Evolutionary history of <Emphasis Type="Italic">Wolbachia</Emphasis> infections in the fire ant <Emphasis Type="Italic">Solenopsis invicta</Emphasis>

Background  

Wolbachia are endosymbiotic bacteria that commonly infect numerous arthropods. Despite their broad taxonomic distribution, the transmission patterns of these bacteria within and among host species are not well understood. We sequenced a portion of the wsp gene from the Wolbachia genome infecting 138 individuals from eleven geographically distributed native populations of the fire ant Solenopsis invicta. We then compared these wsp sequence data to patterns of mitochondrial DNA (mtDNA) variation of both infected and uninfected host individuals to infer the transmission patterns of Wolbachia in S. invicta.     

Evidence for mating plugs in the fire ant <Emphasis Type="Italic">Solenopsis invicta</Emphasis>

Summary Male inhibition of female re-mating is com-mon in many insects. Mating plugs, used by males to con-trol female re-mating, have been postulated in several ant species. Recent studies of bumblebees have described re-mating inhibition by male accessory gland secretions. Fire ants Solenopsis invicta possess accessory glands containing the same four fatty acids as do the bumblebees. Furthermore it appears that some of these acids are transferred to the female at mating. Thus, it is possible that single mating of fire ant females may be enforced by male mating plugs.     

Thermoregulatory brood transport in the fire ant, <Emphasis Type="Italic">Solenopsis invicta</Emphasis>

Nest structure in ants is often designed to optimize the colony’s ability to thermoregulate, and this specialization is most highly developed in mound-building ant species. Solenopsis invicta invest a large amount of energy in building mounds and transporting their brood up and down in their nests as a means of thermoregulation. Because few ant species build true mounds, we wanted to determine the effectiveness of these mounds in harvesting solar heat as well as to distinguish what factors (temperature vs. circadian rhythm) govern where fire ants place their brood in the mound and when they place it. We measured temperature patterns in the mound over several days at different depths and under different conditions (under direct sunlight or shade), and then conducted a series of field experiments to manipulate the orientation and time of heating. On cool mornings in spring or fall, surface temperatures of the mound rise at the fastest rate on the side receiving the most direct sunlight (usually the south side). This heating causes a temperature gradient through different depths in the mound, and shows little difference from outside ground temperature at a depth greater than ~40 cm inside the nest. In the morning, fire ants move their brood up into the mound on the side most directly heated, and when temperatures exceed optimal (~32°C) they move their brood down the temperature gradient to lower depths in the nest. In addition to this, mound temperature does not only increase due to direct sunlight, but temperature also increases higher than ground temperatures when the mound is in the shade due to its low specific heat. Experiments in which sunlight was mirrored to the normally shaded side of the mound, or when mounds were heated at night, revealed that S. invicta primarily track temperature patterns and do not rely on behavioral habits or circadian rhythms for the thermoregulatory transport of their brood. When mounds were shaded, S. invicta brood was evenly distributed directly under the surface of the mound rather than aggregating towards a specific side. The fire ant mound is important for thermoregulation because, compared to moundless subterranean nests, it absorbs heat more rapidly both in direct sunlight and shady conditions. Temperature tracking within the nest is key to understanding thermoregulatory placement of fire ant brood, as well as insight into the production of sexual brood and reproduction. Received 9 August 2007; revised 31 January 2008; accepted 7 February 2008.     

Phenology and parasitism rates in introduced populations of <Emphasis Type="Italic">Pseudacteon tricuspis</Emphasis>, a parasitoid of <Emphasis Type="Italic">Solenopsis invicta</Emphasis>

We documented patterns of seasonal abundance and rates of parasitism in introduced populations of Pseudacteon tricuspis Borgmeier, a phorid parasitoid of the red imported fire ant, Solenopsis invicta Buren. Adult P. tricuspis populations were censused at monthly intervals for 1 year at three sites in northern Florida. Censuses were conducted by aspirating phorids attracted to disturbed S. invicta mounds. Pseudacteon tricuspis adults were present in every month at all sites, although abundances varied greatly among sites and over time. The highest densities of flies (up to 453 censused at 10 disturbed S. invicta mounds in 30 min) were observed in November, and changes in abundance over time were positively correlated among sites. Sex ratios were usually male biased. Parasitism rates were evaluated by collecting workers from field colonies and monitoring them in the laboratory for evidence of parasitism. Parasitism rates were very low – always less than 1%. The average parasitism rate per colony over 16 colonies and 2 years was 0.058%. No pupariation occurred within the first 8 days of collection, suggesting parasitism by P. tricuspis induced behavioral changes in parasitized workers that precluded such workers from our collections. If so, true field parasitism rates may be several times higher than measured here, yet still low in an absolute sense. These low parasitism rates can be reconciled with observed adult phorid densities by considering the large number of host ants present at the study sites.     

