Food supplementation to optimize inoculative release of the predatory bug Macrolophus pygmaeus in sweet pepper

Biological control is widespread in management of greenhouse sweet pepper crops. Several species of predatory mites, bugs, and parasitoids are used against a wide range of pest species. However, biological control of particular pests like aphids, caterpillars, and the tobacco whitefly, Bemisia tabaci Gennadius, remains problematic. Macrolophus pygmaeus Rambur (Hemiptera: Miridae) is a generalist predatory bug which is used on a large scale in Western European tomato greenhouses. It has already been demonstrated that M. pygmaeus is a valuable biocontrol option in sweet pepper crops, but it has yet to find its way into common practice. Macrolophus pygmaeus should be introduced at the start of the growing season and determining an optimal release strategy is a key step in this process. In tomato crops, M. pygmaeus requires supplemental food releases to reach sufficient population numbers and dispersal levels. In this study, the need for food supplementation in sweet pepper is investigated. Three strategies were tested: (1) no food supplementation, (2) local food supplementation, and (3) full field food supplementation. Both population numbers and dispersal rates of the second generation were higher under the third strategy. Macrolophus pygmaeus oviposits near food sources, therefore dispersal rates are higher when food is more spread out. Pest control was achieved in all treatments, but faster and at lower pest levels under the full field strategy.     

Discrimination of the closely related biocontrol agents Macrolophus melanotoma (Hemiptera: Miridae) and M. pygmaeus using mitochondrial DNA analysis

The separation of the closely related predatory species Macrolophus melanotoma Costa (= M. caliginosus Wagner) and Macrolophus pygmaeus (Rambur) based exclusively on the different colour pattern of the first antennal segment (white central band in M. melanotoma and entirely black in M. pygmaeus) is rather precarious and their taxonomic status is still in doubt. In the present study their taxonomic status was evaluated by DNA confirmatory analysis and hybridization experiments between M. pygmaeus and a Macrolophus taxon, resembling M. melanotoma, with a first antennal segment entirely black or with a white central band collected from Dittrichia viscosa (L.) W. Greuter (Asteraceae) in southern Greece. Adult females from Dittrichia plants hybridized with males of M. pygmaeus and vice versa did not produce viable eggs. The Macrolophus species from Dittrichia irrespective of the first antennal segment coloration differed from M. pygmaeusin digestive patterns generated by AseI, XbaI, and MseI on 16S rRNA and in RAPD profiles produced by the primers OPA-18 and OPA-20. These results demonstrate that on Dittrichia plants there is a distinct dimorphic taxon, M. melanotoma, as it is the only species of the genus Macrolophus bearing a first antennal segment with a central white band. Given the limitation of the coloration pattern, the mtDNA genetic markers are the appropriate method for the identification of M. melanotomaand M. pygmaeus.     

Suitability of greenbugs, cotton aphids, andHeliothis virescens eggs for development and reproduction ofOrius insidiosus

Development, fecundity, and longevity of the predator,Orius insidiosus (Say) (Heteroptera: Anthocoridae), when reared on greenbug [Schizaphis graminum (Rondani)], cotton aphid (Aphis gossypii Glover), or eggs of tobacco budworm [Heliothis virescens (F.)] with green beans or water were examined. Developmental time was shortest when predators were reared onH. virescens eggs and beans and longest when reared on cotton aphids and water. Predators were most fecund when fedH. virescens eggs. The inclusion of beans in the nymphal diet further enhanced fecundity when fed eggs. Longevity of both females and males was significantly shorter when reared on aphids than on eggs. Beans in the nymphal diet enhanced longevity of female predators only in combination with budworm eggs. FemaleO. insidiosus were largest when reared onH. virescens eggs. Addition of green beans in aphid treatments resulted in increased size ofO. insidiosus when compared to aphids and free water.     

A method for laboratory studies on the polyphagous predator Macrolophus pygmaeus (Hemiptera: Miridae)

A method for studying the life history parameters of Macrolophus pygmaeus Rambur was developed and described. Fecundity of the predator was studied by placing one female and one male in a plastic cylinder in which a stem piece was used as an ovipositional substrate. Smaller cylinders were used to study the percentage of egg hatch of M. pygmaeus. Results demonstrated that this method is suitable for studying the fecundity and egg eclosion of M. pygmaeus. Allowing for easy modification, the method offers suitable conditions for maintenance of both insect and plant material and can be inexpensively modified for different experiments and the parts are easily constructed. The proposed method could also be employed for similar studies on other predatory hemipterans such as mirids and anthocorids and very likely on other phytophagous insect species that lay their eggs in or on plant stems.     

