Biological control potential of Turkish entomopathogenic nematodes against the Colorado potato beetle,Leptinotarsa decemlineata

Three Turkish isolates of the entomopathogenic nematodes Steinernema carpocapsae, Steinernema feltiae and Heterorhabditis bacteriophora were evaluated under laboratory conditions (in different temperatures and doses) for their biocontrol efficiency against last instar Leptinotarsa decemlineata. Herein, the effects of infective juveniles in aqueous suspension against L. decemlineata were evaluated. S. feltiae appeared to be the most pathogenic nematode among the tested species.     

Effect of an entomopathogenic nematode,Steinernema carpocapsae on haemocyte profile and phenoloxidase activity of the Colorado potato beetle,Leptinotarsa decemlineata

The Colorado potato beetle (CPB), Leptinotarsa decemlineata Say is the most destructive insect pest of potato in many areas of the world. Little is known about the haemocyte types of the CPB and its plasma phenoloxidase (PO). In this regard, we investigated the haemocyte profile and PO of CPB and its immune response to the entomopathogenic nematode, Steinernema carpocapsae. Five types of haemocytes, the plasmatocytes (~67.4%), granulocytes (~23.5%), oenocytoids (~2.4%), spherulocytes (~0.25%) and prohaemocytes (~6.5%) were identified in fourth instar CPB larvae. Total haemocyte counts (THCs) were significantly reduced in nematode-injected insects compared with control groups (P < 0.05). Nematode cellular encapsulation observed in haemolymph of nematode-injected insects may partially explain decreased THCs. Plasma PO assay showed increased PO activity in nematode-injected insects compared with control groups (P < 0.05). Plasma PO assay on native polyacrylamide gel electrophoresis (PAGE) assay with L-3, 4-dihydroxyphenylalanine as substrate showed five bands (with molecular weights of approximately 200, 118, 68.5, 62.5 and 58.75 kDa).     

Efficacy of some medicinal plant extracts and essential oils against Colorado potato beetle,Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae)

The Colorado potato beetle (CPB), Leptinotarsa decemlineata Say is one of the most important defoliator pests of potato in the world and it often causes extremely large potato yield losses. Potatoes are the preferred hosts for the pest, but it may feed and reproduce on a number of other plants in the Solanaceae family. Public concern related to pesticides and their residues in and on the foods had prompted a rise of consumer interest in organically produced foods. There have been growing efforts to detect and introduce suitable plant compounds that they have insecticidal properties. However, discovering of plant extracts for possible use in control of this pest requires more studying about plant extracts and compounds. Since resistance of CPB to common chemical insecticides is well documented and potato is one of the most prominent nutritious food products for many people in many countries, we examined the effect of essential oils (EOs) of European pennyroyal, lavander, mint, oregano and savory and methanolic extracts of fumitory, licorice and oregano on the pest. These plants were selected because they have medicinal properties and they are safe to human and environment. Adult CPBs were exposed to mentioned plant extracts and essential oils. LC₅₀ values for EOs of lavander and European pennyroyal were 4154 and 3561 ppm, respectively. The results demonstrated that essential oil of European pennyroyal (Mentha longifolia) would be suitable compound to control the pest, but essential oil of mint (Mentha spicata) was not effective against the pest. Also it is notable that at all treatments, the amount of adult feeding was very low.     

Intraspecific mitochondrial DNA variation in the Colorado potato beetle,Leptinotarsa decemlineata (Coleoptera: Chrysomelidae)

Restriction endonuclease analyses of mitochondrial DNA (mtDNA) were used to examine genetic variability and population structure inLeptinotarsa decemlineata (Say). A group of three enzymes, EcoRI, HpaI, and PstI, was used to reveal polymorphism both within and among some of the 10 populations tested, yielding 16 haplotypes in combination. The frequencies of these 16 haplotypes differed significantly across geographic regions, indicating some partitioning of mtDNA haplotypes. Estimates of mtDNA sequence divergence (δ) between haplotypes ranged from 0.016 to 0.135%, suggesting local differentiation of mtDNA in some populations. Analysis of these data suggests that Texas was colonized by more than one mtDNA lineage, most likely originating in Mexico. We hypothesize that a larger founder size for the initial introductions or high levels of variability in the parent population at the edge of the CPB expanding range led to the initial partitioning of haplotypes observed in samples from Texas.     

