Late Pupal Stage Research Articles

A cDNA encoding diazepam-binding inhibitor/acyl-CoA-binding protein in Helicoverpa armigera: Molecular characterization and expression analysis associated with pupal diapause

The diazepam binding inhibitor (DBI) or the acyl-CoA-binding protein (ACBP) is a 9–10 kDa highly conserved multifunctional protein that plays important roles in GABA A receptor activity regulation, lipid absorption and steroidogenesis in various organisms. To study the functions of DBI/ACBP in insect development or diapause, we cloned the cDNA from Helicoverpa armigera (Har) utilizing rapid amplification of cDNA ends (RACE). By homology search, Har-DBI/ACBP is conserved with the DBI/ACBPs known from other insects. Northern blot analysis showed that DBI/ACBP gene expressed in nonneural and neural tissues. RT-PCR combined Southern blot analysis revealed that DBI/ACBP mRNA in the brain of nondiapause individual was much higher than that in the brain of diapausing insects. At early and middle stages of 6th instar larvae, the level of DBI/ACBP mRNA was higher in the midgut of diapause type than that in nondiapause type and low at late 6th instar larval stage and early pupal stage in both types. In the prothoracic gland (PG), DBI/ACBP expression appeared at a high level at middle and late stages of 6th larval instar in both nondiapause and diapause types, and declined after pupation. In vitro experiments revealed that DBI/ACBP mRNA in PG could be stimulated by synthetic H. armigera diapause hormone (Har-DH), suggesting that Har-DH may stimulate the PG to produce ecdysteroids by the DBI/ACBP signal pathway. By in vitro assay, we also found that FGIN-1-27, which has similar functions to DBI/ACBP in ecdysteroidogenesis, could induce PG ecdysteroidogenesis effectively, suggesting that DBI/ACBP regulates biosynthesis of ecdysteroids in PG. Thus, DBI/ACBP indeed plays a key role in metabolism and development in H. armigera.

Stage-specific control of grape berry moth, Endopiza viteana (Clemens) (Lepidoptera: Tortricidae), by selective and broad-spectrum insecticides

The insect growth regulators (IGRs) tebufenozide and methoxyfenozide and the broad-spectrum insecticides azinphosmethyl, carbaryl, and fenpropathrin were compared for their activity against adult, egg, and larval stages of the grape berry moth, Endopiza viteana (Clemens) (Lepidoptera: Tortricidae), under laboratory and vineyard conditions. Adult mortality was not affected by exposure to field-equivalent rates of tebufenozide or methoxyfenozide on grape clusters, whereas all the broad-spectrum compounds significantly reduced adult survival, compared with the untreated controls. Surviving adult moths laid significantly more eggs on berries treated with the IGRs than on berries treated with any of the broad-spectrum insecticides. Survival of these eggs through to late larval and pupal stages was significantly lower on methoxyfenozide-treated grapes than on untreated grapes, and no pupae were found when grapes were treated with azinphosmethyl or fenpropathrin. Neither of the growth regulator insecticides limited egg eclosion or larval development by E. viteana when insecticides were applied before egg laying, whereas broad-spectrum insecticides were effective against both eggs and neonates at this timing. When applied after egg eclosion, all insecticide treatments significantly reduced survival of grape berry moth larvae. Under vineyard conditions, berries with 1-d-old residues of tebufenozide or methoxyfenozide received more E. viteana eggs than berries treated with broad-spectrum compounds. After aging for 7 or 14 d, no significant effects on E. viteana survival were detected among treatments. Whereas broad-spectrum insecticides provide control of multiple life stages of E. viteana, integration of tebufenozide or methoxyfenozide into vineyard management programs for control of this pest will be most successful if applications are timed for egg hatch.

