Effect of Bt broccoli and resistant genotype of <Emphasis Type="Italic">Plutella xylostella</Emphasis> (Lepidoptera: Plutellidae) on development and host acceptance of the parasitoid <Emphasis Type="Italic">Diadegma insulare</Emphasis> (Hymenoptera: Ichneumonidae)

The ecological implications on biological control of insecticidal transgenic plants, which produce crystal (Cry) proteins from the soil bacterium Bacillus thuringiensis (Bt), remain a contentious issue and affect risk assessment decisions. In this study, we used a unique system of resistant insects, Bt plants and a parasitoid to critically evaluate this issue. The effects of broccoli type (normal or expressing Cry1Ac protein) and insect genotype (susceptible or Cry1Ac-resistant) of Plutella xylostella L. (Lepidoptera: Plutellidae) were examined for their effects on the development and host foraging behavior of the parasitoid, Diadegma insulare (Cresson) (Hymenoptera: Ichneumonidae) over two generations. Parasitism rate and development of D. insulare were not significantly different when different genotypes (Bt-resistant or susceptible) of insect host larvae fed on non-Bt broccoli plants. D. insulare could not discriminate between resistant and susceptible genotypes of P. xylostella, nor between Bt and normal broccoli plants with different genotypes of P. xylostella feeding on them. No D. insulare could emerge from Bt broccoli-fed susceptible and heterozygous P. xylostella larvae because these larvae were unable to survive on Bt broccoli. The parasitism rate, developmental period, pupal and adult weights of D. insulare that had developed on Bt broccoli-fed Cry1Ac-resistant P. xylostella larvae were not significantly different from those that developed on non-Bt broccoli-fed larvae. Female D. insulare emerged from Cry1Ac-resistant P. xylostella that fed on Bt plants could successfully parasitize P. xylostella larvae. The life parameters of the subsequent generation of D. insulare from P. xylostella reared on Bt broccoli were not significantly different from those from non-Bt broccoli. The Cry1Ac protein was detected in P. xylostella and in D. insulare when hosts fed on Bt broccoli. These results are the first to indicate that Cry1Ac did not harm the development or host acceptance of an important endoparasitoid after two generations of exposure. We suggest that using other Bt crops and resistant insect species would likely lead to similar conclusions about the safety of the presently used Bt proteins on parasitoids.     

Parasitism and developmental parameters of the parasitoid <Emphasis Type="Italic">Diadegma</Emphasis><Emphasis Type="Italic">majale</Emphasis> (Gravenhorst) in control of <Emphasis Type="Italic">Plutella xylostella</Emphasis> (L.) on selected cultivars of canola

The diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), is one of the most economically significant pests of canola, Brassica napus L., in Ardabil region, Iran. Use of host plant resistance integrated with biocontrol agents such as Diadegma majale (Gravenhorst) (Hymenoptera: Ichneumonidae) is an essential component of integrated management of P. xylostella. In this study, we investigated the parasitism by D. majale on six selected cultivars of canola under field conditions and preference and performance of the parasitoid on P. xylostella larvae under laboratory conditions. In field experiments, the highest larval density of P. xylostella was observed on Zarfam during 2008 and 2009. Larval densities were not significantly different among Opera, Hyola 401, Okapi, and Option 500 and Elite in 2008, but the lowest larval density was observed on Opera in 2009. No significant differences were observed among the rate of parasitized larvae on tested cultivars in 2008, while in 2009 the parasitism rate was significantly higher on Opera than on Zarfam. In free-choice situations, the percentage of parasitized larvae was significantly highest on Opera (88.7%) and lowest on Zarfam (62.95%). Developmental time from egg to adult, body mass, length of forewings and hindwings, length of hind femur and hind tibia of D. majale females reared on larvae of P. xylostella fed on Opera did not differ from other cultivars. Our results suggest that cultivation of Opera integrated with D. majale could provide effective and sustainable management of P. xylostella in the region.     

