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Tebufenozide targeted against codling moth (Lepidoptera: Tortricidae) adults, eggs, and larvae.

The effectiveness of tebufenozide applied against the adult, egg, and larvae of codling moth, Cydia pomonella (L.), was evaluated. Significant reductions in fecundity and egg hatch occurred after 1-h and 24-h exposures of females and 24-h exposures of males-only to residues in plastic plates. A significant reduction in egg hatch was also found after a 1-h exposure of males. The ovicidal effects of tebufenozide in field trials did not significantly differ for eggs laid on residues or treated topically. Corrected egg mortality exceeded 95% for cohorts laid <130 degree-days after sprays were applied. Fecundity and egg hatch were measured after either a 24-h exposure of moths or a 10-d exposure of moths and eggs on apple trees. Significant reductions in both fecundity and fertility occurred compared with an untreated control up to 7 d in the 24-h assays and accounted for 60-70% fewer larvae produced per mated female. The mean numbers of larvae produced per mated female after the 10-d exposure were reduced 100-75% in assays started on day 0-21 after the spray application compared with the untreated control. Fecundity was significantly reduced for 7 d and egg hatch was reduced for the entire 21-d test period in these trials. Residues had a 14-d half-life on apple foliage. Residues applied to foliage or to foliage and fruit did not significantly increase the proportion of uninjured fruit compared with the untreated control in bioassays where neonates were placed on foliage 10 cm from fruit. However, the proportion of injured fruits with shallow stings versus deep entries was higher on the treated versus the untreated plants. Field applications of tebufenozide reduced fruit injury >90% when applied early during emergence. Plots treated after the beginning of egg hatch had a larger proportion of injured fruits with shallow larval feeding.

Animals↗

Evaluation of a nonconventional insecticide and appropriate application timing for destruction of gypsy moth (Lepidoptera: Lymantriidae) egg masses.

Two field studies were conducted in 2001-2002 and 2003 to evaluate the effectiveness and appropriate application timing of Golden Pest Spray Oil (GPSO) for destruction of gypsy moth, Lymantria dispar (L.) (Lepidoptera: Lymantriidae), egg masses in Wisconsin. GPSO is a commercially available, registered pesticide that is predominantly comprised of a soybean-oil base (93%); its primary mode of action is by means of suffocation. Because gypsy moth spends the majority (>75%) of its life cycle in the egg stage (August-April), the potential utility of this product by arborists, city foresters, landscapers, and homeowners is high, especially because GPSO is a United States Environmental Protection Agency registered, nonconventional pesticide that is considered relatively nontoxic. When GPSO was applied at a 1:1 ratio with water, >96% control of gypsy moth egg masses was achieved, regardless of application timing (October, 3 d before egg hatch).

Animals↗

No evidence of fine-scale local adaptation of winter moths to variable tree phenology.

Spatial variation in plant phenology can impose strong selective pressures on herbivorous insects whose fitness relies on synchrony with host plants, promoting local adaptation to host timing. Winter moths (Operophtera brumata) have been shown to synchronize egg hatching with host budburst, but whether this reflects local adaptation remains unclear. We used three complementary approaches to assess small-scale local adaptation of winter moths to oak phenology in Wytham Woods, UK, a 385-hectare woodland with repeatable variation in individual oak budburst phenology. We experimentally investigated whether host tree phenology predicts hatch timing using common gardens across multiple temperatures, evaluated fitness benefits of synchrony using translocations, and assessed population structure and gene-environment associations using whole-genome sequencing. We found no support for local adaptation to individual trees. Common garden experiments revealed systematic differences in hatch timing which were unrelated to host budburst, while translocations indicated no fitness consequences of asynchrony. Genetic analyses showed no detectable population structure or association with budburst timing. Local adaptation to host phenology therefore appears not to arise on individual trees but may instead occur at broader spatial scales. Understanding the scale of local adaptation is essential for predicting how insect-plant synchrony will respond to environmental change across heterogeneous landscapes.