DDP Is Needed for Normal Cell Proliferation

To gain insight into the essential functions of E2F, we have examined the phenotypes caused by complete inactivation of E2F and DP family members in Drosophila. Our results show that dDP requires dE2F1 and dE2F2 for DNA-binding activity in vitro and in vivo. In tissue culture cells and in mutant animals, the levels of dE2F and dDP proteins are strongly interdependent. In the absence of dDP, the levels of dE2F1 and dE2F2 decline dramatically, and vice versa. Accordingly, the cell cycle and transcriptional phenotypes caused by targeting dDP mimic the effects of targeting both dE2F1 and dE2F2 and are indistinguishable from the effects of inactivating all three proteins. Although trans-heterozygous dDP mutant animals develop to late pupal stages, the analysis of somatic mutant clones shows that dDP mutant cells are at a severe proliferative disadvantage when compared directly with wild-type neighbors. Strikingly, the timing of S-phase entry or exit is not delayed in dDP mutant clones, nor is the accumulation of cyclin A or cyclin B. However, the maximal level of bromodeoxyuridine incorporation is reduced in dDP mutant clones, and RNA interference experiments show that dDP-depleted cells are prone to stall in S phase. In addition, dDP mutant clones contain reduced numbers of mitotic cells, indicating that dDP mutant cells have a defect in G2/M-phase progression. Thus, dDP is not essential for developmental control of the G1-to-S transition, but it is required for normal cell proliferation, for optimal DNA synthesis, and for efficient G2/M progression.

Differential sequestration of storage proteins by various fat body tissues during post-larval development in silkworm, Bombyx mori L.

Summary During the final larval stadium of the silkworm, Bombyx mori, storage proteins SP1 and SP2 are synthesized mainly by peripheral and dorsal perivisceral fat body tissues. No further synthesis occurs during the non-feeding pupal stage. The ventral perivisceral tissue has no role in this process, but is specialized for the selective storage of the proteins. Storage proteins are selectively sequestered mainly during the pre-pupal period, over a span of approximately 96 h, and subsequently released back into the hemolymph and metabolized during late pupal stages.

Comparison of the circadian eclosion rhythm between non-diapause and diapause pupae in the onion fly, Delia antiqua: the change of rhythmicity

When pupae of Delia antiqua were transferred to constant darkness (DD) from light–dark (LD) cycles or constant light (LL), the sensitivity to light of the circadian clock controlling eclosion increased with age. The daily rhythm of eclosion appeared in both non-diapause and diapause pupae only when this transfer was made during late pharate adult development. When transferred from LL to DD in the early pupal stage, the adult eclosion was weakly rhythmic in non-diapause pupae but arrhythmic in diapause pupae. However, the sensitivity of the circadian clock to temperature cycles or steps was higher in diapause pupae than in non-diapause pupae; in the transfer to a constant 20 °C from a thermoperiod of 25 °C (12 h)/20 °C (12 h) on day 10 after pupation or from chilling (7.5 °C) in DD, the adult eclosion from diapause pupae was rhythmic but that from non-diapause pupae arrhythmic. In a transfer to 20 °C from the thermoperiod after the initiation of eclosion, rhythmicity was observed in both types of pupae. The larval stage was insensitive to the effect of LD cycle initiating the eclosion rhythm. In D. antiqua pupae in the soil under natural conditions, therefore, the thermoperiod in the late pupal stage would be the most important ‘Zeitgeber’ for the determination of eclosion timing.

Morphological Comparison of Pupal Wing Cuticle Patterns in Butterflies

Butterfly wing color-patterns are determined in the prospective wing tissues during the late larval and early pupal stages. To study the cellular differentiation process of wings, morphological knowledge on pupal wings is prerequisite. Here we systematically examined morphological patterns of the pupal wing cuticular surface in a wide variety of nymphalid butterflies in relation to adult color-patterns. Several kinds of pupal wing patterns corresponding to particular adult color-pattern elements were widely observed in many species. Especially noteworthy were the pupal "focal" spots corresponding to the adult border ocelli system, which were detected in many species of Nymphalinae, Apaturinae, Argynninae, Satyrinae, and Danainae. Striped patterns on the pupal wing cuticle seen in some species of Limenitinae, Ariadnae, and Marpesiinae directly corresponded to those of the adult wings. In Vanessa cardui, eyespot-like pattern elements were tentatively produced during development in the wing tissue underneath the pupal spots and subsequently erased, suggesting a mechanism for producing novel color-patterns in the course of development and evolution. The pupal focal spots reasonably correlated with the adult eyespots in size in Precis orithya and Ypthima argus. We physically damaged the pupal focal spots and their corresponding cells underneath in these species, which abolished or inhibited the formation of the adult eyespots. Taken together, our results clarified that pupal cuticle patterns were often indicative of the adult color-patterns and apparently reflect molecular activity of organizing centers for the adult color-pattern formation at least in nymphalid butterflies.

