Multicentered masked placebo-controlled phase 2 clinical study of an extended duration transdermal buprenorphine solution for postoperative pain in cats.

A prospective, double-masked, placebo-controlled, multicentered phase 2 clinical study was conducted to select the transdermal buprenorphine solution (TBS) dosage for the control of postoperative pain in cats. One-hundred fifteen (115) cats were randomized to a single topical dose of placebo solution, low-TBS dosage (1.91-2.07 mg/kg) or high-TBS dosage (4.27-4.88 mg/kg) prior to surgical reproductive sterilization in conjunction with forelimb onychectomy. The low- and high-TBS doses were applied 2-4 and 1-2 hours prior to surgery. Interactive pain assessments and physiological variables were quantified through 96 hours post-anesthetic recovery and rescue analgesia was administered any time that analgesia was considered inadequate. Cats requiring rescue analgesia were considered treatment failures. The estimated overall treatment success rates from generalized linear mixed effects model analysis were 0.10 (95% CI: [0.02-0.36]), 0.56 (95% CI: [0.25-0.83]), 0.71 (95% CI: [0.38-0.91]) in the placebo-, low-, and high-TBS dose groups, respectively. Success rates for both TBS treatment groups were superior to placebo. Adverse events were infrequent in all treatment groups although the postoperative body temperatures over the duration of the study were on average 0.31 (95% CI: [0.08-0.55]) and 0.30 (95% CI: [0.05-0.53]) °C higher in low- and high-TBS dose cats, respectively, compared to placebo. It is concluded that both the low- and high-TBS dosages were safe and effective. The high-TBS dosage resulted in a greater proportion of treatment successes over 96 h, had a more acceptable preoperative application time of 1-2 h prior to surgery, and was therefore selected for further study.

Multicentered masked placebo-controlled phase 3 clinical study of an extended duration transdermal buprenorphine solution for post-operative pain in cats.

A prospective, double masked, placebo-controlled, multicentered phase 3 clinical study was conducted to evaluate the safety and effectiveness of transdermal buprenorphine solution (TBS) for the control of post-operative pain in cats. A total of 228 cats from 12 US investigational sites met the enrollment criteria of which 107 placebo- and 112 TBS-treated cats were included into the per protocol efficacy analysis. The dose of TBS was 8 mg (0.4 ml) to cats 1.2 to 3 kilograms and 20 mg (1 ml) to cats >3 to 7.5 kilograms applied topically to the dorsal unclipped cervical skin 1-2 h prior to the undergoing elective surgical reproductive sterilization in conjunction with forelimb onychectomy. Interactive pain assessments and physiological variables were quantified through 96 h following recovery from anesthesia, and rescue analgesia was administered any time that pain control was scored inadequate. Cats requiring rescue analgesia or experiencing an adverse event suspected to be treatment related were considered treatment failures. Sixty-five and 23 cats were considered treatment failures in the placebo and TBS groups, respectively, with most occurring on the day of surgery. The treatment success rates were 0.40 (95% confidence interval [CI]: [0.28-0.53]) and 0.81 (95% CI: [0.70-0.89]) in the placebo and TBS groups, respectively, and the difference was significant (p < .05). Adverse events occurred at a similar frequency and were not clinically meaningful in either treatment group. The post-operative body temperatures over the duration of the study were on average 0.35 (95% CI: [0.20-0.50]) °C higher than baseline in TBS-treated cats and were not clinically meaningful, an observation typical of opioids in cats. These results serve as substantial evidence that TBS is safe and effective for the control of orthopedic and soft tissue post-operative pain in cats when a single topical dose is applied 1-2 h prior to surgery.

Margin of safety of extended-duration transdermal buprenorphine solution following multiple-dose administrations to cats.

