Psychometric properties of two implementation measures: Normalization MeAsure Development questionnaire (NoMAD) and organizational readiness for implementing change (ORIC).

Background: Effective interventions need to be implemented successfully to achieve impact. Two theory-based measures exist for measuring the effectiveness of implementation strategies and monitor implementation progress. The Normalization MeAsure Development questionnaire (NoMAD) explores the four core concepts (Coherence, Cognitive Participation, Collective Action, Reflexive Monitoring) of the Normalization Process Theory. The Organizational Readiness for Implementing Change (ORIC) is based on the theory of Organizational Readiness for Change, measuring organization members' psychological and behavioral preparedness for implementing a change. We examined the measurement properties of the NoMAD and ORIC in a multi-national implementation effectiveness study. Method: Twelve mental health organizations in nine countries implemented Internet-based cognitive behavioral therapy (iCBT) for common mental disorders. Staff involved in iCBT service delivery (n = 318) participated in the study. Both measures were translated into eight languages using a standardized forward-backward translation procedure. Correlations between measures and subscales were estimated to examine convergent validity. The theoretical factor structures of the scales were tested using confirmatory factor analysis (CFA). Test-retest reliability was based on the correlation between scores at two time points 3 months apart. Internal consistency was assessed using Cronbach's alpha. Floor and ceiling effects were quantified using the proportion of zero and maximum scores. Results: NoMAD and ORIC measure related but distinct latent constructs. The CFA showed that the use of a total score for each measure is appropriate. The theoretical subscales of the NoMAD had adequate internal consistency. The total scale had high internal consistency. The total ORIC scale and subscales demonstrated high internal consistency. Test-retest reliability was suboptimal for both measures and floor and ceiling effects were absent. Conclusions: This study confirmed the psychometric properties of the NoMAD and ORIC in multi-national mental health care settings. While measuring on different but related aspects of implementation processes, the NoMAD and ORIC prove to be valid and reliable across different language settings.

Why was the study done?: Effective interventions need to be implemented successfully to achieve impact. Reliable measurement instruments are needed to determine if an implementation was successful or not. Two theory-based instruments exist for measuring the effectiveness of implementation strategies and monitor progress. The NoMAD measures aspects of normalization related to sense-making, willingness to implement, the work people do, and reflection. The Organizational Readiness for Implementing Change (ORIC) measures organization members' preparedness for implementing a change. What did we do?: This study examined whether the NoMAD and ORIC measure what they are supposed to measure. We translated the instruments from English to eight languages (Albanian, Danish, Dutch, French, German, Italian, and Spanish/Catalan) We applied various statistical methods to confirm the measurement properties, including correlations of scales, factor structures, test­retest reliability, consistency and floor and ceiling effects. 318 mental health professionals from nine countries participated in the study. What did we find?: For both instruments, total scores can be used as well as the subscale scores. Internal consistency for ORIC was high and for NoMAD adequate. Test­retest reliability was demonstrated, and floor and ceiling effects were rare. What does this mean?: NoMAD and ORIC are reliable instruments for measuring implementation processes and outcomes across mental health care settings in different countries and languages. They measure related but different aspects of implementation processes and outcomes. The measures are brief, and theory supported. However, more work is to be done on interpreting scores in relation to implementation success and regarding changes over time.

Transition to a first suicide attempt among young and middle-aged males with a history of suicidal thoughts: A two-year cohort study.