Laboratory and greenhouse evaluation of a synthetic host volatile attractant for Colorado potato beetle, Leptinotarsa decemlineata (Say)

Abstract 1 The attractiveness of potato plants treated with a synthetic host volatile blend [(Z)‐3‐hexenyl acetate (+/–)‐linalool, and methyl salicylate] to newly emerged and 5‐day‐old adult Colorado potato beetle, Leptinotarsa decemlineata (Say), was compared at four doses against untreated control plants and plants treated with an azadirachtin‐based antifeedant in greenhouse cage arenas. 2 Attractant‐treated plants (derived release rates of 0, 5.7, 17.1 or 57 µg/h) were significantly more attractive than untreated control plants to newly emerged and 5‐day‐old adults only at 57 µg/h. 3 Attractant‐treated plants were significantly more attractive than antifeedant‐treated plants to newly emerged and 5‐day‐old adults at the 5.7 µg/h treatment level and higher. Mean insect density on attractant‐treated plants in the attractant/antifeedant study was significantly higher than in the attractant/control study. 4 Habituation to the synthetic attractant was evaluated by exposing adult beetles to the synthetic attractant for 0, 1, 2.5, 4, 8, 12 or 16 h, before release into a wind tunnel in which an attractant‐baited plant model was placed at the upwind end. Insects exposed to the synthetic host attractant for ≤ 8 h moved to the synthetic attractant‐baited plant model whereas insects exposed to the synthetic host attractant for 12 and 16 h did not. Furthermore, beetles exposed to the synthetic attractant for 0 and 1 h moved at rates greater than, or equal to, the median whereas beetles exposed for longer time periods moved at rates significantly less than the median. 5 These results demonstrate the potential for using the synthetic plant attractant and an antifeedant as components in a stimulo‐deterrent strategy for management of the Colorado potato beetle as shown by us in another study.     

Diet-related plasticity of the digestive proteolytic system in larvae of the Colorado potato beetle (Leptinotarsa decemlineata Say)

Quantitative and qualitative changes in digestive proteolytic activities were monitored in fourth-instar larvae of the Colorado potato beetle (Leptinotarsa decemlineata Say) subjected to three different leaf diets. Depending on the diet, the larvae exhibited variable growth rates, similar for potato (Solanum tuberosum) and eggplant (Solanum melongena) diets but lower for the tomato (Lycopersicon esculentum) diet. Interestingly, these growth rates were not associated with total protease activity in the midgut. While growth of tomato-fed insects was negligible, midgut protease activity in these insects was 1.5 and 4.2 times higher than that measured for potato- and eggplant-fed insects, respectively. As seen on gelatin-containing polyacrylamide gels, midgut extracts from insects that ingested eggplant leaves contained only a few proteinase forms, while numerous forms were observed in extracts of potato- and tomato-fed larvae. Although several forms were common to the three diets, their relative importance in the insect midgut varied. This diet-related plasticity of the digestive proteolytic system in Colorado potato beetle larvae leads one to question the potential for control approaches based on the inhibition of digestive proteases. Arch. Insect Biochem. Physiol. 36:241–250, 1997. © 1997 Wiley-Liss, Inc.     

Biocontrol potential of the entomopathogenic nematodes Heterorhabditis bacteriophora and Steinernema carpocapsae on cucurbit fly,Dacus ciliatus (Diptera: Tephritidae)

