Cryopreservation of Mediterranean fruit fly embryos.

In this paper we present a procedure to cryopreserve the embryos of a tephritid, the Mediterranean fruit fly (Ceratitis capitata), by vitrification. Developmental stages between 24 and 32 hours after oviposition were examined for tolerance to cryopreservation. Embryos, 27-hr-old and incubated at 29 C, were found to be at the most suitable stage for treatment. Effects of the previtrification steps of our protocol, dechorionation, permeabilization, cryoprotectant loading, and dehydration, on survival to hatching were also assessed. Dechorionation did not affect viability, while isopropanol and a hexane treatment used in the permeabilization step of the protocol reduced hatching by about 15%. This reduction was dependent on the amount of isopropyl alcohol carried over into the hexane rinse. The remaining previtrification steps reduced hatching by an additional 10%. After optimization of the procedure, normalized hatching was 44% after vitrification in liquid nitrogen vapor followed by storage under liquid nitrogen for a test period of 7 days. Post cryopreservation larval diets containing wheat bran, corncob grits, or agar as the base were examined for survival to pupation and emergence. A yield of 34% egg to adult emergence was obtained when the agar-based diet was used for rearing larvae that had experienced cryopreservation during the embryonic stage.

The piggyBac transposon mediates germ-line transformation in the Oriental fruit fly and closely related elements exist in its genome.

Germ-line transformation of a white eye strain of the Oriental fruit fly, Bactrocera dorsalis, was achieved with the piggyBac vector, derived from a transposon originally isolated from the cabbage looper moth, Trichoplusia ni. The vector was marked with the medfly white+ gene cDNA, and three transgenic lines were identified at a frequency of approximately 2% per fertile G0. Vector integrations were verified by Southern DNA hybridization, which also revealed the presence of endogenous genomic elements closely related to piggyBac. Approximately 10-20 elements per genome were evident in several B. dorsalis strains, and sequence analysis of 1.5 kb gene amplification products from two wild strains and the white eye host strain indicated 95% nucleotide and 92% amino acid sequence identity among resident elements and the T. ni element. PiggyBac was not evident by hybridization in other tephritid species, or insects previously transformed with the transposon. This is the first discovery of piggyBac beyond T. ni, and its existence in a distantly related species has important implications for the practical use of the vector and insects transformed with it.

The lepidopteran transposon vector, piggyBac, mediates germ-line transformation in the Mediterranean fruit fly.

The piggyBac (IFP2) short inverted terminal repeat transposable element from the cabbage looper Trichoplusia ni was tested for gene transfer vector function as part of a bipartite vector-helper system in the Mediterranean fruit fly Ceratitis capitata. A piggyBac vector marked with the medfly white gene was tested with a normally regulated piggyBac transposase helper at two different concentrations in a white eye host strain. Both experiments yielded transformants at an approximate frequency of 3-5%, with a total of six lines isolated having pigmented eyes with various levels of coloration. G1 transformant siblings from each line shared at least one common integration, with several sublines having an additional second integration. For the first transformant line isolated, two integrations were determined to be stable for 15 generations. For five of the lines, a piggyBac-mediated transposition was verified by sequencing the insertion site junctions isolated by inverse PCR that identified a characteristic piggyBac TTAA target site duplication. The efficient and stable transformation of the medfly with a lepidopteran vector represents transposon function over a relatively large evolutionary distance and suggests that the piggyBac system will be functional in a broad range of insects.

Linkage analysis of five new genetic markers of the oriental fruit fly, Bactrocera dorsalis (Diptera: Tephritidae).

Five new autosomal recessive genes are described in the oriental fruit fly, Bactrocera dorsalis (Hendel). These genetic markers are associated into three linkage groups. The matte (mt) gene is linked to the previously described mandarin red (ma) gene, and the white puparium (wp) gene is linked to the white eye (we) and amethyst (am) loci. The third designated linkage group has the yellow eye (ye) marker. The we/we homozygote is epistatic to ye/ye, and each is epistatic to am/am and ma/ma.