INTRODUCTION: Although many studies have examined the risk and protective factors associated with suicidal behavior, little is known about the probability of transition from suicidal thoughts to suicidal attempts and the factors that distinguish those who have suicidal thoughts from those who progress to a suicide attempt. OBJECTIVES: To determine the probability and predictors of transition to a suicide attempt among young and middle-aged males with a history of suicidal thoughts but no prior history of attempting suicide. METHODS: We used data from the first two waves of the Australian Longitudinal Study on Male Health, approximately two years apart. We followed the cohort of males aged 18-55 years who, at wave 1, reported a lifetime history of suicidal ideation but no history of a prior suicide attempt. We report transition probabilities to a first suicide attempt at Wave 2 and used logistic regression models to examine baseline predictors of transition to a first suicide attempt over the two-year period among males aged 18 years and older. RESULTS: From the 1,564 males with suicidal thoughts at wave 1,140 participants (8.9%; 95% CI:7.6,10.5) reported to have had their first suicide attempt in the two-year period. In multivariate analyses, males aged 30-39 (OR=0.31; 95% CI: 0.16,0.60), 40-49 (OR=0.47; 95% CI:0.24,0.91) and 50-55 (OR=0.31; 95% CI: 0.13,0.73) all had lower odds of a first suicide attempt compared to males aged 18-29 years. The odds of a first suicide attempt were significantly higher for males who were: living in inner regional areas (ref: major cities) (OR=2.32; 95% CI: 1.33,4.04); homosexual or bisexual (OR=2.51; 95% CI: 1.17,5.36); working night shift as their main job (OR=1.75; 95% CI: 1.05,2.91); and, living with a disability (OR=1.99; 95% CI: 1.07,3.65). Clinical indicators such as symptoms of depression and illicit substance use were not significant predictors of transition to a first suicide attempt in multivariate models, nor were indicators of social connection. CONCLUSION: We estimated that 8.9% of Australian males aged 15-55 years with a history of suicidal thoughts and no prior history of suicide attempts will progress to a first suicide attempt within two-years. Neither psychological distress, illicit substance use nor social connection indicators were correlated with transition to a first suicide attempt. Rather, it was socio-demographic indicators that were associated with transition to a first suicide attempt.

Protocol for a randomised controlled trial evaluating the effect of a CBT-I smartphone application (Sleep Ninja®) on insomnia symptoms in children.

BACKGROUND: Sleep is necessary for healthy development and mental wellbeing. Despite this, many children do not get the recommended duration of sleep each night, and many experience sleep problems. Although treatable, existing interventions for sleep disturbance are time-consuming, burdensome for families, and focus on providing behavioural strategies to parents rather than upskilling children directly. To address this gap, we modified Sleep Ninja®, an evidence-based cognitive behavioural therapy for insomnia (CBT-I) smartphone app for adolescent sleep disturbance, to be appropriate for 10 to 12 year olds. Here, we describe the protocol for a randomised controlled trial to evaluate the effect of Sleep Ninja on insomnia and other outcomes, including depression, anxiety, sleep quality, and daytime sleepiness, and explore effects on the emergence of Major Depressive Disorder (MDD), compared to an active control group. METHODS: We aim to recruit 214 children aged 10 to 12 years old experiencing disturbed sleep. Participants will be screened for inclusion, complete the baseline assessment, and then be randomly allocated to receive Sleep Ninja, or digital psychoeducation flyers (active control) for 6-weeks. The primary outcome, insomnia symptoms, along with depression, anxiety, sleep quality, and daytime sleepiness will be assessed at 6-weeks (primary endpoint), 3-months, and 9-months post-baseline (secondary and tertiary endpoints, respectively). A mixed model repeated measures analytic approach will be used to conduct intention-to-treat analyses to determine whether reductions in insomnia and secondary outcomes are greater for those receiving Sleep Ninja relative to the control condition at the primary and secondary endpoints. The difference in relative risk for MDD onset will be explored at 9-months and compared between conditions. DISCUSSION: This is the first clinical trial examining the effects of a CBT-I smartphone app in children experiencing sleep disturbance. Results will provide empirical evidence about the effects of Sleep Ninja on insomnia and other mental health outcomes. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry (ACTRN12623000587606). UNIVERSAL TRIAL NUMBER: U1111-1294-4167.

Protocol for a randomised controlled trial evaluating the effectiveness of a CBT-based smartphone application for improving mental health outcomes in adolescents: the MobiliseMe study.

