A DsRed fluorescent protein marker under <Emphasis Type="Italic">polyubiquitin</Emphasis> promoter regulation allows visual and amplified gene detection of transgenic Caribbean fruit flies in field traps

Field population surveillance of a targeted insect pest species is critical in evaluating management programs such as the sterile insect technique. Fluorescent powder dyes currently used to distinguish released tephritids from the field population are not optimal in terms of reliability and human health issues. Genetically transformed tephritid species present the possibility of using fluorescent transgenes for marking. Here we studied the stability of DsRed fluorescence in transgenic flies maintained in aqueous torula yeast borax and propylene glycol. DsRed was stable in both solutions for three weeks by visual microscopic observations and could be used to unambiguously distinguish them from non-fluorescent wild type flies. To compensate for any potential ambiguity in visual identification a diagnostic PCR was developed that could specifically amplify the exotic heterologous marker gene. Therefore, the use of sterile transgenic insect strains carrying stably integrated fluorescent protein marker genes in biologically-based control programs could greatly improve released fly identification in pest management programs.     

Transformed bacterial symbionts re-introduced to and detected in host gut

Strains of Enterobacter agglomerans and Klebsiella pneumoniae isolated from Rhagoletis completa Cresson were engineered to express transgenic fluorescent proteins (ECFP, DsRed). These bacteria were introduced into flies by feeding the flies a sucrose solution in which the bacteria were suspended. The transgenic and heterologous marker protein was expressed and visible in the bacteria after they were ingested by WHF and while they were in the fly gut. We describe the plasmids used to transform these bacteria and demonstrate expression of heterologous proteins from the transforming plasmids and discuss the implications for future pest control strategies. Received: 14 September 2001 / Accepted: 22 October 2001     

Pyrosequencing-Based Analysis of the Microbiome Associated with the Horn Fly,Haematobia irritans

The horn fly, Haematobia irritans, is one of the most economically important pests of cattle. Insecticides have been a major element of horn fly management programs. Growing concerns with insecticide resistance, insecticide residues on farm products, and non-availability of new generation insecticides, are serious issues for the livestock industry. Alternative horn fly control methods offer the promise to decrease the use of insecticides and reduce the amount of insecticide residues on livestock products and give an impetus to the organic livestock farming segment. The horn fly, an obligatory blood feeder, requires the help of microflora to supply additional nutrients and metabolize the blood meal. Recent advancements in DNA sequencing methodologies enable researchers to examine the microflora diversity independent of culture methods. We used the bacterial 16S tag-encoded FLX-titanium amplicon pyrosequencing (bTEFAP) method to carry out the classification analysis of bacterial flora in adult female and male horn flies and horn fly eggs. The bTEFAP method identified 16S rDNA sequences in our samples which allowed the identification of various prokaryotic taxa associated with the life stage examined. This is the first comprehensive report of bacterial flora associated with the horn fly using a culture-independent method. Several rumen, environmental, symbiotic and pathogenic bacteria associated with the horn fly were identified and quantified. This is the first report of the presence of Wolbachia in horn flies of USA origin and is the first report of the presence of Rikenella in an obligatory blood feeding insect.     

Advancements in management of major fruit flies (Diptera: Tephritidae) in North Africa and future challenges: A review

Fruit flies are one of the most economically damaging pests of fleshy fruits worldwide. Two species of highest concern for fruit production in North Africa are the Mediterranean fruit fly (Medfly) Ceratitis capitata (Wied.) and the peach fruit fly (PFF) Bactrocera zonata (Saunders) (in Egypt and Libya only). Currently, both fruit fly species are mainly targeted by chemical applications of broad-spectrum contact insecticides. Despite the disparities in control efforts among North African countries, government and research are focused on reducing chemical reliance and adopt more environmentally friendly technologies. In this review, advances in integrated pest management (IPM) implementation against fruit flies are outlined for each country. In addition, challenges for future efforts are identified with emphasis on the efficacy of trapping for monitoring and control.     

Insecticide resistance in the horn fly: alternative control strategies

Abstract.  The horn fly, Haematobia irritans (Linnaeus 1758) (Diptera: Muscidae) is one of the most widespread and economically important pests of cattle. Although insecticides have been used for fly control, success has been limited because of the development of insecticide resistance in all countries where the horn fly is found. This problem, along with public pressure for insecticide-free food and the prohibitive cost of developing new classes of compounds, has driven the investigation of alternative control methods that minimize or avoid the use of insecticides. This review provides details of the economic impact of horn flies, existing insecticides used for horn fly control and resistance mechanisms. Current research on new methods of horn fly control based on resistant cattle selection, semiochemicals, biological control and vaccines is also discussed.     

