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A number of important physiological processes in insects is controlled by the titer of juvenile hormone (JH). The juvenile (larval) stage is maintained at a high JH titer, whereas the onset of metamorphosis is induced by a low JH titer. Reproduction by adults requires often a high JH titer. Through synchronization of the reproductive phase with favorable environmental conditions, insects are able to adapt to adverse conditions and to enhance the chance for survival. The Colorado potato beetle uses daylength (photoperiod) as key stimulus for seasonal adaptation.In temperate areas the beetle hibernates in the soil and development is arrested. If the adult beetle is exposed to short-day conditions it digs into the soil after 11-12 days, which marks the onset of diapause. This photoperiodic effect is mediated by JH. Long-day conditions result in a high JH titer leading to reproduction. Short-day conditions, however, result in a low JH titer leading to diapause. The JH titer in the hemolymph is the result on the one hand by JH synthesis in the corpora allata and on the other hand by JH degradation in the hemolymph. Hydrolysis of the JH ester by JH esterase (JHE) is the main JH degradation pathway in the beetle. The highest JHE activity occurs in the hemolymph (Chapter 1).Surprisingly, the JHE from the Colorado potato beetle (Coleoptera) was found to be a dimer, consisting of two subunits of 57 kDa, whereas lepidopteran JHEs were characterized as monomers. The fact that the beetle JHE occurs as a dimer, is also the reason why the enzyme could be separated from monomeric general carboxyl esterases by nondenaturing polyacrylamide gel electrophoresis (PAGE). After PAGE purification, narrow-range isoelectric focusing and SDS-PAGE were employed to purify JHE from hemolymph of the last larval instar of the Colorado potato beetle. Two forms were found with isoelectric points of 5.5 and 5.6, respectively (Chapter 2).To clone the encoding cDNA, the purified JHE was subjected to endoproteinase Lys-C digestion. Based on the amino acid sequence of the separated peptides, degenerate primers were designed to perform RT-PCR in order to clone an internal fragment of the encoding cDNA. The 3'-end was found by screening a cDNA library and the 5'-end by using the 5'RACE technique. The size of the transcript was 1.7 kilobase. The deduced amino acid sequence (515 residues) of the putative JHE cDNA showed limited homology to some functional peptide motifs of other insect esterases (Chapter 3).RT-PCR on mRNA and analysis of genomic DNA provided evidence for the existence of two JHE-related genes, JHE.A and JHE.B. Both are intronless in the coding regions. JHE.A with a predicted pI of 5.5 probably codes for hemolymph JHE. The function of JHE.B with a predicted pI of 6.9 is unknown yet, but it may code for cytosolic JHE in the fat body. Both genes lack a conserved motif with a catalytic serine, typical for serine esterases. It was hypothesized that dimerization of the 57 kDa subunits can generate a catalytic site for enzyme activity (Chapter 4).In the beetle, the JHE transcript was mainly synthesized during the feeding stage of the last larval instar as well as in pre-diapausing animals (short-day adults). At these stages the JH titer is low and induces pupation and diapause, respectively. JHE activity in the hemolymph correlated with JHE-mRNA levels in the fat body during the development of the beetle. This observation is a strong indication that the probe used to follow JHE-mRNA levels indeed codes for hemolymph JHE. Also after photoperiodic and JH analog treatment the positive correlation was found between JHE-mRNA levels and JHE activity.JH analog (pyriproxifen) application to early 4th-instar larvae resulted in a negative feedback as JHE synthesis was enhanced to maintain the JH titer low. In contrast, if the JH analog was applied to early short-day adult beetles, JHE gene expression was suppressed to reprogram the beetles. Instead of preparation for diapause with a low JH titer and a high JHE level, the treated beetles showed reproductive development with a low JHE level to maintain a high JH titer (Chapter 5).The putative JHE cDNAs, JHE.A and JHE.B, were expressed in a baculovirus-insect cell system. The JHE-specific transcripts were clearly present in the Spodoptera frugiperda cells, as detected by Northern hybridization. Also a 57 kDa protein was synthesized, as visualized by Coomassie Brilliant Blue staining, although it was at a low level. However, JHE enzyme activity could not be detected. During translation or post-translational modification, other unknown factors may be required for the formation of an enzymatically active dimeric JHE (Chapter 6).Finally, following expression of specific genes gives us accurate information about the initiation of a developmental program. In long-day beetles, the JHE gene is switched off and the JH titer increases, leading to reproduction with vitellogenin expression. In contrast, short-days result in switching on of the JHE gene and a low JH titer. This induces the alternative developmental program, diapause, with the expression of diapause proteins. How signal transduction occurs from photoperiodic perception to gene activity is unknown yet (Chapter 7). |
