Evaluation of some potential target genes and methods for RNAi-mediated pest control of the corn earworm Helicoverpa zea.

In this study, we explored the efficacy of knockdown four genes required for proper nervous system function by RNAi, in the corn earworm Helicoverpa zea (Boddie). Three of these genes encode components of validated insecticide target sites. We synthesized cDNA sequences orthologous to the Drosophila melanogaster genes Para (paralyticts), TipE (temperature-induced paralysis locus E), GluCl (glutamate-gated chloride channel), and Notch, and used these fragments to synthesize double-stranded RNAs (dsRNAs). We then performed experiments in an attempt to induce RNAi-mediated effects on gene expression and viability using three modes of delivery of the dsRNAs: microinjection of eggs, soaking of eggs and feeding of larvae. Microinjection of dsRNAs into eggs induced reduced hatch rates and knockdown of target gene expression for GluCl, para and TipE, but not for Notch. However, neither feeding nor soaking eggs in dsRNA solutions resulted in discernable RNAi effects. These results demonstrated the susceptibility to RNAi effects of the expression of H. zea genes encoding insecticide target sites, which suggests future avenues of research toward practical applications.

Effects of Embryonic Development Stage and Cryoprotective Agents on Galleria Mellonella Embryo Cryopreservation.

BACKGROUND: The increased research use of the honeycomb pest Galleria mellonella has created the need for cryopreservation. The diverse characteristics of G. mellonella embryos at different stages may affect embryo survival upon cryopreservation due to differential dehydration, cold resistance, cryoprotectant toxicity and permeability. OBJECTIVE: The study aimed to determine the ability of G. mellonella to survive cryogenic condition in the early and late embryonic developmental stages (24h and 75h post-oviposition, PO). MATERIALS AND METHODS: A modified protocol was used to select the proper embryonic stage by comparing the early and late embryonic stages under two procedures of dechorionation-permeabilization (D/P) dilutions. The embryos at the early stage were used for testing the toxicity and viscosity effects of various cryoprotective agents. Various cryoprotectant treatments for improving the hatch rate were studied. RESULTS: The survival rates of embryos at the early stages (24h PO) were 51.5% and 69.5% respectively after cryoprotectant loading in 12% ethylene glycol (EG) for 30 min and dehydration in vitrification solution for for 10min. These survival rates of embryos at the late stages (75h PO), however, decreased to 22.9% and 34.0%, respectively. D/P treatment with Tween 20 and Tween 80 (2:6) slightly improved the survival of embryos at the 24h PO stage after immersion in liquid nitrogen (LN) from 0.1% to 0.6%. The increased exposure time in dehydration treatment from 14 to 30min in two vitrification solutions, EG and methanol (MeOH), slightly increased the survival rate of cryopreserved embryos from 0% and 0.3% to 1.1% and 1.4%, respectively, while the vitrification solution containing dimethyl sulfoxide (Me2SO or DMSO) slightly decreased the hatch rate from 1% to 0.6%. CONCLUSION: The survival rate of G. mellonella embryos after cryopreservation was affected by the oviposition period, eggshell permeability to cryoprotectant, cryoprotectant type, as well as their concentration and exposure times.

Embryogenesis of Heliconius erato (Lepidoptera, Nymphalidae): a contribution to the anatomical development of an evo-devo model organism.

This study reports on the embryogenesis of Heliconius erato phyllis between blastoderm formation and the prehatching larval stage. Syncytial blastoderm formation occurred approximately 2 h after egg laying (AEL) and at about 4 h, the cellular blastoderm was formed. The germ band arose from the entire length of the blastoderm, and rapidly became compacted occupying approximately two-thirds of the egg length. At about 7 h AEL, protocephalon and protocorm differentiation occurred. Continued proliferation of the germ band was followed by penetration into the yolk mass, forming a C-shaped embryo at about 10 h. Approximately 12 h AEL, the gnathal, thoracic and abdominal segments became visible. The primordium of the mouthparts and thoracic legs formed as paired evaginations, while the prolegs formed as paired lobes. At about 30 h, the embryo reversed dorsoventrally. Approximately 32 h AEL, the protocephalon and gnathal segments fused, shifting the relative position of the rudimentary appendages in this region. At about 52 h, the embryo was U-shaped in lateral view and at approximately 56 h, the bristles began evagination from the larval cuticle. Larvae hatched at about 72 h. We found that H. erato phyllis followed an embryonic pattern consistent with long-germ embryogenesis. Thus, we believe that H. erato phyllis should be classified as a long-germ lepidopteran. The study of H. erato phyllis embryogenesis provided a structural glimpse into the morphogenetic events that occur in the Heliconius egg period. This study could help future molecular approaches to understanding the evolution of Heliconius development.