Animals↗

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

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

Amino Acid Sequence↗

Identification of cuticular hydrocarbons and the alkene precursor to the pheromone in hemolymph of the female gypsy moth, Lymantria dispar.

Hydrocarbons were extracted from the surface of the cuticle and from the hemolymph of adult female gypsy moths. GC and GC/MS analysis indicated that the cuticular hydrocarbons with chain lengths >21 carbons were the same as those found in the hemolymph. These consisted of mostly saturated straight chain hydrocarbons with heptacosane the major component. Methyl branched hydrocarbons were also identified including a series of tetramethylalkanes with chain lengths of 30, 32, and 34 carbons. In addition to those found on the cuticle surface, the hemolymph contained the alkene pheromone precursor, 2-methyl-Z7-octadecene and two saturated analogues, 2-methyl-octadecane and 2-methyl-hexadecane. No evidence was obtained for the presence of the pheromone 2-methyl-7, 8-epoxy-octadecane in the hemolymph. Pheromone gland extracts indicated that small amounts (<1 ng) of the alkene precursor were also present in the gland. Relatively larger amounts of the alkene precursor were found in the hemolymph at the time when pheromone titers were higher on the gland. The presence of the hydrocarbon pheromone precursor in the hemolymph is discussed in relation to possible biosynthetic pathways for producing the gypsy moth pheromone.

Alkanes↗

Periodicity of sex pheromone biosynthesis, release and degradation in the lightbrown apple moth, Epiphyas postvittana (Walker).

Pheromone titer in moths is a product of three processes occurring in or at the surface of the pheromone gland: biosynthesis, release, and intraglandular degradation, of pheromone. Changes in titers of sex pheromone, the fatty acyl pheromone analog (FAPA), and tetradecanoate, a pheromone biosynthetic intermediate, were studied in detail in the lightbrown apple moth, Epiphyas postvittana (Walker). Although changes in the pheromone titers in a day were relatively small, with the peak titer being 2-3 times greater than that at the trough, pheromone titer did show a distinct diel periodicity. Titer of the FAPA showed a similar, but less variable, diel pattern, but tetradecanoate titer showed little or no diel pattern. The pattern of pheromone titer suggested that females biosynthesize pheromone at two different rates during the photoperiod: a high rate during the latter half of the photophase and most of the scotophase, which is associated with a high pheromone titer, and a low rate throughout the first half of the photophase, which is associated with a low titer. Consistent with data on commencement of copulation, pheromone was released from the second hour of the scotophase through to the eighth hour. Pheromone release rate during this period appeared to be similar to the rate of pheromone biosynthesis. In contrast to the other two processes, pheromone degradation did not appear to have a diel pattern. Females decapitated at different times of the photoperiod showed a similar decline in pheromone titer, consistent with the reaction kinetics being first order in pheromone titer.

Animals↗

Optimum timing of insecticide applications against diamondback moth Plutella xylostella in cole crops using threshold catches in sex pheromone traps.

Field trials were conducted in cabbage (Brassica oleracea var capitata), cauliflower (B oleracea var botrytis) and knol khol (B oleracea gongylodes) crops at two different locations in Karnataka State (India) to optimize the timing of insecticide applications to control the diamondback moth, Plutella xylostella, using sex pheromone traps. Our results indicate that applications of cartap hydrochloride as insecticide during a 12-24 h period after the pheromone traps had caught on average 8, 12 and 16 males per trap per night in cabbage, cauliflower and knol khol, respectively, were significantly more effective than regular insecticide sprays at 7, 9, 12 or 15 days after transplantation. This was demonstrated by estimation of the mean number of eggs and larvae per plant, the percentage of holes produced, as well as the marketable yield of the three crops at each location. A good correlation between the immature stages, infestation level, the estimated crop yield and the number of moths caught in pheromone traps was also found, indicating the usefulness of pheromone-based monitoring traps to predict population densities of the pest.

