Three-dimensional visualisation of the feto-placental vasculature in humans and rodents.

Adequate development of the feto-placental circulation is critical for placental exchange function and healthy fetal growth. Understanding the structure of this circulation and how it informs fetal outcomes is important both in the human placenta, and the rodent, a purported comparative experimental model. Vascular casting and micro-CT imaging approaches enable detailed quantification of the complex vascular relationships in the feto-circulation, and provide detailed data to parameterise in silico models. Here, to assist researchers to apply these technically challenging methods we provide detailed approaches to cast and image; 1) human placentas at the cotyledon-level, and 2) whole rodent placentas.

Role of Ni substitution on structural, magnetic and electronic properties of epitaxial CoCr2O4 spinel thin films.

Cubic spinel CoCr2O4 has recently attained attention due to its multiferroic properties. However, the Co site substitution effect on the structural and magnetic properties has rarely been studied in thin film form. In this work, the structural and magnetic properties of Co1-x Ni x Cr2O4 (x= 0, 0.5) epitaxial thin films deposited on MgAl2O4 (100) and MgO (100) substrates to manipulate the nature of strain in the films using pulsed laser deposition (PLD) technique are presented. The epitaxial nature of the films was manifested through x-ray diffraction (XRD), reciprocal space mapping (RSM) and Rutherford backscattering spectrometry (RBS) measurements. Raman measurements revealed a disappearance of characteristic A 1 g and F 2 g modes of the CoCr2O4 with increase in the Ni content. Atomic force microscopy (AFM) and field emission scanning electron microscopy (FE-SEM) studies show a modification of the surface morphology upon Ni substitution. Magnetic measurements disclose that the ferrimagnetic Curie temperature (T C) of the CoCr2O4 in thin film grown on MgAl2O4 (100) and MgO (100) substrates were found to be 100.6 ± 0.5 K and 93.8 ± 0.2 K, respectively. With Ni substitution the T C values were found to be enhanced to 104.5 ± 0.4 K for MgAl2O4 (100) and 108.5 ± 0.6 K for MgO (100) substrates. X-ray photoelectron spectroscopy (XPS) suggests Cr3+ oxidation states in the films, while Co ions are present in a mixed Co2+/Co3+ oxidation state. The substitution of Ni at Co site significantly modifies the line shape of the core level as well as the valence band. Ni ions are also found to be in a mixed 2+/3+ oxidation state. O 1s core level display asymmetry related to possible defects like oxygen vacancies in the films.

Physiologically relevant screening of polyphenol-rich commercial preparations for bioactivity in vascular endothelial cells and application to healthy volunteers: A viable workflow and a cautionary tale.

This study describes the screening of 13 commercially-available plant extracts for pharmacological activity modulating vascular function using an endothelial cell model. A French maritime pine bark extract (FMPBE) was found to have the greatest effect upon nitric oxide availability in control (181% ± 36% of untreated cells) and dysfunctional cells (132% ± 8% of untreated control cells). In healthy volunteers, the FMPBE increased plasma nitrite concentrations 8 h post-consumption compared to baseline (baseline corrected median 1.71 ± 0.38 (25% IQR) and 4.76 (75% IQR) µM, p < 0.05). This was followed by a placebo-controlled, healthy volunteer study, which showed no effects on plasma nitrite. It was confirmed that different batches of extract had been used in the healthy volunteer studies, and this second batch lacked bioactivity, assessed using the in vitro model. No difference in plasma catechin levels was seen at 8 h following supplementation between the studies (252 ± 194 nM versus 50 ± 64 nM, p > 0.05), however HPLC-UV fingerprinting showed that the new batch had a 5-15% in major constituents (including procyanidins A2, B1 and B2) compared to the original batch. This research describes a robust mechanism for screening bioactive extracts for vascular effects. It also highlights batch variability as a significant limitation when using complex extracts for pharmacological activity, and suggests the use of in vitro systems as a tool to identify this problem in future studies.

Autosomal dominant tubulointerstitial kidney disease (ADTKD) in Ireland.

