Donald L. Silhacek

INSECT GROWTH REGULATORS

Biologists in the early part of the 20th century believed that since the secondary sexual characteristics of insects were unaffected by castration, insects did not secrete hormones. This view was challenged by the conclusive experiments of Stephan Kopec in 1917, who showed by surgical maneuver, that insect metamorphosis was under the hormonal control of the brain. Over the next half- century, significant discoveries in insect endocrinology were made by such pioneers as Wigglesworth, Bodenstein, Fukuda, and Williams, among others (see review by Granger and Bollenbacher 1981). The classical scheme that was developed by these scientists was that a hormone from the brain, now known as prothracicotropic hormone (PTTH), stimulated the prothoracic glands to release a molting hormone (ecdysteroid), which caused either molting or metamorphosis depending upon the stage of the insect, and in particular, on whether a third hormone, juvenile hormone (JH), was present.

Identification and purification of storage proteins in tissues of the greater wax moth Galleria Mellonella (L.)

Four high mol. wt storage proteins have been identified in Galleria mellonella (L.) larvae using sodium dodecyl sulphate polyacrylamide gel electrophoresis. These proteins initially accumulated in haemolymph of final instar larvae and later in development comprised the bulk of the proteins found in fat body from late larvae and pharate pupae. Three of the four proteins have been purified to homogeneity and their peptide patterns immunochemical identity compared. Three of the four proteins were found to contain conjugated carbohydrate on the basis of lectin-agarose chromatography and biosynthetic incorporation of radiolabelled N-acetylglucosamine. One of the polypeptides also contained lipid.

The synthesis and uptake of haemolymph storage proteins by the fat body of the greater wax moth Galleria mellonella (L.)

Abstract The four storage proteins of Galleria mellonella (L.) exhibit stage-dependent alterations in distribution between haemolymph and fat body during the final larval instar. Autoradiograms of newly synthesized fat body proteins separated on sodium dodecyl sulphate polyacrylamide gels demonstrated that this tissue both synthesizes and releases all four storage proteins until pharate pupal development. Although the capacity for protein biosynthesis by fat bodies fluctuated during this period of development, qualitative alterations in the synthesis of the storage proteins were not found. Fat body from wandering through to spinning stage larvae sequestered radiolabelled storage proteins which had been injected into the haemolymph. This provides evidence for the direct resorption of haemolymph storage proteins by fat body; however, it may only in part account for the storage protein pool accumulated by pharate pupal and pupal fat body tissues.

Nucleotide sequence and structure of the arylphorin gene from Galleria mellonella

Abstract The complete nucleotide sequence and the structure of the arylphorin gene of Galleria mellonella have been determined. This gene contains four introns, at positions exactly corresponding to the four introns in other reported lepidopteran storage protein genes. The gene encodes a preprotein of 702 amino acids, including a signal peptide of 16 amino acids and 131 aromatic residues in the mature protein. The deduced amino acid sequence has been confirmed by determination of the 26 N-terminal residues of purified Lhp76. Thus, Lhp76 is a typical member of the arylphorin family of lepidopteran storgae proteins and is here renamed as the Galleria arylphorin. The deduced arylphorin protein sequence includes two potential N-glycosyation sites, in keeping with previous data that Lhp76 is an N-glycosylated protein. Although Galleria arylphorin gene espression is repressed by ecdysone in vivo and in vitro , no exact matches were found between 5′ flanking DNA sequences and known Drosophila ecdysone response elements.

Binding of riboflavin to lipophorin and a hexameric protein in the hemolymph of Heliothis virescens

Abstract The hemolymph of late final instar Heliothis virescens larvae was analyzed for riboflavin and its metabolic derivatives. Riboflavin levels in the hemolymph were found to be relatively high at this developmental stage ( ca 25 μg/ml hemolymph) and all of the flavin was associated with high molecular weight proteins. Based on the recovery of riboflavin with purified hemolymph protein preparations, approx. 15% of the total was bound to lipophorin and the remainder to a hexameric storage protein comprised of 82 kDa subunits, designated p82. The recovery of p82-bound riboflavin was not quantitative, a result attributable to the effects of sample dilution during purification. The p82 hexamer (but not lipophorin, arylphorin or other hemolymph proteins) was bound by a flavinyl-agarose affinity matrix, which provided independent confirmation of its identity as a flavin-binding protein. In addition to the large hemolymph pool of protein-bound riboflavin, H. virescens larvae also contained substantial amounts of free riboflavin in malpighian tubules ( ca 19μg/g tissue) and in testes ( ca 25 μg/g tissue). These findings suggest a complex pattern of riboflavin binding and transport mediated by specific pathways of flavin metabolism, storage and excretion depending upon the target tissue.