Entomopathogenic nematodes (EPNs) from the families Steinernematidae and Hererorhabditidae are considered excellent biological control agents against many insects that damage the roots of crops. In a regional survey, native EPNs were isolated, and laboratory and greenhouse experiments were conducted to determine the infectivity of EPNs against the cucurbit fly, Dacus ciliatus Loew (Diptera: Tephritidae). Preliminary experiments showed high virulence by a native strain of Heterorhabditis bacteriophora Poinar (Rhabditida: Heterorhabditidae) and a commercial strain of Steinernema carpocapsae Weiser (Rhabditida: Steinernematidae). These two strains were employed for further analysis while another native species, Steinernema feltiae, was excluded due to low virulence. In laboratory experiments, larvae and adult flies were susceptible to nematode infection, but both nematode species induced low mortality on pupae. S. carpocapsae had a significantly lower LC₅₀ value against larvae than H. bacteriophora in filter paper assays. Both species of EPNs were effective against adult flies but S. carpocapsae caused higher adult mortality. When EPN species were applied to naturally infested fruit (150 and 300 IJs/cm²), the mortality rates of D. ciliatus larvae were 28% for S. carpocapsae and 12% for H. bacteriophora. Both EPN strains successfully reproduced and emerged from larvae of D. ciliates. In a greenhouse experiment, H. bacteriophora and S. carpocapsae had similar effects on fly larvae. Higher rates of larval mortality were observed in sandy loam and sand soils than in clay loam. The efficacy of S. carpocapsae and H. bacteriophora was higher at 25 and 30°C than at 19°C. The results indicated that S. carpocapsae had the best potential as a biocontrol agent of D. ciliatus, based on its higher virulence and better ability to locate the fly larvae within infected fruits.     

Field efficacy of entomopathogenic nematodes against the beetle Maladera matrida (Coleoptera: Scarabaeidae)

Single, double and triple releases of the entomopathogenic nematode Heterorhabditis bacteriophora Poinar, reduced the population of the beetle Maladera matrida Argaman, infesting peanuts (’Shulamit’ cv.) by 70, 75 and 93% respectively in microplot tests. Simultaneous and late (2 weeks after infestation) applications reduced beetle numbers by 63 and 79% respectively, in the microplots, while early application (2 weeks prior to infestation) did not reduce the beetle population. In a field trial, reductions in insect population and damage to the crop were achieved by early treatment with the nematode as well as by Heptachlor, leading to reductions in the insect population of 60 and 90% respectively, when recorded 4 weeks after nematode application. However, the nematode treatment did not maintain its effectiveness for a longer period and pest damage increased to the same level as the untreated control after 7 weeks. When the nematodes were applied at different concentrations (0.25–1.0 x 10⁶ infective juveniles (IJs) m^‐2) their effectiveness was not related to the concentration level. The only significant (P < 0.05) reduction in insect levels was recorded in the treatment with 0.5 X 10⁶ IJs m^‐2. In a second field trial, both H. bacteriophora and Steinernema glaseri reduced insect populations significantly (P < 0.05) by approximately 50% in comparison to the control. In the third trial, treatment with H. bacteriophora resulted in a decrease in insect population of 90% while treatment with S. carpocapsae reduced the grub numbers by 40% in comparison to the control. A differential susceptibility of various grub developmental stages was recorded in the field. The small grubs (I‐4 mm long, lst‐2nd larval stage) were not affected by the nematode treatments while the numbers of medium and large size grubs were reduced by 2‐ and 3‐fold respectively in the various tests. Nematodes were recovered by ‘nematode traps’ containing Galleria mellonella larvae from treated field plots 78 days after application. The implications of the results from the present studies on the use of entomopathogenic nematodes are discussed in relation to the development of an integrated pest management programme.     

Lethal and sublethal effects of Iranian isolates of Steinernema feltiae and Heterorhabditis bacteriophora on the Colorado potato beetle, Leptinotarsa decemlineata

To determine the LC₅₀ values of two entomopathogenic nematodes against Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae) prepupae, different concentrations of the nematodes were tested in soil. Because of the different temperature requirements of the two nematode species, bioassay experiments were conducted at 20 ± 1°C and 27 ± 2°C for Steinernema feltiae Filipjev (Rhabditida: Steinernematidae) and Heterorhabditis bacteriophora Poinar (Rhabditida: Heterorhabditidae), respectively. Both the isolates were effective against L. decemlineata. LC₅₀ values of H. bacteriophora against progeny of field-collected adults and laboratory-reared adults were estimated as 8.5 and 7.6 IJ per prepupa, respectively. For S. feltiae the value was calculated as 51.2 IJ per prepupa against offspring of laboratory-reared adults of L. decemlineata only. Cellular encapsulation of both nematode species was observed. Sublethal nematode concentrations caused wing deformation and delayed metamorphosis which may affect Colorado potato beetle adult fitness.     