[Antijuvenile influence of the precocene on the development of adult antennae in the apple surface eating tortricid Archips podana Scop. (Lepidoptera: Tortricidae)].

The influence ofprecocene II, an antijuvenile agent, on the development of adult antennae in the apple surface eating tortricid A. podana Scop was demonstrated. Treatment of the fifth instar larvae and prepupae with different doses of precocene proved to cause different sensitivity of the specimens to the juvenile hormone deficit. Treatment with 450 and 600 microg precocene per specimen during the first days after ecdysis to the fifth instar caused the death of larvae. Treatment with 300, 450, and 600 microg per specimen on the third day of the fifth instar larvae and prepupae caused a delay in the development of adult antennae. The results are discussed with respect to the role of the juvenile hormone in the development of imaginal structures during metamorphosis.

Cold tolerance of field-collected and laboratory reared larvae of Sesamia nonagrioides (Lepidoptera: Noctuidae).

Sesamia nonagrioides Lefébvre (Lepidoptera: Noctuidae) is considered one of the most destructive pests of corn in the Mediterranean region. The purpose of the present study was to investigate some aspects of the cold tolerance of non-diapausing and diapausing laboratory reared larvae of S. nonagrioides, as well as of field-collected larvae, taking into consideration various parameters, such as supercooling ability, mean lethal temperature and accumulation of cryoprotectant substances, in relation to diapause. Our results provide evidence that S. nonagrioides has limited cold tolerance as it displays a low ability of supercooling. This is strongly supported by the fact that mortality of the individuals occurred after extended exposure to subzero temperatures, equivalent or slightly lower to their mean supercooling point. However, lethal temperatures of diapausing larvae were significantly lower in relation to that of non-diapausing larvae, indicating the existence of a direct link between diapause and cold tolerance. Regarding the role of cryoprotectant substances, accumulation of glycerol seems to be closely related to diapause, in contrast to accumulation of trehalose, which is more related to exposure to low temperatures slightly higher than 0 degree C. Finally, non-diapausing larvae of different instars displayed a similar ability of supercooling and tolerance to low temperatures as well as accumulation of cryoprotectant substances. The ecological significance of our findings on cold tolerance of this species is being discussed with particular reference to the microclimate observed in northern Greece.

The effect of Tween 80 on eggshell permeabilization in Galleria mellonella (L.) (Lepidoptera, Pyralidae).

The development of a species-specific protocol for dechorionation and permeabilization of insect eggs is a necessary prerequisite to cryopreserve the embryos. Here we tested different procedures based on heptane or the surfactant Tween 80 as an alternative to alkane, evaluating their efficacy and toxicity on the early (24 h post-oviposition) and late (75 h post-oviposition) stage embryos. Heptane efficiently permeabilized the eggs of G. mellonella but the hatching rate ranged from 0.1 to 4.2 percent in the early stage and from 4.3 to 11.2 percent in the late stage. The embryos treated with 1.25 percent NaOCl + 0.08 percent Tween 80 for 2 min showed the same shrinkage and reswelling percentages as eggs exposed to heptane for 10 sec, with a significantly higher hatching percentage in the early (68.2 +/- 1.5 percent) and late stages (22.4 +/- 3.7 percent). Thus, 0.08 percent Tween 80 allows sufficient permeabilization of G. mellonella embryos without the high toxicity of alkane.

Analysis of Cry1Ah Toxin-Binding Reliability to Midgut Membrane Proteins of the Asian Corn Borer.

