Karl J. Kramer

High-energy-resolution scintillator: Ce3+ activated LaBr3

The scintillation properties of LaCl3 doped with 10% Ce3+ are presented. Under optical and gamma ray excitation, Ce3+ emission is observed to peak at 330 and 352 nm. The scintillation light output is 46 000±1000 photons/MeV at 662 keV. Forty percent is emitted with a decay time of 26 ns, 30% with 210 ns, and 30% with about 1000 ns. An energy resolution (full width at half maximum over the peak position) of 3.3±0.3% was observed for the 662 keV full absorption peak.

Insect chitinases: molecular biology and potential use as biopesticides.

Chitin, an insoluble structural polysaccharide that occurs in the exoskeletal and gut linings of insects, is a metabolic target of selective pest control agents. One potential biopesticide is the insect molting enzyme, chitinase, which degrades chitin to low molecular weight, soluble and insoluble oligosaccharides. For several years, our laboratories have been characterizing this enzyme and its gene. Most recently, we have been developing chitinase for use as a biopesticide to control insect and also fungal pests. Chitinases have been isolated from the tobacco hornworm, Manduca sexta, and several other insect species, and some of their chemical, physical, and kinetic properties have been determined. Also, cDNA and genomic clones for the chitinase from the hornworm have been isolated and characterized. Transgenic plants that express hornworm chitinase constitutively have been generated and found to exhibit host plant resistance. A transformed entomopathogenic virus that produces the enzyme displayed enhanced insecticidal activity. Chitinase also potentiated the efficacy of the toxin from the microbial insecticide, Bacillus thuringiensis. Insect chitinase and its gene are now available for biopesticidal applications in integrated pest management programs. Current knowledge regarding the molecular biology and biopesticidal action of insect and several other types of chitinases is described in this mini-review.

Sequence of a cDNA and expression of the gene encoding epidermal and gut chitinases of Manduca sexta

Insects use chitinolytic enzymes to digest chitin in the exoskeleton during the molting process. We have isolated and sequenced a chitinase-encoding cDNA from the tobacco hornworm, Manduca sexta, compared its sequence with genes encoding chitinolytic enzymes from other sources, and studied chitinase gene expression and hormonal regulation during the larval-pupal transformation. The insert DNA in this clone is 2452 nucleotides long with an open reading frame of 1662 nucleotides that encodes a protein of 554 amino acids with a molecular weight of 62 kDa. Several regions of the amino acid sequence in this protein are similar to sequences in yeast, cucumber and bacterial endo-beta-N-acetylglucosaminidases. Hybrid-selection of mRNA and in vitro translation yielded an immunoreactive protein with an apparent molecular mass of 75 kDa, which is similar to the size of a chitinase present in pharate pupal molting fluid. Southern blot analysis indicated that one or two genes related to the cDNA clone are encoding chitinases in the Manduca genome. The major tissues expressing chitinase genes were the epidermis and gut with mRNA levels highest on c. days 5-7 during the fifth larval instar. Injection of 20-hydroxyecdysone into ligated fifth instar abdomens caused about a 10-fold increase in mRNA levels in both epidermis and gut, and topical application of the juvenile hormone mimic, fenoxycarb, suppressed the ecdysteroid-induced accumulation of chitinase RNA.

The Tribolium chitin synthase genes TcCHS1 and TcCHS2 are specialized for synthesis of epidermal cuticle and midgut peritrophic matrix

Functional analysis of the two chitin synthase genes, TcCHS1 and TcCHS2, in the red flour beetle, Tribolium castaneum, revealed unique and complementary roles for each gene. TcCHS1‐specific RNA interference (RNAi) disrupted all three types of moult (larval–larval, larval–pupal and pupal–adult) and greatly reduced whole‐body chitin content. Exon‐specific RNAi showed that splice variant 8a of TcCHS1 was required for both the larval‐pupal and pupal‐adult moults, whereas splice variant 8b was required only for the latter. TcCHS2‐specific RNAi had no effect on metamorphosis or on total body chitin content. However, RNAi‐mediated down‐regulation of TcCHS2, but not TcCHS1, led to cessation of feeding, a dramatic shrinkage in larval size and reduced chitin content in the midgut.

