Molecular detection of knockdown resistance (kdr) in Blattella germanica (Blattodea: Blattellidae) from northwestern Iran.

Pyrethroid insecticides are highly insecticidal compounds that are widely used against the German cockroach, a significant household insect pest. In several insect species, there is a point mutation in the para-type sodium channel gene associated with knockdown resistance (kdr). In the current study, genomic DNA was analyzed in the region where the kdr and super-kdr (an enhanced form of pyrethroid resistance) mutations reside in Blatella germanica (L., 1767) (Blattodea: Blattellidae) collected from Iran. Studies on the extracted DNA from hand-captured German cockroach specimens were conducted by polymerase chain reaction and sequencing to detect related mutations. The kdr mutation, substitution of G for C (L1014F), which results in amino acid replacement (leucine with phenylalanine), was detected in all 18 sequenced specimens from three different locations. However, the super-kdr mutation (M918T), which is detected in super-kdr house flies, was not found in the sequences of the current study. The high ratio of the kdr mutation in a field population of B. germanica in Urmia confirms that the individuals are homozygous. These data should be helpful in designing and implementing a control program and resistance management.

Anopheles peditaeniatus (Leicester) new to the Iranian mosquito fauna with notes on Anopheles hyrcanus group in Iran.

Anopheles peditaeniatus is reported for the first time in Iran. Species of the Hyrcanus Group previously reported in Iran, including An. hyrcanus, An. pseudopictus, and An. nigerrimus, are reviewed.

The Anopheles stephensi odorant binding protein 1 (AsteObp1) gene: a new molecular marker for biological forms diagnosis.

Anopheles (Cellia) stephensi Liston 1901 is known as an Asian malaria vector. Three biological forms, namely "mysorensis", "intermediate", and "type" have been earlier reported in this species. Nevertheless, the present morphological and molecular information is insufficient to diagnose these forms. During this investigation, An. stephensi biological forms were morphologically identified and sequenced for odorant-binding protein 1 (Obp1) gene. Also, intron I sequences were used to construct phylogenetic trees. Despite nucleotide sequence variation in exon of AsteObp1, nearly 100% identity was observed at the amino acid level among the three biological forms. In order to overcome difficulties in using egg morphology characters, intron I sequences of An. stephensi Obp1 opens new molecular way to the identification of the main Asian malaria vector biological forms. However, multidisciplinary studies are needed to establish the taxonomic status of An. stephensi.

Evaluation of three methods for detecting permethrin resistance in adult and nymphal Blattella germanica (Dictyoptera: Blattellidae).

The insecticide resistance status of the German cockroach, Blattella germanica (L.), against permethrin was evaluated with three different bioassay methods. The LT50 and KT50 in adults were measured by glass jar test, the LT50 and KT50 in nymphs were assessed by glass petri dish-test, and the LD50 in adults and nymphs was measured by topical application. The results indicate that in all three bioassays, cockroaches showed different degrees of resistance to permethrin. Resistance ratios of 6.7-, 12.8-, and 7.5-fold for KT50, LD50, and LT50, respectively, were obtained for adult cockroaches compared with those from a susceptible strain. Similarly, resistance ratio values of 17-fold for KT50, 8-fold for LD50, and 6.4-fold for LT50 were obtained for nymphs compared with those from a susceptible strain. Although both adults and nymphs exhibited a similar level of susceptibility to permethrin, adults showed a higher degree of susceptibility to topical application than to the other exposure method. The order of sensitivity (based on resistance ratio values) was topical application (LD50) > LT50 > KT50 in adults. For nymphs, the resistance ratio in the knockdown test was higher than in other tests (resistance ratio = 17-fold). Therefore, the order of test sensitivity based on comparisons of resistance ratio values was KT50 > topical application (LD50) > LT50 in nymphs. Topical application exhibited a higher sensitivity than the LT and KT methods in adults. Thus, the results suggest that topical application and knockdown testing methods are the most sensitive assays for measuring resistance of adult and nymphal German cockroaches, respectively, to permethrin.

Field application of Romanomermis culicivorax (Mermithidae: Nematoda) to control anopheline larvae in southern Iran.

The efficacy of the parasitic nematode Romanomermis culcivorax in controlling anopheline larvae, including malaria vectors, was studied in Fars and Baluchistan provinces. Twenty sites were treated once with the preparastic stage of R. culicivorax in 1984 with 3,000, 5,000 or 10,000 preparasites per m2 surface area, depending on larval density. The average parasitism of anopheline larvae ranged from 56 to 69% based on 24 hr posttreatment dissections. No correlation was found between the level of parasitism and the density of mosquito larvae present in a site. About 61% parasitism was obtained when different rates of preparasites were released in 14 larval breeding sites in 1985. No apparent difference was observed in the rate of parasitism in 10 sites receiving one treatment compared with four sites receiving two treatments, with a seven day interval between each treatment. Romanomermis culcivorax was established in the release sites but caused only minor reductions in anopheline larval populations. The nematode would be of limited use in antimalaria campaigns in southern Iran.

