Grazyna Zimowska

Post-integration stabilization of a transposon vector by terminal sequence deletion in Drosophila melanogaster

Germline transformation systems for nearly 20 insect species have been derived from transposable elements, allowing the development of transgenic insects for basic and applied studies. These systems use a defective nonautonomous vector that results in stable vector integrations after the disappearance of transiently provided transposase helper plasmid, which is essential to maintain true breeding lines and consistent transgene expression that would otherwise be lost after vector remobilization. The risk of remobilization by an unintended transposase source has so far not been a concern for laboratory studies, but the prospective use of millions of transgenic insects in biocontrol programs will likely increase the risk, therefore making this a critical issue for the ecological safety of field release programs. Here we describe an efficient method that deletes a terminal repeat sequence of a transposon vector after genomic integration. This procedure prevents transposase-mediated remobilization of the other terminal sequence and associated genes, ensuring their genomic stability.

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.

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

Characterization of the proteasomeβ2 subunit gene and its mutant allele in the tephritid fruit fly pest, Anastrepha suspensa.

In Drosophila melanogaster the β2 proteasome subunit gene, Prosβ2, was first identified as a dominant temperature sensitive mutant, DTS‐7, that causes pupal lethality at 29 °C but allows survival to adulthood at 25 °C. To explore the use of proteasome mutations for a conditional lethal system in insect pests, we identified and isolated the β2 subunit gene of the 20S proteasome from the Caribbean fruit fly, Anastrepha suspensa. The caribfly ortholog AsProsβ2 was isolated from pupal cDNA by 5′ and 3′ RACE. The AsProsβ2 protein has high amino acid sequence similarity to predicted insect Prosβ2 subunits and homologs from yeast and mammals, and it contains the well conserved amino acids that confer catalytic activity and substrate specificity. AsProsβ2 is a single copy gene and its RNA accumulates throughout all developmental stages of the caribfly. For functional studies a point mutation, analogous to the Prosβ21 mutation in D. melanogaster, was introduced into AsProsβ2 to create an aberrant protein with a Gly170Arg substitution. Consistent with the DTS‐7 mutation, transgenic insects carrying the mutant allele undergo normal metamorphosis at the permissive temperature (25 °C) but at the non‐permissive temperature (29 °C) they exhibit effective pupal lethality. This is the first report of a functional characterization of a Prosβ2 cognate based on the creation of a dominant temperature‐sensitive mutation. This type of temperature‐dependent lethality could be used for biological control, where transgenic insects are reared to adulthood at 25 °C or lower and then released into the field where ambient temperatures averaging 29 °C or greater cause lethality in their progeny.

An α-crystallin protein cognate in germ cells of the moth, Plodia interpunctella

Previously we had reported the production of an antiserum to an antigen found primarily in germ cells of the Indianmeal moth, Plodia interpunctelh (Zimowska et al., 1991). The antigen, molecular weight 25 000 kDa, and a related protein, molecular weight 21000 kDa, co-purified with the follicular epithelium yolk protein. Antisera to the two proteins were raised, and they both reacted with the same four small polypeptides, which had molecular weights of 20 000, 21000, 25 000 and 28 000 kDa, that were present in the eggs throughout embryogenesis. A 30 amino acid sequence of an internal fragment of the 25 000 kDa molecular weight polypeptide showed sequence similarity with the a-crystallin A chain polypeptides from the lenses of vertebrate eyes and, to a lesser extent, with small heat shock proteins. Based on the sequence similarity with the a-crystallins, we suggest that this family of polypeptides from the germ cells of this moth be considered as cognates of the cu-crystallins, and the 25 000 molecular weight polypeptide described here be given the designation ac25. Using immuno-gold labeling with antiserum to ac25, the a-crystallins were shown to be distributed throughout the cytoplasm and nucleoplasm of the oocyte and nurse cells, but not present within yolk spheres or other organelles of the oocyte or nurse cells. Immunofluorescent staining of males showed antigenic material in the sperm bundles within the testes. Oenocytes of the pupal and adult stages also contained cross-reactive material. Published by Elsevier Science Ltd