Late Third-instar Larvae Research Articles

CYP314A1-dependent 20-hydroxyecdysone biosynthesis is involved in regulating the development of pupal diapause and energy metabolism in the Chinese citrus fruit fly, Bactrocera minax.

Diapause is an environmentally preprogrammed period of arrested development, and characterized by metabolic depression that can occur during any development stage of insect. The insect steroid hormone 20-hydroxyecdysone (20E), is converted from ecdysone by the cytochrome P450 enzyme shade (CYP314A1), and it exerts a potent effect on the induction and maintenance of diapause in obligatory diapause insects. However, the regulatory mechanism of 20E in obligatory diapause development remains unclear. In this study, the function of 20E in the pupal diapause of Bactrocera minax was investigated. We determined the expression pattern of Halloween P450 genes from larval to adult B.minax, and found differential expression of CYP314A1 from other P450 genes, with a high level in larvae and a low level in pupae. Dysfunction of CYP314A1 by dsCYP314A1 microinjection in third-instar larvae caused significant larval mortality or abnormal pupae. Compared with dsGFP and DEPC-water, dsCYP314A1-injected larvae had significantly reduced 20E titer and altered energy metabolism, and many individuals failed to pupate. Exogenous 20E microinjected into late third-instar larvae or 20E fed to early third-instar larvae both caused similar energy metabolism changes. The 20E-treated larvae of B.minax had reduced total lipids and increased amounts of trehalose and glycogen. Furthermore, 20E-treated diapause individuals showed rapid pupal development. The 20E biosynthesis was regulated by the expression of CYP314A1, and was involved in the induction and termination phase of obligate diapause by regulating energy metabolism in B.minax. © 2022 Society of Chemical Industry.

Expression of Hey marks a subset of enteroendocrine cells in the Drosophila embryonic and larval midgut.

Hey is a conserved transcription factor of the bHLH-Orange family that participates in the response to Notch signaling in certain tissues. Whereas three Hey paralogues exist in mammalian genomes, Drosophila possesses a single Hey gene. Fly Hey is expressed in the subset of newborn neurons that receive a Notch signal to differentiate them from their sibling cells after the asymmetric division of precursors called ganglion-mother-cells. We used a polyclonal anti-Hey serum and a GFP-tagged transgenic duplication of the Hey locus to examine its expression in tissues outside the nervous system in embryos and larvae. We detected robust Hey expression in the embryonic midgut primordium at the time of birth of enteroendocrine cells, identified by expression of Prospero. Approximately half of the Pros-positive cells were also Hey positive at mid-embryogenesis. By the end of embryogenesis, most enteroendocrine cells had downregulated Hey expression, although it was still detectable at low levels after hatching. Low levels of Hey were also detected in subsets of the epithelial enterocytes at different times. Embryo enteroendocrine Hey expression was found to be Notch dependent. In late third-instar larvae, when few new enteroendocrine cells are born, novel Hey expression was detected in one cell of each sibling pair. In conclusion, Hey is strongly expressed in one of each pair of newly-born enteroendocrine cells. This is consistent with a hypothesis that embryonic enteroendocrine cells are born by an asymmetric division of a precursor, where Notch/Hey probably distinguish between the subtypes of these cells upon their differentiation.

Alterations in the expression of certain midgut genes of Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae) larvae and midgut histopathology in response to Bacillus thuringiensis Cry1C toxin