The utility of microsatellite DNA markers for the evaluation of area-wide integrated pest management using SIT for the fruit fly, Bactrocera dorsalis (Hendel), control programs in Thailand

The oriental fruit fly, Bactrocera dorsalis (Hendel), is a key pest that causes reduction of the crop yield within the international fruit market. Fruit flies have been suppressed by two Area-Wide Integrated Pest Management programs in Thailand using Sterile Insect Technique (AW-IPM-SIT) since the late 1980s and the early 2000s. The projects’ planning and evaluation usually rely on information from pest status, distribution, and fruit infestation. However, the collected data sometimes does not provide enough detail to answer management queries and public concerns, such as the long term sterilization efficacy of the released fruit fly, skepticism about insect migration or gene flow across the buffer zone, and the re-colonisation possibility of the fruit fly population within the core area. Established microsatellite DNA markers were used to generate population genetic data for the analysis of the fruit fly sampling from several control areas, and non-target areas, as well as the mass-rearing facility. The results suggested limited gene flow (m < 0.100) across the buffer zones between the flies in the control areas and flies captured outside. In addition, no genetic admixture was revealed from the mass-reared colony flies from the flies within the control area, which supports the effectiveness of SIT. The control pests were suppressed to low density and showed weak bottleneck footprints although they still acquired a high degree of genetic variation. Potential pest resurgence from fragmented micro-habitats in mixed fruit orchards rather than pest incursion across the buffer zone has been proposed. Therefore, a suitable pest control effort, such as the SIT program, should concentrate on the hidden refuges within the target area.     

Potential for insecticide resistance in populations of Bactrocera dorsalis in Hawaii: spinosad susceptibility and molecular characterization of a gene associated with organophosphate resistance

The potential for populations to become resistant to a particular insecticide treatment regimen is a major issue for all insect pest species. In Hawaii, for example, organophosphate (OP)‐based cover sprays have been the chemical treatment most commonly applied against oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), populations since the 1950s. Moreover, bait spray treatments using spinosad were adopted as a major control tactic in the Hawaii area‐wide fruit fly pest management program beginning in the year 2000. To determine the current level of spinosad and OP tolerance of wild B. dorsalis populations, bioassays were conducted on flies collected from a range of geographic localities within the Hawaiian islands. Adult B. dorsalis flies were tested (1) for the level of susceptibility to spinosad using LC₅₀ diagnostic criteria, and (2) for the presence of alleles of the ace gene previously shown to be associated with OP resistance. Regarding spinosad tolerance, only flies from Puna, the one area lacking prior exposure to spinosad, showed any significant difference compared to controls, and here the difference was only in terms of non‐overlap of 95% fiducial limit values. With respect to OP tolerance, specific mutations in the ace gene associated with resistance to these insecticides were found in only two populations, and in both cases, these alleles occurred at relatively low frequencies. These results suggest that at the present time, populations of B. dorsalis in Hawaii show no evidence for having acquired resistance to the insecticides widely used in control programs.     

Modelling selection for resistance to methods of insect pest control in combination

The development of resistance to insecticides is now widespread among insects. Other methods of pest control are also potentially at risk of encountering resistance. A modelling approach is presented here to evaluate the effects of combining methods of insect pest control on the selection for resistance to the control methods. This analysis is based on partitioning the total mortality acting on a population into its constituent components from all known sources, and these are related to selection for resistance. When two control methods are used in combination, selection for resistance against the two is a linear function if the two don't interact, otherwise it may be sublinear or supralinear. A specific example is presented using a model of the Olive fruit fly (Dacus oleae Gmel.) and employing food-baited and pheromone-baited traps for control. The control methods that appear least likely to encounter resistance are natural enemies and the use of pheromone traps for male annihilation. These should be integrated into a control program where possible to minimize the development of resistance to other control methods being used.     

Managing the horn fly (Diptera: Muscidae) using an electric walk-through fly trap

An electric walk-through fly trap was evaluated for the management of the horn fly, Hematobia irritans (L.), on dairy cattle in North Carolina over 2 yr. The trap relies on black lights and electrocution grids to attract and kill flies that are brushed from the cattle passing through. During the first season, horn fly densities were reduced from >1,400 to <200 flies per animal. Horn fly density averaged 269.2 +/- 25.8 on cattle using the walk-through fly trap twice daily, and 400.2 +/- 43.5 on the control group during the first year. The second year, seasonal mean horn fly density was 177.3 +/- 10.8 on cattle using the walk-through fly trap compared with 321.1 +/- 15.8 on the control group. No insecticides were used to control horn flies during this 2-yr study.     

