Felix D. Guerrero

Assessment of bacterial diversity in the cattle tick Rhipicephalus (Boophilus) microplus through tag-encoded pyrosequencing

BackgroundTicks are regarded as the most relevant vectors of disease-causing pathogens in domestic and wild animals. The cattle tick, Rhipicephalus (Boophilus) microplus, hinders livestock production in tropical and subtropical parts of the world where it is endemic. Tick microbiomes remain largely unexplored. The objective of this study was to explore the R. microplus microbiome by applying the bacterial 16S tag-encoded FLX-titanium amplicon pyrosequencing (bTEFAP) technique to characterize its bacterial diversity. Pyrosequencing was performed on adult males and females, eggs, and gut and ovary tissues from adult females derived from samples of R. microplus collected during outbreaks in southern Texas.ResultsRaw data from bTEFAP were screened and trimmed based upon quality scores and binned into individual sample collections. Bacteria identified to the species level include Staphylococcus aureus, Staphylococcus chromogenes, Streptococcus dysgalactiae, Staphylococcus sciuri, Serratia marcescens, Corynebacterium glutamicum, and Finegoldia magna. One hundred twenty-one bacterial genera were detected in all the life stages and tissues sampled. The total number of genera identified by tick sample comprised: 53 in adult males, 61 in adult females, 11 in gut tissue, 7 in ovarian tissue, and 54 in the eggs. Notable genera detected in the cattle tick include Wolbachia, Coxiella, and Borrelia. The molecular approach applied in this study allowed us to assess the relative abundance of the microbiota associated with R. microplus.ConclusionsThis report represents the first survey of the bacteriome in the cattle tick using non-culture based molecular approaches. Comparisons of our results with previous bacterial surveys provide an indication of geographic variation in the assemblages of bacteria associated with R. microplus. Additional reports on the identification of new bacterial species maintained in nature by R. microplus that may be pathogenic to its vertebrate hosts are expected as our understanding of its microbiota expands. Increased awareness of the role R. microplus can play in the transmission of pathogenic bacteria will enhance our ability to mitigate its economic impact on animal agriculture globally. This recognition should be included as part of analyses to assess the risk for re-invasion of areas like the United States of America where R. microplus was eradicated.

Toxicological and molecular characterization of pyrethroid-resistant horn flies, Haematobia irritans: identification of kdr and super-kdr point mutations.

Two pyrethroid-resistant strains of horn flies were found to be 17- and 688-fold more resistant to permethrin and 17- and 11,300-fold more resistant to cyhalothrin than a susceptible control strain. Synergism experiments with piperonyl butoxide showed that both target site insensitivity and metabolic resistance mechanisms were present in the Super Resistant strain. Using the reverse transcriptase-polymerase chain reaction (RT-PCR), a 0.9 kb fragment of the putative sodium channel gene from susceptible and resistant flies was cloned and sequenced. Two sequence variants were detected, presumably arising from alternative splicing of transcripts. The amino acid sequences deduced from the resistant and susceptible fly gene fragments were identical except for three amino acid substitutions, two of which have been associated with resistance in house flies. A leucine to phenylalanine substitution associated with knockdown resistance (kdr) was found in both resistant strains. A methionine to threonine substitution associated with super-kdr was found in the Super Resistant strain. Translation of poly(A)+ RNA followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) detected translation products whose concentrations increased in association with pyrethroid resistance. Random-amplified polymorphic DNA (RAPD)-PCR of genomic DNA with over 260 DNA oligomers yielded one resistance-associated marker, designated HF-77, which was not detected in any susceptible flies but was present in 16% of the resistant individuals.

Molecular and biochemical survey of acaricide resistance mechanisms in larvae from Mexican strains of the southern cattle tick, Boophilus microplus.

We examined the larvae of several organophosphate and pyrethroid-resistant Mexican strains of Boophilus microplus using biochemical and molecular tests to investigate the mechanisms conferring acaricide resistance. The electrophoretic profiles of esterase activity in protein extracts from coumaphos and permethrin-resistant strains compared to the susceptible strain revealed distinct differences, which inhibitor studies attributed to carboxylesterases. Esterase hydrolysis assays showed significant enhancement of both total and permethrin hydrolysis in one pyrethroid-resistant strain, with no enhancement in two other strains with very high resistance to pyrethroids. Sequence analysis of sodium channel mRNA fragments in all pyrethroid-resistant strains determined that they did not possess the classic kdr and super-kdr mutations known to confer pyrethroid resistance in several insect species. Using reverse transcriptase-polymerase chain reaction (RT-PCR) with degenerate primers designed from conserved regions of insect esterase amino acid sequences, a B. microplus larval cDNA fragment was isolated whose deduced amino acid sequence was significantly similar to esterases from a wide range of species. In Northern blot RNA analysis the cDNA hybridized to a 2.1 kb mRNA that was abundant in all resistant strains except one, in which a very low abundance could provide a marker for the mechanism conferring resistance in this strain.

