Emergency department imaging utilization of cancer patients treated with bevacizumab: single-institution 8-year experience.

PURPOSE: This study aims to highlight the presentations, imaging, and clinical outcomes of cancer patients presenting to the emergency department (ED) while receiving bevacizumab (Avastin) therapy. METHODS: Our retrospective study was based on data from a single institution to identify cancer patients who presented acutely to the ED between 2014 and 2021 within 3 months of beginning bevacizumab who subsequently received diagnostic imaging with CT, MRI, ultrasound, and/or nuclear medicine ventilation/perfusion (VQ) scans. Data gathered included presenting symptoms grouped by body system, imaging impressions, and clinical outcomes, including hospitalization and discontinuation of bevacizumab after each ED visit. Imaging examinations and patient charts were reviewed by a team of fellowship-trained radiologists, radiology residents, and medical students. RESULTS: A total of 84 patients who presented to the ED were included for analysis. This included 32 (38.1%) males and 52 (61.9%) females, with a mean age of 61.2 years and an age range of 29-91 years. Neurological symptoms were the most common presenting symptoms, followed by abdominal symptoms and respiratory symptoms. Head imaging with CT and MRI was the most common imaging ordered with 55 total examinations, followed by abdominal imaging with 37 CT abdomen/pelvis (A/P) examinations, and then CT chest imaging with 22 examinations. Imaging revealed a serious adverse drug reaction in 21 (25.0%) patients, disease progression in 19 (22.6%), and no acute imaging findings in 44 (52.4%) patients. Imaging diagnoses were significantly associated with treatment planning, with a positive determination of bevacizumab-related serious adverse reaction on imaging leading to discontinuation of bevacizumab (p = 0.001). CONCLUSION: Multimodality imaging was a commonly used assessment tool for cancer patients receiving bevacizumab who presented to the ED. Imaging played a crucial role in diagnosis in these patients, especially of treatment-related serious adverse reactions and disease progression. Positive imaging findings of serious adverse reactions affected patient management including discontinuation of bevacizumab.

Abdominopelvic CT Imaging Findings in the Emergency Department in Patients With HIV Positive Status: Single Institute Experience.

OBJECTIVE: To assess emergency department (ED) abdominopelvic computed tomography (CT) imaging utilization and findings in patients with known human immunodeficiency virus (HIV) positive status. MATERIALS AND METHODS: A retrospective chart review of imaging, clinical, and laboratory data was performed for HIV positive patients who demonstrated HIV-related findings on abdominopelvic CT imaging performed within the ED. RESULTS: One hundred and eighty-eight patients with 522 CT scans of the abdomen and/or pelvis were reviewed. 47 patients with HIV presenting to the ED on 82 separate occasions were included in this study (mean age 43.3 years). Patients presented to the ED with infectious/inflammatory disease (n = 54) or history of HIV-related malignancy or new/worsening HIV-related malignancy (n = 28). The most common findings on abdominopelvic CT were anorectal pathology including anorectal abscess or proctitis (n = 22), followed by colitis (n = 19). Findings of HIV-associated malignancy were less common, including anal/rectal cancer (n = 7), Kaposi's sarcoma (n = 4), and lymphoma (n = 2). At the time of ED visit, 25.6% (n = 21) of patients had acquired immunodeficiency syndrome (AIDS). Higher WBC counts were found in the infectious/inflammatory group (P = 0.021) and patients without AIDS (P = 0.0159), while lower WBC counts were associated with new or worsening malignancy (P = 0.007) and AIDS (P = 0.0000). Patients with AIDS were more likely to be deceased at the time of our study. CONCLUSIONS: The majority of ED visits within our population were attributed to infectious/inflammatory etiologies. CT findings demonstrated predominantly infectious/inflammatory processes, with anorectal pathology being the most common. Findings of malignancy on CT were less common, while opportunistic infections and AIDS-defining malignancies were uncommon.

Chemiluminescent sensors for quantitation of the bacterial second messenger cyclic di-GMP.

Chemiluminescent biosensors have been developed and broadly applied to mammalian cell systems for studying intracellular signaling networks. For bacteria, biosensors have largely relied on fluorescence-based systems for quantitating signaling molecules, but these designs can encounter issues in complex environments due to their reliance on external illumination. In order to circumvent these issues, we designed the first ratiometric chemiluminescent biosensors for studying a key bacterial second messenger, cyclic di-GMP. We have shown recently that these biosensors function both in vitro and in vivo for detecting changes in cyclic di-GMP levels. In this chapter, we present a practical and broadly applicable method for high-throughput quantitation of cyclic di-GMP in bacterial cell extracts using the high affinity biosensor tVYN-TmΔ that could serve as the "Bradford assay" equivalent for this bacterial signaling molecule.

