Smoking behavior and motivation to quit among chronic pain patients initiating multidisciplinary pain treatment: a prospective study.

OBJECTIVE: The primary aim of this study was to assess smoking characteristics and cessation motivation prior to and after initiation of multidisciplinary chronic pain treatment. A secondary aim was to identify predictors of cessation motivation among smokers initiating treatment for chronic pain. DESIGN: We used a prospective, nonrandomized, repeated measures design. SETTING: The study was conducted in a multidisciplinary specialty pain treatment program at a veterans hospital. PATIENTS: Smokers (N = 90) referred to a multidisciplinary pain program for the treatment of chronic pain. MEASURES: Patients completed questionnaires assessing pain-related and smoking-related factors prior to (baseline) and 8 weeks post (follow-up) specialty pain treatment initiation. Primary outcome measures were the Contemplation Ladder and the Stages of Change (SOC) algorithm. RESULTS: At baseline, patients reported moderate levels of cessation motivation, and 69% were in the contemplation stage or higher on the SOC. Motivation to quit smoking was higher at follow-up compared with baseline on both continuous, t(89) = 2.11, P < 0.05, and stage-based, z = 3.69, P < 0.01, measures. At follow-up, participants reported greater interest in receiving cessation interventions, and 7.8% of patients had quit smoking. Pain-related predictors of motivation (e.g., pain intensity) were subsumed by more general predictors (e.g., nicotine dependence). CONCLUSIONS: Patients in this sample were more motivated to quit smoking a few weeks after, as compared with before initiating specialty pain treatment. Future research into pain-specific predictors of cessation motivation is warranted to inform the development of interventions that address pain patients' unique needs.

Chronic pain and traumatic brain injury in OEF/OIF service members and Veterans.

The co-occurrence of chronic pain and traumatic brain injury (TBI) are 2 of the most common concerns among the Operations Enduring Freedom and Iraqi Freedom population and present unique challenges for evaluation and treatment. Previous research suggests that almost half the cohort report clinically significant pain, while up to 1 in 4 experiences some form of TBI. There is limited information regarding how TBI affects the presence and course of pain, and how pain impacts TBI and its symptoms. The present paper provides an overview of the range and degree of TBIs as well as a brief summary of current knowledge regarding the interaction between chronic pain and TBI, particularly in light of the numerous variables impacting it. Information on ways to best assess for and treat pain in the TBI population, including in those with multiple system injuries or associated affective symptoms, is provided. In addition, several innovative approaches for addressing the needs of this complex cohort of patients are described, which may stimulate further research and clinical innovation for this important subgroup.

Access to the US Department of Veterans Affairs health system: self-reported barriers to care among returnees of Operations Enduring Freedom and Iraqi Freedom.

BACKGROUND: The U.S. Department of Veterans Affairs (VA) implemented the Polytrauma System of Care to meet the health care needs of military and veterans with multiple injuries returning from combat operations in Afghanistan and Iraq. Studies are needed to systematically assess barriers to use of comprehensive and exclusive VA healthcare services from the perspective of veterans with polytrauma and with other complex health outcomes following their service in Afghanistan and Iraq. These perspectives can inform policy with regard to the optimal delivery of care to returning veterans. METHODS: We studied combat veterans (n = 359) from two polytrauma rehabilitation centers using structured clinical interviews and qualitative open-ended questions, augmented with data collected from electronic health records. Our outcomes included several measures of exclusive utilization of VA care with our primary exposure as reported access barriers to care. RESULTS: Nearly two thirds of the veterans reported one or more barriers to their exclusive use of VA healthcare services. These barriers predicted differences in exclusive use of VA healthcare services. Experiencing any barriers doubled the returnees' odds of not using VA exclusively, the geographic distance to VA barrier resulted in a 7 fold increase in the returnees odds of not using VA, and reporting a wait time barrier doubled the returnee's odds of not using VA. There were no striking differences in access barriers for veterans with polytrauma compared to other returning veterans, suggesting the barriers may be uniform barriers that predict differences in using the VA exclusively for health care. CONCLUSIONS: This study provides an initial description of utilization of VA polytrauma rehabilitation and other medical care for veteran returnees from all military services who were involved in combat operations in Afghanistan or Iraq. Our findings indicate that these veterans reported important stigmatization and barriers to receiving services exclusively from the VA, including mutable health delivery system factors.

Non-coding changes cause sex-specific wing size differences between closely related species of Nasonia.