Animals↗

Cloning and expression of male-specific protein (MSP) from the hemolymph of greater wax moth, Galleria mellonella L.

Male-specific protein (MSP) is a soluble protein that accumulates in high amounts in the hemolymph and other organs of adult male wax moth. The MSP was purified from adult male wax moth by gel filtration and reversed phase column chromatography, and its amino acid sequence was determined. Because of blocked N-terminus, several internal amino acid sequences of MSP were obtained by the in-gel digestion method using trypsin. RT-PCR was conducted using degenerate primers designed from the internal amino acid sequences. 5'-RACE PCR was used to obtain the complete coding region and 5'-UTR sequence. The full length MSP cDNA sequence encodes a 239 amino acid polypeptide with an 18 amino acid signal peptide. The putative mature MSP has a molecular mass of 24,317 Da and an isoelectric point (pI) of 6.00, but shows a molecular mass of 27 kDa on SDS-PAGE. Sequence alignment showed a significant similarity between MSP and juvenile hormone binding proteins (JHBPs) of several lepidopteran species, including G. mellonella.

Amino Acid Sequence↗

Isolation and partial characterization of gypsy moth BTR-270, an anionic brush border membrane glycoconjugate that binds Bacillus thuringiensis Cry1A toxins with high affinity.

BTR-270, a gypsy moth (Lymantria dispar) brush border membrane molecule that binds Bacillus thuringiensis (Bt) Cry1A toxins with high affinity, was purified by preparative gel electrophoresis. Rabbit antibodies specific for the Bt toxin-binding molecule were raised. Attempts to label BTR-270 by protein-directed techniques were futile, but it was degraded by proteases with broad specificity indicating the presence of a peptide. Carbohydrate was detected by labeling with digoxigenin hydrazide following periodate oxidation. Mild alkaline hydrolysis destroyed toxin and antibody binding, suggesting O-linked glycans are involved in the activity. GC/MS composition analysis showed that the predominant sugars were galactose, glucose, and N-acetyl galactosamine with lesser amounts of N-acetyl glucosamine, glucuronic acid, xylose, and fucose. The carbohydrate moiety accounted for 73% of its total mass. Amino acid analysis showed a high content of aspartic/asparagine, threonine, and serine residues in the protein moiety. The purified glycoconjugate was not visualized using Coomassie or silver staining procedures, but stained "blue" using the cationic dye Stains-all. BTR-270 was labeled with biotin and used as a diagnostic probe for screening and identifying toxins that bind to the receptor. Toxin-binding kinetics obtained using a biosensor demonstrated that the receptor binds Cry1Aa and Cry1Ab toxins with high affinity, and displays a weaker affinity for Cry1Ac, in correlation with the toxicity of these toxins towards gypsy moth. Arch.

Amino Acids↗

Pheromonotropic and pheromonostatic activity in moths.

Pheromone biosynthesis in many species of moths requires a pheromonotropic neurosecretion, the pheromone biosynthesis activating neuropeptide (PBAN), from the brain-subesophageal ganglion-corpora cardiaca complex. Some investigations suggest that PBAN is released into the hemolymph and acts directly on sex pheromone glands (SPG) via a Ca++/calmodulin-dependent adenylate cyclase. Others suggest, however, that PBAN acts via octopamine that is released by nerves from the terminal abdominal ganglion innervating the SPG. These findings suggest that there are controversies on the mode of action of PBAN and other pheromonotropic factors, sometimes even within the same species. Mating in many insects results in temporary or permanent suppression of pheromone production and/or receptivity. Such a suppression may result from physical blockage of the gonopore or deposition of pheromonostatic factor(s) by the male during copulation that result in suppressed pheromone production and/or receptivity in females either directly or by a primer effect. In several species of insects, including moths, a pheromonostatic factor is transferred in the seminal fluid of males. Similar to the controversies associated with the pheromonotropic activity of PBAN, sometimes even within the same species, there appear to be controversies in pheromonostasis in heliothines as well. This paper reviews these conflicting findings and presents some data on pheromonostatic and pheromonotropic activity in Heliothis virescens that support and conflict with current information, raising further questions. Answers to some of the questions are partly available; however, they remain to be answered unequivocally.

