Molecular modeling of Bt Cry1Ac (DI–DII)–ASAL (Allium sativum lectin)–fusion protein and its interaction with aminopeptidase N (APN) receptor of Manduca sexta

Genetic engineering of Bacillus thuringiensis (Bt) Cry proteins has resulted in the synthesis of various novel toxin proteins with enhanced insecticidal activity and specificity towards different insect pests. In this study, a fusion protein consisting of the DI–DII domains of Cry1Ac and garlic lectin (ASAL) has been designed in silico by replacing the DIII domain of Cry1Ac with ASAL. The binding interface between the DI–DII domains of Cry1Ac and lectin has been identified using protein–protein docking studies. Free energy of binding calculations and interaction profiles between the Cry1Ac and lectin domains confirmed the stability of fusion protein. A total of 18 hydrogen bonds was observed in the DI–DII–lectin fusion protein compared to 11 hydrogen bonds in the Cry1Ac (DI–DII–DIII) protein. Molecular mechanics/Poisson–Boltzmann (generalized-Born) surface area [MM/PB (GB) SA] methods were used for predicting free energy of interactions of the fusion proteins. Protein–protein docking studies based on the number of hydrogen bonds, hydrophobic interactions, aromatic–aromatic, aromatic–sulphur, cation–pi interactions and binding energy of Cry1Ac/fusion proteins with the aminopeptidase N (APN) of Manduca sexta rationalised the higher binding affinity of the fusion protein with the APN receptor compared to that of the Cry1Ac–APN complex, as predicted by ZDOCK, Rosetta and ClusPro analysis. The molecular binding interface between the fusion protein and the APN receptor is well packed, analogously to that of the Cry1Ac–APN complex. These findings offer scope for the design and development of customized fusion molecules for improved pest management in crop plants.

Are insect frugivores always plant pests? The impact of fruit fly (Diptera: Tephritidae) larvae on host plant fitness

Herbivory is generally regarded as negatively impacting on host plant fitness. Frugivorous insects, which feed directly on plant reproductive tissues, are predicted to be particularly damaging to hosts. We tested this prediction with the fruit fly, Bactrocera tryoni, by recording the impact of larval feeding on two direct (seed number and germination) and two indirect (fruit decay rate and attraction/deterrence of vertebrate frugivores) measures of host plant fitness. Experiments were done in the laboratory, glasshouse and tropical rainforest. We found no negative impact of larval feeding on seed number or germination for three test plants: tomato, capsicum and eggplant. Further, larval feeding accelerated the initiation of decay and increased the final level of fruit decay in tomatoes, apples, pawpaw and pear, a result considered to be beneficial to the fruit. In rainforest studies, native rodents preferred infested apple and pears compared to uninfested control fruit; however, there were no differences observed between treatments for tomato and pawpaw. For our study fruits, these results demonstrate that fruit fly larval infestation has neutral or beneficial impacts on the host plant, an outcome which may be largely influenced by the physical properties of the host. These results may contribute to explaining why fruit flies have not evolved the same level of host specialization generally observed for other herbivore groups.

Taxonomic review of the chironomid genus Cricotopus v.d. Wulp (Diptera: Chironomidae) from Australia : keys to males, females, pupae and larvae, description of ten new species and comments on Paratrichocladius Santos Abreu

The Australian species of the Orthocladiinae genus Cricotopus Wulp (Diptera: Chironomidae) are revised for larval, pupal, adult male and female life stages. Eleven species, ten of which are new, are recognised and keyed, namely Cricotopus acornis Drayson & Cranston sp. nov., Cricotopus albitarsis Hergstrom sp. nov., Cricotopus annuliventris (Skuse), Cricotopus brevicornis Drayson & Cranston sp. nov., Cricotopus conicornis Drayson & Cranston sp. nov., Cricotopus hillmani Drayson & Cranston, sp. nov., Cricotopus howensis Cranston sp. nov., Cricotopus parbicinctus Hergstrom sp. nov., Cricotopus tasmania Drayson & Cranston sp. nov., Cricotopus varicornis Drayson & Cranston sp. nov. and Cricotopus wangi Cranston & Krosch sp. nov. Using data from this study, we consider the wider utility of morphological and molecular diagnostic tools in untangling species diversity in the Chironomidae. Morphological support for distinguishing Cricotopus from Paratrichocladius Santo-Abreu in larval and pupal stages appears lacking for Australian taxa and brief notes are provided concerning this matter.

