Rickettsia felis is an emerging human pathogen associated with cat fleas: A review of findings in Taiwan.

Rickettsia felis is an emerging rickettsial agent principally associated with cat fleas (Ctenocephalides felis), formerly discovered in 1990. Since then, clinical cases of R. felis infection have been identified globally by specific DNA sequences in patients with undifferentiated febrile illness, including in Taiwan, but such evidence is limited. R. felis rickettsiosis is self-limiting and easily treated with doxycycline, but its diagnosis remains a challenge. Environmental risk factors for R. felis rickettsiosis have yet to be clearly demonstrated, and its transmission biology is incompletely understood. Cat fleas are naturally infected with R. felis at varying rates, and vector competence in the transmission of R. felis has been demonstrated in animal models, including dogs, which may serve as reservoir hosts. In northern Taiwan, despite ∼20% of cat fleas infesting companion animals consistently found to be infected with R. felis, only a few cases of potential R. felis infection have been identified through a retrospective serological investigation, though without molecular confirmation. Ecological studies have identified divergent R. felis-like organisms in different arthropod hosts, but these strains appear to serve as nonpathogenic endosymbionts. Although its association with disease is limited, we believe cat flea-borne R. felis warrants increased recognition in an aging population due to immunosenescence and the proximity of companion animals to the elderly. Adopting a One Health approach involving collaboration and communication between clinicians, veterinarians, public health practitioners, and environmental scientists will improve our knowledge about this neglected pathogen and promote the prevention and control of vector-borne diseases.

Investigation of Ctenocephalides felis on domestic dogs and Rickettsia felis infection in the Democratic Republic of Sao Tome and Principe.

Rickettsia felis is an obligate intracellular Gram-negative bacterium which causes flea-borne spotted fever in humans. In the past decades, R. felis has been detected worldwide in Ctenocephalides felis fleas and various other arthropods. However, due to its shared symptoms with other common vector-borne diseases, human infection is prone to be underestimated or misdiagnosed, especially in the malaria-endemic areas including sub-Saharan Africa, where confirmatory laboratory diagnoses are not usually available. In this study, a 'One Health' approach was adopted to explore potential vector-borne and zoonotic pathogens in the Democratic Republic of Sao Tome and Principe (DRSTP), an island nation in the Gulf of Guinea. By collaborating with local veterinarians, 1,187 fleas were collected from 95 domestic dogs across the country and later identified as Ct. felis using taxonomic keys. A cytochrome oxidase gene-based phylogenetic analysis revealed that all collected fleas belonged to a single haplotype and were identical to isolates from Ivory Coast and Brazil that clustered into a clade of tropical distribution. Additional samples of 14 chigoe fleas (Tunga penetrans) were collected from the surrounding environment of the dogs' resting spots. Rickettsia felis infection in fleas was examined by molecular methods targeting the citrate synthase (gltA)- and outer membrane protein A (ompA)-coding genes as well as the R. felis-specific pRF plasmid. The bacterial DNA was detected in 21.01% (146/695) of cat fleas but none of the chigoe fleas. Microimmunofluorescence assay was then performed to assess pathogen exposure of the residents. Of 240 dried blood spots from participants with dog contacts, 8 (3.33%) exhibited R. felis antibodies. Our findings demonstrated the presence of R. felis in DRSTP. Further extensive epidemiological studies regarding its prevalence and its role in causing febrile illness while the nation is entering pre-elimination stage of malaria will be carried out.

A new millipede-parasitizing horsehair worm, Gordius chiashanus sp. nov., at medium altitudes in Taiwan (Nematomorpha, Gordiida).

Gordius chiashanus sp. nov., a newly described horsehair worm that parasitizes the Spirobolus millipede, is one of the three described horsehair worm species in Taiwan. It is morphologically similar to G. helveticus Schmidt-Rhaesa, 2010 because of the progressively broadening distribution of bristles concentrated on the male tail lobes, but it is distinguishable from G. helveticus because of the stout bristles on the mid-body. In addition, a vertical white stripe on the anterior ventral side and areoles on the inside wall of the cloacal opening are rarely mentioned in other Gordius species. Free-living adults emerged and mated on wet soil under the forest canopy in the winter (late November to early February) at medium altitudes (1100-1700 m). Mucus-like structure covering on the body surface, which creates a rainbow-like reflection, might endow the worm with high tolerance to dehydration. Although Gordius chiashanus sp. nov. seems to be more adaptive to the terrestrial environment than other horsehair worm species, cysts putatively identified as belonging to this hairworm species found in the aquatic paratenic host, Ephemera orientalis McLachlan, 1875, suggest the life cycle of Gordius chiashanus sp. nov. could involve water and land. The free-living adults emerged from the definitive hosts might reproduce in the terrestrial environment or enter an aquatic habitat by moving or being washed away by heavy rain instead of manipulating the behavior of their terrestrial definitive hosts.