Development of Ratiometric Bioluminescent Sensors for in Vivo Detection of Bacterial Signaling.

Second messenger signaling networks allow cells to sense and adapt to changing environmental conditions. In bacteria, the nearly ubiquitous second messenger molecule cyclic di-GMP coordinates diverse processes such as motility, biofilm formation, and virulence. In bacterial pathogens, these signaling networks allow the bacteria to survive changing environmental conditions that are experienced during infection of a mammalian host. While studies have examined the effects of cyclic di-GMP levels on virulence in these pathogens, it has not been possible to visualize cyclic di-GMP levels in real time during the stages of host infection. Toward this goal, we generate the first ratiometric, chemiluminescent biosensor scaffold that selectively responds to c-di-GMP. By engineering the biosensor scaffold, a suite of Venus-YcgR-NLuc (VYN) biosensors is generated that provide extremely high sensitivity (KD < 300 pM) and large changes in the bioluminescence resonance energy transfer (BRET) signal (up to 109%). As a proof-of-concept that VYN biosensors can image cyclic di-GMP in tissues, we show that the VYN biosensors function in the context of a tissue phantom model, with only ∼103-104 biosensor-expressing E. coli cells required for the measurement. Furthermore, we utilize the biosensor in vitro to assess changes in cyclic di-GMP in V. cholerae grown with different inputs found in the host environment. The VYN sensors developed here can serve as robust in vitro diagnostic tools for high throughput screening, as well as genetically encodable tools for monitoring the dynamics of c-di-GMP in live cells, and lay the groundwork for live cell imaging of c-di-GMP dynamics in bacteria within tissues and other complex environments.

Second messengers and divergent HD-GYP phosphodiesterases regulate 3',3'-cGAMP signaling.

3',3'-cyclic GMP-AMP (cGAMP) is the third cyclic dinucleotide (CDN) to be discovered in bacteria. No activators of cGAMP signaling have yet been identified, and the signaling pathways for cGAMP have been inferred to display a narrow distribution based upon the characterized synthases, DncV and Hypr GGDEFs. Here, we report that the ubiquitous second messenger cyclic AMP (cAMP) is an activator of the Hypr GGDEF enzyme GacB from Myxococcus xanthus. Furthermore, we show that GacB is inhibited directly by cyclic di-GMP, which provides evidence for cross-regulation between different CDN pathways. Finally, we reveal that the HD-GYP enzyme PmxA is a cGAMP-specific phosphodiesterase (GAP) that promotes resistance to osmotic stress in M. xanthus. A signature amino acid change in PmxA was found to reprogram substrate specificity and was applied to predict the presence of non-canonical HD-GYP phosphodiesterases in many bacterial species, including phyla previously not known to utilize cGAMP signaling.

The Transcriptional Repressor BS69 is a Conserved Target of the E1A Proteins from Several Human Adenovirus Species.

Early region 1A (E1A) is the first viral protein produced upon human adenovirus (HAdV) infection. This multifunctional protein transcriptionally activates other HAdV early genes and reprograms gene expression in host cells to support productive infection. E1A functions by interacting with key cellular regulatory proteins through short linear motifs (SLiMs). In this study, the molecular determinants of interaction between E1A and BS69, a cellular repressor that negatively regulates E1A transactivation, were systematically defined by mutagenesis experiments. We found that a minimal sequence comprised of MPNLVPEV, which contains a conserved PXLXP motif and spans residues 112⁻119 in HAdV-C5 E1A, was necessary and sufficient in binding to the myeloid, Nervy, and DEAF-1 (MYND) domain of BS69. Our study also identified residues P113 and L115 as critical for this interaction. Furthermore, the HAdV-C5 and -A12 E1A proteins from species C and A bound BS69, but those of HAdV-B3, -E4, -D9, -F40, and -G52 from species B, E, D, F, and G, respectively, did not. In addition, BS69 functioned as a repressor of E1A-mediated transactivation, but only for HAdV-C5 and HAdV-A12 E1A. Thus, the PXLXP motif present in a subset of HAdV E1A proteins confers interaction with BS69, which serves as a negative regulator of E1A mediated transcriptional activation.