<Emphasis Type="Italic">Cotesia plutellae</Emphasis> bracovirus suppresses expression of an antimicrobial peptide,cecropin, in the diamondback Moth, <Emphasis Type="Italic">Plutella xylostella</Emphasis>, challenged by bacteria

An endoparasitoid wasp, Cotesia plutellae, induces significant immunosuppression of host insect, Plutella xylostella. This study was focused on suppression in humoral immune response of P. xylostella parasitized by C. plutellae. An EST database of P. xylostella provided a putative cecropin gene (PxCec) which is 627 bp long and encodes 66 amino acids. A signal peptide (22 amino acids) is predicted and two putative O-glycosylation sites in threonine are located at positions 58 and 64. Without bacterial infection, PxCec was expressed in pupa and adult stages but not in the egg and larval stages. Upon bacterial challenge, however, the larvae expressed PxCec as early as 3 h post infection (PI) and maintained high expression levels at 12–24 h PI. By 48 h PI, its expression noticeably diminished. All tested tissues of bacteria-infected P. xylostella showed PxCec expression. However, other microbes, such as virus and fungus, did not induce the PxCec expression. Parasitization by C. plutellae suppressed the expression of PxCec in response to bacterial challenge. Among the parasitic factors of C. plutellae, its symbiotic virus (C. plutellae bracovirus: CpBV) alone was able to inhibit the expression of PxCec of P. xylostella challenged by bacteria. These results indicate that PxCec expression is regulated by both immune and developmental processes in P. xylostella. The parasitization by C. plutellae inhibited the expression of PxCec by the wasp’s symbiotic virus.     

Do laboratory studies predict parasitoid interaction outcomes in the field?

Studying competitive interactions among natural enemies is important to elucidate the success and non-target impact of candidate biological control agents. Increased regulation of new introductions requires that studies on non-target species be carried out in confined conditions. Hypotheses about potential impacts of biological control agents in the field are based on data from Petri dish or small cage experiments conducted in the laboratory. This study compared the performance of the diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), parasitoids Diadegma insulare (Cresson) (Hymenoptera: Ichneumonidae) and Microplitis plutellae (Muesebeck) (Hymenoptera: Braconidae) in experiments conducted in small cages in the laboratory and in large cages in the field. Results showed no significant differences between laboratory and field outcomes for D. insulare alone and when D. insulare and M. plutellae were combined. For M. plutellae alone, parasitism in the laboratory cages was significantly less than in the field cages. These results demonstrate that laboratory studies may be useful to develop hypotheses on competitive interactions of candidate parasitoid biological control agents.     

Host plant nutritional quality affects the performance of the parasitoid Diadegma insulare

The well documented biochemical profile of Brassicaceae, oligophagy of the herbivore Plutella xylostella (L.) (Lepidoptera: Plutellidae), and host specialization of the parasitoid Diadegma insulare (Cresson) (Hymenoptera: Ichneumonidae) provide an ideal system for investigating tritrophic interactions mediated by nutritional quality of plants. We evaluated the bottom-up effects of five soil fertility regimes on nutritional quality of canola (Brassica napus L.) and then on several fitness correlates of female and male D. insulare as mediated through P. xylostella. Variation in soil fertility influenced the nutritional quality of host plants and this in turn affected the performance of D. insulare. In general, D. insulare performed best on plants grown with 3.0 g fertilizer pot⁻¹; these plants had 2.06-, 3.77-, and 1.02-fold more nitrogen, phosphorous and potassium, respectively than ones grown without any added fertilizer. P. xylostella escape from D. insulare was highest (32%) on plants grown at 1.0 g fertilizer, and this could be attributed to both physical and physiological defense mechanisms mediated by host plant nutritional quality. Plant stress and plant vigor are competing paradigms pertaining to the performance of herbivorous insects on their host plants. These hypotheses were originally proposed to predict responses of herbivores, but may also explain the effects of plant quality on koinobiont parasitoids, such as D. insulare.     

