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Molecular characterization of the insect immune protein hemolin and its high induction during embryonic diapause in the gypsy moth, Lymantria dispar.

During the embryonic (pharate first instar) diapause of the gypsy moth, Lymantria dispar, a 55 kDa protein is highly up-regulated in the gut. We now identify that protein as hemolin, an immune protein in the immunoglobulin superfamily. We isolated a gypsy moth hemolin cDNA and demonstrated a high degree of similarity with hemolins from three other moth species. Hemolin mRNA levels increased at the time of diapause initiation and remained high throughout the mandatory period of chilling required to terminate diapause in this species, and then dropped in late diapause. This mRNA pattern reflects the pattern of protein synthesis. These results suggest that hemolin is developmentally up-regulated in the gut during diapause. Diapause in this species can be prevented using KK-42, an imidazole derivative known to inhibit ecdysteroid biosynthesis, and gypsy moths treated in this manner failed to elevate hemolin mRNA. Conversely, this diapause appears to be initiated and maintained by the steroid hormone, 20-hydroxyecdysone, and the addition of 20-hydroxyecdysone to the culture medium elevated hemolin mRNA in the gut. Our results thus indicate a role for 20-hydroxyecdysone in the elevation of hemolin mRNA during diapause. Presumably, hemolin functions to protect the gypsy moth from microbial infection during its long, overwintering diapause.

Amino Acid Sequence↗

A divergent gene family encoding candidate olfactory receptors of the moth Heliothis virescens.

The antennae of moths have been an invaluable model for studying the principles of odour perception. In spite of the enormous progress in understanding olfaction on the molecular level, for the moth one of the key elements in olfactory signalling, the odourant receptors, are still elusive. We have assessed a genome database of a heliothine moth (Heliothis virescens, Noctuidae) and employed exon-specific probes to screen an antennal cDNA library of this species. Analysis of isolated cDNA-clones led to the discovery of a divergent gene family encoding putative seven-transmembrane domain proteins. The notion that they may encode candidate olfactory receptors of the moth, was supported by a tissue-specific expression; several of the subtypes were exclusively expressed in antennae. By means of double-labelling in situ hybridization studies it was demonstrated that the receptors are indeed expressed in antennal sensory neurons; moreover, each receptor subtype appears to be expressed in a distinct population of sensory cells. The results strongly suggest that the newly discovered gene family indeed encodes olfactory receptors of moth.

Amino Acid Sequence↗

Protein engineering of Bacillus thuringiensis delta-endotoxin: mutations at domain II of CryIAb enhance receptor affinity and toxicity toward gypsy moth larvae.

Substitutions or deletions of domain II loop residues of Bacillus thuringiensis delta-endotoxin CryIAb were constructed using site-directed mutagenesis techniques to investigate their functional roles in receptor binding and toxicity toward gypsy moth (Lymantria dispar). Substitution of loop 2 residue N372 with Ala or Gly (N372A, N372G) increased the toxicity against gypsy moth larvae 8-fold and enhanced binding affinity to gypsy moth midgut brush border membrane vesicles (BBMV) approximately 4-fold. Deletion of N372 (D3), however, substantially reduced toxicity (> 21 times) as well as binding affinity, suggesting that residue N372 is involved in receptor binding. Interestingly, a triple mutant, DF-1 (N372A, A282G and L283S), has a 36-fold increase in toxicity to gypsy moth neonates compared with wild-type toxin. The enhanced activity of DF-1 was correlated with higher binding affinity (18-fold) and binding site concentrations. Dissociation binding assays suggested that the off-rate of the BBMV-bound mutant toxins was similar to that of the wild type. However, DF-1 toxin bound 4 times more than the wild-type and N372A toxins, and it was directly correlated with binding affinity and potency. Protein blots of gypsy moth BBMV probed with labeled N372A, DF-1, and CryIAb toxins recognized a common 210-kDa protein, indicating that the increased activity of the mutants was not caused by binding to additional receptor(s). The improved binding affinity of N372A and DF-1 suggest that a shorter side chain at these loops may fit the toxin more efficiently to the binding pockets. These results offer an excellent model system for engineering delta-endotoxins with higher potency and wider spectra of target pests by improving receptor binding interactions.

