Phase variation as a major mechanism of adaptation in Mycobacterium tuberculosis complex.

Phase variation induced by insertions and deletions (INDELs) in genomic homopolymeric tracts (HT) can silence and regulate genes in pathogenic bacteria, but this process is not characterized in MTBC (Mycobacterium tuberculosis complex) adaptation. We leverage 31,428 diverse clinical isolates to identify genomic regions including phase-variants under positive selection. Of 87,651 INDEL events that emerge repeatedly across the phylogeny, 12.4% are phase-variants within HTs (0.02% of the genome by length). We estimated the in-vitro frameshift rate in a neutral HT at 100× the neutral substitution rate at [Formula: see text] frameshifts/HT/year. Using neutral evolution simulations, we identified 4,098 substitutions and 45 phase-variants to be putatively adaptive to MTBC (P < 0.002). We experimentally confirm that a putatively adaptive phase-variant alters the expression of espA, a critical mediator of ESX-1-dependent virulence. Our evidence supports the hypothesis that phase variation in the ESX-1 system of MTBC can act as a toggle between antigenicity and survival in the host.

Analysis of Genome-Wide Mutational Dependence in Naturally Evolving Mycobacterium tuberculosis Populations.

Pathogenic microorganisms are in a perpetual struggle for survival in changing host environments, where host pressures necessitate changes in pathogen virulence, antibiotic resistance, or transmissibility. The genetic basis of phenotypic adaptation by pathogens is difficult to study in vivo. In this work, we develop a phylogenetic method to detect genetic dependencies that promote pathogen adaptation using 31,428 in vivo sampled Mycobacterium tuberculosis genomes, a globally prevalent bacterial pathogen with increasing levels of antibiotic resistance. We find that dependencies between mutations are enriched in antigenic and antibiotic resistance functions and discover 23 mutations that potentiate the development of antibiotic resistance. Between 11% and 92% of resistant strains harbor a dependent mutation acquired after a resistance-conferring variant. We demonstrate the pervasiveness of genetic dependency in adaptation of naturally evolving populations and the utility of the proposed computational approach.

BioPlexR and BioPlexPy: integrated data products for the analysis of human protein interactions.

SUMMARY: The BioPlex project has created two proteome scale, cell-line-specific protein-protein interaction (PPI) networks: the first in 293T cells, including 120k interactions among 15k proteins; and the second in HCT116 cells, including 70k interactions between 10k proteins. Here, we describe programmatic access to the BioPlex PPI networks and integration with related resources from within R and Python. Besides PPI networks for 293T and HCT116 cells, this includes access to CORUM protein complex data, PFAM protein domain data, PDB protein structures, and transcriptome and proteome data for the two cell lines. The implemented functionality serves as a basis for integrative downstream analysis of BioPlex PPI data with domain-specific R and Python packages, including efficient execution of maximum scoring sub-network analysis, protein domain-domain association analysis, mapping of PPIs onto 3D protein structures and analysis of BioPlex PPIs at the interface of transcriptomic and proteomic data. AVAILABILITY AND IMPLEMENTATION: The BioPlex R package is available from Bioconductor (bioconductor.org/packages/BioPlex), and the BioPlex Python package is available from PyPI (pypi.org/project/bioplexpy). Applications and downstream analyses are available from GitHub (github.com/ccb-hms/BioPlexAnalysis).

Gene signature discovery and systematic validation across diverse clinical cohorts for TB prognosis and response to treatment.