Evidence of Cannibalism in Macrolophus pygmaeus, a Natural Enemy of Whiteflies

Cannibalism and the effects of host plant, sex, time and food resources on its expression were studied for the zoophagous mirid Macrolophus pygmaeus Wagner (Hemiptera: Miridae). Cannibalistic behaviour was studied by offering 5 conspecific larvae (first instar) to newly emerging adults. Four treatments were studied: without water, with water only, with a host plant (tobacco) and with both a host plant and prey (eggs of Ephestia kuehniella). Cannibalism was observed in all treatments. In the “host plant + eggs of E. kuehniella” treatment, very few individuals displayed cannibalistic behaviour. The proportion of cannibalism was only reduced when eggs of E. kuehniella were offered. Water (free or via a host plant) was very important for both survival and feeding. The cannibalistic behaviour of M. pygmaeus should be taken into account when planning a release strategy in the context of biological control.     

Macrolophus pygmaeus (Hemiptera: Miridae) population parameters and biological characteristics when feeding on eggplant and tomato without prey

The population parameters and biological characteristics of the predator Macrolophus pygmaeus Rambur when feeding on eggplant, Solanum melongena L., and tomato, Lycopersicon esculentum (Miller) (both Solanaceae), without access to prey, were investigated at several temperatures. The experiments were conducted at 15, 20, 25, and 30 degrees C, 65 +/- 5% RH, and a photoperiod of 16:8 (L:D) h. Females oviposited on both plant species at all temperatures, with the exception of tomato at 30 degrees C. Fecundity was highest at 20 degrees C (21.55 and 8.28 eggs per female on eggplant and tomato, respectively). Adult longevity on both host plants was greatest at 15 degrees C, reaching 38.72 and 34.20 d for females and 92.88 and 62.80 d for males, on eggplant and tomato, respectively. The estimated values of population parameters showed that this predator increased its numbers on eggplant at all temperatures tested, but the highest intrinsic rate of increase occurred at 25 degrees C (0.0401 d(-1)). On tomato, this predator could not increase in number, although it could survive for a relatively long period. Results demonstrated that M. pygmaeus can survive on both host plants in periods of prey scarcity, particularly on eggplant. Therefore, phytophagy can positively contribute to the effectiveness of this biological control agent. The importance of eggplant as a reservoir for the predator in mixed crops that are less suitable host plants is discussed.     

Effects of diet on longevity and fecundity of the spined stilt bug, Jalysus wickhami

We evaluated the suitability of selected food items for the adult spined stilt bug, Jalysus wickhami Van Duzee (Hemiptera: Berytidae), by providing one of seven diet treatments: (1) prepupae of the parasitoid, Cotesia congregata (Say) (Hymenoptera: Braconidae), on a leaf of tobacco (Nicotiana tabacum L., NC 2326) (Solanaceae); (2) C. congregata prepupae alone (i.e., no tobacco leaf); (3) eggs of the parasitoid's host, Manduca sexta (L.) (Lepidoptera: Sphingidae), on a tobacco leaf; (4) M. sexta eggs alone; (5) tobacco aphids, Myzus nicotianae Blackman (Homoptera: Aphididae), on a tobacco leaf; (6) a tobacco leaf alone; and (7) no prey and no tobacco. A tobacco leaf was necessary for the long-term survival of stilt bugs, but prey source did not affect longevity. Regardless of the prey type, stilt bugs with access to a tobacco leaf lived 21–23 days, while stilt bugs without access to tobacco lived less than 12 days. Animal prey provided a protein source required for egg deposition in J. wickhami, and prey species differed in their relative nutritive values. Female stilt bugs that fed on M. sexta eggs or on C. congregata prepupae were significantly more fecund (102 and 106 nymphs per female, respectively) than females that fed on tobacco aphids (24 nymphs per female). Results suggest that stilt bugs may feed on tobacco aphids and C. congregata prepupae when other prey are unavailable.     

