Detection of Changuinola virus (Reoviridae: Orbivirus) in field-caught sand flies in southern Thailand.

BACKGROUND: Phlebotomine sand flies are vectors for several pathogenic bacteria, parasites and viruses that have significant impacts on public health. Sand fly-associated viruses that cause diseases in humans and animals have recently received more attention. This study aimed to detect pathogenic viruses belonging to the Orbivirus genus, Phlebovirus genus, Flavivirus genus and family Rhabdoviridae in several field-caught sand fly species in southern Thailand. METHODS: Sand flies were collected in southern Thailand using CDC light traps. Each sample was processed individually for virus screening using RT-PCR and sequencing. RESULTS: Seven out of 60 sand fly samples (two samples of Idiophlebotomus spp., three of Phlebotomus papatasi and two of Sergentomyia khawi) were positive for the Orbivirus genus, which is closely related to Changuinola virus (CGLV). Phlebovirus genus, Flavivirus genus and family Rhabdoviridae were negative in all samples. CONCLUSIONS: CGLV causes Changuinola virus disease or Changuinola fever, a febrile illness in Central and South America. The virus has never been reported in Thailand. This study is the first report of the detection of CGLV in sand flies from Thailand. An extensive study of sand flies from other regions of the country and the associations between sand flies, viruses and vertebrate hosts in Thailand should be undertaken.

Synergistic Toxicity of Plant Essential Oils Combined with Pyrethroid Insecticides against Blow Flies and the House Fly.

Blow flies (Diptera: Calliphoridae) and the house fly (Diptera: Muscidae) are filth flies of medical importance, and control of their population is needed. As insecticide applications have resulted in fly resistance, and the exploration of plant essential oils (EOs) has increased against filth flies, this study assessed the combination of EOs with pyrethoids to enhance toxic efficacy. The EOs of five effective plants were screened initially against the house fly (Musca domestica L.). Their chemical constituent was performed using gas chromatography-mass spectrometry (GC-MS) analysis. The main components of Boesenbergia rotunda (Zingiberaceae) rhizome, Curcuma longa (Zingiberaceae) rhizome, Citrus hystrix (Rutaceae) fruit peel, Ocimum gratissimum (Lamiaceae) seed, and Zanthoxylum limonella (Rutaceae) fruit were δ-3-caren (35.25%), ß-turmerone (51.68%), ß-pinene (26.56%), p-cumic aldehyde (58.21%), and dipentene (60.22%), respectively. The screening test revealed that the three most effective plant EOs were from B. rotunda, C. longa and O. gratissimum, which were selected for the combination with two pyrethroid insecticides (permethrin and deltamethrin), in order to enhance their synergistic efficacy against the blow flies, Chrysomya megacephala Fabricius, Chrysomya rufifacies Macquart, and Lucilia cuprina Wiedemann, and the house fly. Synergistic action was presented in almost all of the flies tested with permenthrin/deltamethrin/EOs mixtures. It was interesting that the combination of deltamethrin with three EOs showed a synergistic effect on all of the tested flies. However, an antagonistic effect was observed in C. megacephala and M. domestica treated with permethrin-B. rotunda and C. megacephala treated with permethrin-O. gratissimum. The LD50 of insecticides decreased when combined with plant EOs. This alternative strategy will be helpful in developing a formula for effective fly control management.

Vertical transmission of Zika virus in Culex quinquefasciatus Say and Aedes aegypti (L.) mosquitoes.

