Dynamics of the endosymbiont Rickettsia in an insect pest.

A new heritable bacterial association can bring a fresh set of molecular capabilities, providing an insect host with an almost instantaneous genome extension. Increasingly acknowledged as agents of rapid evolution, inherited microbes remain underappreciated players in pest management programs. A Rickettsia bacterium was tracked sweeping through populations of an invasive whitefly provisionally described as the "B" or "MEAM1" of the Bemisia tabaci species complex, in the southwestern USA. In this population, Rickettsia provides strong fitness benefits and distorts whitefly sex ratios under laboratory conditions. In contrast, whiteflies in Israel show few apparent fitness benefits from Rickettsia under laboratory conditions, only slightly decreasing development time. A survey of B. tabaci B samples revealed the distribution of Rickettsia across the cotton-growing regions of Israel and the USA. Thirteen sites from Israel and 22 sites from the USA were sampled. Across the USA, Rickettsia frequencies were heterogeneous among regions, but were generally very high, whereas in Israel, the infection rates were lower and declining. The distinct outcomes of Rickettsia infection in these two countries conform to previously reported phenotypic differences. Intermediate frequencies in some areas in both countries may indicate a cost to infection in certain environments or that the frequencies are in flux. This suggests underlying geographic differences in the interactions between bacterial symbionts and this serious agricultural pest.

Mating behaviour, life history and adaptation to insecticides determine species exclusion between whiteflies.

1. Negative interspecific interactions, such as resource competition or reproductive interference, can lead to the displacement of species (species exclusion). 2. Here, we investigated the effect of life history, mating behaviour and adaptation to insecticides on species exclusion between cryptic whitefly species that make up the Bemisia tabaci species complex. We conducted population cage experiments independently in China, Australia, the United States and Israel to observe patterns of species exclusion between an invasive species commonly referred to as the B biotype and three other species commonly known as biotypes ZHJ1, AN and Q. 3. Although experimental conditions and species varied between regions, we were able to predict the observed patterns of exclusion in each region using a stochastic model that incorporated data on development time, mating behaviour and resistance to insecticides. 4. Between-species variation in mating behaviour was a more significant factor affecting species exclusion than variation in development time. Specifically, the ability of B to copulate more effectively than other species resulted in a faster rate of population increase for B, as well as a reduced rate of population growth for other species, leading to species exclusion. The greater ability of B to evolve resistance to insecticides also contributed to exclusion of other species in some cases. 5. Results indicate that an integrative analysis of the consequences of variation in life-history traits, mating behaviours and adaption to insecticides could provide a robust framework for predicting species exclusion following whitefly invasions.

Transgenic organisms expressing genes from Bacillus thuringiensis to combat insect pests.

Various subspecies (ssp.) of Bacillus thuringiensis (Bt) are considered the best agents known so far to control insects, being highly specific and safe, easily mass produced and with long shelf life.1 The para-crystalline body that is produced during sporulation in the exosporium includes polypeptides named δ-endotoxins, each killing a specific set of insects. The different entomopathogenic toxins of various Bt ssp. can be manipulated genetically in an educated way to construct more efficient transgenic bacteria or plants that express combinations of toxin genes to control pests.2 Joint research projects in our respective laboratories during the last decade demonstrate what can be done by implementing certain ideas using molecular biology with Bt ssp. israelensis (Bti) as a model system. Here, we describe our progress achieved with Gram-negative bacterial species, including cyanobacteria, and some preliminary experiments to form transgenic plants, mainly to control mosquitoes (Diptera), but also a particular Lepidopteran and Coleopteran pest species. In addition, a system is described by which environment-damaging genes can be removed from the recombinants thus alleviating procedures for obtaining permits to release them in nature.

Larvicidal activities against agricultural pests of transgenic Escherichia coli expressing combinations of four genes from Bacillus thuringiensis.

