A survey of mutations in the Cochliomyia hominivorax (Diptera: Calliphoridae) esterase E3 gene associated with organophosphate resistance and the molecular identification of mutant alleles

Cochliomyia hominivorax (Calliphoridae) is one of the most important myiasis-causing flies and is responsible for severe economic losses to the livestock industry throughout the Neotropical region. In Brazil, C. hominivorax has been controlled mainly with organophosphate (OP) insecticides, although the inappropriate use of these chemicals can result in the selection of resistant flies. Changes in carboxylesterase activity have been associated with OP insecticides in some arthopodan species. In this work, we isolated and characterized part of the E3 gene in C. hominivorax (ChE7), which contained the same substitutions responsible for the acquisition of OP hydrolase activity in Lucilia cuprina (Calliphoridae). Digestion of the polymerase chain reaction products with a restriction enzyme that specifically recognized the mutation site unambiguously differentiated wild and mutated esterase alleles. The PCR-RFLP assay therefore provided a fast, reliable DNA-based method for identifying C. hominivorax individuals with a mutation in the esterase gene. Further bioassays to determine the association of this mutation with OP resistance in C. hominivorax should allow the development of more effective strategies for managing this species.     

Identification of screwworms, Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae), with a monoclonal antibody-based enzyme-linked immunosorbent assay (MAb-ELISA).

Myiasis caused by screwworms, Cochliomyia hominivorax (Coquerel), is devastating to warm-blooded animals and economically important to livestock producers. It is difficult to distinguish these pests, immature screwworms, from immatures of other non-pest fly species that often occur in animal wounds; it would be helpful to have tools available that do not rely on morphological characteristics. We developed two monoclonal antibodies (MAbs), highly specific for the screwworm, and used them in an enzyme-linked immunosorbent assay (MAb-ELISA), that differentiated screwworm eggs, larvae, pupae, and adults from those of the closely related secondary screwworm, C. macellaria (Fabricius) as well as Phormia regina (Meigen), Phaenicia sericata (Meigen), Calliphora vicina Robineau-Desvoidy, and Chrysomya rufifacies (Macquart). In a blind study, the microplate MAb-ELISA, which took about 4h to complete, displayed high specificity (99%), sensitivity (92%) and overall accuracy (97%) in distinguishing all life stages of the screwworm. Electrophoresis results suggested that the two monoclonal antibodies recognized identical conformational epitopes present in all screwworm life stages. The screwworm eradication program, successful in eradicating this pest from the US, Mexico, most of Central America and Libya (after an accidental introduction), could benefit in future eradication, surveillance, and exclusion efforts by developing a reliable field identification kit based on MAb-ELISA that accurately and quickly distinguished cases of screwworm myiasis.     

Horn fly (Diptera: Muscidae) resistance to organophosphate insecticides

Insecticidal ear tags impregnated with organophosphate (OP) insecticides were used each year from 1989 to 1998 at Rosepine, LA. Weekly fly counts were conducted to evaluate control efficacy of the treatments, and bioassays were conducted at least twice per year to measure fly susceptibility to OP and pyrethroid insecticides. Between 1989 and 1992, the efficacy of 20% diazinon-impregnated ear tags was reduced from >20 to just 1 week of control. A high risk of control failure was observed when a resistance frequency of approximately 5% was measured in pre-season bioassays. Resistance to diazinon, fenthion, ethion, pirimiphos-methyl, and tetrachlorvinphos was observed. Esterase activity toward alpha-naphthyl acetate was significantly higher in flies collected at Rosepine in 1997 than in flies from a laboratory colony and from a susceptible field population.     

History of the screwworm (Cochliomyia hominivorax) eradication in the Eastern Hemisphere

The screwworm caused by Cochliomyia Hominivorax, attacking warm-blooded animals and man, was discovered for the first time in the history outside of American continent, in the Eastern Hemisphere. After confirming the occurrence of this horrible myiasis in North Africa in 1989, Tripoli sheep import quarantine was traced as primary outbreak locality. The myiasis spread rapidly invading vast territory during several months. The invaded territory of 25000 km2 with more than 2.7 million domestic animals was identified between Mediterranean Sea and Sahara desert threatening territories in Africa and Mediterranean basin. From July 1989 up to eradication in April 1991 were discovered 14,111 cases. Classical control methods were not able to block screwworm spreading and eradicate it. Decision was made to use as the main method the sterile insect technique. Irradiated sterile flies were airlifted from Mexico factory. There were dispersed aerially about 1260 million flies covering invaded territories and 15000 km2 of protective barrier zones. Successful program cost almost 100 million US$. After the eradication Africa as well as Eastern Hemisphere could be declared to be again free of this myiasis. It was the most effective and successful international animal health program in the history of the United Nations Organization.     

