDA ZARAR YAPAN CHRYSOMELA POPULI L. VE CHRYSOMELA TREMULAE F. (CÖL.Z CHRYSOMELIDAE) «NIN KARŞILAŞTIRMALI BİYO-ÖKOLOJ İLERİ VE MÜCADELE YÖNTEMLERİ ÜZERİNDE ARAŞTIRMALAR Huriye ZEKİ Ankara üniversitesi Fen Bilimleri Enstitüsü Bitki Koruıaa Anabil im Dalı Danışman: Prof.Dr. Seval TOROS 1990, sayfa: 25 8 Jüri: Prof.Dr. Seval TOROS Prof. Dr. I. Akif KANSU Prof.Dr. Şeniz KISMALI Bu çalışmada Orta Anadolu Bölgesinde kavakların önemli zararlıları olan Çhrysomela populi L. ve Chrysomela tremulae F. (Col. : Chrysomelidae) ' nın karşılaştırmalı biyo ökoloj ileri ile mücadeleleri araştırılmıştır. Laboratnvar çalışmaları farklı sıcaklıklarda (21-1, 24-1 ve 27İ1°C) Populus nigra L. üzerinde ve değişik konukçu bitkilerde (p.x eura- mericana (Dode) Guiner, P.alba L.ve Sallx babylonica L.) 24il°c sıcaklıkta/ %65±5 orantılı nem, 18 saat gün uzun luğu koşullarında yürütülmüştür, c.populi'de bir dişinin bıraktığı toplam yumurta sayısı sıcaklığa ve konukçu bit kiye göre ortalama 32.90 - 989.18, C tremulae 'da ise 450.36-1320.10 adet arasında değişmiştir. Her iki türde de dişi ve erkek ömrünün sıcaklık artışına bağlı olarak kısal dığı, dişi ömrünün C. populi ve c. tremulae1 da., sırasıyla, ortalama 48.45 - 97.13 ve 42.79 - 78.69, erkek ömrünün iseIV aynı sırayla 63,35 - 111.55 ve 69.64 - 108.29 gün arasında değiştiği belirlenmiştir. Her iki türün dişilerinde farklı sıcaklıklar ve değişik konukçu bitkilere göre doğurganlık tabloları yapılarak biyolojik parametreler bulunmuştur. Yumurta açılım süresi sıcaklığa bağlı olarak Cpopuli'de 3-7, c.tremulae'da 4-9 gün arasında değişmek te ve her iki türün yumurtadan yeni çıkan larvalarının gelişmesi üç larva dönemi geçirdikten sonra tamamlanmak tadır. Yumurta açılımından ergin oluncaya kadar geçen top lam süre Cpopuli'de ortalama 13.08-21.47, C.tremulae'da 21.80 - 33.33 gün arasında değişmiştir, c.populi larvala rının sadece genç yapraklar üzerinde gelişebildiği, buna karşın c.tremu lae larvalarının gelişimi için yaşlı yaprak ların genç yapraklara göre daha uygun olduğu ortaya ko nulmuştur. Çalışmalar sonunda P.x euramericana' nm her iki tür için duyarlı, P.alia'nin ise dayanıklı konukçu bitki olduğu belirlenmiştir. Her iki türün kışı ergin evresinde geçirdiği, Cpopuli'nin yılda 2 ve 3; C.tremu lae' nın ise 2 döl ver diği belirlenmiştir. Doğada c.populi'de bir dişinin bırak tığı toplam yumurta sayısı ortalama 575.33 -800.64, c.tre mulae'da 187.07-453.06 adet arasında değişmiştir. Yumurta açılımından ergin oluncaya kadar geçen toplam süre c.popu li'de ortalama 17.45 - 43.50, c.tremulae'da 27.36-68.17 gün arasında değişmiştir. Gerek doğada gerekse laboratu varda C.populi'de bu sürelerin c.tremu lae' dan daha kısa olduğu belirlenmiştir. Her iki türde de cinsiyetler ora nının 1/1' e yakın olduğu saptanmıştır. c.populi ve c.tremulae'nın ergin ve larvalarının kavakların yapraklarıyla beslenmesi sonucunda ağaçları yap- aksız bırakarak zayıflamasına hattâ genç kavakların ku rumasına neden olabilmektedir. Mücadeleye yönelik çalışma larda, bu zararlıların yoğun olduğu kavaklıklarda kimyasal mücadelenin gerekli olduğu, her iki türün pupalarında gre gar parasitoid olan Schizonotus sieboldi Ratz.(Hym.: Pte rcmalidae) ve bu zararlıların ergin öncesi evrelerini av layan Coccinella saucerotti lutshniki Dobrzh. (Col.: Coccinellidae) 'den ileride uygulanabilecek biyolojik müca delede yararlanılabileceği görüşüne varılmıştır. ANAHTAR KELİMELER: Chrusomela populi L., Chrysomela tremulae F.(Col.: Chrysomelidae), Populus nigra L., Populus x euramericana (Dode) Guiner, Populus a,lba L.,SaIix babylonica L., morfoloji, biyo-ökoloji, mücadele. ABSTRACT PhD Thesis INVESTIGATIONS ON THE COMPARATIVE BIO-ECOLOGIES AND CONTROL METHODS OP CHRYSOMELA POPULI L. AND CHRYSOMELA TREMULAE F.