Evaluation of mirid predatory bugs and release strategy for aphid control in sweet pepper

Zoophytophagous predators of the family Miridae (Heteroptera), which feed both on plant and prey, often maintain a close relationship with certain host plants. In this study, we aimed to select a suitable mirid predatory bug for aphid control in sweet pepper. Four species were compared: Macrolophus pygmaeus (Rambur), Dicyphus errans (Wolff), Dicyphus tamaninii Wagner and Deraeocoris pallens (Reuter). They were assessed on their establishment on sweet pepper plants with and without supplemental food (eggs of the flour moth Ephestia kuehniella Zeller and decapsulated cysts of the brine shrimp Artemia franciscana Kellogg) and on their effects on aphids with releases before and after aphid infestations. None of the predator species was able to control an established population of aphids on sweet pepper plants; however, the predators M. pygmaeus and D. tamaninii could successfully reduce aphid populations when released prior to an artificially introduced aphid infestation. The best results were achieved with M. pygmaeus in combination with a weekly application of supplemental food. Hence, our results demonstrate that the order and level of plant colonization by mirid predators and aphids determines how successful biological control is. Further studies are needed to evaluate the performance of mirid predatory bugs in sweet pepper crops in commercial greenhouses with multiple pests and natural enemies, in particular to understand how increased variation in food sources affects their feeding behaviour and preferences.     

Macrolophus caliginosus (Wagner) as an Intraguild Prey for the Zoophytophagous Dicyphus tamaninii Wagner (Heteroptera: Miridae)

The zoophytophagous predator Macrolophus caliginosus (Wagner) (Heteroptera: Miridae) has previously been identified as a potential prey for the intraguild zoophytophagous predator Dicyphus tamaninii Wagner. Its value as an intraguild prey was tested for D. tamaninii nymphal development and adult survival. In the laboratory, plant (red tomato fruit, green tomato fruit, tomato leaf) and animal (aphids, whiteflies, pyralid eggs) resources were compared to frozen nymphs of M. caliginosus as an intraguild resource for D. tamaninii. M. caliginosus nymphs allowed complete and rapid development of D. tamaninii nymphs and generated low mortality of nymphal and adult stages. Performances were higher with the intraguild M. caliginosus resource than with plant resources. Tomato leaves did not allow the complete nymphal development of D. tamaninii. Tomato fruit generated lighter adults and doubled the nymphal developmental time compared with the M. caliginosus resource. In the animal resource group, there were no differences between the M. caliginosus and the other treatments (aphids, whiteflies, pyralid eggs) for nymphal mortality, average number of molts, nymphal developmental time, adult weight, and adult survival. M. caliginosus should be considered a high-quality resource for D. tamaninii.     

Life table and biological characteristics of Macrolophus pygmaeus when feeding on Myzus persicae and Trialeurodes vaporariorum

The life table and biological characteristics of the predatory bug Macrolophus pygmaeus Rambur (Hemiptera: Miridae) were studied when the bugs were fed with Myzus persicae (Sulzer) (Homoptera: Aphididae) feeding on eggplant and with Trialeurodes vaporariorum Westwood (Homoptera: Aleyrodidae) feeding on tomato plants. The tests were done at five temperatures between 15 and 30 °C, using a L16:D8 photoperiod and 65 ± 5% r.h. Most eggs (range 85 to 90%) hatched at 15 and 20 °C. Incubation period was shortest at 27.5 °C (8.45 and 8.38 days on eggplant and tomato, respectively). Preoviposition was also shortest at 27.5 °C (5.10 and 4.75 days on eggplant and tomato, respectively) whereas fecundity was highest at 20 °C (213.90 and 228.25 eggs on eggplant and tomato, respectively). Maximum longevity of females was at 15 °C (122.40 and 129.35 days on eggplant and tomato, respectively). Mean generation time was longest at 15 °C on both host plants (122.75 and 124.64 days, respectively). The intrinsic rate of increase of M. pygmaeus was highest at 27.5 °C with similar values on eggplant (0.0981 day^–1) and tomato (0.1040 day^–1). Doubling time was shortest at 27.5 °C (7.06 and 6.67 days on eggplant and tomato, respectively) and, also, finite rate of increase was highest at 27.5 °C (1.1031 and 1.1096 on eggplant and tomato, respectively). The results show that the predator M. pygmaeus develops well on the aphid M. persicae or on the whitefly T. vaporariorum, both of which are important pests of vegetable crops. This predator is also well adapted to the temperatures that occur both in greenhouses and in the open field in the Mediterranean region. Compared to other natural enemies of whiteflies, such as Encarsia formosa Gahan (Hymenoptera: Aphelinidae), Macrolophus pygmaeus can increase at relatively low temperatures.     