Evolution of insect resistance to Bt toxins challenges the use of Cry toxins to control agricultural pests. In lepidopterans, Cry toxin affinity towards multiple midgut epithelial receptors has become a matter of dispute. Cry1Ah toxin-binding proteins were identified in the larval midgut of susceptible (ACB-BtS) and resistant (ACB-AhR) strains of the Asian corn borer (ACB). A pull-down assay was performed using biotinylated Cry1Ah toxin, and the binding proteins were identified by employing liquid chromatography-tandem mass spectrometry (LC-MS/MS). This study aimed to find the binding consistency of the midgut epithelial protein to the Cry1Ah toxin. The binding proteins from different fractions of SDS-PAGE showed a different pattern. We observed an isoform of prophenoloxidase PPO1b (UniProt Acc No. A0A1Q1MKI0), which was found only in the ACB-AhR fractions. Prophenoloxidase (proPO) is an extraordinary defense molecule activated in insect species during pathogen invasion and the wound healing process. Importantly, this prophenoloxidase might have direct/indirect interaction with the Cry1Ah toxin. Our data also suggest that factors like techniques, enrichment of binding proteins in the sample and the reversible and irreversible nature of the brush border membrane vesicles (BBMVs) to Cry toxins could cause the inconsistency in the protein-protein interactions. Moreover, inside the larva midgut, the influence of the Cry toxins under physiological conditions might be different from the laboratory procedures.

Induction of invagination in insect epithelium: paradigm for embryonic invagination.

The proposal that adhesive disparities between inpocketing populations of cells and surrounding epithelia drive epithelial invagination was tested in grafting experiments with moth pupal wing epithelium. Evidence exists that a cellular adhesiveness gradient spans the proximodistal axis of the wing. Although pupal wing cells normally do not invaginate or evaginate, epithelial folding can be induced after exchange of grafts from opposite ends of the proximodistal axis. The hypothesis that cytoskeletal elements are the primary agents in epithelial invagination should be reevaluated.

Cloning of Wing-Development-Related Genes and mRNA Expression Under Heat Stress in Chlorpyrifos-Resistant and -Susceptible Plutella xylostella.

Chlorpyrifos-resistant (Rc) Plutella xylostella (DBM) shows higher wing-vein injury than chlorpyrifos-susceptible (Sm) DBM under heat stress in our previous study. To investigate the toxicological mechanisms of the differences in injury of wing vein between Rc- and Sm-DBM collected from Fuzhou, China, total ten cDNA sequences of wing-development-related genes were isolated and characterized in DBM, including seven open reading frame (ORF) (ash1, ah2, ash3, ase, dpp, srf and dll encoded 187 amino acids, 231 aa, 223aa, 397aa, 423aa, 229aa and 299aa, respectively), and three partly sequences (salm, ser and wnt-1 encoded 614aa, 369aa and 388aa, respectively). The mRNA expression of the genes was inhibited in Rc- and Sm-DBM under heat stress, as compared with that an average temperature (25 °C). And, in general, significantly higher down-regulated expressions of the mRNA expression of the wing development-related genes were found in Rc-DBM as compared to those in Sm-DBM under heat stress. The results indicated that Sm-DBM displayed higher adaptability at high temperature because of significantly lower inhibition the mRNA expressions of wing-development-related genes. We suggest that significantly higher injury of wing vein showed in Rc-DBM under heat stress might be associated with the strong down-regulation of wing-development-related genes.

A novel member of the NSF family in the corn earworm, Helicoverpa zea: molecular cloning, developmental expression, and tissue distribution.

N-ethylmaleimide-sensitive fusion protein (NSF) is an ATPase that plays an essential role in intracellular membrane transport events. We report the cloning of a cDNA encoding NSF from the noctuid moth, Helicoverpa zea (Hez), a major agricultural pest. The amino acid sequence deduced from the cDNA indicates that Hez-NSF has 88%, 73%, and 70% identities to NSF from Manduca sexta, Aedes aegypti, and Drosophila melanogaster, respectively. Northern hybridization analysis clearly shows a 4.4 kb mRNA, corresponding in size to the cDNA present in the brain-suboesophageal ganglion (SG) complex of pupae. The NSF transcript is present in components of the central nervous system, including the brain, SG, thoracic ganglion and abdominal ganglion, but is not present in nonneural tissues. A developmental profile of gene expression revealed that Hez-NSF mRNA is present throughout the embryonic, larval, and pupal development.

