Andrew Y. Li

Detection and Characterization of Amitraz Resistance in the Southern Cattle Tick, Boophilus microplus (Acari: Ixodidae)

Abstract Amitraz, a formamidine acaricide, plays an important role in the control of the southern cattle tick, Boophilus microplus (Canestrini), and other tick species that infest cattle, dogs, and wild animals. Although resistance to amitraz in B. microplus was previously reported in several countries, the actual measurement of the level of amitraz resistance in ticks has been difficult to determine due to the lack of a proper bioassay technique. We conducted a survey, by using a newly reported technique that was a modification of the standard Food and Agriculture Organization larval packet test, to measure the levels of resistance to amitraz in 15 strains of B. microplus from four major cattle-producing states in Mexico. Low-order resistance (1.68- to 4.58-fold) was detected in 11 of those strains. Our laboratory selection using amitraz on larvae of the Santa Luiza strain, which originated from Brazil, achieved a resistance ratio of 153.93 at F6, indicating the potential for high resistance to this acaricide in B. microplus. Both triphenylphosphate and piperonyl butoxide significantly synergized amitraz toxicity in both resistant and susceptible tick strains. Diethyl maleate synergized amitraz toxicity in one resistant strain but had no effect on the susceptible strain and had minor antagonistic effects on two other resistant strains. Target site insensitivity, instead of metabolic detoxification mechanisms, might be responsible for amitraz resistance observed in the Santa Luiza strain and possibly in other amitraz resistant B. microplus ticks from Mexico. The Santa Luiza strain also demonstrated high resistance to pyrethroids and moderate resistance to organophosphates. Multiple resistance shown in this strain and other B. microplus strains from Mexico poses a significant challenge to the management of B. microplus resistance to acaricides in Mexico.

Survey of resistance to permethrin and diazinon and the use of a multiplex polymerase chain reaction assay to detect resistance alleles in the horn fly, Haematobia irritans irritans (L.).

Abstract A field survey was conducted in 2001 to evaluate resistance to pyrethroid and organophosphate (OP) insecticides on horn flies, Hematobia irritans irritans (L.), from seven ranches in the state of Tamaulipas, Mexico, and from three locations in central Texas. Filter papers impregnated with either technical permethrin or diazinon were used to measure the levels of resistance to pyrethroids and OPs. A multiplex polymerase chain reaction (PCR) assay was used on individual horn flies from these field populations to detect the presence of the kdr and super-kdr alleles associated with pyrethroid resistance, and a mutated αE7 esterase allele associated with OP resistance. Relative to a susceptible laboratory (Kerrville) strain, horn flies from Mexico exhibited 5.1- to 28.3-fold resistance to permethrin at the LC50, and 23.8- to 136-fold resistance at the LC90. Horn flies from Texas ranches exhibited only two- to five-fold resistance. All field populations of the horn fly were highly susceptible to diazinon, and no mutant αE7 esterase alleles were detected. The super-kdr allele was found only in a single fly from a ranch in Mexico. Results of PCR assays showed that the kdr allele was present at various frequencies in field populations of horn flies. A gender-related bias in distribution of kdr genotypes was found in horn flies from Mexico, but not in horn flies from Texas. The overall kdr allelic frequencies in horn flies from Mexico were 23.2–37.8% higher in females than in males. Regression analysis revealed a significant correlation between kdr allelic frequencies and the levels of knockdown resistance to permethrin among the horn fly populations studied. The results validate the role of the PCR-based molecular assay as a diagnostic tool in monitoring resistance to pyrethroids and also provide useful information on population genetics of horn fly resistance to pyrethroids and OPs.

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.