Host searching behaviour of Diachasmimorpha kraussii (Fullaway) (Hymenoptera: Braconidae: Opiinae), a polyphagous parasitoid of Dacinae fruit flies (Diptera: Tephritidae)

Diachasmimorpha kraussii (Hymenoptera: Braconidae: Opiinae) is a koinobiont larval parasitoid of dacine fruit flies of the genus Bactrocera (Diptera: Tephritidae) in its native range (Australia, Papua New Guinea, Solomon Islands). The wasp is a potentially important control agent for pest fruit flies, having been considered for both classical and inundative biological control releases. I investigated the host searching, selection and utilisation mechanisms of the wasp against native host flies within its native range (Australia). Such studies are rare in opiine research where the majority of studies, because of the applied nature of the research, have been carried out using host flies and environments which are novel to the wasps. Diachasmimorpha kraussii oviposited equally into maggots of four fruit fly species, all of which coexist with the wasp in its native range (Australia), when tested in a choice trial using a uniform artificial diet media. While eggs laid into Bactrocera tryoni and B. jarvisi developed successfully through to adult wasps, eggs laid into B. cucumis and B. cacuminata were encapsulated. These results suggest that direct larval cues are not an important element in host selection by D. kraussii. Further exploring how D. kraussii locates suitable host larvae, I investigated the role of plant cues in host searching and selection. This was examined in a laboratory choice trial using uninfested fruit or fruit infested with either B. tryoni or B. jarvisi maggots. The results showed a consistent preference ranking among infested fruits by the wasp, with guava and peach most preferred, but with no response to uninfested fruits. Thus, it appears the wasp uses chemical cues emitted in response to fruit fly larval infestation for host location, but does not use cues from uninfested fruits. To further tease apart the role of (i) suitable and non-suitable maggots, (ii) infested and uninfested fruits of different plant species, and (iii) adult flies, in wasp host location and selection, I carried out a series of behavioural tests where I manipulated these attributes in a field cage. These trials confirmed that D. kraussii did not respond to cues in uninfested fruits, that there were consistent preferences by the wasps for different maggot infested fruits, that fruit preference did not vary depending on whether the maggots were physiologically suitable or not suitable for wasp offspring development, and finally, that adult flies appear to play a secondary role as indicators of larval infestation. To investigate wasp behaviour in an unrestrained environment, I concurrently observed diurnal foraging behaviours of both the wasp and one of its host fly in a small nectarine orchard. Wasp behaviour, both spatially and temporally, was not correlated with adult fruit fly behaviour or abundance. This study reinforced the point that infested fruit seems to be the primary cue used by foraging wasps. Wasp and fly feeding and mating was not observed in the orchard, implying these activities are occurring elsewhere. It is highly unlikely that these behaviours were happening within the orchard during the night as both insects are diurnal. As the final component of investigating host location, I carried out a habitat preference study for the wasp at the landscape scale. Using infested sentinel fruits, I tested the parasitism rate of B. tryoni in eucalyptus sclerophyll forest, rainforest and suburbia in South East Queensland. Although, rainforest is the likely endemic habitat of both B. tryoni and D. kraussii, B. tryoni abundance is significantly greater in suburban environments followed by eucalyptus sclerophyll forest. Parasitism rate was found to be higher in suburbia than in the eucalyptus sclerophyll forest, while no parasitism was recorded in the rainforest. This result suggests that wasps orient within the landscape towards areas of high host density and are not restricted by habitat types. Results from the different experiments suggest that host searching, selection and utilisation behaviour of D. kraussii are strongly influenced by cues associated with fruit fly larval feeding. Cues from uninfested fruits, the host larvae themselves, and the adult host flies play minimal roles. The discussion focuses on the fit of D. kraussii to Vinson’s classical parasitoid host location model and the implications of results for biological control, including recommendations for host and plant preference screening protocols and release regimes.