Identification and functional characterization of a sex pheromone receptor in the silkmoth Bombyx mori

Sex pheromones released by female moths are detected with high specificity and sensitivity in the olfactory sensilla of antennae of conspecific males. Bombykol in the silkmoth Bombyx mori was the first sex pheromone to be identified. Here we identify a male-specific G protein-coupled olfactory receptor gene, B. mori olfactory receptor 1 (BmOR-1), that appears to encode a bombykol receptor. The BmOR-1 gene is located on the Z sex chromosome, has an eight-exon/seven-intron structure, and exhibits male-specific expression in the pheromone receptor neurons of male moth antenna during late pupal and adult stages. Bombykol stimulation of Xenopus laevis oocytes expressing BmOR-1 and BmGalphaq elicited robust dose-dependent inward currents on two-electrode voltage clamp recordings, demonstrating that the binding of bombykol to BmOR-1 leads to the activation of a BmGalphaq-mediated signaling cascade. Antennae of female moths infected with BmOR-1-recombinant baculovirus showed electrophysiological responses to bombykol but not to bombykal. These results provide evidence that BmOR-1 is a G protein-coupled sex pheromone receptor that recognizes bombykol.

The Drosophila centrosome-associated protein CP190 is essential for viability but not for cell division.

The Drosophila CP190 and CP60 proteins interact with each other and shuttle between the nucleus in interphase and the centrosome in mitosis. Both proteins can bind directly to microtubules in vitro, and have been shown to associate with a specific pattern of loci on salivary gland polytene chromosomes, but their functions are unknown. Here we show that reducing the level of CP190 or CP60 by >90% in tissue culture cells does not significantly interfere with centrosome or microtubule organisation, with cell division, or with cell viability. However, CP190 is an essential protein, as flies homozygous for mutations in the Cp190 gene die at late pupal stages of development. In larval brains of Cp190 mutants, mitosis is not radically perturbed, and a mutated form of CP190 (CP190DeltaM), that cannot bind to microtubules or associate with centrosomes, can rescue the lethality associated with mutations in the Cp190 gene. Thus, CP190 plays an essential role in flies that is independent of its association with centrosomes or microtubules.

The sex-peptide DUP99B is expressed in the male ejaculatory duct and in the cardia of both sexes.

Mating elicits two postmating responses in many insect females: the egg laying rate increases and sexual receptivity is reduced. In Drosophila melanogaster, two peptides of the male genital tract, sex-peptide and DUP99B, elicit these postmating responses when injected into virgin females. Here we show that the gene encoding DUP99B is expressed in the male ejaculatory duct and in the cardia of both sexes. The DUP99B that is synthesized in the ejaculatory duct is transferred, during mating, into the female genital tract. Expression of the gene is first seen in a late pupal stage. Males containing an intact ejaculatory duct, but lacking accessory glands, initiate the two postmating responses in their female partners [Xue, L. & Noll, M. (2000) Proc. Natl Acad. Sci. USA97, 3272-3275]. Although such males synthesize DUP99B in wild-type quantities, they elicit only weak postmating responses in their mating partners. Males lacking the Dup99B gene elicit the two postmating responses to the same extent as wild-type males. These results suggest that both sex-peptide and DUP99B can elicit both responses in vivo. However, sex-peptide seems to play the major role in eliciting the postmating responses, while DUP99B may have specialized for other, as yet unknown, functions.

Intraspecific competition and density dependence in an Ephestia kuehniella–Venturia canescens laboratory system

A model host‐parasitoid system of Ephestia kuehniella and Venturia canescens was used to examine the influence of host and parasitoid density on host and parasitoid life‐history parameters via a two‐way factorial experimental design (5 initial host densities×3 parasitoid densities). In the absence of parasitoids, E. kuehniella experienced scramble‐type competition with reduced growth, diminished adult size and a subsequent fecundity trade‐off for mortality. The mortality that did occur was confined to the late larval and pupal stages. In the presence of parasitoids attacking the late larval stage, competition changed from scramble for food to contest for enemy‐free space, with hosts escaping parasitism being small with low fecundity and reduced egg size, and with parasitoid adult size inversely dependent on host density. Total insect emergence (host+parasitoid), a measure of the influence of host resource competition on survivorship, exhibited a threshold effect as a function of initial host density; the threshold value was increased to a higher initial host density in the presence of parasitoids. Models of host self‐limitation were fitted to the data, with the generalized Beverton‐Holt model that incorporates a threshold effect providing the best fit, and the Ricker model with no threshold providing a very poor fit to the data.