Acetylcholinesterases of blood-feeding flies and ticks

Acetylcholinesterase (AChE) is the biochemical target of organophosphate (OP) and carbamate pesticides for invertebrates, vertebrate nerve agents, and AChE inhibitors used to reduce effects of Alzheimers disease. Organophosphate pesticides (OPs) are widely used to control blood-feeding arthropods, including biting flies and ticks. However, resistance to OPs in pests affecting animal and human health has compromised control efficacy. OP resistance often results from mutations producing an OP-insensitive AChE. Our studies have demonstrated production of OP-insensitive AChEs in biting flies and ticks. Complementary DNA (cDNA) sequences encoding AChEs were obtained for the horn fly, stable fly, sand fly, and the southern cattle tick. The availability of cDNA sequences enables the identification of mutations, expression and characterization of recombinant proteins, gene silencing for functional studies, as well as in vitro screening of novel inhibitors. The southern cattle tick expresses at least three different genes encoding AChE in their synganglion, i.e. brain. Gene amplification for each of the three known cattle tick AChE genes and expression of multiple alleles for each gene may reduce fitness cost associated with OP-resistance. AChE hydrolyzes the neurotransmitter, acetylcholine, but may have additional roles in physiology and development. The three cattle tick AChEs possess significantly different biochemical properties, and are expressed in neural and non-neural tissues, which suggest separation of structure and function. The remarkable complexity of AChEs in ticks suggested by combining genomic data from Ixodes scapularis with our genetic and biochemical data from Rhipicephalus microplus is suggestive of previously unknown gene duplication and diversification. Comparative studies between invertebrate and vertebrate AChEs could enhance our understanding of structure-activity relationships. Research with ticks as a model system offers the opportunity to elucidate structure-activity relationships for AChE that are important for advances in targeted pest control, as well as potential applications for medicine and biosecurity.

Acetylcholinesterase of the sand fly, Phlebotomus papatasi (Scopoli): cDNA sequence, baculovirus expression, and biochemical properties

BackgroundMillions of people and domestic animals around the world are affected by leishmaniasis, a disease caused by various species of flagellated protozoans in the genus Leishmania that are transmitted by several sand fly species. Insecticides are widely used for sand fly population control to try to reduce or interrupt Leishmania transmission. Zoonotic cutaneous leishmaniasis caused by L. major is vectored mainly by Phlebotomus papatasi (Scopoli) in Asia and Africa. Organophosphates comprise a class of insecticides used for sand fly control, which act through the inhibition of acetylcholinesterase (AChE) in the central nervous system. Point mutations producing an altered, insensitive AChE are a major mechanism of organophosphate resistance in insects and preliminary evidence for organophosphate-insensitive AChE has been reported in sand flies. This report describes the identification of complementary DNA for an AChE in P. papatasi and the biochemical characterization of recombinant P. papatasi AChE.MethodsA P. papatasi Israeli strain laboratory colony was utilized to prepare total RNA utilized as template for RT-PCR amplification and sequencing of cDNA encoding acetylcholinesterase 1 using gene specific primers and 3’-5’-RACE. The cDNA was cloned into pBlueBac4.5/V5-His TOPO, and expressed by baculovirus in Sf21 insect cells in serum-free medium. Recombinant P. papatasi acetylcholinesterase was biochemically characterized using a modified Ellman’s assay in microplates.ResultsA 2309 nucleotide sequence of PpAChE1 cDNA [GenBank: JQ922267] of P. papatasi from a laboratory colony susceptible to insecticides is reported with 73-83% nucleotide identity to acetylcholinesterase mRNA sequences of Culex tritaeniorhynchus and Lutzomyia longipalpis, respectively. The P. papatasi cDNA ORF encoded a 710-amino acid protein [GenBank: AFP20868] exhibiting 85% amino acid identity with acetylcholinesterases of Cx. pipiens, Aedes aegypti, and 92% amino acid identity for L. longipalpis. Recombinant P. papatasi AChE1 was expressed in the baculovirus system and characterized as an insect acetylcholinesterase with substrate preference for acetylthiocholine and inhibition at high substrate concentration. Enzyme activity was strongly inhibited by eserine, BW284c51, malaoxon, and paraoxon, and was insensitive to the butyrylcholinesterase inhibitors ethopropazine and iso-OMPA.ConclusionsResults presented here enable the screening and identification of PpAChE mutations resulting in the genotype for insensitive PpAChE. Use of the recombinant P. papatasi AChE1 will facilitate rapid in vitro screening to identify novel PpAChE inhibitors, and comparative studies on biochemical kinetics of inhibition.