While blood gene signatures have shown promise in tuberculosis (TB) diagnosis and treatment monitoring, most signatures derived from a single cohort may be insufficient to capture TB heterogeneity in populations and individuals. Here we report a new generalized approach combining a network-based meta-analysis with machine-learning modeling to leverage the power of heterogeneity among studies. The transcriptome datasets from 57 studies (37 TB and 20 viral infections) across demographics and TB disease states were used for gene signature discovery and model training and validation. The network-based meta-analysis identified a common 45-gene signature specific to active TB disease across studies. Two optimized random forest regression models, using the full or partial 45-gene signature, were then established to model the continuum from Mycobacterium tuberculosis infection to disease and treatment response. In model validation, using pooled multi-cohort datasets to mimic the real-world setting, the model provides robust predictive performance for incipient to active TB risk over a 2.5-year period with an AUROC of 0.85, 74.2% sensitivity, and 78.3% specificity, which approximates the minimum criteria (>75% sensitivity and >75% specificity) within the WHO target product profile for prediction of progression to TB. Moreover, the model strongly discriminates active TB from viral infection (AUROC 0.93, 95% CI 0.91-0.94). For treatment monitoring, the TB scores generated by the model statistically correlate with treatment responses over time and were predictive, even before treatment initiation, of standard treatment clinical outcomes. We demonstrate an end-to-end gene signature model development scheme that considers heterogeneity for TB risk estimation and treatment monitoring.

Benchmarking the empirical accuracy of short-read sequencing across the M. tuberculosis genome.

MOTIVATION: Short-read whole-genome sequencing (WGS) is a vital tool for clinical applications and basic research. Genetic divergence from the reference genome, repetitive sequences and sequencing bias reduces the performance of variant calling using short-read alignment, but the loss in recall and specificity has not been adequately characterized. To benchmark short-read variant calling, we used 36 diverse clinical Mycobacterium tuberculosis (Mtb) isolates dually sequenced with Illumina short-reads and PacBio long-reads. We systematically studied the short-read variant calling accuracy and the influence of sequence uniqueness, reference bias and GC content. RESULTS: Reference-based Illumina variant calling demonstrated a maximum recall of 89.0% and minimum precision of 98.5% across parameters evaluated. The approach that maximized variant recall while still maintaining high precision (<99%) was tuning the mapping quality filtering threshold, i.e. confidence of the read mapping (recall = 85.8%, precision = 99.1%, MQ ≥ 40). Additional masking of repetitive sequence content is an alternative conservative approach to variant calling that increases precision at cost to recall (recall = 70.2%, precision = 99.6%, MQ ≥ 40). Of the genomic positions typically excluded for Mtb, 68% are accurately called using Illumina WGS including 52/168 PE/PPE genes (34.5%). From these results, we present a refined list of low confidence regions across the Mtb genome, which we found to frequently overlap with regions with structural variation, low sequence uniqueness and low sequencing coverage. Our benchmarking results have broad implications for the use of WGS in the study of Mtb biology, inference of transmission in public health surveillance systems and more generally for WGS applications in other organisms. AVAILABILITY AND IMPLEMENTATION: All relevant code is available at https://github.com/farhat-lab/mtb-illumina-wgs-evaluation. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Role of Epistasis in Amikacin, Kanamycin, Bedaquiline, and Clofazimine Resistance in Mycobacterium tuberculosis Complex.

Antibiotic resistance among bacterial pathogens poses a major global health threat. Mycobacterium tuberculosis complex (MTBC) is estimated to have the highest resistance rates of any pathogen globally. Given the low growth rate and the need for a biosafety level 3 laboratory, the only realistic avenue to scale up drug susceptibility testing (DST) for this pathogen is to rely on genotypic techniques. This raises the fundamental question of whether a mutation is a reliable surrogate for phenotypic resistance or whether the presence of a second mutation can completely counteract its effect, resulting in major diagnostic errors (i.e., systematic false resistance results). To date, such epistatic interactions have only been reported for streptomycin that is now rarely used. By analyzing more than 31,000 MTBC genomes, we demonstrated that the eis C-14T promoter mutation, which is interrogated by several genotypic DST assays endorsed by the World Health Organization, cannot confer resistance to amikacin and kanamycin if it coincides with loss-of-function (LoF) mutations in the coding region of eis. To our knowledge, this represents the first definitive example of antibiotic reversion in MTBC. Moreover, we raise the possibility that mmpR (Rv0678) mutations are not valid markers of resistance to bedaquiline and clofazimine if these coincide with an LoF mutation in the efflux pump encoded by mmpS5 (Rv0677c) and mmpL5 (Rv0676c).

A cascade of destabilizations: Combining Wolbachia and Allee effects to eradicate insect pests.