BackgroundBacillus thuringiensis (Bt) utilization as a biological control agent is highly recommended due to its safety, specificity, and efficiency. Importance of the entomocidal Cry proteins secreted by Bt is dramatically increased subsequent Cry genes transformation into a number of economic crops, rendering them protection against insect attack. In the last decade, insect resistance against transgenic Bt crops is gradually raised in several lepidopteran pests. A better understanding of the processing of Bt Cry1C toxin in the larval midgut of the lepidopteran pest species, the cotton leaf worm, Spodoptera littoralis (Boisd.), is very important to characterize the main regulatory elements of Bt tolerance.ResultsThe present study aimed to define factors that are involved in insect tolerance toward Bt Cry1C through evaluating the mRNA level of trypsin (Try), aminopeptidase N (APN), alkaline phosphatase (ALP), cadherin (Cad), and cytochrome P450 (CYP) in both susceptible and cry1C tolerant strains of S. littoralis. Total RNAs were extracted from susceptible and tolerant strains to construct cDNAs. Quantitative real-time polymerase chain reaction (qPCR) showed a significant upregulation of CYP gene in tolerant strain. In contrast, the levels of expression of Try, ALP, and Cad were significantly downregulated in tolerant strain. APN relative mRNA expression did not show significant differences between susceptible and tolerant strains. Histologically, the midgut of late third-instar larvae of tolerant population S. littoralis showed vacuolization of the epithelium and disruption of both the peritrophic membrane and the striated boarder compared to the susceptible strain.ConclusionsObtained data indicated a relationship between exposing to Bt Cry1C toxin and alteration of CYP, Try, ALP, and Cad expression in midgut of S. littoralis. These results may be an evidence for the important roles of CYP, Try, ALP, and Cad in the resistance development and toxicity to Bt Cry1C. The results are useful for further illustrating of Bt Cry1C processing and S. littoralis tolerance.

Effects of blunt force injuries in third-instar Drosophila larvae persist through metamorphosis and reduce adult lifespan.

Blunt force injuries are a significant cause of disability and death worldwide. Here, we describe a Drosophila melanogaster model of blunt force injury that can be used to investigate cellular and molecular mechanisms that underlie the short-term and long-term effects of injuries sustained at a juvenile stage of development. Injuries inflicted in late third-instar larvae using the spring-based High-Impact Trauma (HIT) device robustly activated the humoral defense response process of melanization and caused larval and pupal lethality. Additionally, adults that developed from injured larvae had reduced lifespans, indicating that cellular and molecular mechanisms activated by blunt force injuries in larvae persist through metamorphosis and adult development. Previously, the HIT device has been used to investigate genetic and environmental factors underlying mechanisms that contribute to consequences of blunt force injuries incurred in adult flies. This work expands use of the HIT device to a juvenile stage of development, offering the opportunity to investigate whether the consequences of blunt force injuries involve different factors and mechanisms at different stages of development.

Fruit Fly Helps Elucidate the Biological Effects of Proton Therapy: In Vivo Model of Normal Tissue Response

While the biological effects of proton beams on tumors have been well studied at the cellular and molecular levels, the effects on normal tissues have not been sufficiently investigated. Drosophila melanogaster , an organism well-suited for genetic analysis, is commonly used as a screening tool in basic medical science. To our knowledge, however, no studies have used this organism to elucidate biological aspects of proton beams. The purpose of this study was to demonstrate the usefulness of Drosophila melanogaster as a non-malignant model organism to investigate the biological effects of proton beams. Late third-instar larvae of wild-type Drosophila melanogaster (Oregon-R) were irradiated with 6-MV X rays (X group) or passive scattering proton beams at the center of the spread-out Bragg peak (P group). The larvae were exposed to single doses of 0, 10, 20, 30, 40 and 50 Gy to the total body and were observed for up to 48 hours after eclosion. All adults that subsequently emerged were collected and counted. Real-time quantitative PCR was performed to find mRNA levels of genes associated with angiogenesis, apoptosis, autophagy, inflammation and DNA repair response, using 40-Gy irradiated samples obtained at 2 or 24 hours after irradiation. The surviving fraction of flies at 48 hours after irradiation was: control (0 Gy), 0.87; 10 Gy, 0.75/0.92 (photon/proton); 20 Gy, 0.81/0.73; 30 Gy, 0.73/0.37; 40 Gy, 0.52/0.30; and 50 Gy, 0.12/0.05. Two-way ANOVA revealed significant differences between the radiation types (f1) and doses (f2); p -values were 0.023 for f1, <0.001 for f2, and 0.015 for f1f2. In the analysis of mRNA expression levels of 26 genes, remarkable differences were observed between the X and P groups in genes associated with DNA repair response such as Irbp, the homolog of Ku70 (mean number of folds changed at 2 hours, X 3.5 vs. P 8.0, p = 0.008; at 24 hours, X 3.1 vs. P 2.2, p = 0.11), Ku80 (2 hours, X 3.1 vs. P 4.9, p =0.015; 24 hours, X 2.4 vs. P 1.6, p =0.012), spn-A, the homolog of Rad51 (2 hours, X 1.3 vs. P 2.1, p =0.14; 24 hours, X 1.6 vs. P 1.4, p =0.39) and BRCA2 (2 hours, X 1.4 vs. P 2.0, p =0.23; 24 hours, X 2.1 vs. P 1.5, p =0.52). To date, this is the first study using Drosophila to investigate the biological effects of proton beams. We demonstrated the difference between photons and protons in both the surviving fractions and the levels of mRNA expression in the in vivo model. Due to the recent rapid increase of proton therapy facilities, biological evaluation from multiple aspects is needed. Since various research methods can be used for Drosophila, this experimental model may be useful in elucidating biological effects of proton therapy.

