Functional characterization of the transcriptional regulatory elements of three highly expressed constitutive genes in the jewel wasp, Nasonia vitripennis.

The jewel wasp, Nasonia vitripennis Ashmead (Hymenoptera: Pteromalidae), is an easily reared parasitoid that is providing an ever increasingly malleable model for examining the biology and genetics of Hymenoptera. Utilizing genomic and transcriptome resources, 5' upstream transcriptional regulatory sequences (TREs) from three highly expressed genes were identified and cloned. Criteria for TRE selection included the presence of an adjacent gene 5' of the translation initiation site. One gene was methylated whereas the other two were nonmethylated. Each TRE, heat-shock protein 70 (hsp70), activator of 90 kDa hsp ATPase protein 1 (hsp90A), and lipid storage droplet surface-binding protein 1 (lsdp) was linked with enhanced green fluorescent protein (EGFP) coding sequence and cloned into both pDP9e somatic and piggyBac germline transformation vectors. EGFP expression patterns under control of each TRE were compared with patterns of DsRed fluorescence produced from the transformation vector cassette. Functional activity of each TRE was observed in cultured Spodoptera frugiperda 9 (Sf9) cells and Drosophila melanogaster as well as in N. vitripennis embryos demonstrating that all three sequences had functional transcriptional regulatory activity in three different insect orders. Identification and functional characterization of these three TREs will provide critical and necessary resources for further genetic analyses of N. vitripennis, Hymenoptera and other insects.

Three Halloween genes from the Varroa mite, Varroa destructor (Anderson & Trueman) and their expression during reproduction.

The ecdysteroid biosynthetic pathway involves sequential enzymatic hydroxylations by a group of enzymes collectively known as Halloween gene proteins. Complete sequences for three Halloween genes, spook (Vdspo), disembodied (Vddib) and shade (Vdshd), were identified in varroa mites and sequenced. Phylogenetic analyses of predicted amino acid sequences for Halloween orthologues showed that the acarine orthologues were distantly associated with insect and crustacean clades indicating that acarine genes had more ancestral characters. The lack of orthologues or pseudogenes for remaining genes suggests these pathway elements had not evolved in ancestral arthropods. Vdspo transcript levels were highest in gut tissues, while Vddib transcript levels were highest in ovary-lyrate organs. In contrast, Vdshd transcript levels were lower overall but present in both gut and ovary-lyrate organs. All three transcripts were present in eggs removed from gravid female mites. A brood cell invasion assay was developed for acquiring synchronously staged mites. Mites within 4 h of entering a brood cell had transcript levels of all three that were not significantly different from mites on adult bees. These analyses suggest that varroa mites may be capable of modifying 7-dehydro-cholesterol precursor and hydroxylations of other steroid precursors, but whether the mites directly produce ecdysteroid precursors and products remains undetermined.

Genomic organization and reproductive regulation of a large lipid transfer protein in the varroa mite, Varroa destructor (Anderson & Trueman).

The complete genomic region and corresponding transcript of the most abundant protein in phoretic varroa mites, Varroa destructor (Anderson & Trueman), were sequenced and have homology with acarine hemelipoglycoproteins and the large lipid transfer protein (LLTP) super family. The genomic sequence of VdLLTP included 14 introns and the mature transcript coded for a predicted polypeptide of 1575 amino acid residues. VdLLTP shared a minimum of 25% sequence identity with acarine LLTPs. Phylogenetic assessment showed VdLLTP was most closely related to Metaseiulus occidentalis vitellogenin and LLTP proteins of ticks; however, no heme binding by VdLLTP was detected. Analysis of lipids associated with VdLLTP showed that it was a carrier for free and esterified C12 -C22 fatty acids from triglycerides, diacylglycerides and monoacylglycerides. Additionally, cholesterol and ß-sitosterol were found as cholesterol esters linked to common fatty acids. Transcript levels of VdLLTP were 42 and 310 times higher in phoretic female mites when compared with males and quiescent deutonymphs, respectively. Coincident with initiation of the reproductive phase, VdLLTP transcript levels declined to a third of those in phoretic female mites. VdLLTP functions as an important lipid transporter and should provide a significant RNA interference target for assessing the control of varroa mites.

