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Biomedical subjects

R A Leopold

Publications and source records attributed to R A Leopold.

At least 19 recordsLinked to original sources
PubMed2006

Cryopreservation of Mexican fruit flies by vitrification: stage selection and avoidance of thermal stress.

This report presents details of a vitrification methodology for the cryopreservation of embryos of the Mexican fruit fly, Anastrepha ludens. The overall summary of the data indicates that selecting the correct developmental stage for cryopreservation is the most important criterion. The key aspect in selection of the correct stage is to balance depletion of the gut yolk content against development of the embryonic cuticle. Embryogenesis was divided into four stages between 90 and 120 h after incubation at 21.7 degrees C. The classification was based on the intestinal yolk content and the initial development of mandibular-maxillary complex. Stages having low mid-gut yolk content and the appearance of mouth hooks were found to be the most suitable for cryopreservation. Embryos developing at 30 degrees C had premature cuticle formation relative to gut development and significantly lower hatching after cryopreservation. Vitrification of embryos by direct quenching in liquid nitrogen was less effective than quenching after annealing the samples in liquid nitrogen vapor. Quenched samples of vitrification solutions containing 1,2-ethanediol as the major component exhibited fractures. Fracturing occurred less frequently when the solutions were annealed and when containing polyethylene glycol. Hatching of vitrified embryos stored in liquid nitrogen for over 12 months was not statistically different from those held for only 15 min. Our protocol yielded normalized hatching rates that ranged as high as 61%. Selecting the exact stage for cryopreservation from a population of embryos obtained by collection from ovipositing females during a span of just 30 min resulted in nearly 80% of the embryos hatching into larvae.

Animals↗
PubMed2000

Cryopreservation of Musca domestica (Diptera: Muscidae) embryos.

Prior studies on cryopreserving embryos of several non-drosophilid flies established that two Drosophila melanogaster embryo cryopreservation protocols were not directly suitable for use with these species. This paper describes our work on developing a protocol for cryopreservation of embryos of the housefly, Musca domestica. Significant progress was made when permeabilization of the vitelline membrane was optimized, a vitrification solution containing ethylene glycol, polyethylene glycol, and trehalose was formulated, and when cooling and recovery of the cryopreservation protocol included a step which passed the embryos through liquid nitrogen vapor. More than 70% of housefly embryos withstand treatments of dechorionation, permeabilization, loading with cryoprotectant, and dehydration in vitrification solution, but the cooling, warming, and poststorage rearing steps still cause a considerable reduction in survival. About 53% of the vitrified M. domestica embryos hatched into larvae. Relative to the percentage of the control adult emergence, about 13% of the embryos stored in liquid nitrogen developed into fertile adults. Hatching of the F(1) progeny of adults having been cryopreserved as embryos was similar to control levels.

2-Propanol↗
PubMed2000

Permeabilization of Cochliomyia hominivorax (Diptera: Calliphoridae) embryos.

Embryos of the primary screwworm, Cochliomyia hominivorax (Coquerel), were successfully permeabilized for use in subsequent cryopreservation studies. Mortality was greater for eggs incubated for < 5 h before treatment. The mean survival of embryos to first instars was 55.7, 61.1, and 62.6% when the embryos were incubated for 5, 5.5, and 6 h before treatment, respectively. The survival to the pupal and adult stages was low. An improved media for culturing the embryos during and immediately after treatment needs to be devised and the procedure for rearing the larval stages also needs to be altered to improve survival for emerging adults.

Animals↗
PubMed1998

Post pupariation cold storage of three species of flies: increasing chilling tolerance by acclimation and recurrent recovery periods.

Three species of flies were examined for chilling tolerance from the end of the pupariation to the onset of adult eclosion for the purpose of determining applicability for cold storage. For the housefly, Musca domestica (Diptera, Muscidae), survival to eclosion was affected by developmental stage/age, length of storage, and storage temperature. Houseflies that were closest to having experienced pupariation or the onset of eclosion were the least cold tolerant. For flies stored at 7 or 10 degrees C, age groups placed directly into storage at 36-72 and 36-48 h postpupariation, respectively, had significantly higher poststorage eclosion than younger or older age groups. Death was observed after only 2 or 3 days with the chilling intolerant groups while mid-aged groups survived 10-12 days under cold storage conditions. Damage could be reduced by giving the mid-aged groups chilled at 7 or 10 degrees C recurrent 2- or 3-h recovery periods every 4 days at 28 degrees C. Examination of O2 consumption throughout the housefly pupal and pharate adult stages showed that the most chilling tolerant groups also had the lowest metabolic activity. Combining a prestorage 17-h slow cooling acclimation interval with the recurrent recovery periods increased eclosion of adults 20% for the 12- to 24-h group after 21 days storage at 7 degrees C. Two blowfly species, Lucilia cuprina and Lucilia sericata (Diptera, Calliphoridae), survived 10 degrees C storage best when given a weekly 4-h recovery period. Survival at 90 days poststorage was nearly 50% for L. cuprina and 80% for L. sericata. Increasing survival under subambient temperatures for all three species appears to relate to whether there is a stage of development which allows cold-induced lowering of metabolism that results in a dormant status best characterized as hibernal quiescence. This study indicates that periodic warming during cold storage increases survival by allowing a chilling intolerant stage to develop to a more tolerant stage and/or by eliminating accumulated toxic metabolites.