The genes cry1Ac and cry1Ca from Bacillus thuringiensis subsps. kurstaki HD-73 and aizawai 4J4, respectively, encoding delta-endotoxins against lepidopteran larvae were isolated, cloned and expressed in Escherichia coli, with and without cyt1Aa (encoding cytolytic protein) and p20 (accessory protein) from subsp. israelensis. Nine combinations of the genes under control of an early T7, P A1 inducible promoter, produced the encoding proteins. Toxicities were examined against larvae of three major agricultural pests: Pectinophora gossypiella, Helicoverpa armigera and Spodoptera littoralis. The clones expressing cyt1Aa, with or without p20, were not toxic. The clone expressing cry1Ac (pBt-1A) was the most toxic to P. gossypiella (LC50 of 0.27 x 10(8) cells g(-1)). Clone pBt-1CA expressing cry1Ca and cry1Ac displayed the highest toxicity (LC50 of 0.12 x 10(8) cells ml(-1)) against S. littoralis. Clone pBt-1CARCy expressing all four genes (cry1Ca, cry1Ac, p20, cyt1Aa) in tandem exhibited the highest toxicity to H. armigera (LC50 of 0.16 x 10(8) cells ml(-1)). Cyt1Aa failed to raise the toxicity of these Cry toxins against P. gossypiella and S. littoralis but significantly enhanced toxicity against H. armigera. Two additional clones expressing either cry1Ac or cry1Ca under tandem promoters, P A1 and P psbA (constitutive), displayed significantly higher toxicities (7.5- to 140-fold) than their counterparts with P A1 alone, reducing the LC50 values to below 10(7) cells ml(-1).

Reversal of resistance to pyriproxyfen in the Q biotype of Bemisia tabaci (Hemiptera: Aleyrodidae).

Pyriproxyfen, a juvenile hormone (JH) mimic, is a biorational insecticide that disrupts insect development. It is one of the principal insecticides being used to control Bemisia tabaci (Gennadius) on cotton, and has many environmentally positive attributes that make it compatible with integrated pest management (IPM) programs. In Israel, a high level of resistance to pyriproxyfen has been observed in several isolated regions. Here, tests were conducted to establish whether temporal refuges from exposure to pyriproxyfen could be useful for restoring the effectiveness of the compound. Resistance was found to decrease by a factor of 8 when exposure to pyriproxyfen was ceased for 13 generations. Reversal of resistance was accompanied with increased biotic fitness of the revertant colony. By incorporating experimental estimates of nymph survival, sex ratio, fecundity, egg hatching rate and developmental time, the seasonal cost per generation for resistant insects was estimated to be 25%. A genetic simulation model, optimized by empirical data from bioassays, predicted fitness cost per generation of 19% for resistant homozygous (RR) females and hemizygous (R) males, and produced rates of reversal similar to the experimental results. The model also predicted that, even after 5 years ( approximately 55 generations) without pyriproxyfen treatments, the frequency of the resistance allele (R) will still remain high (0.02). It is therefore concluded, on the basis of experimental and modeling results, that the effectiveness of temporal refuges for reversing development of resistance to pyriproxyfen in B. tabaci may be limited.

Effect of pyriproxyfen on developing stages and embryogenesis of California red scale (CRS), Aonidiella aurantii.

During the past decade, the insect growth regulator pyriproxyfen has been the most used insecticide for controlling the California red scale (CRS), Aonidiella aurantii (Maskell), an important pest in citrus groves worldwide. The aim of the present research was to develop bioassays for determining the effect of pyriproxyfen on developing stages and embryogenesis of CRS that can serve as baselines for monitoring resistance. Pyriproxyfen was very effective against first- and second-instar nymphs of a susceptible strain of CRS; 0.1 and 0.2 mg AI L(-1), resulting in 80 and 100% mortality of first-instar nymphs, and in 62.5 and 91.7% mortality of second-instar nymphs respectively. According to probit analysis, the LC(50) and LC(90) values of pyriproxyfen for first-instar nymphs were 0.03 and 0.15 mg AI L(-1), and for second-instar nymphs they were 0.05 and 0.25 mg AI L(-1) respectively. Pyriproxyfen was quite effective on first-instar nymphs of a CRS field strain originating from a citrus grove where two successive applications of the compound had been applied. Mortality rates were 86 and 100% at concentrations of 0.2 and 0.4 mg AI L(-1) respectively. Pyriproxyfen suppressed embryogenesis when CRS females were treated with pyriproxyfen. Young CRS females treated with 1.0 and 5.0 mg AI L(-1) pyriproxyfen produced 6.6 N(1)/F and 2.4 N(1)/F as compared with 10.9 N(1)/F in untreated control.

