Wen-Jer Wu

The genome of the fire ant Solenopsis invicta

Ants have evolved very complex societies and are key ecosystem members. Some ants, such as the fire ant Solenopsis invicta, are also major pests. Here, we present a draft genome of S. invicta, assembled from Roche 454 and Illumina sequencing reads obtained from a focal haploid male and his brothers. We used comparative genomic methods to obtain insight into the unique features of the S. invicta genome. For example, we found that this genome harbors four adjacent copies of vitellogenin. A phylogenetic analysis revealed that an ancestral vitellogenin gene first underwent a duplication that was followed by possibly independent duplications of each of the daughter vitellogenins. The vitellogenin genes have undergone subfunctionalization with queen- and worker-specific expression, possibly reflecting differential selection acting on the queen and worker castes. Additionally, we identified more than 400 putative olfactory receptors of which at least 297 are intact. This represents the largest repertoire reported so far in insects. S. invicta also harbors an expansion of a specific family of lipid-processing genes, two putative orthologs to the transformer/feminizer sex differentiation gene, a functional DNA methylation system, and a single putative telomerase ortholog. EST data indicate that this S. invicta telomerase ortholog has at least four spliceforms that differ in their use of two sets of mutually exclusive exons. Some of these and other unique aspects of the fire ant genome are likely linked to the complex social behavior of this species.

Global Invasion History of the Fire Ant Solenopsis invicta

Argentine fire ants have spread around the world from a population that was first established a century ago in North America. The fire ant Solenopsis invicta is a significant pest that was inadvertently introduced into the southern United States almost a century ago and more recently into California and other regions of the world. An assessment of genetic variation at a diverse set of molecular markers in 2144 fire ant colonies from 75 geographic sites worldwide revealed that at least nine separate introductions of S. invicta have occurred into newly invaded areas and that the main southern U.S. population is probably the source of all but one of these introductions. The sole exception involves a putative serial invasion from the southern United States to California to Taiwan. These results illustrate in stark fashion a severe negative consequence of an increasingly massive and interconnected global trade and travel system.

Resistance and Synergistic Effects of Insecticides in Bactrocera dorsalis (Diptera: Tephritidae) in Taiwan

Abstract Oriental fruit flies, Bactrocera dorsalis (Hendel), were treated with 10 insecticides, including six organophosphates (naled, trichlorfon, fenitrothion, fenthion, formothion, and malathion), one carbamate (methomyl), and three pyrethroids (cyfluthrin, cypermethrin, and fenvalerate), by a topical application assay under laboratory conditions. Subparental lines of each generation treated with the same insecticide were selected for 30 generations and were designated as x-r lines (x, insecticide; r, resistant). The parent colony was maintained as the susceptible colony. The line treated with naled exhibited the lowest increase in resistance (4.7-fold), whereas the line treated with formothion exhibited the highest increase in resistance (up to 594-fold) compared with the susceptible colony. Synergism bioassays also were carried out. Based on this, S,S,S-tributyl phosphorotrithioate displayed a synergistic effect for naled, trichlorfon, and malathion resistance, whereas piperonyl butoxide displayed a synergistic effect for pyrethroid resistance. All 10 resistant lines also exhibited some cross-resistance to other insecticides, not only to the same chemical class of insecticides but also to other classes. However, none of the organophosphate-resistant or the methomyl-resistant lines exhibited cross-resistance to two of the pyrethroids (cypermethrin and fenvalerate). Overall, the laboratory resistance and cross-resistance data developed here should provide useful tools and information for designing an insecticide management strategy for controlling this fruit fly in the field.

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.

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 worlds 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 Illuminas 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.

Loss of microbial (pathogen) infections associated with recent invasions of the red imported fire ant Solenopsis invicta

Loss of natural enemies during colonization is a prominent hypothesis explaining enhanced performance of invasive species in introduced areas. Numerous studies have tested this enemy release hypothesis in a wide range of taxa but few studies have focused on invasive ants. We conducted extensive surveys for the presence of six microbes in recently established populations (California, Australia, New Zealand, Taiwan, and China) of the invasive fire ant Solenopsis invicta. These microbes include Wolbachia, two microsporidia (Kneallhaziasolenopsae and Vairimorpha invictae) and three RNA viruses (SINV-1, -2 and -3), all of which previously have been reported in native South American populations of S. invicta. These surveys showed that the total number of enemy species is lower in the recently invaded areas compared with both South American and US populations. Only two microbes were found in any of these recently invaded areas: SINV-1 was detected in all surveyed populations except Australia and New Zealand, and SINV-2 was detected in California and Taiwan only. These results support the general prediction that invasive species lose many of their natural enemies during invasion. Further, the conspicuous absence of some of these microbes in these areas may result from strong selection against founders due to fitness costs associated with harboring detrimental infections rather than the alternative hypothesis that they simply were absent among the original founders. While the successful invasion of S. invicta in these recently invaded areas may be explained partly by the absence of natural enemies, other factors likely have been important as well.