微小花蝽对小菜蛾捕食控制的能力

为了明确微小花蝽以小菜蛾卵做为饲料的适宜性及其对小菜蛾的控制能力,在室内以桃蚜作为参比猎物,研究捕食小菜蛾卵对微小花蝽生长发育、繁殖的影响,并结合捕食功能反应评价微小花蝽对小菜蛾的捕食能力.结果表明: 微小花蝽取食小菜蛾卵能够完成世代发育,并能够正常繁育后代.取食小菜蛾卵时,微小花蝽雌、雄若虫的历期(♀: 12.3 d,♂: 12.2 d)、成虫体长[♀: (2.13±0.01) mm,♂: (1.91±0.00) mm]、体宽[♀: (0.87±0.01) mm,♂: (0.71±0.01) mm]、单雌产卵量(12.7±1.1)、产卵前期[(5.1±0.6) d]和产卵期[(3.7±0.4) d]均与桃蚜处理组无显著差异;雌、雄成虫寿命[♀: (10.7±1.4) d,♂: (9.1±1.3) d]显著长于桃蚜处理组[♀: (8.5±0.5) d,♂: (6.4±0.3) d];若虫存活率[(65.0±6.8)%]不及桃蚜处理组[(80.0±8.2)%],且雌性比例偏低.微小花蝽对小菜蛾的捕食功能反应符合HollingⅡ型方程.微小花蝽1～5龄若虫对小菜蛾卵的日均最大捕食量(N_max)分别为7.5、16.3、23.3、29.1和38.7粒;雌、雄成虫的日均最大捕食量分别为39.0和26.9粒;5龄若虫对小菜蛾低龄幼虫的日均最大捕食量为41.3头;雌、雄成虫的日均最大捕食量分别为40.8和23.9头.单头雌、雄微小花蝽一生中最多可捕食小菜蛾卵(711.3±58.1)和(535.4±30.6)粒,小菜蛾低龄幼虫(371.9±52.0)和(253.9±32.3)头.微小花蝽以小菜蛾卵饲养可行,且对小菜蛾具有良好的控制作用.     

Efficiency of four heteroptera as predators ofAphis gossypii andMacrosiphum euphorbiae (Hom.: Aphididae)

Predation rate and nymphal development time were determined for four predatory bugs (Dicyphus tamaninii Wagner andMacrolophus caliginosus Wagner [Het.:Miridae],Orius laevigatus (Fieber) andO. majusculus (Reuter) [Het.:Anthocoridae], on cucumber withAphis gossypii Glover (Hom.:APhididae) as prey and for both mirids also on tomato withMacrosiphum euphorbiae (Thomas) as prey. All four predator species were able to achieve adulthood feeding on both aphids. The developmental period ofD. tamaninii nymphs was the longest and the shortest was forO. majusculus. During nymphal developmentD. tamaninii consumed greater numbers of both aphids thanM. caliginosus, whileOrius’ consumption ofA. gossypii was intermediate. Average daily predation was higher forD. tamaninii than forM. caliginosus, but was not different from bothOrius species. FemaleD. tamaninii exhibited a typical type-II functional response when preying on varying densities ofA. gossypii nymphs.     

Fitness and predation potential of Macrolophus pygmaeus reared under artificial conditions

Abstract The biological parameters of Macrolophus pygmaeus Rambur after prolonged rearing in the absence of plant materials were compared with those of conventionally plant‐reared predators. When eggs of Ephestia kuehniella Zeller were provided as food, developmental and reproductive fitness of M. pygmaeus reared for over 30 consecutive generations using artificial living and oviposition substrates was similar to that of predators kept on tobacco leaves. Plantless‐reared fifth instars of the predator also had similar predation rates on second instars of the tobacco aphid, Myzus persicae nicotianae Blackman, as their peers maintained on plant materials. In a further experiment, predation on aphid prey by fifth instar M. pygmaeus fed one of two egg yolk‐based artificial diets was compared with that of nymphs fed E. kuehniella eggs. Despite their lower body weights, predators produced on either artificial diet killed similar numbers of prey as their counterparts reared on lepidopteran eggs. Our study indicates that artificial rearing systems may be useful to further rationalize the production of this economically important biological control agent.     

Nectarivory by the plant-tissue feeding predator Macrolophus pygmaeus Rambur (Heteroptera: Miridae): Nutritional redundancy or nutritional benefit?