BACKGROUND: Depression is a leading cause of disability in adolescents, however few receive evidence-based treatment. Despite having the potential to overcome barriers to treatment uptake and adherence, there are very few CBT-based smartphone apps for adolescents. To address this gap, we developed ClearlyMe®, a self-guided CBT smartphone app for adolescent depression and anxiety. ClearlyMe® consists of 37 brief lessons containing core CBT elements, accessed either individually or as part of a 'collection'. Here, we describe the protocol for a randomised controlled trial aiming to evaluate the effect of ClearlyMe® on depressive symptoms and secondary outcomes, including engagement, anxiety and wellbeing, when delivered with and without guided support compared to an attention matched control. METHODS: We aim to recruit 489 adolescents aged 12-17 years with mild to moderately-severe depressive symptoms. Participants will be screened for inclusion, complete the baseline assessment and are then randomly allocated to receive ClearlyMe® (self-directed use), ClearlyMe® with guided SMS support (guided use) or digital psychoeducation (attention-matched control). Depressive symptoms and secondary outcomes will be assessed at 6-weeks (primary endpoint) and 4-months post-baseline (secondary endpoint). Engagement, conceptualised as uptake, adherence and completion, will also be assessed 6-weeks post-baseline. Mixed-effects linear modelling will be used to conduct intention-to-treat analyses to determine whether reductions in depressive symptoms and secondary outcomes are greater for conditions receiving ClearlyMe® relative to control at 6-weeks and 4-months post-baseline and greater for intervention adherers relative to non-adherers. To minimise risk, participants will be encouraged to use the Get Help section of the app and can also opt to receive a call from the team clinical psychologist at baseline, and at the 6-week and 4-month post-baseline assessments when reporting suicidal ideation. DISCUSSION: This is the first clinical trial examining a CBT smartphone app specifically designed for adolescent depression. It will provide empirical evidence on the effects of ClearlyMe® on depressive symptoms when used with and without guided support. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry (ACTRN12622000131752). UNIVERSAL TRIAL NUMBER: U1111-1271-8519.

Antidepressant use and its relationship with current symptoms in a population-based sample of older Australians.

BACKGROUND: Australia has the second highest per capita users of antidepressants globally, and their use is pronounced in older Australians. A better understanding of antidepressant use in older adults is important because the elderly are often prescribed multiple drugs, without review, for long periods. METHODS: This study analysed questionnaire data obtained from the Personality and Total Health through life project. Individual respondent data was linked to Pharmaceutical Benefits Scheme (PBS) records. Associations between self-reported medicine use and current symptoms with antidepressant dispensing were examined. RESULTS: 1275 participants aged over 65 were included in the final analysis. One hundred and forty-six (11.5%) participants were dispensed an antidepressant within the specified timeframe. Of those, 38.4% self-reported that they use medicine for depression, 12.3% for anxiety, 17.8% for both depression and anxiety, 6.2% for sleep problems and 3.4% for pain. One fifth of those dispensed an antidepressant did not self-report use of the medicine. Being female or reporting symptoms of depression, anxiety or suicidality were significant predictors of being on an antidepressant. Increasing pain severity was also associated with increased likelihood of being on an antidepressant. LIMITATIONS: We have presented a cross-sectional analysis, which can only provide associations between current symptoms and medicine use. We have only assessed respondents who received their scripts with PBS concession, which limits generalizability. CONCLUSION: Our analysis highlights the high use of antidepressants in the elderly for various reasons. Our findings also uncovered a high amount of under-reporting of antidepressant use by respondents.

A blended face-to-face and smartphone intervention for suicide prevention in the construction industry: protocol for a randomized controlled trial with MATES in Construction.