Immuno-Fluorescent analysis of follicular growth and development in whole ovaries of the Indianmeal moth

The differentiation and growth of ovaries was analyzed using immuno‐fluorescence microscopy and then correlated with the changes in the external morphology of female pupae during metamorphosis of the Indianmeal moth, Plodia interpunctella (Hübner). Fourteen developmental points coincident with a daily change in the light:dark cycle were chosen for observation to describe the progress of cuticular, ovarian, and follicular development during metamorphosis. Follicular structure was examined in whole mounts of ovaries using an immuno‐fluorescent labelling technique. The growth of oocytes and nurse cell cap in terminal follicles was measured throughout ovarian development. A rapid increase in the relative size of the nurse cells began during the fourth scotophase and continued until the beginning of the sixth scotophase. Following the sixth scotophase, the relative size of the nurse cells decreased until they disintegrated prior to choriogenesis. Oocytes began to grow rapidly during the fifth scotophase, coincident with the initiation of vitellogenesis, and continued to grow until choriogenesis was initiated just after adult eclosion. The rate of follicular growth was related to the position of the follicle in the ovariole; the closer to the terminal position, the greater the rate of growth. Thus, at adult eclosion, each ovariole contained a linear array of follicles in progressive stages of development with the terminal follicles ready to begin choriogenesis.

The effects of tunicamycin on the synthesis and export of fat body proteins and glycoproteins in larvae of the greater wax moth Galleria mellonella (L.)

Treatment of fat body from the greater wax moth, Galleria mellonella (L.), with tunicamycin in vitro caused complex alterations in protein synthesis. These included a 50% inhibition of protein synthesis and a greater than 90% inhibition of both protein release and glycoproteins biosyntheis. The inhibition of glycoprotein biosynthesis can be accounted for by the well-documented effects of tunicamycin on GlcNAc-pyrophosphoryl dolichol synthesis, while the two additional effects are likely to have a complex basis. Synthesis and export of the four storage proteins was also studied in fat body treated with tunicamycin. Exposure to antibiotic for short periods of time (2.5 hr) resulted in the sysnthesis of lower mol. wt variants of at least two of these polypeptides in treated cultures, both of which normally contain carbohydrate. Treated fat body released only putative fully-glycosylated forms of the storage proteins which suggests that the attachment of carbohydrate is in one way necessary for their secretion. Newly synthesized storage proteins were not detected in fat body treated with tunicamycin for longer time periods (approx. 7 hr), which indicates carbohydrate may also increase the intracellular half life of this class of proteins.

Declining ecdysteroid levels are temporally correlated with the initiation of vitellogenesis during pharate adult development in the Indianmeal moth, Plodia interpunctella

Photo-synchronized pupae were used to determine the titer of ecdysteroids during the pupal and pharate adult stages of the Indianmeal moth, Plodia interpunctella (Hiibner). For female pupae that were kept in a 16h 1ight:S h dark (longday) photocycle, there was one major peak of ecdysteroids. The ecdysteroids reached a maximum of 2000-2500 pg/mg wet wt between 28-36 h after pupation. The ecdysteroids declined to 400 pglmg wet wt by 68 h after pupation. For pupae that were kept in continuous darkwss, the ecdysteroid peak became broader with a maximum at 24 h (3130 pg/mg wet wt). The composition of ecdysteroids at various times during pharate adult development was determined usiug gas chromatographass spectrometry with selected ion monitoring (GC-MS (S1M)I. Eedysone was the major component (93%) in samples taken 28 h after pupation, at the maximum of the pupal peak. Thus, high levels of ecdysoue are correlated with the beginning of adult ovarian development. As pharate development progressed, the proportion of 20-hydroxyecdysow increased so that by 100 h after pupation 20-hydroxyecdysone was the only ecdysteroid detected. Previously, the initiation of vitellogenesis was determined to begin between % and 100 h after pupation. In conjuction with previous work, these findings demonstrate that the ecdysteroid titers decline before viteUogenesis is initiated and egg maturation can be completed. Ecdysteroids Vitellogenesis Metamorphosis Radioimrnunoassay GC-MS quantification

