Characterization of a new toxin from the entomopathogenic fungus Metarhizium anisopliae: the ribotoxin anisoplin.

Metarhizium anisopliae is an entomopathogenic fungus relevant in biotechnology with applications like malaria vector control. Studies of its virulence factors are therefore of great interest. Fungal ribotoxins are toxic ribonucleases with extraordinary efficiency against ribosomes and suggested as potential insecticides. Here we describe this ribotoxin characteristic activity in M. anisopliae cultures. Anisoplin has been obtained as a recombinant protein and further characterized. It is structurally similar to hirsutellin A, the ribotoxin from the entomopathogen Hirsutella thompsonii. Moreover, anisoplin shows the ribonucleolytic activity typical of ribotoxins and cytotoxicity against insect cells. How Metarhizium uses this toxin and possible applications are of interest.

Fungal ribotoxins: Natural protein-based weapons against insects.

Ribotoxins are fungal extracellular ribonucleases highly toxic due to their ability to enter host cells and their effective ribonucleolytic activity against the ribosome. The natural role of these proteins in the producing fungi is still unsolved. Nevertheless, recent studies showing the insecticidal properties of two ribotoxins from different origin support their involvement in defense mechanisms. Thus, it seems that not just the entomopathogen Hirsutella thompsonii expresses the ribotoxin hirsutellin A as a virulence factor but also Aspergillus, the main ribotoxin producer, does so. In this review we focus on this little known aspect of this family of proteins, their toxicity against insects, from the point of view of its biological relevance and its potential biotechnological applications.

Lethal and sublethal toxicity of fipronil and imidacloprid on Psyttalia concolor (Hymenoptera: Braconidae).

Psyttalia concolor (Szèpligeti) (Hymenoptera: Braconidae) is a koinobiont endoparasitoid of several species of tephritid (Diptera) larvae, such as Bactrocera oleae (Gmelin) and Ceratitis capitata (Wiedemann). Here, we report on the effects of imidacloprid and fipronil on P. concolor females, when different routes of exposure were evaluated: residual contact (cover and bait sprays) and via treatment of host species. Moreover, the persistence of the bait formulated compound also was studied. For each experiment, lethal (mortality) and sublethal effects (parasitization rate or longevity) were studied. Fipronil produced 100% mortality irrespective of exposure route, and it was very persistent, because 34-d-old residues still produced this high mortality rate, being as toxic or even more toxic than the reference product dimethoate. Toxicity of imidacloprid depends on the mode of exposure, although always remained less toxic than dimethoate. Imidacloprid caused high mortality or sublethal effect to the progeny in cover sprays and when applied via treated host, being harmless in bait sprays application. In conclusion, our results suggest that fipronil should not be used in the field when the parasitoid is present. On the contrary, although imidacloprid is physiologically active against females of P. concolor, ecological selectivity may result through the use of bait treatment.

Long-term effects of methoxyfenozide on the adult reproductive processes and longevity of Spodoptera exigua (Lepidoptera: Noctuidae).

The long-term effects of methoxyfenozide on the longevity and reproductive processes of beet armyworm, Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae), adults were assessed after exposure by ingestion. Methoxyfenozide significantly reduced adult male longevity compared with females by 1.1 and 1.5 d at 75 and 150 mg (AI)/liter, respectively. Fecundity decreased by >60% with both concentrations at 72 and 96 h after treatment, but at 48 h, no significant effect was observed. The carbohydrate, protein, and lipid content in the eggs were determined as representatives of the biochemical effects of methoxyfenozide associated with the disruption of reproductive processes. The content of carbohydrates in the eggs laid 48 h at treatment was similar to that of controls, but it increased by approximately 1.5 and 2-fold in eggs laid after 72 and 96 h, respectively, compared with controls (15 microg per egg). Protein content was reduced approximately 2.5 and approximately 3-fold for each treatment concentration, respectively, compared with the controls (25 and 23 microg per egg for 75 and 150 mg [AI]/liter, respectively) in eggs collected 72 and 96 h after treatment. Lipid content significantly decreased by approximately 1.6-fold in both treatment concentrations in eggs collected at 48 and 96 h after treatment compared with the controls (24 and 21 microg per egg for 48 and 96, respectively), but it was similar to controls (approximately 19 microg per egg) at 72 h (approximately 15 microg per egg) for both concentrations. The biochemical effects of methoxyfenozide on S. exigua egg formation detected in this work are consistent with the reduction in fertility observed, as reported previously.