Analysis of Recent Interception Records Reveals Frequent Transport of Arboreal Ants and Potential Predictors for Ant Invasion in Taiwan.

We uncovered taxonomic diversity, country of origin and commodity type of intercepted ants at Taiwanese borders based on an 8 year database of 439 interception records. We found intercepted ants arrived predominantly via timber, a pattern likely reflecting the high domestic demand for foreign timber in Taiwan. The most frequently intercepted species were either arboreal or wood-dwelling ants, raising a concern of these ants constituting a next wave of ant invasion in Taiwan. Further analyses indicate that the taxonomic composition of intercepted ants does not match that of established non-native ant species, suggesting that interception data alone fails to provide adequate power to predict the establishment success of ants. Yet, interception frequency and selected life-history traits (i.e., flexible colony founding mode and general nesting habits) were shown to jointly serve as a practical predictor of the establishment risk of non-native ants. Consistent with other border interception databases, secondary introduction (i.e., species arriving from their introduced ranges instead of their native ranges) also represents a major pathway for transport of invasive ants into Taiwan, suggesting its role in shaping the global invasion of ants. Our findings offer baseline information for constructing a prediction framework for future ant invasions and assist in the decision-making process of quarantine authorities in Taiwan.

Carriers and cutters: size-dependent caste polyethism in the tropical fire ant (Solenopsis geminata).

Body size is an important life-history trait in eusocial insects which plays a key role in colony fitness. The division of labour, represented by caste polyethism, correlates with divergent morphological traits. Size polymorphism has been noted in the tropical fire ant, Solenopsis geminata; however, little is known regarding the differences in the size distributions of workers performing foraging tasks. In the present study, task partitioning was observed in the foraging activities of S. geminata. Two subgroups among foraging workers of S. geminata were discovered using the Gaussian mixture model: a large worker group (head width ≥ 0.924 mm) and a small worker group (head width < 0.924 mm). The foraging worker population comprised two distinct groups - 25.64% were large workers and 74.36% were small workers. Larger workers delivered heavier seeds faster than smaller workers, but this difference became less apparent when lighter seeds were being carried. When large prey such as crickets was encountered during foraging, S. geminata partitioned their tasks into cutting and transportation. The large workers were observed to cut cricket prey into fragments with their longer mandibles, and the small workers then transported these fragments back to the nest. These results present evidence of task partitioning among tropical fire ants, with different tasks being performed by ants of different castes.

A rapid insect species identification system using mini-barcode pyrosequencing.

BACKGROUND: Rapid and accurate species identification is not only important for biodiversity studies and pest quarantine and management, but in some cases may also influence the results of international trade negotiations. In this study, we developed a rapid species identification system for insects. RESULTS: A universal DNA mini-barcode primer pair was designed to target ∼ 120 bp of the mitochondrial 16S rDNA gene. This primer set can amplify the targeted region from all 300 species of 26 insect orders tested as well as other classes of Arthropoda. Although we found no within-species variation in this region, it provided enough information to separate closely related species or species complexes, in particular Thrips spp. and Bemisia spp. By combining a quick DNA extraction method with pyrosequencing, we were able to generate DNA sequences and complete species identification within 5 h. CONCLUSION: Mini-barcode pyrosequencing of 16S rDNA coupled with the GenBank database provides a rapid, accurate, and efficient species identification system. This system is therefore useful for biodiversity discovery, forensic identification, and quarantine control and management. © 2019 Society of Chemical Industry.

Detection of Male Oriental Fruit Fly (Diptera: Tephritidae) Susceptibility to Naled- and Fipronil-Intoxicated Methyl Eugenol.