Several mosquito species have been described as vectors for the Zika virus (ZIKV), such as those in the Aedes, Anopheles, Mansonia and Culex genera. Our previous survey studies were found the ZIKV RNA positive in both male, female and larvae of Culex quinquefasciatus Say and Aedes aegypti (L.) mosquitoes collected from active ZIKV infected patients' homes in Thailand. Therefore, the aims of this study were to investigate whether ZIKV could be vertically transmitted in Cx. quinquefasciatus, Ae. aegypti and Ae. albopictus. Laboratory and field colonies of these mosquito species were maintained and artificially fed with ZIKV in human blood. Fully engorged mosquitoes (F0) were selected and reared for the vertical transmission study. The subsequent mosquito generations were fed with human blood without the virus. ZIKV in the mosquitoes was detected by hemi-nested RT-PCR and sequencing. C6/36 cells were used to isolate ZIKV from samples that tested positive by hemi-nested RT-PCR. Moreover, ZIKV was identified by immunocytochemical staining 7 days after infection in several organs of infected F0 females, including the salivary glands, midguts, yoke granules and facet cells of the eye. The localization of the ZIKV antigen was identified by the presence of the specific antibody in the salivary glands, midguts, yoke granules and facet cells. ZIKV was detected in female and male Cx. quinquefasciatus until the F6 and F2 generations, respectively. The isolated virus showed cytopathic effects in C6/36 cells by 5 days postinfection. The results suggested that the vertical transmission of ZIKV occurs in Cx. quinquefasciatus in the laboratory. However, we were able to detect the presence of ZIKV in Ae. aegypti in only the F1 generation in both male and female mosquitoes, and Ae. albopictus mosquitoes were not able to vertically transmit the virus at all. Data obtained from this study could be valuable for developing a better understanding of the role of Cx. quinquefasciatus as a potential vector for ZIKV transmission in Thailand and may be useful in creating more effective mosquito vector control strategies in the future.

Detection of an Unknown Trypanosoma DNA in a Phlebotomus stantoni (Diptera: Psychodidae) Collected From Southern Thailand and Records of New Sand Flies With Reinstatement of Sergentomyia hivernus Raynal & Gaschen, 1935 (Diptera: Psychodidae).

Although female sand flies are best known as the vectors of Leishmania parasites and viruses, several previous reports have demonstrated that these insects can also act as vectors for the trypanosomes of bats, lizards, and snakes. In this report, we created an inventory of Phlebotomine sand flies from southern Thailand. A novel trypanosome was found in a specimen of Phlebotomus stantoni, and two sand fly species newly recorded in the country, Sergentomyia khawi and Sergentomyia hivernus, were described. PCR primer pairs specific for the internal transcribed spacer 1 (ITS1) and the small subunit ribosomal DNA (SSU rDNA) gene of trypanosomatids were used to demonstrate the presence of the parasite in the sand fly. In addition, the Cytochrome b (CytB) gene was used to identify the sand fly species. Among the 45 samples of the sand fly that were collected, seven samples were Ph. stantoni sand flies and a single sample was positive for Trypanosoma sp. through PCR analysis. This study represents the first detection of Trypanosoma sp. in a sand fly from Thailand. The ITS1 and SSU rDNA sequences indicated that this specimen is suspected to be a novel Trypanosoma species. Further studies of this suspected new Trypanosoma species, including its vertebrate hosts and pathogenic potential, are therefore necessary.

Vertical transmission of Indian Ocean Lineage of chikungunya virus in Aedes aegypti and Aedes albopictus mosquitoes.

BACKGROUND: The re-emergence of chikungunya (CHIK) fever in Thailand has been caused by a novel lineage of chikungunya virus (CHIKV) termed the Indian Ocean Lineage (IOL). The Aedes albopictus mosquito is thought to be a primary vector of CHIK fever in Thailand, whereas Ae. aegypti acts as a secondary vector of the virus. The vertical transmission is believed to be a primary means to maintain CHIKV in nature and may be associated with an increased risk of outbreak. Therefore, the goal of this study was to analyze the potential of these two Thai mosquito species to transmit the virus vertically and to determine the number of successive mosquito generations for the virus transmission. METHODS: Two-hundred-and-fifty female Ae. aegypti and Ae. albopictus mosquitoes were artificially fed a mixture of human blood and CHIKV IOL. Mosquito larvae and adults were sampled and screened for CHIKV by one-step qRT-PCR. LLC-MK2 cell line was used to isolate CHIKV in the mosquitoes each generation. The virus isolate was identified by immunocytochemical staining and was confirmed by sequencing. Both mosquito species fed on human blood without CHIKV and uninfected LLC-MK2 cells were used as controls. RESULTS: Aedes aegypti and Ae. albopictus mosquitoes were able to transmit CHIKV vertically to F5 and F6 progenies, respectively. The virus isolated from the two mosquito species caused cytopathic effect in LLC-MK2 cells by 2 days post-infection and immunocytochemical staining showed the reaction between CHIKV IOL antigen and specific monoclonal antibody in the infected cells. DNA sequence confirmed the virus transmitted vertically as CHIKV IOL with E1-A226V mutation. No CHIKV infection was observed in both mosquito species and LLC-MK2 cells from control groups. CONCLUSIONS: The study demonstrated that Ae. aegypti and Ae. albopictus mosquitoes from Thailand are capable of transmitting CHIKV IOL vertically in the laboratory. Our results showed that Ae. albopictus is more susceptible and has a greater ability to transmit the virus vertically than Ae. aegypti. This knowledge would be useful for risk assessments of the maintenance of CHIKV in nature, which is crucial for disease surveillance, vector control and the prevention of potential CHIKV epidemics.