Animals↗

Behavioural responses of diamondback moth Plutella xylostella (Lepidoptera: Plutellidae) to extracts derived from Melia azedarach and Azadirachta indica.

The impact of three different doses of botanical insecticide derived from the syringa tree, Melia azedarach and the neem tree, Azadirachta indica was tested on the behaviour of the diamondback moth, Plutella xylostella (Linnaeus). Both botanical insecticides had a significant impact on larval behaviour. At higher doses the extracts showed feeding deterrent activity, with larvae preferring the untreated sides of cabbage leaves and consuming less of the treated half of cabbage leaves. The botanical insecticides had less of an effect on the oviposition behaviour of P. xylostella moths. In laboratory and glasshouse trials, significantly fewer eggs were oviposited on the plants that had been treated with syringa extracts. Therefore, the syringa extracts appear to have a repellent effect. In contrast, when exposed to the neem extracts the moths did not discriminate between control plants and treated plants. Behavioural observation indicated that, despite the lower number of eggs oviposited on cabbage treated with syringa extracts, the moths chose cabbage treated with the highest dose of syringa more often than they chose control cabbage plants. Similar observations were found in cabbage plants treated with neem, moths chose the medium dose more often than they chose the control. Oviposition and feeding deterrent properties are important factors in pest control, and results from this study indicate that botanical insecticides have the potential to be incorporated into control programmes for P. xylostella in South Africa.

Animals↗

Entomopathogenic nematodes for control of diapausing codling moth (Lepidoptera: Tortricidae) in fruit bins.

Fruit bins infested with diapausing larvae of codling moth larvae, Cydia pomonella (L.), are a source of reinfestation of orchards and may jeopardize the success of mating disruption programs and other control strategies. Bins are not routinely treated for control of overwintering codling moth before placing them in orchards. Entomopathogenic nematodes provide a noninsecticidal alternative to methyl bromide that could be applied at the time bins are submerged in dump tanks at the packing house for flotation of fruit. Diapausing codling moth larvae in miniature fruit bins were highly susceptible to infective juveniles of Steinernema carpocapsae (Weiser). Immersion of bins in suspensions of S. carpocapsae ranging from 5 to 100 infective juveniles per milliliter of water resulted in 68-100% mortality. Immersion times of 1 or 5 min in suspensions with 5 infective juveniles of S. carpocapsae per milliliter of water, with and without Tween 80 (0.01%), yielded essentially the same mortality of codling moth larvae. Highest mortalities in codling moth larvae (88%) after treatment of bins in suspensions of 5 infective juveniles of S. carpocapsae per milliliter of water were observed after incubation for 24 h at 25 degrees C and 70% RH. Lowest mortalities (37%) were observed after incubation at 15 degrees C and 35% RH. Comparative tests conducted with Heterorhabditis marelatus Liu & Berry, Steinernema kraussei (Steiner), and S. carpocapsae with 5 infective juveniles per milliliter of water resulted in 21.7, 53.9, and 68.7% mortality, respectively. The use of miniature fruit bins as described in this article provides an effective means of assessing nematode efficacy without the cumbersome size of commercial bins.

Animals↗

Moth hearing in response to bat echolocation calls manipulated independently in time and frequency.