The management of insect pests has long been dominated by the use of chemical insecticides, with the aim of instantaneously killing enough individuals to limit their damage. To minimize unwanted consequences, environmentally friendly approaches have been proposed that utilize biological control and take advantage of intrinsic demographic processes to reduce pest populations. We address the feasibility of a novel pest management strategy based on the release of insects infected with Wolbachia, which causes cytoplasmic incompatibilities in its host population, into a population with a pre-existing Allee effect. We hypothesize that the transient decline in population size caused by a successful invasion of Wolbachia can bring the population below its Allee threshold and, consequently, trigger extinction. We develop a stochastic population model that accounts for Wolbachia-induced cytoplasmic incompatibilities in addition to an Allee effect arising from mating failures at low population densities. Using our model, we identify conditions under which cytoplasmic incompatibilities and Allee effects successfully interact to drive insect pest populations towards extinction. Based on our results, we delineate control strategies based on introductions of Wolbachia-infected insects. We extend this analysis to evaluate control strategies that implement successive introductions of two incompatible Wolbachia strains. Additionally, we consider methods that combine Wolbachia invasion with mating disruption tactics to enhance the pre-existing Allee effect. We demonstrate that Wolbachia-induced cytoplasmic incompatibility and the Allee effect act independently from one another: the Allee effect does not modify the Wolbachia invasion threshold, and cytoplasmic incompatibilities only have a marginal effect on the Allee threshold. However, the interaction of these two processes can drive even large populations to extinction. The success of this method can be amplified by the introduction of multiple Wolbachia cytotypes as well as the addition of mating disruption. Our study extends the existing literature by proposing the use of Wolbachia introductions to capitalize on pre-existing Allee effects and consequently eradicate insect pests. More generally, it highlights the importance of transient dynamics, and the relevance of manipulating a cascade of destabilizatons for pest management.

Captures of Wild Ceratitis capitata, Bactrocera dorsalis, and Bactrocera cucurbitae (Diptera: Tephritidae) in Traps with Improved Multilure TMR Dispensers Weathered in California.

During 2012­2013, solid Mallet TMR (trimedlure [TML], methyl eugenol [ME], raspberry ketone [RK]) wafers impregnated with DDVP (2, 2-dichlorovinyl dimethyl phosphate) insecticide were weathered during summer (8 wk) and winter (12 wk) in five California citrus-growing counties (Kern, Ventura, Orange, Tulare, and Riverside). In addition, TMR wafers without DDVP and with a Hercon Vaportape II insecticidal strip were compared with TMR dispensers with DDVP at Exeter and Riverside. Weathered treatments were shipped every week (overnight delivery) to Hawaii and frozen for a later bioassay in a 1,335-ha coffee plantation near Numila, Kauai Island, HI, where Mediterranean fruit fly, Ceratitis capitata (Wiedemann), oriental fruit fly, Bactrocera dorsalis Hendel, and melon fly, Bactrocera cucurbitae Coquillett, were all present. We compared trap captures of the three species, C. capitata, B. dorsalis, and B. cucurbitae, for the five different weathering locations. Captures of C. capitata, B. dorsalis, and B. cucurbitae with Mallet TMR dispensers (with DDVP) were not significantly different for the five locations. Captures with the Mallet TMR dispenser without DDVP and Vaportape were similar to those for Mallet TMR with DDVP, although there were some slight location differences. In conclusion, based on these results, the Mallet TMR dispenser could potentially be used in California habitats where large numbers of detection traps are currently deployed. Use of Vaportape with dispensers would not require them to be registered with US Environmental Protection Agency (EPA). Dispensers for use as Male Annihilation Technique (MAT) devices will be tested further in Hawaii.

Ammonium Acetate Enhances the Attractiveness of a Variety of Protein-Based Baits to Female Ceratitis capitata (Diptera: Tephritidae).