Interactions of ecdysteroid and juvenoid agonists in Plodia interpunctella (Hübner)

The influence of non-steroidal ecdysteroid agonists on Indianmeal moth larvae was assessed by rearing last instar larvae on diet treated with RH-5992 (tebufenozide) or RH-2485 (methoxyfenozide). Larvae were monitored for effects of the ecdysteroid agonists on weight, metamorphosis and mortality. Larvae treated with either of the ecdysteroid agonists at a concentration of 5 ppm or higher gained less weight and had greater mortality than did larvae reared on control diet. For example, the weights of control larvae increased approximately 400% by day 2, compared with only a 50% increase in weight when the larvae were treated with 25 ppm of RH-2485 or RH-5992. Similarly, mortality in control larvae was less than 10%, but was as much as 90–100% in larvae reared on diet treated with one of the ecdysteroid agonists. We also examined the effects of simultaneous treatment with a juvenile hormone (JH) mimic, either methoprene or fenoxycarb. The JH mimics prevented adult emergence, and the larvae continued to feed throughout the month-long observation period. However, larvae treated with a juvenile hormone mimic gained weight despite the presence of an ecdysteroid agonist in the diet. On diets treated with 0.1 ppm of RH-2485 or RH-5992, JH-treated larvae gained even more weight than did untreated controls. Interestingly, although the addition of a JH mimic to ecdysteroid-treated diet resulted in increased weight, it did not lead to reduced mortality. In fact, combinations of a JH mimic with 10 ppm RH 2485 or RH 5992 resulted in nearly 100% mortality compared with 40–70% mortality without the JH compounds. These results indicate that JH mimics overcome the inhibitory effects of ecdysteroid agonists on weight gain; however, they also resulted in increased mortality compared with moderate doses of ecdysteroid agonists alone. One specific action of these compounds at the cellular level was noted in that RH 5992 mimicked ecdysteroids by increasing uptake of 14C-GlcNAc in a Plodia interpunctella cell line, while fenoxycarb was inhibitory. Arch. Insect Biochem. Physiol. 38:91–99, 1998. Published 1998 Wiley-Liss, Inc.

Yolk sphere formation is initiated in oocytes before development of patency in follicles of the moth,Plodia interpunctella

We describe a provitellogenic stage, a previously unrecognized stage of follicle development in moths, and show that oocytes begin yolk sphere formation prior to the development of patency by the follicular epithelium. The vitellogenic activities of follicles from pharate adult femalePlodia interpunctella (Hübner) were determined by visualizing the subunits of vitellin (YP1 and YP3) and the follicular epithelium yolk protein (YP2 and YP4) using monospecific antisera to each subunit to immunolabel whole-mounted ovaries or ultrathin sections. At 92 h after pupation, yolk spheres that contained only YP2 began to proliferate in the oocytes. The inter-follicular epithelial cell spaces were closed at 92 h making vitellogenin inaccessible to the oocyte, and consequently, the vitellin subunits were not observed in the yolk spheres. YP2 uptake most likely occurred across the brush border from the follicular epithelial cells to the oocyte at this time. At 105 h, the inter-follicular epithelial cell spaces appeared closed yet trace amounts of labeling for vitellin were observed in the spaces and also in the yolk spheres along with YP2. Equivalent labeling for all four YPs in yolk spheres was finally observed at 112 h after pupation when the follicular epithelium had become patent. These data indicate that the provitellogenic stage is an extended transition period between the previtellogenic and vitellogenic stages that lasts for approximately 13 h, and it is marked at the beginning by YP2 yolk sphere formation in the oocyte and at the end by patency in the follicular epithelium.