We measured the auditory responses of the noctuid moth Noctua pronuba to bat echolocation calls which were manipulated independently in time and frequency. Such manipulations are important in understanding how insect hearing influences the evolution of echolocation call characteristics. We manipulated the calls of three bat species (Rhinolophus hipposideros, Myotis nattereri and Pipistrellus pipistrellus) that use different echolocation call features by doubling their duration or reducing their frequency, and measured the auditory thresholds from the A1 cells of the moths. Knowing the auditory responses of the moth we tested three predictions. (i) The ranking of the audibility of unmanipulated calls to the moths should be predictable from their temporal and/or frequency structure. This was supported. (ii) Doubling the duration of the calls should increase their audibility by ca. 3 dB for all species. Their audibility did indeed increase by 2.1-3.5 dB. (iii) Reducing the frequency of the calls would increase their audibility for all species. Reducing the frequency had small effects for the two bat species which used short duration (2.7-3.6 ms) calls. However, the relatively long-duration (50 ms), largely constant-frequency calls of R. hipposideros increased in audibility by 21.6 dB when their frequency was halved. Time and frequency changes influence the audibility of calls to tympanate moths in different ways according to call design. Large changes in frequency and time had relatively small changes on the audibility of calls for short, largely broadband calls. Channelling energy into the second harmonic of the call substantially decreased the audibility of calls for bats which use long-duration, constant-frequency components in echolocation calls. We discuss our findings in the contexts of the evolution of both bat echolocation call design and the potential responses of insects which hear ultrasound.

Animals↗

Interactions among moths, crossbills, squirrels, and lodgepole pine in a geographic selection mosaic.

Repeated patterns among biological communities suggest similar evolutionary and ecological forces are acting on the communities. Conversely, the lack of such patterns suggests that similar forces are absent or additional ones are present. Coevolution between a seed predator, the red crossbill (Loxia curvirostra complex), and lodgepole pine (Pinus contorta var. latifolia) exemplifies the ecological and evolutionary predictions for coevolving systems. In the absence of another seed predator and preemptive competitor (pine squirrels Tamiasciurus hudsonicus), natural selection by crossbills results in the evolution of larger cones with thicker distal scales, while relaxation of selection by squirrels results in the evolution of cones with more seeds and a greater ratio of seed mass to cone mass. However, in one range, the Little Rocky Mountains, distal scale thickness has diverged as expected but cone size has not. In these mountains seed predation by lodgepole pine cone borer moths (Eucosma recissoriana) was about 10 times greater than in other ranges lacking squirrels. We quantified moth predation and cone traits and found that moths select for smaller cones with fewer seeds. Thus, selection by moths in the Little Rocky Mountains counters both selection by crossbills for large cone size and relaxation of selection by squirrels favoring more seeds per cone and accounts for the relatively small and few-seeded cones in these mountains. It is also apparent that selection by crossbills changes seed defenses in a manner that favors seed predation by moths, whereas selection by squirrels likely reduces such predation. These results demonstrate the importance of considering the evolutionary consequences of community context in locally evolved (coevolved) traits and interactions.

Animals↗

Cospeciation analysis of an obligate pollination mutualism: have Glochidion trees (Euphorbiaceae) and pollinating Epicephala moths (Gracillariidae) diversified in parallel?

Species-specific obligate pollination mutualism between Glochidion trees (Euphorbiaceae) and Epicephala moths (Gracillariidae) involves a large number of interacting species and resembles the classically known fig-fig wasp and yucca-yucca moth associations. To assess the extent of parallel cladogenesis in Glochidion-Epicephala association, we reconstruct phylogenetic relationships of 18 species of Glochidion using nuclear ribosomal DNA sequences (internal and external transcribed spacers) and those of the corresponding 18 Epicephala species using mitochondrial (the cytochrome oxidase subunit I gene) and nuclear DNA sequences (the arginine kinase and elongation factor-1alpha genes). Based on the obtained phylogenies, we determine whether Glochidion and Epicephala have undergone parallel diversification using several different methods for investigating the level of cospeciation between phylogenies. These tests indicate that there is generally a greater degree of correlation between Glochidion and Epicephala phylogenies than expected in a random association, but the results are sensitive to selection of different phylogenetic hypotheses and analytical methods for evaluating cospeciation. Perfect congruence between phylogenies is not found in this association, which likely resulted from host shift by the moths. The observed significant discrepancy between Glochidion and Epicephala phylogenies implies that the one-to-one specificity between the plants and moths has been maintained through a complex speciation process or that there is an underestimated diversity of association between Glochidion trees and Epicephala moths.