Morbidity Rate Prediction of Dengue Hemorrhagic Fever (DHF) Using the Support Vector Machine and the Aedes aegypti Infection Rate in Similar Climates and Geographical Areas.

BACKGROUND: In the past few decades, several researchers have proposed highly accurate prediction models that have typically relied on climate parameters. However, climate factors can be unreliable and can lower the effectiveness of prediction when they are applied in locations where climate factors do not differ significantly. The purpose of this study was to improve a dengue surveillance system in areas with similar climate by exploiting the infection rate in the Aedes aegypti mosquito and using the support vector machine (SVM) technique for forecasting the dengue morbidity rate. METHODS AND FINDINGS: Areas with high incidence of dengue outbreaks in central Thailand were studied. The proposed framework consisted of the following three major parts: 1) data integration, 2) model construction, and 3) model evaluation. We discovered that the Ae. aegypti female and larvae mosquito infection rates were significantly positively associated with the morbidity rate. Thus, the increasing infection rate of female mosquitoes and larvae led to a higher number of dengue cases, and the prediction performance increased when those predictors were integrated into a predictive model. In this research, we applied the SVM with the radial basis function (RBF) kernel to forecast the high morbidity rate and take precautions to prevent the development of pervasive dengue epidemics. The experimental results showed that the introduced parameters significantly increased the prediction accuracy to 88.37% when used on the test set data, and these parameters led to the highest performance compared to state-of-the-art forecasting models. CONCLUSIONS: The infection rates of the Ae. aegypti female mosquitoes and larvae improved the morbidity rate forecasting efficiency better than the climate parameters used in classical frameworks. We demonstrated that the SVM-R-based model has high generalization performance and obtained the highest prediction performance compared to classical models as measured by the accuracy, sensitivity, specificity, and mean absolute error (MAE).

Molecular survey of the head louse Pediculus humanus capitis in Thailand and its potential role for transmitting Acinetobacter spp.

BACKGROUND: Head louse infestation, which is caused by Pediculus humanus capitis, occurs throughout the world. With the advent of molecular techniques, head lice have been classified into three clades. Recent reports have demonstrated that pathogenic organisms could be found in head lice. Head lice and their pathogenic bacteria in Thailand have never been investigated. In this study, we determined the genetic diversity of head lice collected from various areas of Thailand and demonstrated the presence of Acinetobacter spp. in head lice. METHODS: Total DNA was extracted from 275 head louse samples that were collected from several geographic regions of Thailand. PCR was used to amplify the head louse COI gene and for detection of Bartonella spp. and Acinetobacter spp. The amplified PCR amplicons were cloned and sequenced. The DNA sequences were analyzed via the neighbor-joining method using Kimura's 2-parameter model. RESULTS: The phylogenetic tree based on the COI gene revealed that head lice in Thailand are clearly classified into two clades (A and C). Bartonella spp. was not detected in all the samples, whereas Acinetobacter spp. was detected in 10 samples (3.62%), which consisted of A. baumannii (1.45%), A. radioresistens (1.45%), and A. schindleri (0.72%). The relationship of Acinetobacter spp. and the head lice clades showed that Acinetobacter spp. was found in clade A and C. CONCLUSIONS: Head lice in Thailand are classified into clade A and B based on the COI gene sequences. Pathogenic Acinetobacter spp. was detected in both clades. The data obtained from the study might assist in the development of effective strategies for head lice control in the future. Detection of pathogenic bacteria in head lice could raise awareness of head lice as a source of nosocomial bacterial infections.