Most predators and parasitoids feed on plant-provided food (nectar, pollen) or engage in herbivory during at least part of their life stages. Plant feeding by these insects plays an important role in driving predator-herbivore dynamics. Thus, understanding the effects of plant feeding on omnivores could be an important element in improving biological control strategies. The mirid Macrolophus pygmaeus is an omnivorous heteropteran predator of whitefly and other pests. Unlike other predators that need to seek out accessible nectar to meet their carbohydrate requirements, mirid bugs can access the plant's carbohydrate resources by feeding directly on plant tissues. Leaf and stem feeding could be seen as a nutritional surrogate that allows mirids to become independent of nectar availability. However, to date feeding experiments have not yet considered nectar feeding by these mirid predators. In this study we demonstrate that M. pygmaeus survival is prolonged on broad bean plants featuring extrafloral nectar as compared to broad bean with extrafloral nectaries removed, irrespective of the presence of cattail pollen. Survival on extrafloral nectar was comparable to the survival by individuals kept on broad bean provided with eggs of Ephestia kuehniella as prey. Also, a greater proportion of mirid females laid eggs when extrafloral nectar was available as compared to those confined on nectariless plants without supplemental food.     

Nettle extract effects on the aphid Myzus persicae and its natural enemy, the predator Macrolophus pygmaeus (Hem., Miridae)

Abstract:  The effect of water extract of Urtica urens L. on the biological characteristics and population parameters of Myzus persicae (Sulzer) and its natural enemy, the polyphagous predator Macrolophus pygmaeus (Rambur) was investigated on eggplants. Plant material was extracted in deionized water (1 : 5 w/w) for 24 h. Three applications were made at 5-day intervals using the nettle extract, with deionized water as the control. The effect of the extract on survival, duration of nymphal development, pre-reproductive period, fecundity and longevity of both insects was studied at conditions of 25 ± 1°C, 65 ± 5% relative humidity and photoperiod of 16 : 8 h (light : dark). The results showed that the application of nettle extract significantly reduced the fecundity of M. persicae (20% on average) but did not result in a substantial lowering in its intrinsic rate of population increase. However, the predator's biological characteristics and population parameters were not affected by the application of this plant extract. The importance of these results for the use of nettle extract in the management of M. persicae along with its compatibility with the use or conservation of M. pygmaeus is discussed.     

Microbial community of predatory bugs of the genus <Emphasis Type="Italic">Macrolophus</Emphasis>(Hemiptera: Miridae)

BACKGROUND: The predatory mirids of the genus Macrolophus are key natural enemies of various economically important agricultural pests. Both M. caliginosus and M. pygmaeus are commercially available for the augmentative biological control of arthropod pests in European greenhouses. The latter species is known to be infected with Wolbachia -inducing cytoplasmic incompatibility in its host- but the presence of other endosymbionts has not been demonstrated. In the present study, the microbial diversity was examined in various populations of M. caliginosus and M. pygmaeus by 16S rRNA sequencing and denaturing gradient gel electrophoresis. RESULTS: Besides Wolbachia, a co-infection of 2 Rickettsia species was detected in all M. pygmaeus populations. Based on a concatenated alignment of the 16S rRNA gene, the gltA gene and the coxA gene, the first is phylogenetically related to Rickettsia bellii, whereas the other is closely related to Rickettsia limoniae. All M. caliginosus populations were infected with the same Wolbachia and limoniae-like Rickettsia strain as M. pygmaeus, but did not harbour the bellii-like Rickettsia strain. Interestingly, individuals with a single infection were not found. A PCR assay on the ovaries of M. pygmaeus and M. caliginosus indicated that all endosymbionts are vertically transmitted. The presence of Wolbachia and Rickettsia in oocytes was confirmed by a fluorescence in situ hybridisation. A bio-assay comparing an infected and an uninfected M. pygmaeus population suggested that the endosymbionts had minor effects on nymphal development of their insect host and did not influence its fecundity. CONCLUSION: Two species of the palaearctic mirid genus Macrolophus are infected with multiple endosymbionts, including Wolbachia and Rickettsia. Independent of the origin, all tested populations of both M. pygmaeus and M. caliginosus were infected with three and two endosymbionts, respectively. There was no indication that infection with endosymbiotic bacteria had a fitness cost in terms of development and fecundity of the predators.     

