Field-collected Colonies Research Articles

Metabolic resistance to deltamethrin is mediated by P450 and esterases in common bed bugs Cimex lectularius L. (Heteroptera: Cimicidae)

The infestations of Cimex lectularius L. (Heteroptera: Cimicidae) registered in the last decades have been influenced by several human activities, including international tourism and commerce. Moreover, the development of insecticide resistance and careless pest control strategies contributed to the dispersal of bed bugs. Given the complexity of the topic, distinguishing physiological and molecular mechanisms involved in resistance can help design proper control tools and limit the resistance spread. Here we determined the susceptibility to deltamethrin and imidacloprid in bed bugs collected in Italy. Also, we assessed the role of esterases and P450 monooxygenases by direct enzymatic activity measurement and inhibition by synergism bioassays. Our results showed that the field-collected colony exhibited high resistance ratios to imidacloprid and deltamethrin (757 and >60,000 times, respectively) compared to the susceptible colony. Moreover, resistant bed bugs showed increased activity of esterases and P450 monooxygenases. The synergistic effect of piperonyl butoxide (PBO) suggests the significant contribution of both enzymatic groups as detoxification pathways implicated in pyrethroid-resistant bed bugs. Further investigations are needed to unravel the biochemical and molecular basis involved in the resistant phenotype for developing novel strategies for pest control.

Evaluation of Resistance to Different Insecticides and Metabolic Detoxification Mechanism by Use of Synergist in the Common Bed Bug (Heteroptera: Cimicidae)

Bed bugs have become a common urban pest with consequences on human health and economic costs to the hotel and tourism sectors. Insecticide resistance is considered an important factor in the current bed bug resurgence, and multiple resistance mechanisms could be working in the resistant bed bug populations. In the present study, we determined the resistance profile to four insecticides with a different mode of action in Cimex lectularius L. (Heteroptera: Cimicidae) field-collected colonies from Argentina. Furthermore, the synergism effect of piperonyl butoxide (PBO) with deltamethrin was investigated to explore the contribution of detoxification metabolism to resistance. Our results showed that most of the field-collected colonies are extremely resistant to deltamethrin and propoxur, much more than to azametiphos and imidacloprid. The differences in resistance ratios among field-collected colonies could be associated with different modes of action of insecticides used in control pest and the mechanisms involved in the resistance. PBO pretreatment led to a significantly decreased RR in pyrethroid-resistant colonies, suggesting an upturn of monooxygenase activity for deltamethrin detoxification. However, the high RR detected could involve other mechanisms as part of the whole resistant phenotype in colonies of C. lectularius resistant to pyrethroids.

Isolation and identification of Asaia sp. in Anopheles spp. mosquitoes collected from Iranian malaria settings: steps toward applying paratransgenic tools against malaria

BackgroundIn recent years, the genus Asaia (Rhodospirillales: Acetobacteraceae) has been isolated from different Anopheles species and presented as a promising tool to combat malaria. This bacterium has unique features such as presence in different organs of mosquitoes (midgut, salivary glands and reproductive organs) of female and male mosquitoes and vertical and horizontal transmission. These specifications lead to the possibility of introducing Asaia as a robust candidate for malaria vector control via paratransgenesis technology. Several studies have been performed on the microbiota of Anopheles mosquitoes (Diptera: Culicidae) in Iran and the Middle East to find a suitable candidate for controlling the malaria based on paratransgenesis approaches. The present study is the first report of isolation, biochemical and molecular characterization of the genus Asaia within five different Anopheles species which originated from different zoogeographical zones in the south, east, and north of Iran.MethodsMosquitoes originated from field-collected and laboratory-reared colonies of five Anopheles spp. Adult mosquitoes were anesthetized; their midguts were isolated by dissection, followed by grinding the midgut contents which were then cultured in enrichment broth media and later in CaCO3 agar plates separately. Morphological, biochemical and physiological characterization were carried out after the appearance of colonies. For molecular confirmation, selected colonies were cultured, their DNAs were extracted and PCR was performed on the 16S ribosomal RNA gene using specific newly designed primers.ResultsMorphological, biochemical, physiological and molecular results indicated that all isolates are members of the genus Asaia.ConclusionsContrary to previous opinions, our findings show that Asaia bacteria are present in both insectary-reared colonies and field-collected mosquitoes and can be isolated by simple and specific methods. Furthermore, with respect to the fact that we isolated Asaia within the different Anopheles specimens from distinct climatic and zoogeographical regions, it is promising and may be concluded that species of this genus can tolerate the complicated environmental conditions of the vector-borne diseases endemic regions. Therefore, it can be considered as a promising target in paratransgenesis and vector control programs. However, we suggest that introducing the new technologies such as next generation sequencing and robust in silico approaches may pave the way to find a unique biomarker for rapid and reliable differentiation of the Asaia species.