Serotonin-like immunoreactivity in the central nervous system of two ixodid tick species

Immunocytochemistry was used to describe the distribution of serotonin-like immunoreactive (5HT-IR) neurons and neuronal processes in the central nervous system (CNS), the synganglion, of two ixodid tick species; the winter tick, Dermacentor albipictus and the lone star tick, Amblyomma americanum. 5HT-IR neurons were identified in the synganglion of both tick species. D. albipictus had a significantly higher number of 5HT-IR neurons than A. americanum. The labeling pattern and number of 5HT-IR neurons were significantly different between sexes in D. albipictus, but were not significantly different between sexes in A. americanum. 5HT-IR neurons that were located in the cortex of the synganglion projected processes into the neuropils, invading neuromeres in the supraesophageal ganglion including the protocerebrum, postero-dorsal, antero-dorsal and cheliceral neuromeres. In the subesophageal ganglion, dense 5HT-IR neuronal processes were found in the olfactory lobes, pedal, and opisthosomal neuromeres. Double-labeling with neurobiotin backfilled from the first leg damaged at the Haller’s organ revealed serotoninergic neuronal processes surrounding the glomeruli in the olfactory lobes. The high number of the 5HT-IR neurons and the extensive neuronal processes present in various regions of the synganglion suggest that serotonin plays a significant role in tick physiology.

Better than DEET Repellent Compounds Derived from Coconut Oil

Hematophagous arthropods are capable of transmitting human and animal pathogens worldwide. Vector-borne diseases account for 17% of all infectious diseases resulting in 700,000 human deaths annually. Repellents are a primary tool for reducing the impact of biting arthropods on humans and animals. N,N-Diethyl-meta-toluamide (DEET), the most effective and long-lasting repellent currently available commercially, has long been considered the gold standard in insect repellents, but with reported human health issues, particularly for infants and pregnant women. In the present study, we report fatty acids derived from coconut oil which are novel, inexpensive and highly efficacious repellant compounds. These coconut fatty acids are active against a broad array of blood-sucking arthropods including biting flies, ticks, bed bugs and mosquitoes. The medium-chain length fatty acids from C8:0 to C12:0 were found to exhibit the predominant repellent activity. In laboratory bioassays, these fatty acids repelled biting flies and bed bugs for two weeks after application, and ticks for one week. Repellency was stronger and with longer residual activity than that of DEET. In addition, repellency was also found against mosquitoes. An aqueous starch-based formulation containing natural coconut fatty acids was also prepared and shown to protect pastured cattle from biting flies up to 96-hours in the hot summer, which, to our knowledge, is the longest protection provided by a natural repellent product studied to date.

Surface Polar Lipids Differ in Male and Female Phlebotomus papatasi (Diptera: Psychodidae)

ABSTRACT The polar lipids on the surface of the Old World sand fly, Phlebotomus papatasi (Scopoli), were analyzed by high-resolution mass spectrometry. Blood-fed females and nonblood-fed females and males were separately analyzed and compared. The major polar lipids were found to be long-chain diols and fatty acids. Relatively high levels of diacylglycerols were found in blood-fed females and in males. A wide variety of lipids were found at low levels, including esters, sterols, monoacylglycerols, and hydroxy fatty acids. Blood-fed females had several lyso lipids and N-acyl amino acids that were not found on unfed females or males. These substances may be surfactants used in blood feeding. Heneicosenoic acid was found on females at more than twice the level of males, suggesting it could be a component of a female pheromone. Four substances were identified on males at twofold higher levels than on females: tetradienoic acid, methoxyhexadecasphinganine, butyl octadecanoate, and diacylglycerol(14:1/12:0/0:0). These could be short-range pheromones involved in courtship, and they will be further analyzed in future behavioral bioassays.