Transgenic <Emphasis Type="Italic">Brassica napus</Emphasis> L. lines carrying a two gene construct demonstrate enhanced resistance against <Emphasis Type="Italic">Plutella xylostella</Emphasis> and <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis>

Plant diseases and insect pests are serious threat to the growth and yield of oilseed rape. In this study, a binary vector carrying sporamin and chitinase PjChi-1 genes in tandem was introduced into Brassica napus cv. ZS 758 via Agrobacterium tumefaciens for dual resistance against disease and insect attack. Thirty-two regenerated plantlets exhibiting hygromycin resistance were selected following Agrobacterium-mediated transformation of 600 leaf petiole explants. Of these, 27 transformants were confirmed to carry the two transgenes as detected by polymerase chain reaction (PCR) with 4.5% transformation efficiency. Eight plantlets were randomly selected for further confirmation by Southern and northern blot hybridization analyses. Four plants carried single copy of the transgenes, while the remaining four plants carried either two or three copies of the transgenes. Moreover, expression of the sporamin transgene was detected by northern blot hybridization in transgenic lines, but not in wild-type plants. These eight T₀ plants were grown in vitro, and inoculated with the Lepidoptera larvae of Plutella xylostella and with spores of the fungal pathogen of Sclerotinia sclerotiorum. Transgenic plants exhibited high levels of resistance to P. xylostella and S. sclerotiorum when compared to untransformed wild-type plants. Genetic analysis of T₁ progeny confirmed Mendelian segregation of the introduced genes. Therefore, these transgenic lines demonstrate a promising potential for variety development of oilseed rape lines with enhanced resistance against both P. xylostella and S. sclerotiorum.     

Characterization of directly transformed weedy <Emphasis Type="Italic">Brassica rapa</Emphasis> and introgressed <Emphasis Type="Italic">B. rapa</Emphasis> with Bt <Emphasis Type="Italic">cry1Ac</Emphasis> and <Emphasis Type="Italic">gfp</Emphasis> genes

Crop to weed transgene flow, which could result in more competitive weed populations, is an agricultural biosafety concern. Crop Brassica napus to weedy Brassica rapa hybridization has been extensively characterized to better understand the transgene flow and its consequences. In this study, weedy accessions of B. rapa were transformed with Bacillus thuringiensis (Bt) cry1Ac- and green fluorescence protein (gfp)-coding transgenes using Agrobacterium to assess ecological performance of the wild biotype relative to introgressed hybrids in which the transgenic parent was the crop. Regenerated transgenic B. rapa events were characterized by progeny analysis, Bt protein enzyme-linked immunosorbent assay (ELISA), Southern blot analysis, and GFP expression assay. GFP expression level and Bt protein concentration were significantly different between independent transgenic B. rapa events. Similar reproductive productivity was observed in comparison between transgenic B. rapa events and B. rapa × B. napus introgressed hybrids in greenhouse and field experiments. In the greenhouse, Bt transgenic plants experienced significantly less herbivory damage from the diamondback moth (Plutella xylostella). No differences were found in the field experiment under ambient, low, herbivore pressure. Directly transformed transgenic B. rapa plants should be a helpful experimental control to better understand crop genetic load in introgressed transgenic weeds.     

Natural enemies ofPlutella xylostella (Lep.: Plutellidae) on crucifers in Honduras

Three primary parasitoids in three genera were reared fromPlutella xylostella (L.) larvae and pupae collected in various crucifer producing regions of Honduras. The ichneumonidDiadegma insulare (Cresson) was by far the most abundant species. TwoSpilochalcis species, facultative hyperparasitoids attackingP. xylostella andD. insulare, were encountered as well as eleven species in nine genera of obligate hyperparasitoids attackingD. insulare. Three vespid predators are noted as predating on larvae.      