A Place to Grow? Host Choice and Larval Performance of Microplitis similis (Hymenoptera: Braconidae) in the Host Spodoptera litura (Lepidoptera: Noctuidae).

Host selection is a key stage in the lifecycle of parasitoids, and is critical to both their function in control and to the maintenance of their population. The solitary endoparasitoid Microplitis similis (Hymenoptera: Braconidae) is a potential biological control agent of Spodoptera litura (F.) larvae (Lepidoptera: Noctuidae). In this study, we examined the preference M. similis exhibits for different instars of the host, host instar effects on parasitoid development, and the weight gain and food consumption of different instars of parasitized larvae. In no-choice tests, parasitization rates were highest in second- and early third-instar larvae, and no fourth- or fifth-instar hosts were parasitized. When provided with a choice of first- to late third-instar host larvae, M. similis preferred to parasitize early third-instar host larvae (41%) with a selection coefficient of 0.37. All morphometric features of wasp offspring increased with increasing age of the host at parasitization. A lower proportion of females emerged from first-instar larvae than any other instar. Parasitized S. litura larvae showed a pronounced reduction in food consumption and weight gain. Microplitis similis may have the potential to significantly suppress population growth and the damage caused by S. litura.

A Drosophila model to investigate the neurotoxic side effects of radiation exposure.

ABSTRACTChildren undergoing cranial radiation therapy (CRT) for pediatric central nervous system malignancies are at increased risk for neurological deficits later in life. We have developed a model of neurotoxic damage in adult Drosophila following irradiation during the juvenile stages with the goal of elucidating underlying neuropathological mechanisms and of ultimately identifying potential therapeutic targets. Wild-type third-instar larvae were irradiated with single doses of γ-radiation, and the percentage that survived to adulthood was determined. Motor function of surviving adults was examined with a climbing assay, and longevity was assessed by measuring lifespan. Neuronal cell death was assayed by using immunohistochemistry in adult brains. We also tested the sensitivity at different developmental stages by irradiating larvae at various time points. Irradiating late third-instar larvae at a dose of 20 Gy or higher impaired the motor activity of surviving adults. A dose of 40 Gy or higher resulted in a precipitous reduction in the percentage of larvae that survive to adulthood. A dose-dependent decrease in adult longevity was paralleled by a dose-dependent increase in activated Death caspase-1 (Dcp1) in adult brains. Survival to adulthood and adult lifespan were more severely impaired with decreasing larval age at the time of irradiation. Our initial survey of the Drosophila Genetic Reference Panel demonstrated that differences in genotype can confer phenotypic differences in radio-sensitivity for developmental survival and motor function. This work demonstrates the usefulness of Drosophila to model the toxic effects of radiation during development, and has the potential to unravel underlying mechanisms and to facilitate the discovery of novel therapeutic interventions.

Do Late‐Instar Drosophila Larvae Experience Functional Oxygen Limitations?