Introduction: Autosomal dominant tubulointerstitial kidney disease (ADTKD) is a rare genetic cause of renal impairment resulting from mutations in the MUC1, UMOD, HNF1B, REN, and SEC61A1 genes. Neither the national or global prevalence of these diseases has been determined. We aimed to establish a database of patients with ADTKD in Ireland and report the clinical and genetic characteristics of these families. Methods: We identified patients via the Irish Kidney Gene Project and referral to the national renal genetics clinic in Beaumont Hospital who met the clinical criteria for ADTKD (chronic kidney disease, bland urinary sediment, and autosomal dominant inheritance). Eligible patients were then invited to undergo genetic testing by a variety of methods including panel-based testing, whole exome sequencing and, in five families who met the criteria for diagnosis of ADTKD but were negative for causal genetic mutations, we analyzed urinary cell smears for the presence of MUC1fs protein. Results: We studied 54 individuals from 16 families. We identified mutations in the MUC1 gene in three families, UMOD in five families, HNF1beta in two families, and the presence of abnormal MUC1 protein in urine smears in three families (one of which was previously known to carry the genetic mutation). We were unable to identify a mutation in 4 families (3 of whom also tested negative for urinary MUC1fs). Conclusions: There are 4443 people with ESRD in Ireland, 24 of whom are members of the cohort described herein. We observe that ADTKD represents at least 0.54% of Irish ESRD patients.

Menopause in Latin America: Symptoms, attitudes, treatments and future directions in Costa Rica.

Similar to their US counterparts, Costa Rican women enter menopause at ∼50 years of age, have similar symptoms, including hot flashes and night sweats, as well as an overall negative attitude toward the menopausal transition. One study of rural women in Monteverde reported that women knew little about the menopausal transition, as the subject was not discussed. Similar to other Latin American women, the use of hormone therapy by Costa Rican women is low and instead they use alternative therapies, including massage, dietary changes and herbal medicines. A wide variety of herbal therapies are used, and some of these herbs have estrogenic activities in vitro. However, clinical data on the safety and efficacy of any of these treatments is lacking. Recently, a disturbing increase in the incidence of human papilloma virus infections in menopausal women has been reported, due in part to more sexual freedom after menopause. Fortunately, the strain of HPV infecting these women is not associated with cervical cancer. Overall, there is a significant lack of scientific and medical research on menopausal women in Costa Rica. Considering the aging population, the high use of herbal medicines by menopausal women and the lack of clinical studies on these treatments, future research should focus on gaining a better understanding of menopause in this population. Furthermore, new educational programs for these women and the health professionals who serve them are necessary, as well as investigations of the safety and efficacy of the herbal supplements women use to manage their menopausal symptoms.

Facing herbivory on the climb up: Lost opportunities as the main cost of herbivory in the wild yam Dioscorea praehensilis.

Plants with simple architecture and strong constraints on their growth may offer critical insights into how growth strategies affect the tolerance of plants to herbivory. Although Dioscorea praehensilis, a wild yam of African forests, is perennial, both aerial apparatus and tuber are annually renewed. Each year, the tuber produces a single stem that climbs from the ground to the forest canopy. This stem bears no leaves and no branches until it reaches optimal light conditions. Once in the canopy, the plant's production fuels the filling of a new tuber before the plant dies back to the ground. We hypothesized that if deprived of ant defense, the leafless growth phase is a vulnerable part of the cycle, during which a small amount of herbivory entails a high cost in terms of loss of opportunity. We compared the growth of stems bearing ants or not as well as of intact stems and stems subjected to simulated or natural herbivory. Ants reduce herbivory; herbivory delays arrival to the canopy and shortens the season of production. Artificially prolonging the stem growth to the canopy increased plant mortality in the following year and, in surviving plants, reduced the stem diameter and likely the underground reserves produced. Tuber size is a key variable in plant performance as it affects both the size of the aerial apparatus and the duration of its single season of production. Aerial apparatus and tuber are thus locked into a cycle of reciprocal annual renewal. Costs due to loss of opportunity may play a major role in plant tolerance to herbivory, especially when architectural constraints interact with ecological conditions to shape the plant's growth strategy.

Validation of carcass lesions as indicators for on-farm health and welfare of pigs.