Animals↗

Extinction of the acoustic startle response in moths endemic to a bat-free habitat.

Most moths use ears solely to detect the echolocation calls of hunting, insectivorous bats and evoke evasive flight manoeuvres. This singularity of purpose predicts that this sensoribehavioural network will regress if the selective force that originally maintained it is removed. We tested this with noctuid moths from the islands of Tahiti and Moorea, sites where bats have never existed and where an earlier study demonstrated that the ears of endemic species resemble those of adventives although partially reduced in sensitivity. To determine if these moths still express the anti-bat defensive behaviour of acoustic startle response (ASR) we compared the nocturnal flight times of six endemic to six adventive species in the presence and absence of artificial bat echolocation sounds. Whereas all of the adventive species reduced their flight times when exposed to ultrasound, only one of the six endemic species did so. These differences were significant when tested using a phylogenetically based pairwise comparison and when comparing effect sizes. We conclude that the absence of bats in this habitat has caused the neural circuitry that normally controls the ASR behaviour in bat-exposed moths to become decoupled from the functionally vestigial ears of endemic Tahitian moths.

Animals↗

Fossil-calibrated molecular phylogenies reveal that leaf-mining moths radiated millions of years after their host plants.

Coevolution has been hypothesized as the main driving force for the remarkable diversity of insect-plant associations. Dating of insect and plant phylogenies allows us to test coevolutionary hypotheses and distinguish between the contemporaneous radiation of interacting lineages vs. insect 'host tracking' of previously diversified plants. Here, we used nuclear DNA to reconstruct a molecular phylogeny for 100 species of Phyllonorycter leaf-mining moths and 36 outgroup taxa. Ages for nodes in the moth phylogeny were estimated using a combination of a penalized likelihood method and a Bayesian approach, which takes into account phylogenetic uncertainty. To convert the relative ages of the moths into dates, we used an absolute calibration point from the fossil record. The age estimates of (a selection of) moth clades were then compared with fossil-based age estimates of their host plants. Our results show that the principal radiation of Phyllonorycter leaf-mining moths occurred well after the main radiation of their host plants and may represent the dominant associational mode in the fossil record.

Animals↗

Viral and host cellular transcription in Autographa californica nuclear polyhedrosis virus-infected gypsy moth cell lines.

Infection of two gypsy moth cell lines (IPLB-Ld652Y and IPLB-LdFB) by the Autographa californica multiple-enveloped nuclear polyhedrosis virus (AcMNPV) is characterized by extremely attenuated viral protein synthesis followed by a total arrest of all viral and cellular protein production. In this study, AcMNPV- and host cell-specific transcription were examined. Overall levels of viral RNAs in infected gypsy moth cells were, at most measured times, comparable to RNA levels from an infected cell line (TN-368) permissive for AcMNPV replication. Northern blot (RNA) analyses using viral and host gene-specific probes revealed predominantly normal-length virus- and cell-specific transcripts postinfection. Transport of viral RNAs from the nucleus to the cytoplasm and transcript stability in infected gypsy moth cells also appeared normal compared with similar parameters for AcMNPV-infected TN-368 cells. Host cellular and viral mRNAs extracted from gypsy moth and TN-368 cells at various times postinfection and translated in vitro yielded similar spectra of host and viral proteins. Treatment of infected gypsy moth cells with the DNA synthesis inhibitor aphidicolin eliminated the total protein synthesis shutoff in infected IPLB-LdFB cells but had no effect on protein synthesis inhibition in infected IPLB-Ld652Y cells. The apparent selective block in the translation of viral transcripts early in infection and the absence of normal translation or transcription of host cellular genes at later times is discussed.

Animals↗

Her odours make him deaf: crossmodal modulation of olfaction and hearing in a male moth.