Cuticular hydrocarbons and wax esters of the ectoparasitoid Habrobracon hebetor: Ontogenetic, reproductive, and nutritional effects.

Hydrocarbon and wax ester components of cuticular lipids of the braconid parasitoid Habrobracon hebetor Say reared at 25 degrees C on larvae of a pyralid moth have been identified by GC-MS and analyzed with respect to adult age, mating status, and diet. The hydrocarbons range in carbon number from C(21) to C(45) and consist of a homologous series of n-alkanes, 11-, 13-, and 15-methyl alkanes, 13,17-dimethyl alkanes, and Z-5, Z-7, and Z-9-alkenes. The wax esters found in the cuticular lipid fraction are a series of homologous compounds with the acid portion being short chain, unbranched, even carbon number acids from C(8) to C(20) (predominately C(8) to C(16)). The alcohol portions of the esters are secondary alcohols with carbon number from C(22) to C(25) (predominately C(23) and C(25)) with the hydroxyl function located at C(6), C(7), C(8), and C(9). Gender, age, and nutritional states were significant factors for variation in several of the individual esters, but mating status did not affect wax ester composition. Ontogenetic examinations indicated that prepupal, and early pupal cuticular lipids contain only hydrocarbons. Low levels of wax esters are detectable in late stage pupae, and somewhat greater quantities of wax esters are present on newly eclosed adults. When pharate adults emerge from the cocoon, however, their cuticular lipids consist of approximately equal amounts of hydrocarbons and wax esters, and 6d post emergence from the cocoon, wax esters are the predominant lipid component.

The vitellogenin of the honey bee, Apis mellifera: structural analysis of the cDNA and expression studies

The cDNA of Apis mellifera vitellogenin was cloned and sequenced. It is 5440 bp long and contains an ORF of 1770 amino acids (including a putative signal peptide of 16 residues). The deduced amino acid sequence shows significant similarity with other hymenopteran vitellogenins (58% with Pimpla nipponica and 54% with Athalia rosae). The alignment with 19 insect vitellogenins shows a high number of conserved motifs; for example, close to the C-terminus there is a GL/ICG motif followed by nine cysteines, as occurs in all hymenopteran species, and, as in other insect vitellogenins, a DGXR motif is located 18 residues upstream the GL/ICG motif. Phylogenetic analysis of vitellogenin sequences available in insects gave a tree that is congruent with the currently accepted insect phylogenetic schemes. Using two fragments of the vitellogenin cDNA as probes, we analyzed by Northern blot the sex- and caste-specific patterns of vitellogenin expression in pupae and adults of A. mellifera. In queens, vitellogenin mRNA was first detected in mid-late pupal stage, whereas in workers it was first detected in late pupal stage. Vitellogenin mRNA was also observed in drones, although it was first detected not in pupae but in freshly molted adults.

Evaluation of Entomopathogenic Nematodes to Manage Root Weevil Larvae in Washington State Cranberry, Strawberry, and Red Raspberry

The ability of entomopathogenic nematodes to reduce abundance of subterranean root weevils was evaluated in three small fruit crops of Washington State. In cranberry, the entomopathogenic nematodes Steinernema carpocapsae (Weiser) and S. glaseri Steiner applied at 30 IJ (infective juveniles) per square centimeter reduced abundance of black vine weevil, Otiorhynchus sulcatus (F.), by 96 or 100% respectively compared with the untreated control. According to a waxmoth [Galleria mellonella (L.)] bait bioassay, S. carpocapsae persisted at higher levels than S. glaseri (0.48 versus 0.04% infested waxmoth per applied infective juvenile at 30 d after treatment) and for a longer interval (at least 60 d after treatment), but neither species dispersed beyond the treatment plots. At one of two strawberry sites, S. carpocapsae and Heterorhabditis marelatus Liu OH10 significantly reduced the abundance of black vine weevil (all stages) and pupal strawberry root weevil, O. ovatus (L.), compared with the untreated control. The same treatments were applied at the other site, but only pupal black vine weevils were significantly affected. Neither S. carpocapsae nor H. bacteriophora Poinar significantly reduced the abundance of black vine weevil in two trials conducted in red raspberry. Entomopathogenic nematodes were recovered from larval waxmoth Galleria mellonella baits placed on soil from the red raspberry sites 6 wk after treatment. Efficacy of entomopathogenic nematodes in all trials was likely most limited by cool soil temperatures that are typical to Pacific Northwest small fruit crops during spring when root weevils are in late larval and pupal stages, but soil structure and crop characteristics, particularly dense strawberry foliage and large crowns, were also important.