Incorporating indicators for pig health and welfare at meat inspection could reduce the need for on-farm assessments. Skin and tail lesions are important welfare indicators in pigs with good potential to record during meat inspection and could possibly function as iceberg indicators of on farm welfare. The aim of this study was to validate the use of these carcass lesions at meat inspection for the assessment of pig health and welfare on farm. Thirty-one farrow-to-finish pig farms (∼12% of Irish herds) were assessed using an adapted version of the Welfare Quality protocol by inspecting 6 randomly selected pens of pigs in the first weaner (4 to 8 wk), second weaner (8 to 13 wk) and finisher stage (13 to 23 wk). The average prevalence of welfare outcomes for each stage was calculated. One batch of pigs was observed at slaughter and skin and tail lesions were scored according to severity for each carcass. The average prevalence of carcass lesion outcomes was calculated for each farm. Linear regression models were developed to predict the prevalence of each welfare outcome in each stage based on the prevalence of the different carcass lesions. The welfare outcomes of different welfare aspects that were best predicted by abattoir information (highest ) were poor body condition (first weaner stage), bursitis (second weaner stage), huddling (first weaner stage), severe tail lesions (finisher stage) and coughing (second weaner stage). Regression trees and receiver-operating curves (ROC) were used to evaluate the usefulness of carcass lesions as monitoring tools. Receiver-operating curves were created using the 75th percentile to classify farms as a problem farm for these welfare outcomes. Cut-off values of predictive carcass lesion prevalence were similar using both techniques. Models for predicting problem farms with poor body condition, bursitis and severe tail lesions were moderately accurate. Sensitivity and specificity ranged from 75 to 100% and 70 to 87%, respectively at the optimal cut-off value of the predictive carcass lesion prevalence. Results show potential for using carcass skin and tail lesions as iceberg indicators of pig health and welfare on farm. Future work is needed to evaluate the cost of including carcass lesion recording at meat inspection, the cost of failing to identify problem farms and the cost of incorrectly visiting or penalizing problem farms before carcass lesions can be used as welfare indicators in a commercial setting.

Present-day African analogue of a pre-European Amazonian floodplain fishery shows convergence in cultural niche construction.

Erickson [Erickson CL (2000) Nature 408 (6809):190-193] interpreted features in seasonal floodplains in Bolivia's Beni savannas as vestiges of pre-European earthen fish weirs, postulating that they supported a productive, sustainable fishery that warranted cooperation in the construction and maintenance of perennial structures. His inferences were bold, because no close ethnographic analogues were known. A similar present-day Zambian fishery, documented here, appears strikingly convergent. The Zambian fishery supports Erickson's key inferences about the pre-European fishery: It allows sustained high harvest levels; weir construction and operation require cooperation; and weirs are inherited across generations. However, our comparison suggests that the pre-European system may not have entailed intensive management, as Erickson postulated. The Zambian fishery's sustainability is based on exploiting an assemblage dominated by species with life histories combining high fecundity, multiple reproductive cycles, and seasonal use of floodplains. As water rises, adults migrate from permanent watercourses into floodplains, through gaps in weirs, to feed and spawn. Juveniles grow and then migrate back to dry-season refuges as water falls. At that moment fishermen set traps in the gaps, harvesting large numbers of fish, mostly juveniles. In nature, most juveniles die during the first dry season, so that their harvest just before migration has limited impact on future populations, facilitating sustainability and the adoption of a fishery based on inherited perennial structures. South American floodplain fishes with similar life histories were the likely targets of the pre-European fishery. Convergence in floodplain fish strategies in these two regions in turn drove convergence in cultural niche construction.

Quantitative resonant soft x-ray reflectivity of ultrathin anisotropic organic layers: Simulation and experiment of PTCDA on Au.

Resonant soft X-ray reflectivity at the carbon K edge, with linearly polarized light, was used to derive quantitative information of film morphology, molecular arrangement, and electronic orbital anisotropies of an ultrathin 3,4,9,10-perylene tetracarboxylic dianhydride (PTCDA) film on Au(111). The experimental spectra were simulated by computing the propagation of the electromagnetic field in a trilayer system (vacuum/PTCDA/Au), where the organic film was treated as an anisotropic medium. Optical constants were derived from the calculated (through density functional theory) absorption cross sections of the single molecule along the three principal molecular axes. These were used to construct the dielectric tensor of the film, assuming the molecules to be lying flat with respect to the substrate and with a herringbone arrangement parallel to the substrate plane. Resonant soft X-ray reflectivity proved to be extremely sensitive to film thickness, down to the single molecular layer. The best agreement between simulation and experiment was found for a film of 1.6 nm, with flat laying configuration of the molecules. The high sensitivity to experimental geometries in terms of beam incidence and light polarization was also clarified through simulations. The optical anisotropies of the organic film were experimentally determined and through the comparison with calculations, it was possible to relate them to the orbital symmetry of the empty electronic states.