Identification of polygyne and monogyne fire ant colonies (<Emphasis Type="Italic">Solenopsis invicta</Emphasis>) by multiplex PCR of <Emphasis Type="Italic">Gp-9</Emphasis> alleles

Summary. Oligonucleotide primers were designed to discriminate between the Gp-9^B and Gp-9^b alleles found in monogyne and polygyne colonies of fire ant, Solenopsis invicta Buren. Primers specific for the Gp-9^B allele produced a 517 bp amplicon and primers specific for Gp-9^b allele produced a 423 bp amplicon. When both sets of primers were multiplexed, homozygous monogyne ants produced a single 517 bp amplicon (specific for Gp-9^B), whereas heterozygous polygyne ants produced one 517 bp amplicon and one 423 bp amplicon (specific for Gp-9^B and Gp-9^b, respectively) which allowed the Gp-9 alleles to be discerned in a single reaction. This method was tested on ants from 20 monogyne colonies and 20 polygyne colonies and was 100% accurate in discriminating the two forms.     

Genetic Transformation of Midgut Bacteria from the Red Imported Fire Ant (<Emphasis Type="Italic">Solenopsis invicta</Emphasis>)

In our previous study we isolated 10 bacterial species from fourth-instar larval midguts of the red imported fire ant, Solenopsis invicta. Here we report the genetic transformation and reintroduction of three species (Kluyvera cryocrescens, Serratia marcescens, and isolate 38) into the fire ant host. All three species were transformed with the plasmid vector, pZeoDsRed. High expression levels of DsRed were observed and the plasmid is maintained in these bacteria at 37°C in the absence of antibiotic selection for at least 9 days of subculturing. The transformed bacteria were successfully reintroduced into fire ant larvae and survived in the fire ant gut for at least 7 days. Upon pupal emergence, 7 days after reintroduction, transformed bacteria can still be isolated, however, most were passed out in the meconium. We further demonstrated that the engineered bacteria could be spread within the colony by feeding this meconium to naive larvae with the aid of worker fire ants. Freder Medina and Haiwen Li have contributed equally to this work.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Diversity and recombination in <Emphasis Type="Italic">Wolbachia</Emphasis> and <Emphasis Type="Italic">Cardinium</Emphasis> from <Emphasis Type="Italic">Bryobia</Emphasis>spider mites

BACKGROUND: Wolbachia and Cardinium are endosymbiotic bacteria infecting many arthropods and manipulating host reproduction. Although these bacteria are maternally transmitted, incongruencies between phylogenies of host and parasite suggest an additional role for occasional horizontal transmission. Consistent with this view is the strong evidence for recombination in Wolbachia, although it is less clear to what extent recombination drives diversification within single host species and genera. Furthermore, little is known concerning the population structures of other insect endosymbionts which co-infect with Wolbachia, such as Cardinium. Here, we explore Wolbachia and Cardinium strain diversity within nine spider mite species (Tetranychidae) from 38 populations, and quantify the contribution of recombination compared to point mutation in generating Wolbachia diversity. RESULTS: We found a high level of genetic diversity for Wolbachia, with 36 unique strains detected (64 investigated mite individuals). Sequence data from four Wolbachia genes suggest that new alleles are 7.5 to 11 times more likely to be generated by recombination than point mutation. Consistent with previous reports on more diverse host samples, our data did not reveal evidence for co-evolution of Wolbachia with its host. Cardinium was less frequently found in the mites, but also showed a high level of diversity, with eight unique strains detected in 15 individuals on the basis of only two genes. A lack of congruence among host and Cardinium phylogenies was observed. CONCLUSIONS: We found a high rate of recombination for Wolbachia strains obtained from host species of the spider mite family Tetranychidae, comparable to rates found for horizontally transmitted bacteria. This suggests frequent horizontal transmission of Wolbachia and/or frequent horizontal transfer of single genes. Our findings strengthens earlier reports of recombination for Wolbachia, and shows that high recombination rates are also present on strains from a restrictive host range. Cardinium was found co-infecting several spider mite species, and phylogenetic comparisons suggest also horizontal transmission of Cardinium among hosts.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

Behavioral and Electroantennogram Responses of Phorid fly <Emphasis Type="Italic">Pseudacteon tricuspis</Emphasis> (Diptera: Phoridae) to Red Imported Fire Ant <Emphasis Type="Italic">Solenopsis invicta</Emphasis> Odor and Trail Pheromone