Bean seed fly (Delia platura, Delia florilega) and onion fly (Delia antiqua) incidence in England and an evaluation of chemical and biological control options

Bean seed fly and onion fly are significant pests of alliaceous crops in the UK. Their activity was monitored using yellow water traps at three field sites in England in 2002 and 2003. Bean seed fly were not split between Delia platura or Delia florilega because from the growers point of view control measures are independent of species. The traps were effective at catching bean seed fly, which was present from April until September. A total of 1729 bean seed fly were trapped in 2002 and 4501 in 2003, with peak activity in May in both years. In 2003, there appeared to be three to four peaks in abundance of the pest. Only 113 onion flies were trapped in 2002 and 23 in 2003. More male onion fly were trapped than females. Pot experiments were carried out to evaluate efficacy of a range of insecticides, garlic and two parasitic nematode species (Steinernemafeltiae and Steinernema kraussei) against bean seed fly and onion fly. Pots of salad onions were exposed to natural oviposition by bean seed fly, but the onion fly experiment was carried out in a glasshouse with eggs of the pest being inoculated into the pots. Tefluthrin seed treatment appeared to be especially effective at preventing bean seed fly damage and produced the most robust seedlings but did not appear to kill the larvae. A drench of chlorpyrifos at the ‘crook’ stage gave 100% control of bean seed fly larvae. A chlorpyrifos drench was the only treatment to give effective control of onion fly. There was some evidence that the parasitic nematode S. feltiae reduced numbers of bean seed fly larvae by about 50%. Guidelines for control of both bean seed fly and onion fly are discussed.     

Dividing the pie: differential dung pat size utilization by sympatric Haematobia irritans and Musca autumnalis

Horn flies [Haematobia irritans (Diptera: Muscidae) (L.)] and face flies [Musca autumnalis (Diptera: Muscidae) De Geer] use the same larval resource, but their interactions are poorly studied. Dung pats (n = 350) were core sampled in the summers of 2012 and 2013 from irrigated pastures in Pomona, California, U.S.A. (34°03′N, 117°48′W) and held for face fly and horn fly emergence. Surface areas and estimated weights were recorded for each whole pat. Almost half (42.0%) of the pat cores yielded neither fly, 29.7% yielded horn flies only, 12.9% yielded face flies only and 15.4% yielded both flies. Of the fly‐positive pats, surface area and mass were larger for face fly‐occupied pats, whereas horn fly‐occupied pats were smaller. Pats shared by the two species were intermediate. Horn flies per positive core were unaffected by the absence/presence of face flies, but half as many face flies emerged when pats were co‐inhabited by horn flies. Face flies inhabited larger pats, which might better resist heating and drying, to which they are susceptible; horn flies inhabited a broad pat size range. Horn fly tolerance of lower dung moisture probably allows horn flies to colonize and survive in a wide range of pats in dry areas like southern California.     

Dynamics of cypermethrin resistance in the field in the horn fly,Haematobia irritans

The toxicity of cypermethrin to the horn fly Haematobia irritans (L.) (Diptera: Muscidae) was determined for samples collected from untreated herds at a farm in central Argentina from October 1997 to May 2001. Field tests of the efficacy of cypermethrin against horn flies were first carried out at this farm in 1993, when the fly was shown to be susceptible to pyrethroids. Subsequently the horn fly populations on this farm were shown to have become resistant and, since 1997, the use of cypermethrin has been restricted to experimental purposes. In this study, fly samples collected in 1999, 2000 and 2001 were subjected to a polymerase chain reaction (PCR) to detect the presence of a specific nucleotide substitution in the sodium channel gene sequence, which has been associated with target site insensitivity to pyrethroids. This analysis showed that the level of cypermethrin resistance had diminished between 1997 and 2001. However, this was not sufficient to restore the efficacy of this pyrethroid to the level found prior to the onset of resistance. Heterozygous and homozygous resistant flies were detected in all samples of flies subjected to PCR diagnosis of alleles conferring target site resistance.     