Assessment of clinical occupational dose reduction effect of a new interventional cardiology shield for radial access combined with a scatter reducing drape.

OBJECTIVES: To assess the occupational dose reduction effect of a new interventional cardiology shield for radial access combined with a scatter reducing drape. BACKGROUND: Transradial access for catheterization has been shown to increase occupational radiation dose. Current shielding techniques are primarily based on the femoral access. This article looks at the clinical occupational combined dose reduction effect of a commercially available shield and drape which is specific to access type. METHODS: The evaluation took place in a busy interventional cardiology laboratory, with a single plane 30×40 cm flat panel detector (Siemens Artis Zee, Germany). Radiation exposure to staff was measured using electronic personal dosimeters (Unfors RaysafeAB, Sweden) placed at the collar. Patient radiation exposure was assessed using screening time and dose area product per case. Both staff and patient radiation exposure were monitored for a number of case types and operators before, during, and after deployment of the new shield and drapes. RESULTS: The cardiologists' overall median collar badge reading per case reduced from 15.4 µSv per case without the shield/drape combination to 7.3 µSv per case with the shield drape combination in situ (P<0.001). The radiographers badge reading was reduced from 4.2 µSv per case without to 2.5 µSv per case with the shield drape combination in situ (P<0.001). There was no statistical difference in the cardiac technician's badge reading. Patient's dose area product was not significantly affected by the placement of the shield and drape combination. CONCLUSIONS: The shield/drape combination can significantly reduce operator exposure in a cardiac catheterization laboratory.

Red cell alloimmunisation following intrauterine transfusion and the feasibility of providing extended phenotype-matched red cell units.

OBJECTIVES: To analyse the incidence of additional alloantibody formation following intrauterine red cell transfusion and to evaluate the feasibility of providing extended phenotype-matched red cells in future intrauterine transfusion (IUT). BACKGROUND: IUT is performed in severe, life-threatening fetal anaemia, usually in alloimmunised pregnancies. Its complications include the formation of additional alloantibodies to other red cell antigens. MATERIALS AND METHODS: This was an 11-year retrospective, observational study of additional alloantibody formation in patients receiving IUT in the National Maternity Hospital, Dublin. The study included evaluation of the donor population in the Republic of Ireland (RoI) with regards to the feasibility of providing extended phenotype-matched units in future IUT. RESULTS: Following IUT, 22% of mothers formed additional red cell alloantibodies. In 67% of cases, the transfused donor red cells expressed the cognate antigen. Suitable donors are available for most combinations of Fy, Jk and Ss antigens. CONCLUSIONS: In our population, it is feasible to provide more extensively phenotype-matched red cells for future IUT. These can be supplied from the current donor pool with no significant extra phenotyping required. We consider their provision to be a reasonable proactive step in a known at-risk group.

Neurosurgical capacity building in the developing world through focused training.

OBJECT: In Tanzania, there are 4 neurosurgeons for a population of 46 million. To address this critical shortage of neurosurgical care, the authors worked with local Tanzanian health care workers, neurosurgeons, the Ministry of Health and Social Welfare, and the Office of the President of Tanzania to develop a train-forward method for sustainable, self-propagating basic and emergency neurosurgery in resource-poor settings. The goal of this study was to assess the safety and effectiveness of this method over a 6-year period. METHODS: The training method utilizes a hands-on bedside teaching technique and was introduced in 2006 at a remote rural hospital in northern Tanzania. Local health care workers were trained to perform basic and emergency neurosurgical procedures independently and then were taught to train others. Outcome information was retrospectively collected from hospital records for the period from 2005 (1 year before method implementation) through 2010. Analysis of de-identified data included descriptive statistics and multivariable assessment of independent predictors of complications following a patient's first neurosurgical procedure. RESULTS: By 2010, the initial Tanzanian trainee had trained a second Tanzanian health care worker, who in turn had trained a third. The number of neurosurgical procedures performed increased from 18 in 2005 to an average of 92 per year in the last 3 years of the study period. Additionally, the number of neurosurgical cases performed independently by Tanzanian health care providers increased significantly from 44% in 2005 to 86% in 2010 (p < 0.001), with the number of complex cases independently performed also increasing over the same time period from 34% to 83% (p < 0.001). Multivariable analysis of clinical patient outcome information to assess safety indicated that postoperative complications decreased significantly from 2005 through 2010, with patients who had been admitted as training progressed being 29% less likely to have postoperative complications (OR 0.71, 95% CI 0.52-0.96, p = 0.03). CONCLUSIONS: The Madaktari Africa train-forward method is a reasonable and sustainable approach to improving specialized care in a resource-poor setting.

