Cyclommatus metallifer), juvenile hormone (JH) titers are correlated with the extreme growth of an exaggerated weapon of sexual selection. We then investigate the putative role of JH in the development of the nutritionally-dependent, phenotypically plastic mandibles, by increasing hemolymph titers of JH with application of the JH analog fenoxycarb during larval and prepupal developmental periods. Increased JH signaling during the early prepupal period increased the proportional size of body parts, and this was especially pronounced in male mandibles, enhancing the exaggerated size of this trait. The direction of this response is consistent with the measured JH titers during this same period. Combined, our results support a role for JH in the nutrition-dependent regulation of extreme mandible growth in this species. In addition, they illuminate mechanisms underlying the evolution of trait proportion, the most salient feature of the evolutionary diversification of the insects.The morphological diversity of insects is one of the most striking phenomena in biology. Evolutionary modifications to the relative sizes of body parts, including the evolution of traits with exaggerated proportions, are responsible for a vast range of body forms. Remarkable examples of an insect trait with exaggerated proportions are the mandibular weapons of stag beetles. Male stag beetles possess extremely enlarged mandibles which they use in combat with rival males over females. As with other sexually selected traits, stag beetle mandibles vary widely in size among males, and this variable growth results from differential larval nutrition. However, the mechanisms responsible for coupling nutrition with growth of stag beetle mandibles (or indeed any insect structure) remain largely unknown. Here, we demonstrate that during the development of male stag beetles ( Insects display an astounding variety of forms. Much of this diversity can be understood as the result of evolutionary changes in the proportional sizes of body parts Stag beetles are complete metamorphic developers. Larvae undergo three instars and increase in weight over a combined feeding period of several months. At this point they have completed all growth in body size, and they begin the complex process of metamorphosis including gut purge, regression of larval-specific tissues, and rapid growth of adult structures. Eyes, wings, legs, genitalia and mouthparts all undergo rapid growth during this prepupa period, and in males, mandible growth is especially prolific at this time. Histological examination of the mandibles of both large and small males showed huge differences in epithelial proliferation during development . Under tManduca sextaIt is during the prepupa period that physiological signals are predicted to modulate the amount of growth in adult structures in response to nutrition. One proposed mechanism suggests that JH acts as a nutrition-sensitive regulator of trait growth In this study, we provide correlative evidence that JH regulates the exaggerated growth of male stag beetle mandibles. To test the role of JH during adult mandible development, we increased hemolymph titers of JH by application of the JH analog (JHA) fenoxycarb during both larval and prepupal development. Because growth in overall body size has ceased by the prepupal period, we predicted that perturbation of JH signaling during the prepupal period would affect growth of the adult structures without altering body size –i.e. it would alter the proportional sizes of body parts. As predicted, increasing JH signaling during the early prepupal period increased the proportional size of body parts, and this was especially pronounced in male mandibles  and a high nutrition group (reared in 430 ml cups). They were kept within an incubator under constant darkness at 25±0.5°C. Growth cup size affects the amount of available food and limits space for growth, affecting larval and adult body size. Growth between the two conditions was measured as time to pupation, pupal weight, and adult left-mandible length. Statistical analyses between the two groups were carried out with Student's t-test.The structure of the newly-formed mandibles of large males, small males, and females was observed by scanning electron microscopy (SEM) as described in The juvenile hormone analog (JHA) fenoxycarb , were diluted in acetone and topically applied to male third instar larva 3 days after molting in doses of 50 and 100 µg/larva). Ten µl of the JHA solution was applied to the dorsal thorax surface of each larva. Immediately after JHA application, the treated larvae were placed into individual 430 ml plastic cups filled with decaying wood flakes. They were reared in an incubator under 24-hour darkness at 25±0.5°C until pupation. Larval period (in days), weight (g) and mandible length of pupae and adults (prothorax width and left mandible length) were recorded. The control groups included an untreated group and a group that received an application of 10 µl of acetone.Male and female individuals in the early half of the prepupal period  and males in the late half of the prepupal period (staged from termination of gut purge to pupation) were treated with 10 µl of 5 µg fenoxycarb diluted in acetone as described above . Control groups were untreated, and acetone-only, early and late prepupae. Mandible length and total weight of all pupae was recorded. Statistical analyses for the relationship of mandible length to pupal weights among all groups were conducted using ANCOVA .Hemolymph was collected from the early prepupal periods of experimentally manipulated small and large individuals of both sexes. As mentioned, low food rearing containers with a limited food supply produce small beetles whereas larvae reared in large containers with over three times as much food develop into significantly larger individuals. Prepupae were anaesthetized on ice and hemolymph was collected by dissection at the base of the legs, with a glass Pasteur pipette. Aliquots of collected hemolymph were extracted by using methanol and iso-octane with 30 ng fenoxycarb  as an internal standard. See the 2 C18 reverse-phased column  protected by a guard column  with gradient elution of water/methanol  at a flow rate of 0.2 ml/min, utilizing an Agilent 1100 HPLC system with autosampler. Mass Spectrometry analysis was performed by electrospray ionization (ESI) in the positive mode on a microTOF-HS  under the following conditions: the electrospray capillary was set at 4.5 kV and the dry temperature was 200°C. The nitrogen pressure was 1.2 Bar for the nebulizer and the drying gas nitrogen low-rate was 4 L/min. The Ionization of standard JH III generated [M+CH2O]+, [M+OH]+, [M+H]+ and [M+Na]+ ions. In hemolymph samples, [M+H]+ and [M+Na]+ were the major ions observed. To confirm these ions were JHIII generated, the HRMS (high resolution of mass spectrum) of these ions were compared with the HRMS of JHIII (C16H26O3) with DataAnalysis analysis software . In order to diagnose the presence of additional JH homologs, JHI  and JHII  were also investigated. There were two candidate peaks for JH homologs in the chromatogram of the hemolymph extracts. One peak of m/z  = 295, 317, corresponded with JHI and the other m/z  = 267, 289 to JH III. HRMS analysis indicated that m/z  = 295, 317 was not JH I (C18H30O3) (error > 500 ppm), but m/z  = 267, 289 was closely matched with the exact-mass of JHIII (C16H26O3) (error <5 ppm). In addition, the retention time (RT) of the candidate m/z  = 267, 289 was the same as that of JHIII standard . There were no clear peaks of m/z  = 281, 303 that correspond to JH II. Therefore, JH III was the only JH homolog found in C. metallifer. Quantification of JH III, fenoxycarb was therefore performed by monitoring the [M+H]+ and [M+Na]+ ions. A calibration curve of JH III was made, containing the same concentration of the internal standard fenoxycarb as for the hemolymph samples. The JH III titer from each sample was then calculated after analysis of the chromatogram data with QuantAnalysis software .To quantify juvenile hormone titers in the stag beetle, the experimental methods of LC-MS were modified based on Westerlund et al. 2004 and Cornette et al. 2008 Figure S1The relationship between body weight and mandible length of male pupae treated with acetone or fenoxycarb (JHA) at the late prepupal period.(DOC)Click here for additional data file.Figure S2Comparison of the duration of the third instar among males treated with fenoxycarb (JHA) or acetone only (mean ± SE).(DOC)Click here for additional data file.Figure S3The relationship between prothorax width and left-mandible length of adult males treated with acetone or fenoxycarb (JHA) during the larval period compared with normally reared males.(DOC)Click here for additional data file.Materials and Methods S1Materials and Methods for insect husbandry, scanning electron microscopy (SEM) and hemolymph collection and JH extraction.(DOC)Click here for additional data file.