Test of the enemy release hypothesis: the native magpie moth prefers a native fireweed (Senecio pinnatifolius) to its introduced congener (S.Madagascariensis)

Abstract The enemy release hypothesis predicts that native herbivores will either prefer or cause more damage to native than introduced plant species. We tested this using preference and performance experiments in the laboratory and surveys of leaf damage caused by the magpie moth Nyctemera amica on a co-occuring native and introduced species of fireweed (Senecio) in eastern Australia. In the laboratory, ovipositing females and feeding larvae preferred the native S. pinnatifolius over the introduced S. madagascariensis. Larvae performed equally well on foliage of S. pinnatifolius and S. madagascariensis: pupal weights did not differ between insects reared on the two species, but growth rates were significantly faster on S. pinnatifolius. In the field, foliage damage was significantly greater on native S. pinnatifolius than introduced S. madagascariensis. These results support the enemy release hypothesis, and suggest that the failure of native consumers to switch to introduced species contributes to their invasive success. Both plant species experienced reduced, rather than increased, levels of herbivory when growing in mixed populations, as opposed to pure stands in the field; thus, there was no evidence that apparent competition occurred.

Converse Bergmann cline in a eucalyptus herbivore, paropsis atomaria olivier (coleoptera: chrysomelidae) : phenotypic plasticity or local adaptation?

Aim To measure latitude-related body size variation in field-collected Paropsis atomaria Olivier (Coleoptera: Chrysomelidae) individuals and to conduct common-garden experiments to determine whether such variation is due to phenotypic plasticity or local adaptation. Location Four collection sites from the east coast of Australia were selected for our present field collections: Canberra (latitude 35°19' S), Bangalow (latitude 28°43' S), Beerburrum (latitude 26°58' S) and Lowmead (latitude 24°29' S). Museum specimens collected over the past 100 years and covering the same geographical area as the present field collections came from one state, one national and one private collection. Methods Body size (pronotum width) was measured for 118 field-collected beetles and 302 specimens from collections. We then reared larvae from the latitudinal extremes (Canberra and Lowmead) to determine whether the size cline was the result of phenotypic plasticity or evolved differences (= local adaptation) between sites. Results Beetles decreased in size with increasing latitude, representing a converse Bergmann cline. A decrease in developmental temperature produced larger adults for both Lowmead (low latitude) and Canberra (high latitude) individuals, and those from Lowmead were larger than those from Canberra when reared under identical conditions. Main conclusions The converse Bergmann cline in P. atomaria is likely to be the result of local adaptation to season length.

Conifer defence against insects: microarray gene expression profiling of Sitka spruce (Picea sitchensis) induced by mechanical wounding or feeding by spruce budworms (Choristoneura occidentalis) or white pine weevils (Pissodes strobi) reveals large