Transdermal buprenorphine solution (TBS) is approved for the control of postoperative pain in cats where a single preoperative dose provides 4 days of analgesia. It is administered as a unit dose of 8 mg to cats weighing 1.2-3 kg and 20 mg to cats weighing to >3-7.5 kg, which is equivalent to a dosage on a bodyweight basis of 2.7-6.7 mg/kg. In this safety study, the 1X dose was defined as 6.7 mg/kg. Thirty-two cats (16 males and 16 females) were randomly allocated to placebo, 1, 2, and 3X TBS administered topically to the dorsal cervical skin every 4 days for 3 doses. Clinical observations, behavioral scores, mydriasis score (yes/no), and physiological variables were assessed or measured prior to each dose administration (0 h) and at 1, 2, 4, 8, 12, 24, 36, 48, and 72 h following each treatment and prior to euthanasia on Day 12 or 13. Blood samples for clinical pathology were collected on Days - 1, 4, 8, and prior to euthanasia. There was little evidence of respiratory, cardiovascular, or gastrointestinal effects. Respiratory rates were above the reference range in all groups and lower by 10 breaths/min in the 3X group during the third dosing interval compared to placebo. There were no differences in heart rates. Constipation was transiently observed in approximately equal numbers in placebo- and TBS-treated cats. Behavioral scores showed sedation or euphoria was transient in the first dosing interval but became more prolonged with each dosing interval. Mydriasis was prolonged in the first dosing interval and diminished by the third dosing interval consistent with accommodation. Mean body temperatures in TBS-treated cats were up to 0.6°C (1.8°F) greater than placebo-treated cats. There were no clinically relevant changes to serum chemistry, hematology, or urinalysis outcomes nor gross or microscopic observations attributable to TBS. These data demonstrate that TBS is safe and well-tolerated when administered to 16-week-old cats at multiples of the approved dose and duration and supports clinical safety in the event of delayed buprenorphine metabolism, medication errors, or alterations in the dosing regimen.

Single-dose pharmacokinetics and bioavailability of a novel extended duration transdermal buprenorphine solution in cats.

A novel transdermal buprenorphine solution (TBS) was developed for evaluation in order to make available an extended duration opioid analgesic for cats. Healthy adult cats were administered a single TBS dose of 10 mg (1.57-4.35 mg/kg), 30 mg (4.72-13.0 mg/kg), or 50 mg (7.87-21.7 mg/kg) (4 cats per group) applied topically to the unclipped dorsal cervical skin and plasma buprenorphine concentrations were evaluated through 7 days. To determine the absolute bioavailability of TBS, healthy cats were administered single TBS dose of 20 mg (3.33-4.76 mg/kg) or 0.05 mg (0.008-0.011 mg/kg) IV buprenorphine (6 cats per group). The mean ± standard deviation maximum plasma buprenorphine concentrations (Cmax ) were 10.5 ± 6.28, 18.6 ± 8.68, and 22.5 ± 4.47 ng/ml following 10, 30, and 50 mg doses, respectively, with the time of Cmax occurrence (tmax ) typically occurring at 2-12 h post-dosing. Mean plasma buprenorphine terminal half-lives ranged between 78.3 and 91.2 h. Increasing the dose threefold and fivefold from the 10 mg dose increased the exposure by 2.8- and 3.6-fold, respectively, indicating that plasma buprenorphine exposure increased in a less than proportional manner at doses >30 mg. Transient sedation, mydriasis, and euphoria were observed within 4 h post-dosing. Mean rectal temperatures were increased 0.6-0.9°C greater than baseline (37.4-37.8°C) through 168 h post-dosing. The absolute bioavailability was 16.0% (90% CI: [11.8%-21.7%]). Flip-flop pharmacokinetics were observed with a terminal elimination half-life of 0.82 ± 0.13 and 64.9 ± 15.0 h for IV buprenorphine and 20 mg of TBS, respectively. A single administration of TBS over a range of doses resulted in extended plasma buprenorphine concentrations and opioid physiological and behavioral effects.

Immunoassay-Amplified Responses Using a Functionalized MoS2-Based SPR Biosensor to Detect PAPP-A2 in Maternal Serum Samples to Screen for Fetal Down's Syndrome.