Bose–Einstein condensation of the triplet states in the magnetic insulator TlCuCl3

Bose–Einstein condensation denotes the formation of a collective quantum ground state of identical particles with integer spin or intrinsic angular momentum. In magnetic insulators, the magnetic properties are due to the unpaired shell electrons that have half-integer spin. However, in some such compounds (KCuCl3 and TlCuCl3), two Cu2+ ions are antiferromagnetically coupled to form a dimer in a crystalline network: the dimer ground state is a spin singlet (total spin zero), separated by an energy gap from the excited triplet state (total spin one). In these dimer compounds, Bose–Einstein condensation becomes theoretically possible. At a critical external magnetic field, the energy of one of the Zeeman split triplet components (a type of boson) intersects the ground-state singlet, resulting in long-range magnetic order; this transition represents a quantum critical point at which Bose–Einstein condensation occurs. Here we report an experimental investigation of the excitation spectrum in such a field-induced magnetically ordered state, using inelastic neutron scattering measurements of TlCuCl3 single crystals. We verify unambiguously the theoretically predicted gapless Goldstone mode characteristic of the Bose–Einstein condensation of the triplet states.

Oxidative conjugation of catechols with proteins in insect skeletal systems

Abstract Cuticle sclerotization or tanning is a vital process that occurs during each stage of insect development to harden and stabilize the newly secreted exoskeleton. The structural polymers protein and chitin make up the bulk of the cuticle, and chemical interactions between these biopolymers with quinonoid tanning agents are largely responsible for the physical properties of the mature exoskeleton. The oxidative conjugation of catechols with cuticular proteins plays an important role in this metabolism. The main hypothesis for cuticle sclerotization involves the formation of adducts and cross-links between nucleophilic imidazole nitrogens of histidyl residues in the proteins and electrophilic ring or side-chain carbons of ortho-quinones and para-quinone methides derived from the catechols, N-acetyldopamine, N-beta-alanyldopamine, and 3,4-dihydroxyphenylethanol. C–N and C–O linkages between these quinone tanning agents and proteins in cuticles from a variety of insects from several orders have been elucidated. cDNAs for both the tyrosinase and laccase types of phenoloxidases that catalyze the cross-linking reactions have been isolated and sequenced. The sequences of laccase cDNAs from two insect species were more similar to fungal laccases than to those from plants. These results provide insights into how insects use structural proteins, catechols, and oxidative enzymes to form catechol–amino acid adducts during sclerotization.

Insect resistance of transgenic tobacco expressing an insect chitinase gene.

Chitinase expression in the insect gut normally occurs only during moulting, where the chitin of the peritrophic membrane is presumably degraded. Thus, insects feeding on plants that constitutively express an insect chitinase gene might be adversely affected, owing to an inappropriately timed exposure to chitinase. This hypothesis was tested by introducing a cDNA encoding a tobacco hornworm (Manduca sexta) chitinase (EC 3.2.1.14) into tobacco via Agrobacterium tumefaciens-mediated transformation. A truncated but enzymatically active chitinase was present in plants expressing the gene. Segregating progeny of high-expressing plants were compared for their ability to support growth of tobacco budworm (Heliothis virescens) larvae and for feeding damage. Both parameters were significantly reduced when budworms fed on transgenic tobacco plants expressing high levels of the chitinase gene. In contrast, hornworm larvae showed no significant growth reduction when fed on the chitinase-expressing transgenics. However, both budworm and hornworm larvae, when fed on chitinase-expressing transgenic plants coated with sublethal concentrations of a Bacillus thuringiensis toxin, were significantly stunted relative to larvae fed on toxin-treated non-transgenic controls. Foliar damage was also reduced. Plants expressing an insect chitinase gene may have agronomic potential for insect control

Juvenile hormone-specific esterases in the haemolymph of the tobacco hornworm, Manduca sexta