Conifers are resistant to attack from a large number of potential herbivores or pathogens. Previous molecular and biochemical characterization of selected conifer defence systems support a model of multigenic, constitutive and induced defences that act on invading insects via physical, chemical, biochemical or ecological (multitrophic) mechanisms. However, the genomic foundation of the complex defence and resistance mechanisms of conifers is largely unknown. As part of a genomics strategy to characterize inducible defences and possible resistance mechanisms of conifers against insect herbivory, we developed a cDNA microarray building upon a new spruce (Picea spp.) expressed sequence tag resource. This first-generation spruce cDNA microarray contains 9720 cDNA elements representing c. 5500 unique genes. We used this array to monitor gene expression in Sitka spruce (Picea sitchensis) bark in response to herbivory by white pine weevils (Pissodes strobi, Curculionidae) or wounding, and in young shoot tips in response to western spruce budworm (Choristoneura occidentalis, Lepidopterae) feeding. Weevils are stem-boring insects that feed on phloem, while budworms are foliage feeding larvae that consume needles and young shoot tips. Both insect species and wounding treatment caused substantial changes of the host plant transcriptome detected in each case by differential gene expression of several thousand array elements at 1 or 2 d after the onset of treatment. Overall, there was considerable overlap among differentially expressed gene sets from these three stress treatments. Functional classification of the induced transcripts revealed genes with roles in general plant defence, octadecanoid and ethylene signalling, transport, secondary metabolism, and transcriptional regulation. Several genes involved in primary metabolic processes such as photosynthesis were down-regulated upon insect feeding or wounding, fitting with the concept of dynamic resource allocation in plant defence. Refined expression analysis using gene-specific primers and real-time PCR for selected transcripts was in agreement with microarray results for most genes tested. This study provides the first large-scale survey of insect-induced defence transcripts in a gymnosperm and provides a platform for functional investigation of plant-insect interactions in spruce. Induction of spruce genes of octadecanoid and ethylene signalling, terpenoid biosynthesis, and phenolic secondary metabolism are discussed in more detail.

Genetic resources in wild and cultured stocks of the Asian mud crab, Scylla paramamosain

The mud crab (Scylla spp.) aquaculture industry has expanded rapidly in recent years in many countries in the Indo - West Pacific (IWP) region as an alternative to marine shrimp culture because of significant disease outbreaks and associated failures of many shrimp culture industries in the region. Currently, practices used to produce and manage breeding crabs in hatcheries may compromise levels of genetic diversity, ultimately compromising growth rates, disease resistance and stock productivity. Therefore, to avoid “genetic pollution” and its harmful effects and to promote further development of mud crab aquaculture and fisheries in a sustainable way, a greater understanding of the genetic attributes of wild and cultured mud crab stocks is required. Application of these results can provide benefits for managing wild and cultured Asian mud crab populations for multiple purposes including for commercial production, recreation and conservation and to increase profitability and sustainability of newly emerging crab culture industries. Phylogeographic patterns and the genetic structure of Asian mud crab populations across the IWP were assessed to determine if they were concordant with those of other widespread taxa possessing pelagic larvae of relatively long duration. A 597 bp fragment of the mitochondrial DNA COI gene was amplified and screened for variation in a total of 297 individuals of S. paramamosain from six sampling sites across the species’ natural geographical distribution in the IWP and 36 unique haplotypes were identified. Haplotype diversities per site ranged from 0.516 to 0.879. Nucleotide diversity estimates among haplotypes were 0.11% – 0.48%. Maximum divergence observed among S. paramamosain samples was 1.533% and samples formed essentially a single monophyletic group as no obvious clades were related to geographical location of sites. A weak positive relationship was observed however, between genetic distance and geographical distance among sites. Microsatellite markers were then used to assess contemporary gene flow and population structure in Asian mud crab populations sampled across their natural distribution in the IWP. Eight microsatellite loci were screened in sampled S. paramamosain populations and all showed high allelic diversity at all loci in sampled populations. In total, 344 individuals were analysed, and 304 microsatellite alleles were found across the 8 loci. The mean number of alleles per locus at each site ranged from 20.75 to 28.25. Mean allelic richness per site varied from 17.2 to 18.9. All sites showed high levels of heterozygosity as average expected heterozygosities for all loci ranged from 0.917 – 0.953 while mean observed heterozygosity ranged from 0.916 – 0.959. Allele diversities were similar at all sites and across all loci. The results did not show any evidence for major differences in allele frequencies among sites and patterns of allele frequencies were very similar in all populations across all loci. Estimates of population differentiation (FST) were relatively low and most probably largely reflect intra – individual variation for very highly variable loci. Results from nDNA analysis showed evidence for only very limited population genetic structure among sampled S. paramamosain, and a positive and significant association for genetic and geographical distance among sample sites. Microsatellite markers were then employed to determine if adequate levels of genetic diversity has been captured in crab hatcheries for the breeding cycle. The results showed that all microsatellite loci were polymorphic in hatchery samples. Culture populations were in general, highly genetically depauperate, compared with comparable wild populations, with only 3 to 8 alleles recorded for the same loci set per population. In contrast, very high numbers of alleles per locus were found in reference wild S. paramamosain populations, which ranged from 18 to 46 alleles per locus per population. In general, this translates into a 3 to 10 fold decline in mean allelic richness per locus in all culture stocks compared with wild reference counterparts. Furthermore, most loci in all cultured S. paramamosain samples showed departures from HWE equilibrium. Allele frequencies were very different in culture samples from that present in comparable wild reference samples and this in particular, was reflected in a large decline in allele diversity per locus. The pattern observed was best explained by significant impacts of breeding practices employed in hatcheries rather than natural differentiation among wild populations used as the source of brood stock. Recognition of current problems and management strategies for the species both for the medium and long-term development of the new culture industry are discussed. The priority research to be undertaken over the medium term for S. paramamosain should be to close the life cycle fully to allow individuals to be bred on demand and their offspring equalised to control broodstock reproductive contributions. Establishing a broodstock register and pedigree mating system will be required before any selection program is implemented. This will ensure that sufficient genetic variation will be available to allow genetic gains to be sustainably achieved in a future stock improvement program. A fundamental starting point to improve hatchery practices will be to encourage farmers and hatchery managers to spawn more females in their hatcheries as it will increase background genetic diversity in culture stocks. Combining crablet cohorts from multiple hatcheries into a single cohort for supply to farmers or rotation of breeding females regularly in hatcheries will help to address immediate genetic diversity problems in culture stocks. Application of these results can provide benefits for managing wild and cultured Asian mud crab populations more efficiently. Over the long-term, application of data on genetic diversity in wild and cultured stocks of Asian mud crab will contribute to development of sustainable and productive culture industries in Vietnam and other countries in the IWP and can contribute towards conservation of wild genetic resources.