Identification of putative kdr mutations in the tropical bed bug, Cimex hemipterus (Hemiptera: Cimicidae).

BACKGROUND: Bed bugs [both Cimex hemipterus (F.) and Cimex lectularius L.] are highly resistant to pyrethroids worldwide. An important resistance mechanism known as 'knockdown resistance' (kdr) is caused by genetic point mutations on the voltage-gated sodium channel (VGSC) gene. Previous studies have identified two point mutations (V419L and L925I) on the VGSC gene in C. lectularius that are responsible for kdr-type resistance. However, the kdr mutations in C. hemipterus have not been investigated. RESULTS: Four novel mutations, L899V (leucine to valine), M918I (methionine to isoleucine), D953G (aspartic acid to glycine) and L1014F (leucine to phenylalanine), were identified in the domain II region of the C. hemipterus VGSC gene. This region has been widely investigated for the study of kdr-type resistance to pyrethroids in other insect pests. The V419L and L925I kdr mutations as previously identified in C. lectularius were not detected in C. hemipterus. CONCLUSION: M918I and L1014F are considered to be probable kdr mutations and may play essential roles in kdr-type resistance to pyrethroids in C. hemipterus. Further studies are under way in the authors' laboratory to determine the non-kdr-type resistance mechanisms in C. hemipterus.

Wolbachia supergroups A and B in natural populations of medically important filth flies (diptera: muscidae, calliphoridae, and sarcophagidae) in Thailand.

Filth flies, belonging to suborder Brachycera (Family; Muscidae, Calliphoridae and Sarcophagidae), are a major cause of nuisance and able to transmit pathogens to humans and animals. These insects are distributed worldwide and their populations are increasing especially in sub-tropical and tropical areas. One strategy for controlling insects employs Wolbachia, which is a group of maternally inherited intracellular bacteria, found in many insect species. The bacteria can cause reproductive abnormalities in their hosts, such as cytoplasmic incompatibility, feminization, parthenogenesis, and male lethality. In this study we determined Wolbachia endosymbionts in natural population of medically important flies (42 females and 9 males) from several geographic regions of Thailand. Wolbachia supergroups A or B were detected in 7 of female flies using PCR specific for wsp. Sequence analysis of wsp showed variations between and within the Wolbachia supergroup. Phylogenetics demonstrated that wsp is able to diverge between Wolbachia supergroups A and B. These data should be useful in future Wolbachia-based programs of fly control.

Efficacy and safety of topical Trikatu preparation in, relieving mosquito bite reactions: a randomized controlled trial.

INTRODUCTION: Trikatu is composed of dried fruits of Piper nigrum L and Piper retrofractum Vahl, and dried rhizomes of Zingiber officinale R. Although this preparation has been used to relieve pruritis, pain, and inflammation for a long time, there is no clinical evidence to confirm its efficacy and safety. Therefore, we performed a double-blind, within person-randomized controlled study of 30 healthy volunteers to determine efficacy and safety of topical Trikatu on mosquito bite reactions. METHODS: All subjects were bitten by Aedes aegypti laboratory mosquitoes on their forearms and they were randomly assigned arms to apply either Trikatu or reference product on the mosquito bite papule. The main outcome was the difference of papule size reduction at 30 min, measured by a caliper, between the Trikatu and reference arms. Pruritis, redness, pain, and patient satisfaction were assessed at 15, 30, 60, 180, and 360 min as secondary outcomes. RESULTS: There were no significant differences between treatment and reference arms on any outcome at any time of measurement. CONCLUSION: Trikatu did not show additional effects for relieving mosquito bite reaction as compared with the reference product containing camphor, menthol, and eucalyptus. For further study, it is very important to consider a proper selection of subjects, comparator product, and concentration of extract when Trikatu preparation is investigated.

