Host Preferences of Trichogramma pretiosum and the Influence of Prior Ovipositional Experience on the Parasitism of Plutella xylostella and Pseudoplusia includens Eggs

Trichogramma wasps are generalist egg parasitoids used in biological control efforts. In a multi host situation they may preferentially parasitize a non-target host species to the detriment of the control program. Plutella xylostella Linnaeus (Lepidoptera: Plutellidae) is a very serious pest of cabbage, but is only one in a number of species in the ‘cabbageworm’ complex. We investigated the host preferences of Trichogramma pretiosum Riley (Hymenoptera: Chalcidoidea) when offered the eggs of Plutella xylostella and the eggs of Pseudoplusia includens Hübner (Lepidoptera: Noctuidae), another species in the ‘cabbageworm’ complex. Trichogramma pretiosum reared on the eggs of the factitious host Ephestia kuehniella Zeller (Lepidoptera: Pyralidae) parasitized both Plutella xylostella and Pseudoplusia includens eggs under laboratory conditions. For both choice and no-choice experiments, T. pretiosum parasitized significantly more P. xylostella eggs than P. includens eggs. Prior ovipositional experience with one or other of the two host species had no effect on the subsequent parasitism levels of the two host species. The preference for P. xylostella eggs was also not affected by this prior ovipositional experience.     

Nocturnal parasitism of moth eggs by Trichogramma wasps

Parasitoid wasps of the genus Trichogramma are used worldwide as biological control agents against lepidopteran pests. Trichogramma wasps develop inside eggs of a wide range of host species, most of them moths. They are generally considered as diurnal insects. Here, we investigated whether Trichogramma wasps can also successfully parasitise host eggs at night under controlled laboratory conditions. Eggs of the moth Ephestia kuehniella were offered under dark conditions (scotophase) to females of Trichogramma brassicae and Trichogramma evanescens either from 9:00 PM to 9:00 AM or from 11:00 AM to 5:00 PM at four different temperatures (5°C, 10°C, 15°C and 20°C). Both species are known to parasitise E. kuehniella eggs in the photophase during daytime. The results show that T. brassicae did not parasitise eggs in the scotophase at night and only very few in the artificially induced scotophase during daytime from 10°C to 20°C. In contrast, T. evanescens parasitised more eggs in the dark both at night and artificially induced scotophase during daytime. Parasitism in the scotophase already started at 5°C, with more eggs being parasitised and more offspring being produced at higher temperatures. T. evanescens displayed higher parasitism activity in the induced scotophase during daytime than in the scotophase at night. The present study suggests that Trichogramma are capable of successfully parasitising host eggs at night, even at low temperatures, but that nocturnal activity with respect to parasitism varies between wasp species.     

Fertility life table of Trichogramma pretiosum (Hym., Trichogrammatidae) in eggs of Tuta absoluta and Phthorimaea operculella (Lep., Gelechiidae) at different temperatures

Abstract: The aim of this work was to study the reproductive potential of Trichogramma pretiosum reared on Tuta absoluta and Phthorimaea operculella eggs through fertility life tables at different temperatures. The development cycle and the parasitization capacity of this parasitoid was determined in order to calculate the net reproductive rate ( R _o), the infinitesimal increase ratio ( r _m), the finite increase rate ( λ ) and the mean duration of the generation ( T ). The mean duration of one generation of T. pretiosum kept on both eggs was observed to show an inverse relation with the increase of temperature. The net reproduction rate varied according to the temperature variation for both species. The maximum increase in capacity of T. pretiosum on the first host ( T. absoluta ) was reached at 22°C and on the second host ( P. operculella ) between 22 and 25°C. The infinitesimal increase rate and the finite increase rate for both moths had a relationship with the increase of temperature ranging from 18 to 30°C. The highest value of λ for both moths occurred at 30 and 32°C according to the lesser duration of a generation.     

Pseudoplusia includens Densovirus Genome Organization and Expression Strategy

The genome of a densovirus of a major phytophagous pest, Pseudoplusia includens, was analyzed. It contained 5,990 nucleotides (nt) and included inverted terminal repeats of 540 nt with terminal Y-shaped hairpins of 120 nt. Its DNA sequence and ambisense organization with 4 typical open reading frames demonstrated that it belonged to the genus Densovirus in the subfamily Densovirinae of the family Parvoviridae.     