Differential expression of mitochondrial energy metabolism profiles across the metaplasia-dysplasia-adenocarcinoma disease sequence in Barrett's oesophagus.

Contemporary clinical management of Barrett's oesophagus has highlighted the lack of accurate predictive markers of disease progression to oesophageal cancer. This study aims to examine alterations in mitochondrial energy metabolism profiles across the entire disease progression sequence in Barrett's oesophagus. An in-vitro model was used to screen 84 genes associated with mitochondrial energy metabolism. Three energy metabolism genes (ATP12A, COX4I2, COX8C) were significantly altered across the in-vitro Barrett's disease sequence. In-vivo validations across the Barrett's sequence demonstrated differential expression of these genes. Tissue microarrays demonstrated significant alterations in both epithelial and stromal oxidative phosphorylation (ATP5B and Hsp60) and glycolytic (PKM2 and GAPDH) protein markers across the in-vivo Barrett's sequence. Levels of ATP5B in sequential follow up surveillance biopsy material segregated Barrett's non progressors and progressors to HGD and cancer. Utilising the Seahorse XF24 flux analyser, in-vitro Barrett's and adenocarcinoma cells exhibited altered levels of various oxidative parameters. We show for the first time that mitochondrial energy metabolism is differentially altered across the metaplasia-dysplasia-adenocarcinoma sequence and that oxidative phosphorylation profiles have predictive value in segregating Barrett's non progressors and progressors to adenocarcinoma.

A correlation between severe haemolytic disease of the fetus and newborn and maternal ABO blood group.

OBJECTIVE: To analyse anti-D quantification levels and frequency of intrauterine transfusion (IUT), per maternal ABO blood group. BACKGROUND: Maternally derived red cell allo-antibodies can target fetal red cell antigens in utero leading to haemolytic disease and fetal anaemia. When a clinically significant allo-antibody is formed the priority is ascertaining the risk to the fetus and maternal ABO blood groups are not considered relevant. MATERIALS AND METHODS: This was a 10-year retrospective, observational study carried out on women referred for anti-D quantification (n = 1106), and women whose fetuses required an IUT to treat fetal anaemia (n = 62) due to anti-D, in the Republic of Ireland. RESULTS: Relative to the overall incidence of RhD allo-immunisation by blood group, women of blood group A were more likely to require IUT compared with those who were blood group O (P = 0.002). CONCLUSION: It is known that ABO feto-maternal compatibility can influence the incidence and level of red cell allo-antibodies in pregnancy; however, it does not account for the significantly high rate of severe haemolytic disease requiring IUT seen in blood group A women.

Debt crisis ahead for Irish medical students.

Internationally medical student debt is a cause of concern. A survey of medical students in UCC (response rate of 191 representing 35% of the EU student cohort) reveals that 34 (26%) of direct entry medicine (DEM) students and 36 (61%) graduate entrants (GEM) have a loan with an anticipated average debt of Euro17,300 and Euro80,000 on graduation respectively. Fifty-three (90%) graduate entrants and 75 (57%) direct entrants revealed that they often worry about their current financial situation. Fifty-three (28%) of students have a part-time job and many were concerned about the degree to which this conflicted with their academic workload. 118 (89%) of school leavers and 48 (81%) graduates received financial assistance from their families to fund their college expenses. Student responses recommended the introduction of a government supported low interest rate loan and other incentives to help service high levels of debt associated with medical education.

Reassessing critical maternal antibody threshold in RhD alloimmunization: a 16-year retrospective cohort study.