Expression pattern of a butterfly achaete-scute homolog reveals the homology of butterfly wing scales and insect sensory bristles.

BACKGROUND: Lepidopteran wing scales are the individual units of wing color patterns and were a key innovation during Lepidopteran evolution. On the basis of developmental and morphological evidence, it has been proposed that the sensory bristles of the insect peripheral nervous system and the wing scales of Lepidoptera are homologous structures. In order to determine if the developmental pathways leading to Drosophila sensory bristle and butterfly scale formation use similar genetic circuitry, we cloned, from the butterfly Precis coenia, a homolog of the Drosophila achaete-scute (AS-C) genes--which encode transcription factors that promote neural precursor formation--and examined its expression pattern during development. RESULTS: During embryonic and larval development, the expression pattern of the AS-C homolog, ASH1, forecasted neural precursor formation. ASH1 was expressed both in embryonic proneural clusters--within which an individual cell retained ASH1 expression, enlarged, segregated, and became a neural precursor--and in larval wing discs in putative sensory mother cells. ASH1 was also expressed in pupal wings, however, in evenly spaced rows of enlarged cells that had segregated from the underlying epidermis but, rather than give rise to neural structures, each cell contributed to an individual scale. CONCLUSIONS: ASH1 appears to perform multiple functions throughout butterfly development, apparently promoting the initial events of selection and formation of both neural and scale precursor cells. The similarity in the cellular and molecular processes of scale and neural precursor formation suggests that the spatial regulation of an AS-C gene was modified during Lepidopteran evolution to promote scale cell formation.

[Influence of precocene II on the sensory system of antennae and mouthparts in larvae of the fruit tree tortricid Archips podana Scop. (Lepidoptera: Tortricidae)].

The influence of precocene II, an antijuvenile agent, on morphological characters of the chemoreceptor apparatus of antennae and mouthparts was studied in fifth instar A. podana larvae. Treatment with different doses of precocene was performed at the egg stage. It proved to cause changes in the form and number of basiconic sensilla on the maxillary palps and galea and in the size of basiconic sensilla on the second and third antennal segments. The results are discussed with respect to the influence of precocenes on the insect sensory system and the role of the juvenile hormone in regulation of its development.

Comparative embryogenesis of Mecoptera and Lepidoptera with special reference to the abdominal prolegs.

The eruciform larvae of holometabolous insects are primarily characterized by bearing a varying number of abdominal prolegs in addition to three pairs of thoracic legs. However, whether the prolegs are evolutionarily homologous among different insect orders is still a disputable issue. We examined the embryonic features and histological structure of the prolegs of the scorpionfly Panorpa byersi Hua and Huang (Mecoptera: Panorpidae) and the Oriental armyworm Mythimna separata (Walker) (Lepidoptera: Noctuidae) to investigate whether the prolegs are homologous between these two holometabolous insect orders. In the scorpionfly, paired lateral process primordia arise on abdominal segments I-VIII (A1-A8) in line with the thoracic legs in early embryonic stages, but degenerate into triangular protuberances in later stages, and paired medial processes appear along the midventral line before dorsal closure and eventually develop into unjointed, cone-shaped prolegs. Histological observation showed that the lumina of the prolegs are not continuous with the hemocoel, differing distinctly from that of the basic appendicular plan of thoracic legs. These results suggest that the prolegs are likely secondary outgrowths in Mecoptera. In the armyworm, lateral process primordia appear on A1-A10 in alignment with the thoracic legs in the early embryonic stages, although only the rudiments on A3-A6 and A10 develop into segmented prolegs with the lumina continuous with the hemocoel and others degenerate eventually, suggesting that the prolegs are true segmental appendages serially homologous with the thoracic legs in Lepidoptera. Therefore, we conclude that the larval prolegs are likely not evolutionarily homologous between Mecoptera and Lepidoptera.

Patterns of postzygotic isolation in Lepidoptera.