Animals↗

Digital atlases of the antennal lobe in two species of tobacco budworm moths, the Oriental Helicoverpa assulta (male) and the American Heliothis virescens (male and female).

The antennal lobe of the moth brain is the primary olfactory center processing information about pheromones and plant odors. We present here a digital atlas of the glomerular antennal lobe structures in the male of Helicoverpa assulta and the male and female of Heliothis virescens, based on synaptic antibody staining combined with confocal microscopy. The numbers of the glomeruli in the three specimens were similar, 65, 66, and 62, respectively. Whereas the male antennal lobe has a macroglomerular complex consisting of three and four units in the two species, the female lobe has two enlarged glomeruli at a corresponding position, near the entrance of the antennal nerve. Another large glomerulus, showing homology in the three specimens, is ventrally located. The small size of the heliothine moths is advantageous for confocal microscopy because the entire brain can be visualized as a single image stack. The maps are freely accessible on the internet, and the digital form of the data allows each atlas to be rotated and sectioned at any angle, providing for the identification of glomeruli in different preparations.

Animals↗

Olfactory protocerebral pathways processing sex pheromone and plant odor information in the male moth Agrotis segetum.

We investigated protocerebral processing of behaviorally relevant signals in the turnip moth, Agrotis segetum. Single neurons were studied both physiologically and morphologically using intracellular recording techniques. In moth pheromone communication systems, the presence of the complete, female-produced pheromone blend is necessary for male attraction. We predicted that more protocerebral neurons, compared with AL, would display blend interactions. However, only a few protocerebral neurons responded differently to the blend than could be deduced from the response to single components. The majority of the pheromone-sensitive protocerebral neurons identified in this study responded to the major pheromone component. In coding time, most AL neurons can follow a 5-Hz odor stimulus, whereas most protocerebral neurons failed at higher frequencies than 1 Hz. The majority of neurons that responded to the odorants tested innervated one or both of the protocerebral lateral accessory lobes. If only one of these was innervated, then the innervation always displayed a varicose appearance, suggesting a presynaptic function. Thus, information seems to be transferred from other protocerebral areas to the lateral accessory lobes. Into these, descending neurons sent smooth, postsynaptic branches. A majority of the neurons innervating the superior medial protocerebrum were found to display single-component specificity. Few additional correlations between odor specificity and structural characteristics were apparent.

Animals↗

Projections of male-specific receptor neurons in the antennal lobe of the Oriental tobacco budworm moth, Helicoverpa assulta: a unique glomerular organization among related species.

The macroglomerular complex in the primary olfactory center of male moths receives information from numerous pheromone-detecting receptor neurons housed in specific sensilla located on the antennae. We investigated the functional organization of the three glomeruli constituting this complex in Helicoverpa assulta, a unique species among heliothine moths as concerns the composition of the pheromone blend. By tip recordings from the male-specific receptor neurons combined with cobalt-lysine stainings, the axon terminals in the brain were traced and subsequently reconstructed by camera lucida drawings. Some were also reconstructed in a digital form. The results showed that the sensilla could be classified into two functional types. A major category housed two colocalized receptor neurons, one responding to the primary pheromone component cis-9-hexadecenal and the other to the behavioral antagonists cis-9-tetradecenal and cis-9-hexadecenol. Cobalt-lysine applied to this sensillum type consistently resulted in two stained axons, each terminating in one of the two large subunits of the macroglomerular complex: the cumulus or the dorsomedial glomerulus. The second, less frequently appearing sensillum type contained a receptor neuron responding to the second pheromone component, cis-11-hexadecenal. Dye applied to this type resulted in stained axon projections in the ventral glomerulus. In an evolutionary context it is particularly interesting that differences of related heliothine species are reflected in the functional organization of the MGC compartments.