Ammonia and its derivatives are used by female fruit flies (Diptera: Tephritidae) as volatile cues to locate protein-rich food needed to produce their eggs. This need for external protein sources has led to the development of behaviorally based control strategies such as food-based lures and insecticidal baits targeting pestiferous fruit fly species. In field cage studies conducted in Hawaii, we examined the behavioral response of laboratory-reared male and female Mediterranean fruit fly, Ceratitis capitata (Wiedemann), to seven commercially available protein baits and to beer waste, a relatively inexpensive and readily available substance. Each material was tested alone or in combination with either ammonium acetate or ammonium carbonate. For the majority of baits evaluated, the presence of ammonium acetate, but not ammonium carbonate, elicited a significantly greater level of response of female C. capitata compared with the protein baits alone. The addition of ammonium acetate to selected baits increased bait attractiveness to a level comparable with that elicited by the most widely used spinosad-based protein bait, GF-120. Our findings indicate that the addition of ammonium acetate to commercially available proteinaceous baits and to beer waste can greatly improve their attractiveness to C. capitata, potentially increasing the bait's effectiveness for fruit fly monitoring and suppression.

Weathering and Chemical Degradation of Methyl Eugenol and Raspberry Ketone Solid Dispensers for Detection, Monitoring, and Male Annihilation of Bactrocera dorsalis and Bactrocera cucurbitae (Diptera: Tephritidae) in Hawaii.

Solid male lure dispensers containing methyl eugenol (ME) and raspberry ketone (RK), or mixtures of the lures (ME + RK), and dimethyl dichloro-vinyl phosphate (DDVP) were evaluated in area-wide pest management bucket or Jackson traps in commercial papaya (Carica papaya L.) orchards where both oriental fruit fly, Bactrocera dorsalis (Hendel), and melon fly, Bactrocera cucurbitae (Coquillett), are pests. Captures of B. dorsalis with fresh wafers in Jackson and bucket traps were significantly higher on the basis of ME concentration (Mallet ME [56%] > Mallet MR [31.2%] > Mallet MC [23.1%]). Captures of B. cucurbitae with fresh wafers in Jackson and bucket traps were not different regardless of concentration of RK (Mallet BR [20.1%] = Mallet MR [18.3%] = Mallet MC [15.9%]). Captures of B. dorsalis with fresh wafers, compared with weathered wafers, were significantly different after week 12; captures of B. cucurbitae were not significantly different after 16 wk. Chemical analyses revealed presence of RK in dispensers in constant amounts throughout the 16-wk trial. Degradation of both ME and DDVP over time was predicted with a high level of confidence by nonlinear asymptotic exponential decay curves. Results provide supportive data to deploy solid ME and RK wafers (with DDVP) in fruit fly traps for detection programs, as is the current practice with solid TML dispensers placed in Jackson traps. Wafers with ME and RK might be used in place of two separate traps for detection of both ME and RK responding fruit flies and could potentially reduce cost of materials and labor by 50%.

Deconstructing the surrogate species concept: a life history approach to the protection of ecosystem services.

The use of the surrogate species concept is widespread in environmental risk assessment and in efforts to protect species that provide ecosystem services, yet there are no standard protocols for the choice of surrogates. Surrogates are often chosen on the basis of convenience or vague resemblances in physiology or life history to species of concern. Furthermore, our ability to predict how species of concern will fare when subjected to disturbances such as environmental contaminants or toxicants is often based on woefully misleading comparisons of static toxicity tests. Here we present an alternative approach that features a simple mathematical model parameterized with life history data applied to an assemblage of species that provide an important ecosystem service: a suite of parasitoid wasps that provide biological control of agricultural pests. Our results indicate that these parasitoid wasp species have different population responses to toxic insult--that is, we cannot predict how all four species will react to pesticide exposure simply by extrapolating from the response of any one species. Furthermore, sensitivity analysis of survivorship and reproduction demonstrates that the life stage most sensitive to pesticide disturbance varies among species. Taken together, our results suggest that the ability to predict the fate of a suite of species using the response of just one species (the surrogate species concept) is widely variable and potentially misleading.

Attraction and mortality of Bactrocera dorsalis (Diptera: Tephritidae) to STATIC Spinosad ME weathered under operational conditions in California and Florida: a reduced-risk male annihilation treatment.