Functional response of Macrolophus pygmaeus (Rambur) and Nesidiocoris tenuis (Reuter) feeding on two different prey species

Functional response type and predatory parameters of Macrolophus pygmaeus and Nesidiocoris tenuis (Hemiptera: Miridae), the two important predators widely used in IPM programmes at tomato greenhouses, were investigated. The predators fed on Tuta absoluta (Lepidoptera: Gelechiidae) or Ephestia kuehniella (Lepidoptera: Pyralidae) eggs. Different densities of prey eggs including 1, 2, 3, 4, 5, 8, 10, 15, 20, 30 and 40 (latest only for E. kuehniella) were used at laboratory conditions. The results showed that both predatory bugs had type II functional response. Also, predation indices were significantly different between the predators. Although N. tenuis was more efficient against T. absoluta eggs than M. pygmaeus, M. pygmaeus predatory indices showed that this predator was more efficient on E. kuehniella eggs. Thus, minimum and maximum attack rate were observed in N. tenuis fed on E. kuehniella (0.0871 h^?1) and T. absoluta (0.2514 h^?1) eggs, respectively. Whilst, the minimum and maximum handling time were observed in M. pygmaeus fed on E. kuehniella (1.8695 h) and T. absoluta (2.7415 h) eggs, respectively.     

Observations onCheyletus cacahuamilpensis,a predator of the tenuipalpid miteDolichotetranychus floridanus

Aspects of the biology ofCheyletus cacahuamilpensis Baker a predatory mite of the associated lawn grass pestDolichotetranychus floridanus (Banks) were studied. Apart from a larval stage immature females ofC. cacahuamilpensis pass through 2 nymphal stages, while the male has only 1 before reaching adulthood. At 27°C the life cycle and adult longevity respectively averaged 30.1 & 34.4 days for the female and 22.8 & 16.4 days for the male. In a 20.4 days oviposition period 28 eggs were laid. Immature and adult females devoured an average of 80.4 & 143.2D. floridanus, while those of the males consumed 40.8 & 63.6 individuals respectively. Thus the total number ofD. floridanus attacked during the predator's life span averaged 223.6 per female and 104.4. per male.     

Multiple mating in the traumatically inseminating Warehouse pirate bug, Xylocoris flavipes: effects on fecundity and longevity

Optimal mating frequencies differ between sexes as a consequence of the sexual differentiation of reproductive costs per mating, where mating is normally more costly to females than males. In mating systems where sexual reproduction is costly to females, sexual conflict may cause both direct (i.e. by reducing female fecundity or causing mortality) and indirect (i.e. increased risk of mortality, reduced offspring viability) reductions in lifetime reproductive success of females, which have individual and population consequences. We investigated the direct and indirect costs of multiple mating in a traumatically inseminating (TI) predatory Warehouse pirate bug, Xylocoris flavipes (Reuter) (Hemiptera: Anthocoridae), where the male penetrates the female's abdomen during copulation. This study aimed to quantify the effects of TI on female fecundity, egg viability, the lifetime fecundity schedule, longevity and prey consumption in this cosmopolitan biocontrol agent. We found no difference in the total reproductive output between mating treatments in terms of total eggs laid or offspring viability, but there were significant differences found in daily fecundity schedules and adult longevity. In terms of lifetime reproduction, female Warehouse pirate bugs appear to be adapted to compensate for the costs of TI mating to their longevity.     

The influence of acclimation, endosymbionts and diet on the supercooling capacity of the predatory bug Macrolophus pygmaeus

The generalist predator Macrolophus pygmaeus Rambur (Hemiptera: Miridae) is a key biological control agent in European greenhouses. The influence of acclimation, infection with endosymbiotic bacteria and diet on the cold tolerance of the Mediterranean biocontrol population was assessed by determining the supercooling point, i.e. the temperature at which the insect’s body fluids freeze. This parameter provides a first indication of an insect’s establishment potential in a new region and of its possible geographical range. Allowing the predatory bugs to adapt to lower temperatures resulted in an increase in supercooling ability. Macrolophus pygmaeus bugs exposed to antibiotics in their artificial diet and hence cured from their infection with the endosymbiotic bacteria Wolbachia pipientis, Rickettsia bellii and R. limoniae were more tolerant to freezing than infected bugs. The diet of the predators also affected the freezing temperature of the body fluids. Predators fed an artificial diet based on egg yolk were less resistant to freezing than those fed Ephestia kuehniella Zeller (Lepidoptera: Pyralidae) eggs. These findings illustrate that several factors may affect the cold hardiness of a biocontrol agent and may thus complicate the evaluation of its establishment potential in the framework of an environmental risk assessment.