Tapirus terrestris) and muriquis (Brachyteles arachnoides) are potential candidates for functional redundancy on seed dispersal effectiveness. Here we provide a comparison of the quantitative, qualitative and spatial effects on seed dispersal by these megafrugivores in a continuous Brazilian Atlantic forest.Functional redundancy has been debated largely in ecology and conservation, yet we lack detailed empirical studies on the roles of functionally similar species in ecosystem function. Large bodied frugivores may disperse similar plant species and have strong impact on plant recruitment in tropical forests. The two largest frugivores in the neotropics, tapirs (We found a low overlap of plant species dispersed by both muriquis and tapirs. A group of 35 muriquis occupied an area of 850 ha and dispersed 5 times more plant species, and 13 times more seeds than 22 tapirs living in the same area. Muriquis dispersed 2.4 times more seeds in any random position than tapirs. This can be explained mainly because seed deposition by muriquis leaves less empty space than tapirs. However, tapirs are able to disperse larger seeds than muriquis and move them into sites not reached by primates, such as large forest gaps, open areas and fragments nearby. Based on published information we found 302 plant species that are dispersed by at least one of these megafrugivores in the Brazilian Atlantic forest.Our study showed that both megafrugivores play complementary rather than redundant roles as seed dispersers. Although tapirs disperse fewer seeds and species than muriquis, they disperse larger-seeded species and in places not used by primates. The selective extinction of these megafrugivores will change the spatial seed rain they generate and may have negative effects on the recruitment of several plant species, particularly those with large seeds that have muriquis and tapirs as the last living seed dispersers. It has been hypothesized that in biodiversity-rich ecosystems, the extinction of some species will not cause a substantial loss in ecosystem function if remnant species play equivalent roles and are capable of taking over the functions played by extinct species  Ateles, Brachyteles, Logothrix), for instance, can disperse millions of seeds per year Tapirus spp.) eat large amounts of fruits, disperse large quantities of seeds, often at long distances, and are able to disperse very large seeds even across heavily disturbed areas Large vertebrates are particularly important, because they remove a larger amount of seeds, disperse them for longer distances, and are able to disperse larger seeds than smaller frugivores Brachyteles arachnoides) and the tapir (Tapirus terrestris)  .Pinus and Eucalyptus, pastures, and other small-scale agricultural crops. The Paranapiacaba massif comprises more than 120,000 ha of Atlantic Forest distributed through four protected parks and several private properties and holds an almost complete assemblage of threatened mammals, including jaguars (Panthera onca), bush dogs (Speothos venaticus), tapirs (Tapirus terrestris) and muriquis (Brachyteles arachnoides), although white-lipped peccaries (Tayassu pecari) are locally extinct Brachyteles arachnoides (hereafter muriquis), corresponding to approximately 850 ha at about 750 m a.s.l, located at the extreme north of CBSP This study was carried out at Carlos Botelho State Park (hereafter CBSP), a 37,644 ha Protected Area in a continuous massif named “Serra de Paranapiacaba” in São Paulo state, southeastern Brazil  were sampled in equal proportions. We defined as “latrine” a clump of tapir feces with different ages inside a 5-meter radius During the same period, we followed individuals of the sub-groups of muriquis habituated to human presence, from sunrise (5∶15 h) to sunset (18∶30 h) totaling 246,7 km walked with an average of 17,5 km per month. Observations totaled 432 h of direct contact, of which 382 h (89%) were on adults. We totaled 31 complete days of observation following the same sub-group from sunrise (waking-up site) to sunset (sleeping site) and 20 incomplete days (i.e. observation interrupted for more than 1 hour). To verify the sub-group size we collected data on the number of muriquis surrounding the focal individual (about 15 m) three times a day . Points along daily routes were geopositioned every 15 minutes. To determine the feces deposition pattern, each observed defecation event, here defined as a defecation of a single individual at the same place within 1 minute, was also geopositioned. The deposition pattern was defined as “clumped” if the feces fell in less than 30 cm radius, or “scattered” if, while falling, the feces collided with the leaves and branches of the understory and reached the ground in more than 30 cm radius Coussapoa sp.) were collected in a plate under the sieve and whenever possible were identified and the number of seeds estimated. We also collected fallen fruits under fruiting trees, removed the pulp and measured the seeds for comparison with dispersed seeds. To estimate the minimum seeds dispersed by month by muriquis we used the average of 30.6 seeds dispersed by day, obtained from five complete days where we were able to collect at least 4 defecation of the same individual  and multiplied by 30 days. For tapirs, we used the average of 140 seeds per individual per month. This was based on the number of new seeds found per month in specific latrines that were monitored monthly.Muriqui and tapir feces were collected and analyzed in the laboratory. When tapir feces were located in water we only collected them if the boluses were whole (i.e. not broken or dissolved). For all samples, we recorded the topographical feature , distance (m) from the nearest tree with DBH >40 cm and all possible parental fruiting trees in an area of 25 m radius. All feces were sieved through a mesh of 2×2 mm and all remaining seeds were identified with the aid of guides Hieronyma alchorneoides, Phyllanthaceae) and one large-seeded species . The fruits collected on the ground or directly from trees were manually defleshed and seeds cleaned. For C. mandioccana we also used the whole fruits (i.e. with pulp). We just used seeds with no sign of insect damage or predation. The seeds of H. alchorneoides are dispersed by muriquis, tapirs, and small birds, while the seeds of C. mandioccana are dispersed by primates (muriquis and howlers monkeys), tapirs, and piping guans (Aburria jacutinga) C. mandioccana, 25 from tapir feces, 25 from muriqui feces, 25 seeds cleaned manually and 25 whole fruits (with pulp), and 75 of H. alchorneoides with the same methods as C. mandioccana, excepting whole fruits. The seeds were placed individually in plastic bags with sterilized soil and placed in a greenhouse located right at the border of the CBSP forest. The greenhouse is covered with a shadow mesh of 50% of sunlight retention and all seeds were watered and monitored daily.We conducted germination tests to determine the effect of the passage through tapir and muriqui guts on the germination success  and time to germination (average days for germination) of the seeds. Due to the difficulty in finding large enough quantities of seeds for the trials, we chose one small-seeded  for the three situations mentioned above. T-tests were applied to test statistic significances between empty space estimates for paired seed disperser situations. Seed deposition quantity (i.e. the number of seeds within a determined radius per any random position) was estimated for muriquis, tapirs and muriquis+tapirs using interpolation methods available within spatstat R package. We estimated the number of seeds dispersed within a search radius of 250 m. This value was adopted because it is close to the average empty space accounted for feces deposition recorded for the species with the largest empty space (i.e. tapir). Student T-tests were applied to test statistic significances for seed deposition quantity for paired seed disperser situations.To verify the redundancies and complementarities of the seed dispersal by tapirs and muriquis, here defined as the spatial (deposition pattern in space), quantitative (number of seeds dispersed) and qualitative (germination) overlap, we adopted the following criteria and indicators To compare the intraspecific sizes of seeds dispersed by muriquis and tapirs, we used a nested ANOVA with disperser nested within the specific identity of the plant. The disperser factor had three levels, muriquis, tapirs (seeds collected from dung) and control, i.e., a random sample of seeds collected from trees and ground. We used post hoc t-test based on standard errors estimated from the linear predictor of the ANOVA model to uncover particular differences.Germination velocity was analyzed through a Cox proportional hazards regression model To compare the overlap in number of species with different seed size we used a contingency table together with residual analysis and a randomization test to uncover particular significant differences across the table. Randomization was done within each seed size level.Coussapoa microcarpa, Urticaceae with more than 80 seeds). Of all seeds found in tapir feces, just 12 seeds of Cryptocarya mandioccana  were mechanically predated. Two species were predated by muriquis  but were not found in their feces. Muriquis defecated on average 10 times a day  but we were not able to define tapirs defecation rate.We analyzed 106 defecations of muriquis and 49 of tapirs. Muriquis dispersed 28 seed species, with an average of 23.3±17.5 (13–86) seeds larger than 2 mm per fecal sample, while tapirs dispersed six seed species, with an average of 72.5±48.8 (13–183) seeds larger than 2 mm per fecal sample. Muriquis dispersed 2.04±1.25 (mean ± SD) species with a maximum of five species in a single defecation, while tapirs dispersed 1.26±0.45 species with a maximum of 2 species in a single defecation. Several seed species smaller than 2 mm were deposited in a single defecation  in the assemblages of plant species dispersed by muriquis and tapirs, and highly asymmetric complementarity between the two frugivorous. Muriquis complemented tapirs by 79% while tapirs complemented muriquis only with 3%. Only five species of 28 species recorded were dispersed by both tapirs and muriquis . The palm We conservatively estimated that one muriqui could disperse at least 918 seeds >2 mm per month, or 11,016 seeds per year. One tapir can disperse at least 140 seeds >2 mm per month or 1,680 seeds per year. Therefore, a muriqui troop with 35 individuals is able to disperse at least 385,000 seeds >2 mm/year. In the home range of muriquis studied group (850 ha), 22 individuals of tapirs were identified based on molecular analysis , which could disperse 36,960 seeds/year.Miconia cabussu (Melastomataceae) with less than 2 mm to Parinari excelsa  with 23.4 mm seed diameter  with 3.1 mm, and the largest was Eugenia sp1 with 29.2 mm diameter. In general muriquis selected a subset of relatively small seeds in plant species  with seeds over 15 mm of diameter . Exceptions to this pattern were Parinaria excelsa and Vantanea compacta for which we collected only two and ten seeds, respectively, from muriquis feces. In contrast, in 8 out of 10 species with small seeds (<13 mm of diameter) muriquis did not select for a subset of seed size but in Byrsonimia sp.  and C. guaviroba  muriquis selected for a subset of large seeds. Overall tapirs did not seem to select fruit/seed sizes except for C. mandioccana for which seeds in tapir dung were on average significantly smaller than the average of the whole set of available seeds .Muriquis dispersed seeds from a wide range of sizes, from diameter . For tapFurther, from an extended review of the literature on seeds dispersed by muriquis and tapirs in the Atlantic forest , we founC. mandioccana .Germination time of dioccana  was signOut of the 266 registered muriqui defecation events, 55 (21%) feces reached the ground in a cohesive block (<0.3 m), while in 211 cases (79%) feces were fragmented as they hit the understory, generating a locally widespread seed rain, from 0.3 m to more than 3 m radius, especially when on steep slopes. Fifty-nine feces (22%) fell in the water, 26 (10%) on dry valleys, 85 (32%) on