Characterization of two Bacillus thuringiensis ssp. morrisoni strains isolated from Thaumetopoea pityocampa (Lep., Thaumetopoeidae)

The pine processionary moth Thaumetopoea pityocampa Den. and Schiff. (Lep., Thaumetopoeidae) is one of the most harmful insect pests for pine species in Mediterranean countries including Turkey. Two Bacillus thuringiensis isolates obtained from T. pityocampa were identified and characterized in terms of crystal shape using electron microscopy, SDS–PAGE analysis, cry gene contents, H-serotype and insecticidal activity. Examination by a scanning electron microscope showed that Tp6 and Tp14 isolates have flat square and bipyramidal crystal shapes, respectively. PCR analysis showed that Tp6 contains cry3 gene and Tp14 isolate contains cry1 and cry2 genes. On the other hand, the presence of Cry3 and Cry1 proteins were confirmed by observation of approximately 65- and 130-kDa proteins by SDS–PAGE in Tp6 and Tp14 isolates, respectively. According to H-serotype results, these isolates were identified as Bacillus thuringiensis ssp. morrisoni (H8a8b). Toxicity tests were performed against six insect species belonging to Lepidoptera and Coleoptera. The highest insecticidal activity was 100% for Tp6 isolate on larvae of Agelastica alni and Leptinotarsa decemlineata and 100% for Tp14 isolate on larvae of Malacosoma neustria. Our results indicate that isolates Tp6 and Tp14 may be valuable biological control agents for various coleopteran and lepidopteran pests.     

Pine processionary moth (Thaumetopoea pityocampa,Lepidoptera: Thaumetopoeidae) larvae are highly susceptible to the entomopathogenic fungi Metarhizium brunneum and Beauveria bassiana

The larvae of the pine processionary moth (PPM), Thaumetopoea pityocampa, feed on the needles of pine and cedar. The urticating hairs of older instars pose a threat to human and animal health. Strains of the entomopathogenic fungi, Metarhizium brunneum (V275, ARSEF 4556) and Beauveria bassiana (KTU-24), were assayed against first to fourth instar T. pityocampa using doses ranging from 1?×?10⁵ to 1?×?10⁸ conidia mL^?1. The three strains differed slightly in their virulence but caused 100% mortality of all instars at the highest dose. The newly emerged or first instar larvae were extremely susceptible with 100% mortality being achieved 2–4 days post inoculation with V275 at all but the lowest dose. The fourth instar larvae appeared to be less susceptible than earlier instars. There was good horizontal transmission of conidia from treated to un-inoculated larvae. However, mortality was higher in third and fourth instars and where the ratio of inoculated versus untreated larvae was high. This we presume is due to spores being more readily trapped by the urticating hairs found on third and older instar larvae. Injection of the nests offers a simple and environmentally friendly way of controlling the pest with reduced risk to operators.     

Characterization of five microsatellite loci in the Pine Processionary Moth Thaumetopoea pityocampa (Lepidoptera Notodontidae Thaumetopoeinae)

Five microsatellite markers were developed for the lepidopteran species Thaumetopoea pityocampa using an enrichment protocol. All loci could be amplified with no evidence of null alleles and will be useful for population genetic studies. The number of alleles ranged from three to 12 for a population of 30 individuals. Observed heterozygosities ranged from 0.53 to 0.80. No significant heterozygote deficiency was detected. Four markers might be of interest for Th. wilkinsoni.     

Incipient allochronic speciation in the pine processionary moth (Thaumetopoea pityocampa, Lepidoptera, Notodontidae)

A plausible case of allochronic differentiation, where barrier to gene flow is primarily attributable to a phenological shift, was recently discovered in Portugal for the pine processionary moth Thaumetopoea pityocampa. Previous results suggested that the observed 'summer population' (SP) originated from the sympatric winter population (WP). Our objectives were to finely analyse these patterns and test their stability in time, through field monitoring and genetic analyses of larvae and adults across different years. Reproductive activity never overlapped between SP and WP. Microsatellites showed a clear differentiation of the SP, consistent with a strong reduction in gene flow owing to the phenological shift. Assignment tests suggested that some individuals shift from the SP to the WP phenology, causing some hybridization. We discuss these patterns and their maintenance over time. This could be a first stage of allochronic speciation, and SP should be considered as a distinct phenological race.     