Variable induction of vitellogenin genes in the varroa mite, Varroa destructor (Anderson & Trueman), by the honeybee, Apis mellifera L, host and its environment.

Transcript levels of vitellogenins (Vgs) in the varroa mite, Varroa destructor (Anderson & Trueman), were variably induced by interactions between the developing honeybee, Apis mellifera L, as a food source and the capped honeybee cell environment. Transcripts for two Vgs of varroa mites were sequenced and putative Vg protein products characterized. Sequence analysis of VdVg1 and VdVg2 proteins showed that each had greater similarity with Vg1 and Vg2 proteins from ticks, respectively, than between themselves and were grouped separately by phylogenetic analyses. This suggests there was a duplication of the ancestral acarine Vg gene prior to the divergence of the mites and ticks. Low levels of transcript were detected in immature mites, males and phoretic females. Following cell invasion by phoretic females, VdVg1 and VdVg2 transcript levels were up-regulated after cell capping to a maximum at the time of partial cocoon formation by the honeybee. During oviposition the two transcripts were differentially expressed with higher levels of VdVg2 being observed. A bioassay based on assessing the transcript levels was established. Increases in VdVg1 and VdVg2 transcripts were induced experimentally in phoretic females when they were placed inside a cell containing an early metamorphosing last instar bee but not when exposed to the metamorphosing bee alone. The variable response of Vg expression to the food source as well as environmental cues within the capped cell demonstrates that perturbation of host-parasite interactions may provide avenues to disrupt the reproductive cycle of the varroa mites and prevent varroasis.

Regulation of Junonia coenia densovirus P9 promoter expression.

Transcriptional activity of the Junonia coenia densovirus (JcDNV) P9 promoter depends on a 557-bp sequence located within the overlapping 3' sequences for viral capsid and nonstructural genes. Utilizing a somatic transformation assay to assess JcDNV promoter activity in Drosophila melanogaster and Plodia interpunctella, viral sequences were subjected to deletional analysis. Removal of a 685-bp fragment reduced P9-driven expression to background levels. Inclusion of a second expression cassette demonstrated vector persistence and confirmed somatic transformation. P9 promoter-driven expression was restored by insertion of a 557-bp JcDNV fragment or by inclusion of a heterologous baculovirus hr5 enhancer. Consensus polycomb transcriptional factor binding sites were identified within the 557-bp fragment, which suggests a potential role in regulating densoviral transcription.

Somatic transformation efficiencies and expression patterns using the JcDNV and piggyBac transposon gene vectors in insects.

A somatic transformation gene vector that exploits the genomic integration properties of Junonia coenia lepidopteran densovirus (JcDNV) sequences in vivo has been developed. JcDNV somatic transformation vectors are derivatives of plasmids containing an interrupted genome of JcDNV that provide efficient, robust vectors that can be used to examine regulation of chromosomally integrated transgenes in insects. Microinjection of JcDNV plasmids into syncytial embryos of Drosophila melanogaster or the lepidopterans Plodia interpunctella, Ephestia kuehniella or Trichoplusia ni resulted in persistent transgene expression throughout development. Inclusion of transgenes with tissue-specific promoters resulted in expression patterns canonical with phenotypes of piggyBac germline transformants. Somatic transformation required the presence of the viral inverted terminal repeat in cis only and did not depend upon non-structural viral proteins.

Development of the larval ovary in the moth, Plodia interpunctella.