All animals have to cope with sensory conflicts arising from simultaneous input of incongruent data to different sensory modalities. Nocturnal activity in moths includes mate-finding behaviour by odour detection and bat predator avoidance by acoustic detection. We studied male moths that were simultaneously exposed to female sex pheromones indicating the presence of a potential mate, and artificial bat cries simulating a predation risk. We show that stimulation of one sensory modality can modulate the response to information from another, suggesting that behavioural thresholds are dynamic and depend on the behavioural context. The tendency to respond to bat sounds decreased as the quality and/or the amount of sex pheromone increased. The behavioural threshold for artificial bat cries increased by up to 40 dB when male moths where simultaneously exposed to female sex pheromones. As a consequence, a male moth that has detected the pheromone plume from a female will not try to evade an approaching bat until the bat gets close, hence incurring increased predation risk. Our results suggest that male moths' reaction to sensory conflicts is a trade-off depending on the relative intensity of the input to CNS from the two sensory modalities.

Acoustic Stimulation↗

Tussock moth dermatitis: evidence for histamine involvement.

Moth hairs shed by the few tussock moths caught during the first documented outbreak of pruritic dermatitis in Singapore were investigated to provide evidence for the presence of histamine. Aqueous extract of the moth hairs was used for the test and analyses. In the isolated guinea pig ileum, the extract, like histamine, produced responses that were blocked by mepyramine. The high performance liquid chromatographic (HPLC) analysis of the fluorescamine derivative of the moth extract resulted in a fluorescent peak with exactly the same retention. time as the histamine fluorescamine derivative. The mass spectrum of the pentafluoropropionic acid (PFP) derivative of the extract was also identical to that obtained from the reference histamine-PFP derivative. We concluded that histamine was present in the tussock moth hairs and was involved as one of the inflammatory mediators responsible for the pruritic skin rashes.

Animals↗

Roles of alpha-farnesene in the behaviors of codling moth females.

Reproduction and olfactory behavioral responses of codling moth, Cydia pomonella (L.), females to synthetic alpha-farnesene were observed in the laboratory as well as their reproduction behaviors in an apple orchard. Calling levels were lifted and ovipositional peaks were advanced in codling moth females at presence of 1 microg and 0.1 microg of alpha-farnesene, respectively. Mated females of codling moth more actively responded to 0.01 microg alpha-farnesene with walking and wing-fanning while walking than to other doses (0.001, 0.1, 1, 10 microg) and control. The results show that alpha-farnesene plays important roles in the behaviors of codling moth females. However, the differences between responses to alpha-farnesene and those to apple volatiles by codling moth females indicate that components other than alpha-farnesene in apple volatiles also have biological activities.

Animal Communication↗

Ionizing irradiation quarantine treatment against oriental fruit moth (Lepidoptera: Tortricidae) in ambient and hypoxic atmospheres.

Oriental fruit moth, Grapholita molesta (Busck), is a pest of many rosaceous temperate fruits, including pomes, Malus spp., and stone fruits, Prunus spp., in much of the world. However, some areas are free of the pest, and shipments of fruit hosts from infested to noninfested areas may be regulated. Current quarantine treatments for oriental fruit moth include methyl bromide fumigation and cold storage for several weeks. Methyl bromide use is being restricted because it is a stratospheric ozone-depleting substance, and alternatives are sought. Cold is not tolerated by many hosts of oriental fruit moth. The objective of this research was to develop irradiation quarantine treatments against the pest under ambient and hypoxic storage conditions because some hosts of oriental fruit moth are stored in hypoxic atmospheres, and hypoxia is known to lessen the effects of irradiation. In ambient atmospheres, no adults emerged from 58,779 fifth instars (the most radiotolerant stage present in fruit) irradiated with a target dose of 200 Gy (195-232 Gy measured). In atmospheres flushed with nitrogen, 5.3% of adults emerged from 44,050 fifth instars irradiated with a target dose of 200 Gy (194-230 Gy measured), but they died at a faster rate than control adults and without laying eggs. A dose of 232 Gy (the maximum recorded when 200 Gy was targeted) is recommended to disinfest any fruit of oriental fruit moth under ambient and hypoxic atmospheres.