Studies were conducted in 2013-2014 to quantify attraction, feeding, and mortality of male oriental fruit flies, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), to STATIC Spinosad ME a reduced-risk male annihilation treatment (MAT) formulation consisting of an amorphous polymer matrix in combination with methyl eugenol (ME) and spinosad compared with the standard treatment of Min-U-Gel mixed with ME and naled (Dibrom). Our approach used a behavioral methodology for evaluation of slow-acting reduced-risk insecticides. ME treatments were weathered for 1, 7, 14, 21, and 28 d under operational conditions in California and Florida and shipped to Hawaii for bioassays. In field tests using bucket traps to attract and capture wild males, and in toxicity studies conducted in 1-m(3) cages using released males of controlled ages, STATIC Spinosad ME performed equally as well to the standard formulation of Min-U-Gel ME with naled for material aged up to 28 d in both California and Florida. In laboratory feeding tests in which individual males were exposed for 5 min to the different ME treatments, mortality induced by STATIC Spinosad ME recorded at 24 h did not differ from mortality caused by Min-U-Gel ME with naled at 1, 7, 14, and 21 d in California and was equal to or higher for all weathered time periods in Florida during two trials. Spinosad has low contact toxicity, and when mixed with an attractant and slow release matrix, offers a reduced-risk alternative for eradication of B. dorsalis and related ME attracted species, without many of the potential negative effects to humans and nontargets associated with broad-spectrum contact insecticides such as naled.

Understanding the movement and dispersal patterns of released Fopius arisanus (Hymenoptera: Braconidae) parasitoids in a papaya orchard.

Implementation of augmentative biological control requires estimates of parasitoid dispersal from the release point to determine appropriate release density, spacing, and timing. This study evaluated the movement patterns of Fopius arisanus Sonan (Hymenoptera: Braconidae) parasitoids, which have historically been used to control invasive tephritid fruit flies. The wasps were released from the central point, and dispersal was monitored over time using parasitism in sentinel fruit and trap captures at 40 points radiating out from the center (15-240 m). The releases were conducted 4 times during June, July, September, and November 2006. The data showed that there were large declines in dispersal by distance. Parasitism was greatest closest to the release point, within 30 m. Parasitism was also greatest within the first 24 h of the release. After 1 wk, parasitism decreased from 41% to 1.5% within 30 m. These data correlated strongly with trap capture data, which also showed that parasitoid movement favored the SE region of our release site, roughly corresponding to the overall prevailing winds. Wind speed, relative humidity, and temperature all affected parasitoid movement during our trial, indicating the complex environmental factors that can affect release success. This is the first report of dispersal metrics for F. arisanus. Our findings are in agreement with other similar studies on braconid movement generally and suggest that frequent, high-density releases are most effective since the dispersal of F. arisanus is limited and retention in the environment is low. We discuss our results in the context of international augmentative biological control release programs.

Single nucleotide variation catalog from clinical isolates mapped on tertiary and quaternary structures of ESX-1-related proteins reveals critical regions as putative Mtb therapeutic targets.

Proteins encoded by the ESX-1 genes of interest are essential for full virulence in all Mycobacterium tuberculosis complex (Mtbc) lineages, the pathogens causing the highest mortality worldwide. Identifying critical regions in these ESX-1-related proteins could provide preventive or therapeutic targets for Mtb infection, the game changer needed for tuberculosis control. We analyzed a compendium of whole genome sequences of clinical Mtb isolates from all lineages from >32,000 patients and identified single nucleotide polymorphisms. When mutations corresponding to all non-synonymous single nucleotide polymorphisms were mapped on structural models of the ESX-1 proteins, fully conserved regions emerged. Some could be assigned to known quaternary structures, whereas others could be predicted to be involved in yet-to-be-discovered interactions. Some mutants had clonally expanded (found in >1% of the isolates); these mutants were mostly located at the surface of globular domains, remote from known intra- and inter-molecular protein-protein interactions. Fully conserved intrinsically disordered regions of proteins were found, suggesting that these regions are crucial for the pathogenicity of the Mtbc. Altogether, our findings highlight fully conserved regions of proteins as attractive vaccine antigens and drug targets to control Mtb virulence. Extending this approach to the whole Mtb genome as well as other microorganisms will enhance vaccine development for various pathogens. IMPORTANCE: We mapped all non-synonymous single nucleotide polymorphisms onto each of the experimental and predicted ESX-1 proteins' structural models and inspected their placement. Varying sizes of conserved regions were found. Next, we analyzed predicted intrinsically disordered regions within our set of proteins, finding two putative long stretches that are fully conserved, and discussed their potential essential role in immunological recognition. Combined, our findings highlight new targets for interfering with Mycobacterium tuberculosis complex virulence.