RNA interference from a CAX trinucleotide repeat.

The use of RNA interference (RNAi) techniques, combined with a spatially controlled expression of doublestranded RNA (dsRNA), provides a convenient method for producing cell-type-specific perturbations of gene function (Fortier and Belote, 2000; Martinek et al., 2000). While studying the function of the Drosophila miniature-dusky (m-dy) gene complex (DiBartolomeis et al., 2002), we produced transgenic flies expressing a dsRNA that would permit silencing of the dy gene (P{UAS-dy-dsRNA}; Fig. 1a). The m and dy genes share sequence similarity and both encode fly Cuticulin proteins (DiBartolomeis et al., 2002) that are required at the pupal stage of development (Newby and Jackson, 1995) for normal wing differentiation. In the absence of m-dy products, the wing is reduced in size, due to an autonomous effect on the size and/or shape of wing epidermal cells (Newby et al., 1991). Although a reduction in wing size is the only obvious morphological phenotype in m-dy flies, certain mutants exhibit reduced viability and/or fertility (Newby et al., 1991). To examine the phenotypic effects of the UAS-dy-dsRNA transgene, we ectopically expressed it within wing tissues, taking advantage of four different existing Drosophila GAL4 driver strains: P{GawB}30A, P{GawB}34B, MS1096, and A9. The first two strains are described in Flybase, the last two were obtained from M. O’Connor (Univ. of Minnesota). All four strains provide expression in wing and other imaginal discs. Previously characterized dy mutants have small wings of normal proportion ( 2/3 of the wild-type size), whereas m mutants show a more extreme reduction in wing size and the wings are malproportioned compared to the wild type. The relatively less severe phenotype of dy mutants approximates the null phenotype for this gene, as dy/dy and dy/Df flies exhibit similar wing defects (Newby et al., 1991). Expression of UAS-dy-dsRNA with three out of four of the GAL4 drivers (P{GawB}34B, MS1096, and A9) was associated with a wing phenotype, the severity of which was dependent on the particular driver (Table 1). The fourth driver, P{GawB}30A, caused complete lethality at the late pupal stage (Table 1) and pharate adults were not dissected for examination. We were surprised that ectopic expression of UAS-dydsRNA, using either MS1096 or A9 as a driver, produced a wing phenotype more severe than that observed in existing single dy or m mutants (Table 1; Fig. 1; data not shown). Both of these GAL4 transgenes are known to drive expression in the developing wings, although MS1096 has a slightly broader pattern of expression (M. O’Connor, pers. commun.) that might explain the more severe wing phenotype compared to A9. A wing phenotype was observed with these two drivers using three independent UAS-dy-dsRNA insertions (19-4, 28-1, and 38-1), so the phenotype is not a consequence of transgene insertion. In addition, wing abnormalities were not observed in control siblings carrying any one of the GAL4 drivers or the UAS-dy-dsRNA transgene alone (not shown). Interestingly, the size of the wing in MS1096 or A9; UAS-dy-dsRNA flies is similar to that of a m-dy double mutant called Df(1)m (Fig. 1f), which is known to carry a chromosomal deletion removing only m and dy (Roberts and Jackson, 1997). Given the known sequence similarity between m and dy, such a result suggests that the UAS-dy-dsRNA transgene might target both m and dy RNAs for destruction (see below). Other abnormalities, in addition to wing phenotypes, were observed in our GAL4/UAS-dy-dsRNA crosses. Lethality was observed in populations carrying UAS-dydsRNA with any of the four GAL4 transgenes (Table 1) and the degree of lethality was dependent on the GAL4 driver (see Table 1 notes). In many crosses, much of this lethality appeared to occur at the late pupal stage, although we cannot exclude death at earlier developmental stages. As indicated, the combination of P{GawB}30A and UAS-dy-dsRNA was completely lethal, whereas the other drivers were semilethal in combination with the UAS transgene. Some of this observed lethality is attributable to elimination of m-dy function(s), as Df(1)m also exhibits poor viability (Roberts and Jackson, 1997). Although the general morphology of surviving GAL4; UAS-dy-dsRNA flies is relatively normal, and females are fertile (not shown), these flies nonetheless exhibit additional phenotypes that might reflect targeting of genes other than m and dy. A comparison of m and GAL4; UAS-dy-dsRNA flies, for example, indicates that the mor-

A genetic analysis of the porin gene encoding a voltage-dependent anion channel protein in Drosophila melanogaster.