Impact of phagostimulants on effectiveness of OMRI‐listed insecticides used for control of spotted‐wing drosophila (Drosophila suzukii Matsumura)

Spotted‐wing drosophila, Drosophila suzukii Matsumura, is an invasive pest in the United States that causes considerable damage to fruit crops. It is responsible for many millions of dollars of revenue loss. The female D. suzukii has a heavily sclerotized ovipositor and can lay eggs in ripening or ripe fruit. The arrival of this invasive species has disrupted existing integrated pest management programmes, and growers rely on repeated insecticide applications to protect fruit. Organic growers have few chemical control options, and their reliance on spinosad increases the risk of developing insecticide resistance. We hypothesized that combining phagostimulants with insecticides would increase insecticide efficacy by prompting flies to spend more time in contact with residues. Therefore, the objective of this study was to evaluate the effectiveness of sucrose and the yeast Saccharomyces cerevisiae as phagostimulants in combination with organic biopesticides against D. suzukii in blueberries. Adding sucrose with or without yeast did not improve insecticide efficacy in terms of adult fly mortality or fruit infestation. Spinosad was very effective in all experiments, and for this product, there is little room for improvement. The phagostimulants had no effect on residual activity of any insecticide. The addition of sucrose with or without yeast did not improve the effectiveness of organic insecticides for D. suzukii. Concentrations of these phagostimulants in our experiments (0.36%) may have been too low to elicit a response. Further research is recommended to test different types and concentrations of phagostimulants.     

A real‐time PCR toolbox for accurate identification of invasive fruit fly species

Significant plant pests such as fruit flies that travel with fresh produce between countries as eggs or larvae pose a great economic threat to the agriculture and fruit industry worldwide. Time‐limited and expensive quarantine decisions require accurate identification of such pests. Immature stages are often impossible to identify, making them a serious concern for biosecurity agencies. Use of COI barcoding PCR, often the only molecular identification resource, is time‐consuming. We assess the suitability of the COI barcoding region for real‐time PCR assays to identify four pest fruit fly species (Family: Tephritidae), in a diagnostic framework. These species, namely Mediterranean fruit fly (Ceratitis capitata), Queensland fruit fly (Bactrocera tryoni), African invader fly (Bactrocera invadens) and Island fly (Dirioxa pornia) each provide a different set of genetic species delimitation problems. We discuss the benefits and limitations of using a single‐gene TaqMan^? real‐time approach for such species. Our results indicate that COI‐based TaqMan^? real‐time PCR assays, in particular for genetically distinct species, provide an accurate, sensitive and rapid diagnostic tool.     

Suppression of female melon fly,Zeugodacus cucurbitae,with cue‐lure and fipronil bait stations through horizontal insecticide transfer

Melon fly, Zeugodacus cucurbitae (Coquillett) (Diptera: Tephritidae), is an important quarantine tephritid fruit fly with resident populations established in Hawai'i, USA. In the male‐annihilation approach, male flies are targeted using dispensers with cue‐lure (C‐L) and insecticides, typically organophosphates. The efficacy of the male annihilation approach is thought to be limited to individual male flies, contacting the lure and the pesticide, after which they die. Alternative classes of insecticides, such as fipronil, have been investigated for use in male‐annihilation. We hypothesized that ingestion of fipronil by male flies could lead to horizontal transfer and mortality in female flies. Horizontal insecticide transfer extends pesticide control beyond the individual contacting the toxicant through indirect contact via food sharing or other mechanisms. We tested the possibility for horizontal transfer of fipronil from male to female Z. cucurbitae through field and laboratory studies. Two repeated field trials were conducted to compare the numbers of female flies collected in fields treated with Amulet C‐L (0.34% fipronil active ingredient) bait stations, sanitation, and spot treatments of GF‐120 Fruit Fly Bait to numbers collected in fields where sanitation and spot‐treatments were used without Amulet C‐L. In fields with Amulet C‐L bait stations in conjunction with sanitation and weekly protein bait spot treatments of GF‐120 Fruit Fly Bait, female captures were significantly lower than those in field plots treated with weekly protein bait spot treatments and sanitation. In subsequent laboratory studies, all females died within 6 h after direct exposure to male flies that had access to Amulet C‐L for 1–4 min. The possibility that male regurgitant could be a mechanism for horizontal transfer and subsequent female mortality was determined by collecting regurgitated droplets from fipronil‐fed male flies and feeding them to males and females. Both male and female flies exposed to regurgitant from fipronil‐fed male flies or droplets containing fipronil had higher mortality than the male and female flies that were exposed to regurgitant or droplets with only the C‐L compound or sugar solution. Thus, female flies do experience mortality from exposure to regurgitant from males that have fed on fipronil laced solutions. This provides evidence of at least one mechanism of horizontal transfer of insecticide in tephritid fruit flies. These findings are discussed in the context of Z. cucurbitae integrated pest management programs in Hawai'i.     

Ecological and Control Techniques for Sand Flies (Diptera: Psychodidae) Associated with Rodent Reservoirs of Leishmaniasis

Background

Leishmaniasis remains a global health problem because of the substantial holes that remain in our understanding of sand fly ecology and the failure of traditional vector control methods. The specific larval food source is unknown for all but a few sand fly species, and this is particularly true for the vectors of Leishmania parasites. We provide methods and materials that could be used to understand, and ultimately break, the transmission cycle of zoonotic cutaneous leishmaniasis.