Estimation of country-specific tuberculosis resistance antibiograms using pathogen genomics and machine learning.

BACKGROUND: Global tuberculosis (TB) drug resistance (DR) surveillance focuses on rifampicin. We examined the potential of public and surveillance Mycobacterium tuberculosis (Mtb) whole-genome sequencing (WGS) data, to generate expanded country-level resistance prevalence estimates (antibiograms) using in silico resistance prediction. METHODS: We curated and quality-controlled Mtb WGS data. We used a validated random forest model to predict phenotypic resistance to 12 drugs and bias-corrected for model performance, outbreak sampling and rifampicin resistance oversampling. Validation leveraged a national DR survey conducted in South Africa. RESULTS: Mtb isolates from 29 countries (n=19 149) met sequence quality criteria. Global marginal genotypic resistance among mono-resistant TB estimates overlapped with the South African DR survey, except for isoniazid, ethionamide and second-line injectables, which were underestimated (n=3134). Among multidrug resistant (MDR) TB (n=268), estimates overlapped for the fluoroquinolones but overestimated other drugs. Globally pooled mono-resistance to isoniazid was 10.9% (95% CI: 10.2-11.7%, n=14 012). Mono-levofloxacin resistance rates were highest in South Asia (Pakistan 3.4% (0.1-11%), n=111 and India 2.8% (0.08-9.4%), n=114). Given the recent interest in drugs enhancing ethionamide activity and their expected activity against isolates with resistance discordance between isoniazid and ethionamide, we measured this rate and found it to be high at 74.4% (IQR: 64.5-79.7%) of isoniazid-resistant isolates predicted to be ethionamide susceptible. The global susceptibility rate to pyrazinamide and levofloxacin among MDR was 15.1% (95% CI: 10.2-19.9%, n=3964). CONCLUSIONS: This is the first attempt at global Mtb antibiogram estimation. DR prevalence in Mtb can be reliably estimated using public WGS and phenotypic resistance prediction for key antibiotics, but public WGS data demonstrates oversampling of isolates with higher resistance levels than MDR. Nevertheless, our results raise concerns about the empiric use of short-course fluoroquinolone regimens for drug-susceptible TB in South Asia and indicate underutilisation of ethionamide in MDR treatment.

Leg impairment magnifies reproductive costs in male Mediterranean fruit flies, Ceratitis capitata.

Injuries frequently accumulate with age in nature. Despite the commonality of injury and the resulting impairment, there are limited experimental data for the effects of impairment on life history trade-offs between reproduction and survival in insects. We tested the effects of artificial injury and the resulting impairment on the reproductive costs and behavior of male medflies, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae). Treatment flies were impaired by amputating tarsomere segments 2-5 from the right foreleg at either eclosion or age 22 days. The effect of impairment and age on the cost of reproduction was tested by varying the timing of female availability among the treatments. Courtship behavior and copulation rates were observed hourly from age 2-5 days to determine the effects of impairment on reproductive behavior. Female access combined with the impairment reduced the life expectancy of males more than the impairment alone, whereas the health effect of amputation was influenced by age. Conversely, the risk of death due to impairment was not influenced by the males' mating status prior to amputation. The males' copulation success was reduced due to impairment, whereas courtship behavior was not affected. Impairment does not reduce the males' impulse to mate but decreases the females' receptivity to copulation, while also increasing the cost of each successful mating. Overall, minor impairment lowers the reproductive success of males and reduces longevity.

Field trials of solid triple lure (trimedlure, methyl eugenol, raspberry ketone, and DDVP) dispensers for detection and male annihilation of Ceratitis capitata, Bactrocera dorsalis, and Bactrocera cucurbitae (Diptera: Tephritidae) in Hawaii.