Arthropods gain considerable mass between molts, increasing oxygen demand while parts of the respiratory system are fixed in size. We hypothesized that internal hypoxia could be a cue triggering molting in arthropods and/or be a functional reason that the animal needs to molt. In support of this hypothesis, hypoxic rearing (10 kPa) triggered an ecdysone peak and molting to adulthood at a smaller body size in Drosophila, and hyperoxia induced earlier pupariation (JEB 216:4334). We tested for evidence that late‐third instar Drosophila larvae show signs of functional oxygen‐limitation. The dorsal longitudinal tracheae increased in diameter through the instar, scaling with mass0.38, approximately as predicted to conserve oxygen delivery. Respiratory exchange ratios declined through the instar, indicating that metabolism remains aerobic and shifts away from carbohydrate catabolism. Lactate levels did not increase later in the instar and were unaffected by rearing in hyperoxia. However, hyperoxia (30 kPa) increased late‐instar metabolic rates, suggesting functional oxygen limitation. Late‐instar larvae had greater capacities to generate lactate in response to anoxia, suggesting past selection for coping with late‐instar oxygen limitation. Most late‐ but not early‐stage larvae unregulated HIF signaling. Together these data support the conclusion that Drosophila, and likely other arthropods, experience some functional oxygen limitations later in the instar that trigger HIF signaling, potentially linked to the molting cascade. This research was supported by NSF IOS 1256745.

CDNA CLONING AND TRANSCRIPTIONAL REGULATION OF THE CECROPIN AND ATTACIN FROM THE ORIENTAL FRUIT FLY, Bactrocera dorsalis (DIPTERA: TEPHRITIDAE).

We described the cDNA cloning of two antimicrobial peptides (AMPs), cecropin (BdCec), and attacin C (BdAttC), from the oriental fruit fly, Bactrocera dorsalis (Hendel), a serious insect pest of fruit trees. Using rapid amplification of cDNA ends, fragments encompassing the entire open reading frames of BdCec and BdAttC were cloned and sequenced. The complete 425 bp cDNA of BdCec encodes a protein of 64 amino acids with a predicted molecular weight of 6.84 kDa. The 931 bp cDNA of BdAttC encodes a protein of 239 residues with a predicted molecular weight of 24.97 kDa. Real-time quantitative RT-PCR demonstrated that the developmental transcription profiles of BdCec and BdAttC were similar in each larvae, pupae, and adults. The constitutive expression levels of both AMPs were high in the first-instar and late third-instar larvae, suggesting that their antimicrobial activity is active in the newly hatched larvae and just before pupation. The basal expression levels were not significant different in adult fat bodies. The expression of BdCec and BdAttC was upregulated after bacterial challenge in adult fat bodies. The ratio of inducible expression to constitutive expression was lower in males compared to females.

Low-cost and eco-friendly green synthesis of silver nanoparticles using Feronia elephantum (Rutaceae) against Culex quinquefasciatus, Anopheles stephensi, and Aedes aegypti (Diptera: Culicidae).

Mosquitoes transmit serious human diseases, causing millions of deaths every year. The use of synthetic insecticides to control vector mosquitoes has caused physiological resistance and adverse environmental effects in addition to high operational cost. Insecticides of synthesized natural products for vector control have been a priority in this area. In the present study, the larvicidal activity of silver nanoparticles (AgNPs) synthesized using Feronia elephantum plant leaf extract against late third-instar larvae of Anopheles stephensi, Aedes aegypti, and Culex quinquefasciatus was determined. The range of concentrations of synthesized AgNPs (5, 10, 15, 20, and 25 μg mL(-1)) and aqueous leaf extract (25, 50, 75, 100, and 125 μg mL(-1)) were tested against the larvae of A. stephensi, A. aegypti, and C. quinquefasciatus. Larvae were exposed to varying concentrations of aqueous crude extract and synthesized AgNPs for 24 h. Considerable mortality was evident after the treatment of F. elephantum for all three important vector mosquitoes. The synthesized AgNPs from F. elephantum were highly toxic than crude leaf aqueous extract to three important vector mosquito species. The results were recorded from UV-visible spectroscopy, Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and energy-dispersive X-ray spectroscopy analysis (EDX). Synthesized AgNPs against the vector mosquitoes A. stephensi, A. aegypti, and C. quinquefasciatus had the following LC50 and LC90 values: A. stephensi had LC50 and LC90 values of 11.56 and 20.56 μg mL(-1); A. aegypti had LC50 and LC90 values of 13.13 and 23.12 μg mL(-1); and C. quinquefasciatus had LC50 and LC90 values of 14.19 and 24.30 μg mL(-1). No mortality was observed in the control. These results suggest that the green synthesis of silver nanoparticles using F. elephantum has the potential to be used as an ideal eco-friendly approach for the control of A. stephensi, A. aegypti, and C. quinquefasciatus. This is the first report on the mosquito larvicidal activity of the plant extracts and synthesized nanoparticles.