Synthetic host volatiles increase efficacy of trap cropping for management of Colorado potato beetle, Leptinotarsa decemlineata (Say)

Abstract 1 The attractiveness of pitfall traps baited with a synthetic host volatile attractant to colonizing adult Colorado potato beetle, Leptinotarsa decemlineata (Say) was evaluated in a field setting. 2 Significantly more postdiapause, colonizing adult L. decemlineata were captured in baited than unbaited pitfall traps. 3 The potential for this synthetic kairomone to enhance the efficacy of trap cropping as a management tool was evaluated by comparing conventionally managed plots with like‐sized plots bordered by either attractant‐treated trap crop or untreated trap crop. 4 More postdiapause, colonizing adults, egg masses and small larvae were present in attractant‐treated trap crops than in untreated trap crops. 5 There were no significant differences in egg mass and small larvae densities between plots bordered by attractant‐treated trap crops and conventionally managed plots, but there were significantly fewer large larvae and adult beetles in conventionally managed plots. 6 Plant canopy area of conventionally managed plots was significantly greater than in plots bordered by either type of trap crop. 7 Yields for conventionally managed plots and plots bordered by attractant‐treated trap crops did not differ, and less insecticide (44%) was applied to plots bordered by attractant‐treated trap crops.     

Host density effects on efficacy of entomopathogenic nematodes against white grub (Coleoptera: Scarabaeidae) species

We tested the effect of host density on entomopathogenic nematode efficacy in 1-L pots with grass and soil. In four experiments, combinations ranged from somewhat resistant hosts (oriental beetle, Anomala orientalis, or northern masked chafer, Cyclocephala borealis, with Heterorhabditis bacteriophora) over more susceptible hosts (Japanese beetle, Popillia japonica, with Steinernema glaseri) to a highly susceptible host (P. japonica and S. scarabaei). In each experiment, four larval densities were exposed to two nematode rates over a 14-day period. A significant effect of host density on nematode efficacy occurred only in the A. orientalis–H. bacteriophora combination, but there was no clear trend in the data. This suggests that an exhaustion of available nematode populations to less lethal levels by high host numbers was counteracted by other factors such as increased chances for nematode-host contact and increased host susceptibility due to stress via reduced food resources and increased aggression between larvae.     

新疆北部马铃薯甲虫幼虫对常规杀虫剂的抗药性（英文）

为了了解并掌握马铃薯甲虫Leptinotarsa decemlineata (Say)在新疆的抗性水平及动态, 为科学用药提供理论依据, 利用点滴法测定了新疆北部马铃薯甲虫6个田间种群(乌鲁木齐、 阜康、 昌吉、 阿勒泰、 尼勒克和特克斯) 的4龄幼虫对14种常规药剂的敏感性, 并测试了磷酸三苯酯（TPP）、 顺丁烯二酸二乙酯（DEM）和氧化胡椒基丁醚（PBO）的增效作用。结果表明: 与已报道的基线相比, 特克斯种群对几种药剂都敏感, 因此确定特克斯田间种群为敏感品系。与特克斯敏感品系相比, 昌吉种群对三氟氯氰菊酯、 溴氰菊酯、 克百威及丁硫克百威的抗性倍数分别为108.8, 30, 17.6和24.7, 而尼勒克种群对这4种药剂的抗性倍数分别为29.4, 42.5, 10.1和89.9。此外, 昌吉和阜康种群对高效氯氰菊酯也分别产生8.9和13.0倍的中等抗性。增效测定结果显示: 对于尼勒克种群, PBO对三氟氯氰菊酯有明显增效作用; 对于昌吉种群, PBO, TPP和DEM对克百威有明显增效作用。因此推断解毒酶活性增强可能是新疆马铃薯甲虫对三氟氯氰菊酯和克百威的抗性机制之一。由于增效作用不能完全消除抗性, 因此新疆马铃薯甲虫的抗药性还可能涉及到其他机制。     