slopes, and 90 (34%) on hilltops. We found that for the 49 tapir feces, 11 (22%) were in the water, five (10%) on dry valley, 16 (33%) on slopes and 17 (35%) on hilltops.Eugenia sp. 1 seeds were dispersed 169 and 693 m from the parent tree. On 16 occasions, it was possible to measure the distance of muriqui-defecated seeds from a conspecific adult, with average of 12.7±9.2 m (2–25 m). For tapirs we never identified a conspecific adult inside the 25 m radius circle searched.For muriquis, the average distance of the defecations from trees with DBH >40 cm was 2.9±1.9 m  and for tapirs 2.5±1.7 m , showing a random pattern with no preference in defecating near tree trunks. On just two occasions, we were able to accurately measure the seed dispersal distance for muriquis and found that vs. muriquis only , muriquis+tapirs vs. tapirs only  and muriquis only vs. tapirs only . Seed deposition at any random position within study site were estimated as 68.2±0.3 seeds for muriquis only, 28.4±0.6 for tapirs only and 78.5±0.4 for muriquis+tapirs , muriquis+tapirs vs. tapirs only  and muriquis only vs. tapirs only .When analyzing the spatial distribution of feces for muriquis only, tapirs only and muriquis+tapirs, we observed that the spatial distribution of seeds deposited by muriquis was more scattered. The average empty space for muriquis was 122.6±2.7 m, while for tapirs it was almost double (202.1±4.4 m). But if we combine both dispersers the average empty space decreases to 106.6±2.3 m . The t-ts+tapirs . The t-tTapirs and muriquis have distinct seed dispersal effectiveness , and differ in the diversity of plant species, total number of seeds, aggregation and seed sizes dispersed . The quaAttalea dubia, Eugenia sp. 1, Pouteria sp., Pouteria bullata and Parinari excelsa, all with more than 15 mm of average width. All but Attalea dubia rely exclusively on tapirs and muriquis for seed dispersal and are heavily predated by squirrels and bruchids. Although other studies report Attalea seeds being dispersed by tapirs Attalea seed in tapir feces in our study area.The seed size (width) is typically employed as the main characteristic that defines the array of dispersers Maximiliana maripa, is attributed to tapir seed dispersal The tapir behavior of defecating in latrines can reduce seed predation by bruchids and rodents Lagothrix lagotricha) in Colombia In terms of seed size selection, in general muriquis do not select seeds of small-seeded species (<13 mm), as they ingested the whole range of sizes available in the plant community, while in plant species with seeds larger than 15 mm muriquis selected for a subset of relatively small seeds. The seed size limit that an adult muriqui can ingest is 23 mm, 22% larger than the seed size limit swallowed by woolly monkeys  and defecate mostly in latrines or in water bodies, while muriquis occur in relatively higher density , have a fission-fusion strategy with sub-groups consisting of about 5 to 20 or more individuals, which means a higher probability of seed dispersal across different microsites. So, while tapirs clearly generate a clumped distribution of seeds, muriquis generate both scattered and clumped seed dispersal, a pattern also reported for Ateles paniscus in Peru Seed shadow in rainforests basically consists of the spatial distribution of the seeds generated by animal dispersers, which is mainly influenced by the gut passage time, defecation pattern (frequency), movement and habitat use C. mandioccana, indicating that pulp removal is relevant for germination in this species, and probably also in other species dispersed by muriquis C. mandioccana by 5 to 8 days compared with seeds manually removed from the pulp and seeds remaining in the pulp. Although this may seem a small improvement in germination for C. mandioccana, it can become quite relevant if seed predation and decay are minimized via shorter exposure to seed predators and microbes, and also via removal of pulp that may act as an incubator for fungus and bacteria likely to rot seed tissues. This may be particularly relevant if the main demographic sieve controlling C. mandioccana population is at the seed stage. Rodents and invertebrates prey heavily upon C. mandioccana seeds, particularly beneath the parent tree .We found no difference in the germination success of seeds that were dispersed by tapirs and muriquis. The gut treatment of these frugivores shortened the germination times of Muriquis and tapirs are the last representatives of a formerly rich neotropical frugivore megafauna Although we did not evaluate post-dispersal seed fate, muriqui feces attract higher abundance and diversity of secondary seed dispersers, such as dung beetles, than tapir feces , increasing the probability of germination and escape from seed predation. On the other hand, some authors have found that post-dispersal seed predation on seeds in tapir dung is lower than seeds not immersed in latrines We found that muriquis dispersed 28 species during 18 mo of this study, but it is likely they eat a much higher diversity of fruits. Studies on muriqui diet in neighboring sites recorded another 36 fruit species that were not observed in muriquis feces in our study but are known to occur in our study area Large-bodied primates and ungulates are threatened by habitat loss and poaching all over the Neotropics Alouatta spp.) and ungulates (peccaries and deer) do not replace the seed dispersal services by muriquis and tapirs. Howlers are highly folivorous, have a much smaller home range and defecate mostly beneath the sleeping trees The extinction of tapirs may also disrupt long-distance dispersal and colonization of large seeded species into open areas or fragments Therefore, the extinction of these last megafrugivores may translate into a series of impoverished plant-animal interactions, whereby the persistence of many plant species will have to rely on human interventions. Our study demonstrates that both megafrugivores are essential for promoting distinct seed dispersal services in tropical forests.Figure S1Comparative of seed size (diameter) of plant species eaten by muriquis (Brachyteles arachnoides and B. hypoxanthus) and tapirs (Tapirus terrestris) in the Atlantic forest .(TIF)Click here for additional data file.Table S1Species dispersed and number of seeds per scat by tapirs (Tapirus terrestris) and muriquis (Brachyteles arachnoides) in Carlos Botelho State Park, Atlantic Forest, Brazil.(DOC)Click here for additional data file.Table S2Compiled information from a literature review of seed size of species dispersed by muriquis (Brachyteles arachnoides and hypoxanthus) and tapirs (Tapirus terrestris) in the Brazilian Atlantic forest. Plant names of eaten fleshy fruits follow (DOC)Click here for additional data file.


NOS3) variants and risk of primary open-angle glaucoma (POAG) depend on female reproductive factors.To investigate whether associations with the nitric oxide synthase gene  were evaluated in a nested case-control study from the Nurses’ Health Study (women followed 1980 – 2002). Participants were aged ≥40 years and Caucasian, who were followed biennially with update information on reproductive factors. We included 374 Nurses’ Health Study (NHS) cases and 1,085 controls, matched on age and eye exam at the matched cases’ diagnosis dates. Relative risks (RRs) were estimated using multivariable conditional logistic regression.Glu298Asp SNP were at increased risk, while among parous women with 1–2 children, they were not (p-interaction=0.003). No significant interactions between NOS3 SNPs and oral contraceptive use in POAG were detected.Among women with age at menarche <13 years, compared with the CC homozygotes of the rs3918188 tagging SNP, the wild-type AA homozygotes were at significantly reduced risk of POAG ; however, for women with age at menarche ≥13 years, the SNP was not associated with POAG (p-interaction=0.0007). Among parous women with 3+ children, carriers of the minor variant (T) allele of the functional NOS3 genotype - female reproductive health interactions are important in POAG pathogenesis.These data provide further support for the notion that  Nitric oxide synthase 3 (NOS3) is an enzyme that catalyzes the production of nitric oxide (NO), which modulates the tone of luminal structures with adjacent smooth muscle tissue . NOS3 maDysfunction in the ocular vascular endothelium and in the trabecular meshwork cells could lead to primary open angle glaucoma (POAG). Feke and Pasquale  documentNOS3, thereby enhancing the release of nitric oxide [NOS3) variants that were significantly stronger in women than in men and evidence of stronger associations with NOS3 variants among current users of postmenopausal hormones (PMH) compared to non-current users in relation to high-tension POAG [Reproductive hormones such as estrogen may play a role in modulating IOP and the risk of POAG in women -19. One ic oxide -23 to reic oxide . Also, wion POAG .NOS3 variants and the risk of POAG depended on the status of reproductive factors of age at menarche, parity, oral contraceptive use. We used data from a large case-control study nested within the Nurses’ Health Study (NHS) where for cases, the exposure information was collected before any diagnoses of POAG.In this study, we evaluated whether the associations with The NHS was initiated in 1976, when 121,700 female US registered nurses (aged 30–55 years) returned a questionnaire on health-related information . ParticiIn 1989–1990, blood samples were collected from 32,826 (27%) women. In 2001–2004, among 29,684 women who did not provide a blood sample, buccal cell samples were collected.Blood samples were collected using sodium heparin as the anticoagulant. Samples were mailed back within 26 h of blood draw, immediately centrifuged, aliquoted into buffy coat components, red blood cells, and plasma, and stored in liquid nitrogen freezers for later retrieval. Buccal cell samples were collected using a single “swish-and-spit” procedure . Within a week of receipt, samples were processed and DNA was extracted.We first identified POAG cases with the biennial questionnaires, where we asked whether participants received an eye exam with a diagnosis of “glaucoma.” For participants self-reporting glaucoma, we sought permission to retrieve the medical records. We contacted the diagnosing eye care provider for all prior visual field (VF) tests and for the completion of a glaucoma questionnaire; this questionnaire included items for maximal IOP, the status of the filtration apparatus, optic nerve structural information, prior ophthalmic surgery, and any VF loss. Relevant medical records were accepted in lieu of questionnaires. All confirmatory information from questionnaires/medical records and VFs were evaluated in a standardized manner by a glaucoma specialist (L.R.P.). We previously demonstrated our case definition is valid by showing appropriate positive associations with a family history of glaucoma and with African heritage .We included as cases only participants determined to have “definite” or “probable” POAG. For definite POAG cases (70% of case), slit lamp biomicroscopy showed no sign of pigment dispersion syndrome, uveitis, exfoliation syndrome, trauma, or rubeosis in either eye; gonioscopy demonstrated that the angles were open in both eye; and ≥2 reliable VFs showing reproducible defects consistent with glaucoma had to be available. For probable POAG cases, the same slit lamp exam and VF criteria were also required, but documentation of pupil dilation without subsequent adverse events was accepted if gonioscopy was not performed. For VFs, there was no requirement for the type of perimetry performed; however, in 95% of cases, full static threshold testing was completed, and in <1% of cases, kinetic VFs were used. For static threshold or suprathreshold tests, we regarded the VF reliable if the fixation loss rate was ≤33%, the false positive rate was ≤20% and the false negative rate was ≤20%. For kinetic VFs, we considered the field reliable unless the examiner noted otherwise.We included 374 cases and 1,085 control subjects who were at least 40 years in age and Caucasian. The controls were matched on sample type (blood or cheek cell), year of birth, ethnicity (Latino