The voltage-dependent anion channel (VDAC, also known as porin) is an abundant protein in the outer mitochondrial membrane that forms transmembrane channels permeable to solutes. While in mammals at least three different porin genes have been found, only one VDAC-encoding gene, porin, has been described so far in Drosophila melanogaster. It produces transcripts with alternative untranslated sequences. Here we report the identification of two PlacW insertions in the porin gene among a set of P-element insertions that have been mapped to the 32B3-4 region on the second chromosome. Homozygotes, as well as trans-heterozygotes for these insertions, lack VDAC, and die during the late pupal stage. Function can be restored by precise excision of the P transposon, while most deletions in the porin locus, produced by imprecise excisions, display the recessive lethal effect of the original mutant alleles. However, one of the deletions was found to be a hypomorphic male-sterile allele producing low levels of the VDAC protein, indicating that the product of the porin gene is also essential for male fertility. Analysis of the new mutant alleles also showed that the untranslated exon 1B of the porin gene is not required for VDAC synthesis. In the course of our investigation, we found that immediately adjacent to the porin gene are three more genes encoding proteins that share homology with the VDAC protein. The possible evolutionary and functional relationships of the porin-like genes at 32B3-4 are discussed.

Cloning, partial purification and in vivo developmental profile of expression of the juvenile hormone epoxide hydrolase of Ctenocephalides felis.

cDNAs encoding two different epoxide hydrolases (nCfEH1 and nCfEH2) were cloned from a cDNA library prepared from the wandering larval stage of the cat flea, Ctenocephalides felis. Predicted translations of the open reading frames indicated the clones encoded proteins of 464 (CfEH1) and 465 (CfEH2) amino acids. These proteins have a predicted molecular weight of 53 kDa and a putative 22 amino acid N-terminal hydrophobic membrane anchor. The amino acid sequences are 77% identical, and both are homologous to previously isolated epoxide hydrolases from Manduca sexta, Trichoplusia ni, and Rattus norvegicus. Purification of native juvenile hormone epoxide hydrolase (JHEH) from unfed adult cat fleas generated a partially pure protein that hydrolyzed juvenile hormone III to juvenile hormone III-diol. The amino terminal sequence of this;50-kDa protein is identical to the deduced amino terminus of the protein encoded by the nCfEH1 clone. Affinity-purified rabbit polyclonal antibodies raised against Escherichia coli-expressed HisCfEH1 recognized a approximately 50-kDa protein present in the partially purified fraction containing JHEH activity. Immunohistochemistry experiments using the same affinity-purified rabbit polyclonal antibodies localized the epoxide hydrolase in developing oocytes, fat body, and midgut epithelium of the adult flea. The presence of JHEH in various flea life stages and tissues was assessed by Northern blot and enzymatic activity assays. JHEH mRNA expression remained relatively constant throughout the different flea larval stages and was slightly elevated in the unfed adult flea. JHEH enzymatic activity was highest in the late larval, pupal, and adult stages. In all stages and tissues examined, JHEH activity was significantly lower than juvenile hormone esterase (JHE) activity, the other enzyme responsible for JH catalysis.

Developmental changes in the density of ionic currents in antennal-lobe neurons of the sphinx moth, Manduca sexta.

Early in metamorphic adult development, action potentials elicited from Manduca sexta antennal lobe neurons are small in amplitude, long in duration, and calcium dependent. As development proceeds, the action potential waveform becomes larger in amplitude, shorter in duration, and increasingly sodium dependent. Whole cell voltage-clamp analysis of Manduca antennal-lobe neurons in vitro has been used to identify voltage-activated currents that contribute to developmental changes in the electrical excitability of these cells. Proximal Branching neurons [putative projection (output) neurons] and Rick Rack neurons (putative local antennal-lobe interneurons) are examined in detail early (pupal stage 5) and late (pupal stage 14) in adult metamorphosis. In both cell types, four voltage-gated and two calcium-dependent ionic currents have been identified. Cell-type-specific changes in the density of sodium, calcium, and potassium currents correlate temporally with changes in cell excitability and spike waveform. Developmental changes in ionic current profiles are accompanied also by the emergence of cell-type-specific response characteristics in the cells. Together with the accompanying paper, this study provides an important foundation for examining the impact of developmental changes in electrical excitability on the growth, electrical properties and connectivity of neurons in central olfactory pathways of the moth.