BACKGROUND: Construction workers are at elevated risk of suicide. MATES in Construction (MATES) is one of the few suicide prevention programs that explicitly address this problem. The MATES program includes an integrated system of services that supports prevention, early intervention and recovery (i.e., primary, secondary and tertiary prevention) for mental health problems among construction workers. In this protocol, we describe a proposed evaluation of MATESmobile, an electronic platform which will be accessed by workers who have undergone MATES training. METHODS/DESIGN: In this protocol, we describe a Randomised Controlled Trial (RCT) which seeks to assess whether MATESmobile results in improved literacy regarding suicide prevention, and improved help-seeking and help-offering attitudes among those who have attended MATES training. Secondary outcomes include changes in suicide ideation, suicide attempt and psychological distress. Workers will be recruited prior to MATES face-to-face training. In total, 295 workers will be randomly assigned to the intervention condition (MATESmobile + face-to-face training) and 295 will be randomly allocated to the control (face-to-face training). The intervention will run for 8 weeks. Assessments will be run immediately post intervention, and at 3, 6, and 12 months DISCUSSION: MATESmobile offers the potential to reinforce and enhance the effects of face-to-face training, resulting in greater skills and knowledge in suicide prevention, as well as a reduction in suicidality and distress. TRIAL REGISTRATION: This trial is registered with the Australian New Zealand Clinical Trials Registry ( ACTRN12619000625178 ; 26 April 2019).

Validity of the PROMIS depression and anxiety common metrics in an online sample of Australian adults.

PURPOSE: Recent US-based studies have utilised item response theory (IRT) to equate several self-report scales for depression and anxiety using the PROMIS depression and anxiety common metrics. The current study reports on the validity of the US-based equating procedures for the Patient Health Questionnaire-9 (PHQ-9), Generalized Anxiety Disorder-7 (GAD-7) and Kessler 6 psychological distress scale (K6) to equate scores in a large online sample of Australian adults. METHODS: Data comprised 3175 Australians recruited online. Each participant provided responses to the PROMIS depression and anxiety item banks, the PHQ-9, the GAD-7 and the K6. Two scoring methods were used to convert the scores on the PHQ-9, GAD-7 and K6 to the PROMIS depression and anxiety metrics. The converted scores were compared to the PROMIS depression and anxiety scores using intraclass correlations, mean difference, mean of absolute differences and Bland-Altman limits of agreement. RESULTS: Statistically significant mean differences were identified in five out of eight equated scores, albeit the effect sizes were small (Cohen's dz ≤ 0.25). The correlations were uniformly high (ICC ≥ 0.86). The mean of absolute differences between observed and equated scores for each metric and across scoring methods ranged between 4.23 and 5.33. CONCLUSIONS: The results demonstrate the validity of generating PROMIS depression and anxiety scores from the PHQ-9, GAD-7 and K6 in an independent sample of Australians. The agreement between equated scores provides some assurance that researchers and clinicians can utilise the converted PHQ-9, GAD-7 and K6 scores on the PROMIS metrics without a substantial decrease in accuracy and precision at the group level.

Assessing distress in the community: psychometric properties and crosswalk comparison of eight measures of psychological distress.

BACKGROUND: Many measures are available for measuring psychological distress in the community. Limited research has compared these scales to identify the best performing tools. A common metric for distress measures would enable researchers and clinicians to equate scores across different measures. The current study evaluated eight psychological distress scales and developed crosswalks (tables/figures presenting multiple scales on a common metric) to enable scores on these scales to be equated. METHODS: An Australian online adult sample (N = 3620, 80% female) was administered eight psychological distress measures: Patient Health Questionnaire-4, Kessler-10/Kessler-6, Distress Questionnaire-5 (DQ5), Mental Health Inventory-5, Hopkins Symptom Checklist-25 (HSCL-25), Self-Report Questionnaire-20 (SRQ-20) and Distress Thermometer. The performance of each measure in identifying DSM-5 criteria for a range of mental disorders was tested. Scale fit to a unidimensional latent construct was assessed using Confirmatory Factor Analysis (CFA). Finally, crosswalks were developed using Item Response Theory. RESULTS: The DQ5 had optimal performance in identifying individuals meeting DSM-5 criteria, with adequate fit to a unidimensional construct. The HSCL-25 and SRQ-20 also had adequate fit but poorer specificity and/or sensitivity than the DQ5 in identifying caseness. The unidimensional CFA of the combined item bank for the eight scales showed acceptable fit, enabling the creation of crosswalk tables. CONCLUSIONS: The DQ5 had optimal performance in identifying risk of mental health problems. The crosswalk tables developed in this study will enable rapid conversion between distress measures, providing more efficient means of data aggregation and a resource to facilitate interpretation of scores from multiple distress scales.