or not), and they were required to have had an eye exam in the same period as when the matched case was diagnosed. Approximately three controls per case were matched to each case, using incidence density sampling.rs2070744 and Glu298Asp: rs1799983) and two tagging SNPs . The tagging SNPs corresponded to the NOS3 linkage disequilibrium (LD) blocks, and were selected using Haploview (version 4.1) according to the HapMap data (release 22) from the Utah residents with Northern and Western European ancestry (CEU) population [rs3918188 and rs7830) captured the majority (88%) of alleles at r2 greater than 0.8 across the whole gene, including the 5′- and 3′-untranslated regions.We genotyped two functional single nucleotide polymorphisms (SNPs) , 96 spin blood kit protocol. A quantitative PCR approach  was used for genotyping, according to the manufacturer’s instructions. The RT–PCR amplification of whole genome amplified DNA was performed in 96 well plates with a sequence detection system . The thermal cycler  was set at the following parameters: 50 °C for 2 min, 95 °C for 10 min, 92 °C for 15 s, and 58 °C for 1 min for a total of 60 cycles. The genotyping success rate was over 90%. Plates that passed quality control measures (including Hardy–Weinberg equilibrium tests) were included, and in 5% of samples that underwent repeat genotyping, there was >95% concordance on genotyping calls.From 1976, participants were asked about the age at menarche and when menstrual periods ceased permanently. Additional questions were asked of menopausal women, including type of menopause  and, if surgical menopause, the number of ovaries removed. Menopausal status was updated biennially.Current and past OC use was asked at 1976 and updated biennially until 1984, at which point no women reported current use. Women were also asked to report the time periods for which they used OCs and reasons for stopping.Parity, which was defined as pregnancy lasting at least 6 months, was assessed in 1976 and biennially updated through 1984 and updated again in 1996.We used conditional logistic regression to estimate relative risks (RRs) and 95% confidence intervals (CIs), adjusting for potential confounders. We used SAS  for analyses, and a value of p<0.05 was considered statistically significant. To address the multiple testing issue, in secondary analyses, we calculated adjusted p-values using an optimized false discovery rate approach .2), physical activity (quartiles of activity intensity/day), self reported history (yes/no) of diabetes, and cumulatively updated caffeine intake , age at menopause , PMH status , age at menarche (linear years), parity (linear number of children), and oral contraceptive use .Information on exposures and potential confounders was obtained from the biennial questionnaires and was updated through the questionnaire immediately before the diagnosis date of the index case. Potential confounders were family history of glaucoma, body mass index  and “normal-tension” POAG, defined as those with maximum IOP<22 mmHg before VF loss.To evaluate effect modification, we tested the significance of the estimates of the interaction terms from the multivariable conditional logistic regression models, using Wald tests, where the term was constructed as a product of the SNP variables that were coded as 0, 1, 2, and the reproductive factor of interest.The characteristics of POAG cases and their matched controls as of the index case’s date of diagnosis were similar . Cases hNOS3 polymorphisms in relation to POAG have been previously reported [rs3918188, where the variant was significantly (p=0.04) inversely associated with overall POAG only among the women ; p-trend with increasing variant allele=0.0008). In relation to normal-tension glaucoma, for the T-786C polymorphism, the “pooled” RR  for the CC homozygote was 0.44  and the p for trend was 0.03.The main effects of the reported . Brieflyhe women . In secoNOS3 SNP rs3918188 and age at menarche in POAG. Specifically, the inverse association between the rs3918188 polymorphism and POAG was more evident among those with age at menarche <13 years, after adjustment for age at menopause and other factors ; however, among those with age at menarche ≥13 years, the SNP was not associated with POAG. On the other hand, we did not find a significant interaction between the functional NOS3 Glu298Asp SNP and age at menarche. Among women with age at menarche <13 years, the adverse association with the Glu298Asp polymorphism was more apparent  than among those with older age at menarche. This interaction was borderline significant (p=0.05), and was no longer significant after multiple testing correction.We observed a significant interaction between rs3918188 SNP and parity in POAG. Among those with 3 or more children, the protective association with the rs3918188 SNP was more apparent  than among those with 1 or 2 children, but this interaction was borderline significant (p=0.04) and was no longer significant after multiple testing correction.Because 80% of our participants were parous, we did not have the power to examine nulliparity; thus, we examined having 3 or more children versus having 1–2 children for evaluating effect modification. We did observe a significant crossover interaction between the Glu298Asp SNP and parity in POAG. Using women with multiparity who were homozygous for the common Gl298Asp NOS3 variant (GG) as the reference group, both homozygosity for the minor Glu298Asp variant (TT) among those with 3 or more children and homozygosity for the common variant (GG) among women with 1–2 children were adversely related to POAG . Interactions were not observed with oral contraceptives in relation to POAG overall. Because this is the first study to examine the interaction between NOS3 SNPS and female reproductive factors, these results need to be confirmed in other studies.In this nested case-control, we observed that the associations with NOS3 SNPs, the functional consequences of the rs3918188 tagging SNP are unknown; however, we previously found that the minor allele (A) was associated with a reduced risk of POAG in women (p-trend=0.04) but not men (p-trend=0.58). We observed a significant interaction between the minor allelic variant of the rs3918188 tagging SNP and age at menarche in POAG: women with homozygosity for the minor allele and age at menarche <13 years had a 69% reduction in POAG risk compared to those with homozygosity for the wild-type allele and older age at menarche. Biologically, this is plausible as increased age at menarche may be related to reduced NOS3 activity [These findings should be interpreted in the context of previous cohort studies describing the relation between reproductive factors and POAG ,25,31. Factivity , which mWe also observed that the association between the functional Glu298Asp SNP and POAG was significantly modified by parity. For parity, in the NHS, we reported a null association with POAG , althougNOS3 SNPs and reproductive factors that reflect exposure to estrogen, which potently modulates NOS3 activity, provides further support for the important role of endothelial cell dysfunction in POAG etiology. Such dysfunction in the trabecular meshwork and ocular vascular endothelium may lead to increased IOP and retinal ganglion cell vulnerability, resulting in glaucomatous visual loss. Recent emerging data from animal models, studies of POAG genetics and clinical trials of agents related to vascular tone also support this hypothesis. In a mouse model, there is evidence that knocking out soluble guanylate cyclase, the downstream mediator of nitric oxide, produces modest increases in IOP and considerable loss of retinal ganglion cells and nerve fiber layer dropout [The associations between the  dropout . In an I dropout . A rando dropout -43. Othe dropout ,45. ThusThere are limitations to this work that should be considered. First, our glaucoma definition was based on self-report with confirmation with medical records and visual fields. This definition has very high specificity, as we required documentation of reproducible defect on at least 2 reliable visual field tests and we have demonstrated strong associations with established risk factors, such as African-American heritage and family history . Given tNOS3 SNPs and POAG depended on parity and age at menarche. Previously we showed the associations between NOS3 SNPs and POAG depended on PMH use. These data indicate that NOS3 gene variants interact with sex hormone levels in ways to influence the risk of POAG. In the future we hope to validate these interactions in animal model systems.In conclusion, the associations between


Dairy proteins, in particular the whey fraction, exert insulinogenic properties and facilitate glycemic regulation through a mechanism involving elevation of certain plasma amino acids, and stimulation of incretins. Human milk is rich in whey protein and has not been investigated in this respect.Nine healthy volunteers were served test meals consisting of human milk, bovine milk, reconstituted bovine whey- or casein protein in random order. All test meals contributed with 25g intrinsic or added lactose, and a white wheat bread (WWB) meal was used as reference, providing 25g starch. Post-prandial levels in plasma of glucose, insulin, incretins and amino acids were investigated at time intervals for up to 2 h.All test meals elicited lower postprandial blood glucose responses, expressed as iAUC 0–120 min compared with the WWB (P < 0.05). The insulin response was increased following all test meals, although only significantly higher after whey. Plasma amino acids were correlated to insulin and incretin secretion (iAUC 0–60 min) (P ≤ 0.05). The lowered glycemia with the test meals (iAUC 0–90 min) was inversely correlated to GLP-1 (iAUC 0–30 min) (P ≤ 0.05).. Despite its lower protein content, the human milk was a potent GLP-1 secretagogue and showed insulinogenic properties similar to that seen with reconstituted bovine whey-protein, possibly due to the comparatively high proportion of whey in human milk.This study shows that the glycemic response was significantly lower following all milk/milk protein based test meals, in comparison with WWB. The effect appears to originate from the protein fraction and early phase plasma amino acids and incretins were involved in the insulin secretion The prevalence of type 2 diabetes (T2D) is increasing around the world, with sedentary lifestyles and inadequate dietary habits as the major causing factors. Epidemiological evidence suggests, that a diet characterized by a low glycemic index (GI), protects against the development of T2D . The GI In a previous study, we have shown that whey protein resulted in a higher insulin response than cheese (casein) or milk (whey and casein), respectively . The majThe protein concentration in human milk is considerably lower than in bovine milk (8.5 g/L vs. 34.5 g/L) . Also thThe aim of the present study was to investigate the glycemic and insulinemic effects of human and bovine milk in comparison with white wheat bread (WWB). In addition, casein and whey protein fractions from bovine milk were included. For this purpose, human milk, bovine milk, bovine casein and whey proteins, respectively, were standardized with respect to lactose content and provided as breakfast drinks to healthy adults. The study was performed in a glycemic index measuring setting, and a carbohydrate equivalent amount of white wheat bread (WWB) was therefore used as reference meal. In addition to blood glucose and insulin, plasma levels of amino acids and the incretins GIP and GLP-1 were measured in the postprandial phase.Four test drinks were included in the study; human and bovine milk and reconstituted bovine whey and casein drinks. The nutritional compositions of the meals are shown in Table 2 (mean ± SD) and without drug treatment participated in the study. All subjects had normal fasting blood glucose levels (4.4 ± 0.05 mmol/l). The meals were served in random order as breakfast meals at 5 different occasions, at least 2 days apart. When arriving at the laboratory in the morning (8 am), a peripheral catheter was inserted into the antecubital vein and a fasting blood sample was drawn. At all sampling time points, samples were taken in one tube for serum and one for plasma (EDTA), respectively. The subjects were requested to consume the test and reference meals steadily over a 12 min period. All subjects were conscious of the possibility to withdraw from the study at any time they desired. The study was approved by the Ethics Committee of the Faculty of Medicine at Lund University.Nine healthy, non-smoking, volunteers, (6M: 3F), aged 22–30 years, with body mass index 25.8 ± 3.4 kg/ met al.