Activation of Bacillus thuringiensis Cry1I to a 50 kDa stable core impairs its full toxicity to Ostrinia nubilalis.

Bacillus thuringiensis Cry1I insecticidal proteins are structurally similar to other three-domain Cry proteins, although their size, activity spectrum, and expression at the stationary phase are unique among other members of the Cry1 family. The mode of action of Cry1 proteins is not completely understood but the existence of an activation step prior to specific binding is widely accepted. In this study, we attempted to characterize and determine the importance of the activation process in the mode of action of Cry1I, as Cry1Ia protoxin or its partially processed form showed significantly higher toxicity to Ostrinia nubilalis than the fully processed protein either activated with trypsin or with O. nubilalis midgut juice. Oligomerization studies showed that Cry1Ia protoxin, in solution, formed dimers spontaneously, and the incubation of Cry1Ia protoxin with O. nubilalis brush border membrane vesicles (BBMV) promoted the formation of dimers of the partially processed form. While no oligomerization of fully activated proteins after incubation with BBMV was detected. The results of the in vitro competition assays showed that both the Cry1Ia protoxin and the approx. 50 kDa activated proteins bind specifically to the O. nubilalis BBMV and compete for the same binding sites. Accordingly, the in vivo binding competition assays show a decrease in toxicity following the addition of an excess of 50 kDa activated protein. Consequently, as full activation of Cry1I protein diminishes its toxicity against lepidopterans, preventing or decelerating proteolysis might increase the efficacy of this protein in Bt-based products. KEY POINTS: • Processing Cry1I to a 50 kDa stable core impairs its full toxicity to O. nubilalis • Partially processed Cry1Ia protoxin retains the toxicity of protoxin vs O. nubilalis • Protoxin and its final processed forms compete for the same functional binding sites.

Biological responses of Tetranychus urticae (Acari: Tetranychidae) to sub-lethal concentrations of the entomopathogenic fungus Beauveria bassiana.

Tetranychus urticae (Acari: Tetranychidae) is one of the most important pests of agricultural crops with worldwide distribution causing considerable damage to different products. Application of chemical acaricides is one of the most important strategies used for the control of this pest. Entomopathogenic fungi, however, have been proposed as alternative control agents. In this study, sub-lethal effects (LC10 = 6.76 × 102, LC20 = 8.74 × 103, and LC30 = 55.38 × 103 conidia ml-1) of Beauveria bassiana strain TV on the life table parameters of T. urticae were evaluated under laboratory conditions. The results demonstrated that by increasing the concentration, a significant decline was observed in adult longevity of both male and female individuals. Total fecundity of T. urticae was calculated as 45.16, 36.28, 23.98, and 18.21 eggs in control, LC10, LC20, and LC30 treatments, respectively. Sub-lethal concentrations drastically affected the population parameters of this mite pest. The intrinsic rate of increase (r) ranged from 0.1983 to 0.1688 day-1 for the mites treated with distilled water and LC20 treatments, respectively. The net reproductive rate (R0) was affected by the sub-lethal concentrations (lower value at LC30 concentration: 11.19 offspring/individual). Considering the detrimental effects of B. bassiana on some biological parameters of T. urticae, it can be concluded that this product can be used to develop targeted interventions aimed at integrated pest management of this pest.

Behavior manipulation of mosquitoes by a mermithid nematode.

We examined manipulation of mosquito behavior by the parasitic mermithid nematode, Strelkovimermis spiculatus. This nematode species typically infects early instar host larvae and emerges after parasitic development to kill last-instar larvae. Parasitized adults, however, have occasionally been reported from field collections. We obtained low rates (1.7-11.5%) of parasitized adults in laboratory exposures only when Culex pipiens pipiens fourth-instar larvae nearing pupation were exposed to infective nematodes. This did not allow an adequate interval for parasitic development in immature host stages. Parasitized adult females in a multiple-choice assay were three times more likely to seek water than a blood source (63.1 vs. 20.5%), whereas uninfected females were twice as likely to seek blood than water (64%3.9 vs. 32.6%). This altered host behavior benefits the parasite by providing the only mechanism for dispersal and colonization of new host habitats while concurrently avoiding risks from the defensive behaviors associated with blood-feeding. Behavioral alternation in Cx. p. pipiens larval hosts was also examined using larvae infected as second instars to allow for a normal duration of parasitic development. As larvae neared pupation and parasite emergence, parasitized larvae became more spatially aggregated than unparasitized larvae. This altered host behavior benefits the parasite by providing a corresponding increase in post-parasite aggregation, which facilitates formation of large mating clusters and concomitantly reproductive success. Parasites derive fitness gains by overriding host autonomy, whereas hosts have zero fitness once parasitism is established, suggesting a coevolutionary response is inoperative and that the behavioral modifications may be adaptive.