The genetic basis of morphological differences among species is still poorly understood. We investigated the genetic basis of sex-specific differences in wing size between two closely related species of Nasonia by positional cloning a major male-specific locus, wing-size1 (ws1). Male wing size increases by 45% through cell size and cell number changes when the ws1 allele from N. giraulti is backcrossed into a N. vitripennis genetic background. A positional cloning approach was used to fine-scale map the ws1 locus to a 13.5 kilobase region. This region falls between prospero (a transcription factor involved in neurogenesis) and the master sex-determining gene doublesex. It contains the 5'-UTR and cis-regulatory domain of doublesex, and no coding sequence. Wing size reduction correlates with an increase in doublesex expression level that is specific to developing male wings. Our results indicate that non-coding changes are responsible for recent divergence in sex-specific morphology between two closely related species. We have not yet resolved whether wing size evolution at the ws1 locus is caused by regulatory alterations of dsx or prospero, or by another mechanism. This study demonstrates the feasibility of efficient positional cloning of quantitative trait loci (QTL) involved in a broad array of phenotypic differences among Nasonia species.

Resistance mechanisms to targeted therapy in BRAF-mutant melanoma - A mini review.

BACKGROUND: The introduction of targeted therapies for the treatment of BRAF-mutant melanomas have improved survival rates in a significant proportion of patients. Nonetheless, the emergence of resistance to treatment remains inevitable in most patients. SCOPE OF REVIEW: Here, we review known and emerging molecular mechanisms that underlay the development of resistance to MAPK inhibition in melanoma cells and the potential strategies to overcome these mechanisms. MAJOR CONCLUSIONS: Multiple genetic and non-genetic mechanisms contribute to treatment failure, commonly leading to the reactivation of the MAPK pathway. A variety of resistance mechanisms are enabled by the underlying heterogeneity and plasticity of melanoma cells. Moreover, it has become apparent that resistance to targeted therapy is underpinned by early functional adaptations involving the rewiring of cell states and metabolic pathways. GENERAL SIGNIFICANCE: The evidence presented suggest that the use of a combinatorial treatment approach would delay the emergence of resistance and improve patient outcomes.

The mushroom habitat as an ecological arena for global exchange of Wolbachia.

Wolbachia infect a variety of arthropod and nematode hosts, but in arthropods, host phylogenetic relationships are usually poor predictors of strain similarity. This suggests that new infections are often established by horizontal transmission. To gain insight into the factors affecting the probability of horizontal transmission among host species, we ask how host phylogeny, geographical distribution and ecology affect patterns of Wolbachia strain similarity. We used multilocus sequence typing (MLST) to characterize Wolbachia strain similarity among dipteran hosts associated with fleshy mushrooms. Wolbachia Supergroup A was more common than Supergroup B in Diptera, and also more common in mycophagous than non-mycophagous Diptera. Within Supergroup A, host family within Diptera had no effect on strain similarity, and there was no tendency for Wolbachia strains from sympatric host species to be more similar to one another than to strains from hosts in different biogeographical realms. Supergroup A strains differed between mycophagous and non-mycophagous Diptera more than expected by chance, suggesting that ecological associations can facilitate horizontal transmission of Wolbachia within mycophagous fly communities. For Supergroup B, there were no significant associations between strain similarity and host phylogeny, biogeography, or ecology. We identified only two cases in which closely related hosts carried closely related Wolbachia strains, evidence that Wolbachia-host co-speciation or early introgression can occur but may not be a major contributor to overall strain diversity. Our results suggest that horizontal transmission of Wolbachia can be influenced by host ecology, thus leading to partial restriction of Wolbachia strains or strain groups to particular guilds of insects.

Comparison of Circulating Tumour DNA and Extracellular Vesicle DNA by Low-Pass Whole-Genome Sequencing Reveals Molecular Drivers of Disease in a Breast Cancer Patient.

There is increasing recognition of circulating tumour DNA (ctDNA) as a non-invasive alternative to tumour tissue for the molecular characterisation and monitoring of disease. Recent evidence suggests that cancer-associated changes can also be detected in the DNA contained within extracellular vesicles (EVs). As yet, there has been limited investigation into the relationship between EV DNA and ctDNA, and no studies have examined the EV DNA of breast cancer patients. The aim of this study was to use low-pass whole-genome sequencing to identify copy number variants (CNVs) in serial samples of both ctDNA and EV DNA from a patient with breast cancer. Of the 52 CNVs identified in tumour DNA, 36 (69%) were detected in at least one ctDNA sample and 13 (25%) in at least one EV DNA sample. The number of detectable variants in ctDNA and EV DNA increased over the natural history of the patient's disease, which was associated with progression to cerebral metastases. This case study demonstrates that, while CNVs are detectable in patient EV DNA, ctDNA has greater sensitivity than EV DNA for serial monitoring of breast cancer.

