Statistical tools to interpret risks that arise from rare events in host specificity testing

Host specificity testing to predict host range is one of the key steps to predicting the risk a biological control agent will present to non-target organisms in the new environment. When host specificity testing data contain discrepancies, or unacceptable levels of uncertainty, it can be difficult for decision-makers to adequately address this uncertainty. To better understand the uncertainty in host specificity testing, we used a range of statistical tools to examine a data set associated with the leaf weevil Cleopus japonicus (Curculionidae), a biological control agent for the weed Buddleja davidii (Buddlejaceae) in New Zealand. Significant uncertainty arose during the early stages of host specificity testing when one C. japonicus larva reared to pupation on a culturally important native plant. Further trials were conducted to evaluate the suitability of C. japonicus as a biological control agent, and despite the uncertainty, C. japonicus was released in New Zealand in 2006, and has since established populations at each release site. However, the possibility of larvae completing their life cycle on the native plant initiated this evaluation of the statistics associated with testing biological control agents. We present results from analyses of the C. japonicus survival data using confidence intervals, equivalence testing, power analyses and survival curves to highlight the appropriateness of each of these tools for interpreting host specificity tests in biological control.     

Quantitative continuous assay for hyaluronan synthase

A rapid, continuous, and convenient three-enzyme coupled UV absorption assay was developed to quantitate the glucuronic acid and N-acetylglucosamine transferase activities of hyaluronan synthase from Pasteurella multocida (PmHAS). Activity was measured by coupling the UDP produced from the PmHAS-catalyzed transfer of UDP-GlcNAc and UDP-GlcUA to a hyaluronic acid tetrasaccharide primer with the oxidation of NADH. Using a fluorescently labeled primer, the products were characterized by gel electrophoresis. Our results show that a truncated soluble form of recombinant PmHAS (residues 1-703) can catalyze the glycosyl transfers in a time- and concentration-dependent manner. The assay can be used to determine kinetic parameters, inhibition constants, and mechanistic aspects of this enzyme. In addition, it can be used to quantify PmHAS during purification of the enzyme from culture media.     

Role of central nervous system and ovarian insulin receptor substrate 2 signaling in female reproductive function in the mouse

Insulin receptor signaling regulates female reproductive function acting in the central nervous system and ovary. Female mice that globally lack insulin receptor substrate (IRS) 2, which is a key mediator of insulin receptor action, are infertile with defects in hypothalamic and ovarian functions. To unravel the tissue-specific roles of IRS2, we examined reproductive function in female mice that lack Irs2 only in the neurons. Surprisingly, these animals had minimal defects in pituitary and ovarian hormone levels, ovarian anatomy and function, and breeding performance, which indicates that the central nervous system IRS2 is not an obligatory signaling component for the regulation of reproductive function. Therefore, we undertook a detailed analysis of ovarian function in a novel Irs2 global null mouse line. Comparative morphometric analysis showed reduced follicle size, increased numbers of atretic follicles, as well as impaired oocyte growth and antral cavity development in Irs2 null ovaries. Granulosa cell proliferation was also defective in the Irs2 null ovaries. Furthermore, the insulin- and eCG-stimulated phosphoinositide-3-OH kinase signaling events, which included phosphorylation of Akt/protein kinase B and glycogen synthase kinase 3-beta, were impaired, whereas mitogen-activated protein kinase signaling was preserved in Irs2 null ovaries. These abnormalities were associated with reduced expression of cyclin D2 and increased CDKN1B levels, which indicates dysregulation of key components of the cell cycle apparatus implicated in ovarian function. Our data suggest that ovarian rather than central nervous system IRS2 signaling is important in the regulation of female reproductive function.     

Body temperature variation of free-ranging and captive southern brown bandicoots Isoodon obesulus (Marsupialia: Peramelidae)

To investigate patterns of thermoregulation in free-ranging and captive southern brown bandicoots Isoodon obesulus, we measured abdominal body temperature (T_b) of five free-ranging bandicoots over 42 days using implanted data loggers and T_b of three captive bandicoots over 3 months using implanted temperature-sensitive radio transmitters. Bandicoots in the wild had a mean T_b of 36.5±1.0 °C (range 33.4–39.8 °C) and showed a pronounced nychthemeral pattern with two distinct temperature phases. T_b increased at 13:30±2.6 h each day and remained high for 10.65±2.07 h, suggesting a crepuscular and early evening activity pattern. Daily T_b variation of I. obesulus would save considerable energy by reducing daytime thermoregulatory costs in the wild. Captive bandicoots had a similar mean body temperature (36.9±0.2°C) and range (33.0–39.9°C) as free-ranging bandicoots. However, the nychthemeral T_b pattern of captive bandicoots was different from free-ranging bandicoots, with a less pronounced daily cycle and the nocturnal rise in T_b occurring mainly at sunset and the daily decline occurring mainly at dawn.     