Effect of temperature and social environment on worker size in the ant Temnothorax nylanderi

Warm temperatures decrease insect developmental time and body size. Social life could buffer external environmental variations, especially in large social groups, either through behavioral regulation and compensation or through specific nest architecture. Mean worker size and distribution of worker sizes within colonies are important parameters affecting colony productivity as worker size is linked to division of labor in insect societies. In this paper, we investigate the effect of stressful warm temperatures and the role of social environment (colony size and size of nestmate workers) on the mean size and size variation of laboratory-born workers in the small European ant Temnothorax nylanderi. To do so, we reared field-collected colonies under medium or warm temperature treatments after having marked the field-born workers and removed the brood except for 30 first instar larvae. Warm temperature resulted in the production of fewer workers and a higher adult mortality, confirming that this regime was stressful for the ants. T. nylanderi ants followed the temperature size rule observed in insects, with a decreased developmental time and mean size under warm condition. Social environment appeared to play an important role as we observed that (i) larger colonies buffered the effect of temperature better than smaller ones (ii) colonies with larger workers produced larger workers whatever the rearing temperature and (iii) the coefficient of variation of worker size was similar in the field and under medium laboratory temperature. This suggests that worker size variation is not primarily due to seasonal environmental fluctuations in the field. Finally, we observed a higher coefficient of variation of worker size under warm temperature. We propose that this results from a disruption of social regulation, i.e. the control of nestmate workers over developing larvae and adult worker size, under stressful conditions.

Chemical microenvironments and single-cell carbon and nitrogen uptake in field-collected colonies of Trichodesmium under different pCO2

Gradients of oxygen (O2) and pH, as well as small-scale fluxes of carbon (C), nitrogen (N) and O2 were investigated under different partial pressures of carbon dioxide (pCO2) in field-collected colonies of the marine dinitrogen (N2)-fixing cyanobacterium Trichodesmium. Microsensor measurements indicated that cells within colonies experienced large fluctuations in O2, pH and CO2 concentrations over a day–night cycle. O2 concentrations varied with light intensity and time of day, yet colonies exposed to light were supersaturated with O2 (up to ~200%) throughout the light period and anoxia was not detected. Alternating between light and dark conditions caused a variation in pH levels by on average 0.5 units (equivalent to 15 nmol l−1 proton concentration). Single-cell analyses of C and N assimilation using secondary ion mass spectrometry (SIMS; large geometry SIMS and nanoscale SIMS) revealed high variability in metabolic activity of single cells and trichomes of Trichodesmium, and indicated transfer of C and N to colony-associated non-photosynthetic bacteria. Neither O2 fluxes nor C fixation by Trichodesmium were significantly influenced by short-term incubations under different pCO2 levels, whereas N2 fixation increased with increasing pCO2. The large range of metabolic rates observed at the single-cell level may reflect a response by colony-forming microbial populations to highly variable microenvironments.

Midgut-based resistance to oral infection by a nucleopolyhedrovirus in the laboratory-selected strain of the smaller tea tortrix, Adoxophyes honmai (Lepidoptera: Tortricidae).

A strain of Adoxophyes honmai resistant to Adoxophyes honmai nucleopolyhedrovirus (AdhoNPV) was established from a field-collected colony by repeated selection. Fifth-instar larvae of this resistant strain (R-strain) had over 66 666-fold greater resistance in terms of 50 % lethal concentration values to oral infection of AdhoNPV than non-selected strain larvae (susceptible for AdhoNPV; S2-strain). In this study, the mechanism of resistance to AdhoNPV was determined in R-strain larvae. An assessment of viral genome replication in AdhoNPV-infected S2- and R-strain larvae by quantitative PCR showed no viral genome replication occurring in R-strain larvae. Transcription of AdhoNPV ie-1, vp39 and polyhedrin genes was also not detected in R-strain midgut cells. Besides, a fluorescent brightener had no effect on AdhoNPV infection in either S2- or R-strain. However, binding and fusion of occlusion-derived virus with R-strain were significantly lower than those of S2-strain. These findings suggest that R-strain Adoxophyeshonmai larvae possess a midgut-based resistance to oral infection by AdhoNPV in which midgut epithelial cells are infected less efficiently.