The role of topography in structuring the demographic history of the pine processionary moth,Thaumetopoea pityocampa (Lepidoptera: Notodontidae)

Aim We investigated the Quaternary history of the pine processionary moth, Thaumetopoea pityocampa, an oligophagous insect currently expanding its range. We tested the potential role played by mountain ranges during the post‐glacial recolonization of western Europe. Location Western Europe, with a focus on the Pyrenees, Massif Central and western Alps. Methods Maternal genetic structure was investigated using a fragment of the mitochondrial cytochrome c oxidase subunit I (COI) gene. We analysed 412 individuals from 61 locations and performed maximum likelihood and maximum parsimony phylogenetic analyses and hierarchical analysis of molecular variance, and we investigated signs of past expansion. Results A strong phylogeographic pattern was found, with two deeply divergent clades. Surprisingly, these clades were not separated by the Pyrenees but rather were distributed from western to central Iberia and from eastern Iberia to the Italian Peninsula, respectively. This latter group consisted of three shallowly divergent lineages that exhibited strong geographic structure and independent population expansions. The three identified lineages occurred: (1) on both sides of the Pyrenean range, with more genetically diverse populations in the east, (2) from eastern Iberia to western France, with a higher genetic diversity in the south, and (3) from the western Massif Central to Italy. Admixture areas were found at the foot of the Pyrenees and Massif Central. Main conclusions The identified genetic lineages were geographically structured, but surprisingly the unsuitable high‐elevation areas of the main mountainous ranges were not responsible for the spatial separation of genetic groups. Rather than acting as barriers to dispersal, mountains appear to have served as refugia during the Pleistocene glaciations, and current distributions largely reflect expansion from these bottlenecked refugial populations. The western and central Iberian clade did not contribute to the northward post‐glacial recolonization of Europe, yet its northern limit does not correspond to the Pyrenees. The different contributions of the identified refugia to post‐glacial expansion might be explained by differences in host plant species richness. For example, the Pyrenean lineage could have been trapped elevationally by tracking montane pines, while the eastern Iberian lineage could have expanded latitudinally by tracking thermophilic lowland pine species.     

Study of the δ-endotoxins produced by three recently isolated strains of Bacillus thuringiensis

Abstract Three different strains of Bacillus thuringiensis , subsp. toumanoffi, sotto and kurstaki , producing parasporal inclusion crystals, have recently been isolated in Tunisia. The δ-endotoxins produced by the different strains gave distinct patterns on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Polymerase chain reaction screening of these three strains, using oligonucleotides specific for the genes cryIA, cryIII and cryIV , did not generate amplified fragment profiles characteristic of these genes. For each of the strains, the presence of one or more δ-endotoxin coding genes having partial sequence similarities to one or more genes of these three groups was found.     

The relationship between pheromone trap catch and local population density of the oak processionary moth Thaumetopoea processionea (Lepidoptera: Thaumetopoeidae)

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Natural biological control of the pine processionary moth Thaumetopoea pityocampa (Den. & Schiff.) by the Argentine ant Linepithema humile (Mayr) in Portugal

1 Defoliation by larvae of the pine processionary moth, Thaumetopoea pityocampa, is negligible in stands of Pinus pinaster colonized by the Argentine ant Linepithema humile which preys fiercely on the young moth larvae. In contrast, such damage is widespread where pine plantations are colonized by native ants, predominantly Tapinoma nigerrimum and Lasius niger, which seemingly disregard the larvae. 2 Where L. humile- and native ant-occupied sectors adjoin, there is a 20–50 m overlap in the transition area between L. humile- and native ant-occupied pines. This was most evident in a > 500 ha plantation where there was severe or very severe T. pityocampa attack in native ant sectors contrasting with none in adjoining L. humile sectors. 3 Predation by L. humile is no doubt enhanced by its existence as super-colonies over very large areas, by its foraging activity and recruitment on trees throughout the time when T. pityocampa and other prey are present, and by honeydew- producing Homoptera which help retain foraging L. humile workers in pine tree crowns. 4 The role of L. humile could be enhanced by cultivations that disturb the soil and restrain ground vegetation.     

Thaumetopoein, an urticating protein of the processionary hairs of the caterpillar (Thaumetopoea pityocampa Schiff) (Lepidoptera, Thaumetopoeidae)

The irritating fraction extracted from processionary caterpillar hairs contains soluble proteins which were separated by various electrophoretic and immuno-electrophoretic techniques. Some of these proteins are present also in cuticle and haemolymph. One protein of 28,000 daltons, formed of two subunits (13,000 and 15,000 daltons) is hair specific and causes a reaction in pig skin identical to that produced by hair extract. It is therefore an urticating protein and which we have named "Thaumetopoein".     

Efficacy and molecular studies of a Lebanese isolate of Beauveria for control of Thaumetopoea wilkinsoni (Lepidoptera: Thaumetopoeidae)

A Lebanese isolate of a Beauveria species originally isolated from a hymenopterous insect pest was found to be very effective against the pine processionary moth Thaumetopoea wilkinsoni. In laboratory bioassays, high mortality rates of the five larval stages were attained with spore concentrations of about 5000–50,000 spores/larva. The efficacy of kill on the first three larval stages was similar to the growth regulator diflubenzuron, but it was significantly higher on the fourth and fifth larval stages. Sequences of the DNA lyase gene and the EF-1α gene were used for molecular characterisation of this Beauveria isolate. The DNA lyase gene showed more polymorphism than the previously reported ITS region and EF-1α gene. This constitutes the first report on the possibility of using the DNA lyase gene as a molecular tool in fungal taxonomy.     