Patterning Axon Targeting of Olfactory Receptor Neurons by Coupled Hedgehog Signaling at Two Distinct Steps

We present evidence for a coupled two-step action of Hedgehog signaling in patterning axon targeting of Drosophila olfactory receptor neurons (ORNs). In the first step, differential Hedgehog pathway activity in peripheral sensory organ precursors creates ORN populations with different levels of the Patched receptor. Different Patched levels in ORNs then determine axonal responsiveness to target-derived Hedgehog in the brain: only ORN axons that do not express high levels of Patched are responsive to and require a second step of Hedgehog signaling for target selection. Hedgehog signaling in the imaginal sensory organ precursors thus confers differential ORN responsiveness to Hedgehog-mediated axon targeting in the brain. This mechanism contributes to the spatial coordination of ORN cell bodies in the periphery and their glomerular targets in the brain. Such coupled two-step signaling may be more generally used to coordinate other spatially and temporally segregated developmental events.

Chronic lead exposure alters presynaptic calcium regulation and synaptic facilitation in Drosophila larvae

Prolonged exposure to inorganic lead (Pb 2+) during development has been shown to influence activity-dependent synaptic plasticity in the mammalian brain, possibly by altering the regulation of intracellular Ca 2+ concentration ([Ca 2+] i). To explore this possibility, we studied the effect of Pb 2+ exposure on [Ca 2+] i regulation and synaptic facilitation at the neuromuscular junction of larval Drosophila. Wild-type Drosophila (CS) were raised from egg stages through the third larval instar in media containing either 0 μM, 100 μM or 250 μM Pb 2+ and identified motor terminals were examined in late third-instar larvae. To compare resting [Ca 2+] i and the changes in [Ca 2+] i produced by impulse activity, the motor terminals were loaded with a Ca 2+ indicator, either Oregon Green 488 BAPTA-1 (OGB-1) or fura-2 conjugated to a dextran. We found that rearing in Pb 2+ did not significantly change the resting [Ca 2+] i nor the Ca 2+ transient produced in synaptic boutons by single action potentials (APs); however, the Ca 2+ transients produced by 10 Hz and 20 Hz AP trains were larger in Pb 2+-exposed boutons and decayed more slowly. For larvae raised in 250 μM Pb 2+, the increase in [Ca 2+] i during an AP train (20 Hz) was 29% greater than in control larvae and the [Ca 2+] i decay τ was 69% greater. These differences appear to result from reduced activity of the plasma membrane Ca 2+ ATPase (PMCA), which extrudes Ca 2+ from these synaptic terminals. These findings are consistent with studies in mammals showing a Pb 2+-dependent reduction in PMCA activity. We also observed a Pb 2+-dependent enhancement of synaptic facilitation at these larval neuromuscular synapses. Facilitation of EPSP amplitude during AP trains (20 Hz) was 55% greater in Pb 2+-reared larvae than in controls. These results showed that Pb 2+ exposure produced changes in the regulation of [Ca 2+] i during impulse activity, which could affect various aspects of nervous system development. At the mature synapse, this altered [Ca 2+] i regulation produced changes in synaptic facilitation that are likely to influence the function of neural networks.

MSL Complex Is Attracted to Genes Marked by H3K36 Trimethylation Using a Sequence-Independent Mechanism

In Drosophila, X chromosome dosage compensation requires the male-specific lethal (MSL) complex, which associates with actively transcribed genes on the single male X chromosome to upregulate transcription approximately 2-fold. We found that on the male X chromosome, or when MSL complex is ectopically localized to an autosome, histone H3K36 trimethylation (H3K36me3) is a strong predictor of MSL binding. We isolated mutants lacking Set2, the H3K36me3 methyltransferase, and found that Set2 is an essential gene in both sexes of Drosophila. In set2 mutant males, MSL complex maintains X specificity but exhibits reduced binding to target genes. Furthermore, recombinant MSL3 protein preferentially binds nucleosomes marked by H3K36me3 in vitro. Our results support a model in which MSL complex uses high-affinity sites to initially recognize the X chromosome and then associates with many of its targets through sequence-independent features of transcribed genes.