Acetylcholinesterase of Stomoxys calcitrans (L.) (Diptera: Muscidae): cDNA sequence, baculovirus expression, and biochemical properties

A 2193-nucleotide cDNA encoding acetylcholinesterase (AChE) of the stable fly, Stomoxys calcitrans (L.) was sequenced and expressed in the baculovirus system. The open reading frame encoded a 91 amino acid secretion signal peptide and a 613 amino acid mature protein with 96% and 94% identity to the AChEs of Haematobia irritans (L.) and Musca domestica (L.), respectively. Structural characteristics of M. domestica, H. irritans, and Drosophila melanogaster AChEs were conserved in the S. calcitrans AChE. The recombinant enzyme was inhibited by eserine, coroxon, and paraoxon and exhibited K_m values of 63.9 μM for acetylthiocholine and 96.7 μM for butyrylthiocholine, confirming its biochemical identity as an acetylcholinesterase (EC 3.1.1.7). These data will enable rapid identification and assay for mutations that reduce AChE sensitivity to organophosphate (OP) pesticides, potentially aiding resistance management efforts to prevent fixation of the mutations in pest populations.     

Chrysomya bezziana (Diptera: Calliphoridae) infestation in two Malaysian cats treated with oral lotilaner

The most common fly species associated with screwworm myiasis in Southeast Asia is Chrysomya bezziana (Ch. bezziana), the Old‐World screwworm. Treatment of screwworm myiasis in cats traditionally has comprised subcutaneous injection of ivermectin or oral administration of nitenpyram, combined with aggressive tissue debridement and larval removal under general anaesthesia. Two cats diagnosed with cutaneous myiasis caused by the larvae of Ch. bezziana were treated with lotilaner. In both cats, a single dose of lotilaner at 6–26 mg/kg, killed all larvae within 24 h, negating the need for general anaesthesia. Both cats were simultaneously infested with Lynxacarus radovskyi (L. radovskyi) which also was eradicated with lotilaner. No adverse reactions were observed and both cats recovered without complications.     

Acetylcholinesterase mutation in diazinon-resistant Haematobia irritans (L.) (Diptera: Muscidae)

Acetylcholinesterase (AChE) cDNA from individual field-collected diazinon-resistant horn flies was amplified by RT-PCR. Sequencing of the amplification products revealed that 8/12 of the diazinon-resistant horn flies contained a point mutation previously associated with resistance to organophosphates in house flies and Drosophila, strongly suggesting that this cDNA encodes the AChE that is the target site for organophosphate (OP) pesticide. The point mutation (G262A) resulted in a shift from glycine to alanine in the mature HiAChE amino acid sequence at position 262. Allele-specific PCR and RLFP assays were developed to diagnose the presence or absence of the G262A mutation in individual flies. Use of the allele-specific assays each demonstrated the presence of the G262A mutation in 10 of 12 individual field-collected flies, demonstrating higher sensitivity than direct sequencing of RT-PCR amplification products. The G262A mutation was found in additional fly populations previously characterized as OP-resistant, further supporting that this AChE is the target site for OP pesticide. The allele-specific assay is a useful tool for quantitative assay of the resistance allele in horn fly populations.     

Seasonal abundance of carrion-frequenting blow-flies (Diptera: Calliphoridae) in the Kruger National Park

Monthly population fluctuations of carrion-frequenting blow-flies over a 24-month period were monitored using 2 carrion-baited traps in the southern Kruger National Park (KNP) and 3 in the northern KNP. All species displayed a clear seasonality. Chrysomyia marginalis and Chrysomyia albiceps were by far the most abundant. C. marginalis attained maximum abundance between November and March, with relatively low numbers present between May and September. C. albiceps maintained high population numbers between January and March in the northern KNP, with minimum numbers between May and August. In the southern KNP, C. albiceps became abundant from November to February, with low population levels between April and September. Although present only in relative low numbers, populations of Lucilia cuprina showed a clear increase in winter. Chrysomyia chloropyga, Chrysomyia putoria and Chrysomyia bezziana were trapped in significant numbers in the southern KNP, the latter 2 species reaching relative abundance in the warmer months, whereas C. chloropyga increased in cooler months from June to September. Graphic illustrations of monthly abundance are provided for all species.     