I present patterns characterizing the evolution of intrinsic postzygotic isolation in Lepidoptera by analyzing data from the literature on genetic distance, strength of hybrid sterility and inviability, biogeography, and natural hybridization. Using genetic distance as a proxy for time, I investigate the time-course of the evolution of postzygotic isolation and the waiting times to particular hybrid fitness problems. The results show that postzygotic isolation increases gradually as species diverge, but that hybrid sterility evolves faster than hybrid inviability. The overwhelming preponderance of female-specific hybrid problems in Lepidoptera shows that Haldane's rule (the preferential sterility or inviability of the heterogametic sex) is well obeyed. Together the rates and patterns characterizing the accumulation of postzygotic isolation allow several tests of the composite theory of Haldane's rule. Interestingly, comparing these data with those from Drosophila reveals that Haldane's rule for sterility evolves as fast (if not faster) in Lepidoptera. Finally, I show that a substantial fraction of sympatric species hybridizes in nature and that the majority of these suffer some level of hybrid sterility or inviability.

Cloning, characterization, and genetics of the juvenile hormone esterase gene from Heliothis virescens.

The gene for juvenile hormone esterase (JHE) was cloned from Heliothis virescens (Lepidoptera: Noctuidae). A genomic library was constructed from embryonic DNA and screened with a homologous N-terminal probe from the JHE cDNA. Five genomic clones were isolated and analyzed by dot blot hybridization using regions of the JHE cDNA as probes. Clone C hybridized to both 5' and 3' probes from the JHE cDNA, suggesting that clone C contains both ends of JHE gene. This was verified by sequencing the ends of the JHE gene from clone C using primers from both the 5' and 3' ends of the JHE cDNA. Additional sequencing and restriction mapping were used to characterize the gene. The gene is c. 8 kb long and contains four introns with consensus intron-exon junctions. One of the introns is relatively large (4 kb) and is situated near the extreme 5' end of the gene. Genetic analysis of RFLP variation in interspecific and intraspecific crosses shows that the JHE locus is single-copy with no closely related paralogs and is autosomally encoded in Heliothis. Therefore the developmental pattern of expression of this gene and the previously documented sequence variation in cDNA clones is not explainable by reference to a JHE gene family with distinct structural loci for the different forms.

Mortality staging of dominant lethals induced in the F1 generation of the sugarcane borer; Diatraea saccharalis (f.).

Comparative egg maturation was observed between fertile eggs laid by unirradiated females, by unirradiated females mated with irradiated males, by irradiated females mated with normal males, and by virgin unirradiated females. Haploid eggs turn yellow and dry by the fifth day. Eggs from irradiated parents develop more slowly than normal eggs. Most embryos from parents irradiated as P generation adults at 16.8 kilorads for males and 12.0 kR for females die early in embryonic development. A description of egg development is included.

Development and distribution of serotonin in the central nervous system of Manduca sexta during embryogenesis. II. The ventral ganglia.

The distribution of serotonin (5-HT) immunoreactive cells and their projections was mapped in the ganglia of the ventral nerve cord of the tobacco hornworm, Manduca sexta, during embryonic development, using an antiserum to 5-HT. Immunoreactive cells were first seen at 60% development. By 75% of embryonic development, a total of 94 immunoreactive cells were found in the ventral ganglia, including the suboesophageal ganglion. This number decreased to 80 neurons by 100% of development. About 50% of these cells were arranged in the abdominal ganglia and the rest were located in both the suboesophageal and thoracic ganglia. The suboesophageal ganglion exhibited immunoreactive segmental interneurons in areas corresponding to the mandibular, maxillary and labial neuromeres. Two pairs of immunoreactive interneurons were also observed to occur bilaterally in each of the thoracic and abdominal ganglia, with the exception of the prothoracic ganglion. This ganglion contained three pairs of bilaterally arranged immunoreactive neurons as early as 60% of embryonic development. Serotonin immunoreactivity was also found in a number of efferent neurons in the mandibular and labial neuromeres of the suboesophageal ganglion and in the prothoracic, mesothoracic and posterior abdominal ganglia. The occurrence of 5-HT in these efferent neurons suggests an involvement of serotonin in fore- and hindgut function via its effect on the visceral muscles. Immunoreactive lateral longitudinal fibers extended along the entire length of the ventral nerve cord together with dense segmental arborizations. The latter had regressed by the time the embryo was fully developed. This regression of the arborizations in the ganglia at the end of embryonic development indicates that a reorganization of 5-HT innervation occurs to support new larval functions. The time of appearance of 5-HT immunoreactive cells and fibers suggests that serotonin may play a role in the development of the ventral nerve cord.