Control of photoreceptor cell morphology, planar polarity and epithelial integrity during Drosophila eye development.

We report that the hindsight (hnt) gene, which encodes a nuclear zinc-finger protein, regulates cell morphology, cell fate specification, planar cell polarity and epithelial integrity during Drosophila retinal development. In the third instar larval eye imaginal disc, HNT protein expression begins in the morphogenetic furrow and is refined to cells in the developing photoreceptor cell clusters just before their determination as neurons. In hnt mutant larval eye tissue, furrow markers persist abnormally posterior to the furrow, there is a delay in specification of preclusters as cells exit the furrow, there are morphological defects in the preclusters and recruitment of cells into specific R cell fates often does not occur. Additionally, genetically mosaic ommatidia with one or more hnt mutant outer photoreceptor cells, have planar polarity defects that include achirality, reversed chirality and misrotation. Mutants in the JNK pathway act as dominant suppressors of the hnt planar polarity phenotype, suggesting that HNT functions to downregulate JUN kinase (JNK) signaling during the establishment of ommatidial planar polarity. HNT expression continues in the photoreceptor cells of the pupal retina. When an ommatidium contains four or more hnt mutant photoreceptor cells, both genetically mutant and genetically wild-type photoreceptor cells fall out of the retinal epithelium, indicating a role for HNT in maintenance of epithelial integrity. In the late pupal stages, HNT regulates the morphogenesis of rhabdomeres within individual photoreceptor cells and the separation of the rhabdomeres of adjacent photoreceptor cells. Apical F-actin is depleted in hnt mutant photoreceptor cells before the observed defects in cellular morphogenesis and epithelial integrity. The analyses presented here, together with our previous studies in the embryonic amnioserosa and tracheal system, show that HNT has a general role in regulation of the F-actin-based cytoskeleton, JNK signaling, cell morphology and epithelial integrity during development.

Perspectives on polydnavirus origins and evolution

Publisher Summary This chapter discusses the origin and evolution of Polydnavirus (PDV) that exists in obligate mutualisms with some parasitic Hymenoptera. A given PDV species replicates only in a single wasp species, and all members of that wasp species carry the associated virus. Two genera of PDVs are recognized––the bracoviruses (BVs) and the ichnoviruses (IVs)–– associated with braconid and ichneumonid wasps, respectively. The PDV genera have similar genome organization and replication/transmission pathways but have no known genetic similarity. Viral gene expression in insects parasitized by PDV-carrying wasps causes physiological alterations that are essential for parasitoid survival and development. PDV life cycles have been described as having “two arms.” PDVs replicate from proviral DNA in specialized cells of the wasp oviduct. Virus replication is first detected in the late pupal stage with virus released from calyx cells by budding or cell lysis and accumulating to high concentrations in the oviduct lumen. PDVs provide an interesting system for viral evolutionary studies. The unique life cycle involves coevolution with two hosts––one mutualistic and one pathogenic––thus, imposing both reductive and diversifying selection pressures on viral genes.

Variation in resistance and acclimation to low-temperature stress among three geographical strains of Drosophila melanogaster

Variation in cold resistance was examined in cold acclimated and non-acclimated Drosophila melanogaster from three geographical strains representing Morocco, France and Finland. Resistance was estimated as survival of adults at 0°C; the acclimation treatment involved a long-term exposure to 11°C starting from the late pupal stage and continuing with adults. After the cold stress, two fitness traits, percentage of fertile individuals and the number of adult progeny, were scored in both acclimated and non-acclimated flies. Acclimation dramatically increased survival in all strains, but did not affect the pattern of geographic variation in cold resistance. The European flies tended to be more resistant than the African ones and the ranking from most to least resistant strain was France>Finland>Morocco. In the absence of acclimation, females showed a higher survival than males. Percentage of fertile males in all strains and the number of progeny in the Finnish and French strains were decreased after acclimation. Without cold acclimation, the number of progeny was higher in the European flies as compared with the African ones. The results suggest that populations of D. melanogaster from cold climates are better adapted to low stressful temperatures and among-population variation in cold resistance may be due to non-plastic rather than plastic genetic changes. The deleterious effects of cold pretreatment on the life-history parameters indicate a possibility for acclimation costs in reproduction.