Detection of BRAF splicing variants in plasma-derived cell-free nucleic acids and extracellular vesicles of melanoma patients failing targeted therapy therapies.

The analysis of plasma circulating tumour nucleic acids provides a non-invasive approach to assess disease burden and the genetic evolution of tumours in response to therapy. BRAF splicing variants are known to confer melanoma resistance to BRAF inhibitors. We developed a test to screen cell-free RNA (cfRNA) for the presence of BRAF splicing variants. Custom droplet digital PCR assays were designed for the detection of BRAF splicing variants p61, p55, p48 and p41 and then validated using RNA from cell lines carrying these variants. Evaluation of plasma from patients with reported objective response to BRAF/MEK inhibition followed by disease progression was revealed by increased circulating tumour DNA (ctDNA) in 24 of 38 cases at the time of relapse. Circulating BRAF splicing variants were detected in cfRNA from 3 of these 38 patients; two patients carried the BRAF p61 variant and one the p55 variant. In all three cases the presence of the splicing variant was apparent only at the time of progressive disease. BRAF p61 was also detectable in plasma of one of four patients with confirmed BRAF splicing variants in their progressing tumours. Isolation and analysis of RNA from extracellular vesicles (EV) from resistant cell lines and patient plasma demonstrated that BRAF splicing variants are associated with EVs. These findings indicate that in addition to plasma ctDNA, RNA carried by EVs can provide important tumour specific information.

Comparison of pain and emotional symptoms in soldiers with polytrauma: unique aspects of blast exposure.

OBJECTIVES: To compare the physical and emotional presentation and pain treatment outcomes of service members who sustained polytrauma secondary to blast with those of soldiers injured by other means. DESIGN: Retrospective medical record review. SETTING AND PATIENTS: One of four Veterans Affairs multidisciplinary inpatient Polytrauma Rehabilitation Centers. One hundred twenty-eight Operation Enduring Freedom and Operation Iraqi Freedom military personnel with serious polytrauma injuries and concomitant pain categorized into three groups based on type of injuries: blast injuries, combat injuries other than blast, and noncombat, nonblast injuries. INTERVENTIONS: Intensive, interdisciplinary inpatient rehabilitation and pain treatment. OUTCOME MEASURES: Pain intensity ratings; Functional Independence Measure scores; Rancho Los Amigos levels; and opioid analgesic doses. RESULTS: Service members injured via blast demonstrated a broader spectrum of physical injuries, higher levels of admission and discharge opioid analgesic use, reduced improvement in pain intensity following treatment, and much higher rates of posttraumatic stress disorder and other psychiatric diagnoses than those injured via other means. CONCLUSIONS: Blast injury may be associated with differential physical, emotional, and pain-related symptoms that pose increased challenges for successful treatment.

Assessment and treatment of pain associated with combat-related polytrauma.

Due to the high rates of blast injuries sustained during operations in Iraq and Afghanistan, the number of soldiers returning with massive and multiple wounds is unprecedented. While casualty survival rates have improved dramatically, the extent and impact of these wounds on soldiers' functioning pose unique challenges for their rehabilitation. Pain is highly prevalent in these individuals with polytrauma injuries and is a source of suffering, as well as an impediment to rehabilitation. However, there are a number of obstacles to effective pain treatment in this group of war-injured, including their multiple and severe injuries, the high prevalence of brain injuries, cognitive impairments and emotional distress, the prolonged and intensive rehabilitation process, and the frequent need for repeated follow-up surgeries. As a result, we believe that a comprehensive, interdisciplinary approach to pain treatment is required. In this article we describe the model of pain care that has evolved at the Tampa Polytrauma Rehabilitation Center, which incorporates medical, rehabilitative, cognitive-behavioral, and interventional treatments targeting pain intensity as well as pain-related impairments and coping. We include a case study illustrating some key aspects of our approach.

Systematic review of the literature on pain in patients with polytrauma including traumatic brain injury.