Comparative field efficacy of newly developed formulations of larvicides against Aedes aegypti (L.) (Diptera: Culicidae).

Aedes aegypti (L.) is known as vector of dengue and chikungunya fever. Larvicides are used to control this vector. We evaluated the efficacy of newly developed formulations of larvicides to control Ae. aegypti under field conditions for 24 weeks post single application. Mosdop P and Mosdop TB containing diflubenzuron (2% and 40 mg/tablet, respectively) as the active ingredient, were applied at a dosage of 0.1 mg a.i./1 and Mosquit TB10, Mosquit TB100 and Temecal containing temephos (1%, 10% and 1%, respectively) as the active ingredient were applied at a dosage of 1 mg active ingredent (a.i.) to 200 liter water storage jars. Two water regimens were used in the jars: in one regimen the jar was kept full of water all the time and in the other regimen a full jar had half the volume removed and refilled weekly. The larvicidal efficacy was reported as the level of inhibition of emergence (IE%) calculated based on the pupal skins in the jars versus the original number of larvae added. Mosdop P, Mosdop TB, Mosquit TB10, Mosquit TB100 and Temecal showed complete larvicidal efficacy (100% IE) in the constantly full jars for 16, 17, 14, 20 and 13 weeks posttreatment, respectively; in the jars where half the volum of water was replaced weekly, the larvicides had complete larvicidal efficacy (100% IE) for 19, 20, 17, 24 and 15 weeks post-treatment, respectively. The five larvicide regimens evaluated in this study are effective for controlling Ae. aegypti larvae.

Comparison of allergenic components and biopotency in whole body extracts of wild and laboratory reared American cockroaches, Periplaneta americana.

BACKGROUND AND OBJECTIVE: Most allergen extracts/vaccines used today in clinical practice are derived from natural allergen sources. Therefore, their allergic components may vary as these are prone to natural variation. This study aims to compare the allergenic components and biological potency of crude extracts from wild and laboratory reared American cockroaches. METHODS: Crude extracts of male and female of wild and laboratory reared American CR, were prepared by the same method. Their allergenic components were evaluated by in vitro assays such as protein contents, protein profiles, quantification of major allergens (Per a 1 and Per a 9) and IgE inhibition ELISA assay. RESULTS AND DISCUSSION: There was no statistically significant difference between the protein contents and the concentrations of Per a 1 in the crude extracts from both groups. However, the Per a 9 levels in extracts of wild CR were significantly higher than those from the extracts of laboratory reared CR. The protein patterns of the extracts of laboratory reared CR exhibited more consistency in the number of bands with higher intensity than those of wild CR. Pooled extracts of laboratory reared CR could inhibit IgE binding to that of wild CR up to 78%. The endotoxin content of extracts of laboratory reared CR were ten times less than those of the the wild CR. We have successfully determined the allergenic potency of the extracts of laboratory reared CR versus those of the wild CRs by in vitro assays. Further studies should be performed to determine the biological potency of CR extracts by in vivo assays for clinical application. CONCLUSION: Our finding indicates that the laboratory reared CR would be the better source of material in vaccine production than the wild CR.

Insecticide resistance in bedbugs in Thailand and laboratory evaluation of insecticides for the control of Cimex hemipterus and Cimex lectularius (Hemiptera: Cimicidae).