Mosquito (Diptera: culicidae) fauna of qom province, iran.

BACKGROUND: There is very little information about the mosquito fauna of Qom Province, central Iran. By now only three species; Anopheles claviger, An. multicolor, and An. superpictus have been reported in the province. To study mosquito fauna and providing a primary checklist, an investigation was carried out on a collection of mosquitoes in this province. METHODS: TO STUDY THE MOSQUITO (DIPTERA: Culicidae) fauna, larval collections were carried out from different habitats on 19 occasions using the standard dipping technique during spring and summer 2008 and 2009. RESULTS: In total, 371 mosquito larvae were collected and morphologically identified including 14 species representing four genera: Anopheles claviger, An. marteri, An. turkhudi, An. superpictus, Culex arbieeni, Cx. hortensis, Cx. mimeticus, Cx. modestus, Cx. pipiens, Cx. territans, Cx. theileri, Culiseta longiareolata, Cs. subochrea, and Ochlerotatus caspius s.l. All species except for An. claviger and An. superpictus were collected for the first time in the province. All larvae were found in natural habitats. The association occasions and percentages of the mosquito larvae in Qom Province were discussed. CONCLUSION: There are some potential or proven vectors of different human and domesticated animal pathogens in Qom Province. The ecology of these species and the unstudied areas of Qom Province need to be investigated extensively.

Susceptibility of field populations of Blattella germanica (Blattaria: Blattellidae) to spinosad.

The German cockroach is an important household insect pest worldwide and acts as a mechanical vector and reservoir for pathogenic agents. The aim of this study was to examine the basic laboratory toxicity of Blattella germanica to spinosad. The M, T, A22, AZAR4, BOOSTAN7 and ABAN21 strains were collected from field populations of six infested kitchen student dormitories and the SAMAN strain was collected from a residential area after insecticide spraying control failure in Tehran, Iran. Technical grade spinosad was delivered in 0.5 microL acetone to the first abdominal sternum of briefly CO2-anesthetize adult male cockroaches by topical application bioassay. Treated males monitored for mortality. Mortality data from the replicates was assessed by probit analysis. The average LD50 of susceptible strain was 494.3, 148.8 and 55.1 ng per insect after 24, 48 and 72 h, respectively. The LD50 of spinosad decreased with time in the field population strains. All German cockroach strains showed a similar susceptibility or lower tolerance (1.6-folds) for spinosad compared with the susceptible laboratory strain and the steep slopes of dose-response curves indicated that the field population of these German cockroach strains was homogenous in response to spinosad. These results indicated that the spinosad was relatively slow-acting in topical application bioassay, with LD50 values decreasing until 72 h and becoming stable thereafter. The effectiveness of spinosad against susceptible and the field population German cockroach strains in laboratory condition showed that spinosad probably could be useful for the control of the German cockroach.

Insecticide resistance and synergism of three field-collected strains of the German cockroach Blattella germanica (L.) (Dictyoptera: Blattellidae) from hospitals in Kermanshah, Iran.

The development of insecticide resistance in the German cockroach, Blattella germanica (L.) is a serious problem in controlling this medically important household pest. The insecticide resistance status in three hospital-collected strains of the German cockroach using four commonly used insecticides from different classes (permethrin, cypermethrin, bendiocarb and chlorpyrifos) was detected by topical bioassay method and preliminary information on possible involvement of monooxygenases in permethrin resistant strains employing synergist piperonyl butoxide (PBO) was obtained. For each insecticide, four to six concentrations resulting in >0% and <100% mortality were used. Three to six replicates of 10 cockroaches per concentration were conducted. For synergism studies, 100 µg PBO per gram body weight of cockroach as the maximum sublethal dose was administered to the first abdominal segment 1 h before insecticide treatment. The differences between LD50 (µg/g) values were considered statistically significant only when the 95% confidence intervals did not overlap. The resistance ratio and synergism ratio were calculated for each insecticide. All three hospital-collected strains of the German cockroach showed different levels of resistance to permethrin and cypermethrin based on resistance ratios compared with SUS strain. Permethrin and cypermethrin resistance ratios ranged from 11.61 to 17.64 and 11.45 to 26.45 at LD50 levels, respectively. Low to moderate levels of bendiocarb resistance and low level of chlorpyrifos resistance were also observed in the hospital-collected strains under study. The synergist piperonyl butoxide (PBO) significantly enhanced the toxicity of permethrin to all strains with different degrees of synergist ratio, 2.45-, 1.87-, 2.51- and 2.38-fold, suggesting monooxygenase involvement in permethrin resistance.