Solid Mallet TMR (trimedlure [TML], methyl eugenol [ME], raspberry ketone [RK]) wafers and Mallet CMR (ceralure, ME, RK, benzyl acetate) wafers impregnated with DDVP (2,2-dichlorovinyl dimethyl phosphate) insecticide were measured in traps as potential detection and male annihilation technique (MAT) devices. Comparisons were made with 1) liquid lure and insecticide formulations, 2) solid cones and plugs with an insecticidal strip, and 3) solid single and double lure wafers with DDVP for captures of Mediterranean fruit fly, Ceratitis capitata (Wiedemann); oriental fruit fly, Bactrocera dorsalis Hendel; and melon fly, B. cucurbitae Coquillett. Bucket and Jackson traps were tested in a coffee plantation near Eleele, Kauai Island, HI (trials at high populations) and avocado orchards near Kona, HI Island, HI (trials at low populations). Captures of all three species with Mallet TMR were not different from Mallet CMR; therefore, subsequent experiments did not include Mallet CMR because of higher production costs. In MAT trials near Eleele, HI captures in AWPM traps with Mallet TMR wafers were equal to any other solid lure (single or double) except the Mallet ME wafer. In survey trials near Kona, captures of C. capitata, B. cucurbitae, and B. dorsalis with Mallet TMR wafers were equal to those for the standard TML, ME, and C-L traps used in FL and CA. A solid Mallet TMR wafer is safer, more convenient to handle, and may be used in place of several individual lure and trap systems, potentially reducing costs of large survey and detection programs in Florida and California, and MAT programs in Hawaii.

Morphological features of the ovaries during oogenesis of the Oriental fruit fly, Bactrocera dorsalis, in relation to the physiological state.

Determination of physiological state in insects is useful in furthering the understanding of how insect behavior changes with age. Central to this determination is the identification of characters that allow assessment of physiological age. While non-destructive measures produce the most desired outcomes, internal markers may be more diagnostic and reliable. In this study, key morphological characters during previtellogenesis through vitellogenesis and ovulation were assessed as markers to determine physiological states of the oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae). Ovary length and width, ovarian index (length × width), and egg load of laboratory-reared B. dorsalis females recorded daily from eclosion up to 80 days old suggested significant differences in the ovarian index and egg load between females from each oogenesis stage. Parity status determined by the presence of follicular relics was found to provide high-accuracy classifications for B. dorsalis females. The presence of follicular relics with distinct morphological features provides a reliable identification tool to determine the physiological state of wild female oriental fruit fly. The potential applications of this technique to identify the physiological age of female fruit flies to study behavioral attributes in their natural habitat, and also the potential applications in relation to field control, are discussed.

Response of female Ceratitis capitata (Diptera: Tephritidae) to a spinosad bait and polymer matrix mixture with extended residual effect in Hawaii.

The effectiveness of foliar applications of protein baits against pestiferous fruit flies (Tephritidae) can be adversely affected by a rapid loss of attractive volatile compounds and by rainfall due to the high water solubility of the baits. In a large coffee, Coffea arabica L., plantation in Hawaii with high and low populations of Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), the relative attractiveness of GF-120 NF Naturalyte Fruit Fly Bait as either a 40% (vol:vol) spray solution (= GF-120 NF) or as a formulated proprietary amorphous polymer matrix (= GF-120 APM) was compared. The GF-120 APM formulations contained either, 25, 50, or 75% of GF-120 NF (wt:wt). All baits were tested in association with visually attractive yellow bait stations as a way of standardizing the evaluations. With both high and low C. capitata populations, significantly more females were attracted to the fresh sprayed GF-120 NF than to any of the three fresh GF-120 APM formulations. The attractiveness of GF-120 sprayed decreased significantly after 1 wk, whereas 1-wk-old GF-120 APM formulations were as attractive as similar fresh formulations. GF-120 APM 75% aged for 3 wk outperformed similarly-aged sprayed GF-120 NF with comparatively high C. capitata populations. With low populations, both GF-120 APM 75% and GF-120 APM 50% aged for 2 wk outperformed the similarly aged sprayed GF-120 NF. Combined findings indicate that APM mixed with either 50 or 75% GF-120 applied to bait stations can be attractive to female C. capitata for up to 3 wk longer than the standard sprayed GF-120 NF.