Naled-intoxicated methyl eugenol (ME) is commonly used to control oriental fruit flies, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), in Taiwan. However, non-responsiveness to ME and pesticide resistance in oriental fruit flies may reduce control efficacy. In this study, mark-recapture experiments were used to analyze the effects of naled-intoxicated ME on field and naled-resistant fly strains. ME non-responsiveness was tested in field, naled-resistant, and susceptible strains and pyrosequencing techniques were used to detect frequencies of point mutations on organophosphate resistant alleles in field strains. Finally, the effects of fipronil-intoxicated ME were analyzed to determine whether control efficiency could be enhanced through the use of alternate pesticides. Control efficiency of naled-intoxicated ME was found to be significantly lower in the field and resistant strains compared to the susceptible strain. ME non-responsiveness was found to be 1.7-1.9% in our lab-reared strains (both naled-resistant and susceptible) and 3.4-4.3% in field strains. Results of our pyrosequencing study found frequency of resistant alleles in captured male field flies to be significantly lower than that of the original population, indicating that it is highly probable that resistant flies may escape from traps. Finally, capture rates of naled-resistant flies increased when naled was replaced with fipronil in attractants, showing that use of pesticides with different modes of action could possibly increase control efficiency of intoxicated ME attractants.

A new orthopteran-parasitizing horsehair worm, Acutogordius taiwanensis sp. n., with a redescription of Chordodes formosanus and novel host records from Taiwan (Nematomorpha, Gordiida).

A description of a new species of horsehair worm, Acutogordius taiwanensissp. n., a redescription of Chordodes formosanus, and novel host records for the latter are provided. Acutogordius taiwanensissp. n. is morphologically similar to A. protectus with moderately flat areoles on its tail tips, but is distinguishable by small mid-body ornamentations. Despite the distinct differences in the post-cloacal crescents between 14 male samples, their conspecific status, along with that of nine female samples, was upheld by a phylogenetic comparison of partial cytochrome oxidase subunit I (COI) sequences. Chordodes formosanus is another common horsehair worm species in Taiwan, which was previously believed to specifically parasitize Hierodula mantids. However, in this study, five C. formosanus were observed emerging from an Acromantis mantid, and two long-horned grasshopper hosts (Leptoteratura sp. and Holochlora japonica). These five worms showed high degrees of similarity in COI sequences and morphology, but one of these individuals bore abnormal crowned areoles, which has never been observed in C. formosanus, and may be attributed to the incomplete development of this particular individual.

Annual Survey of Horsehair Worm Cysts in Northern Taiwan, with Notes on a Single Seasonal Infection Peak in Chironomid Larvae (Diptera: Chironomidae).

The life cycle of the freshwater horsehair worm typically includes a free-living phase (adult, egg, larva) and a multiple-host parasitic phase (aquatic paratenic host, terrestrial definitive host). Such a life cycle involving water and land can improve energy flow in riparian ecosystems; however, its temporal dynamics in nature have rarely been investigated. This study examined seasonal infection with cysts in larval Chironominae (Diptera: Chironomidae) in northern Taiwan. In the larval chironomids, cysts of 3 horsehair worm species were identified. The cysts of the dominant species were morphologically similar to those of Chordodes formosanus. Infection with these cysts increased suddenly and peaked 2 mo after the reproductive season of the adult horsehair worms. Although adult C. formosanus emerged several times in a year, only 1 distinct infection peak was detected in September in the chironomid larvae. Compared with the subfamily Chironominae, samples from the subfamilies Tanypodinae and Orthocladiinae were less parasitized. This indicates that the feeding behavior of the chironomid host likely affects horsehair worm cyst infections; however, bioconcentration in predatory chironomids was not detected.

Prevalence of antibodies against Ehrlichia spp. and Orientia tsutsugamushi in small mammals around harbors in Taiwan.

BACKGROUND: Tick-borne ehrlichiosis and mite-borne scrub typhus represent important emerging zoonotic rickettsial diseases. Although scrub typhus has been recognized by the Taiwanese public health system, information on ehrlichial infections is scarce in Taiwan. In this study, the risk of spread of ectoparasites on rodents through aerial and marine transportation was assessed in international and domestic harbors. Here, we report the first systematic surveillance of seroprevalence against Ehrlichia spp. in small mammals on the main island of Taiwan. METHODS: In total, 1648 small mammals were trapped from 8 international ports, 18 domestic fishing harbors, and 7 local public health centers around Taiwan from November 2004 to December 2008. Sera were analyzed using indirect immunofluorescence assays to detect IgG antibodies against Ehrlichia chaffeensis and Orientia tsutsugamushi. A serum titer of ≧1:80 was considered positive. RESULTS: Antibodies against Ehrlichia spp. and O. tsutsugamushi were detected in 3.28% and 4.92% of small mammals active around harbors, respectively. The seropositive rate against Ehrlichia was higher in northern Taiwan from 2005 to 2008. However, O. tsutsugamushi infections increased in southern Taiwan during this period. The serological evidence of ehrlichial and O. tsutsugamushi infections in all international ports were included in the study. No significant differences were found among the seropositive rates of Ehrlichia spp. and O. tsutsugamushi in small mammals trapped between international and local harbors. CONCLUSIONS: The overall prevalence of Ehrlichia spp. and O. tsutsugamushi infections in small mammals active around harbors was 3.28% and 4.92%, respectively. The results provided serological evidence supporting the potential risks of transporting pathogens through air and maritime traffic. This study highlights serious issues of the emergence and spread of rickettsial diseases in Taiwan. The incidence of human ehrlichiosis requires further investigation.