The morphogenesis of ovaries and the organization of germ cells within them were visualized during the larval stages of the moth, Plodia interpunctella. The germ cells were observed by utilizing confocal microscopy coupled with immuno-fluorescent staining for the alpha-crystallin protein 25 (alphaCP25). The alphaCP25 was previously shown to be specific to germ cells of pupae and adults, and this study shows that alphaCP25 is present in larval germ cells as well. A cluster of 28 germ cells that stain for alphaCP25 was found in the gonads of newly hatched first instar larvae. The founding germ cells became segregated into four clusters, most likely by somatic cell intrusion, around the beginning of the second instar. Division of the primary germ cells began by the end of the second instar and the formation of all cystoblasts appeared to be completed within the four ovarioles by the end of the third instar. Within the ovarioles of third instar larvae, the germ cells were organized with a distal cap of seven germ cells which was segregated from the majority of the germ cells. The main body of germ cells was arranged around a central germ cell-free core as a spiral. Divisions of the cystoblasts to form cystocyte clusters were nearly completed during the fourth (last) larval instar. These features suggest that the strategy to produce follicles in moths is fundamentally different from the fruitfly, Drosophila. It appears that during the initial stages of ovary development in P. interpunctella, the primary germ cells undergo stage-complete divisions that are completed prior to the onset of the next set of divisions, which results in a complete complement of follicles available by the time of adult eclosion, while in Drosophila the primary germ cell divisions are initiated in the adult stage, and follicles are produced individually as resources are available.

Fat body expressed yolk protein genes in Hyphantria cunea are related to the YP4 follicular epithelium yolk protein subunit gene of pyralid moths.

cDNA clones for two of the yolk proteins, YP1 and YP2, produced by the fat body of the moth, Hyphantria cunea, were sequenced and found to be homologous to the follicular epithelium yolk proteins of pyralid moths. Both cDNA clones coded for polypeptides of 290 residues and the deduced amino acid sequence identity between YP1 and YP2 was very high (79.0%). Analysis of the secondary structure of the predicted polypeptides suggests that YP1 and YP2 do not form heteromeric proteins because of differences in secondary structure due to the lack of alpha helices in YP1. Northern blot analysis showed that the transcripts for YP1 (1.2 kb) and YP2 (1.1 kb) were present primarily in the female fat body with only trace levels detectable in the ovary of the adult female. In a developmental study, the YP1 and YP2 transcripts were first detectable in 10-day-old pupae and increased into the adult stage. These results suggest that the YP1 and YP2 genes in H. cunea have been recruited to replace the vitellogenin gene as the primary source of yolk proteins. During this process they have acquired a modified pattern of expression that is different from homologous genes reported in pyralid moths. The assessment of the evolution of proteinaceous yolk in these moths should serve as an excellent model for the evolution of gene recruitment.

Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector.

We have developed a system for stable germline transformation in the silkworm Bombyx mori L. using piggyBac, a transposon discovered in the lepidopteran Trichoplusia ni. The transformation constructs consist of the piggyBac inverted terminal repeats flanking a fusion of the B. mori cytoplasmic actin gene BmA3 promoter and the green fluorescent protein (GFP). A nonautonomous helper plasmid encodes the piggyBac transposase. The reporter gene construct was coinjected into preblastoderm eggs of two strains of B. mori. Approximately 2% of the individuals in the G1 broods expressed GFP. DNA analyses of GFP-positive G1 silkworms revealed that multiple independent insertions occurred frequently. The transgene was stably transferred to the next generation through normal Mendelian inheritance. The presence of the inverted terminal repeats of piggyBac and the characteristic TTAA sequence at the borders of all the analyzed inserts confirmed that transformation resulted from precise transposition events. This efficient method of stable gene transfer in a lepidopteran insect opens the way for promising basic research and biotechnological applications.

cDNA of YP4, a follicular epithelium yolk protein subunit, in the moth, Plodia interpunctella.

YP4, a subunit of the follicular epithelium yolk protein in the moth, Plodia interpunctella, is produced in the follicle cells during vitellogenesis and after secretion is taken up into the oocyte and stored in the yolk spheres for utilization during embryogenesis. In order to identify the cDNA clones for YP4, a degenerate PCR primer was designed to six amino acid residues identified in the NH2-terminal sequence of mature YP4. The YP4 degenerate primer plus T7 reverse PCR primer produced a PCR product from a cDNA library for the majority of the YP4 coding sequence. Combined cDNA and 5' RACE sequencing showed the YP4 transcript to be 991 bp in length with a single open reading frame for a predicted polypeptide of 299 amino acids. Northern analysis showed a single YP4 transcript was present in ovarian RNA that was approximately 1 kb in length. The predicted amino acid sequence for YP4 from P. interpunctella was most closely related to the predicted YP4 protein from the moth, Galleria mellonella, and the spherulin 2a protein from the slime mold, Physarum polycephalum.