Hematopoiesis in larval Pseudoplusia includens and Spodoptera frugiperda

Maintenance of circulating hemocytes in larval Lepidoptera has been attributed to both mitosis of hemocytes already in circulation and the release of hemocytes from hematopoietic organs. In this study, we compared hematopoiesis in the noctuids Pseudoplusia includens and Spodoptera frugiperda. For both species, hemocyte densities per microl of blood increased with instar. Differential hemocyte counts indicated that plasmatocytes were the most abundant hemocyte type during early instars but granular cells were the most abundant hemocyte type in the last instar. Hematopoietic organs were located in the meso- and metathorax of S. Frugiperda and P. Includens. These organs contained large numbers of hemocytes in S. Frugiperda, but contained few hemocytes in P. Includens. The majority of the hemocytes recovered from hematopoietic organs were identified as plasmatocytes. Using hemocyte type-specific markers and bromodeoxyuridine (BrdU) incorporation experiments, we determined that all hemocyte types with the exception of oenocytoids synthesize DNA. BrdU labeling indices for both species also fluctuated with the molting cycle. Ligation experiments suggested that hematopoietic organs are an important source of circulating plasmatocytes in S. Frugiperda but not in P. Includens. Injection of heat killed bacteria into larvae induced higher levels of BrdU labeling than injection of sterile saline, suggesting that infection and wounding induce different levels of hemocyte proliferation. Arch.     

The genetics and evolution of obligate reproductive parasitism in Trichogramma pretiosum infected with parthenogenesis-inducing Wolbachia

Parthenogenesis-inducing (PI) Wolbachia belong to a class of intracellular symbionts that distort the offspring sex ratio of their hosts toward a female bias. In many PI Wolbachia-infected species sex ratio distortion has reached its ultimate expression-fixation of infection and all-female populations. This is only possible with thelytokous PI symbionts as they provide an alternative form of reproduction and remove the requirement for males and sexual reproduction. Many populations fixed for PI Wolbachia infection have lost the ability to reproduce sexually, even when cured of the infection. We examine one such population in the species Trichogramma pretiosum. Through a series of backcrossing experiments with an uninfected Trichogramma pretiosum population we were able to show that the genetic basis for the loss of female sexual function could be explained by a dominant nuclear effect. Male sexual function had not been completely lost, though some deterioration of male sexual function was also evident when males from the infected population (created through antibiotic curing of infected females) were mated to uninfected females. We discuss the dynamics of sex ratio selection in PI Wolbachia-infected populations and the evolution of non-fertilizing mutations.     

拟澳洲赤眼蜂和短管赤眼蜂在米蛾多卵上的种间竞争

1引言 小菜蛾(Plutella xylostella)是十字花科蔬菜的一种重要害虫.20世纪70年代以来,化学杀虫剂的大量使用和抗性的产生.使小菜蛾成为南亚、东南亚和中国南方蔬菜生产中最为严重的害虫[8,22].因此,寻求安全有效的生物防治措施是防治小菜蛾的必要途径[23].赤眼蜂(Trichogramma)是一类多食性的卵寄生蜂,近百年来已被成功地用于防治为害多种作物及森林的害虫[21],但研究和利用赤眼蜂防治小菜蛾只有10来年的历史[12,23].     

Regulation of melanization by glutathione in the moth Pseudoplusia includens

The phenoloxidase (PO) cascade regulates the melanization of hemolymph, which serves as a conserved humoral immune response in insects and other arthropods. The reductant glutathione (GSH) has long been used to inhibit melanization of hemolymph from insects but whether GSH levels in hemolymph are sufficient to play a physiological role in regulating melanization is unknown. Here, we characterized the abundance and effects of GSH on the melanization of plasma from larval stage Pseudoplusia includens (Lepidoptera: Noctuidae). GSH concentration in newly collected plasma from day two fifth instars ranged from 50 to 115 μM, while the titer of tyrosine, a substrate for the PO cascade, was 141 μM. GSH titers rapidly declined in plasma after collection from larvae, but no melanin formation occurred until GSH levels fell below 20 μM. Added GSH dose-dependently blocked melanization while PO substrates overrode GSH inhibition. Experiments conducted in the absence of oxygen and presence of PO cascade inhibitors further suggested that depletion of GSH from plasma was primarily due to formation of reactive intermediates produced by activated PO. Additional studies identified hemocytes as a potential source of plasma GSH. Hemocyte lysates recycled oxidized glutathione (GSSG) into GSH using NADPH, while intact hemocytes released GSH into the medium. These results suggest that in addition to protease cascade-releated mechanisms that regulate phenoloxidase, GSH exerts another level of control on melanization of insect hemolymph.     