Effects of mixed cropping on population densities and parasitism rates of the diamondback moth, <Emphasis Type="Italic">Plutella xylostella</Emphasis> (Lepidoptera: Plutellidae)

Effects of mixed cropping and barrier crops on the population density and parasitism of the diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), were evaluated in field plots of cabbage grown in Bali, Indonesia. The densities of P. xylostella at larval and pupal stages, as well as the overall density at larval plus pupal stages, were significantly lower in cabbage/coriander mixed cropping subplots than in cabbage monoculture subplots. Parasitism of P. xylostella by the larval parasitoid Diadegma semiclausum (Hellen) (Hymenoptera: Ichneumonidae) was not significantly different between the mixed and monocultural cropping systems. These results do not support the so-called enemies hypothesis, but suggest that disruption of the host searching behavior of female moths by neighboring non-host plants is the mechanism behind the associational resistance observed in the coriander mixed cropping system. The inclusion of a Napier grass barrier between mixed crop and monoculture subplots did not affect the influence of mixed cropping on larval and pupal densities. Therefore, Napier grass, which is used locally as a fence for preventing livestock invasion of fields, would not obstruct the pest-reducing effect of coriander/cabbage mixed cropping.     

Yeheb (<Emphasis Type="Italic">Cordeauxia</Emphasis><Emphasis Type="Italic">edulis</Emphasis>) extract deters feeding and oviposition of <Emphasis Type="Italic">Plutella xylostella</Emphasis> and attracts its natural enemy

The effects of yeheb (Cordeauxia edulis Hemsl.) leaf extract on feeding and oviposition by diamondback moth (DBM) (Plutella xylostella L.) and the behavior of DBM parasitoid, Cotesia vestalis (Haliday), were studied. Volatile organic compounds (VOCs) from the headspace of intact and DBM-damaged broccoli plants sprayed with yeheb extracts (YE) were also analyzed. Larval feeding and growth, and oviposition by adult DBM were strongly inhibited by the extract. Cotesia vestalis were attracted to volatile blends from intact or DBM-damaged broccoli plants sprayed with YE over intact plants sprayed with water or methanol. Analyses of VOCs in the headspace of broccoli plants revealed that both intact and DBM-damaged plants sprayed with YE showed remarkable differences in sesquiterpene compounds compared to intact control treatments. These combined negative effects of YE on DBM fitness together with positive effects on the parasitoid show that yeheb is a potential source of compounds for use in integrated pest management to control damage caused by DBM.     

Iranian <Emphasis Type="Italic">Trichogramma</Emphasis>: ITS2 DNA characterization and natural <Emphasis Type="Italic">Wolbachia</Emphasis> infection

Egg parasitoids of the genus Trichogramma (Hymenoptera: Trichogrammatidae) are important natural enemies of many lepidopterous pests in agricultural and forest areas. Because the use of indigenous Trichogramma populations/species can significantly increase the biological control efficiency, the characterization of endemic populations and precise species identification is important. In this study, Trichogramma wasps were collected from parasitized eggs of several lepidopteran pests in Northern and Central Iran. Seven Trichogramma species were identified based on the morphology and the nucleotide sequence, size and restriction profile (PCR-RFLP) of the internal transcribed spacer 2 (ITS2) region of the rDNA of T. brassicae Bezdenko, T. cacoeciae Marchal, T. embryophagum Hartig, T. evanescens Westwood, T. euproctidis Girault, T. pintoi Voegelé and T. tshumakovae Sorokina. This is the first report of T. euproctidis in Iran. Additionally, natural populations were surveyed for the prevalence of Wolbachia. Two populations of T. brassicae were found to be infected by a single Wolbachia strain which was identified by using a multi-locus sequencing approach.     

Fitness and life history parameters of <Emphasis Type="Italic">Leptomastix epona</Emphasis> and <Emphasis Type="Italic">Pseudaphycus flavidulus</Emphasis>, two parasitoids of the obscure mealybug <Emphasis Type="Italic">Pseudococcus viburni</Emphasis>

