The speciation continuum: population structure, gene flow, and maternal ancestry in the Simulium arcticum complex (Diptera: Simuliidae).

Comparative analyses of populations at different stages of divergence can yield insights into the process of speciation. We assess population structure, gene flow, and maternal ancestry at five locations containing sympatric members of the Simulium arcticum complex at different stages of chromosome divergence. We analyze both nuclear and mitochondrial DNA markers, including 11 microsatellite loci, as well as COI, COII, cytb, and ND4 gene sequences. Simulium negativum, representing the later stages of divergence, shows both nuclear and mitochondrial differentiation when compared with allopatric and sympatric chromosomal forms, as well as both low contemporary and historical gene flow in sympatry. At intermediate stages of chromosome divergence, populations differ at nuclear, but not mitochondrial, loci in allopatry and sympatry. In one comparison of intermediate stage chromosomal forms (S. arcticum sensu stricto and S. apricarium), populations demonstrate low contemporary, but higher historical, gene flow in sympatry. In a second sympatric comparison (S. arcticum s. s. and S. brevicercum), both contemporary and historical gene flow are high. All analyses of sympatric populations at the earliest stages of chromosome divergence demonstrate panmixia; yet, some nuclear differentiation in allopatry is apparent. These findings suggest that molecular divergence is tracking chromosome divergence along a chromosomally-defined continuum of speciation in black flies.

Research Note: Comparative effects of liquid and dry applications of a combination of lysolecithin, synthetic emulsifier, and monoglycerides on growth performance, nutrient digestibility, and litter moisture in broilers fed diets of differing energy density.

Supplementation of a combination of lysolecithin, a synthetic emulsifier, and monoglycerides (LEX) in liquid and dry form to broiler diets with different energy levels was investigated to determine their effect on performance, litter quality and subsequent occurrence of footpad lesions. One thousand two hundred and forty-eight-day-old Ross 308 broilers were assigned to 1 of 6 treatments for a 42-day study: a basal diet with a normal energy content (NE); NE + 300 g/t LEX in liquid form (LEL); NE + 500 g/t LEX in dry form (LED); a basal diet with low energy (LE, -90 kcal/kg starter, -100 kcal/kg grower, finisher), LE + 300 g/t LEL and a LE + 500 g/t LED. Each treatment consisted of 13 pens of 16 birds each. Diets were fed in 3 phases (starter d 0-10, grower d 11-21, finisher d 22-42). Feed intake and weight were measured on d 0, 10, 21, and 42. On d 42 a litter sample was collected from each pen and 2 birds per pen were assessed for footpad lesions and breast scald. Data were analyzed using JMP 16, with means separation achieved using Tukey's HSD; significance was assumed at P < 0.05. Results showed a higher (P < 0.05) cumulative bodyweight gain with LEX supplementation (NE CON = 2,718 g, NE+LED = 2,829, NE+LEL = 2,895, LE CON = 2,722, LE+LED = 2,787, LE+LEL = 2,893; P = 0.0027). An increased feed intake was observed for the LE diets, however cumulative FCR of LE+LED and LE+LEL remained equal to the NE control (1.657 NE CON, 1.657 LE+LED, 1.623 LE+LEL; P > 0.05), suggesting LEX enabled the birds to compensate for the energy gap. Litter dry matter was significantly improved with both LED and LEL supplementation compared to the control groups, and resulted in lower (P < 0.05) occurrence and severity of footpad lesions and breast scalds. Considering the income over feed cost (IOFC) of the NE treatment as the reference point for comparison, all other treatments improved profitability, with NE+LEL and LE+LEL achieving the greatest IOFC with 154.58 and 175.96 €/1,000 birds respectively. In conclusion, feeding broilers a combination of lysophospholipids, a synthetic emulsifier and monoglycerides resulted in improved bird performance. The use of the LEX also improved litter quality and footpad health, therefore improving animal welfare indicators such as breast scald and footpad measurements.

Good species behaving badly: Non-monophyly of black fly sibling species in the Simulium arcticum complex (Diptera: Simuliidae).