BACKGROUND: Due to educational, social and economic reasons, more and more women are delaying childbirth. However, advanced maternal age is associated with several adverse pregnancy outcomes, and in particular a high risk of Down's syndrome (DS). Hence, it is increasingly important to be able to detect fetal Down's syndrome (FDS). METHODS: We developed an effective, highly sensitive, surface plasmon resonance (SPR) biosensor with biochemically amplified responses using carboxyl-molybdenum disulfide (MoS2) film. The use of carboxylic acid as a surface modifier of MoS2 promoted dispersion and formed specific three-dimensional coordination sites. The carboxylic acid immobilized unmodified antibodies in a way that enhanced the bioaffinity of MoS2 and preserved biorecognition properties of the SPR sensor surface. Complete antigen pregnancy-associated plasma protein-A2 (PAPP-A2) conjugated with the carboxyl-MoS2-modified gold chip to amplify the signal and improve detection sensitivity. This heterostructure interface had a high work function, and thus improved the efficiency of the electric field energy of the surface plasmon. These results provide evidence that the interface electric field improved performance of the SPR biosensor. RESULTS: The carboxyl-MoS2-based SPR biosensor was used successfully to evaluate PAPP-A2 level for fetal Down's syndrome screening in maternal serum samples. The detection limit was 0.05 pg/mL, and the linear working range was 0.1 to 1100 pg/mL. The women with an SPR angle >46.57 m° were more closely associated with fetal Down's syndrome. Once optimized for serum Down's syndrome screening, an average recovery of 95.2% and relative standard deviation of 8.5% were obtained. Our findings suggest that carboxyl-MoS2-based SPR technology may have advantages over conventional ELISA in certain situations. CONCLUSION: Carboxyl-MoS2-based SPR biosensors can be used as a new diagnostic technology to respond to the increasing need for fetal Down's syndrome screening in maternal serum samples. Our results demonstrated that the carboxyl-MoS2-based SPR biosensor was capable of determining PAPP-A2 levels with acceptable accuracy and recovery. We hope that this technology will be investigated in diverse clinical trials and in real case applications for screening and early diagnosis in the future.

Development of a bioaffinity SPR immunosensor based on functionalized graphene oxide for the detection of pregnancy-associated plasma protein A2 in human plasma.

BACKGROUND: Graphene-like material such as functionalized carboxyl-graphene oxide (carboxyl-GO) can be intelligently tuned to achieve particular properties for biological and chemical sensing applications. METHODS: In this study, we propose a method to improve interference of non-specific proteins for use in human plasma assays. The highly specific interactions between molecules are an advantage of carboxyl-GO-based surface plasmon resonance (SPR) immunoassays, and this can be applied to spiked plasma samples with pregnancy-associated plasma protein A2 (PAPPA2). RESULTS: The experiment results showed that carboxyl-GO could be used to modulate the plasmon resonance energy, work function and conductivity properties. In addition, carboxyl groups could be used to enhance the conduction of electrons between carboxyl-GO and Au electrodes due to the excellent conductivity and electron transfer rate. The carboxyl-GO-based SPR chip exhibited high sensitivity based on the electric field amplification effects of the composite dielectric material. Therefore, the surface electric field could be enhanced by electron transfer, thereby greatly improving the sensitivity of the sensing system. Enhanced electric field intensity was generated around the carboxyl-GO of 63.58 V/m, and the measured work function was 4.95 eV. The results showed that the carboxyl-GO-based SPR biosensor had high sensitivity, affinity and selective ability for PAPPA2 protein with a high association rate constant (ka) of 3.1 ×109 M-1 S-1 and a limit of detection of 0.01 pg/mL in spiked human plasma. CONCLUSION: The results showed a detection accuracy of protein in spiked plasma of >90% compared to PBS buffer, suggesting that the carboxyl-GO-based SPR biosensor could be used in assays of human plasma for early and late gynecological diseases. The future of this technology will be useful for the diagnosis and evaluation of the risk of early maternal preeclampsia and potentially in clinical applications for gestational diseases.