Effect of Carbohydrate Supplementation on Investment into Offspring Number, Size, and Condition in a Social Insect.

Resource availability can determine an organism’s investment strategies for growth and reproduction. When nutrients are limited, there are potential tradeoffs between investing into offspring number versus individual offspring size. In social insects, colony investment in offspring size and number may shift in response to colony needs and the availability of food resources. We experimentally manipulated the diet of a polymorphic ant species (Solenopsis invicta) to test how access to the carbohydrate and amino acid components of nectar resources affect colony investment in worker number, body size, size distributions, and individual percent fat mass. We reared field-collected colonies on one of four macronutrient treatment supplements: water, amino acids, carbohydrates, and amino acid and carbohydrates. Having access to carbohydrates nearly doubled colony biomass after 60 days. This increase in biomass resulted from an increase in worker number and mean worker size. Access to carbohydrates also altered worker body size distributions. Finally, we found a negative relationship between worker number and size, suggesting a tradeoff in colony investment strategies. This tradeoff was more pronounced for colonies without access to carbohydrate resources. The monopolization of plant-based resources has been implicated in the ecological success of ants. Our results shed light on a possible mechanism for this success, and also have implications for the success of introduced species. In addition to increases in colony size, our results suggest that having access to plant-based carbohydrates can also result in larger workers that may have better individual fighting ability, and that can withstand greater temperature fluctuations and periods of food deprivation.

Investigation of Age Polyethism in Food Processing of the Fungus-Growing Termite Odontotermes formosanus (Blattodea: Termitidae) Using a Laboratory Artificial Rearing System.

Laboratory rearing systems are useful models for studying Rhinotermitid behavior. Information on the biology of fungus-growing termites, however, is limited because of the difficulty of rearing colonies in the laboratory settings. The physical structure of termite nests makes it impossible to photograph or to observe colonies in the field. In this study, an artificial rearing system for field-collected colonies of the fungus-growing termite Odontotermes formosanus (Shiraki) was developed to facilitate observation in the laboratory. We recorded colony activity within the artificial rearing system and documented a variety of social behaviors that occurred throughout the food processing of the colony. This complex miniature ecosystem was cooperatively organized via division of labor in the foraging and processing of plant materials, and the observed patterns largely resembled the caste and age-based principles present in Macrotermes colonies. This work extends our insights into polyethism in the subfamily Macrotermitinae.

Immunolocalisation of microcystins in colonies of the cyanobacterium Rivularia in calcareous streams

The cyanobacterium Rivularia is often the dominant genus in unpolluted stretches of many calcareous streams. Previous studies have detected microcystins in field-collected colonies from Mediterranean streams in Spain. Because sheaths and mucilage represent a substantial part of the colonies, the localisation of microcystins within Rivularia colonies was tested with immunological methods to elucidate the role of mucilage in toxicity. Microcystins were localised inside the trichomes, in the filament sheaths and in the colonial mucilage. The presence of microcystins was also shown in some heterocysts, but no mircocystins were detected in multicellular hairs. We suggest that microcystins are important for a benthic organism growing slowly for much of the time, that some labour division may exists between the cells in the colony and that these immunological methods may be a useful alternative for microcystin detection.

Effect of group size and caste ratio on individual survivorship and social immunity in a subterranean termite

Individuals living within social groups may benefit from the efficiencies of division of labour, but on the other hand render themselves vulnerable to socially transmitted disease. This cost to social living should promote cooperative barriers to disease transmission, especially in eusocial taxa where spatial and genetic proximity to nestmates are characteristically pronounced. Termites are eusocial yet little is known about how their sociality is deployed to resist contagion. In this study, we manipulate two variables that are expected to affect the number and nature of social interactions and measure the ability of individuals within groups to resist fungal infection. From laboratory experiments on field-collected colonies, we report that both group size and caste composition directly affect the survivorship of individuals within groups, but only caste composition moderates survivorship upon immune challenge. Our study therefore provides no statistical evidence that individual Eastern subterranean termites (Reticulitermes flavipes) have increased resistance to disease in crowded groups—that is, there is no evidence for a density-dependent social immune response. Our results do suggest, however, that the caste-specific nature of interactions may be important for controlling disease in a social context.