The impact of suicidality on health-related quality of life: A latent growth curve analysis of community-based data.

OBJECTIVE: The subjective burden of suicidality on mental and physical health-related quality of life (HRQoL) remains to be examined. Eight-year trajectories of mental and physical components of HRQoL were compared for suicidal and non-suicidal participants at baseline. The effect of poor mental and/or physical HRQoL on subsequent suicidality was also investigated. METHOD: Randomly-selected community data (W1=7485; W2=6715; W3=6133) were analysed with multivariate latent growth curve (LGC) and logistic regression models. RESULTS: Adjusted LGC modelling identified that baseline ideation was associated with poorer mental, but better physical HRQoL at baseline (b=-3.93, 95% CI=-4.75 to -3.12; b=1.38, 95% CI=0.53-2.23, respectively). However, ideation was associated with a declining physical HRQoL trajectory over 8 subsequent years (b=-0.88, 95% CI=-1.42 to -0.35). Poorer mental HRQoL was associated with higher odds of ideation onset (OR=0.98, 95% CI=0.96-0.99). LIMITATIONS: Frequency of data collection was four-yearly, while suicidality was reported for the previous 12-months; analyses did not control for physical health problems at baseline, baseline depression may have influenced physical QoL; suicidality was assessed with binary measures; and, prior analyses of attrition over time showed those with poorer health were less likely to continue participating in the study. CONCLUSIONS: Suicidality has differential longitudinal effects on mental and physical HRQoL. Findings emphasise the considerable subjective HRQoL burden upon suicidal individuals. HRQoL may be useful to compare relative social and economical impacts.

Predictors of personal, perceived and self-stigma towards anxiety and depression.

BACKGROUND: Stigma towards individuals experiencing a mental illness is associated with a range of negative psychological, social and financial outcomes. Factors associated with stigma remain unclear; the relationship between stigma and various personal factors may depend on both the type of disorder being stigmatised and what type of stigma is assessed. Different forms of stigma include personal stigma (negative attitudes towards others), perceived stigma (perceived attitudes of others) and self-stigma (self-attribution of others' negative attitudes). METHOD: Three hundred and fifty university students and members of the general public completed an online survey assessing contact with and knowledge of both depression and anxiety, age, gender, current depression and anxiety symptoms, and personal, perceived and self-stigma for both depression and anxiety. RESULTS: Greater contact with, and knowledge of that illness predicted lower personal stigma for both anxiety and depression. Participants with greater levels of current depression symptomatology and females, reported higher perceived stigma towards depression. Males reported higher personal stigma for anxiety. For both anxiety and depression, higher current symptomatology was associated with greater levels of self-stigma towards the illness. CONCLUSIONS: Findings confirm the role of contact and knowledge in personal stigma for both disorders, consistent with previous findings. This finding also supports evidence that interventions addressing these factors are associated with a decline in personal stigma. However, lack of relationship between contact with, and knowledge of a mental illness and perceived and self-stigma for either depression or anxiety suggests that these factors may not play a major role in perceived or self-stigma. The identification of symptomatology as a key factor associated with self-stigma for both anxiety and depression is significant, and has implications for community-wide interventions aiming to increase help-seeking behaviour, as well as individual treatment strategies for clinicians. Further research should examine whether these relationships hold for groups with clinically diagnosed depression and anxiety disorders.

Control of the sheep blowfly in Australia and New Zealand--are we there yet?