Effects of Bacillus thuringiensis kurstaki on Malpighian tubule cells of Thaumetopoea pityocampa (Lepidoptera: Thaumetopoeidae) larvae

In this study effects of Bacillus thuringiensis kurstaki (Btk) on Malpighian tubule cells of Thaumetopoea pityocampa (Lepidoptera: Thaumetopoeidae) larvae was investigated by electron microscopy. 3 mg/l Btk was given with food. After Btk administration, the Malpighian tubule cells were investigated and compared with a control group. 3 and 6 hrs after Btk administration swelling in Malpighian tubule cells was observed. Swelling of mitochondria and separation of their cristae was seen after 12 hrs. After 24 hrs dissolution of the basal cytoplasm, swelling and vacuolization of all mitochondria, partial dissolution of the nucleoplasm, and swelling and separation ofmicrovilli was documented. A membrane-body in the nucleus was seen after 48 hrs. The nucleoplasm was completely dissolved after 72 hrs and after 96 hrs large vacuoles appeared in the cytoplasm and shortening of microvilli was observed.     

Prepupal diapause synchronizes adult emergence in the pine processionary moth Thaumetopoea pityocampa (Lepidoptera: Notodontidae)

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Efficacy of entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae against Thaumetopoea pityocampa Shiff. (Lepidoptera: Thaumatopoeidae)

Abstract

In this study, Metarhizium anisopliae TR 106 and Beauveria bassiana TR 217 was tested against fourth instar larvae of Thaumetopoea pityocampa. The LT₅₀ and LT₉₀ of 1?×?10⁶ concentration of M. anisopliae against T. pityocampa were 3.60 and 4.11 for direct application, while these were 2.87 and 3.60?days, respectively in leaves application. The LT₅₀ and LT₉₀ of the 1?×?10⁸ concentration of the same isolate were 2.50 and 2.95?days for direct application, and 2.98 and 3.74?days for leaves application. The LT₅₀ of insect and leaves application for 1?×?10⁶ of B. bassiana were 3.75 and 3.49?days, respectively. The LT₉₀ of same concentration for insect application was 4.48?days, while LT₉₀ for leaves application was 4.63?days. Similarly, LT₅₀ of insect and leaves application for 1?×?10⁸ of B. bassiana were 3.03 and 3.31?days, while LT₉₀ were 3.68 and 4.29?days, respectively. Approximate 100% mycosis was observed in all treatments.     

Isolation and identification of bacteria from <Emphasis Type="Italic">Thaumetopoea pityocampa</Emphasis> Den. and Schiff. (Lep., Thaumetopoeidae) and determination of their biocontrol potential

The pine processionary moth Thaumetopoea pityocampa (Den. and Schiff.) is one of the most harmful insect pest for pine species in Mediterranean countries including Turkey. The objective of the present study is to find a more effective and safe biological control agent against T. pityocampa. Thus, we investigated the bacterial flora of the pest insect, collected from the Middle Black Sea Region of Turkey from 2003 to 2004. Based on morphological, physiological, biochemical and molecular methods, 14 different bacterial isolates were determined. The identified bacterial flora of T. pityocampa consisted of bacteria belonging to the Enterobacteriaceae (Tp1), Arthrobacter sp. (Tp2), Staphylococcus spp. (Tp3 and 10), Bacillus subtilis (Tp4), Serratia liquefaciens (Tp5), Bacillus thuringiensis subsp. morrisoni (Tp6 and 14), an acrystalliferous form Bacillus thuringiensis (Tp7), Staphylococcus cohnii (Tp8), Bacillus licheniformis (Tp9), Bacillus pumilus (Tp11), Brevibacterium sp. (Tp12) and Bacillus simplex (Tp13). After analysing the conclusions of conventional and molecular tests Tp1 (Enterobacteriaceae), Tp2 (Arthrobacter sp.) and Tp12 (Brevibacterium sp.) were assigned as novel bacterial species. Isolate Tp5 had a promising insecticidal effect on third instar larvae of T. pityocampa (up to 70% mortality within 10 days).     

Daily cycles for emission of urticating hairs from the pine processionary caterpillar (Thaumetopoea pityocampa S.) and the brown tail moth (Euproctis chrysorrhoea L) (Lepidoptera) in laboratory conditions

Summary In laboratory conditions, urticating hairs from the pine processionary caterpillar (Thaumetopoea pityocampa S.) and from the brown tail moth (Euproctis chrysorrhoea L.) are detectable in the air using an apparatus designed for the capture of airborne microorganisms and pollen research studies. The hairs produced by the caterpillars of these two species are distributed either via air currents or moths (only forEuproctis). Daily cycles of hair emission were observed and were in relation with locomotion and feeding activities of the caterpillars and with flying and reproductive activities of moths.     