Mitochondrial based phylogenetic reconstructions often show deviations from species-level monophyly. We used the Simulium arcticum species complex (Diptera: Simuliidae) as a model system for interpreting non-monophyly in light of chromosomal data supporting species status of siblings. For cytogenetic identification of morphologically indistinguishable black fly sibling species, larvae must be preserved in Carnoy's solution, a fixative known to degrade DNA. Consequently, we reconstructed phylogenetic relationships based on 12S, COII, cyt b, and ITS-1 gene sequences obtained from larvae sampled from presumed taxon-pure localities. As species composition at 'taxon-pure' sites may have changed at the time of sampling, we performed a second study that aimed to: (1) assess phylogenetic relationships among cytologically verified members of the S. arcticum species complex using COI and COII gene sequences; (2) determine whether useable genetic information could be gleaned from Carnoy's fixed specimens; and (3) determine the extent to which Carnoy's fixative degrades DNA over time. We consistently obtained genetic data from material stored in Carnoy's solution for two to three months. Genetic analysis of samples fixed in Carnoy's solution for up to six years indicates that larvae preserved for a maximum of five years can provide useable information for molecular analysis. Our preliminary and cytologically confirmed phylogenetic analyses demonstrate that mitochondrial DNA fails to resolve species-level monophyly of chromosomally distinct S. arcticum taxa. As results of analyses based on cytologically verified larvae mirror those of our preliminary study, we rule out imperfect taxonomy as the reason for species-level non-monophyly. Although we cannot confidently reject either inadequate phylogenetic information or incomplete lineage sorting as the cause of non-monophyly, the sharing of alleles between sympatric siblings suggests introgressive hybridization between taxa. We conclude that the patterns present in the S. arcticum phylogeny likely represent the initial stages of chromosome based sibling speciation.

Simulium (Inaequalium) marins, a new species of black fly (Diptera: Simuliidae) from inselbergs in Brazil.

A new species of black fly, Simulium (Inaequalium) marins (Diptera: Simuliidae), is described based on the male, female, pupa and larva. This new species was collected from two localities: a small stream on the Pico dos Marins, a high mountain with granite outcrops in Piquete County, state of São Paulo, and in a small stream in the Serra dos Orgãos National Park, state of Rio de Janeiro, Brazil.

Austrocnephia, new genus, for five species of 'Paracnephia' (Diptera: Simuliidae), with a key to Australian black fly genera.

A segregate of the so-called Australian 'Paracnephia' (Diptera: Simuliidae) is assigned to a new genus, Austrocnephia. The taxon is fully diagnosed and a key to constituent species presented. Two species-groups are recognized: the aurantiaca species-group, comprised of A. aurantiaca (Tonnoir 1925) and A. strenua (Mackerras Mackerras 1950), and the tonnoiri species-group, comprised of A. fuscoflava (Mackerras Mackerras 1948), A. orientalis (Mackerras Mackerras 1950) and A. tonnoiri (Drummond 1931). Both species-groups are diagnosed and the included species fully redescribed. Detailed locality data is given, as is information about biology, when known. Brief comments are offered about the historical biogeography of Austrocnephia. A key to Australian simuliid genera is also provided.

Diptera of Canada.

The Canadian Diptera fauna is updated. Numbers of species currently known from Canada, total Barcode Index Numbers (BINs), and estimated numbers of undescribed or unrecorded species are provided for each family. An overview of recent changes in the systematics and Canadian faunistics of major groups is provided as well as some general information on biology and life history. A total of 116 families and 9620 described species of Canadian Diptera are reported, representing more than a 36% increase in species numbers since the last comparable assessment by JF McAlpine et al. (1979). Almost 30,000 BINs have so far been obtained from flies in Canada. Estimates of additional number of species remaining to be documented in the country range from 5200 to 20,400.

Nothogreniera new genus, for two species of Australian "Paracnephia" (Diptera: Simuliidae).