Chemiluminescent Biosensors for Detection of Second Messenger Cyclic di-GMP.

Bacteria colonize highly diverse and complex environments, from gastrointestinal tracts to soil and plant surfaces. This colonization process is controlled in part by the intracellular signal cyclic di-GMP, which regulates bacterial motility and biofilm formation. To interrogate cyclic di-GMP signaling networks, a variety of fluorescent biosensors for live cell imaging of cyclic di-GMP have been developed. However, the need for external illumination precludes the use of these tools for imaging bacteria in their natural environments, including in deep tissues of whole organisms and in samples that are highly autofluorescent or photosensitive. The need for genetic encoding also complicates the analysis of clinical isolates and environmental samples. Toward expanding the study of bacterial signaling to these systems, we have developed the first chemiluminescent biosensors for cyclic di-GMP. The biosensor design combines the complementation of split luciferase (CSL) and bioluminescence resonance energy transfer (BRET) approaches. Furthermore, we developed a lysate-based assay for biosensor activity that enabled reliable high-throughput screening of a phylogenetic library of 92 biosensor variants. The screen identified biosensors with very large signal changes (∼40- and 90-fold) as well as biosensors with high affinities for cyclic di-GMP ( KD < 50 nM). These chemiluminescent biosensors then were applied to measure cyclic di-GMP levels in E. coli. The cellular experiments revealed an unexpected challenge for chemiluminescent imaging in Gram negative bacteria but showed promising application in lysates. Taken together, this work establishes the first chemiluminescent biosensors for studying cyclic di-GMP signaling and provides a foundation for using these biosensors in more complex systems.

Mendel's law reveals fatal flaws in Bateman's 1948 study of mating and fitness.

Bateman's experimental study of Drosophila melanogaster produced conclusions that are now part of the bedrock premises of modern sexual selection. Today it is the most cited experimental study in sexual selection, and famous as the first experimental demonstration of sex differences in the relationship between number of mates and relative reproductive success. We repeated the experimental methodology of the original to evaluate its reliability. The results indicate that Bateman's methodology of visible mutations to assign parentage and reproductive success to subject adults is significantly biased. When combined in offspring, the mutations decrease offspring survival, so that counts of mate number and reproductive success are mismeasured. Bateman's method overestimates the number of subjects with no mates and underestimates the number with one or more mates for both sexes. Here we discuss why Bateman's paper is important and present additional analyses of data from our monogamy trials. Monogamy trials can inform inferences about the force of sexual selection in populations because in monogamy trials male-male competition and female choice are absent. Monogamy trials also would have provided Bateman with an a priori test of the fit of his data to Mendel's laws, an unstated, but vital assumption of his methodology for assigning parentage from which he inferred the number of mates per individual subject and their reproductive success. Even under enforced monogamous mating, offspring frequencies of double mutant, single mutant and no mutant offspring were significantly different from Mendelian expectations proving that Bateman's method was inappropriate for answering the questions he posed. Double mutant offspring (those with a mutation from each parent) suffered significant inviability as did single mutant offspring whenever they inherited their mother's marker but the wild-type allele at their father's marker locus. These inviability effects produced two important inaccuracies in Bateman's results and conclusions. (1) Some matings that actually occurred were invisible and (2) reproductive success of some mothers was under-estimated. Both observations show that Bateman's conclusions about sex differences in number of mates and reproductive success were unwarranted, based on biased observations. We speculate about why Bateman's classic study remained without replication for so long, and we discuss why repetition almost 60 years after the original is still timely, necessary and critical to the scientific enterprise. We highlight overlooked alternative hypotheses to urge that modern tests of Bateman's conclusions go beyond confirmatory studies to test alternative hypotheses to explain the relationship between mate number and reproductive success.

No evidence of sexual selection in a repetition of Bateman's classic study of Drosophila melanogaster.