OBJECTIVE: To review the literature addressing the assessment and management of pain in patients with polytraumatic injuries including traumatic brain injury (TBI) and blast-related headache, and to identify patient, clinician and systems factors associated with pain-related outcomes. DESIGN: Systematic review. METHODS: We conducted searches in MEDLINE of literature published from 1950 through July 2008. Due to a limited number of studies using controls or comparators, we included observational and rigorous qualitative studies. We systematically rated the quality of systematic reviews, cohort, and case-control design studies. RESULTS: One systematic review, 93 observational studies, and one qualitative research study met inclusion criteria. The literature search yielded no published studies that assessed measures of pain intensity or pain-related functional interference among patients with cognitive deficits due to TBI, that compared patients with blast-related headache with patients with other types of headache, or that assessed treatments for blast-related headache pain. Studies on the association between TBI severity and pain reported mixed findings. There was limited evidence that the following factors are associated with pain among TBI patients: severity, location, and multiplicity of injuries; insomnia; fatigue; depression; and post-traumatic stress disorder. CONCLUSIONS: Very little evidence is currently available to guide pain assessment and treatment approaches in patients with polytrauma. Further research employing systematic observational as well as controlled intervention designs is clearly indicated.

Traumatic brain injury, polytrauma, and pain: challenges and treatment strategies for the polytrauma rehabilitation.

PROBLEM: Chronic pain conditions are common sequelae of traumatic brain injury (TBI). Unfortunately, the incidence of TBI among personnel deployed for Operations Enduring Freedom and Iraqi Freedom (OEF/OIF) is significant, and there is growing evidence that ongoing pain, particularly headaches, will be a primary concern for these individuals. OBJECTIVE: This article synthesizes empirical data from civilian and veteran populations and clinical experience with OEF/OIF personnel with polytrauma to provide recommendations for the assessment and treatment of chronic pain among those with TBI. CONCLUSIONS: The available data signal the need for the incorporation of early and aggressive pain management strategies into existing treatment models. Challenges to providing effective pain management for OEF/OIF veterans are numerous and include comorbid cognitive, medical, and emotional impairments that complicate readjustment to civilian life. It is likely that the problem of polytrauma pain and associated comorbid conditions such as posttraumatic stress disorder and postconcussive syndrome will require the development of integrated approaches to clinical care which bridge traditional subspecialty divisions. A proposed model of treatment is presented.

Cluster analysis of the pain outcomes questionnaire.

OBJECTIVE: The purpose of this study was to derive and describe subtypes of Pain Outcomes Questionnaire (POQ) profiles produced by a large, heterogeneous multisite sample of chronic pain patients (N = 672). METHODS: The POQ core scales were subjected to Ward's hierarchical agglomerative cluster analyses in order to establish the optimal number of clusters in validation (N = 336) and cross-validation (N = 336) subsamples. Based on the consistency of the solutions derived from the two subsamples, a third Ward's procedure was performed on the entire sample and, finally, a K-means iterative partitioning procedure was used to assign cases to the cluster groups. The resulting cluster solution was validated using a broad range of demographic, pain, and treatment outcomes data. RESULTS: The cluster analytic procedure revealed the presence of eight unique subgroups of POQ profiles. The adequacy of the final solution was demonstrated using a multivariate analysis of variance and a discriminant function analysis. Group differences on the demographic and pain variables were generally consistent with expectations based on mean profiles, and therefore, provided evidence of the validity of the cluster solution. Brief descriptions of each cluster group were generated based on the correlate data. The clinical utility of the solution was in part demonstrated by differential treatment response rates among the groups. DISCUSSION: The results of this study are generally consistent with those of previous cluster analytic investigations of pain patients and suggest that psychosocial/behavioral classification systems may serve a useful heuristic function in the assessment and treatment of chronic pain that is not provided by current diagnostic taxonomies.

Induced paternal effects mimic cytoplasmic incompatibility in Drosophila.