Apolipoprotein D3 and LOX product play a role in immune-priming of a lepidopteran insect, Spodoptera exigua.

Immune-priming occurs in insects after a prior pathogen exposure. However, its underlying mechanism in insects remains elusive. In the present work, immune-priming was detected in a lepidopteran insect, Spodoptera exigua. Specifically, a prior infection with a heat-killed pathogenic bacterium, Escherichia coli, led to increased survival upon the second infection of different pathogens. Plasma collected from larvae with the prior infection possessed the immune-priming factor(s) that significantly up-regulated cellular and humoral immune responses of naïve larvae. Our study also finds that variations in the timing of plasma collection for priming larvae resulted in distinct impacts on both cellular and humoral responses. However, when the active plasma exhibiting the immune-priming was heat-treated, it lost this priming activity, therefore suggesting that protein factor(s) play a role in this immune-priming. An immunofluorescence assay showed that the hemocytes collected from the immune-primed larvae highly reacted to a polyclonal antibody specific to a vertebrate lipocalin, apolipoprotein D (ApoD). Among 27 ApoD genes (Se-ApoD1 âˆ¼ Se-ApoD27) of S. exigua, Se-ApoD3 was found to be highly induced during the immune-priming, in which it was shown to be expressed in hemocytes and fat body from a fluorescence in situ hybridization analysis. RNA interference of Se-ApoD3 expression significantly impaired the immune-priming of S. exigua larvae. Moreover, the inhibition of eicosanoid biosynthesis suppressed the immune-priming, in which treatment with a lipoxygenase (LOX) inhibitor-and not treatment with a cyclooxygenase inhibitor-suppressed immune-priming. Further, an addition of LOX product such as lipoxin A4 or lipoxin B4 significantly rescued the lost immune-priming activity. Taken together, these results suggest that a complex of ApoD3 and LOX product mediates the immune-priming activity of S. exigua.

Evaluation of the Entomopathogenic Fungus Beauveria bassiana on Different Stages of Phlebotomus papatasi (Diptera: Psychodidae), Vector of Zoonotic Cutaneous Leishmaniasis in Iran.

Background: Zoonotic cutaneous leishmaniasis is a major public health problem in Iran with the main vector of Phlebotomus papatasi. The use of entomopathogenic fungi for biological control of the vector is a potential substitute for the current methods which are being used. The purpose of the current study was to assess the virulence of two local isolates of Beauveria bassiana (OZ2 and TV) against Ph. papatasi. Methods: To perform the bioassay test, fungal suspensions were applied for every stage of the sand fly life cycle. The mortality rate, longevity, and number of eggs laid were determined. Also, the probability of fungal survival on the surface of rodent's body was assessed. Results: The longevity of infected adult sand flies with both isolates of B. bassiana was significantly lower (P< 0.05) in comparison to the negative control. The estimated Lethal concentration 50 (LC50) values for adult female and male sand flies treated with OZ2 isolate were 1.4×106 and 2.2×107 conidia/ml, respectively, while they were 6.8×106 and 2.3×108 conidia/ml for TV isolate, respectively. Both isolates of B. bassiana exhibited nonsignificant mortality rates in sand fly larvae and pupae and fecundity rate (P> 0.05). According to our findings for both isolates, the fungus continued to spread throughout the surface of the rodent's body for 144 hours after spraying. Conclusion: The current study demonstrated that both isolates of B. bassiana have considerable biological control capacity against adult sand flies.

Ontogeny and reproductive biology of Diadegma semiclausum (Hym.: Ichneumonidae), a larval endoparasitoid of Diamondback Moth, Plutella xylostella (Lep.: Plutellidae).