The last 50 years of research into infections in Australia and New Zealand caused by larvae of the sheep blowfly, Lucilia cuprina, have significantly advanced our understanding of this blowfly and its primary host, the sheep. However, apart from some highly effective drugs it could be argued that no new control methodologies have resulted. This review addresses the major areas of sheep blowfly research over this period describing the significant outcomes and analyses, and what is still required to produce new commercial control technologies. The use of drugs against this fly species has been very successful but resistance has developed to almost all current compounds. Integrated pest management is becoming basic to control, especially in the absence of mulesing, and has clearly benefited from computer-aided technologies. Biological control has more challenges but natural and perhaps transformed biopesticides offer possibilities for the future. Experimental vaccines have been developed but require further analysis of antigens and formulations to boost protection. Genetic technologies may provide potential for long-term control through more rapid indirect selection of sheep less prone to flystrike. Finally in the future, genetic analysis of the fly may allow suppression and perhaps eradication of blowfly populations or identification of new and more viable targets for drug and vaccine intervention. Clearly all these areas of research offer potential new controls but commercial development is perhaps inhibited by the success of current chemical insecticides and certainly requires a significant additional injection of resources.

Predictors and moderators of response to internet-delivered Interpersonal Psychotherapy and Cognitive Behavior Therapy for depression.

BACKGROUND: By identifying which predictors and moderators lead to beneficial outcomes, accurate selection of the best initial treatment will have significant benefits for depressed individuals. METHOD: An automated, fully self-guided randomized controlled internet-delivered noninferiority trial was conducted comparing two new interventions (Interpersonal Psychotherapy [IPT; n=620] and Cognitive Behavioral Therapy [CBT; n=610]) to an active control intervention (MoodGYM; n=613) over a period of 4 weeks to spontaneous visitors of an internet-delivered therapy website (e-couch). A range of putative predictors and moderators (socio-demographic characteristics [age, gender, marital status, education level], clinical characteristics [depression/anxiety symptoms, disability, quality of life, medication use], skills [mastery and dysfunctional attitudes] and treatment preference) were assessed using internet-delivered self-report measures at baseline and immediately following treatment and at six months follow-up. Analyses were conducted using Mixed Model Repeated Measures (MMRM). RESULTS: Female gender, lower mastery and lower dysfunctional attitudes predicted better outcome at post-test and/or follow-up regardless of intervention. No overall differential effects for condition on depression as a function of outcome were found. However, based on time-specific estimates, a significant interaction effect of age was found. For younger people, internet-delivered IPT may be the preferred treatment choice, whereas older participants derive more benefits from internet-delivered CBT programs. LIMITATIONS: Although the sample of participants was large, power to detect moderator effects was still lacking. CONCLUSIONS: Different e-mental health programs may be more beneficial for specific age groups. The findings raise important possibilities for increasing depression treatment effectiveness and improving clinical practice guidelines for depression treatment of different age groups.

Exon-primed intron-crossing (EPIC) PCR markers of Helicoverpa armigera (Lepidoptera: Noctuidae).

Applying microsatellite DNA markers in population genetic studies of the pest moth Helicoverpa armigera is subject to numerous technical problems, such as the high frequency of null alleles, occurrence of size homoplasy, presence of multiple copies of flanking sequence in the genome and the lack of PCR amplification robustness between populations. To overcome these difficulties, we developed exon-primed intron-crossing (EPIC) nuclear DNA markers for H. armigera based on ribosomal protein (Rp) and the Dopa Decarboxylase (DDC) genes and sequenced alleles showing length polymorphisms. Allele length polymorphisms were usually from random indels (insertions or deletions) within introns, although variation of short dinucleotide DNA repeat units was also detected. Mapping crosses demonstrated Mendelian inheritance patterns for these EPIC markers and the absence of both null alleles and allele 'dropouts'. Three examples of allele size homoplasies due to indels were detected in EPIC markers RpL3, RpS6 and DDC, while sequencing of multiple individuals across 11 randomly selected alleles did not detect indel size homoplasies. The robustness of the EPIC-PCR markers was demonstrated by PCR amplification in the related species, H. zea, H. assulta and H. punctigera.

Molecular markers to discriminate among four pest species of Helicoverpa (Lepidoptera: Noctuidae).

The four significant pest species in the Helicoverpa genus (H. armigera, H. assulta, H. punctigera and H. zea) are morphologically similar and can only be reliably distinguished through dissection of adult genitalia. Two partial regions of the mitochondrial DNA (mtDNA), the cytochrome oxidase subunit I (COI) and the cytochrome b (Cyt b) genes were amplified by PCR and digested with restriction endonucleases. The restriction patterns, generated by the endonucleases BstZ17I and HphI, demonstrated reliable differentiation of the four Helicoverpa pest species. This technique is fast, reliable and effective at distinguishing specimens irrespective of their life stages and offers support to conventional taxonomic differentiation based on morphological characters.