Bedbugs are found in many countries around the world, and in some regions they are resistant to numerous insecticides. This study surveyed bedbugs in Thailand and determined their resistance to insecticides. The surveys were carried out in six provinces that attract large numbers of foreign tourists: Bangkok, Chonburi, Chiang Mai, Ubon Ratchathani, Phuket, and Krabi. Bedbugs were collected from hotels and colonized in the laboratory to evaluate their resistance to insecticides. Cimex hemipterus (F.) was found in some hotels in Bangkok, Chonburi, Phuket, and Krabi, whereas Cimex lectularius L. was found only in hotels in Chiang Mai. No bedbugs were found in Ubon Ratchathani. The colonized bedbugs showed resistance to groups of insecticides, including organochlorines (dichlorodiphenyl trichloroethane, dieldrin), carbamates (bendiocarb, propoxur), organophosphates (malathion, fenitrothion), and pyrethroids (cyfluthrin, deltamethrin, permethrin, lambda-cyhalothrin, etofenprox) in tests using World Health Organization insecticide-impregnated papers. The new insecticides imidacloprid (neonicotinoid group), chlorfenapyr (pyrrole group), and fipronil (phenylpyrazole group) were effective against the bedbugs; however, organophosphate (diazinon), carbamates (fenobucarb, propoxur), and pyrethroids (bifenthrin, cypermethrin, esfenvalerate, etofenprox) were ineffective. Aerosols containing various pyrethroid insecticides with two to four different active ingredients were effective against the bedbugs. The results obtained from this study suggested that both species of bedbugs in Thailand have developed marked resistance to various groups of insecticides, especially those in the pyrethroid group, which are the most common insecticides used for pest control. Therefore, an integrated pest management should be implemented for managing bedbugs in Thailand.

Competitive suppression between chikungunya and dengue virus in Aedes albopictus c6/36 cell line.

Aedes albopictus C6/36 cell line was used to evaluate dengue virus serotype-3 (DENV-3) and chikungunya virus (CHIKV) co-infection. Virus infection was determined using a one-step duplex reverse transcriptase polymerase chain reaction (D- RT-PCR). D-RT-PCR was positive for both viruses when equal multiplicity of infection (MOI) was utilized. Co-infection with different titers between a higher CHIKV titer (MOI = 1.0) than DENV-3 titer (MOI = 0.1) showed similar results with that of equal titer. However, co-infection with a lower CHIKV titer (MOI = 0.1) than DENV-3 titer (MOI = 1.0) revealed positive results for only dengue virus infection, suggesting DENV competitive suppression of CHIKV. This competitive suppression occurred in mixed-infection samples but not in individually double infection (super infection) samples which produced both dengue and chikungunya virus progenies. Both virus replications depend on the amount of virus titer rather than serial infection. These findings have provided information regarding pathogen-pathogen interaction in host cell, which could be used to predict outbreaks, and to develop virus detection and vector control.

Identification of blood meal of field caught Aedes aegypti (L.) by multiplex PCR.

Laboratory bred female Aedes aegypti (L.) was used to determine sensitivity of multiplex PCR for detecting human blood meal. Human blood DNA was detected in live fully fed mosquitoes until 3 days after blood feeding, and for 4 weeks when stored at -20 degrees C. Among 890 field caught female mosquito samples examined for vertebrate DNA by multiplex PCR, results were positive for human, pig, dog, cow and mixture of 2 host DNA at 86.1, 3.4, 2.1, 1.0 and 3.6%, respectively, while 3.9% of the samples were negative. Blood feeding pattern must be considered when mosquito control strategies become employed.

Mitochondrial DNA-based identification of some forensically important blowflies in Thailand.

Accurate identification of insects collected from death scenes provides not only specific developmental data assisting forensic entomologists to determine the postmortem interval more precisely but also other kinds of forensic evidence. However, morphological identification can be complicated due to the similarity among species, especially in the early larval stages. To simplify and make the species identification more practical and reliable, DNA-based identification is preferentially considered. In this study, we demonstrate the application of partial mitochondrial cytochrome oxidase I (COI) and cytochrome oxidase II (COII) sequences for differentiation of forensically important blowflies in Thailand; Chrysomya megacephala, Chrysomya rufifacies and Lucilia cuprina by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The PCR yields a single 1324bp-sized amplicon in all blowfly specimens, followed by direct DNA sequencing. Taq(α)I and VspI predicted from the sequencing data provide different RFLP profiles among these three species. Sequence analysis reveals no significant intraspecific divergence in blowfly specimens captured from different geographical regions in Thailand. Accordingly, neighbor-joining tree using Kimura's 2-parameter model illustrates reciprocal monophyly between species. Thus, these approaches serve as promising tools for molecular identification of these three common forensically important blowfly species in Thailand.