alpha-Crystallin protein cognates in eggs of the moth, Plodia interpunctella: possible chaperones for the follicular epithelium yolk protein.

alpha-Crystallin protein cognates were found in germ cells of the Indianmeal moth, Plodia interpunctella (Shirk and Zimowska, 1997). A cDNA clone of 674 bp with a single open reading frame was isolated for a 25,000 molecular weight polypeptide member of this family, alpha CP25, and a single transcript of approximately 700 bp was found in the ovary of vitellogenic females. Both the DNA sequence and predicted amino acid sequence showed considerable homology with the embryonic lethal gene, l(2)efl, in Drosophila melanogaster. In addition to the sequence for l(2)efl, the predicted amino acid sequence for acp25 also showed significant sequence similarly with the alpha-crystallin A chain polypeptides from the lenses of vertebrae eyes. An N-terminal hydrophobic aggregation site and a C-terminal protective binding site common to alpha-crystallin proteins were present in the predicted acp25 and l(2)efl amino acid sequences, while only the C-terminal protective binding site was present in the small heat shock protein sequences from D. melanogaster. This evidence suggests that although the alpha-crystallin protein cognates in P. interpunctella evolved from a gene common with small heat shock protein genes, the amino acid sequence has converged on a structure similar to that of alpha-crystallin proteins. Native immunoblot analysis showed that the alpha-crystallin proteins formed high molecular weight complexes with the follicular epithelium yolk protein (FEYP) but not vitellin in yolk. An electroblot binding assay was used to show that the germ-cell alpha-crystallins of P. interpunctella bind specifically with the FEYP and that the binding was reversible in the presence of ATP or low pH. This evidence in conjunction with the evidence that the alpha-crystallins and FEYP form a stable complex that co-purifies from native egg proteins suggests that the alpha-cystallin cognates function as chaperones for the follicular epithelium yolk proteins in the embryos of P. interpunctella.

5' coding region of the follicular epithelium yolk polypeptide 2 cDNA in the moth, Plodia interpunctella, contains an extended coding region.

The 5' region of YP2 cDNA, a follicular epithelium yolk protein subunit in the moth, Plodia interpunctella, shows that the polypeptide contains an extended internal coding region. Partial cDNA clones for YP2 were isolated from a pharate adult female ovarian cDNA expression library in Lambda Zap II by screening with antigen selected YP2 antiserum. The 5' sequence of the YP2 transcript was determined by 5' RACE PCR of ovarian mRNA using YP2 sequence-specific nested primers. The combined cDNA and 5' RACE sequencing showed the YP2 transcript to be 1971 bp in length up to the poly(A) tail with a single open reading frame for a predicted polypeptide of 616 amino acids. Northern analysis showed a single YP2 transcript to be present in ovarian RNA that was approximately 2 kb in length. The predicted amino acid sequence for YP2 from P. interpunctella is most closely related to egg specific protein (ESP) from Bombyx mori and the partial YP2 sequence from Galleria mellonella. YP2 from P. interpunctella also is similar to vertebrate lipases and contains a conserved lipid binding region. However, the 5' coding region of YP2 from P. interpunctella contains an in-frame insert of approximately 438 bp that had replaced an approximately 270-bp region as compared with ESP from B. mori and YP2 of G. mellonella. This suggests that the insert occurred by a recombinational event internal to the YP2 structural gene of P. interpunctella.

An alpha-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 interpunctella (Zimowska et al., 1991). The antigen, molecular weight 25,000 kDa, and a related protein, molecular weight 21,000 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, 21,000, 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 alpha-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 alpha-crystallins, we suggest that this family of polypeptides from the germ cells of this moth be considered as cognates of the alpha-crystallins, and the 25,000 molecular weight polypeptide described here be given the designation ac25. Using immuno-gold labeling with antiserum to ac25, the alpha-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.

Activity-related changes in protein phosphorylation in an identified Aplysia neuron.