We are unique in reporting a repetition of Bateman [Bateman AJ (1948) Heredity (Edinb) 2:349-368] using his methods of parentage assignment, which linked sex differences in variance of reproductive success and variance in number of mates in small populations of Drosophila melanogaster. Using offspring phenotypes, we inferred who mated with whom and assigned offspring to parents. Like Bateman, we cultured adults expressing dramatic phenotypes, so that each adult was heterozygous-dominant at its unique marker locus but had only wild-type alleles at all other subjects' marker loci. Assuming no viability effects of parental markers on offspring, the frequencies of parental phenotypes in offspring follow mendelian expectations: one-quarter will be double-mutants who inherit the dominant gene from each parent, the offspring from which Bateman counted the number of mates per breeder; half of the offspring must be single mutants inheriting the dominant gene of one parent and the wild-type allele of the other parent; and one-quarter would inherit neither of their parent's marker mutations. Here we show that inviability of double-mutant offspring biased inferences of mate number and number of offspring on which rest inferences of sex differences in fitness variances. Bateman's method overestimated subjects with zero mates, underestimated subjects with one or more mates, and produced systematically biased estimates of offspring number by sex. Bateman's methodology mismeasured fitness variances that are the key variables of sexual selection.

Sexual isolation between North American and Bogota strains of Drosophila pseudoobscura.

Sexual isolation, the reduced ability of organisms of different species to successfully mate, is one of the reproductive barriers that prevent gene flow between different taxa. Various species-specific signals during courtship are attributed to the sexual isolation between them. Drosophila pseudoobscura has been widely used to study the behavioral and genetic mechanisms underlying selection for sexual isolation, as a model system for speciation. D. pseudoobscura and its sibling species, D. persmilis, live together in many locations but are reproductively isolated from one another. North American geographic strains of D. pseudoobscura from the American West mate at random. Several decades ago, D. pseudoobscura was collected in the vicinity of Bogota, Colombia, and later named the subspecies D. pseudoobscura bogotana. Nearly 5,000 matings were observed in this study. We analyzed mating behavior and cuticular hydrocarbon profiles as well as courtship within and between North American and Bogota strains of D. pseudoobscura. Here we report for the first time that Bogota strains of D. pseudoobscura do show statistically significant sexual isolation from North American strains. In addition, there are quantitative variations in cuticular hydrocarbons as well as in courtship behavior between Bogota and North American strains, and females of both North American and Bogota strains show strong preference for North American strain males having high mating propensities, suggesting that the Bogota strains are at an early stage that could lead to a separate species.

Preference status does not indicate intrinsic quality differences in Drosophila pseudoobscura.

The good genes hypothesis states that choosers prefer individuals of high intrinsic quality to individuals of lower intrinsic quality. Variation in longevity is thought to reflect, in part, intrinsic quality differences of individuals in addition to the costs of mating and reproduction. Here we report longevity variation of Drosophila pseudoobscura, a species in which previous experiments have demonstrated that individual mate preferences (pre-touching mate assessments) of females and males are associated with enhanced numbers of eclosed (adult) offspring and higher egg-to-adult survival (offspring viability). Using mate assessment arenas and protocols similar to those in a previous experiment that demonstrated fitness benefits to breeders and their offspring of mating with individuals they preferred, we tested the following predictions: (i) preferred discriminatees live longer than non-preferred discriminatees; (ii) males live longer than females; and (iii) virgins live longer than mated individuals. The experiment yielded 938 individuals for longevity analysis. Sex and mating status affected longevity: males lived longer than females, virgin females lived longer than mated females, but there were no differences in longevity for mated and virgin males. Non-preferred discriminatees of both sexes survived as long as preferred discriminatees, a result inconsistent with the prediction of the good genes hypothesis for mate preferences. To our knowledge, this is the first demonstration of the cost of reproduction for females in D. pseudoobscura and of longevity variation of preferred and non-preferred discriminatees.

Polytene chromosomal maps of 11 Drosophila species: the order of genomic scaffolds inferred from genetic and physical maps.

The sequencing of the 12 genomes of members of the genus Drosophila was taken as an opportunity to reevaluate the genetic and physical maps for 11 of the species, in part to aid in the mapping of assembled scaffolds. Here, we present an overview of the importance of cytogenetic maps to Drosophila biology and to the concepts of chromosomal evolution. Physical and genetic markers were used to anchor the genome assembly scaffolds to the polytene chromosomal maps for each species. In addition, a computational approach was used to anchor smaller scaffolds on the basis of the analysis of syntenic blocks. We present the chromosomal map data from each of the 11 sequenced non-Drosophila melanogaster species as a series of sections. Each section reviews the history of the polytene chromosome maps for each species, presents the new polytene chromosome maps, and anchors the genomic scaffolds to the cytological maps using genetic and physical markers. The mapping data agree with Muller's idea that the majority of Drosophila genes are syntenic. Despite the conservation of genes within homologous chromosome arms across species, the karyotypes of these species have changed through the fusion of chromosomal arms followed by subsequent rearrangement events.