Two species of Australian Simuliidae known only from adult females and currently assigned to "Paracnephia" are re-described, as are their now-known males and immature stages. Morphological character states of "Paracnephia" fergusoni (Tonnoir) and "P." fergusoni var. (Mackerras Mackerras) reveal that they are markedly distinct from all other Australian species, and are here assigned to the new genus-Nothogreniera-the most plesiomorphic Gondwanan Australian simuliid. Structural variation among populations of N. fergusoni suggests that this entity comprises a species complex.

A new genus, Protaustrosimulium, for four species of Australian black flies (Diptera: Simuliidae).

Protaustrosimulium n. gen. is described for four species: two previously named species from southeastern Australia-Paracnephia pilfreyi (Davies Györkös 1988) and Paracnephia terebrans (Tonnoir 1925)-plus two newly described ones from the southwestern-most corner of Western Australia-Prot. amphorum n. sp. and Prot. opscurum n. sp. Molecular and morphological data suggest a close relationship between members of the new genus and Austrosimulium Tonnoir 1925. Monophyly of Protaustrosimulium is supported mainly by characters of adult females, as two of the four species are known only in that life stage. Two species groups are recognized: the pilfreyi-group for Prot. pilfreyi and Prot. amphorum, and the terebrans-group for Prot. terebrans and Prot. opscurum. The constituent species in each group are distributed vicariously in southeastern and southwestern Australia-a common biogeographical pattern in Australian simuliids.

Reassignment of Western Australia Paracnephia gladiator Moulton Adler to a new genus, Bunyipellum (Diptera: Simuliidae).

With new material available of most stages of many known Australian Paracnephia, including new species, it is now clear that certain segregates warrant assignment to new genera. This applies to Paracnephia gladiator Moulton Adler, a Western Australia simuliid with numerous unique character states. The species is fully redescribed and assigned to Bunyipellum nov. gen. A diagnosis is provided and relationships discussed, as is historical biogeography. Bunyipellum appears to be more closely related to elements of the South American simuliid fauna than to any other Gondwanan Australian species.

Tolerance of Broilers to Dietary Supplementation with High Levels of the DHA-Rich Microalga, Aurantiochytrium Limacinum: Effects on Health and Productivity.

It is well established that the docosahexaenoic acid (DHA) content of commonly consumed meats, such as chicken, can be increased through dietary supplementation with DHA-rich ingredients. The purpose of this study was to investigate the tolerance of broilers to dietary supplementation with the unextracted biomass of a DHA-rich microalgae Aurantiochytrium limacinum, so as to ensure its safety, since it is accumulated in broiler meat. Healthy day-old male Ross 308 chicks (n = 1120) were evenly distributed to 32 pens (35 chicks per pen), with pens randomly allocated to one of four dietary treatments, each having eight replicates. The dietary groups included one untreated control and three treatments corresponding to three inclusion levels (0.5, 2.5, and 5.0%) of All-G-Rich®, with the birds receiving the experimental diets ad libitum during the study (day 0⁻42). Bird survival, blood parameters, productivity, and breast and thigh DHA content were determined after 42 days of feeding. Supplementation at up to 10 times the intended use level had no negative effects on the mortality, blood parameters or productivity of the birds, while significant increases in the meat DHA content were observed. These results indicate that supplementation with Aurantiochytrium limacinum is a safe and effective way to increase broiler tissue DHA content.

Precipitation and ectoparasitism reduce reproductive success in an arctic-nesting top-predator.

Indirect impacts of climate change, mediated by new species interactions (including pathogens or parasites) will likely be key drivers of biodiversity reorganization. In addition, direct effects of extreme weather events remain understudied. Simultaneous investigation of the significance of ectoparasites on host populations and extreme weather events is lacking, especially in the Arctic. Here we document the consequences of recent black fly outbreaks and extreme precipitation events on the reproductive output of an arctic top predator, the peregrine falcon (Falco peregrinus tundrius) nesting at the northern range limit of ornithophilic black flies in Nunavut, Canada. Overall, black fly outbreaks and heavy rain reduced annual nestling survival by up to 30% and 50% respectively. High mortality caused by ectoparasites followed record-breaking spring snow precipitation, which likely increased stream discharge and nutrient runoff, two key parameters involved in growth and survival of black fly larvae. Using the RCP4.5 intermediate climate scenario run under the Canadian Global Climate Model, we anticipate a northward expansion of black fly distribution in Arctic regions. Our case study demonstrates that, in the context of climate change, extreme weather events can have substantial direct and indirect effects on reproductive output of an arctic top-predator population.