Genetic diversity of the allodeterminant alr2 in Hydractinia symbiolongicarpus.

Hydractinia symbiolongicarpus, a colonial cnidarian (class Hydrozoa) epibiont on hermit crab shells, is well established as a model for genetic studies of allorecognition. Recently, two linked loci, allorecognition (alr) 1 and alr2, were identified by positional cloning and shown to be major determinants of histocompatibility. Both genes encode putative transmembrane proteins with hypervariable extracellular domains similar to immunoglobulin (Ig)-like domains. We sought to characterize the naturally occurring variation at the alr2 locus and to understand the origins of this molecular diversity. We examined full-length cDNA coding sequences derived from a sample of 21 field-collected colonies, including 18 chosen haphazardly and two laboratory reference strains. Of the 35 alleles recovered from the 18 unbiased samples, 34 encoded unique gene products. We identified two distinct structural classes of alleles that varied over a large central region of the gene but both possessed highly polymorphic extracellular domains I, similar to an Ig-like V-set domain. The discovery of structurally chimeric alleles provided evidence that interallelic recombination may contribute to alr2 variation. Comparisons of the genomic region encompassing alr2 from two field-derived haplotypes and one laboratory reference sequence revealed a history of structural variation at the haplotype level as well. Maintenance of large numbers of equally rare alleles in a natural population is a hallmark of negative frequency-dependent selection and is expected to produce high levels of heterozygosity. The observed alr2 allelic diversity is comparable with that found in immune recognition molecules such as human leukocyte antigens, B cell Igs, or natural killer cell Ig-like receptors.

Effects of Selfing on Offspring Survival and Reproduction in a Colonial Simultaneous Hermaphrodite (Bugula stolonifera, Bryozoa)

Understanding the consequences of selfing in simultaneous hermaphrodites requires investigating potential deleterious effects on fitness at all stages of life. In this study, I examined the effects of selfing throughout the life cycle of the marine bryozoan Bugula stolonifera, a colonial simultaneous hermaphrodite. In 2008, larvae from field-collected colonies were cultured through metamorphosis to reproductively mature colonies either in the presence of one other colony, the paired treatment, or alone, the solitary treatment. Results demonstrated that selfing in this species is possible, in that colonies in the solitary treatment produced viable larvae that successfully completed metamorphosis. On average, however, these colonies released significantly fewer larvae, which experienced reduced rates of metamorphic initiation and completion compared to the paired treatment. These experiments were extended in 2009, when metamorphs from colonies reared in the solitary (n = 58) and paired (n = 61) treatments were transferred to the field for growth to reproductive maturity and then brought back to the laboratory for larval collection. Results revealed additional deleterious effects associated with selfing, as no viable larvae were recovered from colonies deriving from the solitary treatment. In contrast, offspring from the paired treatment released 1030 larvae and 99% initiated metamorphosis, 97% of which completed metamorphosis. Overall, selfed larvae not only had significantly decreased chances of survival, but those that did survive did not successfully reproduce.

Microbial Community Structure of Leaf-Cutter Ant Fungus Gardens and Refuse Dumps

BackgroundLeaf-cutter ants use fresh plant material to grow a mutualistic fungus that serves as the ants' primary food source. Within fungus gardens, various plant compounds are metabolized and transformed into nutrients suitable for ant consumption. This symbiotic association produces a large amount of refuse consisting primarily of partly degraded plant material. A leaf-cutter ant colony is thus divided into two spatially and chemically distinct environments that together represent a plant biomass degradation gradient. Little is known about the microbial community structure in gardens and dumps or variation between lab and field colonies.Methodology/Principal FindingsUsing microbial membrane lipid analysis and a variety of community metrics, we assessed and compared the microbiota of fungus gardens and refuse dumps from both laboratory-maintained and field-collected colonies. We found that gardens contained a diverse and consistent community of microbes, dominated by Gram-negative bacteria, particularly γ-Proteobacteria and Bacteroidetes. These findings were consistent across lab and field gardens, as well as host ant taxa. In contrast, dumps were enriched for Gram-positive and anaerobic bacteria. Broad-scale clustering analyses revealed that community relatedness between samples reflected system component (gardens/dumps) rather than colony source (lab/field). At finer scales samples clustered according to colony source.Conclusions/SignificanceHere we report the first comparative analysis of the microbiota from leaf-cutter ant colonies. Our work reveals the presence of two distinct communities: one in the fungus garden and the other in the refuse dump. Though we find some effect of colony source on community structure, our data indicate the presence of consistently associated microbes within gardens and dumps. Substrate composition and system component appear to be the most important factor in structuring the microbial communities. These results thus suggest that resident communities are shaped by the plant degradation gradient created by ant behavior, specifically their fungiculture and waste management.