[et al.[Lactose content of the test meals was analyzed as galactose and glucose following enzymatic hydrolysis with β-galactosidase as described by Nilsson et al..The peptl.[et al. startingl.[et al.. The crul.[et al..125I human GIP (70 MBq/nmol) were used for standards and tracers. The concentration of plasma GLP-1 was measured [Venous blood samples were taken at fasting (0) and, at 7.5, 15, 30, 45, 60, 75, 90, 105 and 120 min after the meal were initiated, for analysis of blood glucose, serum insulin and GIP and GLP-1 analysis. Additionally, plasma was collected at 0, 7.5, 15, 30, 45 and 60 min for amino acids analysis. The plasma tubes were allowed to rest for 30 min before being centrifuged at 3000 rpm during 6 min. 800 μl plasma was separated for free amino acids analysis and the remaining plasma was frozen at −20°C before analysis of GIP and GLP-1. Serum tubes were centrifuged for 15 min at 4000 rpm and serum was separated and frozen at −20°C for insulin analysis. Whole blood glucose concentrations were determined with a glucose oxidase-peroxidase reagent. The serum insulin determination was performed on an integrated immunoassay analyzer, CODA™ Open Microplate System  using an enzyme immunoassay kit . Plasma GIP and GLP-1 concentrations were measured after extraction of plasma with 70% ethanol . For the GIP radioimmunoassay , we usedeasured  against The results are expressed as mean ± SEM. The incremental areas under the curves (iAUC) for glucose, insulin, GIP, GLP-1 and plasma amino acids were calculated for each test subject and meal, using a trapezoid model . All areas below the baseline were excluded from the calculations and each subject was their own reference. Statistical comparisons for iAUC-values were performed in MINITAB® Statistical Software (release 14.13 for windows). Significances were evaluated with the general linear model (ANOVA), followed by Tukey’s multiple comparisons test. Statistical significances were considered for values of P < 0.05. Differences between the products at different time points were analyzed by using a mixed model  with repeated measures and an autoregressive covariance structure. When significant interactions between treatment and time were found, Tukey’s multiple comparison test were performed for each time point . Correlations between dependent measures were conducted using Spearman's partial correlation coefficients controlling for subjects and corresponding baseline values (two-tailed test), . Due to missing values the bovine milk had to be withdrawn from the GLP-1 and GIP statistical calculations, and the amino acid isoleucine (ile) had to be excluded from the calculations on free amino acids in plasma. In addition, there were only eight test subjects on the WWB and whey, and seven test subjects on the casein and the human milk respectively included in the GIP and GLP-1 calculations, due to missing values.The iAUCs for plasma glucose and serum insulin are shown in Table The amino acid concentrations of the different meals are presented in Table The 0–60 min iAUC for plasma amino acids are displayed in Table The iAUCs 0–120 min for GLP-1 and GIP are shown in Table Correlations between plasma amino acids, serum insulin and plasma incretins are displayed in Table To the best of our knowledge, this is the first study where human milk is evaluated with respect to glycemic and insulinemic effects. The study was performed in healthy adults with a GI setting, thus post prandial glycemic and hormonal responses to the human and bovine milk were compared to an equicarbohydrate amount of WWB. In the present study, we found that human milk displayed insulinogenic properties and resulted in a postprandial glycemic response (GI 57) that was in the same range as that to bovine milk (GI 43) or the whey and casein fractions, respectively (GI 61 and 44). The human milk showed the lowest insulin response (iAUC 0–120 min) in comparison to all the other meals, thus indicating that human milk display superior insulin economy. Worth emphasizing is that the protein content of the human milk meal was markedly lower than that of bovine milk, whey and casein meals. According to our analysis, the human milk meal contained 3.5g protein as calculated from nitrogen analysis using 6.38 as conversion factor. However, it has been has reported that the true protein content of human milk is overestimated from nitrogen analysis due to its high non protein nitrogen (NPN) content  ,17. Consin vitro that the amino acids ile, leu, val, lys and thr induce strong insulin secreting properties in mouse pancreatic islets [All the test products in our study yielded higher postprandial plasma amino acid responses compared to the WWB reference meal, although not reaching significance for all products. The bovine milk and the reconstituted whey and casein meals were especially prone to affect responses of the BCAA; val and leu, and also lys and thr. We also noticed a tendency to a rise in these amino acids following human milk in comparison to the WWB. We recently showed  islets . In part islets ,26. More islets ,28, may  islets ,30. In tStrong positive correlations were found between the early GLP-1 response iAUC 0–15 min) and postprandial insulin release up to 30 min, as well as negative correlations to blood glucose response (iAUC 0–90 min), an indication that early GLP-1 release may be important for the modulation of glycemia following both human and bovine milk. In support of such an opinion, we recently observed that ingestion of whey protein and free amino acid caused an early GLP-1 secretion that reduced postprandial glycemia in absence of a hyper-insulinemic peak . In addi min and In contrast to whey, the casein meal resulted in a small incremental insulin response and had the lowest glycemic response compared to the other test meals. This differences in glycemic and insulinemic responses when, comparing the casein and whey meal, is in contrast with observations by Hall et al. , who fouThere were some limitations of the present study. All the test meals resulted in lower blood glucose increments compared to the WWB reference meal. It should, however, be noted that lactose was the carbohydrate source in all the test meals, whereas the reference WWB meal contained starch. In previous studies, we have shown that lactose has a lower glycemic index (GI 68) and insulinemic index (II 50) than the starchy WWB (GI 100 and II 100) . ConsequThe present study showed that human milk as well as bovine milk and whey and casein protein lowered the glycemia compared to the WWB reference meal. We also demonstrated that the human milk exerted insulinogenic properties and the effect appeared to originate from the protein fraction. Consequently, all the test meals increased plasma amino acid levels, and the amino acids correlated positively with both insulin secretion and incretin responses as well as inversely to the glycemic response. Interestingly, the human milk appeared to be a particularly potent GLP-1 secretagogue. The relative potency of human milk to stimulate incretins is noteworthy and the impact of incretins for metabolism and appetite regulation is an interesting field for further research and should be taken into consideration when producing baby formulas based on bovine milk.iAUC: Incremental area under the curve; BCAA: Branched chain amino acids; GI: Glycemic index; GIP: Glucose-dependent insulinotropic polypeptide; GLP-1: Glucagon-like peptide 1; II: Insulinemic index; NPN: Non-protein nitrogen; T2D: Type 2 diabetes; WWB: White wheat bread.The authors declare that they have no competing interest.UG was responsible for sample collection and analysis of the data, statistical analysis and for writing the paper. JJH was responsible for the incretin analysis. EÖ was involved in interpretation of data and in writing the paper. IB was responsible for the study design and coordination of the study, securing the funding and was involved in the evaluation and in writing the paper. All authors read and approved the final manuscript.


Acropora millepora and Montipora tuberculosa) were kept in coastal seawater, or coastal seawater that was further organically and nutrient enriched (OE), and/or enriched with nitrate. Batches of OE were created daily, facilitating nutrient uptake, plankton succession and organic enrichment as observed in coastal waters. After 10 days of acclimation, 67% of the colonies had their temperature gradually increased from 27° to 31.2°C. After 3–7 weeks of heat stress, colonies of both species had significantly greater reductions in fluorescence yields and lower survival in OE than without addition of OE. Furthermore, photophysiological recovery was incomplete 31–38 days after ending the heat stress only in the OE treatments. Nitrate alone had no measurable effect on survival, bleaching and recovery in either species. Skeletal growth rates were reduced by 45% in heat-stressed A. millepora and by 24% in OE-exposed M. tuberculosa. We propose a conceptual trophic framework that resolves some of the apparently contradictory outcomes revealed by the review. Our study shows that management actions to reduce coastal eutrophication can improve the resistance and resilience of vulnerable coastal coral reefs to warming temperatures.Global warming, and nutrient and sediment runoff from coastal development, both exert increasing pressures on coastal coral reefs. The objective of this study was to resolve the question of whether coastal eutrophication may protect corals from thermal stress by improving their nutritional status, or rather diminish their thermal tolerance through the synergy of dual stressors. A review of previous studies on the topic of combined trophic status and heat exposure on the thermal tolerance of corals reveals a broad range of outcomes, including synergistic, additive and antagonistic effects. We conducted a 90-day long experiment exposing corals to realistic levels of elevated nutrients and sediments, and heat stress. Colonies of two common scleractinian corals ( Periods of high sea surface temperatures and light intensity have severe detrimental effects on scleractinian corals, causing bleaching, mortality and reduced calcification Artemia salina nauplii, and reduced water clarity. The studies suggest at least four different ways in which bleaching risk during temperature and insolation stress can be ameliorated in corals exposed to increased nutrients:The following review of the literature shows that it has remained equivocal as to whether corals exhibit synergistic, additive or antagonistic responses when simultaneously exposed to heat stress and elevated nutrients. Previous studies have used a wide range of field and controlled laboratory methods to investigate the issue, with treatments ranging from starvation in filtered water or removal of trace elements, to the addition of dissolved inorganic nutrients, suspended particulate matter, zooplankton or Stylophora pistillata and Galaxea fascicularis) maintained higher photosynthetic efficiencies and cell division rates of algal endosymbionts (mitotic index) when fed with Artemia salina nauplii, while starved corals suffered progressive declines in photosynthetic efficiency and endosymbiont cell division rates It is now clearly established that corals are more resistant  when they are well-fed rather than experimentally starved. For example, two species of temperature-stressed corals  Acropora intermedia had lower bleaching and higher survival rates when supplied with suspended particulate matter compared to those not exposed to this source of food and shading Well-fed corals are also more resilient  compared to starved corals. Bleaching reduces photosynthetic carbon fixation, yet some species can prevent starvation