Mitch – a rapidly evolving component of the Ndc80 kinetochore complex required for correct chromosome segregation inDrosophila

We identified an essential kinetochore protein, Mitch, from a genetic screen in D. melanogaster. Mitch localizes to the kinetochore, and its targeting is independent of microtubules (MTs) and several other known kinetochore components. Animals carrying mutations in mitch die as late third-instar larvae; mitotic neuroblasts in larval brains exhibit high levels of aneuploidy. Analysis of fixed D. melanogaster brains and mitch RNAi in cultured cells, as well as video recordings of cultured mitch mutant neuroblasts, reveal that chromosome alignment in mitch mutants is compromised during spindle formation, with many chromosomes displaying persistent mono-orientation. These misalignments lead to aneuploidy during anaphase. Mutations in mitch also disrupt chromosome behavior during both meiotic divisions in spermatocytes: the entire chromosome complement often moves to only one spindle pole. Mutant mitotic cells exhibit contradictory behavior with respect to the spindle assembly checkpoint (SAC). Anaphase onset is delayed in untreated cells, probably because incorrect kinetochore attachment maintains the SAC. However, mutant brain cells and mitch RNAi cells treated with MT poisons prematurely disjoin their chromatids, and exit mitosis. These data suggest that Mitch participates in SAC signaling that responds specifically to disruptions in spindle microtubule dynamics. The mitch gene corresponds to the transcriptional unit CG7242, and encodes a protein that is a possible ortholog of the Spc24 or Spc25 subunit of the Ndc80 kinetochore complex. Despite the crucial role of Mitch in cell division, the mitch gene has evolved very rapidly among species in the genus Drosophila.

The biology of Bracon celer as a parasitoid of the olive fruit fly

A series of laboratory experiments was conducted on a colony of Bracon celer Szepligeti (Hymenoptera: Braconidae) reared on the olive fly, Bactrocera oleae (Rossi) (Diptera: Tephritidae). Female B. celer preferentially probe and oviposit into olives containing late third-instar fly larvae. The parasitoid develops as a solitary, ectoparasitic idiobiont. Mean development time (oviposition to adult eclosion) at 22 °C was, for females, 36±1 (SE) days, and for males, 34±1 days. The mean longevity of adult female wasps when provided honey and water was significantly greater than when they were provided water alone, or nothing. The females produced an average of 9.7±7.2 progeny during their lifetimes, but production levels in the insectary colony suggested that this level of fecundity was artificially low and could be improved. The discrepancy may be a consequence of constraints on oviposition behavior imposed by the experimental design. The results are discussed with respect to insectary production methods and the potential use of B. celer as a biological control agent for olive fly in California.

Depth of pupation and survival of the Oriental fruit fly, Bactrocera dorsalis (Diptera: Tephritidae) pupae at selected soil moistures

Estimates of pupation depth and survival of the Oriental fruit fly, Bactrocera dorsalis (Hendel), are important for optimizing soil control and for better understanding its natural mortality in the agricultural system of Guangzhou, China. Late third-instar larvae were placed in soils having relative moistures of 0-100% for pupation. No pupae were found on the surface at soil moistures of 0-70%. Instead, more than 50% of the larvae pupated on the surface at soil moistures of 80, 90, and 100%. Most of the larvae preferred to pupate in less than 4cm of the soils, while relatively few larvae moved more than 4 cm when the soils received too little water or too much water. The survival rate of pupae at 70% moisture level was low, and the pupae were unable to survive at soil moistures of 0, 80, 90, and 100%, while emergence rates exceeded 90% at the conditions of 10-60% moisture levels. Moreover, soil moistures had an influence on the average time to emergence (average time between the larvae release and the emergence of adults). Adult flies at 30% moisture level emerged earlier than those at the other moisture levels, whereas the average time to emergence at 70% moisture level was the longest.

