Joel P. Siegel

Safety and ecotoxicology of entomopathogenic bacteria

Bacillus entomopathogens, especially Bacillus thuringiensis, have been used extensively for control of insect pests in crops, forests, and the aquatic environment. Their safety for vertebrates and nontarget invertebrates has been thoroughly documented in a myriad of studies. Their short term effects on nontarget organisms that are unrelated to target insects is negligible. However, the effect of repeated applications on most ecosystems is relatively unknown. It is highly probable that any regular disruption of large insect communities, due to chemical or microbial insecticides or natural disaster, could have long term deleterious effects on higher trophic levels and ecosystem structure. The more diversified the food web, the less likely that complete or partial removal of a single species will result in catastrophic consequences. The more species a given intervention affects, the greater the likelihood of altering ecosystem structure. The safety and environmental impact of entomopathogenic bacteria should be evaluated in light of the risk for nontarget organisms in comparison with other interventions and the effect no treatment at all will have on an ecosystem.

A substrate-specific cytochrome P450 monooxygenase, CYP6AB11, from the polyphagous navel orangeworm (Amyelois transitella)

The navel orangeworm Amyelois transitella (Walker) (Lepidoptera: Pyralidae) is a serious pest of many tree crops in California orchards, including almonds, pistachios, walnuts and figs. To understand the molecular mechanisms underlying detoxification of phytochemicals, insecticides and mycotoxins by this species, full-length CYP6AB11 cDNA was isolated from larval midguts using RACE PCR. Phylogenetic analysis of this insect cytochrome P450 monooxygenase established its evolutionary relationship to a P450 that selectively metabolizes imperatorin (a linear furanocoumarin) and myristicin (a natural methylenedioxyphenyl compound) in another lepidopteran species. Metabolic assays conducted with baculovirus-expressed P450 protein, P450 reductase and cytochrome b(5) on 16 compounds, including phytochemicals, mycotoxins, and synthetic pesticides, indicated that CYP6AB11 efficiently metabolizes imperatorin (0.88 pmol/min/pmol P450) and slowly metabolizes piperonyl butoxide (0.11 pmol/min/pmol P450). LC-MS analysis indicated that the imperatorin metabolite is an epoxide generated by oxidation of the double bond in its extended isoprenyl side chain. Predictive structures for CYP6AB11 suggested that its catalytic site contains a doughnut-like constriction over the heme that excludes aromatic rings on substrates and allows only their extended side chains to access the catalytic site. CYP6AB11 can also metabolize the principal insecticide synergist piperonyl butoxide (PBO), a synthetic methylenedioxyphenyl compound, albeit slowly, which raises the possibility that resistance may evolve in this species after exposure to synergists under field conditions.

The impact of mixed infection of three species of microsporidia isolated from the gypsy moth, Lymantria dispar L. (Lepidoptera: Lymantriidae).

The outcome of mixed infection by three species of microsporidia in the genera Endoreticulatus, Nosema, and Vairimorpha, isolated from different populations of Lymantria dispar in Bulgaria, was evaluated in the laboratory. All possible combinations of two species were administered either simultaneously or sequentially to larvae, and mortality, duration of development, and larval weight at 20 days post-infection (simultaneous inoculation) or 23 days post-infection (sequential inoculation) were chosen as the outcome variables. Larvae were also dissected and the presence of each species of microsporidia and the tissues infected were recorded for each treatment. Effects of infection were dependent on both host sex and the type of exposure. Infected larvae were more likely to die than uninfected larvae, but there were no differences in mortality between single and mixed infections. Addition of Endoreticulatus to infections of Nosema or Vairimorpha significantly increased duration of development to the fourth ecdysis; this effect was additive. Addition of Nosema or Vairimorpha to an existing infection had no such effect. When Nosema was administered simultaneously with Endoreticulatus or Vairimorpha, infected larvae weighed more than larvae that had single infections with either pathogen. Nosema was displaced from the silk glands by Vairimorpha and Nosema suppressed octospore formation by Vairimorpha in fat body. The histological evidence combined with the data on larval weight supports the hypothesis that competition occurred in mixed infections.

Variable development rate and survival of navel orangeworm (Lepidoptera: Pyralidae) on wheat bran diet and almonds.

ABSTRACT A series of laboratory and field studies were conducted using three lines of navel orangeworm, Amyelois transitella (Walker) (Lepidoptera: Pyralidae), reared on wheat bran diet and almonds, Prunus dulcis (Mill.) D.A. Webb, at constant and fluctuating temperature. The duration of development on wheat bran diet at constant temperature differed significantly among the three lines. Development was as much as 40% faster at constant temperature than at fluctuating temperatures, consequently the developmental duration determined at constant temperature was not an absolute measure. When the maximum temperature in fluctuating regimes exceeded 43°C, survival decreased by 50% compared with the constant temperature control. In almonds held at constant temperature, the developmental rate on new-crop nuts was variety-dependent and was fastest on ‘Nonpareil’ almonds and slowest on the experimental selection ‘23–122’, Development and survival were also variety-dependent on unharvested (mummy) almonds, and navel orangeworm average emergence was earliest from Nonpareil and latest from ‘Carmel’ nuts, differing by 529 degree-days, whereas survival was the highest on ‘Butte’, 35.7%, and the lowest on Carmel nuts, 7.2%, In our trials, both the speed of development and survival depended on host age, variety and quality, indicating that almonds were a dynamic rather than a static nutrient source for navel orangeworm, Identifying the factors responsible for variation in development and survival will give insight into improving control strategies.