Affinity capture surface carboxyl-functionalized MoS2 sheets to enhance the sensitivity of surface plasmon resonance immunosensors.

The development of functionalized molybdenum disulfide (MoS2) has led to a new trend in the biosensing field, owing to its high sensitivity and bio-affinity characteristics with regards to the simple synthesis of carboxyl-functionalized MoS2 nanocomposites. In this study, we used monochloroacetic acid (MCA) to successfully modify carboxyl-MoS2. The efficiency of this MCA modification method showed a higher -COOH group content of 30.1%, mainly due to chlorine atoms occupying the MoS2 sulfur vacancy to allow for the formation of a strong bonding effect. This then enhanced the surface area of -COOH and improved the formation of covalent bonds between proteins. We demonstrated that MoS2-COOH-based surface plasmon resonance (SPR) chips can provide excellent sensitivity and high affinity for immunoassay biomolecules detected in a low sample volume of 20 µl. With respect to the shifts of the SPR angles of the chips, the high binding affinity at a BSA concentration of 14.5 nM for a MoS2-COOH chip, a MoS2 chip and a traditional SPR chip are 4.69 m°, 2.49 m° and 1.53 m°, respectively. In addition, the MoS2-COOH chip could amplify the SPR angle response by 3.1 folds and enhance the high association rate of ka by 212 folds compared to MoS2 and traditional SPR chips. The results thus obtained revealed that the overall affinity binding value, KA, of the MoS2-COOH chip can be significantly enhanced by up to ∼ 6.5 folds that of the MoS2 chip. In summary, the excellent binding affinity, biocompatible and high sensitivity suggest the potential of the clinical application of this MoS2-COOH-based SPR chip detection method for in vitro diagnostic and point-of-care testing devices.

Ultra-high sensitivity of the non-immunological affinity of graphene oxide-peptide-based surface plasmon resonance biosensors to detect human chorionic gonadotropin.

Specific peptide aptamers can be used in place of expensive antibody proteins, and they are gaining increasing importance as sensing probes due to their potential in the development of non-immunological assays with high sensitivity, affinity and specificity for human chorionic gonadotropin (hCG) protein. We combined graphene oxide (GO) sheets with a specific peptide aptamer to create a novel, simple and label-free tool to detect abnormalities at an early stage of pregnancy, a GO-peptide-based surface plasmon resonance (SPR) biosensor. This is the first binding interface experiment to successfully demonstrate binding specificity in kinetic analysis biomechanics in peptide aptamers and GO sheets. In addition to the improved affinity offered by the high compatibility with the target hCG protein, the major advantage of GO-peptide-based SPR sensors was their reduced nonspecific adsorption and enhanced sensitivity. The calculation of total electric field intensity (ΔE) in the GO-based sensing interfaces was significantly enhanced by up to 1.2 times that of a conventional SPR chip. The GO-peptide-based chip (1mM) had a high affinity (KA) of 6.37×1012M-1, limit of detection of 0.065nM and ultra-high sensitivity of 16 times that of a conventional SPR chip. The sensitivity of the slope ratio of the low concentration hCG protein assay in linear regression analysis was GO-peptide (1mM): GO-peptide (0.1mM): conventional chip (8-mercaptooctanoic acid)-peptide (0.1mM)=8.6: 3.3: 1. In summary, the excellent binding affinity, low detection limit, high sensitivity, good stability and specificity suggest the potential of this GO-peptide-based SPR chip detection method in clinical application. The development of real-time whole blood analytic and diagnostic tools to detect abnormalities at an early stage of pregnancy is a promising technique for future clinical application.

Extralenticular and lenticular aspects of accommodation and presbyopia in human versus monkey eyes.