Alterations of the acetylcholinesterase enzyme in the oriental fruit fly Bactrocera dorsalis are correlated with resistance to the organophosphate insecticide fenitrothion

Alterations of the structure and activity of the enzyme acetylcholinesterase (AChE) leading to resistance to organophosphate insecticides have been examined in the oriental fruit fly, Bactrocera dorsalis (Hendel), an economic pest of great economic importance in the Asia-Pacific region. We used affinity chromatography to purify AChE isoenzymes from heads of insects from lines showing the phenotypes of resistance and sensitivity to insecticide treatments. The AChE enzyme from a strain selected for resistance to the insecticide fenitrothion shows substantially lower catalytic efficiency for various substrates and 124-, 373- and 5810-fold less sensitivity to inhibition by paraoxon, eserine and fenitroxon, respectively, compared to that of the fenitrothion susceptible line. Using peptide mass fingerprinting, we also show how specific changes in the structure of the AChE enzymes in these lines relate to the resistant and sensitive alleles of the AChE (ace) gene characterized previously in this species (described in Hsu, J.-C., Haymer, D.S., Wu, W.-J., Feng, H.-T., 2006. Mutations in the acetylcholinesterase gene of Bactrocera dorsalis associated with resistance to organophosphorus insecticides. Insect Biochem. Mol. Biol. 36, 396-402). Polyclonal antibodies specific to the purified isoenzymes and real-time PCR were also used to show that both the amount of the isoenzyme present and the expression levels of the ace genes were not significantly different between the R and S lines, indicating that quantitative changes in gene expression were not significantly contributing to the resistance phenotype. Overall, our results support a direct causal relationship between the mutations previously identified in the ace gene of this species and qualitative alterations of the structure and function of the AChE enzyme as the basis for the resistance phenotype. Our results also provide a basis for further comparisons of insecticide resistance phenomena seen in closely related species, such as Bactrocera oleae, as well as in a wide range of more distantly related insect species.

Bartonella Infection in Shelter Cats and Dogs and Their Ectoparasites

Mainly through vector transmission, domestic cats and dogs are infected by several Bartonella spp. and represent a large reservoir for human infections. This study investigated the relationship of prevalences of Bartonella infection in shelter dogs and cats and various ectoparasite species infesting them (fleas, ticks, and lice). Moreover, relationships between Bartonella infection and animal gender and age and presence of ectoparasites were analyzed. Blood samples were collected from 120 dogs and 103 cats. There were 386 ticks and 36 fleas harvested on these dogs, and 141 fleas, 4 ticks, and 2 lice harvested on these cats. Isolation/detection of Bartonella sp. was performed by culture, polymerase chain reaction (PCR), and partial sequencing. Bartonella was isolated from 21 (20.4%) cats and detected by PCR from 20 (19.4%) cats, 2 (1.7%) dogs, 55 (39%) fleas collected from cats, 28 (10%) ticks DNA samples, and 1 (2.8%) flea collected from dogs. When combining culture and PCR data, 27 cats and 55 fleas collected on cats were positive for Bartonella henselae or Bartonella clarridgeiae, but none were coinfected. Approximately half of the B. henselae isolates from 21 cats were B. henselae type I. Moreover, B. henselae, Bartonella phoceensis, Bartonella queenslandensis, Bartonella rattimassiliensis, Bartonella elizabethae DNA was detected in ticks collected from dogs and one flea was B. clarridgeiae PCR positive. This is the first report of such a wide variety of Bartonella spp. detected in Rhipicephalus sanguineus. Further studies are required to understand the relative importance of these ectoparasites to transmit Bartonella spp. in dogs and cats.

Effects of Multiple Mating on Female Reproductive Output in the Cat Flea (Siphonaptera: Pulicidae)

Abstract Multiple mating behavior of female cat fleas, Ctenocephalides felis (Bouché), was confirmed in this study, and its effects on fecundity and fertility were investigated as well. The number of fertile eggs produced by mated females was close to nil within 7 d after removal of males, but it was resumed when females were exposed to males again on day 7. Multiple-mated females displayed significantly higher fecundity (400.3 eggs per female) and fertility (182.8 viable eggs per female) than single-mated females (61.7 and 19.0, respectively) in the 24-d period, suggesting that multiple mating by females is an advantageous strategy for cat fleas. The duration of first mating averaged 63.1 min. The high ratio (55.56%) and short duration (34.0 min) of impotent mating suggested that cryptic female choice may be involved during copulation.

Off-host observations of mating and postmating behaviors in the cat flea (Siphonaptera: Pulicidae)

Abstract A blood meal was necessary for a male cat flea, Ctenocephalides felis (Bouché), to display the mating attempts to an unfed or fed female. More mating pairs resulted when both sexes were fed. Copulation occurred when fed fleas were placed on surfaces with temperatures from 34 to 42°C. This article not only describes the mating and postmating behaviors of cat fleas, but also compares them with those of other fleas. The sequence of mating behavior began when a male approached a female ventrally, and the male’s antennae and claspers became erect to attach to the abdomen of the female. Clasper attachment lasted until copulation ended, whereas the male retrieved his antennae immediately after genitalia linkage. The male generally grasped the female’s tarsi with his claws during mating. The length of the mating interval terminated by the male ranged from 25 to 110 min and was significantly longer than that terminated by the female (averaging 12.11 min). After the mating pair separated, the male displayed a series of postmating behaviors discussed herein. This article documents grasping and postmating behaviors of the male cat flea.