Laboratory observation of siblicide with hatching asynchrony in an insect with parental provisioning

We report laboratory observation of siblicide with hatching asynchrony in the passalid beetle Cylindrocaulus patalis. Parents construct a breeding gallery in rotten logs and show complex care of offspring, including provisioning. Clutch sizes are extremely small, and only a single offspring is raised in a colony in each breeding season. We introduced field-collected colonies into an artificial gallery and recorded the behaviour of colony members. Eggs hatched at 2- to 10-day intervals in each colony. Fatal fighting invariably occurred among the larvae soon after hatching of the junior larva, and a single larva, usually the senior one, survived.

The interplay between genetic and environmental effects on colony insularity in the clonal invasive little fire ant Wasmannia auropunctata

The little fire ant, Wasmannia auropunctata, constitutes one clonal supercolony throughout Israel, providing an opportunity to examine the effects of genotype versus environment on nestmate recognition. Intraspecific encounters among field-collected or among laboratory-maintained colonies were nonaggressive, but encounters between freshly collected and laboratory-maintained colonies were highly aggressive. Analyses of cuticular hydrocarbons revealed that freshly field-collected colonies had distinguishable profiles. Moreover, freshly collected colonies had profiles disparate from those of the same colonies after 4 months in the laboratory. These results indicate a strong interplay between genetic-based and environmentally based effects on the recognition cues. We propose that in the field the ants’ diet breadth is broad and consequently the incorporation of diet-borne substances is insufficient to mask the genetically determined cues. In the laboratory, however, the restricted diet promoted the incorporation of alien hydrocarbons at high levels, thus altering the genetically based cues to the point of alienation. These results shed a new light on the mechanisms by which environmental cues may affect label and/or template formation in ants.

Isolation and Structure Determination of Malevamide E, a Dolastatin 14 Analogue, from the Marine Cyanobacterium Symploca laete-viridis

A new depsipeptide, malevamide E (1), was isolated from field-collected colonies of the filamentous cyanobacterium Symploca laete-viridis. The gross structure of 1 was determined by spectroscopic analyses, including one- and two-dimensional NMR and accurately measured MS/MS. Chiral HPLC analyses of an acid hydrolysate of 1 allowed the stereochemical assignments of its amino acid residues, which include N-methyl-L-alanine, alpha-N,gamma-N-dimethyl-L-asparagine, N-methyl-L-phenylalanine, L-proline, D-valine, and N-methyl-L-valine. LC-MS/MS analysis of S. laete-viridis fractions established the co-occurrence of malevamide E (1) and its homologue dolastatin 14 (2), which was previously reported in low yield from the sea hare Dolabella auricularia. Malevamide E (1) demonstrated a dose-dependent (2-45 microM) inhibition of store-operated Ca(2+) entry in thapsigargin-treated human embryonic kidney (HEK) cells, indicating an inhibitory effect on Ca(2+) release-activated Ca(2+) (CRAC) channels.

Geographical variation in Argentine ant aggression behaviour mediated by environmentally derived nestmate recognition cues

Social insects use a complex of recognition cues when discriminating nestmates from non-nestmate conspecifics. In the Argentine ant, Linepithema humile, recognition cues can be derived from exogenous sources, and L. humile acquires prey-derived hydrocarbons that are used in nestmate discrimination. We studied Argentine ant population-level distinctions in response to external recognition cues. Ants belonging to a California population were strongly affected by the imposition of prey-derived hydrocarbons, with spatially isolated colony fragments that had been fed different cockroach prey (Blattella germanica or Supella longipalpa) showing high and injurious intracolony aggression when reunited. In contrast, colonies of Argentine ants from the southeastern U.S. showed only moderate and noninjurious aggression when subjected to the same treatment. Field-collected colonies of L. humile had hydrocarbons in the range of those provided by S. longipalpa, and colonies from the southeastern U.S. had significantly higher initial levels of Supella-shared hydrocarbons. When fed cockroaches, Argentine ants from both regions acquired additional amounts of Supella- and Blattella-specific hydrocarbons, with a significant increase in levels of Blattella-specific hydrocarbons. Therefore, diet partitioning produced a greater change in the proportion of prey hydrocarbons in the California than in the southeastern U.S. populations, which may be responsible for the altered behaviour observed in the California population. Identifying factors underlying geographical variation in cue expression and/or perception may bring us closer to elucidating the selective forces driving nestmate recognition systems.