Comparative susceptibility of two different hosts to genotypic variants of the Anticarsia gemmatalis nuclear polyhedrosis virus

The susceptibility of third instar larvae of Anticarsia gemmatalis (Lepidoptera: Noctuidae) and Diatraea saccharalis (Lepidoptera: Pyralidae) to ten distinct plaque purified genotypic variants of a selected isolate of the Anticarsia gemmatalis multiple-embedded nuclear polyhedrosis virus (AgMNPV), was compared. Despite the fact that this isolate, AgMNPV-Ds20, represents a wild strain of the AgMNPV selected for higher virulence to D. saccharalis, an alternate host, most of the variants are much more virulent to the original host Anticarsia than to Diatraea. Bioassays have shown an over one hundred-fold variation in LD₅₀ values ranging from 1700 polyhedron inclusion bodies (PIBs) to more than 200 000 PIBs/larva. The PIB production in infected larvae increased with the pathogenicity of the variant to the host, showing an average ten-fold reduction in Diatraea when compared to Anticarsia for the same variant. The virus particle yield ranged from 6×10⁷ to more than 10⁹ PIBs/g of infected larvae in Diatraea and from 8×10⁸ to more than 10¹⁰ PIBs/g of infected Anticarsia larvae. The data show a clear difference of the pathogenicity of the genotypic variants of AgMNPV in vivo both between the original and alternate host and between the individual variants for the same host. These differences found in vivo indicate that monitoring of shifts in variant frequency of wild and laboratory-propagated viral isolates in these highly heterogeneous populations would help ensure the efficacy of biological control programs.     

Parasitism of cotton leafworm Alabama argillacea eggs by Trichogramma pretiosum in commercial cotton fields

Release of natural enemies in commercial fields is challenging and has been inconsistent in the results achieved. This work discusses the augmentative releases of Trichogramma pretiosum to control the cotton leafworm (CLW) Alabama argillacea and also examines the parasitoid–host interaction under grower field conditions. The treatments consisted of fields with and without releases of T. pretiosum set up in Primavera do Leste and Campo Verde Counties, MT, Brazil, during three different seasons (2003 and 2004 dry and 2004 regular summer seasons). Trichogramma wasps were weekly released in the treated fields throughout the entire sampling period (14–15 week period) at a rate of 100 000 wasps per hectare. One‐way repeated measures analysis of variance was performed for the number of parasitized eggs, followed by a meta‐analysis procedure to determine the contribution of T. pretiosum release on overall parasitism. In addition, regression analysis was conducted with each season’s data sets to study the relationship of the host density and parasitism response by T. pretiosum. The overall results of Trichogramma augmentative releases did not result in significant increase of CLW egg parasitism beyond the natural parasitism in the areas studied. However, based on Cohen’s d effect sizes from the meta‐analysis, the parasitism rate was greater in fields under T. pretiosum releases during four out of 15 weeks surveyed. The parasitism of CLW eggs by T. pretiosum exhibited host density‐dependence only in one out of three seasons surveyed. These findings are encouraging as they are evidence that T. pretiosum is able to maintain a considerable level of parasitism under commercial field conditions, highlighting their potential value in large‐scale commercial areas of cotton as previously found at the small and diverse farming scale. Future studies should address the potential of early‐season, low density releases of the parasitoid.     

Methods for Detection of Anticarsia gemmatalis Nucleopolyhedrovirus DNA in Soil

Two methods, phenol-ether and magnetic capture-hybridization (MCH), were developed and compared with regard to their sensitivities and abilities to extract the DNA of the insect baculovirus Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) from soil and to produce DNA amplifiable by PCR. Laboratory experiments were performed with 0.25 g of autoclaved soil inoculated with different viral concentrations to optimize both methods of baculovirus DNA extraction and to determine their sensitivities. Both procedures produced amplifiable DNA; however, the MCH method was 100-fold more sensitive than the phenol-ether procedure. The removal of PCR inhibitors from the soil appeared to be complete when MCH was used as the viral DNA isolation method, because undiluted aliquots of the DNA preparations could be amplified by PCR. The phenol-ether procedure probably did not completely remove PCR inhibitors from the soil, since PCR products were observed only when the AgMNPV DNA preparations were diluted 10- or 100-fold. AgMNPV DNA was detected in field-collected soil samples from 15 to 180 days after virus application when the MCH procedure to isolate DNA was coupled with PCR amplification of the polyhedrin region.     