PURPOSE: To determine if the accommodative forward movements of the vitreous zonule and lens equator occur in the human eye, as they do in the rhesus monkey eye; to investigate the connection between the vitreous zonule posterior insertion zone and the posterior lens equator; and to determine which components-muscle apex width, lens thickness, lens equator position, vitreous zonule, circumlental space, and/or other intraocular dimensions, including those stated in the objectives above-are most important in predicting accommodative amplitude and presbyopia. METHODS: Accommodation was induced pharmacologically in 12 visually normal human subjects (ages 19-65 years) and by midbrain electrical stimulation in 11 rhesus monkeys (ages 6-27 years). Ultrasound biomicroscopy imaged the entire ciliary body, anterior and posterior lens surfaces, and the zonule. Relevant distances were measured in the resting and accommodated eyes. Stepwise regression analysis determined which variables were the most important predictors. RESULTS: The human vitreous zonule and lens equator move forward (anteriorly) during accommodation, and their movements decline with age, as in the monkey. Over all ages studied, age could explain accommodative amplitude, but not as well as accommodative lens thickening and resting muscle apex thickness did together. Accommodative change in distances between the vitreous zonule insertion zone and the posterior lens equator or muscle apex were important for predicting accommodative lens thickening. CONCLUSIONS: Our findings quantify the movements of the zonule and ciliary muscle during accommodation, and identify their age-related changes that could impact the optical change that occurs during accommodation and IOL function.

Effects of unilateral topical administration of 0.5% tropicamide on anterior segment morphology and intraocular pressure in normal cats and cats with primary congenital glaucoma.

OBJECTIVE: To determine the effects of topical 0.5% tropicamide on anterior segment morphology (ASM) and intraocular pressure (IOP) in normal and glaucomatous cats. ANIMALS USED: Normal cats and cats with inherited primary congenital glaucoma (PCG). PROCEDURES: Control IOP curves were performed in untreated normal and PCG cats. In the first experiment, tropicamide was applied OD in eight normal and nine PCG cats. IOP and pupillary diameter (PD) were measured at 0, 30, and 60 min, then hourly until 8 h post-treatment. In a second experiment, six normal and seven PCG cats received tropicamide OD. High-resolution ultrasound images were obtained at 0, 1, 5, and 10 h post-treatment to measure ASM changes. IOP and PD were measured OD at 0, 1, 2, 3, 5, 7, and 9 h. RESULTS: In untreated normal cats IOP OU decreased throughout the day. In PCG cats IOP OU had wide fluctuations over time. In normal cats IOP response varied in the treated eye but did not change significantly in untreated eyes. IOP significantly increased from baseline in both eyes of all treated PCG cats. Increases in IOP were associated with some ASM changes. Cats with PCG had a significantly smaller angle recess areas, diminished ciliary clefts and decreased iris-lens contact. ASM changes were not strongly correlated with IOP in all cats. CONCLUSIONS: The ASM of PCG cats is markedly different from normal cats, and clinically significant increases in IOP OU occur in cats with PCG after tropicamide treatment. The mechanism for this increase remains unclear.

Association of intestinal disorders in cats with findings of abdominal radiography.

OBJECTIVE: To compare the radiographic appearance of small and large intestines of cats with various medical conditions and create a quantitative index for interpretation of intestinal diameters on radiographic views of the abdomen. DESIGN: Retrospective cohort study. ANIMALS: 74 cats that underwent abdominal radiography. PROCEDURES: Cats were assigned to 1 of 4 diagnosis categories: no gastrointestinal tract disease (n = 20), nonobstructive gastrointestinal tract disease (32), linear foreign body (LFB; 11), and small intestinal mechanical obstruction not caused by an LFB (11). Abdominal radiographs were evaluated without knowledge of history or diagnosis. Maximum and minimum external small intestine diameter (SID) and colon diameter (CD) were compared; dorsoventral and mediolateral measurements of the cranial end plate of L2 (VEL2) and L5 vertebrae were compared. Dorsoventral height of VEL2 from lateral radiographic views was used to determine maximum-SID:VEL2 and maximum-CD:VEL2 ratios. Gas patterns were evaluated. RESULTS: Nonobstructive gastrointestinal tract disease was more likely than obstruction until a maximum-SID:VEL2 ratio > 2.0. At a maximum-SID:VEL2 ratio of 2.5, probability of a disease not related to the intestinal tract was < 4%. At a maximum-SID:VEL2 ratio of 3.0, probability of a mechanical intestinal obstruction was > 70%. When the maximum-CD:VEL2 ratio was 2.0, probability of LFB was 50%; as the maximum-CD:VEL2 ratio increased beyond 2.0, likelihood of LFB decreased. Both gas pattern and CD correlated with diagnosis category. CONCLUSIONS AND CLINICAL RELEVANCE: Normalizing ratios of maximum-SID:VEL2 and maximum-CD:VEL2 obtained from measurements on lateral radiographic views of the abdomen in cats were related to diagnosis category.