Cloning and sequencing of a cDNA expressing a ribosomal P0 peptide from Culicoides nubeculosus (Diptera)

Insect bite dermal hypersensitivity (IBH) is an allergic dermatitis of horses caused by bites of Culicoides spp. and sometimes Simulium spp. The aim of the investigation presented here was to identify allergens causing IBH. A cDNA library expressing recombinant Culicoides nubeculosus proteins was screened using affinity-purified serum from an IBH-affected horse. Screening of the library resulted in identification of one immunoreactive clone. The sequence of the cDNA insert was determined and revealed a 600 bp insert with an open reading frame coding for a 78 amino acid long protein, called rCul n 1. Analysis of the deduced amino acid sequence revealed an identity of 67-78% to the C-terminal part of the 318 amino acid long ribosomal P0 protein from other Diptera. Furthermore, the 38 C-terminal amino acids displayed an identity of 57% with the C-terminal part of the acidic ribosomal protein P2 from Aspergillus fumigatus. The cDNA insert was subcloned and expressed as a [His]6-tagged protein in Escherichia coli and purified using Ni2(+)-chelate affinity chromatography. The 10kDa recombinant Cul n 1 protein bound the affinity-purified antibody fraction used for screening the expression library. Determination of IgE and IgG levels against rCul n 1 by ELISA in sera from 19 IBH-affected and 18 Swiss control horses and in sera from eight control horses living in Iceland showed no significant differences between the three groups of horses (median IgE levels = 60, 49 and 44 relative ELISA units, respectively). rCul n 1 did not induce sulfidoleukotriene (sLT) release from peripheral blood leukocytes of IBH-affected horses (N = 5), although sLT release was induced with the Culicoides whole body extract.     

Persistent activity of doramectin and ivermectin in the prevention of cutaneous myiasis in cattle experimentally infested with Cochliomyia hominivorax

A study was conducted to evaluate the activity of a single administration of doramectin or ivermectin against severe, induced infestations of Cochliomyia hominivorax. Twenty-four Holstein bull calves were allocated to four groups of six animals each and treated either with saline, doramectin 1%, or either one of two formulations of ivermectin 1% at a dose rate of 200 microg/kg. On Day 12 after treatment, each calf was anesthetized and two wounds were created on the left side of the shoulder and rump of each calf and 2 h later, each wound was implanted with 100 newly hatched larvae of C. hominivorax. On Day 15 after treatment, the procedure was repeated on the right side of each calf. Wounds were examined daily for 5 days and evidence of live larvae was recorded. Doramectin provided reduction in myiasis of 90.9 and 83.3% at 12 and 15 days after treatment, respectively, compared to the saline control treatment (P < 0.0001). In contrast, there were no significant differences in the number of calves with myiasis between those treated with either of the ivermectin formulations and the saline control.     

Cochliomyia hominivorax myiasis in a colony of stray cats (Felis catus Linnaeus, 1758) in Rio de Janeiro, RJ

Cochliomyia hominivorax infestation in domestic cats of an urban colony in the city of Rio de Janeiro is described. The overall prevalence over the period 2001-2005 was 12.5%. Only adult cats were found infested and among these, most cases were observed in males (28%) (p < or = 0.05). The most frequently infested areas of the cats' bodies were the face and nape of the neck. Most lesions were found on the front part of the body of adult males (80%), suggesting that myiasis occur in consequence of competitive fighting wounds.     

Evaluation of efficacy of entomopathogenic nematodes against larvae of Lucilia sericata (Meigen, 1826) (Diptera: Calliphoridae)

The blowfly Lucilia sericata (Meigen, 1826) (Diptera: Calliphoridae) is the primary agent of cutaneous myiasis of sheep in northern Europe, southern Africa, Australia and New Zealand. As the application of chemicals has several disadvantages, alternative control measures of traumatic myiasis of livestock must be developed. In this study, the use of entomopathogenic nematodes (EPNs) as potential biocontrol agents against second instar larvae of Lucilia sericata was considered. The following nematode species were tested: Heterorhabditis bacteriophora (IS 5, HHU 1, Hmol, HNC 1, HAZ 36, Hbrecon, HHU 2, HAZ 29, HHP 88, HHU 3, HHU 4 and HGua), Steinernema intermedia, NC513 strain of S. glaserii, S. anomali, S. riobrave, Steinernema sp. and 5 strains of S. feltiae (22, Vija Norway, HU 1, scp, and IS 6). None of the examined EPN species or strains showed larvicidal efficacy at 37 degrees C (no killing effect was observed in the case of the two heat-tolerant strains--H. bacteriophora and S.feltiae) against L. sericata larvae. At lower temperatures (20 degrees C and 25 degrees C) only strains of S. feltiae were found to be active. The overall odds ratios calculated for L. sericata maggots to contract S. feltiae nematode infection show significant (p < 0.05) effect only in the case of strains HU 1, 22 and IS 6. In the case of strains HU 1 and 22 parasitic forms of S. feltiae could be detected in the dead larvae of L. sericata. Strain IS 6 (and also Vija Norway at 20 degrees C) penetrated and killed fly larvae, but only adult forms of the nematode occurred in the cadavers.     