Characterization and cross-amplification of 13 microsatellite loci in the northern pine processionary moth, Thaumetopoea pinivora (Lepidoptera: Notodontidae)

Thirteen polymorphic microsatellite loci were developed for the northern pine processionary moth (Thaumetopoea pinivora) and tested for cross-amplification in seven other species within the Thaumetopoea family. Number of alleles ranged from two to 10 when at least 28 individuals from one population were screened and one locus, Thapin06, appears to be sex linked. Expected heterozygosity ranged from 0.094 to 0.856 and observed heterozygosity ranged from 0.097 to 0.806. Amplification success varied between sister species, with two up to seven loci being successfully amplified. The described loci will be valuable for studying the population genetic structure and dispersal behaviour of this forest pest.     

Binding of Cry δ-endotoxins to brush border membrane vesicles of Helicoverpa armigera and Helicoverpa punctigera （Lepidoptera： Noctuidae）

The relatively low susceptibility ofHelicoverpa armigera to CrylAc, its history of resistance to chemical insecticides and the seasonal decline in expression of CrylAc in transgenic cotton necessitated the development of cotton expressing two insecticidal proteins to provide sustainable control of this multinational pest. To manage the resistance issue, it was essential that the second insecticidal protein have a significantly different mode of action to CrylAc. A common feature of resistance to CrylA proteins in several species as well as H. armigera has been a change in the binding site. A study of binding sites for some Cry proteins in the brush border membrane vesicles （BBMV） ofH. armigera and Helicoverpa punctigera was undertaken. The binding affinity for CrylAc was higher than for CrylAb, matching their relative toxicities, and CrylAc and CrylAb were found to share at least one binding site in both I-1. armigera and I-1. punctigera. However Cry2Aa did not compete with CrylAc for binding and so could be used in transgenic cotton in combination with CrylAc to control H. armigera and manage resistance. Variation in the susceptibilities of three different H. armigera strains to CrylAc correlated with the parameter Bmax/Kcom.     

Atmospheric pollution of animal origin: the urticating hairs of the processionary caterpillar (Thaumetopoea pityocampa Schiff.) (Insects, Lepidoptera)

Urticating hairs of pine processionary caterpillars are detectable in air using techniques designed for airborne microorganisms and pollen's research. As with pollens, abondance of hairs is correlated with distance of production zone and with local meterological conditions. Collected in Bordeaux, urticating hairs will be considered for allergists as pollens and other allergic particles.     

Interaction of larval age and dietary formaldehyde on the susceptibility of tufted apple budmoth (Lepidoptera: Tortricidae)to Bacillus thuringiensis

Laboratory tests of Bacillus thuringiensis var. kurstaki (strain HD-1) against Platynota idaeusalis reared on a semisynthetic diet showed that the LC₅₀ values for neonates and 9-day-old larvae showed no significant difference in susceptibility; however, 14-day-old larvae were significantly more susceptible than neonates or 9-day-old larvae. There was no evidence of any interaction between 14-day-old larvae reared on the semisynthetic diets with and without formaldehyde and B. thuringiensis toxicity at any assessment reading. Likewise the toxicity of B. thuringiensis to 14-day-old larvae reared on apple leaves did not differ from the B. thuringiensis toxicity of 14-day-old larvae reared on semisynthetic diets with and without formaldehyde.     

Evidence for low‐level hybridization between two allochronic populations of the pine processionary moth,Thaumetopoea pityocampa (Lepidoptera: Notodontidae)

Divergence between populations sharing the same habitat can be initiated by different reproductive times, leading to allochronic differentiation. A spatially localized allochronic summer population (SP) of the pine processionary moth Thaumetopoea pityocampa, recently discovered in Portugal, occurs in sympatry with the local winter population (WP). We examined the level of genetic differentiation between the two populations and estimated the current gene flow within the spatial framework of their co‐occurrence. Mitochondrial data indicated that the two sympatric populations were genetically closer than other WP populations. Conversely, microsatellite genotyping uncovered greater differentiation between the two sympatric populations than between allopatric ones. While male trapping confirmed that reproduction of SP and WP occurred at distinct times, clustering approaches demonstrated the presence of a few LateSP individuals emerging within the WP flight period, although genetically identified as SP. We also identified rare recent hybridization events apparently occurring mainly in the margins of the current SP range. The ongoing gene flow detected between the ancestral and the emerging allochronic populations revealed an incomplete reproductive isolation, which must therefore be taken into account and integrated with studies focussed on ecological drivers, so that a complete understanding of the ongoing speciation process might be achieved.