A Checklist of Chigger Mites (Acari: Trombiculidae and Leeuwenhökiidae) From Taiwan, With Descriptions of Three New Species.

Scrub typhus is a lethal human disease transmitted by larval trombiculid mites (i.e., chiggers) that have been infected with the rickettsia Orientia tsutsugamushi. In total, 21 chigger species are known from Taiwan. We update the checklist of chiggers of Taiwan based on an intensive survey of shrew and rodent hosts in grasslands and agricultural fields in lowland Taiwan, coupled with surveys of forests in one mountainous site and an opportunistic examination of submitted host specimens. Three new species of chiggers, Gahrliepia (Gateria) lieni sp. n., Gahrliepia (Gateria) minuta sp. n., and Gahrliepia (Gateria) yilanensis sp. n., as well as 23 newly recorded chigger species, were discovered. Accordingly, recorded chigger species of Taiwan more than doubled from 21 to 47 species. Two new species and nine newly recorded chigger species were discovered in forests in one mountainous site in northeastern Taiwan, suggesting that many more chigger species may be uncovered, particularly in mountainous Taiwan. Further studies should also investigate O. tsutsugamushi infection in different chigger species to assess its risks to human health.

Morphological allometry and intersexuality in horsehair-worm-infected mantids, Hierodula formosana (Mantodea: Mantidae).

Parasitic castration is a strategy used by parasites to minimize damage to the host by consuming its reproductive system, which results in the morphological alteration of the host. We determined that the forewing shape and density of the antennal sensilla of field-collected adult male mantids (Hierodula formosana), infected by horsehair worms (Chordodes formosanus) was partially feminized (intersexuality), and both male and female mantids infected by horsehair worms exhibited allometric changes in their wings and walking legs. In addition, the testes of most infected male adults disappeared or reduced in size, whereas the number of ovarioles in infected female adults was unaffected. The infection mainly influenced the structures related to host reproduction and locomotion, suggesting unbalanced energy exploitation and the reduction of parasitic virulence. In addition, the intersexuality of infected male adults indicated that sexual differentiation in insects, which researchers have considered to be an autonomous process, was influenced by the infection. The similarity of the antennae of infected male adults with those of last-instar female nymphs suggested that parasitic juvenilization may cause such feminization, but the mechanism of parasitic influence on insect sex characteristics should be studied further.

Ficus (Moraceae) and fig wasps (Hymenoptera: Chalcidoidea) in Taiwan.

Although Ficus-associated wasp fauna have been extensively researched in Australasia, information on these fauna in Taiwan is not well accessible to scientists worldwide. In this study, we compiled records on the Ficus flora of Taiwan and its associated wasp fauna. Initial agronomic research reports on Ficus were published in Japanese in 1917, followed by reports on applied biochemistry, taxonomy, and phenology in Chinese. On the basis of the phenological knowledge of 15 species of the Ficus flora of Taiwan, recent research has examined the pollinating and nonpollinating agaonid and chalcid wasps (Hymenoptera: Chalcidoidea). Updating records according to the current nomenclature revealed that there are 30 taxa (27 species) of native or naturalized Ficus with an unusually high proportion of dioecious species (78%). Four species were observed to exhibit mutualism with more than one pollinating wasp species, and 18 of the 27 Ficus species were reported with nonpollinating wasp species. The number of nonpollinating wasp species associated with specific Ficus species ranges from zero (F. pumila) to 24 (F. microcarpa). Approximately half of the Taiwanese fig tree species have been studied with basic information on phenology and biology described in peer-reviewed journals or theses. This review provides a solid basis for future in-depth comparative studies. This summary of knowledge will encourage and facilitate continuing research on the pollination dynamics of Ficus and the associated insect fauna in Taiwan.

An improved bioassay facilitates the screening of repellents against cat flea, Ctenocephalides felis (Siphonaptera: Pulicidae).