Identification and localization of a Rickettsia sp. in Bemisia tabaci (Homoptera: Aleyrodidae).

Whiteflies (Homoptera: Aleyrodidae) are sap-sucking insects that harbor "Candidatus Portiera aleyrodidarum," an obligatory symbiotic bacterium which is housed in a special organ called the bacteriome. These insects are also home for a diverse facultative microbial community which may include Hamiltonella, Arsenophonus, Fritchea, Wolbachia, and Cardinium spp. In this study, the bacteria associated with a B biotype of the sweet potato whitefly Bemisia tabaci were characterized using molecular fingerprinting techniques, and a Rickettsia sp. was detected for the first time in this insect family. Rickettsia sp. distribution, transmission and localization were studied using PCR and fluorescence in situ hybridizations (FISH). Rickettsia was found in all 20 Israeli B. tabaci populations screened but not in all individuals within each population. A FISH analysis of B. tabaci eggs, nymphs, and adults revealed a unique concentration of Rickettsia around the gut and follicle cells, as well as a random distribution in the hemolymph. We postulate that the Rickettsia enters the oocyte together with the bacteriocytes, leaves these symbiont-housing cells when the egg is laid, multiplies and spreads throughout the egg during embryogenesis and, subsequently, disperses throughout the body of the hatching nymph, excluding the bacteriomes. Although the role Rickettsia plays in the biology of the whitefly is currently unknown, the vertical transmission on the one hand and the partial within-population infection on the other suggest a phenotype that is advantageous under certain conditions but may be deleterious enough to prevent fixation under others.

Effect of Zohar LQ-215, a biorational surfactant, on the sweetpotato whitefly Bemisia tabaci (biotype b).

Zohar LQ-215, a surfactant based on plant oils, able to control nymphs of the whitefly, Bemisia tabaci (Gennadius), under laboratory and field conditions. To evaluate the effects of the surfactant on the nymph stages of whitefly under laboratory conditions, potted cotton seedlings infested with 1st- or 3rd-instars were treated with the compound aqueous solutions. LC90 values of Zohar LQ-215 on 1st and 3rd-nymphs, based on mortality curves, were 0.78% and 1.14%, respectively. Adult mortality of 12% and 19% was obtained at concentrations of 0.5% and 1%, respectively. Under both laboratory and, in some cases, under field conditions, greater mortality was achieved when combining Zohar LQ-215 with the insect growth regulator buprofezin than when either insecticide was applied separately. Our results indicate that Zohar LQ-215 could serve as a potential compound for controlling whiteflies under light to moderate infestation and could be used in combination with other rational insecticides such as buprofezin for controlling whiteflies in integrated pest management programs.

Biorational insecticides: mechanism and cross-resistance.

Potency and cross-resistance of various biorational insecticides, exemplified by the whitefly Bemisia tabaci, have been studied. Bemisia tabaci were exposed to the juvenile hormone mimic pyriproxyfen for the past 12 years resulting in an over 2,000-fold resistance, but there was no appreciable cross-resistance with the benzoylphenyl urea novaluron. Similarly, no cross-resistance was found between pyriproxyfen and the two neonicotinoids, acetamiprid and imidacloprid. On the other hand, a slight cross-resistance of 5-13-fold was observed with another neonicotinoid thiamethoxam. Among the neonicotinoids, a resistant strain of B. tabaci to thiamethoxam (approximately 100-fold) showed no appreciable cross-resistance to either acetamiprid or imidacloprid, while another strain 500-fold resistant to thiamethoxam resulted in a mild of 4-6-fold resistance to acetamiprid and imidacloprid. In other assays, B. tabaci strain resistant to thiamethoxam (approximately 100-fold) had no cross-resistance to pyriproxyfen. Our findings indicate that no appreciable cross-resistance was observed between the benzoylphenyl urea novaluron, the juvenile hormone mimic pyriproxyfen, and the neonicotinoids acetamiprid and imidacloprid. Hence, these compounds could be used as components in insecticide resistance management programs.

Biotypes B and Q of Bemisia tabaci and their relevance to neonicotinoid and pyriproxyfen resistance.