Wolbachia is an intracellular microbe found in a wide diversity of arthropod and filarial nematode hosts. In arthropods these common bacteria are reproductive parasites that manipulate central elements of their host's reproduction to increase their own maternal transmission in one of several ways. Cytoplasmic incompatibility (CI) is one such manipulation where sperm are somehow modified in infected males and this modification must be rescued by the presence of the same bacterial strain in the egg for normal development to proceed. The molecular mechanisms involved in the expression of CI are unknown. Here we show that Wolbachia infection results in increased mRNA and protein expression of the Drosophila simulans nonmuscle myosin II gene zipper. Induced overexpression of zipper in Wolbachia-free transgenic D. melanogaster males results in paternal-effect lethality that mimics the fertilization defects associated with CI. Likewise, overexpression of the tumor suppressor gene, lethal giant larvae [l(2)gl], results in egg lethality and a CI phenotype. Stoichiometric levels of zipper and l(2)gl are required for proper segregation of cellular determinants during neuroblast stem cell division. Taken together these results form the basis of a working hypothesis whereby Wolbachia induces paternal effects in sperm by manipulating the expression of key regulators of cytoskeletal activity during spermatogenesis.

Widespread prevalence of wolbachia in laboratory stocks and the implications for Drosophila research.

Wolbachia is an intracellular microbe harbored by a wide variety of arthropods (including Drosophila) and filarial nematodes. Employing several different strategies including male killing, induced parthenogenesis, cytoplasmic incompatibility, and feminization, and acting by as-yet-unknown mechanisms, Wolbachia alters host reproduction to increase its representation within a population. Wolbachia is closely associated with gametic incompatibility but also interacts with Drosophila in other, little understood ways. We report here significant and widespread infection of Wolbachia within laboratory stocks and its real and potential impact on Drosophila research. We describe the results of a survey indicating that approximately 30% of stocks currently housed at the Bloomington Drosophila Stock Center are infected with Wolbachia. Cells of both reproductive tissues and numerous somatic organs harbor Wolbachia and display considerable variation in infection levels within and between both tissue types. These results are discussed from the perspective of Wolbachia's potential confounding effects on both host fitness and phenotypic analyses. In addition to this cautionary message, the infection status of stock centers may provide further opportunities to study the genetic basis of host/symbiosis.

Validity of Center for Epidemiologic Studies Depression (CES-D) scale in a sample of Iraq and Afghanistan Veterans.

OBJECTIVES: Optimal depression screening necessitates measurement tools that are valid across varied populations and in the presence of comorbidities. METHODS: This study assessed the test properties of two versions of the Center for Epidemiologic Studies Depression scale against psychiatric diagnoses established by the Mini International Neuropsychiatric Interview among a clinical sample of US Veterans deployed during Operations Enduring Freedom, Iraqi Freedom, and New Dawn. Participants (N = 359) recruited from two Department of Veterans Affairs hospitals completed a clinical interview, structured diagnostic interview, and self-reported measures. RESULTS: Based on diagnostic interview and the Diagnostic and Statistical Manual of Mental Disorders 4th Edition criteria, 29.5% of the sample met diagnostic criteria for major depressive disorder and 26.5% met diagnostic criteria for post-traumatic stress disorder. Both Center for Epidemiologic Studies Depression-20 and Center for Epidemiologic Studies Depression-10 scales performed well and almost identically against the Mini International Neuropsychiatric Interview-major depressive disorder in identifying Veterans with major depressive disorder (Center for Epidemiologic Studies Depression-20 area under the Receiver Operating Characteristic curve 91%; Center for Epidemiologic Studies Depression-10 area under the ROC curve 90%). Overall, higher cut points for the Center for Epidemiologic Studies Depression scales performed better in correctly identifying true positives and true negatives for major depressive disorder (Center for Epidemiologic Studies Depression-20 cut point 18+ sensitivity 92% specificity 72%; Center for Epidemiologic Studies Depression-10 cut point 10+ sensitivity 92% specificity 69%). CONCLUSIONS: The specificity of the Center for Epidemiologic Studies Depression scales was poor among Veterans with co-occurring post-traumatic stress disorder (13% and 16%). Veterans with post-traumatic stress disorder who have a positive depression screen should have a more thorough assessment of mental health symptoms and comorbidities, rather than immediate diagnosis of and treatment for depression.

Comparative Genomics of Two Closely Related Wolbachia with Different Reproductive Effects on Hosts.