Age- and diapause-related acid and alkaline phosphatase activities in the intestine and malpighian tubules of the Colorado potato beetle, Leptinotarsa decemlineata (Say)

Specific activities for soluble (s) and membrane (m)-bound acid (ACP) and alkaline phosphatases (ALP) were determined in the midgut, hindgut, and Malpighian tubules for developing, prediapausing, and diapausing adult Colorado potato beetles, Leptinotarsa decemlineata (Say). High ACP activities were found in the hindgut and Malpighian tubules while high ALP activities were found in the Malpighian tubules. Variation in both ACP and ALP activities in each tissue reflects fluctuation in protein synthesis and secretion involved with digestion, excretion, and other unknown functions. Phosphatase activities in the tissues examined show the dynamic nature of diapause in this insect. Diapausing beetles showed increases in phosphatase activity after hormone treatments. JHA treatments increased s-ACP and m-ACP activities in all tissues but 20-HE did not increase activity in any tissue. Allatotropin tended to mimic the effects of JHA treatment. The s-ALP activity was also increased in all tissues whereas m-ALP was increased in the midgut and hindgut by JHA treatment. Malpighian tubule m-ALP activity was only increased by 20-HE treatments. Allatotropin was not as effective in increasing ALP activities as it was with ACP activities.     

Isolation and characterization of Locusta migratoria accessory gland myotropin I (Lom-AG-MT-I) from the brain of the Colorado potato beetle,Leptinotarsa decemlineata

A novel myotropic Colorado potato beetle peptide, active in the Locusta oviduct motility assay, was isolated from a methanolic extract of 9,000 brain complexes of adult Leptinotarsa decemlineata by means of HPLC. Its sequence is Gly-Phe-Lys-Asn-Val-Ala-Leu-Ser-Thr-Ala-Arg-Gly-Phe-NH₂. This peptide is identical to Lom-AG-MT-I, a myotropin previously isolated from the male accessory glands of Locusta migratoria, using the L. migratoria oviduct motility bioassay as a monitoring system. It strongly stimulated the frequency, amplitude, and tonus of the myogenic oviduct contractions, even at low concentrations. © 1996 Wiley-Liss, Inc.     

Effect of soil type on infectivity and persistence of the entomopathogenic nematodes Steinernema scarabaei, Steinernema glaseri, Heterorhabditis zealandica, and Heterorhabditis bacteriophora

We tested the effect of soil type on the performance of the entomopathogenic pathogenic nematodes Steinernema scarabaei, Steinernema glaseri, Heterorhabditis zealandica, and Heterorhabditis bacteriophora. Soil types used were loamy sand, sandy loam, loam, silt loam, clay loam, acidic sand, and a highly organic potting mix. Infectivity was tested by exposing third-instar Anomala orientalis or Popillia japonica to nematodes in laboratory and greenhouse experiments and determining nematode establishment in the larvae and larval mortality. Infectivity of H. bacteriophora and H. zealandica was the highest in potting mix, did not differ among loamy sand and the loams, and was the lowest in acidic sand. Infectivity of S. glaseri was significantly lower in acidic sand than in loamy sand in a laboratory experiment but not in a greenhouse experiment, and did not differ among the other soils. Infectivity of S. scarabaei was lower in silt loam and clay loam than in loamy sand in a greenhouse experiment but not in a laboratory experiment, but was the lowest in acidic sand and potting mix. Persistence was determined in laboratory experiments by baiting nematode-inoculated soil with Galleria mellonella larvae. Persistence of both Heterorhabditis spp. and S. glaseri was the shortest in potting mix and showed no clear differences among the other substrates. Persistence of S. scarabaei was high in all substrates and its recovery declined significantly over time only in clay loam. In conclusion, generalizations on nematode performance in different soil types have to be done carefully as the effect of soil parameters including soil texture, pH, and organic matter may vary with nematode species.     