Resistance monitoring for Bemisia tabaci field populations to the juvenile hormone mimic, pyriproxyfen, was conducted from 1996 to 2003 in commercial cotton fields in two areas of Israel: the Ayalon Valley (central Israel) and the Carmel Coast (northwestern Israel). Although the use of pyriproxyfen ceased in these areas in 1996-1997 (because of the resistance), resistance levels to pyriproxyfen declined to some extent in the fields but remained quite stable, and the susceptibility has not been totally restored. Two strains of B. tabaci collected from the Ayalon Valley in the late 1999 and 2002 cotton seasons (AV99L, AV02L) were assayed for their susceptibility to pyriproxyfen at F1, and subsequently a line of each strain was kept under controlled conditions without exposure to insecticides. After maintenance of more than 20 generations under laboratory conditions, the resistance to pyriproxyfen in the untreated strains substantially declined. This decline was concurrent with a replacement of Q biotype by B-type under non-insecticidal regimes; apparently B biotype was more competitive than the pyriproxyfen-resistant Q-type. Selection under controlled conditions with neonicotinoids on these B. tabaci strains resulted in continued pyriproxyfen resistance, predominantly of Q biotype. Based on our data, applications of either pyriproxyfen or neonicotinoids may select for biotype Q, which would survive to a greater degree where these insecticides are applied.

Dynamics of resistance to the neonicotinoids acetamiprid and thiamethoxam in Bemisia tabaci (Homoptera: Aleyrodidae).

The dynamics of resistance in the sweetpotato whitefly, Bemisia tabaci (Gennadius), to the neonicotinoids acetamiprid and thiamethoxam was studied extensively in cotton fields in Israel during the cotton-growing seasons 1999-2003. Whitefly strains were collected in early and late seasons mainly in three locations in northern, central, and southern Israel. The whiteflies were assayed under laboratory conditions for susceptibility to neonicotinoids, as part of the Israeli cotton insecticide resistance management strategy. Selections to both acetamiprid and thiamethoxam and cross-resistance between them also were conducted in the laboratory. Although no appreciable resistance to acetamiprid was observed up to 2001, a slight increase of approximately five-fold resistance was detected during 2002 and 2003. However, from 2001 to 2003 thiamethoxam resistance increased >100-fold in the Ayalon Valley and Carmel Coast cotton fields. In cross-resistance assays with both neonicotinoids, the strain that had been selected with thiamethoxam for 12 generations demonstrated almost no cross-resistance to acetamiprid, whereas the acetamiprid-selected strain exhibited high cross-resistance of >500-fold to thiamethoxam.

Novaluron (Rimon), a novel IGR: potency and cross-resistance.

The potency of novaluron on laboratory susceptible and field strains of S. littoralis resembles that of chlorfluazuron and both compounds are about 20-fold more potent than teflubenzuron. No appreciable resistance to novaluron or chlorfluazuron was observed in a field strain of Spodoptera littoralis collected from cucumber field in the central part of Israel. On the other hand, the field strain showed a mild resistance of about 4-fold to teflubenzuron as compared to the laboratory susceptible strain. A very resistant colony of Bemisia tabaci to pyriproxyfen (1,200- to 2,000-fold) showed no appreciable cross-resistance to novaluron. Two field colonies of B. tabaci pressurized with acetamiprid or thiamethoxam for 22 generations, resulting in a 30- to 50-fold resistance to acetamiprid and thiamethoxam, has no cross-resistance to novaluron. The above results are of special interest, indicating a possible alternation between novaluron, pyriproxyfen, and neonicotinoids in insecticide-resistance management programs aiming at preventing resistance development to these novel groups of insecticides against important pests such as whitefly and lepidopteran species.

Inheritance of pyriproxyfen resistance in the whitefly, Bemisia tabaci (Q biotype).