Evaluation of the prophylactic effect and curative efficacy of fipronil 1% pour on (Topline) on post-castration scrotal myiasis caused by Cochliomyia hominivorax in cattle

A field trial was carried out during a summer-fall period on a commercial beef cattle farm in Minas Gerais State, located in the Southeast of Brazil. In order to evaluate the prophylactic effect and the curative efficacy of fipronil in a 1% solution, 200 Zebu crossbred bulls, with ages varying from 20 to 30 months and weights from 233 to 362 kg, were selected. The bulls were assigned by ranked pair to an untreated control group (A) or to a treated group (B), resulting in 100 animals per group. All experimental animals were surgically castrated on day 0, following routine procedures. After castration all animals in the group B were treated with 10 mg/kg bw of a 1% fipronil solution, topically on the dorsal mid-line. The wounds were individually inspected on days: 2, 4, 7, 10, 14, 17, 21, 28 and 35. After castration the animals were naturally exposed to Cochliomyia hominivorax and remained in the same pasture throughout the trial. Among the animals in the control group, 83 were observed to harbor C. hominivorax eggs, with a total of 97 ovipositions, and among those 73 animals had active myiasis. In group B (fipronil 1%), 66 animals showed C. hominivorax eggs, with 92 ovipositions and five animals with active myiasis. Most ovipositions and active myiasis were detected until seven days post-castration for both groups. Wound parasite infestation evidenced bleeding, serous purulent exudation and presence of active C. hominivorax larvae. Treatment with fipronil 1% had a prophylactic effect on scrotal wounds against the development of C. hominivorax larvae in more than 95% of the treated animals for up to 17 days after castration. The treatment showed partial protection of 66% and 50% on days 21 and 28 post-treatment (pt), respectively. Three animals from the control group and one from the treated group showed active screwworms on day 21 pt, and one animal from the treated group and two from the control group also presented C. hominivorax larvae on scrotal wounds on day 28 pt. By the end of the observation period (day 35 pt), the castration wound had healed in all animals. All experimental animals presenting scrotal wounds infested with C. hominivorax larvae were treated with a 1% pour-on formulation of fipronil, on the same day that infestation was observed. Active C. hominivorax larvae were not seen during the monitoring period immediately after treatment. The curative efficacy of fipronil 1% against C. hominivorax larvae infestation in castration wounds was 100%.     

Effects of latex from "Amapazeiro"Parahancornia amapa (Apocynaceae) on blowfly Chrysomya megacephala (Diptera: Calliphoridae) post-embryonic development

Nowadays, insect control is usually carried out using chemical insecticides, but insect resistance and other negative side effects have prompted the search for alternatives. Biopesticides provide a positive alternative to synthetic pesticides because they have low impact on the environmental, low toxicity to humans and low costs among other advantages. This research was carried out to evaluate the activity of Parahancornia amapa (Huber) Ducke (Apocynaceae) lyophilized latex on the post embryonic development of Chrysomya megacephala (F.) (Diptera: Calliphoridae). Larvae treated with 1.0% latex showed a shorter post embryonic development period (larval, pupal and newly hatched larvae to adult); whereas larvae treated with 3.0% latex provoked a prolongation of these periods. Viability (53%) was also very low at the newly hatched larvae to adult period for larvae treated with 3.0% latex, indicating that latex from P. amapa at high concentrations could change C. megacephala post embryonic development.     

Dispersal, density and habitat preference of the blow-flies Chrysomyia albiceps (Wd.) and Chrysomyia marginalis (Wd.) (Diptera: Calliphoridae)

16 000 Chrysomyia albiceps and 52 000 C. marginalis adults were radioactively labelled with 32P-orthophosphate and released in the northern Kruger National Park, South Africa. After a 1-week dispersal period 69 baited blow-fly traps were placed in different habitat types and at varying distances around the release point. C. albiceps were subsequently found to have covered up to 37.5 km and C. marginalis 63.5 km, suggesting dispersal rates per day of 2.20 km and 2.35 km for the 2 species, respectively. Calculation of density using the Lincoln Index yielded estimates per hectare of 7.56 C. albiceps and 29.03 C. marginalis. Both species were trapped more numerously in forested environments than in open scrub, and both avoided arid scrubland.     

鸡β-防御素Gal-1 cDNA的克隆及序列分析

利用细胞总RNA提取试剂盒,从1月龄SPF鸡白细胞中分离提取总RNA,经过反转录PCR(RT-PCR)扩增出鸡β-防御素(Gal-1)cDNA片段。PCR产物经凝胶回收纯化后与pMD18-T载体连接,转化感受态JM109细胞,在含X-gal、IPTG的LB平板上筛选阳性克隆,经菌落PCR与质粒限制性酶切鉴定后,对目的片段进行测序。结果表明RT-PCR扩增出的cDNA片段ORF大小为198bp,用Blast程序进行检索比较表明该序列与Genbank中发表的鸡Gal-1 cDNA编码区序列同源性高达99.5%。该cDNA序列编码65个氨基酸,包括信号肽、前片段和成熟肽,成熟肽由40个氨基酸残基组成。