Outbreak of chikungunya fever in Thailand and virus detection in field population of vector mosquitoes, Aedes aegypti (L.) and Aedes albopictus Skuse (Diptera: Culicidae).

We investigated chikungunya fever outbreak in the southern part of Thailand. Human plasma specimens obtained from suspected patients and adult wild-caught mosquitoes were detected for chikungunya virus employing reverse transcriptase-polymerase chain reaction technique. Chikungunya virus was detected in about half of the blood specimens whereas a range of 5.5 to 100% relative infection rate was found in both sexes of the vector mosquitoes, Aedes aegypti (L.) and Ae. albopictus Skuse. The infection rate in Ae. albopictus was higher than in Ae. aegypti, with relative infection rate in male of both species being higher than in female. The appearance of chikungunya virus in adult male mosquitoes of both species reveals a role of transovarial transmission of the virus in field population of the mosquito vectors. These findings have provided further understanding of the relationship among mosquito vectors, chikungunya virus and epidemiology of chikungunya fever in Thailand.

Field evaluation in Thailand of spinosad, a larvicide derived from Saccharopolyspora spinosa (Actinomycetales) against Aedes aegypti (L.) larvae.

Two formulations of spinosad, direct application tablet (DT) and 0.5% granules (GR), at 3 dosages (0.25, 0.5 and 1.0 mg/l) in 200-liter earthen jars were evaluated against the larvae of Aedes aegypti. Two water regimens were used in the jars: jar full all the time and a full jar in which half the volume of the water was removed and replaced at each assessment interval. All treatments and controls were replicated 4 times and challenged with cohorts of 25 third-instar larvae of Ae. aegypti at weekly intervals during the study. The number of pupal skins (indicating successful emergence of adults) in the treated and control regimens were counted 7 days post-addition and they were used to calculate inhibition of emergence (% IE) based on the original number of larvae used. The DT formulation at the highest concentration (1.0 mg/l) yielded 79-100% IE for 34 days in the full jars, efficacy declining beyond this period. However, the longevity of this dosage was much longer with 90-100% IE for 62 days post-treatment in the water exchange regimen. The target and manufacturer-recommended concentration of 0.5 mg/l of DT gave good control (92-100% IE) for 20 days, declining below 92% IE thereafter in full jars. This dose also yielded good control with IE of 97-100% for 27 days in the water exchange regimen. The 0.5% GR formulation at all 3 dosages showed higher efficacy and greater longevity in the jars than the DT. In the full jars, all 3 dosages produced IE of 76-100% for 55 days post-treatment. In the water exchange regimen, the efficacy and longevity were increased by about one week, up to 62 days post-treatment. It is clear that the DT formulation can be used effectively against Ae. aegypti larvae at a target dose of 0.5 mg/l in 200-liter jars. This dose can be increased to 1.0 mg/l if slightly longer residual activity is desired. In containers where water is consumed and more water added, the longevity of efficacy will be longer for the DT than in jars which remain full all the time. GR (0.5%) gave longer control than DT. GR (0.5%) floated on the surface and produced scum and an oily film, features not desirable in stored water.

Intramolecular integration assay validates integrase phi C31 and R4 potential in a variety of insect cells.

Phage phi C31 and R4 integrases are site-specific and unidirectional serine recombinases. We have analyzed the ability of these integrases to mediate intramolecular integration between their attB and attP sites in 7 important insect cell lines as a means of predicting their relative mobility in the corresponding insect species. Both integrases exhibit significantly higher frequencies in Drosophila S2 cells than in the other insect cell lines examined, but do work well in all of the species tested. Our results, coupled with previous results of the activity of phi C31 integrase in D. melanogaster and Aedes aegypti, suggest the family of serine catalyzed integrases will be useful site-specific integration tools for functional genome analysis and genetic engineering in a wide range of insect species.