Sexual differentiation of identified motor terminals inDrosophila larvae

In Drosophila, we have found that some of the motor terminals in wandering third-instar larvae are sexually differentiated. In three out of the four body-wall muscle fibers that we examined, we found female terminals that produced a larger synaptic response than their male counterparts. The single motor terminal that innervates muscle fiber 5 produces an EPSP that is 69% larger in females than in males. This is due to greater release of transmitter from female than male synaptic terminals because the amplitude of spontaneous miniature EPSPs was similar in male and female muscle fibers. This sexual difference exists throughout the third-instar: it is seen in both early (foraging) and late (wandering) third-instar larvae. The sexual differentiation appears to be neuron specific and not muscle specific because the same axon produces Is terminals on muscle fibers 2 and 4, and both terminals produce larger EPSCs in females than males. Whereas, the Ib terminals innervating muscle fibers 2 and 4 are not sexually differentiated. The differences in transmitter release are not due to differences in the size of the motor terminals. For the terminal on muscle fiber 5 and the Is terminal on muscle fiber 4, there were no differences in terminal length, the number of branches, or the number of synaptic boutons in males compared to females. These sexual differences in neuromuscular synaptic physiology may be related to male-female differences in locomotion.

Effect of Short-Term Exposure to Chlorpyrifos on Developmental Parameters and Biochemical Biomarkers in Chironomus riparius Meigen

Chironomus riparius Meigen were exposed to sediment spiked with 0, 0.01, 0.05, and 0.10 mg chlorpyrifos/kg dry sediment for 48 h as late third-instar larvae. Acetylcholinesterase activity, glutathione-S-transferase activity, burrowing behavior, emergence time, and adult dry weight were measured to determine which were the most sensitive biomarkers of exposure and effect for short-term sublethal exposures. There was a significant drop in acetylcholinesterase activity at the two highest concentrations, but no effect on glutathione-S-transferase activity. Behavioral and life history biomarkers were sensitive only to the highest concentration of chlorpyrifos. The association of life history changes (female weight and male emergence times) with a significant inhibition of acetylcholinesterase activity suggests that this can be used as a biomarker of exposure and effect.

The hiiragi Gene Encodes a Poly(A) Polymerase, Which Controls the Formation of the Wing Margin in Drosophila melanogaster

The hiiragi (hrg) gene plays a key role in the development of the wing margin in Drosophila melanogaster. A mutation in the hrg gene resulted in a decrease in the level of the hrg transcript and was associated with a notched wing phenotype. We report here that the hrg gene encodes a poly(A) polymerase (PAP). The bovine cDNA for PAP type II reversed the phenotype due to mutation of the hrg gene, suggesting that hrg might encode a functional homolog of PAP. A mutation that reduced the enzymatic activity of Hrg failed to reverse the phenotype of hrg mutants, suggesting that the enzymatic activity of Hrg was required to rescue the wing phenotype. The levels of expression of wingless and cut at the presumptive wing margins were reduced in the late third-instar larvae of hrg mutants. These results suggest that the product of hrg is required for the normal expression of a series of genes in this region. Our results provide the first evidence that a PAP in Drosophila plays a key role in the early development of the wing margin, acting to regulate the specific expression of a series of genes via, perhaps, control of the processing of the 3′ ends of transcripts.

Blood-eye barrier of the developing drosophila melanogaster (Diptera : Drosophilidae)

For the past quarter century, it has been known that a blood-eye barrier exists in adult insects. However, the life stage at which the barrier arises and the anatomical correlate of the barrier were not known. Compound eye development in Drosophila melanogaster (Diptera : Drosophilidae) is essentially complete at approximately 140 h after pupariation ; or about 20 h prior to eclosion. A search for a blood-eye barrier spanned late third-instar larvae, through late pupal life sampled at 40 and 140 h post-pupariation. No blood-eye barrier is present in the eye discs of last-instar larvae, based on the presence of lanthanum tracer among ommatidial cells and their processes. Pleated-sheet septate junctions, which link larval ommatidial cells, are not yet capable of totally blocking tracer from paracellular passage. The blood-eye barrier is constructed in the early phase (0–60 h) of pupal development in the wake of apoptosis and new cellular reorganization. In the developing compound eye, mature photoreceptor neurons must then be protected from the ionic vagaries of hemolymph to become electrophysically competent. First vestiges of a barrier with occluding septate junctions are seen in the 40-h old pupa, and by 140 h, the barrier is complete. The barrier prevails throughout adult life.