by burning stored energy reserves, reducing metabolic rates, and/or increasing feeding rates (heterotrophic carbon gain) The release of limitation by iron or other trace metals through terrestrial runoff may provide some protection against bleaching, by facilitating the generation of metalloenzymic antioxidants Corals may be more resistant to bleaching conditions on turbid inshore reefs, because clade D algal endosymbionts are usually found in turbid or low light environments on Indo-Pacific coral reefs In summary, according to these studies, corals in waters that are turbid, rich in nutrients and trace metals should be more resistant and resilient to temperature and solar insolation stress, since they have greater scope to maintain energy reserves through heterotrophy after endosymbiont loss, their antioxidant enzymes provide protection against oxygen radicals, and they may be equipped with more thermally tolerant endosymbionts.There are however, at least six other lines of evidence based on physiological or correlative field data, which suggest synergistic or additive effects of temperature stress and nutrients. According to these studies, corals in nutrient-enriched or turbid waters are more vulnerable to temperature stress than those in low nutrient environments:Corals exposed to high levels of dissolved inorganic nitrogen (DIN) experience greater bleaching susceptibility compared with corals in low nutrient environments. The proposed mechanism for this link is that high concentrations of DIN lead to high endosymbiont division rates, resulting in an increased demand for all essential plant nutrients by the proliferating endosymbiont populations, resulting in a relative under-supply of phosphate. The latter leads to altered thylakoid membrane structures with enhanced susceptibility to thermal and light damage Porites cylindrica, standardized by pigment concentration or algal endosymbiont number, declined by ∼30% in corals exposed to either high NO3 or high temperature compared to controls, but declined by ∼60% in corals exposed to both high NO3 and temperature, suggesting an additive effect between both agents Corals with high endosymbiont densities  Calcification in the massive The above review shows that from the existing literature and the theoretical perspective, it is surprisingly unclear as to whether corals exhibit elevated or reduced thermal tolerance when simultaneously exposed to heat stress and eutrophication. In this study, consisting of a 90-day long experiment, we aimed to expand our understanding of the link between chronic exposure of corals to turbid nutrient-enriched waters and their thermal tolerance from episodic heat stress. The objective of the experiment was to resolve whether coastal eutrophication may protect corals from thermal stress due to improved nutritional status Acropora millepora , colony pieces were sourced from Pelorus  and Double Cone Islands , and branchlets were used to form nubbins (length ∼4 cm). For the foliose Montipora tuberculosa , colony pieces were sourced from Magnetic Island , which were cut into ∼9–16 cm2 sized fragments. The water quality conditions of the collection sites are characterized in Colony fragments were sourced from inshore reefs of the central GBR at 2–4 m depth. For the corymbose Symbiodinium types were determined based on sequence differences in the rDNA ITS2 region using denaturing gradient gel electrophoresis A. millepora containing only C2 and M. tuberculosa only “C1-like” types of algal endosymbionts.Both study species are classified as highly susceptible to bleaching −1) were set up outdoors under a 30% light absorbing polycarbonate roof in three 1000 L water baths (8 tanks per water bath). Three Odyssey light loggers were used to record irradiance within the water baths (10-min readings throughout the 90 day period). The median daily maximum photosynthetic irradiance was 1017 µmol photons m−2 s−1 between 12∶00 and 13∶00 pm (range: 167–1340 µmol photons m−2 s−1), equivalent to a median of 25 mol photons m−2 d−1 (range: 5.3–50.7 mol photons m−2 d−1), depending on cloud cover and length of day. The tanks were supplied with 4 L hr−1 of coastal seawater . A small bilge pump  was placed into each tank to provide water flow and reduce particle settling. Six days before the experiment started, A. millepora nubbins were suspended in the water with nylon string from transparent plastic sticks, and four M. tuberculosa fragments were placed on the bottom of each tank . The water column over A. millepora and M. tuberculosa colonies was 10 and 16 cm deep respectively, hence differences in light exposure between enriched and unaltered seawater treatments and between colonies that were suspended and on the bottom were negligible (<3% difference at an estimated mean light diffusive attenuation coefficient Kd of 0.4 and 0.2).The experiment was carried out at the Townsville site of the Australian Institute of Marine Science (AIMS). Light and water quality treatments were designed to be as environmentally relevant as possible. Twenty-four aerated 20 L glass tanks with flow-through seawater (4 L hr3). The ambient temperature treatments contained either Controls  or +OE +NO3. Heat-stress treatments contained all four combinations of nutrient additions: Controls, +NO3, +OE, or +OE +NO3.On Day 1 of the experiment, corals were exposed to their respective nutrient and sediment treatments for acclimatization at ambient temperature. In summary, six treatments were established, each represented by four tanks, with four coral fragments of each of the two species added to each tank. Two levels each of temperature, organically enriched water and nitrate were used (as described in detail below): ambient temperature and heat stress (25°C vs. 31°C); without and with the addition of organically and nutrient enriched water (+OE); and without and with the addition of nitrate (+NO−1) vigorously circulated the warmed water within the water baths to ensure uniform temperature across treatments. Water quality treatments were distributed evenly across these water baths (two of each treatment per water bath), and positions were randomised within water baths. Water temperature was measured daily in all tanks with a digital thermometer (accuracy ±0.1°C). Daily mean temperatures averaged 31.2°C ±0.6 SD in the heated tanks, and 25.3°C ±0.8 SD in the control tanks (range: 26.6°C ±0.6 SD in April to 24.6°C ±0.9 SD in July). The temperature was kept at these levels until the onset of severe decline in photosynthetic yields and visible bleaching in at least one treatment per species. This occurred after 23 days of heat stress for A. millepora and after 49 days for M. tuberculosa. Heat-stressed colonies from all treatments were moved into recovery tanks in the ‘ambient temperature’ water bath, with their exposure to NO3 and/or OE unchanged. The experiment was terminated after 38 days of recovery for A. millepora, and 31 days of recovery for M. tuberculosa.After 10 days of coral acclimatization to the water quality treatments at ∼27°C, the water in 16 of the 24 tanks (two of the three water baths) was gradually increased over a four-day period to 31.2°C (the mean long-term summer maximum temperature in the region is ∼30°C). Submersible titanium heaters were used to warm the water in the water bath, and large bilge pumps (5000 L hr−1), however settlement of the larger particle fraction occurred. Two levels of organically enriched water (OE) were used: 12 tanks were supplied with 2 L hr−1 of the coastal seawater complemented with 2 L hr−1 of water from the incubation batch (+OE), and the remaining 12 tanks were supplied with the coastal seawater at 4 L hr−1, without OE addition.The coastal water around AIMS, located downstream of two major rivers (Burdekin and Haughton Rivers), contains substantial concentrations of nutrients and sediments (‘Control’ concentrations in 3 stock solution diluted with filtered seawater (0.0053∶ 1) was fed continuously into 12 tanks (+NO3: six with and six without OE) by means of a peristaltic pump  with Tygon Tubing  at a rate of 0.25 ml min−1. Nitrate in the +NO3 treatment was nominally increased by 4.0 µmol L−1 (but see below for uptake). The control tanks without nitrate addition had a mean ambient concentration of 0.23 µmol L−1.Nitrate exposure was manipulated to distinguish between the commonly investigated direct effects of nitrate on the endosymbionts and the effects of organical enrichment on the coral holobiont. DIN rather than DIN plus phosphate was added, since an over-supply of DIN has been suggested to be largely responsible for declining thermal tolerance of corals a , and dissolved and particulate nutrients and chlorophyll seawater . Final cnge rate . This inDifferences in heat stress tolerance were quantified as differences in survival, photophysiological stress and recovery, and skeletal growth rates.A. millepora, and Day 90 in M. tuberculosa.Survival of colonies was assessed daily, and mortality was defined as the point where tissue was sloughed off from >50% of the colony surface. Dying corals were removed from the tanks to avoid affecting other colonies. Tissue slothing, once started, was inevitably followed by the death of nubbins within 1–2 days. Survival was expressed as the proportion of colonies within each tank that survived to Day 71 of the experiment in v/Fm = (Fm – F0)/Fm with Fv, Fm and F0 being the variable, maximum and background fluorescence in dark-adapted state −2 s−1) red light to obtain F0, followed by a 1 s pulse of saturating actinic light (>5000 m−2 s−1) to determine Fm. Fluorescence yields were determined as the mean of 5 area readings per fragment. They were measured on the second day of the acclimation period, at the beginning of the heat stress period, and then at 5-day intervals until yields started to decline, upon which measurement frequency was increased to once every 2 to 3 days. During recovery, yields were measured every 6 to 10 days.Photo-physiological responses were assessed using pulse amplitude modulation fluorometry . The effects of the different treatments on the photochemical capacity of photosystem II were explored by determining changes in chlorophyll fluorescence yields , following Three sets of analyses were undertaken.3, +OE, +OE +NO3) were estimated using a generalized linear model with quasibinomial errors and a logistic link function A. millepora and M. tuberculosa, respectively.Survival was expressed as the proportion of colonies that had survived at the end of the recovery period in each tank, and differences in survival between heat-stressed colonies in response to the four nutrient treatments  died on the last day of the heat stress period , and mortality increased to a mean of 59.4% of colonies per tank by the end of the recovery period. Survival varied greatly between tanks, but means were slightly lower in the two heated +OE treatments compared to those without OE addition .In the ambient temperature treatments, no mortality was recorded in either species throughout the 90-day long experiment . Of the Montipora tuberculosa, none had died after 58 days (48 days of heat stress). In the two treatments without OE, survival remained at 100% throughout the recovery period, in contrast to the two +OE treatments where survival varied widely between tanks but declined to a mean of 44% and 56% at the end of the recovery period , while the addition of NO3 did not affect the survival of heat-stressed M. tuberculosa, and there was no interaction between OE and NO3.Of the heat-stressed y period . The difA. millepora showed a minor decline (–0.05 units) over time in both +OE +NO3 and Controls  = 6.4, P = 0.01, with also significant differences between tanks; M. tuberculosa: 0.647±0.021 vs. 0.628±0.014, F = 28.0, P<0.001, no differences between tanks), suggesting a minor photophysiological response to the higher nutrients and slightly reduced light.Chlorophyll fluorescence yields for each of the species were similar across all treatments at the beginning of the 10-day acclimation period P>0.05,  and 3. AControls , while tal trend . After tA. millepora was more susceptible to heat stress than M. tuberculosa. Reductions in yields were observed after 13 days of heat stress in