OBJECTIVE: To determine the critical maternal antibody threshold for specialist referral in cases of RhD alloimmunization. METHODS: This was a retrospective cohort study, covering a 16-year period at the national tertiary fetal medicine center for management of alloimmunization. Data concerning RhD alloimmunized pregnant women were extracted from an institutional database and maternal anti-D antibody levels were cross-checked with the national reference laboratory. Fetal hemoglobin (Hb) levels were determined only at the first intrauterine transfusion (IUT) and were compared with the pretransfusion maternal anti-D antibody level (IU/mL). Sensitivity, specificity and positive and negative predictive values of maternal antibody thresholds for detecting moderate to severe (Hb ≤ 0.64MoM) fetal anemia were calculated. RESULTS: Between 1996 and 2011, 66 women underwent a first IUT for RhD alloimmunization at our institution. The highest serum anti-D antibody level was extracted for 208 RhD alloimmunized women who did not require IUT during the last 10 years of the study period. The traditional maternal antibody threshold of > 15 IU/mL failed to detect 20% of cases of moderate to severe fetal anemia. The ≥ 4 IU/mL threshold had 100% sensitivity but a 45% false-positive rate. The optimal anti-D antibody threshold for specialist referral in our population was ≥ 6 IU/mL; at this level, no case of moderate to severe anemia was missed and specificity was 61%. Use of this threshold would have eliminated 10% of referrals to our fetal medicine unit without compromising fetal outcomes. CONCLUSIONS: Setting the critical maternal RhD antibody level at >15 IU/mL does not provide sufficient sensitivity. The lower threshold of ≥ 4 IU/mL, though sensitive, is associated with a 45% false-positive rate. In our population, a threshold of ≥ 6 IU/mL minimizes false-positive referrals while maintaining 100% sensitivity for moderate to severe fetal anemia.

On the potential of hydrated storage for naturally derived ECMs and associated effects on mechanical and cellular performance.

Tissue engineered acellular vascular grafts are an emerging concept in the development of vascular prostheses for the minimally invasive treatment of cardiovascular diseases. Extracellular matrix (ECM) scaffolds, such as small intestinal submucosa (SIS) and urinary bladder matrix (UBM), offer many advantages over currently available synthetic devices. However, storage of such biomaterials can unduly influence the scaffold properties. This study evaluated the effects of up to 16 weeks hydrated storage on the mechanical and cellular performance of stented and unstented tubular scaffolds. This study aimed to demonstrate the viability, mechanical integrity, and bioactive potential of xenogeneic ECMs as potential off-the-shelf vascular prosthetic devices. Rehydrated ECM samples versus the lyophilized controls showed an increase in UTS and stiffness. The mechanical strength of all samples evaluated was above the average reported aortic tissue failure strength and more compliant than current synthetic materials employed. Post-storage cellular bioactivity investigations indicated that both ECM scaffolds tested were unaffected by increased hydrated storage duration when compared with the controls. Overall, the results indicate that the biomechanical and biologic properties of ECMs are not negatively affected by long-term hydrated storage. Therefore, with further investigations, naturally derived ECM materials may offer potential as an off-the-shelf therapeutic treatment of cardiovascular diseases.

Repeated evolution of fungal cultivar specificity in independently evolved ant-plant-fungus symbioses.

Some tropical plant species possess hollow structures (domatia) occupied by ants that protect the plant and in some cases also provide it with nutrients. Most plant-ants tend patches of chaetothyrialean fungi within domatia. In a few systems it has been shown that the ants manure the fungal patches and use them as a food source, indicating agricultural practices. However, the identity of these fungi has been investigated only in a few samples. To examine the specificity and constancy of ant-plant-fungus interactions we characterised the content of fungal patches in an extensive sampling of three ant-plant symbioses (Petalomyrmex phylax/Leonardoxa africana subsp. africana, Aphomomyrmex afer/Leonardoxa africana subsp. letouzeyi and Tetraponera aethiops/Barteria fistulosa) by sequencing the Internal Transcribed Spacers of ribosomal DNA. For each system the content of fungal patches was constant over individuals and populations. Each symbiosis was associated with a specific, dominant, primary fungal taxon, and to a lesser extent, with one or two specific secondary taxa, all of the order Chaetothyriales. A single fungal patch sometimes contained both a primary and a secondary taxon. In one system, two founding queens were found with the primary fungal taxon only, one that was shown in a previous study to be consumed preferentially. Because the different ant-plant symbioses studied have evolved independently, the high specificity and constancy we observed in the composition of the fungal patches have evolved repeatedly. Specificity and constancy also characterize other cases of agriculture by insects.