Abstract A new sensitive method for determining juvenile hormone (JH) hydrolysis has been developed which measures the release of tritiated methanol from JH labelled in the methyl ester group. Using this assay we investigated the interaction of JH with haemolymph esterases and haemolymph JH-binding protein. Haemolymph from fifth instar larvae of Manduca sexta contains two families of esterases which can be distinguished by their reactivity with diisopropylphosphorofluoridate (DFP). One group consists of general esterases which are capable of hydrolysing free JH but not JH complexed to the binding protein and are completely inhibited by low concentrations of DFP (10 −4 M). The other group (JH-specific esterases), relatively DFP resistant, has little detectable general esterase activity but can hydrolyse JH bound to the binding protein as well as free JH. The major JH-esterase has a sedimentation coefficient of 4·98 S and a diffusion coefficient of 6·4 × 10 −7 cm 2 sec −1 . The molecular weight calculated from these values is 6·7 × 10 4 . The general esterases are present throughout the larval stage, but the JH-specific esterases are barely detectable until the fourth day of the fifth instar when they suddenly appear at a high concentration. Since the general esterases cannot hydrolyse bound JH, one function of the binding protein is to protect JH during transport in the early instars, thus confirming that the binding protein is a true carrier of JH. In the late fifth instar prior to metamorphosis, however, JH-specific esterases appear in the haemolymph resulting in the hydrolysis of JH complexed to the carrier protein. Thus, by lowering JH titre, the JH-esterases play an important role in development in M. sexta .

7 – Chitin Metabolism in Insects

Publisher Summary This chapter highlights some of the recent and important findings obtained from studies conducted on the synthesis, structure, physical state, modification, organization, and degradation of chitin in insect tissues, as well as the interplay of chitin with chitin-binding proteins, the regulation of genes responsible for chitin metabolism, and, finally, the targeting of chitin metabolism for insect-control purposes. Chitin is the major polysaccharide present in insects and many other invertebrates as well as in several microbes, including fungi. It serves as the skeletal polysaccharide of several animal phyla, such as the Arthropoda, Annelida, Molluska, and Coelenterata. In several groups of fungi, chitin replaces cellulose as the structural polysaccharide. In insects, it is found in the body wall or cuticle, gut lining or peritrophic matrix (PM), salivary gland, trachea, eggshells, and muscle attachment points. In the course of evolution, insects have made excellent use of the rigidity and chemical stability of the polymeric chitin to assemble both hard and soft extracellular structures such as the cuticle (exoskeleton) and PM respectively, both of which enable insects to be protected from the environment while allowing for growth, mobility, respiration, and communication. All of these structures are primarily composites of chitin fibers and proteins with varying degrees of hydration and trace materials distributed along the structures.

Functional specialization among insect chitinase family genes revealed by RNA interference

The biological functions of individual members of the large family of chitinase-like proteins from the red flour beetle, Tribolium castaneum (Tc), were examined by using gene-specific RNAi. One chitinase, TcCHT5, was found to be required for pupal–adult molting only. A lethal phenotype was observed when the transcript level of TcCHT5 was down-regulated by injection of TcCHT5-specific dsRNA into larvae. The larvae had metamorphosed into pupae and then to pharate adults but did not complete adult eclosion. Specific knockdown of transcripts for another chitinase, TcCHT10, which has multiple catalytic domains, prevented embryo hatch, larval molting, pupation, and adult metamorphosis, indicating a vital role for TcCHT10 during each of these processes. A third chitinase-like protein, TcCHT7, was required for abdominal contraction and wing/elytra extension immediately after pupation but was dispensable for larval–larval molting, pupation, and adult eclosion. The wing/elytra abnormalities found in TcCHT7-silenced pupae were also manifest in the ensuing adults. A fourth chitinase-like protein, TcIDGF4, exhibited no chitinolytic activity but contributed to adult eclosion. No phenotypic effects were observed after knockdown of transcripts for several other chitinase-like proteins, including imaginal disk growth factor IDGF2. These data indicate functional specialization among insect chitinase family genes, primarily during the molting process, and provide a biological rationale for the presence of a large assortment of chitinase-like proteins.