The phorid fly, Pseudacteon tricuspis is an introduced parasitoid of the red imported fire ant, Solenopsis invicta in the United States. Previous studies show that phorid flies are attracted to host ant workers at disturbed colonies, to colonies exhibiting aggressive interspecific interactions, and to fire ant mating flights. In a series of behavioral and electroantennogram (EAG) experiments, we confirm the possible use of fire ant odor as cues for host location by P. tricuspis. We tested the response of P. tricuspis of different sex and mating status to several host-related odor stimuli including live fire ant workers, extracts of worker whole body, head, thorax, and abdomen, and (E,E)-α-farnesene, a trail pheromone component of Solenopsis fire ants. Results from Y-tube olfactometer bioassays demonstrated the attraction of mated female P. tricuspis to live S. invicta workers. In addition, extracts of S. invicta worker whole body and thorax elicited strong olfactometer response in female flies (mated and unmated) and mated males, but not in unmated males. Pseudacteon tricuspis did not show significant attraction to extracts of S. invicta worker head and abdomen, or to (E,E)-α-farnesene, irrespective of sex and mating status. In EAG experiments, female and male P. tricuspis showed significant EAG response to extracts of worker whole body, head, and abdomen, and to a less extent, thorax extract, but not to (E,E)-α-farnesene. Females showed slightly greater EAG response than males, but EAG response was not affected by mating status. These results suggest that fire ant thorax is likely the source of kairomones used as host location cues by P. tricuspis, and support the hypothesis that fire ant worker trail pheromones are not likely used by P. tricuspis for host location.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

Electrofusion of protoplasts from <Emphasis Type="Italic">Solanum tuberosum</Emphasis>, <Emphasis Type="Italic">S. bulbocastanum</Emphasis> and <Emphasis Type="Italic">S. pinnatisectum</Emphasis>

This paper discusses a number of experiments performed, involving the fusion by an electric field of mesophyll protoplasts from Solanum tuberosum cv. Bintje, S. tuberosum dihaploid clones 243, 299 and the wild tuberous disease-resistant species S. bulbocastanum and S. pinnatisectum. Three fusion experiments (S. bulbocastanum + S. tuberosum dihaploid 243, S. pinnatisectum + S. tuberosum cv. Bintje and S. pinnatisectum + S. tuberosum dihaploid 299) yielded 542 calli, the 52 ones of which produced shoots. Obtained regenerants were estimated by the flow-cytometry (FC) and RAPD analysis to determine hybrid plants.The utilisation of the FC as a useful method for detecting somatic hybrids is also discussed in this paper. The combination S. bulbocastanum + S. tuberosum dihaploid 243 led to the creation of eight somatic hybrids, the combination S. pinnatisectum + S. tuberosum cv. Bintje yielded four somatic hybrids and the combination S. pinnatisectum + S. tuberosum dihaploid 299 resulted in no hybrid regenerants. Morphology in vitro, growth vigour and production of tuber-like structures were evaluated in hybrid plants. Plants were transferred in vivo for further estimation (acclimatization, habitus evaluation and tuberization ability).     

Sex-Ratio Bias in Alates of Polygyne Colonies of the Red Imported Fire Ant <Emphasis Type="Italic">Solenopsis invicta</Emphasis> Buren (Hymenoptera: Formicidae) in China

A sex-ratio bias was studied in alates of natural polygyne colonies of Solenopsis invicta Buren in southern China. The results showed that at the population level, the numbers of male and female alates were nearly equal, even though there was a strong bias toward producing one particular sex at the nest level. For example, 88.89% of the nests sampled were strongly biased toward males or females. In particular, three bias types were observed: extreme female bias, extreme male bias, and a moderate bias. Future studies should address the factors that lead queens to produce strongly biased sex ratios.     

In planta transformation of <Emphasis Type="Italic">Notocactus scopa</Emphasis> cv. <Emphasis Type="Italic">Soonjung</Emphasis> by <Emphasis Type="Italic">Agrobacterium </Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>

Notocactus scopa cv. Soonjung was subjected to in planta Agrobacterium tumefaciens-mediated transformation with vacuum infiltration, pin-pricking, and a combination of the two methods. The pin-pricking combined with vacuum infiltration (20-30 cmHg for 15 min) resulted in a transformation efficiency of 67-100%, and the expression of the uidA and nptII genes was detected in transformed cactus. The established in planta transformation technique generated a transgenic cactus with higher transformation efficiency, shortened selection process, and stable gene expression via asexual reproduction. All of the results showed that the in planta transformation method utilized in the current study provided an efficient and time-saving procedure for the delivery of genes into the cactus genome, and that this technique can be applied to other asexually reproducing succulent plant species.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

New combinations and typifications in <Emphasis Type="Italic">Bistorta</Emphasis>, <Emphasis Type="Italic">Persicaria</Emphasis>, <Emphasis Type="Italic">Polygonum,</Emphasis> and <Emphasis Type="Italic">Rumex</Emphasis> (Polygonaceae)

New combinations are proposed in anticipation of the Polygonaceae treatment in the forthcoming volume of Intermountain Flora: Polygonum kelloggii var. esotericum, P. kelloggii var. watsonii , Rumex densiflorus var. pycnanthus , R. salicifolius var. utahensis, and R. occidentalis var. tomentellus. Typifications are proposed to facilitate ongoing studies in Polygonaceae and to maintain current usage.