Role of the kdr and super-kdr sodium channel mutations in pyrethroid resistance: correlation of allelic frequency to resistance level in wild and laboratory populations of horn flies (Haematobia irritans)

The kdr and super-kdr point mutations found in the insect sodium channel gene are postulated to confer knockdown resistance (kdr) to pyrethroids. Using an allele-specific PCR assay to detect these mutations in individual horn flies, Haematobia irritans (L.), we determined the allelic frequency of the kdr and super-kdr mutations in several wild and laboratory populations. Wild populations with very similar allelic frequencies had resistance levels that ranged widely from 3- to 18-fold relative to a susceptible population. Conversely, the kdr allele frequency in a lab population with 17-fold resistance was nearly double that found in a heavily pressured wild population with 18-fold resistance. We conclude that, although the kdr mutation confers significant levels of pyrethroid resistance, a substantial component of resistance in insecticidally pressured populations is conferred by mechanisms that are PBO-suppressible. High super-kdr allele frequencies were detected in two resistant lab populations, but in wild populations with equivalent resistance the super-kdr allele frequency was very low. Interestingly, in over 1200 individuals assayed, the super-kdr mutation was never detected in the absence of the kdr mutation.

Acaricide resistance mechanisms in Rhipicephalus (Boophilus) microplus

Acaricide resistance has become widespread in countries where cattle ticks, Rhipicephalus (Boophilus) microplus, are a problem. Resistance arises through genetic changes in a cattle tick population that causes modifications to the target site, increased metabolism or sequestration of the acaricide, or reduced ability of the acaricide to penetrate through the outer protective layers of the ticks body. We review the molecular and biochemical mechanisms of acaricide resistance that have been shown to be functional in R. (B.) microplus. From a mechanistic point of view, resistance to pyrethroids has been characterized to a greater degree than any other acaricide class. Although a great deal of research has gone into discovery of the mechanisms that cause organophosphate resistance, very little is defined at the molecular level and organophosphate resistance seems to be maintained through a complex and multifactorial process. The resistance mechanisms for other acaricides are less well understood. The target sites of fipronil and the macrocyclic lactones are known and resistance mechanism studies are in the early stages. The target site of amitraz has not been definitively identified and this is hampering mechanistic studies on this acaricide.

Use of an Allele-Specific Polymerase Chain Reaction Assay to Genotype Pyrethroid Resistant Strains of Boophilus microplus (Acari: Ixodidae)

Abstract A polymerase chain reaction-based assay was developed to detect the presence of a pyrethroid resistance-associated amino acid substitution in Boophilus microplus (Canestrini). The assay uses a simple method for the extraction of genomic DNA from individual larvae and genotypes individuals for the presence of a Phe → Ile amino acid substitution in the S6 transmembrane segment of domain III of the para-like sodium channel, clearly distinguishing heterozygotes from homozygotes. High frequencies for this amino acid substitution were found in the Corrales and San Felipe strains, which have target site insensitivity mechanisms for pyrethroid resistance. The Caporal resistant strain contained lower yet substantial numbers of amino acid-substituted alleles. Low amino acid substitution frequencies were found in the susceptible reference Gonzales strain and the Coatzacoalcos strain, which has metabolic esterase-mediated pyrethroid resistance. The amino acid substitution was not found in six other strains that were susceptible to pyrethroids.

Sequencing a new target genome: the Boophilus microplus (Acari: Ixodidae) genome project.