Evolution of genes and genomes on the Drosophila phylogeny.

Comparative analysis of multiple genomes in a phylogenetic framework dramatically improves the precision and sensitivity of evolutionary inference, producing more robust results than single-genome analyses can provide. The genomes of 12 Drosophila species, ten of which are presented here for the first time (sechellia, simulans, yakuba, erecta, ananassae, persimilis, willistoni, mojavensis, virilis and grimshawi), illustrate how rates and patterns of sequence divergence across taxa can illuminate evolutionary processes on a genomic scale. These genome sequences augment the formidable genetic tools that have made Drosophila melanogaster a pre-eminent model for animal genetics, and will further catalyse fundamental research on mechanisms of development, cell biology, genetics, disease, neurobiology, behaviour, physiology and evolution. Despite remarkable similarities among these Drosophila species, we identified many putatively non-neutral changes in protein-coding genes, non-coding RNA genes, and cis-regulatory regions. These may prove to underlie differences in the ecology and behaviour of these diverse species.

The hypothesis of reproductive compensation and its assumptions about mate preferences and offspring viability.

The Compensation Hypothesis says that parents and prospective parents attempt to make up for lowered offspring viability by increasing reproductive effort to produce healthy, competitive offspring and by increasing investment in less viable, but still-living progeny (parental effects). The hypothesis assumes that offspring viability is lower when individuals are constrained (often through sexual conflict) to breed with individuals they do not prefer. We review results of experimental tests of the offspring-viability assumption in Tanzanian cockroaches, fruit flies, pipefish, wild mallards, and feral house mice. Experimental constraints on mating preferences lowered offspring viability in each of the studies. Females breeding under constraints laid more eggs or gave birth to more young than females breeding without or with fewer constraints on their mating preferences, and males mating under constraints on their mate preferences ejaculated more sperm than males mating without constraints. The number of eggs laid or offspring born was higher when female choosers were experimentally constrained to reproduce with males they did not prefer. Constrained females may increase fecundity to enhance the probability that they produce adult offspring with rarer phenotypes with survival benefits against offspring generation pathogens. Similarly, ejaculation of more sperm when males are paired with females they do not prefer may be a mechanism that provides more variable sperm haplotypes for prospective mothers or that may provide nutritional benefits to mothers and zygotes.

Experimental constraints on mate preferences in Drosophila pseudoobscura decrease offspring viability and fitness of mated pairs.

Using Drosophila pseudoobscura, we tested the hypothesis that social constraints on the free expression of mate preferences, by both females and males, decrease offspring viability and reproductive success of mating pairs. Mate preference arenas eliminated intrasexual combat and intersexual coercion. The time female and male choosers spent in arena tests near either of two opposite-sex individuals measured the preferences of choosers. We placed choosers in breeding trials with their preferred or nonpreferred discriminatee when they met the minimum criteria for showing the same preference in two consecutive tests. There was no statistically significant difference in the frequency of female and male choosers meeting minimal preference criteria. There was a significant difference between female and male choosers for offspring viability, with female choice having the greater effect, but there was not a significant difference in the overall reproductive success of male and female choosers. There were significant differences in fitness between matings to preferred and nonpreferred partners. Female and male choosers paired with their nonpreferred discriminatees had offspring of significantly lower viability, as predicted by the constraints hypothesis. Reproductive success, our measure of overall fitness, was greater when males or females mated with the partner they preferred rather than the one they did not prefer.

Chinch bug (Hemiptera: Blissidae) mouthpart morphology, probing frequencies, and locations on resistant and susceptible germplasm.

Chinch bugs are common pests of many agronomic and horticulturally important crops and turfgrasses. Previous research has indicated that some grasses exhibit resistance to multiple chinch bug species, whereas others are resistant to only one species. The objectives of this research were to document differences in the probing frequencies and locations among Blissus species as well as differences in mouthpart morphology as a first step in understanding the differential responses of grasses to chinch bug feeding. Scanning electron microscopy detected differences in the total lengths of proboscises as well as individual mouthpart segments among the four species studied. Blissus occiduus Barber probed significantly more often on buffalograss, Buchloë dactyloides (Nuttall) Engelmann, than any other plant material. Probing locations of B. occiduus and Blissus leucopterus leucopterus (Say) were similar on both B. occiduus-resistant and susceptible buffalograsses and KS94 sorghum, Sorghum bicolor (L.) Moench (B. occiduus-resistant, B. l. leucopterus-resistant). However, on 'Wheatland' sorghum (B. occiduus-resistant, B. l. leucopterus-susceptible), stylet tracts of B. l. leucopterus most often terminated in the bundle sheath cells, whereas those of B. occiduus generally terminated in the vascular tissues.