Iranian non-responding contact method German cockroach permethrin resistance strains resulting from field pressure pyrethroid spraying.

The present study investigated the probable responsible reasons for non-response the B. germanica to permethrin only in World Health Organization glass jar method after insecticide spraying control failure with pyrethroid insecticide groups in Islamic Republic of Iran. Eleven German cockroach strains were collected from field populations of nine infested kitchen student dormitories and two infested hospitals after insecticide spraying control failure with pyrethroid insecticide groups in Iran. The current study, in World Health Organization glass jar and knock down methods conducted on newly emerged adult males. All feral strains, with various levels of resistance to pyrethroids, in World Health Organization glass jar method at 400 min (6 h) time exposures, was not observed mortality, while the susceptible strain was observed 100% mortality in 25 min time exposures. Susceptible strain at LT50 after assessing on mortality data from the replicates by probit analysis in World Health Organization glass jar method was 15.3 min. In this study, all feral strains in World Health Organization glass jar method after 400 min (6 h) time exposures, was not observed mortality, that showed these strains very high-level resistance to permethrin. In the knock down method, the resistance ratios were 3.6 to 26.1-folds compare with the susceptible reference strain. In a comparison, among this study and previous studies resistance ratios of 8.6 to 17.7-folds for permethrin in topical application, indicated that German cockroach have had under pressure spraying. German cockroach have had in vicinity to pyrethroid insecticides especially permethrin in these locations in long period for non-responding to permethrin insecticide only in World Health Organization glass jar method, and the field evident confirm this subject.

Permethrin resistance ratios compared by two methods of testing nymphs of the German cockroach, Blattella germanica.

For the German cockroach, Blattella germanica L. (Dictyoptera: Blattellidae), the permethrin resistance ratio (RR) was assessed by topical application and by tarsal contact tests, using first-instar nymphs of five strains from Tehran, Iran. Each test was replicated three or four times with 10 nymphs aged 2-3 days; mortality was scored 24h post-treatment. The reference susceptible strain showed LD50 permethrin 0.0175 microl/nymph from topical application, KT50 of 8.41 min and LT50 of 12.82 following tarsal contact with permethrin 15 mg/m2. In four wild strains (F1 generation) the RR varied from 4.14 to 4.7 for mortality after topical application, from 4.2 to 6.45 for mortality and 17-27 for knockdown following tarsal contact tests. Hence, overall knockdown results gave much higher RRs than for mortality data. Resistance ratios based on both methods of treatment were very similar: one strain showed a slightly higher value by topical application (RR 4.6 vs. 4.2, i.e. 1.1-fold difference) whereas the other three strains gave slightly greater RR (1.2-1.4 fold) by tarsal contact. Resistance was abolished by cotreatment with the synergist piperonyl butoxide plus permethrin (ratio 3:1 required for full efficacy), indicating that mixed-function oxidases were inhibited as a major metabolic pathway in all four resistant strains.

Molecular evidence for a kdr-like pyrethroid resistance mechanism in the malaria vector mosquito Anopheles stephensi.

The mosquito Anopheles stephensi Liston (Diptera: Culicidae) is the urban vector of malaria in several countries of the Middle East and Indian subcontinent. Extensive use of residual insecticide spraying for malaria vector control has selected An. stephensi resistance to DDT, dieldrin, malathion and other organophosphates throughout much of its range and to pyrethroids in the Middle East. Metabolic resistance mechanisms and insensitivity to pyrethroids, so-called knockdown resistance (kdr), have previously been reported in An. stephensi. Here we provide molecular data supporting the hypothesis that a kdr-like pyrethroid-resistance mechanism is present in An. stephensi. We found that larvae of a pyrethroid-selected strain from Dubai (DUB-R) were 182-fold resistant to permethin, compared with a standard susceptible strain of An. stephensi. Activities of some enzymes likely to confer pyrethroid-resistance (i.e. esterases, monooxygenases and glutathione S-transferases) were significantly higher in the permethrin-resistant than in the susceptible strain, but the use of synergists--piperonyl butoxide (PBO) to inhibit monooxygenases and/or tribufos (DEF) to inhibit esterases--did not fully prevent resistance in larvae (permethrin LC50 reduced by only 51-68%), indicating the involvement of another mechanism. From both strains of An. stephensi, we obtained a 237-bp fragment of genomic DNA encoding segment 6 of domain II of the para type voltage-gated sodium channel, i.e. the putative kdr locus. By sequencing this 237 bp fragment, we identified one point mutation difference involving a single A-T base change encoding a leucine to phenylalanine amino acid substitution in the pyrethroid-resistant strain. This mutation appears to be homologous with those detected in An. gambiae and other insects with kdr-like resistance. A diagnostic polymerase chain reaction assay using nested primers was therefore designed to detect this mechanism in An. stephensi.