A. millepora, and after 33 days in M. tuberculosa , whereas colonies in the other three treatments had recovered to values similar to their initial values .Changes in colony weights were calculated, excluding corals that had died. For  weights . There wM. tuberculosa, initial and final mean weights were 3.377 g and 3.916 g respectively, giving a mean gain of 0.539 g. Weight gains dropped sharply for initial weights <3 g, but plateaued and were more variable at higher weights .For  weights . There wThis study demonstrates that exposure to organically and nutrient enriched coastal waters at environmentally relevant concentrations reduces the resistance and resilience of corals to temperature stress, leading to greater reductions in fluorescence yields, lower survival and slower recovery in two common coastal coral species.PoritesMontastrea faveolataDiploastrea helioporaPocillopora damicornisA. millepora therefore adds important information on the potential effects of warming seawater temperatures on Acropora growth. This estimate is conservative since the most sensitive individuals had died and were not included in the analysis. In contrast, skeletal growth of the more temperature tolerant M. tuberculosa was unaffected by temperature stress. Instead, the growth of this foliose species was reduced by 24% at +OE, potentially reflecting its exposure to settling particles.There is mounting evidence that heat stress lowers skeletal growth rates, as demonstrated for massive corals such as Terrestrial runoff, and the associated increase in nutrients and sediments, represents a complex and multi-factorial agent of change, affecting corals through many different but related pathways: (1) increased availability of particulate food, (2) increased exposure to dissolved inorganic nutrients, (3) reduced light availability from reduced water clarity, and (4) increased stress from exposure to organically and nutrient enriched sediments Acropora intermedia at high irradiance. In contrast to that study, we incubated muddy sediments enriched with nutrients. The difference strongly suggests that it is the organical enrichment that constitutes a stress factor, which simulated the plankton successions in organical and nutrient enriched inshore waters in the field, which contain bacterio-, phyto- and zooplankton, detrital matter, fecal pellets and other organic and inorganic particles colonized by bacteria and microalgae, as well as dissolved organic and inorganic forms of nutrients. Our experimental design simulated natural and environmentally relevant processes, but the trade-off was that its natural complexity does not allow attributing the additional stress to any specific agent. Stress may have been caused by one or several different mechanisms, including: greater oxygen radical production of the dense endosymbionts 2 and metabolic products of the organic-rich materials, altering the seawater chemistry and causing harm when ingested or when settling on colony surfaces The strong negative effect of organically and nutrient enriched waters on the corals’ thermal tolerance demonstrated in this study suggests that coastal eutrophication produces an additional stress factor that outweighed nutritional benefits in these two coral species. Interestingly, Anthony et al. Artemia in experiments) and other microplankton, and to a more limited extent pico- and nanoplankton and particulate organic matter Our literature review has shown that both from existing empirical data and theoretical perspective, it has remained surprisingly unclear whether corals exhibit synergistic/additive or antagonistic responses when simultaneously exposed to heat stress and eutrophication. Although the complexity of the water quality problem and the diversity of study methods preclude a quantitative meta-analysis, it allows identifying directions of change along environmental gradients that are consistently observed despite the different study species and methods used. The observed commonalities are summarised in a conceptual trophic framework , which iAcanthaster planci when exposed to terrestrial runoff In conclusion, this study adds to mounting evidence that eutrophication can worsen thermal stress on inshore reef communities. Even without heat stress, the exposure to organical enrichment has strong negative effects on the photophysiology and survival of inshore corals


Zoologica Scripta, 41, 617–636.Elven, E., Bachmann, L. & Gusarov V. I. (2012) Molecular phylogeny of the Athetini–Lomechusini–Ecitocharini clade of aleocharine rove beetles (Insecta). —Lomechusa. The two clades formed a sister group to the main Athetini clade, which also included Ecitocharini and the ‘false Lomechusini’, a group of New World genera normally placed in Lomechusini. The following changes in classification are proposed: (i) Geostibina Seevers, 1978 is raised to tribal rank, and 13 Athetini genera are placed in Geostibini; (ii) Ecitodonia Seevers, 1965; Ecitopora Wasmann, 1887, and Tetradonia Wasmann, 1894 are moved from Lomechusini to Athetini; (iii) Ecitocharini Seevers, 1965 is placed in synonymy with Athetini; (iv) Discerota Mulsant & Rey, 1874 is tentatively included in Oxypodini; (v) Actocharina Bernhauer, 1907 is placed in synonymy with Hydrosmecta Thomson, 1858.It has previously been shown that the Aleocharinae tribes Athetini and Lomechusini form a well-supported clade, which also includes the small Neotropical tribe Ecitocharini. However, neither Athetini nor Lomechusini were recovered as monophyletic. In this study, we addressed the basal phylogenetic relationships among the three tribes using sequence data from (i) a mitochondrial fragment covering the COI, Leu2 and COII genes; (ii) a mitochondrial fragment covering part of the 16S gene, the Leu1 gene and part of the NADH 1 gene; and (iii) a part of the nuclear 18S gene, for 68 Athetini, 33 Lomechusini and 2 Ecitocharini species, plus representatives from 10 other tribes. The athetine subtribe Geostibina was recovered as sister group to the ‘true Lomechusini’, which included the type genus  The rove beetles (Staphylinidae) are one of the two largest families of Coleoptera, comprising (with Scydmaeninae) 32 extant subfamilies and more than 55 000 species ] are theIn a recent study, Atheta alone includes 1700 species . The majority of athetines are free living, while a few are associated with ants or termites. The tribe is traditionally diagnosed based on a combination of several characters, for example, galea and lacinia of moderate length, tarsal formula 4-5-5, mesocoxae narrowly or moderately separated, mesoventral process narrow, athetine bridge of aedeagus present. However, none of these characters is unique to Athetini. An older available family group name, Callicerini Jakobson, 1908 , exists for this tribe, but Athetini Casey, 1910 is the name currently in prevailing use . They discovered the ALE clade, consisting of the three tribes Athetini, Lomechusini and Ecitocharini. Within the ALE clade, the athetine genera Geostiba and Earota formed a sister group to the lomechusine genera Pella and Drusilla. This clade in turn formed a sister group to the remaining Athetini (referred to as the ‘main Athetini clade’), which also included the tribe Ecitocharini and the lomechusine genus Myrmedonota. The  clade was an unexpected discovery, but there is at least one tentative morphological synapomorphy for this clade: sensillum a of the epipharynx being reduced  Geostibina and the ‘true Lomechusini’ form a sister group to the main Athetini clade; (iii) All athetine genera that have sensillum a of the epipharynx reduced belong to Geostibina; (iv) Several genera traditionally placed in Lomechusini are not members of the ‘true Lomechusini’ clade, but form a subclade  within the main Athetini clade; and (v) Ecitocharini are monophyletic, nested within the main Athetini clade, and have Stethusa as their sister group.The main goal of this study is to firmly resolve the phylogeny of the major lineages of the ALE clade discovered by a of the epipharynx: Alevonota, Aloconota, Callicerus, Earota, Geostiba and Pelioptera. These genera were hypothesized to belong to a monophyletic Geostibina. We included the type species of the type genera of Athetini, Lomechusini and Geostibina, and the type species of many other genera including Alevonota, Aloconota, Callicerus, Drusilla, Earota and Meronera  could not even be assigned to genera, and their initial tribal placement in Lomechusini is based on an assessment of tribal characters, including tarsal formula 4-5-5, mesocoxae relatively broadly separated and mesoventral process short and broad.Most specimens were collected directly into >96% ethanol and stored at −20 °C prior to processing, but some trap material that had been exposed to high (+20 to +40 °C) temperatures and/or dilution by rain was also included. All specimens used for DNA extraction are labelled as vouchers and deposited at the Natural History Museum, University of Oslo (ZMUN) or the Zoological Museum, Natural History Museum of Denmark (ZMUC). Label information is provided in Table S1.Nucleotide sequences from one nuclear and two mitochondrial regions were targeted. The first mitochondrial region covered most of the cytochrome oxidase 1 and 2 (COI and COII) and the tRNA-Leucine 2 (Leu2) genes. The second mitochondrial region covered the 3′-end of the large ribosomal subunit (16S), the tRNA-Leucine 1 (Leu1) and a small part of the NADH dehydrogenase subunit 1 (NADH1) genes. The nuclear region covered an internal part of the small ribosomal subunit (18S) gene. These markers were used by Actocharina, the whole body was used. DNA extraction was performed on vacuum-dried samples without prior homogenization. Samples were incubated in lysis buffer for 20–30 h. After extraction, the exoskeletons were retrieved for dry mounting with the rest of the voucher.DNA was extracted from the head and prothorax using the Qiagen DNeasy Blood and Tissue Kit  following the manufacturer’s protocol for animal tissue. For the very small specimens of m MgCl2 , 1 × ABI GeneAmp PCR buffer (Applied Biosystems), 0.8 mm GeneAmp dNTPs (Applied Biosystems), 0.5 μm of each primer , 1 U ABI AmpliTaq DNA Polymerase (Applied Biosystems) and 3 μL template DNA extract. Most reactions also included 1.1 mg dimethyl sulfoxide  to improve PCR performance. When amplifying the 16S–Leu1–NADH1 region, the PCR set-up was adjusted to 2 mm MgCl2, 0.96 × PCR buffer, 0.64 mm dNTPs and 0.4 μm of each primer. The amplification profile consisted of an initial denaturation step of 94 °C for 30 s, followed by 30 cycles of 94 °C for 1 min, annealing temperature Ta for 30 s and 72 °C for 2 min, and finally a 10 min extension step at 72 °C. The annealing temperatures are listed in Table S2. For some difficult samples, PCR performance was improved by replacing dimethyl sulfoxide in the reaction mix with 0.4 μg bovine serum albumin , by using alternative primers (listed in Table S2), by lowering the annealing temperature or by using an alternative PCR protocol employing HotStar Taq DNA Polymerase (Qiagen).The targeted regions were amplified using the primers listed in Table S2. The mitochondrial COI–Leu2–COII region was amplified in three overlapping fragments, while the mitochondrial 16S–Leu1–NADH1 region and the nuclear 18S region were each amplified in single fragments. PCRs were set-up in a 25-μL reaction volume containing 2.5 mPCR products were purified using ExoSAP-IT . If secondary products were detected on a standard agarose gel, the PCR product of appropriate size was cut out from 1% agarose gel and purified using the MN NucleoSpin Extract II gel extraction kit . Purified PCR products were sequenced in both directions using the terminal primers with the ABI BigDye Terminator Cycle Sequencing Kit v3.1 (Applied Biosystems) and analysed on the ABI 3730 DNA Analyzer (Applied Biosystems). All DNA sequencing was outsourced to the ABI-lab, Departments of Biology and of Molecular Biosciences, University of Oslo.Apis mellifera  to produce a total of seven partitions  and Bayesian inference. Two analyses were performed with each method; one with all sequences included and one where sequences with more than 20% missing information were excluded (i.e. those lacking