(COL^: C HRY SOME LID AE ) HARMFUL TO POPLARS IN CENTRAL ANATOLIA REGION Huriye ZEKİ Ankara University Graduate School of Natural and Applied Sciences Department of Plant Protection Supervisor: Prof.Dr. Seval TOROS 199 0, page: 258 Jury: Prof.Dr. Seval TOROS Prof.Dr. I. Akif KANSU Prof. Dr. Seniz KISMALI In this study r comparative bio-ecologies and control methods of Chrysomela popull L. and Chrysomela tremulae E. (Col.: Chrysomelidae), important poplar pests in Central Anatolia Region, were investigated. Laboratorial studies were carried on Populus nigra. L. at various temperatures (21-1, 24-1 and 27±1°C) and on other hosts (P.x euramericana (Dode) Guiner. P.alha L. and Sallx babylonlca. L.) at 24±1°C and 65-5% R.H. and l8h photoperiod. Average total number of eggs laid by each female was found to change between 32.90 - 989.18 in c.popvcli, and between 450.36 - 1320.10 in C.tremulae. In both species, the life longevity of the females and males was found to be shortened by increases in temperature and it was avaraged in between 48.45 - 97.13 and 42.79 - 78.69 in females and 63.35 -111.55 and 69.64 - 108.29 days in males in c. popull and c.tremulae, respectively. Fertility tables of females of the two speciesVI were made for different temperatures and hosts, and biological parameters were determined. Egg hatching period differed between 3-7 days in c.populi and 4-9 days in ctremulae according to the temperature, and larval development was completed after three stages in both species. Average period from egg hatching to adult stage was found to change between 13.08-21.47 days in C.populi and 21.80 - 33.33 days in Ctremulae. c.populi larvae could develop only on young leaves whereas old ones were found to be more suitable for Ctremulae. Studies showed that P.x euramericana was sensitive and p. alba was resistant for both species. Both species overwintered in adult stages and C.populi had 2 and 3 generations whereas Ctremulae 2. Average total number of eggs laid by females in nature were found to change between 575.33 - 800.64 for c.populi and 187.07-453.06 for Ctremulae. Average total period from egg hatching to adult stage was found to change between 17.45 - 43.50 days in C.populi, 27.36 - 68.17 days in Ctremulae. These periods were found to be less in c.populi with respect to Ctremulae both in nature and in laboratory conditions. In both species, the sex ratio was about 1/1. Both larvae and adults of C.populi and Ctremulae fed on the foliage of poplars and they strip the leaves, in this way they cause weakness of the trees and the death of young ones. Control studies showed that chemical control is necessary especially in much infestations. Pupal gregar parasitoid Schizonotus sieboldi Ratz. (Hym. : Pteromalidae) and Coccinella saucerotti lutshniki Dobrzh. (Col. : Cocci- nellidae), preys on pre-adult stages of these two pests, were found to have potential to be used in biological control of these species. KEY WORDS: Chrusomela populi L., Chrusomela tremulae F. (Col.: Chrysomelidae), Populus nigra L., Populus x euramericana (Dode) Guiner, populus alba L., Salix babylonica L., morphology, bio-ecology, control. 258

Phratora vitellinae L. and Chrysomela tremulae F. (Chrysomelinae, Coleoptera) feed on Salix or Populus spp. (Salicaceae). Their larvae, as well as the larvae of other chrysomelines feeding on Salicaceae, secrete salicylaldehyde. In this study, we demonstrate that salicylaldehyde is derived from salicin, a phenylglucoside present in the leaves of the host plant. The concentration of salicylaldehyde in the secretion is positively correlated with the amount of salicin in the food of the larvae. The transformation of salicin into salicylaldehyde occurs in the defence glands since the β‐glucosidase activity is 4 times higher in their glands than in the gut. The larvae recover most of the glucose that results from the hydrolysis of salicin. For generalist predators, such as ants, salicylaldehyde is a more potent deterrent than saligenin or salicin. Copyright © 1983, Wiley Blackwell. All rights reserved, SCOPUS: ar.j, FLWNA, info:eu-repo/semantics/published