Nucleotide diversity in the Hsp90 gene in natural populations of Drosophila melanogaster from Australia.

Hsp90 is regarded as one of the best candidates for an evolved mechanism that regulates the expression of genetic and phenotypic variability. We examined nucleotide diversity in both the promoter and coding regions of Hsp90, the gene which encodes Hsp90 in Drosophila, in natural populations of Drosophila melanogaster from eastern Australia. We found that Hsp90 is polymorphic for only two nonsynonymous changes in the coding region, both of which are deletions of a lysine residue. One of these lysine deletions was in complete linkage disequilibrium with the inversion In(3L)P, and showed a significant association with latitude. The other lysine deletion reported here for the first time varied from 0 to 15% in natural populations, but did not show a clinal pattern. The regulatory and coding regions of Hsp90 showed very low nucleotide diversity compared to other nuclear genes, and chromosomes containing In(3L)P had lower levels of nucleotide diversity than the standard arrangements. Non-neutral evolution of Hsp90 was not supported by analyses of either the regulatory or coding regions of the gene. These results are discussed within the context of Hsp90 variation being involved in thermotolerance as well as the expression of genetic and phenotypic variability.

Positional cloning of a cyromazine resistance gene in Drosophila melanogaster.

Cyromazine is an effective insecticide used to control dipteran insects. Its precise mode of action is yet to be determined, although it has been suggested that it interferes with the hormone system, sclerotization of the cuticle, or nucleic acid metabolism. To understand the way in which cyromazine acts, we have positionally cloned a cyromazine resistance gene from Drosophila melanogaster. Six cyromazine resistance alleles had previously been generated by ethyl methanasulphonate treatment. Two of these failed to complement each other and here we identify them as having independent non-sense mutations in CG32743, which is an ortholog of Smg1 of worms and mammals and encodes a phosphatidylinositol kinase-like kinase (PIKK). RNAi experiments confirm that cyromazine resistance can be achieved by knocking down CG32743. These are the first cyromazine resistant mutations identified at the nucleotide level. In mammals Smg1 phosphorylates P53 in response to DNA damage. This finding supports the hypothesis that cyromazine interferes with nucleic acid metabolism.

Dissecting chill coma recovery as a measure of cold resistance: evidence for a biphasic response in Drosophila melanogaster.

Cold resistance in insects has traditionally been measured in terms of survival following a stress, but alternative methods are increasingly being used because of their relevance to the ecology of organisms and their utility in characterizing variation among species, populations and individuals. One such method capable of discriminating among Drosophila species and conspecific Drosophila populations from different environments is adult chill coma recovery time, the time taken for adults to become active again after being knocked down by a cold stress. Here we characterized the chill coma response of D. melanogaster in detail. Adults were exposed to a range of temperatures and stressful periods prior to measuring recovery. Recovery from chill coma in D. melanogaster was biphasic; as flies were stressed under cooler temperatures, recovery times leveled off and then decreased before sharply increasing again as mortality starts to occur. This biphasic response has previously been observed in D. subobscura where it has a somewhat different shape. A second mechanism therefore acts at relatively lower temperatures to ameliorate the effects of the cold stress. When D. melanogaster were reared at 19 and 25 degrees C for two generations, the shape of the curve relating temperature to recovery time was similar, but flies from the warmer temperature had longer recovery times and showed responses that leveled off and then decreased at relatively higher temperatures. As exposure time to cold stress was increased, recovery times also increased except at mild stress levels. Chill coma recovery in D. melanogaster is a complex trait and likely to reflect multiple underlying components.

A single p450 allele associated with insecticide resistance in Drosophila.