BACKGROUND: Repellents are a common method for preventing flea bites, making an effective system for flea repellent screening advantageous. We describe an improved technique to facilitate repellent activity screening of numerous plant-based Ctenocephalides felis (cat flea) repellents. RESULTS: Two long strips of filter paper were impregnated with test compounds (dissolved in ethanol) and ethanol only, respectively. After drying, the two filter papers were glued together along the long side and inserted into a glass tube containing non-fed cat fleas. The distribution of cat fleas in each half of the filter paper was recorded after 30 min to calculate repellency. Results showed that the essential oil of Cinnamomum osmophloeum (from leaf), Taiwania cryptomerioides (from heartwood) and Plectranthus amboinicus (from leaf) exhibits repellent activity against cat fleas in a dose dependent manner. Moreover, the repellent activities against cat fleas of 2% trans-cinnamaldehyde (the main constituent of Ci. osmophloeum essential oil) and 0.5% thymol (the main constituent of P. amboinicus essential oil) are 97.6% and 90.6%, and can persist for up to 4 and 8 h, respectively. These results are comparable to those of 15% DEET. CONCLUSION: The proposed screening technique can facilitate the pre-screening of numerous flea repellents for further evaluation on animal or human subjects.

Genetic and physiological data suggest demographic and adaptive responses in complex interactions between populations of figs (Ficus pumila) and their pollinating wasps (Wiebesia pumilae).

To study interactions between host figs and their pollinating wasps and the influence of climatic change on their genetic structures, we sequenced cytoplasmic and nuclear genes and genotyped nuclear microsatellite loci from two varieties of Ficus pumila, the widespread creeping fig and endemic jelly fig, and from their pollinating wasps, Wiebesia pumilae, found in Taiwan and on nearby offshore islands. Great divergence in the mitochondrial cytochrome c oxidase subunit I (mtCOI) with no genetic admixture in nuclear markers indicated that creeping- and jelly-fig wasps are genetically distinct. Compared with creeping-fig wasps, jelly-fig wasps also showed better resistance under cold (20 °C) than warm (25 and 30 °C) conditions in a survival test, indicating their adaptation to a cold environment, which may have facilitated population expansion during the ice age as shown by a nuclear intron and 10 microsatellite loci. An excess of amino acid divergence and a pattern of too many rare mtCOI variants of jelly-fig wasps as revealed by computer simulations and neutrality tests implied the effect of positive selection, which we hypothesize was associated with the cold-adaptation process. Chloroplast DNA of the two fig plants was completely segregated, with signs of genetic admixture in nuclear markers. As creeping- and jelly-fig wasps can pollinate creeping figs, occasional gene flow between the two figs is thus possible. Therefore, it is suggested that pollinating wasps may be playing an active role in driving introgression between different types of host fig.

An injury equivalency system for establishing a common economic threshold for three species of rice planthoppers (Hemiptera: Delphacidae) in Taiwan.

The economic threshold (ET) for multiple pest species that share the same injury type on host plants (feeding guild) has been proposed for decision-making in integrated management framework of many defoliating insect pests. However, only a few consider agricultural pests with sucking mouthparts. This study presents the first injury equivalency system for the feeding guild made up of three rice (Oryza sativa L.) planthopper (Hemiptera: Delphacidae) species--Nilaparvata lugens (Stål), Sogatella furcifera (Harváth), and Laodelphax striatellus (Fallén)--by using relative amount of honeydew excretion of each species. The intraspecific injury equivalent coefficient was determined; this coefficient provides an exchange rate for different developmental stages in a species. N. lugens was chosen as the standard species to obtain interspecific injury equivalents for other individuals in the guild, allowing estimates of total guild injury feasible. For extension purposes, the injury equivalency was simplified by pooling all nymphs and adults in the guild to mitigate the potential confusion resulting from uncertainty of instars or wing form. A matrix of ETs established on previous studies and incorporating changes of management cost and rice price was used and served as a control decision guide for the guild samples. The validity of the proposed injury equivalency system was tested using several field data sets, and the results are generally promising and meaningfully elevate the accuracy of estimating combined injury and damage to rice, suggesting that the proposed system is a better integrated pest management decision-making system compared with conventional practices.

The insecticidal activities of fire ant (Hymenoptera: Formicidae) venoms against Plutella xylostella (Lepidoptera: Plutellidae) larvae.