Age-related changes in centripetal ciliary body movement relative to centripetal lens movement in monkeys.

The goal was to determine the age-related changes in accommodative movements of the lens and ciliary body in rhesus monkeys. Varying levels of accommodation were stimulated via the Edinger-Westphal (E-W) nucleus in 26 rhesus monkeys, aged 6-27 years, and the refractive changes were measured by coincidence refractometry. Centripetal ciliary process (CP) and lens movements were measured by computerized image analysis of goniovideographic images. Ultrasound biomicroscopy (UBM) at 50 MHz was used to visualize and measure accommodative forward movements of the ciliary body in relation to age, accommodative amplitude, and centripetal CP and lens movements. At approximately 3 diopters of accommodation, the amount of centripetal lens movement required did not significantly change with age (p = 0.10; n = 18 monkeys); however, the amount of centripetal CP movement required significantly increased with age (p = 0.01; n = 18 monkeys), while the amount of forward ciliary body movement significantly decreased with age (p = 0.007; n = 11 monkeys). In the middle-aged animals (12-16.5 years), a greater amount of centripetal CP movement was required to induce a given level of lens movement and thereby a given level of accommodation (p = 0.01), compared to the young animals (6-10 yrs). Collectively, the data suggests that, with age, the accommodative system may be attempting to compensate for the loss of forward ciliary body movement by increasing the amount of centripetal CP movement. This, in turn, would allow enough zonular relaxation to achieve the magnitude of centripetal lens movement necessary for a given amplitude of accommodation.

Effect of daily prolonged ketamine anesthesia on intraocular pressure in monkeys.

PURPOSE: To determine if repeated intramuscular ketamine in monkeys on consecutive days affects intraocular pressure (IOP) and if the ketamine-induced IOP change has any relationship to systemic dehydration and/or changes in mean arterial pressure (MAP) of the animals. METHODS: Nine monkeys were studied per four protocols. IOP was determined hourly for 6 hr by Goldmann tonometry under ketamine anesthesia on 3 (protocol 1) or 5 (protocols 2 and 3) consecutive days, or on alternating days 1, 3, and 5 (protocol 4). Monkeys in protocols 3 and 4, but not in protocols 1 and 2, received subcutaneous Ringer's fluids at the end of each 6-hr session on days 1-4 or days 1, 3, and 5; monkeys in protocols 2 and 3 received intravenous fluid infusion throughout the experiment on day 5. In protocols 2-4, MAP was measured hourly following each IOP measurement. RESULTS: Monkeys receiving ketamine but no Ringer's fluids in protocol 1 or 2 showed significant IOP declines on days 2-3 or 2-4. The IOP declines were greater in magnitude in protocol 1 than in protocol 2. Daily subcutaneous Ringer's fluids appeared to delay IOP declines in protocol 3. Continuous intravenous fluid infusion on day 5 variably prevented IOP declines in protocols 2 and 3. Monkeys receiving ketamine and subcutaneous fluids on alternate days in protocol 4 showed no decline in IOP. No significant relationship between IOP and MAP was observed. CONCLUSIONS: Anesthesia induced by repeated intramuscular ketamine on consecutive days may produce significant IOP declines. Systemic dehydration during the anesthesia seems to be the predominant factor contributing to the IOP reduction. However, inter-individual differences in monkeys indicate that multiple factors may be involved. This study also suggests that fluid supplementation plus alternating anesthesia with recovery days may prevent IOP reduction in monkeys resulting from daily prolonged ketamine anesthesia.