Insecticide resistance is one of the most widespread genetic changes caused by human activity, but we still understand little about the origins and spread of resistant alleles in global populations of insects. Here, via microarray analysis of all P450s in Drosophila melanogaster, we show that DDT-R, a gene conferring resistance to DDT, is associated with overtranscription of a single cytochrome P450 gene, Cyp6g1. Transgenic analysis of Cyp6g1 shows that overtranscription of this gene alone is both necessary and sufficient for resistance. Resistance and up-regulation in Drosophila populations are associated with a single Cyp6g1 allele that has spread globally. This allele is characterized by the insertion of an Accord transposable element into the 5' end of the Cyp6g1 gene.

The acetylcholinesterase gene and organophosphorus resistance in the Australian sheep blowfly, Lucilia cuprina.

Acetylcholinesterase (AChE), encoded by the Ace gene, is the primary target of organophosphorous (OP) and carbamate insecticides. Ace mutations have been identified in OP resistants strains of Drosophila melanogaster. However, in the Australian sheep blowfly, Lucilia cuprina, resistance in field and laboratory generated strains is determined by point mutations in the Rop-1 gene, which encodes a carboxylesterase, E3. To investigate the apparent bias for the Rop-1/E3 mechanism in the evolution of OP resistance in L. cuprina, we have cloned the Ace gene from this species and characterized its product. Southern hybridization indicates the existence of a single Ace gene in L. cuprina. The amino acid sequence of L. cuprina AChE shares 85.3% identity with D. melanogaster and 92.4% with Musca domestica AChE. Five point mutations in Ace associated with reduced sensitivity to OP insecticides have been previously detected in resistant strains of D. melanogaster. These residues are identical in susceptible strains of D. melanogaster and L. cuprina, although different codons are used. Each of the amino acid substitutions that confer OP resistance in D. melanogaster could also occur in L. cuprina by a single non-synonymous substitution. These data suggest that the resistance mechanism used in L. cuprina is determined by factors other than codon bias. The same point mutations, singly and in combination, were introduced into the Ace gene of L. cuprina by site-directed mutagenesis and the resulting AChE enzymes expressed using a baculovirus system to characterise their kinetic properties and interactions with OP insecticides. The K(m) of wild type AChE for acetylthiocholine (ASCh) is 23.13 microM and the point mutations change the affinity to the substrate. The turnover number of Lucilia AChE for ASCh was estimated to be 1.27x10(3) min(-1), similar to Drosophila or housefly AChE. The single amino acid replacements reduce the affinities of the AChE for OPs and give up to 8.7-fold OP insensitivity, while combined mutations give up to 35-fold insensitivity. However, other published studies indicate these same mutations yield higher levels of OP insensitivity in D. melanogaster and A. aegypti. The inhibition data indicate that the wild type form of AChE of L. cuprina is 12.4-fold less sensitive to OP inhibition than the susceptible form of E3, suggesting that the carboxylesterases may have a role in the protection of AChE via a sequestration mechanism. This provides a possible explanation for the bias towards the evolution of resistance via the Rop-1/E3 mechanism in L. cuprina.

A genetic analysis of cyromazine resistance in Drosophila melanogaster (Diptera: Drosophilidae).

Flies resistant to the insect growth regulator cyromazine were selected in the F1 generation from a cyromazine-susceptible strain of Drosophila melanogaster (Meigen) treated with ethyl methanesulfonate. Four resistant strains were isolated by screening with cyromazine at a concentration > LC100 of susceptibles. In each strain, resistance is conferred by a single gene mutation. Cyromazine resistance in two of the mutants (rst(1a)cyr1 and rst(1a)cyr2) localizes to map position 17 of the X chromosome. Evidence is presented that these mutations are alleles of the gene rst(1a)cyr. Cyromazine resistance in another of the mutants (Rst(1b)Cyr) is also X-linked, and localizes to map position 49 of the X chromosome. The location of the gene conferring cyromazine resistance in the other mutant (Rst(2b)Cyr) is map position 66 of chromosome II. This is possibly an allele of a previously characterized cyromazine resistance gene, Rst(2)Cyr. Dosage-mortality analyses demonstrate a low level of cyromazine resistance is conferred in all strains.