Although fire ants frequently have negative impacts on agricultural systems and public health, they have additional beneficial insecticidal effects. To evaluate the potential effect of fire ant venoms on agricultural pests, the compositions of the venoms and their insecticidal activities against Plutella xylostella (L.) larvae were evaluated under laboratory conditions. The alkaloids found in Solenopsis geminata (F.) venom are primarily saturated C11, which occur in both cis and trans forms, whereas the venom of S. invicta Buren contains six principal alkaloids (from trans C1, to C17). Moreover, the proportions of unsaturated alkaloids in the venom of polygynous S. invicta were significantly higher than the corresponding proportions in the monogynous S. invicta, as shown by our previous studies. Fire ant venoms were topically applied to the dorsal thoracic region of fourth-instar larvae of P. xylostella. The results of the experiment showed that the larval symptoms induced by fire ant venom include contractile, flaccid paralysis, black coloration and death. P. xylostella larvae were most susceptible to S. geminata venom. The order of the susceptibilities of the larvae to the venoms was as follows: S. geminata > S. invicta (monogyne form) > S. invicta (polygyne form), as measured by the corresponding LT50 values at 24 h.

Discovery of genes related to insecticide resistance in Bactrocera dorsalis by functional genomic analysis of a de novo assembled transcriptome.

Insecticide resistance has recently become a critical concern for control of many insect pest species. Genome sequencing and global quantization of gene expression through analysis of the transcriptome can provide useful information relevant to this challenging problem. The oriental fruit fly, Bactrocera dorsalis, is one of the world's most destructive agricultural pests, and recently it has been used as a target for studies of genetic mechanisms related to insecticide resistance. However, prior to this study, the molecular data available for this species was largely limited to genes identified through homology. To provide a broader pool of gene sequences of potential interest with regard to insecticide resistance, this study uses whole transcriptome analysis developed through de novo assembly of short reads generated by next-generation sequencing (NGS). The transcriptome of B. dorsalis was initially constructed using Illumina's Solexa sequencing technology. Qualified reads were assembled into contigs and potential splicing variants (isotigs). A total of 29,067 isotigs have putative homologues in the non-redundant (nr) protein database from NCBI, and 11,073 of these correspond to distinct D. melanogaster proteins in the RefSeq database. Approximately 5,546 isotigs contain coding sequences that are at least 80% complete and appear to represent B. dorsalis genes. We observed a strong correlation between the completeness of the assembled sequences and the expression intensity of the transcripts. The assembled sequences were also used to identify large numbers of genes potentially belonging to families related to insecticide resistance. A total of 90 P450-, 42 GST-and 37 COE-related genes, representing three major enzyme families involved in insecticide metabolism and resistance, were identified. In addition, 36 isotigs were discovered to contain target site sequences related to four classes of resistance genes. Identified sequence motifs were also analyzed to characterize putative polypeptide translational products and associate them with specific genes and protein functions.

Truncated transcripts of nicotinic acetylcholine subunit gene Bdα6 are associated with spinosad resistance in Bactrocera dorsalis.

Spinosad-resistance mechanisms of Bactrocera dorsalis, one of the most important agricultural pests worldwide, were investigated. Resistance levels to spinosad in a B. dorsalis strain from Taiwan were more than 2000-fold, but showed no cross resistance to imidacloprid or fipronil. Combined biochemical and synergistic data indicated that target-site insensitivity is the major resistance component. The gene encoding the nAChR subunit alpha 6 (Bdα6), the putative molecular target of spinosad, was isolated using PCR and RACE techniques. The full-length cDNA of Bdα6 from spinosad-susceptible strains had an open reading frame of 1467 bp and codes for a typical nAChR subunit. Two isoforms of exon 3 (3a and 3b) and exon 8 (8a and 8b), and four full-length splicing variants were found in the susceptible strain. All transcripts from the spinosad-resistant strain were truncated and coded for apparently non-functional Bdα6. Genetic linkage analysis further associated spinosad-resistance phenotype with the truncated Bdα6 forms. This finding is consistent with a previous study in Plutella xylostella. Small deletions and insertions and consequent premature stop codons in exon 7 were associated with the truncated transcripts at the cDNA level. Analysis of genomic DNA sequences (intron 2 and exons 3-6) failed to detect exon 5 in resistant flies. In addition, a mutation in Bdα6 intron 2, just before the truncated/mis-splicing region and in same location with a mutation previously reported in the Pxylα6 gene, was identified in the resistant flies. RNA editing was investigated but was not found to be associated with resistance. While the demonstration of truncated transcripts causing resistance was outlined, the mechanism responsible for generating truncated transcripts remains unknown.