Efficacy of entomopathogenic nematodes against potato tuber moth,Phthorimaea operculella (Lepidoptera: Gelechiidae) under laboratory conditions

The susceptibility of potato tuber moth, Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae) to native and commercial strains of entomopathogenic nematodes (EPNs) was studied under laboratory conditions. Native strains of EPNs were collected from northeastern Iran and characterised as Steinernema feltiae and Heterorhabditis bacteriophora (FUM 7) using classic methods as well as analysis of internal transcribed spacer (ITS) and D2/D3 sequences of 28S genes. Plate assays were performed to evaluate the efficiency of five EPN strains belonging to four species including Steinernema carpocapsae (commercial strain), S. feltiae, Steinernem glaseri and H. bacteriophora (FUM 7 and commercial strains). This initial assessment with 0, 75, 150, 250, 375 and 500 IJs/ml concentrations showed that S. carpocapsae and H. bacteriophora caused the highest mortality in both larval and prepupal stages of P. operculella, PTM. Thereafter, these three strains (i.e. S. carpocapsae, H. bacteriophora FUM 7 and the commercial strains) were selected for complementary assays to determine the effects of soil type (loamy, loamy–sandy and sandy) on the virulence of EPNs against the second (L2) and fourth instar (L4) larvae as well as prepupa. A soil column assay was conducted using 500 and 2000 IJs in 2-ml distilled water. Mortality in the L2 larvae was not affected by the EPN strain or soil type, while there was a significant interactive effect of nematode strains and soil type on larval mortality. The results also showed that EPN strains have higher efficiency in lighter soils and caused higher mortality on early larvae than that in loamy soil. In L4 larvae, mortality of PTM was significantly influenced by nematode strain and applied concentrations of infective juveniles. The larval mortality induced by S. carpocapsae was higher than those caused either by a commercial or the FUM 7 strain of H. bacteriophora. Prepupa were the most susceptible stage.     

Pathogenicity,reproduction and foraging behaviours of some entomopathogenic nematodes on a new turf pest,Dorcadion pseudopreissi (Coleoptera: Cerambycidae)

Entomopathogenic nematodes (EPNs) in the families Heterorhabditidae and Steinernematidae have considerable potential as biological control agents of soil-inhabiting insect pests. In the present study, the control potential of the EPNs Steinernema carpocapsae (TUR-S4), S. feltiae (Nemaplus), S. carpocapsae (Nemastar), S. feltiae (TUR-S3) and Heterorhabditis bacteriophora (Nematop) against a new longicorn pest, Dorcadion pseudopreissi Breuning, 1962 (Coleoptera: Cerambycidae), on turf was examined in laboratory studies. Pathogenicity tests were performed at the following doses: 50, 100 and 150 Dauer Juveniles (DJs)/larva at 25°C. Highest mortalities (75–92%) of the larvae were detected at the dose of 150 DJs/larva for all nematodes used. Reproduction capabilities of the used EPNs were examined at doses of 50, 75, 100 and 150 DJs/larva at 25°C. S. carpocapsae (TUR-S4) had the most invasions (32 DJs/larva) and reproduction (28042 DJs/larva) at the dose of 100 DJs, and the highest reproduction (per invaded DJ into a larva) was observed in H. bacteriophora (Nematop) (2402.85 DJs) at a dose of 50 DJs. The foraging behaviour of the nematodes in the presence of D. pseudopreissi and Galleria mellonella L. (Lepidoptera: Galleriidae) larvae was studied using a Petri dish filled with sand at 20°C. All of the used nematodes accumulated near the larvae section of both insect species (32–53% of recovered DJs) with a higher percentage of S. carpocapsae (TUR-S4) (53%) and H. bacteriophora (48%) (Nematop) moving towards larvae of D. pseudopreissi, than the S. feltiae strains.     