Fitness and life table parameters of two endoparasitoids of the obscure mealybug Pseudococcus viburni (Signoret), the solitary Leptomastix epona (Walker) and the gregarious Pseudaphycus flavidulus (Brèthes), were examined in relation to temperature and host size with a view to determine the efficacy of the parasitoids as biocontrol agents of the pest. Three temperature levels (21°C, 26°C and 31°C) and two host sizes classes (small, which mostly comprised third instar nymphs and large, which consisted of female adults) were studied. The lower developmental threshold and thermal constant of the host and the parasitoids were found similar so the coincidence of pest and parasitoids is likely. The rate of development of the parasitoids increased with a linear trend as the temperature increased from 21°C to 31°C. Temperature had a significant effect on mummification in both parasitoid species and on successful parasitism by P. flavidulus. Host size had a significant effect on the mummification caused by L. epona and on the proportion of the male offspring which emerged as well as on the successful parasitism by P. flavidulus. Life table parameters of the parasitoids were estimated in small and large hosts at 26°C in the laboratory. Both parasitoids achieved a greater intrinsic rate of natural increase and gross reproductive rate in addition to a shorter generation and doubling time in large mealybugs compared with small ones. Consequently, large hosts are expected to have a higher impact on the rise of the parasitoids population and the potential of the parasitoids to control the mealybug population improves with the increase of host size. Handling Editor: Torsen Meiners.     

A comparison of the host-searching efficiency of two larval parasitoids of Plutella xylostella

1. A host specialist parasitoid is thought to have greater efficiency in locating hosts or greater ability to overcome host defence than a generalist species. This leads to the prediction that a specialist should locate and parasitise more hosts than a generalist in a given arena. The work reported here tested these predictions by comparing the host‐searching behaviour of Diadegma semiclausum (a specialist) and Cotesia plutellae (an oligophagous species), two parasitoids of larval Plutella xylostella. 2. Both parasitoids employed antennal search and ovipositor search when seeking hosts but D. semiclausum also seemed to use visual perception in the immediate vicinity of hosts. 3. Larvae of P. xylostella avoided detection by parasitoids by moving away from damaged plant parts after short feeding bouts. When they encountered parasitoids, the larvae wriggled vigorously as they retreated and often hung from silk threads after dropping from a plant. 4. These two parasitoids differed in their responses to host defences. Diadegma semiclausum displayed a wide‐area search around feeding damage and waited near the silk thread for a suspended host to climb up to the leaf, then attacked it again. Cotesia plutellae displayed an area‐restricted search and usually pursued the host down the silk thread onto the ground. 5. Diadegma semiclausum showed a relatively fixed behavioural pattern leading to oviposition but C. plutellae exhibited a more plastic behavioural pattern. 6. The time spent by the two parasitoids on different plants increased with increasing host density, but the time spent either on all plants or a single plant by D. semiclausum was longer than that of C. plutellae. Diadegma semiclausum visited individual plants more frequently than C. plutellae before it left the patch, and stung hosts at more than twice the rate of C. plutellae. 7. The results indicated that the host‐location strategies employed by D. semiclausum were adapted better to the host's defensive behaviour, and thus it was more effective at detecting and parasitising the host than was C. plutellae.     

<Emphasis Type="Italic">In vivo</Emphasis> fluorescence observation of parasporal inclusion formation in <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

A recombinant gene expressing a Cry1Ac-GFP fusion protein with a molecular mass of approximately 160 kD was constructed to investigate the expression of cry1Ac, the localization of its gene product Cry1Ac, and its role in crystal development in Bacillus thuringiensis. The cry1Ac-gfp fusion gene under the control of the cry1Ac promoter was cloned into the plasmid pHT304, and this construct was designated pHTcry1Ac-gfp. pHTcry1Ac-gfp was transformed into the crystal-negative strain, HD-73 cry⁻, and the resulting strain was named HD-73⁻(pHTcry1Ac-gfp). The gfp gene was then inserted into the large HD-73 endogenous plasmid pHT73 and fused with the 3′ terminal of the cry1Ac gene by homologous recombination, yielding HD-73Φ(cry1Ac-gfp)3534. Laser confocal microscopy and Western blot analyses showed for the first time that the Cry1Ac-GFP fusion proteins in both HD-73⁻(pHTcry1Ac-gfp) and HD-73Φ(cry1Ac-gfp)3534 were produced during asymmetric septum formation. Surprisingly, the Cry1Ac-GFP fusion protein showed polarity and was located near the septa in both strains. There was no significant difference between Cry1Ac-GFP and Cry1Ac in their toxicity to Plutella xylostella larvae.     