In this article, the ontogeny and reproductive biology of Diadegma semiclausum (Hym.: Ichneumonidae), an important parasitoid of Plutella xylostella (Lep.: Plutellidae) are described in detail. We did dissect parasitized P. xylostella larvae in phosphate-buffered saline and determine the external morphology of its parasitoid at all developmental stages. The developmental duration of its immature stages, adult longevity, total oviposition period and fecundity of the parasitoid are determined at 24 ± 1 °C, 65  ±  5% R.H., and a photoperiod of 16:8 h L:D. The mean duration of egg and larval stages is 9.56 days and the prepupa and pupa stages last for 8.27 days. In average, female longevity is 1.31 times longer than that of males, and females lay 300 eggs in total. The peak of D. semiclausum oviposition is on the eighth day after mating. The egg loading pattern of D. semiclausum was investigated to determine the parasitoid 'ovigeny index' throughout the female's parasitoid lifetime. Initial egg load in immature females (<2 h age) is 1.45 per female and the mean lifetime potential fecundity (total immature and mature oocytes), at four interval ages, is 34. With an ovigeny index value of 0.038, D. semiclausum is considered moderately to strongly synovigenic. In the absence of the host, after 3 days, the number of eggs is decreased in D. semiclausum. Our results demonstrated that there is a negative relation between the ovigeny index and egg resorption in this parasitoid.

Effect of midgut proteolytic activity on susceptibility of lepidopteran larvae to Bacillus thuringiensis subsp. Kurstaki.

Bacillus thuringiensis (Bt) is the most effective microbial control agent for controlling numerous species from different insect orders. All subspecies and strains of B. thuringiensis can produce a spore and a crystalline parasporal body. This crystal which contains proteinaceous protoxins is dissolved in the alkaline midgut, the resulting molecule is then cleaved and activated by proteolytic enzymes and acts as a toxin. An interesting aspect of this activation process is that variations in midgut pH and protease activity have been shown to account for the spectrum of some Bt proteins activity. Thus, an important factor that could be a determinant of toxin activity is the presence of proteases in the midgut microenvironment of susceptible insects. Reciprocally, any alteration in the midgut protease composition of the host can result in resistance to Bt. Here in this paper, we reviewed this processes in general and presented our assays to reveal whether resistance mechanism to Bt in Diamondback Moth (DbM) larvae could be due to the function of the midgut proteases? We estimated LC50 for both probable susceptible and resistant populations in laboratory and greenhouse tests. Then, the midgut protease activities of the B. thuringiensis induced-resistant and susceptible populations of the DbM were assayed on Hemoglubin and on N-alpha-benzoyl-DL-arginine-p-nitroanilide (BapNA) for total and tryptic activities, respectively. Six hours after feeding on Bt treated and untreated canola leaves, the midguts of instar larvae of both populations were isolated. Following related protocols, peptides released through the activity of proteinases on Hemoglubin and BApNA were recorded using microplate reader. Control (Blank) was also considered with adding TCA to reaction mix before adding enzymatic extract. Data analysis indicated that there are significant differences for tryptic activity on BApNA and also for total proteolytic activity on Hemoglubin between susceptible and resistant populations fed on Bt treated leaves. But these differences were not significant for larvae fed on healthy canola leaves between these two populations. These results which supported the role of DbM's proteolytic system in development of resistance to Bt, will be discussed in details.

Insect inducible antimicrobial peptides and their applications.

Antimicrobial peptides (AMPs) are found as important components of the innate immune system (host defense) of all invertebrates. These peptides can be constitutively expressed or induced in response to microbial infections. Indeed, they vary in their amino acid sequences, potency and antimicrobial activity spectra. The smaller AMPs act greatly by disrupting the structure or function of microbial cell membranes. Here, the insect innate immune system with emphasis on inducible antimicrobial peptide properties against microbial invaders has been discussed.

Germination polarity of Beauveria bassiana conidia and its possible correlation with virulence.

Three different germination types of conidia; unidirectional, bidirectional and multidirectional, were revealed through microscopic observations for eight Beauveria bassiana isolates germinated on Sabouraud dextrose agar. Canonical correlation analysis indicated that there is a positive correlation between unidirectional germination and virulence against diamondback moth, Plutella xylostella and the Colorado potato beetle, Leptinotarsa decemlineata. Scanning electron microscopy revealed different in vivo behaviors for unipolar- and bipolar-germinated conidia. Unipolar-germinated conidia produced a strong germ tube with mostly appressorium-like structures while bipolar-germinated conidia continued to invasive hyphal growth without any penetration, indicating that germination polarity in one way or another may be an indicator of pathogenic ability.