data for one of the three targeted regions).ML analyses were performed in RAxML 7.0.3  using thi = auto vs. i = 10) were compared by performing ML optimization on 10 randomized parsimony trees with each setting, and choosing the setting resulting in the highest likelihood score for the optimized trees. Four alternative numbers of rate categories  were then compared in the same way using the best rearrangement setting from above. The same 10 starting trees were used for all comparisons. Based on the results of these comparisons, the analysis that included incomplete sequences was performed using i = auto, while the analysis using only complete sequences was performed using i = 10. Both analyses were performed using 25 rate categories.Prior to the analysis, the rearrangement settings and number of rate categories were optimized following the author’s recommendations . Two alternative rearrangement settings  at the University of Oslo, Norway.Bayesian analyses were performed in MrBayes v3.1.2  using thThe concatenated sequence alignment of 180 samples included 3786 positions after trimming. Complete sequence information was obtained for 150 samples, while 30 lacked sequence data for one of the three target regions . BecauseThe majority-rule consensus trees from the Bayesian analyses are shown in In both Bayesian analyses, the average standard deviation of split frequencies stabilized between 0.01 and 0.05 after about 20 million generations, indicating that stationarity was reached after about 5 million generations. The final standard deviation values after 100 million generations were 0.011 when incomplete sequences were excluded and 0.032 when they were included. The potential scale reduction factor approached 1 for all model parameters, never exceeding 1.009. Most model parameters showed good mixing, but the substitution rates and base frequencies for unpaired mitochondrial sites showed bimodal sampling when incomplete sequences were excluded. The chains swapped frequently between the two distinct optima, and both were thoroughly sampled during the run. The rate multiplier parameters showed alternating periods of good and poor mixing when the proposals tuning parameter value was adjusted to 8000, in contrast to uniformly poor mixing under the default value of 500.The Bayesian and ML analyses produced largely congruent phylogenies . In turn, the  clade formed a sister group to the main Athetini clade , which included Ecitocharini and the ‘false Lomechusini’. The ‘false Lomechusini’ clade, consisting of Ecitodonia, Ecitopora, Meronera, Myrmedonota, Tetradonia and several not yet identified Neotropical taxa, was well supported in the Bayesian analysis (PP = 0.99) but not in the ML analysis (BS < 50%). The Ecitocharini, represented by two genera, were recovered as monophyletic  and had Stethusa as its sister group .The ALE clade was recovered with high statistical support in all analyses (Bayesian posterior probability (PP) = 1.00, ML bootstrap support (BS) = 87%). When incomplete sequences were excluded  and S1, Stethusa, Ecitocharini) clade was not recovered when incomplete sequences were included; Stethusa and Ecitocharini instead formed separate clades with poorly supported or unresolved relationships to other athetine clades. Also, the ‘false Lomechusini’ were not recovered as monophyletic when incomplete sequences were included.The inclusion of 30 incomplete sequences had little impact on the overall tree topology  and S2. a of the epipharynx reduced. Among the other genera included in our study, only Pelioptera shares this character state. When examining the microscope slides of the entomological collection of the Natural History Museum, Oslo, as well as published illustrations of athetine genera, we identified additional genera (not included in our phylogenies) that have sensillum a of the epipharynx reduced. a has been confirmed. In total, 13 genera were confirmed to share this character state. For nine of these, the type species was examined. Six other athetine genera that resemble Geostiba in having the ligula fully bilobed were found to have sensillum a fully developed (i.e. much longer than wide): Boreophilia (slide preparation examined), Liogluta , Saphocallus (see Schistoglossa (slide preparation examined) and Tomoglossa , for which there was only moderate support. The five hypotheses are reviewed below.The molecular phylogeny of the Athetini–Lomechusini–Ecitocharini (ALE) clade of aleocharine rove beetles presented in this study is in line with the main finding of H1: Geostibina are a sister clade to the ‘true Lomechusini’.The Geostibina clade and the ‘true Lomechusini’ clade were both strongly supported, and the sister group relationship between them strongly corroborated.Earota (two species) and Aloconota (four species) was confirmed, but the monophyly of Geostiba (three species) and Alevonota (two species) was not. The inclusion of Callicerus in the Geostibina clade creates a nomenclatural problem. The rarely used family group name Callicerina Jakobson, 1908 has priority over the currently more widely used Geostibina Seevers, 1978; but the first is also a junior homonym of Callicerini Rondani, 1845, which is in use for a tribe of hoverflies (Diptera: Syrphidae). To solve the issue, an application proposing to suppress the name Callicerina Jakobson, 1908 and to keep the more widely used Geostibina Seevers, 1978 has been submitted to the International Commission on Zoological Nomenclature , Orphnebius (four species) and Pella (two species) were recovered as monophyletic with high support. The genus Drusilla (four species) was recovered as paraphyletic with respect to the morphologically distinct Oriental genus Amaurodera (one species). As currently accepted, Drusilla is distributed worldwide and includes almost 200 species . A worldwide revision of Drusilla is needed to divide the genus into diagnosable monophyletic genera. The genus Zyras  was recovered as polyphyletic. The nominotypical subgenus (represented by Z. collaris) was recovered as the sister group to all other ‘true Lomechusini’. Two closely related species of Zyras (Glossacantha) from Thailand were recovered in a separate clade with a very long branch. A third clade included yet another species of Zyras (Glossacantha), one species of Zyras (Rhynchodonia), one Zyras species not yet identified to subgenus and the morphologically very distinct genus Pedinopleurus. Accordingly, even the subgenus Glossacantha is not monophyletic. In the current classification, Zyras is divided into 54 subgenera and includes more than 800 species  have sensillum a of the epipharynx reduced. Among the other genera included in this study, only Pelioptera has this character state. In our analyses, Pelioptera formed a sister group to the  clade. The support for this placement was not strong, however, and is further weakened by the fact that the entire COI–COII region was missing from the Pelioptera sequence. Given that Pelioptera shares additional character states (see below) with the five genera of Geostibina, we hypothesize that Pelioptera is also a member of Geostibina and that the reduced sensillum a is a synapomorphy for this group. In addition to the six genera included in the molecular analyses, seven further athetine genera were confirmed to possess the reduced sensillum a of the epipharynx  were also confirmed to have a normally developed sensillum a. Based on this evidence, we predict that they do not belong to the Geostibina clade.All five genera forming the Geostibina clade  from the New World. Seven of the included genera were unidentified. The clade was well supported only in the Bayesian analysis  are exclusively Neotropical. Two genera (Tetradonia and Meronera) are distributed mostly in the Neotropical region, with a few species also in the Nearctic. The fifth genus, Myrmedonota, is known from both the Nearctic and the Oriental regions (but see below). The geographic distribution of the ‘false Lomechusini’ suggests that the clade may have originated in South America and dispersed into North America. More extensive taxon sampling in both Old and New World tropics is needed to test this hypothesis.Ten of the included genera were collected in Ecuador and two in the USA. Of the five identified genera, two  and geographic distribution. It seems reasonable to assume that the tribe is monophyletic.The type genus of Ecitocharini, r states  and are As the Ecitocharini are nested within the main Athetini clade, their inclusion in Athetini should be uncontroversial. The aim of this study was to resolve the phylogeny of the major lineages of the ALE clade in order to revise the classification of the tribes involved. With strong support for all three subclades within the ALE clade, the ‘true Lomechusini’, the subtribe Geostibina and the main Athetini clade, we can address the issue of classification. A revised tribe-level classification needs to meet the following criteria: (i) all formally recognized taxa should be monophyletic; (ii) the classification should reflect the three main subclades and be compatible with their phylogenetic relationships: ; (iii) the principle of priority should be satisfied; (iv) the recognized family group taxa should be diagnosable using morphological characters, preferably apomorphic states; and (v) the choice of classification should promote stability of scientific names  are removed from Athetini by raising the rank of the subtribe to tribe. The genera of the ‘false Lomechusini’ are moved from Lomechusini to Athetini. Further subdivision of the three tribes into subtribes is still possible.Alternative 2: moving subtribe Geostibina to Lomechusini. Two tribes are recognized in the ALE clade: Athetini and Lomechusini (including subtribe Geostibina). Most of the genera and species currently in Athetini and Lomechusini stay in their respective tribes. The subtribe Geostibina is moved from Athetini to Lomechusini, while the genera of ‘false Lomechusini’ are moved from Lomechusini to Athetini. Subdivision of the two tribes into subtribes is still possible.Alternative 3: expanding the Lomechusini to include all Athetini. Only one tribe is recognized in the ALE clade: Lomechusini. This tribe is further subdivided into three subtribes: Athetina, Geostibina and Lomechusina. This solution is similar to some earlier classifications  formed a well-supported clade with Halobrecta outside the ALE clade. In Halobrecta was removed from Athetini and tentatively placed in Oxypodini. Like Halobrecta, Discerota lacks the athetine bridge of the aedeagus and has both male and female genitalia similar to some Oxypodini. For these reasons, we move Discerota from Athetini and place it tentatively in Oxypodini as well.The athetine genus Compared to Actocharina, Atheta  vilis, Brundinia, Parameotica and Hydrosmecta (represented by five species).Remarkably, many riparian taxa were recovered as members of a strongly supported monophyletic group. This riparian clade included Actocharina, originally a subgenus of Atheta, is distributed in Austria and northern Italy where it inhabits sandy river banks in the Kalkalpen. The beetles are minuscule, only up to 1.4 mm long, have reduced eyes, wings and pigmentation and presumably move in the interstitial space between sand particles like many Hydrosmecta. When describing Atheta (Actocharina) leptotyphloides, Hydrosmecta, H. subtilissima and H. tenuissima. With respect to external morphology, Actocharina is indeed similar to the smallest species of Hydrosmecta, for example, H. delicatula or H. tenuissima  Three genera are moved from Lomechusini to Athetini. (iii) The family group name Ecitocharini Seevers, 1965 is placed in synonymy with the name Athetini Casey, 1910. All 10 genera formerly treated as members of the tribe Ecitocharini are moved to Athetini. (iv) The genus Discerota Mulsant & Rey, 1874 is removed from Athetini and tentatively included in Oxypodini. (v) The genus name Actocharina Bernhauer, 1907 is placed in synonymy with Hydrosmecta Thomson, 1858. The new combination Hydrosmecta leptotyphloides  is established for the species originally described as Atheta (Actocharina) leptotyphloides Bernhauer, 1907.The following changes in classification of Aleocharinae are proposed (see