Evaluation of cool- and warm-season grasses for resistance to multiple chinch bug (Hemiptera: Blissidae) species.

Chinch bugs are common pests of many agronomic and horticulturally important crops and turfgrasses. The extensive overlap of plant hosts and geographic distribution of Blissus leucopterus leucopterus (Say), Blissus leucopterus hirtus Montandon, Blissus insularis Barber, and Blissus occiduus Barber underscores the importance of identifying resistant germplasm. Cool- and warm-season turfgrasses and sorghum, Sorghum bicolor (L.) Moench, were evaluated for resistance to chinch bugs in the Blissus complex, and the presence of multiple resistance was documented. Greenhouse studies established that B. occiduus-resistant ('Prestige', formerly NE91-118) and -susceptible ('378') buffalograsses,, Buchloë dactyloides (Nuttall) Engelmann, were susceptible to all other chinch bug species. KS94 sorghum exhibited resistance to both B. occiduus and B. l. leucopterus, whereas B. insularis-resistant St. Augustinegrass, Stenotaphrum secundatum (Walter) Kuntze ('Floratam'), was also resistant to B. occiduus. B. l. leucopterus-susceptible sorghum ('Wheatland') and B. insularis-susceptible St. Augustinegrasses ('Raleigh' and 'Amerishade') were highly resistant to B. occiduus. Endophyte-free and -enhanced fine fescues (Festuca spp.) were moderately to highly susceptible to B. l. hirtus but moderately to highly resistant to B. occiduus. The results of this research showed the buffalograsses evaluated, including B. occiduus-resistant Prestige, are moderately to highly susceptible to the three other chinch bug species. In contrast, B. occiduus did not cause considerable damage to any of the turfgrasses or sorghum cultivars evaluated, other than buffalograss, irrespective of whether or not they are resistant to another chinch bug species. This information is increasingly important as various grasses become adapted to regions that may possess chinch bug species other than those with which they are typically associated. These levels of Blissus resistance should be included when selecting resistant germplasm for managing Blissus species pests.

Mechanisms of genetic exchange within the chromosomal inversions of Drosophila pseudoobscura.

We have used the inversion system of Drosophila pseudoobscura to investigate how genetic flux occurs among the gene arrangements. The patterns of nucleotide polymorphism at seven loci were used to infer gene conversion events between pairs of different gene arrangements. We estimate that the average gene conversion tract length is 205 bp and that the average conversion rate is 3.4 x 10(-6), which is 2 orders of magnitude greater than the mutation rate. We did not detect gene conversion events between all combinations of gene arrangements even though there was sufficient nucleotide variation for detection and sufficient opportunity for exchanges to occur. Genetic flux across the inverted chromosome resulted in higher levels of differentiation within 0.1 Mb of inversion breakpoints, but a slightly lower level of differentiation in central inverted regions. No gene conversion events were detected within 17 kb of an inversion breakpoint suggesting that the formation of double-strand breaks is reduced near rearrangement breakpoints in heterozygotes. At least one case where selection rather than proximity to an inversion breakpoint is responsible for reduction in polymorphism was identified.

Sexual isolation between sympatric and allopatric populations of Drosophila pseudoobscura and D. persimilis.

According to reinforcement theory, sexual isolation between species in sympatry is strengthened by natural selection against maladaptive hybrids. Drosophila pseudoobscura and D. persimilis from four locations where these species are sympatric, and from three locations where only D. pseudoobscura has been found, were utilized in studies of sexual isolation. Multiple-choice observation chambers were used to record matings between sympatric and allopatric strains of the two species. There was a wide variation in sexual isolation between the two species in the four localities we studied. The average isolation index for sympatric strains of the species was not significantly different from the average index for allopatric strains. There were no meaningful differences between the isolation indices in sympatric and allopatric strains of the species. The failure to find a relationship is likely the result of gene flow among populations within the two species.