Effects of long-term captivity on thermoregulation,metabolism and ventilation of the southern brown bandicoot (Marsupialia: Peramelidae)

Thermoneutral metabolic and ventilatory parameters were measured every 3 months over 2 years for southern brown bandicoots held in captivity, and from a nearby reserve. Captive bandicoots were 130 g (9.9%) heavier than wild bandicoots. Long-term captivity had no effect on body temperature, basal metabolic rate (oxygen consumption), thermal conductance or respiratory ventilation, but there was an effect on carbon dioxide production, respiratory exchange ratio and total evaporative water loss (values were between 15 and 25% higher for captive than for wild bandicoots). Diet may be influencing these aspects of captive bandicoot physiology; the diet of captive bandicoots would be considerably different to that of wild bandicoots. Water availability seems to have a minimal effect. This study has important implications regarding physiological measurement for captive and wild mammals. For bandicoots at least, captive animals are equivalent to wild animals for some physiological parameters at thermoneutrality (body temperature, resting metabolic rate and thermal conductance), but not others.     

Biosynthesis and engineering of isoprenoid small molecules

Isoprenoid secondary metabolites are a rich source of commercial products that have not been fully explored. At present, there are isoprenoid products used in cancer therapy, the treatment of infectious diseases, and crop protection. All isoprenoids share universal prenyl diphosphate precursors synthesized via two distinct pathways. From these universal precursors, the biosynthetic pathways to specific isoprenoids diverge resulting in a staggering array of products. Taking advantage of this diversity has been the focus of much effort in metabolic engineering heterologous hosts. In addition, the engineering of the mevalonate pathway has increased levels of the universal precursors available for heterologous production. Finally, we will describe the efforts to produce to commercial terpenoids, paclitaxel and artemisinin.     

Observations of parasitoid behaviour in both no‐choice and choice tests are consistent with proposed ecological host range

The solitary larval endoparasitoid Eadya daenerys Ridenbaugh (Hymenoptera: Braconidae) is a proposed biocontrol agent of Paropsis charybdis Stål (Coleoptera: Chrysomelidae, Chrysomelinae), a pest of eucalypts in New Zealand. Eadya daenerys oviposition behaviour was examined in two assay types during host range testing, with the aim of improving ecological host range prediction. No‐choice sequential and two‐choice behavioural observations were undertaken against nine closely related species of New Zealand non‐target beetle larvae, including a native beetle, introduced weed biocontrol agents, and invasive paropsine beetles. No behavioural measure was significantly different between no‐choice and two‐choice tests. In sequential no‐choice assays the order of first presentation (target–non‐target) had no significant effect on the median number of attacks or the attack rate while on the plant. Beetle species was the most important factor. Parasitoids expressed significantly lower on‐plant attack rates against non‐targets compared to target P. charybdis larvae. The median number of attacks was always higher towards target larvae than towards non‐target larvae, except for the phylogenetically closest related non‐target Trachymela sloanei (Blackburn) (Coleoptera: Chrysomelidae, Chrysomelinae). Most non‐target larvae were disregarded upon contact, which suggests that the infrequent attack behaviour observed by two individual E. daenerys against Allocharis nr. tarsalis larvae in two‐choice tests and the frass of Chrysolina abchasica (Weise) was probably abnormal host selection behaviour. Results indicate that E. daenerys is unlikely to attack non‐target species apart from Eucalyptus‐feeding invasive paropsines (Chrysomelinae). Non‐lethal negative impacts upon less preferred non‐target larvae are possible if E. daenerys does attack them in the field; however, this is likely to be rare.     

Identification of Novel Genes Associated with Alginate Production in Pseudomonas aeruginosa Using Mini-himar1 Mariner Transposon-mediated Mutagenesis

Pseudomonas aeruginosa is a Gram-negative, environmental bacterium with versatile metabolic capabilities. P. aeruginosa is an opportunistic bacterial pathogen which establishes chronic pulmonary infections in patients with cystic fibrosis (CF). The overproduction of a capsular polysaccharide called alginate, also known as mucoidy, promotes the formation of mucoid biofilms which are more resistant than planktonic cells to antibiotic chemotherapy and host defenses. Additionally, the conversion from the nonmucoid to mucoid phenotype is a clinical marker for the onset of chronic infection in CF. Alginate overproduction by P. aeruginosa is an endergonic process which heavily taxes cellular energy. Therefore, alginate production is highly regulated in P. aeruginosa. To better understand alginate regulation, we describe a protocol using the mini-himar1 transposon mutagenesis for the identification of novel alginate regulators in a prototypic strain PAO1. The procedure consists of two basic steps. First, we transferred the mini-himar1 transposon (pFAC) from host E. coli SM10/λpir into recipient P. aeruginosa PAO1 via biparental conjugation to create a high-density insertion mutant library, which were selected on Pseudomonas isolation agar plates supplemented with gentamycin. Secondly, we screened and isolated the mucoid colonies to map the insertion site through inverse PCR using DNA primers pointing outward from the gentamycin cassette and DNA sequencing. Using this protocol, we have identified two novel alginate regulators, mucE (PA4033) and kinB (PA5484), in strain PAO1 with a wild-type mucA encoding the anti-sigma factor MucA for the master alginate regulator AlgU (AlgT, σ²²). This high-throughput mutagenesis protocol can be modified for the identification of other virulence-related genes causing change in colony morphology.