Host use and crop impacts of Oribius Marshall species (Coleoptera: Curculionidae) in Eastern Highlands Province, Papua New Guinea

Oribius species are small flightless weevils endemic to the island of New Guinea and far northern Cape York, Australia. The adults feed externally on leaves, developing fruit and green bark, but their impact as pests and general host use patterns are poorly known. Working in Eastern Highlands Province, Papua New Guinea, we carried out structured host use surveys, farmer surveys, shade-house growth trials, and on-farm and on-station impact trials to: (i) estimate the host range of the local Oribius species; (ii) understand adult daily activity patterns; (iii) elucidate feeding habits of the soil dwelling larvae; and (iv) quantify the impacts of adult feeding damage. Oribius inimicus and O. destructor accounted for nearly all the Oribius species encountered locally: of these two O. inimicus was the most abundant. Weevils were collected from 31 of 33 plants surveyed in the Aiyura Valley and a combination of farmer interviews and literature records provided evidence for the beetles being pestiferous on 43 crops currently or previously grown in the Highlands. Adult weevils had a distinct diurnal pattern of being in the upper plant canopy early in the morning and, to a lesser extent, again late in the afternoon. For the remainder of the day beetles resided within the canopy, or possibly off the plant. Movement of adults between plants appeared frequent. Pot trials confirmed the larvae are root feeders. Quantified impact studies showed that the weevils are damaging to a range of vegetable and orchard crops (broccoli, capsicum, celery, French bean, Irish potato, lettuce, orange and strawberry), causing average yield losses of around 30-40%, but up to 100% on citrus. Oribius weevils pose a significant and apparently growing problem for Highland’s agriculture.