Testing the pathogenicity and infectivity of entomopathogens to mammals

The purpose of this chapter is to provide a brief overview of mammalian safety testing, as well as short-term testing protocols that can help determine whether a candidate microbial pest control agent (MPCA) is suitable for more detailed study. Mammalian safety screens are a subset of a larger grouping of tests that assess the effects of an MPCA on nontarget organisms (NTO) which include plants, fish, beneficial arthropods, birds, and mammals. Ultimately, NTO testing can be viewed as attempts to manipulate a candidate organism into doing something it would not do in nature, either by providing access to hosts outside its natural range or by varying both the dose and route of exposure in order to produce infection and/or mortality. Tests are conducted with the expectation that the infectivity and the majority of the toxicity tests will be negative, because of the specificity of entomopathogens to arthropods. While it is always possible to kill an animal if the dose is high enough, through suffocation, embolism, or blockage of the gastrointestinal tract, these results have little relevance to the real world and the test results must be judged in the context of the experimental protocol. An adverse outcome in any one screen may not be grounds for automatic rejection but further tests will probably be conducted to quantify the effect. The tests outlined in this chapter for the assessment of oral, dermal, pulmonary and intra-peritoneal toxicity are based on the registration requirements of the United States Environmental Protection Agency and guidelines published by the World Health Organization. These screens may serve as a starting point for the registration of an entomopathogen or a point of comparison for an existing protocol.

Spread of Aspergillus flavus by Navel Orangeworm (Amyelois transitella) on Almond

Navel orangeworm (NOW) damage to almond is correlated with increased incidence of aflatoxin contamination caused by Aspergillus flavus. However, no reports demonstrate a causative relationship between NOW feeding and A. flavus infection. To demonstrate the potential of NOW to act as a vector of A. flavus on almond, NOW eggs were dusted with A. flavus and incubated in microchambers adjacent to but not touching agar plates or almond kernels. Following egg hatch, A. flavus colonies developed on agar along trails left by NOW larvae. Almond kernels damaged with A. flavus-carrying NOW showed higher incidence of A. flavus colonization and aflatoxin contamination than control treatments. Interestingly, levels of aflatoxin in NOW-damaged, A. flavus-infected almond were significantly higher than control treatments, even in the absence of visible fungal growth. Commercial almond orchards had a relatively low level of contamination with Aspergillus section Flavi in spring and early summer and a high level during summer, corresponding with the higher level of NOW infestation of the crop. Our study demonstrates that NOW is capable of vectoring A. flavus to almond, and that monitoring and sorting of almond kernels for insect damage is warranted to limit aflatoxin contamination potential both before and after harvesting.

Effects of a Naturally Occurring and a Synthetic Synergist on Toxicity of Three Insecticides and a Phytochemical to Navel Orangeworm (Lepidoptera: Pyralidae)

ABSTRACT The navel orangeworm, Amyelois transitella (Walker) (Lepidoptera: Pyralidae), is the most destructive lepidopteran pest of almonds [Prunus dulcis (Mill.) D.A.Webb] and pistachios (Pistacia vera L.) in California and is a serious problem in figs (Ficus carica L.) and walnuts (Juglans spp.). In addition to direct damage, larval feeding leaves nuts vulnerable to infection by Aspergillus spp., fungi that produce toxic aflatoxins. A potentially safe and sustainable approach for managing navel orangeworm in orchards may be to use natural essential oil synergists to interfere with this insects ability to detoxify insecticides and phytochemicals. We tested the effects of a naturally occurring plant-derived chemical, myristicin, and a synthetic inhibitor of cytochrome P450 monooxygenases (P450s), piperonyl butoxide, on the toxicity of three insecticides (&agr;-cypermethrin, &tgr;-fluvalinate, and methoxyfenozide [Intrepid]) and a phytochemical (xanthotoxin) to A. transitella. Piperonyl butoxide significantly synergized &agr;-cypermethrin and &tgr;-fluvalinate, whereas myristicin synergized only &agr;-cypermethrin. Piperonyl butoxide synergized the toxicity of xanthotoxin as early as 72 h after exposure, whereas myristicin synergized xanthotoxin after 120 h. In view of these findings and the limited availability of environmentally safe synthetic insecticides for sustainable management, particularly in organic orchards, myristicin is a potential field treatment in combination with insecticides to reduce both navel orangeworm survival and aflatoxin contamination of nuts. In addition, this study demonstrates that in A. transitella the insect growth regulator methoxyfenozide is not detoxified by P450s.