Parasitism capacity of Trichogramma pretiosum Riley (Hymenoptera: Trichogrammatidae) reared under different temperatures on Bonagota salubricola (Meyrick) (Lepidoptera: Tortricidae) eggs

The parasitism capacity of Trichogramma pretiosum Riley strain bonagota on Bonagota salubricola (Meyrick) eggs was studied under the temperatures of 18, 20, 22, 25, 28, 30 and 32 degrees C. The number of days with parasitism, accumulated parasitism, total number of eggs parasitized per female and parasitoid longevity was evaluated. In the first 24h, parasitism ranged from 1.6 (32 degrees C) to 8.8 (22 degrees C) eggs of B. salubricola. Accumulated egg parasitism of B. salubricola reached 80% in 1st to 4th day at 20 degrees C to 32 degrees C, respectively, and in the 7th day at 18 degrees C. Temperatures from 18 degrees C to 22 degrees C were the best suited for the total eggs parasitized for female, resulting in 35.4 and 24.6 eggs/male respectively. T. pretiosum female longevity ranged from 7.8 to 2.5 days, at 18 degrees C and 32 degrees C, respectively. The results showed that T. pretiosum strain bonagota is better adapted to temperatures from 18 degrees C to 22 degrees C.     

Trichogramma pretiosum parasitism and dispersal capacity: a basis for developing biological control programs for soybean caterpillars

In order to succeed in biological control programs, not only is it crucial to understand the number of natural enemies to be released but also on how many sites per area this releasing must be performed. These variables might differ deeply among egg parasitoid species and crops worked. Therefore, these trials were carried out to evaluate the parasitism (%) in eggs of Anticarsia gemmatalis and Pseudoplusia includens after the release of different densities of the egg parasitoid Trichogramma pretiosum. Field dispersal was also studied, in order to determine appropriate recommendations for the release of this parasitoid in soybean fields. The regression analysis between parasitism (%) and densities of the parasitoid indicated a quadratic effect for both A. gemmatalis and P. includens. The maximum parasitism within 24 h after the release was reached with densities of 25.6 and 51.2 parasitoids per host egg, respectively, for the two pests. Parasitism of T. pretiosum in eggs of P. includens decreased linearly as the distance of the pest eggs from the parasitoid release sites increased. For P. includens, the mean radius of T. pretiosum action and the area of parasitoid dispersal in the soybean crop were 8.01 m and 85.18 m2, respectively. We conclude that for a successful biological control program of lepidopteran pests using T. pretiosum in soybean fields, a density of 25.6 parasitoids per host egg, divided into 117 sites per hectare, should be used.     

Separation and behavior in vitro of hemocytes from the moth,Pseudoplusia includens

Hemocytes collected from larvae of Pseudoplusia includens (Lepidoptera: Noctuidae) were separated by centrifugation on Percoll cushions. The procedure resulted in 95% purity of plasmatocytes and greater than 99% purity of granular and spherule cells. Medium supplemented with chicken serum enhanced cell viability and promoted spreading of plasmatocytes. Cell-free plasma and medium preconditioned by plasmatocytes or granular cells stabilized cells in vitro and also accelerated spreading of plasmatocytes relative to medium supplemented with chicken serum. Oenocytoids were the only morphotype that exhibited endogenous phenoloxidase activity, while granular cells and plasmatocytes were the only cells that endocytosed fluorescent beads in vitro. Granular cells and plasmatocytes ingested fluorescently labelled beads, both in mixed populations of hemocytes and after separation. Plasmatocytes were the only morphotype that encapsulated large foreign targets in vitro following separation. Separated granular cells attached and spread on the surface of foreign targets but never formed a multilayered capsule.     

Structural and ultrastructural studies of Anticarsia gemmatalis midgut cells infected with the baculovirus A. gemmatalis nucleopolyhedrovirus

Anticarsia gemmatalis is a lepidopteran insect susceptible to A. gemmatalis nucleopolyhedrovirus (AgNPV), which is being used in a large scale, in Brazil, as a biological control agent against this serious soybean pest. Baculovirus usually infects its insect host through the midgut epithelium. In the midgut, it replicates in the nuclei of epithelial cells, producing progeny virus and establishing systemic infection. The AgNPV infection of A. gemmatalis midgut was studied using light and electron microscopy. It was observed that AgNPV enters the midgut mainly through columnar cells. Although the virus was not found in the nuclei of columnar cells until late on infection, it is believed that these cells are the primary sites of infection and replication. This fact can be explained by the continuous regeneration of the midgut epithelium. Besides, the infection may be occurring in isolated cells, making it more difficult to be visualized by electron microscopy. At 48 h post infection, hemocytes and tracheoblasts are infected and polyhedra are formed later in these cells, which are the secondary sites of infection.     