Influence of azadirachtin and methoxyfenozide on life parameters of Spodoptera littoralis (Lepidoptera: Noctuidae).

Effects on adult longevity, fecundity and fertility, as well as long-term effects on progeny were determined through oral exposure of Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae) adults to azadirachtin and methoxyfenozide. Both compounds reduced adult longevity by 2.3 d at the higher concentrations tested, but no significant differences were observed between sexes. Fecundity and fertility were significantly affected for both insecticides, although this effect was only dose-dependent for azadirachtin. The progeny from adults treated with azadirachtin or methoxyfenozide were only affected in percentage of pupation of eggs that successfully hatched, but no effects were observed in adult emergence of individuals that successfully pupated for either insecticide. In the second part of this study, each sex was exposed separately to methoxyfenozide by topical application or ingestion. Adult fecundity was more affected when moths were treated by ingestion than when treated topically, with a mean number eggs laid per female of 343 +/- 89 and 932 +/- 79, respectively. Finally, azadirachtin applied to pepper plants showed a significant oviposition deterrence activity on S. littoralis adults. However, when fecundity was scored for one additional day in females that had been previously exposed to pepper, Capsicum annum L., plants treated with this insecticide, the number of eggs laid per female did not differ significantly from that of controls. The effects of azadirachtin and methoxyfenozide on S. littoralis suggest changes in population dynamics of this pest in crops treated with these insecticides.

Lethal and sublethal effects of methoxyfenozide and spinosad on Spodoptera littoralis (Lepidoptera: Noctuidae).

Susceptibility to methoxyfenozide of Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae) larvae was determined through exposure of neonate and fourth instars to dipped and sprayed pepper, Capsicum annum L., leaves. Methoxyfenozide and spinosad were tested against adults of this noctuid by oral, residual, and topical application. In larvae, we evaluated five (range, 0.001-10 mg active ingredient [AI] /liter) and six (range, 1-250 mg [AI] /liter) concentration levels by instars, respectively, by using two application methods at three different age leaf residues (0, 3, and 6 d after application). According to LC50 values, no significant differences were observed between the same age leaf residues of both application methods at 96 and 72 h after ingestion treatment on neonates and fourth instars, respectively. Nevertheless, toxicity of methoxyfenozide decreased significantly after time. For both application methods, the LC50 values of the first leaf residue (0 d after application) were significantly lower than those of 6-d-old residues. Furthermore, larval weight of fourth instars fed for 48 h with pepper, Capsicum annum L., leaves containing methoxyfenozide was significantly suppressed. Spinosad and methoxyfenozide reduced in a dose-dependent manner the fecundity and fertility of S. littoralis adults when treated oral and residually. Likewise, when methoxyfenozide was administered orally in three different adult crosses, the fecundity was strongly affected, independently of the treated sex. We conclude that the combination of lethal and sublethal effects of methoxy-fenozide and spinosad might exhibit significant effects on the population dynamics of S. littolaris.

Insecticidal effects of various concentrations of selected extractions of Cestrum parqui on adult and immature Ceratitis capitata.

Aqueous extracts of Cestrum parqui L'Héritier (Solanaceae) were evaluated at different concentrations in several stages of Ceratitis capitata (Wiedemann) (Diptera: Tephritidae). For adults, the study was extended to extracts obtained with several solvents of an increasing degree of polarity. Aqueous extracts from C. parqui showed a high toxicity to neonate larvae when ingested through diet, inhibiting pupation at a concentration above 0.6%. Lower concentrations delayed the larval development and reduced the percentages of pupae formed and adult emergence. An LC50 = 0.9% after 3 d of continuous ingestion of C. parqui aqueous extracts could be calculated, whereas extracts obtained with organic compounds were nearly innocuous except with the use of the solvent methanol/water (80:20), the more polar of those tested, that killed 12.5% of adults. Aqueous extracts were also harmful to adults by diminishing the reproductive potential, which implies a significant effect on the offspring. Egg contact with insecticide or dipping third instars did not cause any adverse effect, supporting the hypothesis that only by means of ingestion can the toxic compounds of C. parqui reach the target. Our results showed that C. parqui components causing C. capitata mortality are mostly dissolved in water and not in organic solvents, which point out their polar character.