Surgical intervention and accommodative responses, I: centripetal ciliary body, capsule, and lens movements in rhesus monkeys of various ages.

PURPOSE: To determine how surgically altering the normal relationship between the lens and the ciliary body in rhesus monkeys affects centripetal ciliary body and lens movement. METHODS: In 18 rhesus monkey eyes (aged 6-27 years), accommodation was induced before and after surgery by electrical stimulation of the Edinger-Westphal nucleus. Accommodative amplitude was measured by coincidence refractometry. Goniovideography was performed before and after intra- and extracapsular lens extraction (ICLE, ECLE) and anterior regional zonulolysis (ARZ). Centripetal lens/capsule movements, centripetal ciliary process (CP) movements, and circumlental space were measured by computerized image analysis of the goniovideography images. RESULTS: Centripetal accommodative CP and capsule movement increased in velocity and amplitude after, compared with before, ECLE regardless of age (n = 5). The presence of the lens substance retarded capsule movement by approximately 21% in the young eyes and by approximately 62% in the older eyes. Post-ICLE compared with pre-ICLE centripetal accommodative CP movement was dampened in all eyes in which the anterior vitreous was disrupted (n = 7), but not in eyes in which the anterior vitreous was left intact (n = 2). After anterior regional zonulolysis (n = 4), lens position shifted toward the lysed quadrant during accommodation. CONCLUSIONS: The presence of the lens substance, capsule zonular attachments, and Wieger's ligament may play a role in centripetal CP movement. The capsule is still capable of centripetal movement in the older eye (although at a reduced capacity) and may have the ability to produce approximately 6 D of accommodation in the presence of a normal, young crystalline lens or a similar surrogate.

Antagonistic bacterial interactions help shape host-symbiont dynamics within the fungus-growing ant-microbe mutualism.

Conflict within mutually beneficial associations is predicted to destabilize relationships, and theoretical and empirical work exploring this has provided significant insight into the dynamics of cooperative interactions. Within mutualistic associations, the expression and regulation of conflict is likely more complex than in intraspecific cooperative relationship, because of the potential presence of: i) multiple genotypes of microbial species associated with individual hosts, ii) multiple species of symbiotic lineages forming cooperative partner pairings, and iii) additional symbiont lineages. Here we explore complexity of conflict expression within the ancient and coevolved mutualistic association between attine ants, their fungal cultivar, and actinomycetous bacteria (Pseudonocardia). Specifically, we examine conflict between the ants and their Pseudonocardia symbionts maintained to derive antibiotics against parasitic microfungi (Escovopsis) infecting the ants' fungus garden. Symbiont assays pairing isolates of Pseudonocardia spp. associated with fungus-growing ants spanning the phylogenetic diversity of the mutualism revealed that antagonism between strains is common. In contrast, antagonism was substantially less common between more closely related bacteria associated with Acromyrmex leaf-cutting ants. In both experiments, the observed variation in antagonism across pairings was primarily due to the inhibitory capabilities and susceptibility of individual strains, but also the phylogenetic relationships between the ant host of the symbionts, as well as the pair-wise genetic distances between strains. The presence of antagonism throughout the phylogenetic diversity of Pseudonocardia symbionts indicates that these reactions likely have shaped the symbiosis from its origin. Antagonism is expected to prevent novel strains from invading colonies, enforcing single-strain rearing within individual ant colonies. While this may align ant-actinomycete interests in the bipartite association, the presence of single strains of Pseudonocardia within colonies may not be in the best interest of the ants, because increasing the diversity of bacteria, and thereby antibiotic diversity, would help the ant-fungus mutualism deal with the specialized parasites.