Is a mango just a mango? Testing within-fruit oviposition site choice and larval performance of a highly polyphagous fruit fly

For fruit flies, fully ripe fruit is preferred for adult oviposition and is superior for offspring performance over unripe or ripening fruit. Because not all parts of a single fruit ripen simultaneously, the opportunity exists for adult fruit flies to selectively choose riper parts of a fruit for oviposition and such selection, if it occurs, could positively influence offspring performance. Such fine scale host variation is rarely considered in fruit fly ecology, however, especially for polyphagous species which are, by definition, considered to be generalist host users. Here we study the adult oviposition preference/larval performance relationship of the Oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), a highly polyphagous pest species, at the “within-fruit” level to see if such a host use pattern occurs. We recorded the number of oviposition attempts that female flies made into three fruit portions (top, middle and bottom), and larval behavior and development within different fruit portions for ripening (color change) and fully-ripe mango, Mangifera indica L. (Anacardiaceae). Results indicate that female B. dorsalis do not oviposit uniformly across a mango fruit, but lay most often in the top (i.e., stalk end) of fruit and least in the bottom portion, regardless of ripening stage. There was no evidence of larval feeding site preference or performance (development time, pupal weight, percent pupation) being influenced by fruit portion, within or across the fruit ripening stages. There was, however, a very significant effect on adult emergence rate from pupae, with adult emergence rate from pupae from the bottom of ripening mango being approximately only 50% of the adult emergence rate from the top of ripening fruit, or from both the top and bottom of fully-ripe fruit. Differences in mechanical (firmness) and chemical (total soluble solids, titratable acidity, total non-structural carbohydrates) traits between different fruit portions were correlated with adult fruit utilisation. Our results support a positive adult preference/offspring performance relationship at within-fruit level for B. dorsalis. The fine level of host discrimination exhibited by B. dorsalis is at odds with the general perception that, as a polyphagous herbivore, the fly should show very little discrimination in its host use behavior.

Elimination of dengue by community programs using Mesocyclops(Copepoda) against Aedes aegypti in central Vietnam

From September 2000 to June 2003, a community-based program for dengue control using local predacious copepods of the genus Mesocyclops was conducted in three rural communes in the central Vietnam provinces of Quang Nam, Quang Ngai, and Khanh Hoa. Post-project, three subsequent entomologic surveys were conducted until March 2004. The number of households and residents in the communes were 5,913 and 27,167, respectively, and dengue notification rates for these communes from 1996 were as high as 2,418.5 per 100,000 persons. Following knowledge, attitude, and practice evaluations, surveys of water storage containers indicated that Mesocyclops spp. already occurred in 3-17% and that large tanks up to 2,000 liters, 130-300-liter jars, wells, and some 220-liter metal drums were the most productive habitats for Aedes aegypti. With technical support, the programs were driven by communal management committees, health collaborators, schoolteachers, and pupils. From quantitative estimates of the standing crop of third and fourth instars from 100 households, Ae. aegypti were reduced by approximately 90% by year 1, 92.3-98.6% by year 2, and Ae. aegypti immature forms had been eliminated from two of three communes by June 2003. Similarly, from resting adult collections from 100 households, densities were reduced to 0-1 per commune. By March 2004, two communes with no larvae had small numbers but the third was negative; one adult was collected in each of two communes while one became negative. Absolute estimates of third and fourth instars at the three intervention communes and one left untreated had significant correlations (P = 0.009-< 0.001) with numbers of adults aspirated from inside houses on each of 15 survey periods. By year 1, the incidence of dengue disease in the treated communes was reduced by 76.7% compared with non-intervention communes within the same districts, and no dengue was evident in 2002 and 2003, compared with 112.8 and 14.4 cases per 100,000 at district level. Since we had similar success in northern Vietnam from 1998 to 2000, this study demonstrates that this control model is broadly acceptable and achievable at community level but vigilance is required post-project to prevent reinfestation.