The encapsidated genome of Microplitis demolitor bracovirus integrates into the host Pseudoplusia includens

Polydnaviruses (PDVs) are symbionts of parasitoid wasps that function as gene delivery vehicles in the insects (hosts) that the wasps parasitize. PDVs persist in wasps as integrated proviruses but are packaged as circularized and segmented double-stranded DNAs into the virions that wasps inject into hosts. In contrast, little is known about how PDV genomic DNAs persist in host cells. Microplitis demolitor carries Microplitis demolitor bracovirus (MdBV) and parasitizes the host Pseudoplusia includens. MdBV infects primarily host hemocytes and also infects a hemocyte-derived cell line from P. includens called CiE1 cells. Here we report that all 15 genomic segments of the MdBV encapsidated genome exhibited long-term persistence in CiE1 cells. Most MdBV genes expressed in hemocytes were persistently expressed in CiE1 cells, including members of the glc gene family whose products transformed CiE1 cells into a suspension culture. PCR-based integration assays combined with cloning and sequencing of host-virus junctions confirmed that genomic segments J and C persisted in CiE1 cells by integration. These genomic DNAs also rapidly integrated into parasitized P. includens. Sequence analysis of wasp-viral junction clones showed that the integration of proviral segments in M. demolitor was associated with a wasp excision/integration motif (WIM) known from other bracoviruses. However, integration into host cells occurred in association with a previously unknown domain that we named the host integration motif (HIM). The presence of HIMs in most MdBV genomic DNAs suggests that the integration of each genomic segment into host cells occurs through a shared mechanism.     

Methods for detection of Anticarsia gemmatalis nucleopolyhedrovirus DNA in soil.

Two methods, phenol-ether and magnetic capture-hybridization (MCH), were developed and compared with regard to their sensitivities and abilities to extract the DNA of the insect baculovirus Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) from soil and to produce DNA amplifiable by PCR. Laboratory experiments were performed with 0. 25 g of autoclaved soil inoculated with different viral concentrations to optimize both methods of baculovirus DNA extraction and to determine their sensitivities. Both procedures produced amplifiable DNA; however, the MCH method was 100-fold more sensitive than the phenol-ether procedure. The removal of PCR inhibitors from the soil appeared to be complete when MCH was used as the viral DNA isolation method, because undiluted aliquots of the DNA preparations could be amplified by PCR. The phenol-ether procedure probably did not completely remove PCR inhibitors from the soil, since PCR products were observed only when the AgMNPV DNA preparations were diluted 10- or 100-fold. AgMNPV DNA was detected in field-collected soil samples from 15 to 180 days after virus application when the MCH procedure to isolate DNA was coupled with PCR amplification of the polyhedrin region.     

Plasmatocytes from the moth Pseudoplusia includens induce apoptosis of granular cells

The primary immune response toward internal parasites and other large foreign objects that enter the insect hemocoel is encapsulation. Prior studies indicated that granular cells and plasmatocytes are the two hemocyte types required for capsule formation by the moth Pseudoplusia includens (Lepidoptera: Noctuidae). Capsules formed by P. includens also have a defined architecture with primarily granular cells attaching directly to the target, multiple layers of plasmatocytes adhering to this inner layer of granular cells, and a monolayer of granular cells attaching to the capsule periphery. Dye-exclusion assays indicated that granular cells die shortly after attaching to the capsule periphery, leaving a basal lamina-like layer around the capsule. In examining the mechanisms underlying granular cell death, we found that culture medium preconditioned by plasmatocytes induced apoptosis of granular cells. Characteristics of plasmatocyte-induced apoptosis included condensation of chromatin, cell surface blebbing and fragmentation of nuclear DNA. Plasmatocyte-conditioned medium did not induce apoptosis of other hemocyte types, and medium conditioned by other hemocyte types did not induce apoptosis of granular cells. The adhesive state of granular cells and plasmatocytes also affected levels of apoptosis. Conditioned medium from spread plasmatocytes induced higher levels of granular cell apoptosis than medium conditioned by unspread plasmatocytes. Reciprocally, spread granular cells underwent significantly higher rates of apoptosis than unspread granular cells in medium conditioned by spread plasmatocytes. In situ analysis indicated that granular cells on the periphery of capsules also undergo apoptosis. Collectively, our results suggest that spread plasmatocytes release one or more factors that induce apoptosis of granular cells, and that this response is important in the final phases of capsule formation.