Development and distribution of serotonin in the central nervous system of Manduca sexta during embryogenesis. I. The brain and frontal ganglion.

Development of the serotonergic system in the brain and frontal ganglion of the Manduca embryo between 35 and 100% of development was studied immunocytochemically with an antiserum to serotonin (5-HT). Serotonin immunoreactivity was initially detectable at 40-45% development in short fibers in the head region, prior to differentiation of the brain. Immunoreactive cell bodies were first seen in the brain at 60% development, located in the protocerebrum and tritocerebrum. Thick fiber tracts crossing the midline (commissures) could also be observed at this early stage. As development of the embryo progressed, eight immunoreactive cell groups, containing a total of about 38-40 cells, and four commissures with terminal arborizations appeared successively in the brain. From 75 to 100% development, no obvious changes occurred in the number or distribution of cells, and the brain exhibited the same pattern of 5-HT immunoreactive cells, fiber tracts and arborizations as in last instar larvae of Manduca. However, an increase in the size of the cells in both the brain and frontal ganglion was noted between 75 and 80% development, followed by a decrease by 100% development. The frontal ganglion was found to contain three 5-HT immunoreactive cells, which appeared to send bilateral projections into the frontal connectives and the recurrent nerve. During embryonic development, the dendritic arborizations of these frontal ganglion cells increased, while the amount of 5-HT immunoreactivity in the cell bodies decreased. Thus, the serotonergic system first appears in the Manduca embryo at an early stage of development, similar to the situation in other insects as well as vertebrates. By the end of the embryonic period, the same number of serotonergic neurons are present in the brain as in larval and adult Manduca, suggesting that once formed, these cells persist through postembryonic development and metamorphosis.

Influence of low temperature on the synthesis of some Galleria mellonella proteins.

In developing Galleria mellonella larvae (reared at 30 degrees C) three proteins of 74, 76 and 81/82 kDa were identified. They represent a group of storage proteins (LHP proteins). In Galleria larvae, the development of which is arrested by low temperature (18 degrees C), accumulation of the 74, 76 and 81/82 kDa proteins was detected in the hemolymph. The synthesis of 74 kDa and 76 kDa proteins started after 24 h, and that of about 80 kDa after 96 h following the transfer of larvae from 30 degrees C to 18 degrees C. 20-Hydroxyecdysone inhibited synthesis of the 74 and 76 kDa proteins in larvae exposed to low temperature. The arrest of development of Galleria larvae is associated with the synthesis and accumulation of storage proteins, and ecdysteroids are involved in these processes.

Programmed cell death in the wing of Orgyia leucostigma (Lepidoptera: Lymantriidae).

Programmed cell death is an integral and ubiquitous phenomenon of development that is responsible for the reduction of wing size in female moths of Orgyia leucostigma (Lymantriidae). Throughout larval and pupal life, cells of the wing epithelium proliferate and interact to form normal imaginal discs and pupal wings in both sexes. But at the onset of adult development, most cells in female O. leucostigma wings degenerate over a brief, 2-day period. Lysosomes and autophagic vacuoles appear in cells of the wing epithelium shortly after it retracts from the pupal cuticle. Hemocytes actively participate in removing the resulting cellular debris. By contrast, epithelial cells in wings of developing adult males of O. leucostigma do not undergo massive cell death. Wing epithelium of female pupae transferred to male pupal hosts behaves autonomously in this foreign environment. By pupation, cells of the female wing apparently are committed to self-destruct even in a male pupal environment. Normal interactions among epithelial cells within the plane of a wing monolayer as well as between the upper and lower monolayers of the wing are disrupted in female O. leucostigma by massive cell degeneration. Despite this disruption, the remaining cells of the wing contribute to the formation of a diminutive, but reasonably proportioned, adult wing with scales and veins.