Action Potentials↗

Consistent organization of glomeruli in the antennal lobes of related species of heliothine moths.

The glomeruli of the antennal lobes in insects reflect the organization of the olfactory system, which is important for species-specific behaviors in response to insect- and plant-produced odorants. We studied the antennal lobes of the polyphagous moth Helicoverpa armigera and the oligophagous H. assulta (Heliothinae; Lepidoptera; Noctuidae) in order to see whether there are any anatomical differences that might elucidate how information about odorants is analyzed. Three-dimensional models of the antennal lobes were made, based on synaptic antibody staining combined with confocal laser scanning microscopy. These showed 65 glomeruli in each sex of H. armigera and 66 glomeruli in females of H. assulta. Sixty-two of the glomeruli were identified in both sexes and species and were given the same numbers. The sex-specific glomeruli included three macroglomerular units in H. armigera males, as well as three and four female-specific glomeruli in H. armigera and H. assulta, respectively. The species specificity of H. assulta females also appeared by the particular large size of two ordinary glomeruli. The accumulating knowledge on how biologically relevant information is encoded in receptor and antennal lobe neurons in heliothines makes these moths particularly interesting for studying the functional organization of the glomeruli. The anatomical atlases of the antennal lobes, as presented here, are prerequisites for identifying glomeruli ascribed to particular functions across sexes and species.

Anatomy, Artistic↗

Metamorphosis of the cerebral neuroendocrine system in the moth Manduca sexta.

We have examined the morphology and neuronal elements of the cerebral neuroendocrine system in the larval, pupal, and adult stages of the moth Manduca sexta with a variety of neuroanatomical techniques. The larval brain contains several discrete groups of neurosecretory and non-neurosecretory cells that project to the associated neurohemal organs (the corpora cardiaca-allata complex, or CC-CA) and to a variety of more peripheral structures. A previously undescribed set of cells in the subesophageal ganglion have also been found that project out the neurosecretory nerves. During metamorphosis, the cerebral neuroendocrine system undergoes a dramatic structural reorganization, including the reduction or loss of many larval nerves and a repositioning of the cell groups and their dendritic fields. Despite these changes, most of its central elements are retained. In addition, by the completion of adult development a new cluster of cells can be found on either side of the dorsal midline of the brain. We have also determined the relative contributions of the different cell groups to the moth neuroendocrine system by intracellular iontophoresis of dye into individual cells. Within the dorsal protocerebrum, five separate morphological types of cells can be recognized, each with a distinctive pattern of dendritic arborization in the brain and terminal neurohemal processes that project to the CC, the CA, the aorta, or to a combination of these regions. The large intrinsic cells of the CC have also been filled, revealing an unusual set of morphological features in these peripheral neurosecretory cells.

Animals↗

Organization of the auditory pathway in the thoracic ganglia of noctuid moths.

We describe the neuroarchitecture of the noctuid thoracic nerve cord and use this framework to interpret the organization of the auditory pathway responsible for escape behaviour in noctuid moths. Noctuid moths possess only two auditory receptors (A1, A2), in each ear. The axon of the A1 cell projects initially to a glomerulus located ventrally and medially in the metathoracic ganglion, where it bifurcates. One branch ascends in the ventral intermediate tract to the brain, the other descends in the ventral intermediate tract into abdominal neuromeres of the metathoracic ganglion. Both axons arborize in the median ventral and ring tracts in each neuromere. The central projections of the A2 cell remain largely within the metathoracic ganglion. The axon bifurcates at the midline and directs arborizations dorsally to the dorsal intermediate and median dorsal tracts, and ventrally into the ring tract where the arborizations overlap those of the A1 afferent. The afferent projections remain ipsilateral to the ear of origin. We describe a posterior auditory association area in the metathoracic ganglion in which the major arborizations of several identified interneurones overlap those of the A1 afferent and make monosynaptic connections with it. These interneurones all respond tonically to sound stimuli. We have also identified the projections of the A1 afferent, interneurones, and motor neurones in the segmentally equivalent anterior auditory association area of the mesothoracic ganglion. An interneurone with major arborizations in the same tracts as the A1 afferent, and receiving monosynaptic input from it, is described. The arborizations of higher order interneurones lie mainly in dorsal tracts along with those of flight motor neurones. All the interneurones in this anterior centre respond phasically or phasic/tonically to sound stimuli. The relevance of this anatomical organization for predator avoidance behaviour is considered and the organization of auditory pathways in tympanate insects compared.