Diet-Related Modification of Cuticular Hydrocarbon Profiles of the Argentine Ant, Linepithema humile, Diminishes Intercolony Aggression

Territorial boundaries between conspecific social insect colonies are maintained through a highly developed nestmate recognition system modulated by heritable and, in some instances, nonheritable cues. Argentine ants, Linepithema humile, use both genetic and environmentally derived cues to discriminate nestmates from nonnestmates. We explored the possibility that intraspecific aggression in the Argentine ant might diminish when colonies shared a common diet. After segregating recently field-collected colony pairs into high or moderate aggression categories, we examined the effect of one of three diets: two hydrocarbon-rich insect prey, Blattella germanica and Supella longipalpa, and an artificial (insect-free) diet, on the magnitude of aggression loss. Aggression diminished between colony pairs that were initially moderately aggressive. However, initially highly aggressive colony pairs maintained high levels of injurious aggression throughout the study, independent of diet type. Each diet altered the cuticular hydrocarbon profile by contributing unique, diet-specific cues. We suggest that acquisition of common exogenous nestmate recognition cues from shared food sources may diminish aggression and promote fusion in neighboring colonies of the Argentine ant.

Predatory wasps learn to overcome the shelter defences of their larval prey

Larvae of Epargyreus clarus (Hesperiidae), the silver-spotted skipper, inhabit leaf-and-silk shelters that they construct on their leguminous host plants. In the field, Polistes spp. (Vespidae) wasps land on the shelters, quickly extracting and killing the larvae within. In marked contrast, wasps that emerge from field-collected colonies maintained in the laboratory visit and examine leaflets bearing sheltered caterpillars, but only rarely do they extract and kill the sheltered larvae. To examine whether learning is involved in the development of the ability of Polistes wasps to forage successfully on sheltered E. clarus larvae, we tested the responses of P. fuscatus and P. dominulus wasps to sheltered E. clarus larvae before and after their exposure to unsheltered larvae that were visible either on an opened host-leaf shelter ( P. fuscatus and P. dominulus) or on a nonhost leaf in the absence of a shelter ( P. fuscatus). After killing and processing an unsheltered larva that was visible on an opened leaf shelter, a majority of foragers subsequently extracted and killed larvae from closed shelters. Wasps that killed and processed an unsheltered larva on a nonhost leaf, on the other hand, generally did not later open shelters. Thus, it seems that experience with an exposed larva in the context of its shelter is necessary for a wasp to be able to prey on sheltered larvae. We conclude that the wasps must learn to associate the taste of the larva with shelter-related cues, such as presence of leaf damage and silk. In nature, this initial exposure may occur when the larva is visible in or near its shelter, perhaps when feeding or constructing a new shelter. Learning opportunities will thus depend on larval density. Our results show that invertebrate predators can learn to overcome their prey's defences, and are therefore able to make use of previously inaccessible prey.

Insecticide resistance of housefly, Musca domestica L. (Diptera : Muscidae) in poultries, with special reference to the yearly changes in resistance levels

Insecticide resistance of houseflies inhabiting a poultry ranch in Hachinohe City, Aomori Prefecture was studied from 1982 to 1998. An outbreak of houseflies in 1989 proved to be highly resistant to permethrin, with the LD_<50> value of 67 times that of the susceptible fly. They were also resistant to organophosphorus (OP) compounds. The LD_<50> value of the field-collected colony in 1989 increased 140 times the resistance ratio for permethrin by succesive treatments of permethrin for 5 generations (Hachinohe R). Houseflies taken from a poultry ranch in Tojo Cho, Hiroshima Prefecture showed drastic development of permethrin-resistance, with the LD_<50> value of 10.1μg per female (673 times that of the susceptible fly). The testing result on the Hachinohe R strain indicates that a recessive kdr gene might participate to develop pyrethroid-resistance. The resistance ratios to Insect Growth Regulators were low or moderate.