Effects of two biorational insecticides, spinosad and methoxyfenozide, on Spodoptera littoralis (Lepidoptera: Noctuidae) under laboratory conditions.

The toxicity of two biorational insecticides, spinosad (Tracer) and methoxyfenozide (RH-2485), was tested against eggs, larvae, and pupae of the noctuid Spodoptera littoralis (Boisduval). In the first experiment, filter paper circles containing egg masses of two different age classes, young (<24 h old) and old (24-48 h old), were dipped in different concentrations of each insecticide diluted in either water or acetone. No ovicidal activity was recorded when insecticides were diluted in water. In contrast, when insecticides were diluted in acetone, both egg age classes generally showed a concentration-dependent response for both compounds. Mortality of larvae that hatched from both egg age classes was significantly increased, compared with control larvae, at all concentrations of both insecticides when diluted in water or acetone alike. The prevalence of mortality was similar with each insecticide. In the second experiment, third instars of S. littoralis were fed semisynthetic diet containing different concentrations of both insecticides. According to LC50 values, no significant differences were observed between spinosad (2.11 mg [AI]/kg diet) and methoxyfenozide (3.98 mg [AI]/kg diet) after 48 h of treatment, based on the overlap of 95% CL. Toxic effects on the mortality of pupae, adult emergence, and the prevalence of deformed adults after topical application on young pupae also were examined. Only methoxyfenozide caused pupal mortality and deformed adults. Our results suggest that spinosad and methoxyfenozide are potentially potent compounds for control of S. littoralis.

Toxicity and kinetics of methoxyfenozide in greenhouse-selected Spodoptera exigua (Lepidoptera: Noctuidae).

Methoxyfenozide (RH-2485) was found to be 7.5-fold less toxic in terms of LD50 values against last-instar larvae of a greenhouse-selected strain of the beet armyworm Spodoptera exigua (Hübner) that was collected in July 2001 in an experimental greenhouse for resistance at Murcia in southern Spain, in comparison with a laboratory susceptible strain. To date, the compound is the newest member of this new group of moulting hormone accelerating IGRs to reach the marketplace against Lepidoptera. To understand this different potency in the greenhouse-selected S exigua, oxidative metabolism and acetylcholinesterase activities were measured in last-instar larvae and adults. In addition, we determined, by the use of 14C-labelled methoxyfenozide, the pattern of absorption in body tissues and excretion via faeces in last-instar larvae of the greenhouse-selected strain and compared the results with those from the laboratory susceptible strain. It was striking that the rate of excretion was about twice as high in the greenhouse-selected strain, resulting in a more rapid clearance of insecticide amounts from the insect body. Data are discussed in relation to mechanisms of lower toxicities for this new group of IGRs.

Significance of penetration, excretion, and transovarial uptake to toxicity of three insect growth regulators in predatory lacewing adults.

Topical treatment of the predatory lacewing adults, Chrysoperla carnea (Stephens) (Neuroptera: Chrysopidae) before and after the onset of oviposition with diflubenzuron (DFB) at doses based on the maximum field recommended concentration resulted in a total inhibition of egg hatch due to death of the embryo. In contrast, pyriproxyfen (PYR) and tebufenozide (TEB) did not affect fecundity and egg fertility. To explain these differences in toxicity, the patterns of penetration through the cuticle, distribution inside the insect body, and excretion were studied using [(14)C]-labeled isotopes of each insect growth regulator (IGR). Penetration of DFB and TEB reached about 16 and 26% in 7 days, whereas 88% of PYR had penetrated in 24 h. However, the rate of excretion for PYR was very high, compared to that of DFB and TEB. Low amounts of absorbed radioactivity were recovered from the female body with the exception of DFB, the ovaries and the eggs deposited during a week. DFB and PYR concentration reached a peak in the eggs deposited at the fourth and second day after treatment, respectively. The current data indicate the importance of penetration through the insect cuticle. However, other mechanisms are likely to be involved in the selectivity of the current IGRs towards this beneficial insect.