Pathogenicity of Heterorhabditis bacteriophora, Steinernema glaseri, and S. scarabaei (Rhabditida: Heterorhabditidae, Steinernematidae) Against 12 White Grub Species (Coleoptera: Scarabaeidae)

We compared the pathogenicity of the entomopathogenic nematodes Heterorhabditis bacteriophora, Steinernema glaseri, and S. scarabaei against third instars of 12 white grub species. The Japanese beetle (Popillia japonica) was highly susceptible to all nematode species. Oriental beetle [Exomala (=Anomala) orientalis], European chafer (Rhizotrogus majalis), Asiatic garden beetle (Maladera castanea), and the May/June beetles Phyllophaga crinita, Ph. congrua, and Ph. (Subgenus Phytalus) georgiana were highly susceptible to S. scarabaei but had mediocre to low susceptibility to H. bacteriophora and S. glaseri. The black turfgrass ataenius (Ataenius spretulus) was very susceptible to H. bacteriophora but had mediocre susceptibility to S. glaseri and S. scarabaei. Northern (Cyclocephala borealis) and southern masked chafer (C. lurida) had mediocre and southwestern masked chafer (C. pasadenae) and green June beetle (Cotinis nitida) had low susceptibility to all nematode species.     

Characterization of cell lines developed from the colorado potato beetle, Leptinotarsa decemlineata say (coleoptera: Chrysomelidae)

In order to isolate new pathogens (viruses, microsporidia, etc.) or to evaluate the efficiency of some pathogens (serovarieties and mutants of Bacillus thuringiensis, fungi, etc.) in the control of Colorado potato beetle, an economically important pest, we established four cell lines from tissues of this insect. One was initiated from embryonated egg fragments in the M3 medium supplemented with 20% fetal bovine serum (FBS) and then transferred after several passages to the Ex-Cell 400 medium with 20% FBS. Another was initiated from larval hemocytes in Ex-Cell 400 with 5% FBS. Finally, two other cell lines were initiated from adult hemocytes: one in the Ex-Cell 400 with 20% FBS and 1% of lipid mixture and the other in the Ex-Cell 400 with 5% FBS only. These cell lines have been characterized by their morphology with light and electron microscopy, their karyotypes, cell growth, and isozyme analysis. Each cell line differed in morphologic, karyologic, growth, and isozyme patterns. The cell line initiated from embryonated eggs was growing slower than the three initiated from hemocytes. The cytotoxicity of solubilized crystal delta-endotoxins from different B. thuringiensis formulations (M-One, Trident, MYX-1806, Teknar-HPD, and Thuricide) and of destruxins, mycotoxins from Metarhizium anisopliae, was tested on these cell lines. They are sensitive to the solubilized toxins of some strains of B. thuringiensis (serovar. San Diego and serovar. tenebrionis) and to destruxins, and they can be used for the bioassay and detection of toxins and for the study of the mechanism of their action on coleopteran cells.     

Differential responses of galeal gustatory sensilla of the adult Colorado potato beetle, Leptinotarsa decemlineata (Say), to leaf saps from host and non-host plants

ABSTRACT Responses of galeal sensilla of the Colorado potato beetle, Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae), to leaf sap from Solanum tuberosum and a number of other solanaceous and non-solanaceous plants are described. A single cell responds vigorously to expressed sap from S.tuberosum , -and even to ten-fold dilutions of expressed sap. The response is characterized by a high spike frequency, low phasic activity and a tonic phase which can last for tens of seconds. At 8 s following stimulus application the primary response is typically 50% of its original activity, indicating slow adaptation to the sap stimulus. The primary response varies little among multiple stimulations of single sensilla and among sensilla on the same animal. There is a secondary response to potato leaf sap from one or more cells which have low spike amplitudes. This is usually of much lower frequency than the response from the primary cell, and it is highly variable.
The prominent and reproducible single cell response to potato sap may be an important component in signalling the presence of a host plant. This hypothesis is tested using saps from two other species of Solanum which are hosts, and several solanaceous and non-solanaceous non-hosts. Responses of galeal sensilla to the three host-plants tested were qualitatively similar and reproducible while responses to the non-hosts were highly variable and qualitatively different from responses to host saps. These results are interpreted and discussed in the context of a possible mechanism for chemosensory coding of complex stimuli in the insect/plant relationship.
Analysis of the raw electrophysiological data required extensive use of a microcomputer and several programs were developed during the course of this study. The computer analyses are briefly described in the paper and additional information and copies of the programs are available from the authors.