Methods and Findings

We demonstrated in laboratory studies that analysis of the stable carbon and nitrogen isotopes found naturally in plant and animal tissues was highly effective for linking adult sand flies with their larval diet, without having to locate or capture the sand fly larvae themselves. In a field trial, we also demonstrated using this technique that half of captured adult sand flies had fed as larvae on rodent feces. Through the identification of rodent feces as a sand fly larval habitat, we now know that rodent baits containing insecticides that have been shown in previous studies to pass into the rodents'' feces and kill sand fly larvae also could play a future role in sand fly control. In a second study we showed that rubidium incorporated into rodent baits could be used to demonstrate the level of bloodfeeding by sand flies on baited rodents, and that the elimination of sand flies that feed on rodents can be achieved using baits containing an insecticide that circulates in the blood of baited rodents.

Conclusions

Combined, the techniques described could help to identify larval food sources of other important vectors of the protozoa that cause visceral or dermal leishmaniasis. Unveiling aspects of the life cycles of sand flies that could be targeted with insecticides would guide future sand fly control programs for prevention of leishmaniasis.     

Sequence and transcript expression of the super-kdr locus of the horn fly,Haematobia irritans

In horn flies, Haematobia irritans irritans (Diptera: Muscidae) (Linnaeus, 1758), target site resistance to pyrethroids can be diagnosed by an allele-specific PCR that genotypes individual flies at both the super-kdr (skdr) and the knock down resistance (kdr) associated loci. When this technique uses genomic DNA as template, modifications, such as alternative RNA splicing and RNA editing are not specifically detected. Alternative splicing at the skdr locus has been reported in Dipterans; thus, the genomic DNA-based allele-specific PCR may not accurately reflect the frequency of the skdr mutation in horn fly field populations. To investigate if alternative splicing occurs at the skdr locus of horn flies, genomic DNA and cDNA sequences isolated from two wild populations and two laboratory-reared colonies with varying degrees of pyrethroid resistance were compared. There was no indication of alternative splicing at the super-kdr locus neither in the wild populations nor in the laboratory-reared colonies.     

Influence ofOnthophagus gazella on hornfly,Haematobia irritans density in irrigated pastures

Haematobia irritans (L.) breeding in flood irrigated pastures of the lower Colorado Desert of southeastern California continues to remain unacceptably high during warm seasons (>1,000 adult flies per bovine head) despite the presence of moderately abundant populations ofOnthophagus gazella F. This study suggests that densities of > 40–70 adult beetles per dung pad and giving pronounced dung shredding activity, caused fly mortality of 38–56 %. The continued high abundance of adult horn flies on cattle suggests that at > 50% mortality, the pasture environment still produces sufficient flies to saturate cattle, although emigration might be reduced. Additional species of scarabs may be necessary to increase fly mortality. However, the dung drying activity of existingO. gazella significantly could interfere with resident staphylinid beetle breeding, which was significantly lower in pastures whereO. gazella reached densities of 40 per dung pad. Scarab beetle activity might also impede the introduction of superior predatory species for biological control.      

The putative‐farnesoic acid O‐methyl transferase (FAMeT) gene of Ceratitis capitata: characterization and pre‐imaginal life expression

Farnesoic acid O‐methyl transferase (FAMeT) is the enzyme involved in the penultimate step of insect juvenile hormone (JH) biosynthesis and is thus a key regulator in insect development and reproduction. We report the characterization of the putative‐FAMeT in the medfly or Mediterranean fruit fly, Ceratitis capitata. This gene was identified by suppressive subtractive hybridization and completely sequenced by the screening of a medfly cDNA library. The obtained sequence was analyzed for conserved protein domain identification and its expression profile was evaluated by quantitative Real‐Time PCR in medfly pre‐imaginal life. The tissue expression of the isolated gene was verified by in situ hybridization on third instar larvae sections. The characterization of the isolated gene pointed out several typical features of methyl transferase genes. The pre‐imaginal putative‐FAMeT expression levels were consistent with JH titer change in Diptera. As recognized in some crustaceans, this gene seems to be widely expressed in the medfly as well. Ceratitis capitata is one of the most relevant agricultural pests against which insecticides and the sterile insect technique (SIT) are extensively used in spite of the well‐known limitations of these approaches. Although results are not conclusive for the physiological role of the isolated gene, they suggest the characterization of a new gene in the Mediterranean fruit fly potentially involved in JH biosynthesis and may, therefore, have implications for pest control. © 2010 Wiley Periodicals, Inc.