Abstract The southern cattle tick, Boophilus microplus (Canestrini), causes annual economic losses in the hundreds of millions of dollars to cattle producers throughout the world, and ranks as the most economically important tick from a global perspective. Control failures attributable to the development of pesticide resistance have become commonplace, and novel control technologies are needed. The availability of the genome sequence will facilitate the development of these new technologies, and we are proposing sequencing to a 4–6X draft coverage. Many existing biological resources are available to facilitate a genome sequencing project, including several inbred laboratory tick strains, a database of ≈45,000 expressed sequence tags compiled into a B. microplus Gene Index, a bacterial artificial chromosome (BAC) library, an established B. microplus cell line, and genomic DNA suitable for library synthesis. Collaborative projects are underway to map BACs and cDNAs to specific chromosomes and to sequence selected BAC clones. When completed, the genome sequences from the cow, B. microplus, and the B. microplus-borne pathogens Babesia bovis and Anaplasma marginale will enhance studies of host–vector–pathogen systems. Genes involved in the regeneration of amputated tick limbs and transitions through developmental stages are largely unknown. Studies of these and other interesting biological questions will be advanced by tick genome sequence data. Comparative genomics offers the prospect of new insight into many, perhaps all, aspects of the biology of ticks and the pathogens they transmit to farm animals and people. The B. microplus genome sequence will fill a major gap in comparative genomics: a sequence from the Metastriata lineage of ticks. The purpose of the article is to synergize interest in and provide rationales for sequencing the genome of B. microplus and for publicizing currently available genomic resources for this tick.

Characterization of Acaricide Resistance in Rhipicephalus sanguineus (Latreille) (Acari: Ixodidae) Collected from the Corozal Army Veterinary Quarantine Center, Panama

Abstract Rhipicephalus sanguineus (Latreille) were collected from the Corozal Army Veterinary Quarantine Center in Panama and characterized for resistance to five classes of acaricides. These ticks were highly resistant to permethrin, DDT, and coumaphos; moderately resistant to amitraz; and not resistant to fipronil when compared with susceptible strains. Resistance to both permethrin and DDT may result from a mutation of the sodium channel. However, synergist studies indicate that enzyme activity is involved. The LC50 estimate for permethrin was lowered further in the Panamanian strain then in susceptible strains with the addition of triphenylphosphate (TPP), but not with the addition of piperonyl butoxide (PBO). This suggests that esterases and not oxidases are responsible for at least some pyrethroid resistance. Elevated esterase activity and its inhibition by TPP were confirmed by native gel electrophoresis. The LC50 estimate obtained for coumaphos in the Panamanian strain was not lowered further than what was observed for susceptible strains by the addition of TPP or PBO. This indicates that enzyme activity might not be involved in coumaphos resistance. Resistance to amitraz was measured through a modification of the Food and Agriculture Organization Larval Packet Test. All tick strains were found to be susceptible to fipronil.

The resistance of varroa mites (Acari: Varroidae) to acaricides and the presence of esterase

Abstract Varroa mites (Varroa destructor Anderson and Trueman, 2000) are becoming resistant to acaricide treatments via metabolic and/or target site desensitivity. Results of a survey of mites from the Carl Hayden AZ lab and from cooperators in five locations (Arizona, California, Florida, Maine, North Dakota) showed that some mites were susceptible to all three acaricides (Amitraz, Coumaphos, Fluvalinate) in the spring of 2003, but by fall most mites were resistant. Mites were resistant to all chemicals, even from beekeepers that do not treat colonies with acaricides. We used esterase native activity gels to test for the presence of specific esterases which might be involved in pesticide resistance in varroa. All mites tested had positive bands for esterase, even those exhibiting susceptibility to some acaricides. Based on the differences between the esterase activity gel profile of the susceptible and cross-resistant V. destructor, it is possible that an esterase-mediated resistance mechanism is operative in the population of the mites we analyzed. However, a combination of other resistance mechanisms may be present which make the esterase activity gel method unreliable for use in identifying varroa mites with multiple resistance.

Proteomic profiling of Rhipicephalus (Boophilus) microplus midgut responses to infection with Babesia bovis

Differences in protein expression in midgut tissue of uninfected and Babesia bovis-infected southern cattle ticks, Rhipicephalus (Boophilus) microplus, were investigated in an effort to establish a proteome database containing proteins involved in successful pathogen transmission. The electrophoretic separation of midgut membrane proteins was greatly improved by using liquid-phase isoelectric focusing combined with one-dimensional or two-dimensional (2-D) gel electrophoresis. A selection of differentially expressed proteins were subjected to analysis by capillary-HPLC-electrospray tandem mass spectrometry (HPLC-ESI-MS/MS). Among the identified Babesia-affected tick midgut proteins were six proteins that are implicated in signaling processes, including three Ca(2+)-binding proteins, a guanine nucleotide-binding protein, a protein with signal peptide activity and a translocon-associated receptor protein. Up-regulation of five metabolic enzymes indicated parasite-induced changes in electron and proton transport, protein processing and retinoic acid metabolism. Among the down-regulated proteins were a molecular chaperone, a cytoskeletal protein and a multifunctional protein of the prohibitin family. Identification of these proteins may provide new insights into the molecular interactions between B. bovis and its tick vector, and could lead to identification of anti-tick and transmission-blocking vaccine candidates.