Animals↗

The mechanoreceptive origin of insect tympanal organs: a comparative study of similar nerves in tympanate and atympanate moths.

A chordotonal organ occurring in the posterior metathorax of an atympanate moth, Actias luna (L.) (Bombycoidea: Saturniidae), appears to be homologous to the tympanal organ of the noctuoid moth. The peripheral anatomy of the metathoracic nerve branch, IIIN1b1 was examined in Actias luna with cobalt-lysine and Janus Green B, and compared to its counterpart, IIIN1b (the tympanal branch), in Feltia heralis (Grt.) (Noctuoidea: Noctuidae). The peripheral projections of IIIN1b1 were found to be similar in both species, dividing into three branches, the second (IIIN1b1b) ending as a chordotonal organ. The atympanate organ possesses three sensory cell bodies and three scolopales, and is anchored peripherally via an attachment strand to the undifferentiated membranous region underlying the hindwing alula, which corresponds to the tympanal region of the noctuoid metathorax. Extracellular recordings of the IIIN1b1 nerve in Actias luna revealed a large spontaneously active unit which fired in a regular pattern (corresponding to the noctuoid B cell) and smaller units (corresponding to the noctuoid acoustic A cells) which responded phasically to low frequency sounds (2 kHz) played at high intensities (83-96 dB, SPL) and also responded phasically to raising and lowering movements of the hindwing. We suggest that the chordotonal organ in Actias luna represents the evolutionary prototype to the noctuoid tympanal organ, and that it acts as a proprioceptor monitoring hindwing movements. This system, in its simplicity (consisting of only a few neurons) could be a useful model for examining the changes to the nervous system (both central and peripheral) that accompanied the evolutionary development of insect tympanal organs.

Acoustic Stimulation↗

Localization of immunoreactive ubiquitin in the nervous system of the Manduca sexta moth.

Selective neuronal death is a normal component of metamorphosis in the moth, Manduca sexta. In particular, the three unfused abdominal ganglia of the ventral nerve cord serve as a useful experimental preparation in which to study the regulation of the molecular mechanisms that mediate programmed cell death. Ubiquitin, a highly conserved 76-amino acid protein found in all eukaryotic cells, has previously been shown to be present in increased amounts in some tissues undergoing programmed cell death (e.g., larval intersegmental muscles in Manduca sexta moths, dying cells in developing tunicates), but not in others (T-cells, Drosophila ommatidial cells, cultured sympathetic neurons deprived of nerve growth factor). It has been hypothesized that the need for ubiquitin-dependent proteolysis is increased in dying cells, and that the accumulation of ubiquitin might serve as an early marker for cells committed to die. Immunohistochemical localization of ubiquitin at the light microscopic level in the abdominal ganglia of Manduca sexta suggests that this protein plays a number of important roles in neuronal physiology and may be associated with the death of some neurons in this tissue. The most intense staining of neuronal cytoplasm, however, was found not in dying neurons, but instead in sets of persisting neurons that may serve a primarily neurosecretory or neuromodulatory function. The staining obtained in these cells with antibodies directed against ubiquitin was developmentally regulated.

Abdomen↗