The relationship between long-term electrical activity and protein phosphorylation was investigated in single, identifiable neurons in the abdominal ganglion of Aplysia californica by the intracellular injection of radiolabeled ATP followed by sodium dodecyl sulfate (SDS) gel electrophoresis. Natural and pharmacological treatments that alter the impulse activity of neurons L6 and R15 for prolonged periods did not appear to affect the phosphorylation of most of the 15 major phosphoproteins examined in these cells. Long-term excitation of L6 induced by the phosphodiesterase inhibitor IBMX correlated with phosphorylation of a 29,000-dalton protein. Long-term inhibition of L6 induced by afterdischarge of peptidergic bag-cell neurons appeared to cause dephosphorylation of a 29,000-dalton protein. Burst augmentation of R15 induced by bag-cell afterdischarge did not cause detectable changes in the phosphorylation of the major proteins we examined. These data are consistent with other studies of neural and nonneural tissues which have found a correlation between activity and the level of phosphorylation of a 29,000-dalton protein.

Identification, synthesis, and characterization of the yolk polypeptides of Plodia interpunctella.

The mature eggs of Plodia interpunctella were found to contain four major polypeptides. These yolk polypeptides (YPs) were found to have approximate molecular weights of 153,000 daltons (YP1), 69,000 daltons (YP2), 43,000 daltons (YP3), and 33,000 daltons (YP4) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In addition, we found YP1 was resolved by a 5% polyacrylamide gel into two separate polypeptides of 153,000 and 147,000 daltons. All of the YPs could be labeled in vivo or in vitro with [35S]-methionine. Yolk peptide 1 and YP3 were synthesized by fat body of pharate adult and adult females and secreted into the hemolymph. Yolk peptide 2 and YP4 were synthesized and secreted into incubation medium by ovaries that contained vitellogenic oocytes, but these polypeptides were not found in the hemolymph. Fat bodies of males synthesized and secreted an immunoprecipitable polypeptide similar to YP3 as well as immunoprecipitable polypeptides larger than 200,000 daltons that had no counterparts in the oocytes. Peptide mapping by protease digestion showed each YP to be cleaved into unique fragments, suggesting that no precursor-product relationship exists between the YPs. Ion exchange chromatography and gel permeation chromatography separated that yolk proteins into two groups with approximate molecular weights of 462,000 and 264,000 daltons. By resolving these peaks on SDS-PAGE, it was found that YP1 and YP3 formed the 462,000-dalton yolk protein and YP2 and YP4 formed the 264,000-dalton yolk protein.

20-Hydroxyecdysone stimulates the accumulation of translatable yolk polypeptide gene transcript in adult male Drosophila melanogaster.

Yolk polypeptide (YP) synthesis is hormonally stimulated during maturation of adult female Drosophila melanogaster. Synthesis of the three YPs is sex specific and occurs in fat body cells and follicle cells of adult females. However, males have been shown to produce YPs when treated with the steroid hormone 20-hydroxyecdysone (20-HE). By using a cell-free translation system as an assay for YP mRNA, we found that 20-HE also causes the accumulation of translatable YP message in males. In addition, hybridization of cloned copies of genes for both YP1 and YP3 to total RNA from males showed that 20-HE caused the appearance of YP gene transcripts in males. Eight hours after treatment of males with 20-HE, YP gene transcript levels had increased at least 25-fold to approximately 2.7 x 10(6) copies of YP1 gene transcript per adult male fly. In normal adult females, there were 42 x 10(6) copies per fly by 24 hr. There was neither detectable YP synthesis nor translatable YP gene transcript in either normal 1- to 3-day-old males or 24-hr-old males treated with a juvenile hormone analogue. This evidence shows that 20-HE acts to regulate the levels of translatable YP mRNA in male Drosophila.

The accessory sex glands as the repository for juvenile hormone in male cecropia moths.

Gas chromatographic determinations, bioassays, and radio-labelling experiments show that the juvenile hormone in adult male Hyalophora cecropia is accumulated exclusively in the accessory sex glands. Moths do not store measurable quantities of juvenile hormone if their accessory sex glands are removed shortly after adult eclosion.