Adaptation, Biological↗
PubMed1996

Gene transfer into corn earworm (Helicoverpa zea) embryos.

Transposable elements with short inverted repeats at their termini have been identified in a number of diverse insect species and have proven to be useful gene delivery vectors for the transformation of Drosophila melanogaster. In this report we examine the ability of the D. melanogaster hobo element to transpose in lepidopteran species. A Trichoplusia ni (cabbage looper) and a Helicoverpa zea (corn earworm) embryonic cell line were found to be capable of supporting productive transposition of the hobo element as measured by a plasmid-based excision assay. Furthermore, hobo transposition was detected in H. zea embryos in a manner consistent with that seen for the cell line. In both cases, transposition/excision was found to be independent of vector-encoded transposase functions, indicating that endogenous genes are involved in hobo mobility. Finally, we demonstrate the stable insertion of the bacterial lacZ gene into the H. zea genome. These data demonstrate that hobo elements are capable of transgressing species boundaries and functioning in non-drosophilid cellular environments. More importantly, this represents the first description of a genetic transformation system for a lepidopteran species.

Animals↗
PubMed1996

Using electroporation and a slot cuvette to deliver plasmid DNA to insect embryos.

Microinjection is the method used almost exclusively to deliver DNA constructs to insect embryos while electroporation is commonly used for DNA delivery to bacteria, cell cultures and certain plant tissues. This communication describes a method using an easily constructed slot cuvette and the electroporation technique for transfer of DNA to insect embryos for possible use in developing methods for germline transformation. This method eliminates time-consuming individual embryo manipulation and thus far has been found to be adaptable for use on several types of insect embryos. Using this method, we show successful transfer of plasmid DNA to embryos of the corn earworm moth, Helicoverpa zea, and the house fly, Musca domestica.

Animals↗
PubMed1992

Transfection of cultured cells of the cotton boll weevil, Anthonomus grandis, with a heat-shock-promoter-chloramphenicol-acetyltransferase construct.

Expression of heat shock proteins (hsp) in the BRL-AG-3C cell line from the cotton boll weevil was examined. It was determined that the maximal expression of endogenous hsp occurred at 41 degrees C. Various transfection methods were then compared using this cell line in conjunction with a transiently expressed bacterial gene marker (chloramphenicol acetyltransferase) which was under the control of the Drosophila hsp 70 gene promoter. The cationic lipid preparation Lipofectin was found to be very efficient at transfecting the boll weevil cells. Polylysine and 20-hydroxyecdysone-conjugated polylysine were moderately effective, whereas polybrene and electroporation, under the conditions reported herein, were ineffective at transfecting this cell line.

Animals↗
PubMed1991

Evidence for an inactive plasma membrane-associated precursor of active cytoplasmic ornithine decarboxylase in developing embryos of Musca domestica.

During embryonic development of Musca domestica inactive ornithine decarboxylase protein appears in the embryos at 6 h postoviposition, increases in concentration and reaches a maximum level at 9 h postoviposition. The inactive enzyme is associated with the plasma membrane and appears to be the precursor for active ornithine decarboxylase, which is associated with the cytosolic fraction just prior to hatching. Both ornithine decarboxylase protein and enzymatic activity disappear during the early larval stage of this insect.

Animals↗
PubMed1970

Cockroach leg regeneration: effects of ecdysterone in vitro.

Regenerating epithelial tissue from the cockroach leg forms two cuticular coverings. The first of these consists of a protein-carbohydrate complex that is replaced before molting. The second covering represents the cuticle containing chitin. The deposition of the cuticle can be induced in vitro by adding ecdysterone to the culture medium.

Animals↗
PubMedDate not supplied

Genetics of house flies. Variability studies with North Dakota, Texas, and Florida populations.

Genetic data were used to compare the structure of native house fly populations collected in North Dakota, Texas, and Florida. Recombination studies with mutant markers on chromosomes 3 and 4 indicated a lack of inversion polymorphism among the three populations in those areas of the genetic map studied. Significant differences were observed among flies from the three regions with regard to the frequency of 1) females that produced only male progeny, and 2) male-determining 3rd chromosomes (IIIm chromosomes). However, the North Dakota and Texas flies were more similar to each other than to the Florida flies since populations from the two former areas possessed a low frequency of both male-producing females and IIIm chromosomes; in contrast, the Florida population was void of females that produced males only and a high percentage if not all Florida males appeared to possess the IIIm male-determining mechanism. Tests for recessive lethal 3rd chromosomes showed that there was no significant difference in the frequency of lethal factors recovered from the North Dakota and Texas flies; the presence of IIIm chromosomes in Florida males precluded the recovery of lethal factors from this population by the method employed. The data suggest that house fly strains to be employed in genetic control programs should 1) originate from target control areas to avoid possible behavioral differences existing among flies from different locales, 2) be initiated with as many flies as possible to provide a background for the maintenance of variability, and 3) be renewed periodically with field-collected material since the genotype may be capable of rapid reorganization in response to laboratory selection pressures.

Animals↗