The inheritance of resistance to pyriproxyfen, an insect growth regulator (a juvenoid, with ovicidal and larvicidal activities), was studied in the whitefly Bemisia tabaci (Gennadius). Two parental strains, both belonging to Q biotype, were assayed with pyriproxyfen; a susceptible strain (ALM-1) originating from Spain and a pyriproxyfen-resistant one (Pyri-R) from Israel. The resistance ratio between the two parental strains was approximately 7,000-fold. Concentration-mortality lines for F(1) heterozygous females from reciprocal crosses (SS female symbol X R male symbol and RR female symbol X S male symbol ) were derived by statistical modelling and proved intermediate to those of the parents. The pooled degree of dominance from both reciprocal crosses was +0.26, indicating that resistance was incompletely or partially dominant. Mortality curves for F(2) males produced by virgin F(1) heterozygous females displayed a broad plateau at 50% mortality, indicating that resistance to pyriproxyfen in B. tabaci is conferred primarily by a mutant allele at a single locus. The role of arrhenotoky in influencing the mode of inheritance of resistance, and its selection in field populations, is discussed.

Emamectin, a novel insecticide for controlling field crop pests.

Emamectin is a macrocyclic lactone insecticide with low toxicity to non-target organisms and the environment, and is considered an important component in pest-management programmes for controlling field crop pests. It is a powerful compound for controlling the cotton bollworm Helicoverpa armigera (Hübner). A spray concentration of 25 mg AI litre-1 in a cotton field resulted in over 90% suppression of H armigera larvae up to day 28 after treatment, while similar mortality of the Egyptian cotton leafworm Spodoptera littoralis Boisduval, under the same conditions, was maintained for 3 days only. Emamectin is a potent compound for controlling the western flower thrips Frankliniella occidentalis (Pergande) under both laboratory and field conditions and its activity on adults was over 10-fold greater than that of abamectin. Spray concentrations of 10 and 50 mg AI litre-1 in Ageratum houstonianum Mill flowers resulted in total suppression of adults up to day 11 and of larvae up to day 20 after treatment. Under standard laboratory conditions, emamectin exhibits a considerable activity on the whitefly Bemisia tabaci (Gennadius) and the leafminer Liriomyza huidobrensis (Blanchard). Further studies are required to evaluate its potential activity on the latter pests under field conditions.

Dynamics of insecticide resistance in Bemisia tabaci: a case study with the insect growth regulator pyriproxyfen.

The dynamics of pyriproxyfen resistance in Bemisia tabaci (Gennadius) have been studied intensively in cotton fields and greenhouses in Israel. High resistance to pyriproxyfen evolved one year after its introduction for use on flowers in greenhouses, after three successive applications. After ten years of pyriproxyfen use in cotton fields, with only one application per season, a high level of resistance was observed, but its rate of development differed among localities. It is hypothesized that these differences reflect temporal and spatial variations in the availability of alternative host plants for B tabaci, and that pyriproxyfen resistance evolved most readily in geographically isolated areas. It is also likely that the occurrence and development of resistance is partially biotype-related. Pyriproxyfen use has ceased in high-resistance localities, but in areas with low frequencies of resistance, cotton growers have continued to achieve acceptable control of B tabaci with pyriproxyfen. Due to the absence of applications of pyriproxyfen in some cotton fields, resistance levels tended to decline between 1998 and 2001. Laboratory experiments support the hypothesis that this decline reflects, in part, fitness costs associated with pyriproxyfen resistance.

Novaluron (Rimon), a novel IGR--mechanism, selectivity and importance in IPM programs.

Novaluron (Rimon), a new IGR, acts by both ingestion and contact. It is a powerful toxicant for controlling lepidopteran larvae resulting in LC-90 values of 0.42 and 0.30 mg a.i./liter for Spodoptera littoralis and S. exigua, respectively. Its residual activity under field conditions ranges between 10 and 30 days depending on environmental conditions. The compound is a powerful toxicant against developing stages of whiteflies, resulting in LC-90 values of 0.68 and 8.56 mg a.i./liter for Bemisia tabaci and Trialeurodes vaporariorum, respectively. It has translaminar activity, enabling the control of the leaf miner Liriomyza huidobrensis at concentrations ranging between 5 and 45 mg a.i./liter. Novaluron has no cross-resistance with other leading compounds for controlling whiteflies such as buprofezin and pyriproxyfen. It is a rain fast compound suitable in the tropics and in rainy seasons. It has no appreciable effect on parasitoids and phytoseiids and a mild effect on other natural enemies and may be considered a potential component in IPM programs. The compound is in the process of commercialization worldwide by Makhteshim Chemical Works for controlling agricultural pests.