Field efficacy and application timing of methoxyfenozide, a reduced-risk treatment for control of navel orangeworm (Lepidoptera: Pyralidae) in almond.

ABSTBACT Large-scale field efficacy trials of methoxyfenozide (Intrepid), a reduced-risk molting agonist insecticide, were conducted in 2004 and 2005 in an orchard containing ‘Nonpareil’ and ‘Sonora’ almonds [Prunus dulcis (Mill.) D.A. Webb] located in Kern County, CA. Methoxyfenozide applied one to three times, the organophosphate phosmet (Imidan) alone or in combination with methoxyfenozide, or the pyrethroid permethrin (Perm-Up) were tested for efficacy against the primary lepidopteran pest navel orangeworm, Amyelois transitella (Walker) (Lepidoptera: Pyralidae), and three other lepidopteran pests of almond: oriental fruit moth, Grapholita molesta (Busck); obliquebanded leafroller, Choristoneura rosaceana (Harris); and peach twig borer, Anarsia lineatella Zeller. Two or three applications of methoxyfenozide (bracketing hull split or spring plus bracketing hull split) were more effective than a single hull split application of phosmet, phosmet combined with permethrin, or methoxyfenozide. In these trials, a spring application followed by a posthull split application was as effective as the applications bracketing hull split. Navel orangeworm accounted for >60% of the total damage, whereas oriental fruit moth and peach twig borer were the dominant secondary pests. In experiments conducted in 2010 to assess the direct toxicity of methoxyfenozide to navel orangeworm eggs under field conditions, exposure to methoxyfenozide reduced survival by 96–99%. We conclude that this reduced-risk insecticide is effective, although its efficacy is maximized with more than one well-timed application.

Protracted emergence of overwintering Amyelois transitella (Lepidoptera: Pyralidae) from pistachios and almonds in California.

ABSTRACT The navel orangeworm, Amyelois transitella (Walker), is the primary insect pest of pistachios and almonds in California. Four years of research (2002–2006) were conducted in Madera and Kern Counties to elucidate the pattern of adult emergence of the overwintering navel orangeworm population. Springtime emergence from unharvested (mummy) nuts was protracted (600 degree-days or more from 1 January of each year) and in 2004 and 2006 extended to mid-July. The population structure, sex ratio, and timing of emergence differed between pistachio and almond mummies, Pistachio populations had a significantly greater proportion of late stage individuals compared with almond mummies, 85.7 versus 34.1%. The sex ratio of adults emerging from pistachio mummies was significantly skewed with a ratio 57:43 male:female compared with 50:50 in almond mummies. Emergence from mummies held outdoors (variable temperature) began in early March and continued through early June in both pistachio mummies and almond mummies. The adult emergence pattern from pistachio mummies contained a single emergence peak, whereas emergence from almond mummies occurred in multiple peaks. These same patterns occurred when mummies were held at constant temperature, and the emergence peak from pistachio mummies occurred sooner. The impact of these findings on understanding navel orangeworm population dynamics and current control recommendations is discussed.

Mechanism of Resistance Acquisition and Potential Associated Fitness Costs in Amyelois transitella (Lepidoptera: Pyralidae) Exposed to Pyrethroid Insecticides.

ABSTRACT The polyphagous navel orangeworm, Amyelois transitella (Walker) (Lepidoptera: Pyralidae), is the most destructive pest of nut crops, including almonds and pistachios, in California orchards. Management of this insect has typically been a combination of cultural controls and insecticide use, with the latter increasing substantially along with the value of these commodities. Possibly associated with increased insecticide use, resistance has been observed recently in navel orangeworm populations in Kern County, California. In studies characterizing a putatively pyrethroid-resistant strain (R347) of navel orangeworm, susceptibility to bifenthrin and &bgr;-cyfluthrin was compared with that of an established colony of susceptible navel orangeworm. Administration of piperonyl butoxide and S,S,S-tributyl phosphorotrithioate in first-instar feeding bioassays with the pyrethroids bifenthrin and &bgr;-cyfluthrin produced synergistic effects and demonstrated that cytochrome P450 monooxygenases and carboxylesterases contribute to resistance in this population. Resistance is therefore primarily metabolic and likely the result of overexpression of specific cytochrome P450 monooxygenases and carboxylesterase genes. Resistance was assessed by median lethal concentration (LC50) assays and maintained across nine generations in the laboratory. Life history trait comparisons between the resistant strain and susceptible strain revealed significantly lower pupal weights in resistant individuals reared on the same wheat bran-based artificial diet across six generations. Time to second instar was greater in the resistant strain than the susceptible strain, although overall development time was not significantly different between strains. Resistance was heritable and may have an associated fitness cost, which could influence the dispersal and expansion of resistant populations in nut-growing areas in California.