Wolbachia pipientis are obligate intracellular bacteria commonly found in many arthropods. They can induce various reproductive alterations in hosts, including cytoplasmic incompatibility, male-killing, feminization, and parthenogenetic development, and can provide host protection against some viruses and other pathogens. Wolbachia differ from many other primary endosymbionts in arthropods because they undergo frequent horizontal transmission between hosts and are well known for an abundance of mobile elements and relatively high recombination rates. Here, we compare the genomes of two closely related Wolbachia (with 0.57% genome-wide synonymous divergence) that differ in their reproductive effects on hosts. wVitA induces a sperm-egg incompatibility (also known as cytoplasmic incompatibility) in the parasitoid insect Nasonia vitripennis, whereas wUni causes parthenogenetic development in a different parasitoid, Muscidifurax uniraptor Although these bacteria are closely related, the genomic comparison reveals rampant rearrangements, protein truncations (particularly in proteins predicted to be secreted), and elevated substitution rates. These changes occur predominantly in the wUni lineage, and may be due in part to adaptations by wUni to a new host environment, or its phenotypic shift to parthenogenesis induction. However, we conclude that the approximately 8-fold elevated synonymous substitution rate in wUni is due to a either an elevated mutation rate or a greater number of generations per year in wUni, which occurs in semitropical host species. We identify a set of genes whose loss or pseudogenization in the wUni lineage implicates them in the phenotypic shift from cytoplasmic incompatibility to parthenogenesis induction. Finally, comparison of these closely related strains allows us to determine the fine-scale mutation patterns in Wolbachia Although Wolbachia are AT rich, mutation probabilities estimated from 4-fold degenerate sites are not AT biased, and predict an equilibrium AT content much less biased than observed (57-50% AT predicted vs. 76% current content at degenerate sites genome wide). The contrast suggests selection for increased AT content within Wolbachia genomes.

Pain and psychiatric comorbidities among two groups of Iraq and Afghanistan era Veterans.

This study aimed to (1) identify the prevalence and severity of pain and psychiatric comorbidities among personnel who had been deployed during Operation Iraqi Freedom (OIF), Operation Enduring Freedom (OEF), and Operation New Dawn (OND) and (2) assess whether the Department of Veterans Affairs (VA) Polytrauma System of Care and an OIF/OEF/OND registry reflect real differences among patients. Participants (N = 359) were recruited from two VA hospitals. They completed a clinical interview, structured diagnostic interview, and self-report measures. Results indicated pain was the most common complaint, with 87 percent experiencing pain during the prior week and 56 percent reporting moderate or severe pain. Eighty percent of participants met criteria for at least one of seven assessed comorbid problems (moderate or severe pain, postconcussional disorder, posttraumatic stress disorder [PTSD], anxiety disorder, mood disorder, substance use disorder, psychosis), and 59 percent met criteria for two or more problems. PTSD and postconcussional disorder rarely occurred in the absence of pain or other comorbidities (0.3% and 0%, respectively). The Polytrauma group had more comorbid psychiatric conditions (χ(2) = 48.67, p < 0.05) and reported greater severity of symptoms (p < 0.05) than the Registry group. This study confirmed the high prevalence of pain and concurrent mental health problems among personnel returning from military deployment.

Cytoplasmic incompatibility and sperm cyst infection in different Drosophila-Wolbachia associations.

Wolbachia are a group of maternally transmitted obligatory intracellular alpha-proteobacteria that infect a wide range of arthropod and nematode species. Wolbachia infection in Drosophila in most cases is associated with the induction of cytoplasmic incompatibility (CI), manifested as embryonic lethality of offspring in a cross between infected males and uninfected females. While the molecular basis of CI is still unknown, it has been suggested that two bacterial functions are involved: mod (for modification) modifies the sperm during spermatogenesis and resc (for rescue) acts in the female germline and/or in early embryos, neutralizing the modification. There is considerable variation in the level of incompatibility in different Wolbachia/host interactions. We examine the relationship between the levels of CI in a number of naturally infected and transinfected Drosophila hosts and the percentage of Wolbachia-infected sperm cysts. Our results indicate the presence of two main groups of Drosophila-Wolbachia associations: group I, which exhibits a positive correlation between CI levels and the percentage of infected sperm cysts (mod(+) phenotype), and group II, which does not express CI (mod(-) phenotype) irrespective of the infection status of the sperm cysts. Group II can be further divided into two subgroups: The first one contains associations with high numbers of heavily Wolbachia-infected sperm cysts while in the second one, Wolbachia is rarely detected in sperm cysts, being mostly present in somatic cells. We conclude that there are three requirements for the expression of CI in a host-Wolbachia association: (a) Wolbachia has to be able to modify sperm (mod(+) genotype), (b) Wolbachia has to infect sperm cysts, and (c) Wolbachia has to be harbored by a permissive host.