<Emphasis Type="Italic">In vitro</Emphasis> flowering of <Emphasis Type="Italic">Perilla frutescens</Emphasis>

This study investigated the factors affecting in vitro flowering of Perilla frutescens. The shoots regenerated from cotyledonary and hypocotyl explants cultured on Murashige and Skoog (MS) medium supplemented with benzyladenine (BA) and indole-3-acetic acid, each at 0.5 mg l⁻¹, were excised and transferred to MS medium containing 30 g l⁻¹ of sucrose, 8.25 g l⁻¹ of ammonium nitrate, and 1.0 mg l⁻¹ of BA. After 40 d of culture, 86.2% of shoots flowered and most of which self-fertilized in vitro and produced mature fruits with viable seeds. These seeds were germinated and plants were grown to maturity and flowered in soil under greenhouse conditions. The in vitro flowering system reported in this study may facilitate rapid breeding of P. frutescens and offers a model system for studying the physiological mechanism of flowering.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

Close-Range Host Searching Behavior of the Stemborer Parasitoids <Emphasis Type="Italic">Cotesia sesamiae</Emphasis> and <Emphasis Type="Italic">Dentichasmias busseolae</Emphasis>: Influence of a Non-Host Plant <Emphasis Type="Italic">Melinis minutiflora</Emphasis>

Studies were conducted on the host searching behavior of the larval parasitoid Cotesia sesamiae (Cameron) (Hymenoptera: Braconidae) and the pupal parasitoid Dentichasmias busseolae Heinrich (Hymenoptera: Ichneumonidae), both of which attack lepidopteran (Crambidae, Noctuidae) cereal stemborers. The behavior of D. busseolae was observed in a diversified habitat that consisted of stemborer host plants (maize, Zea mays L. and sorghum, Sorghum bicolor (L). Moench (Poaceae)) and a non-host plant (molasses grass, Melinis minutiflora Beauv. (Poaceae)), while C. sesamiae was observed separately on host plants and molasses grass. In previous olfactometer studies, C. sesamiae was attracted to molasses grass volatiles while hboxD. busseolae was repelled. The aim of the present study was to investigate the influence of molasses grass on close-range foraging behavior of the parasitoids in an arena that included infested and uninfested host plants. Dentichasmias busseolae strongly discriminated between host and non-host plants, with female wasps spending most of the time on infested host plants and least time on molasses grass. Likewise, C. sesamiae spent more time on uninfested and infested host plants than it did on molasses grass in single choice bioassays. While on infested plants, the wasps spent more time foraging on the stem, the site of damage, than on other areas of the plant. Overall, the results indicate that presence of the non-host plant does not hinder close range foraging activities of either parasitoid.     

<Emphasis Type="Italic">Drosophila</Emphasis><Emphasis Type="Italic">P</Emphasis> transposons of the urochordata <Emphasis Type="Italic">Ciona intestinalis</Emphasis>

P transposons belong to the eukaryotic DNA transposons, which are transposed by a cut and paste mechanism using a P-element-coded transposase. They have been detected in Drosophila, and reside as single copies and stable homologous sequences in many vertebrate species. We present the P elements Pcin1, Pcin2 and Pcin3 from Ciona intestinalis, a species of the most primitive chordates, and compare them with those from Ciona savignyi. They showed typical DNA transposon structures, namely terminal inverted repeats and target site duplications. The coding region of Pcin1 consisted of 13 small exons that could be translated into a P-transposon-homologous protein. C. intestinalis and C. savignyi displayed nearly the same phenotype. However, their P elements were highly divergent and the assumed P transposase from C. intestinalis was more closely related to the transposase from Drosophila melanogaster than to the transposase of C. savignyi. The present study showed that P elements with typical features of transposable DNA elements may be found already at the base of the chordate lineage. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.