Animals↗

Evaluation of potato tuber moth (Lepidoptera: Gelechiidae) resistance in tubers of Bt-cry5 transgenic potato lines.

The potato tuber moth, Phthorimaea operculella (Zeller), in tropical and subtropical countries, is the most destructive pest of potato, Solanum tuberosum L. The larvae attack foliage and tubers in the field and in storage. The purpose of this study was to evaluate the efficacy of a Bt-cry5 transgene to control the potato tuber moth in tuber tissues. Tuber bioassays using stored (11-12 mo old) and newly harvested tubers of Bt-cry5-Lemhi Russet and Bt-cry5-Atlantic potato lines showed up to 100% mortality of 1st instars. Mortality was lowest in the newly harvested tubers of Bt-cry5-Atlantic lines (47.1-67.6%). Potato tuber moth mortality was 100% in the Bt-cry5-Spunta lines that were transformed with Bt-cry5 gene controlled by the CaMV 35S promoter (pBIML5 vector) and in 2 of 3 lines transformed with Bt-cry5 gene controlled by the Gelvin super promoter (pBIML1 vector). The transgenic Spunta lines expressing Bt-cry5 controlled by the patatin promoter (pBMIL2 vector) showed the lowest tuber moth mortality (25.6 and 31.1%). The Bt-cry5 transgenic lines with high tuber expression of B. thuringiensis have value in an integrated pest management system to control potato tuber moth.

Animals↗

Comparison of pheromone-mediated mating disruption and conventional insecticides for management of tufted apple bud moth (Lepidoptera: Tortricidae).

Large-plot studies were used to compare pheromone-mediated mating disruption and conventional insecticide applications for management of tufted apple bud moth, Platynota idaeusalis (Walker), in North Carolina in 1993 and 1994. Pheromone trap catches were reduced in mating disruption blocks, and traps placed in the lower stratum of the canopy had a higher level of trap capture reduction compared with traps placed in the upper stratum. First-generation tufted apple bud moth exposure to either pheromones for mating disruption or insecticides affected second generation pheromone trap catches in the lower and upper canopy. More second generation male moths were caught in pheromone traps placed in the upper compared with the lower canopy in blocks treated with pheromones for mating disruption during the first generation, whereas the opposite was true in blocks treated with insecticides during the first generation. Despite reduced trap catches in pheromone-treated blocks, egg mass densities were not reduced in these blocks compared with insecticide-treated blocks. Furthermore, fruit damage was not significantly different between mating disruption blocks and conventionally treated blocks in orchards with relatively low populations of tufted apple bud moth, but damage was greater in mating disruption blocks in orchards with higher moth densities.

Animals↗

Evaluation of border sprays for managing the codling moth (Tortricidae: Lepidoptera) and the apple maggot (Tephritidae: Diptera) in Ontario apple orchards.

The efficacy of two insecticide control programs for managing the codling moth, Cydia pomonella (L.), and the apple maggot, Rhagoletis pomonella (Walsh), were compared in the Georgian Bay, London, Niagara, and Quinte apple production areas of Ontario during 1995, 1996, and 1997. In the border spray program, an initial cover spray of organophosphorus insecticide was applied to eradicate codling moths that may have colonized a test plot during the previous growing season. Subsequent sprays were applied only to a four-tree-wide zone (approximately 20 wide) around the perimeter of the plot to control immigrating codling moths or apple maggots. In the cover spray program, all sprays of organophosphorus insecticide were applied to the entire plot. Apple maggot injury was significantly greater in border spray program plots than in cover spray program plots only during 1995 in the London production area. There was no significant difference in codling moth injury between border spray and cover spray plots in the four production areas during the three-year study. The elimination of cover sprays from border spray plots during July and August may have left the apple crop more susceptible to damage by second generation larvae of the obliquebanded leafroller, Choristoneura rosaceana (Harris), in the London production area during 1995. There was a trend of increasing codling moth injury from 1995 to 1997 in two border spray plots, and apple maggot injury was detected in these plots during the third year of the study.

Animals↗