Taxonomic revision of Paraustrosimulium Wygodzinsky & Coscarón: reassignment of Austrosimulium colboi and description of P. obcidens n. sp. from Western Australia.

The hitherto monotypic South American genus Paraustrosimulium Wygodzinsky & Coscarón is revised to accommodate two Australian species: Austrosimulium colboi Davies & Györkös and Paraustrosimulium obcidens n. sp. The generic diagnosis is updated and the eastern Australian species Paraustrosimulium colboi (Davies & Györkös) n. stat. is re-described, including the male for the first time. The Western, Australian sister species of P. colbo, namely P. obcidens Craig, Moulton Currie n. sp. is also fully described. The relationship of Paraustrosimulium to other simuliid genera is discussed, as are aspects of historical biogeography.

System-Level Reform in Healthcare Delivery for Patients and Populations Living with Chronic Disease.

Healthcare in Canada has generally not kept pace with the evolving needs of patients since the creation of medicare in the 1960s. Budgets for hospitals, physicians and prescription drugs make up the bulk of spending in health, despite the need for better prevention and management of chronic disease, the needed expansion of home-based care services and the call for reform of front-line primary care. Over the past decade, a number of Canadian health authorities have adopted the US-based Institute for Healthcare Improvement Triple Aim philosophy (better population health, better patient experience and better per capita cost of care) in order to build system-level change. The Atlantic Healthcare Collaboration was one attempt to initiate system-level reform in healthcare delivery for patients living with chronic disease.

The use of electrodialysis to prepare aqueous bread extracts for bromate determination by chemiluminescence.

A cleanup procedure based on electrodialysis is described for the preparation of aqueous bread extracts for bromate determination by chemiluminescence. The technique utilizes electrophoresis with 3 chambers separated by semipermeable membranes. The relative merits of reverse osmosis (RO), ultrafiltration, and nanofiltration membranes with various molecular weight cutoffs were evaluated. The best results were obtained with an RO membrane manufactured from thin-film (composite) polysulfone as support for polyamide. A 0.14 M sodium sulfate solution in the center or collection chamber provides optimum conductivity. Aqueous hydroxylamine sulfate (30 mM) was selected for the anode compartment as a reductant for the anode oxidation products. The constant current mode at 150 mA with a potential of ca 100 volts was used. After electrophoretic separation, the bromate concentration in the collection chamber was typically 2 to 3 times greater than the concentration in the bread extract. The chemiluminescent reaction of bromate with sulfite with hydrocortisone as the enhancer was selected for detection of bromate. The emission, with a wavelength maximum at 575 nm, was found to "glow" rather than "flash" after the reagents were mixed; therefore, it was possible to optimize the light collection period. The method was validated with a variety of commercial bread products. White bread, hot dog buns, hamburger rolls, and a multigrain bread from 7 different manufacturers were studied.

Tissue-specific and dose-related accumulation of arsenic in mouse offspring following maternal consumption of arsenic-contaminated water.

The developmental toxicity of arsenic is not as well characterized as other metals such as lead or mercury. Many previous animal studies have used an acute exposure paradigm, which does not model chronic, low-level human exposure. The following study administered 10, 20, 40, 80 or 100 ppm sodium arsenite in drinking water to pregnant C57BL6/J mice. Adipose, blood, brain, breastmilk in stomach, kidney and liver tissues were collected from male and female offspring on postnatal day (PND) 1 and 21 to allow for disposition comparisons between tissues, sexes and across time. The 100 ppm dose was foetotoxic. Significantly fewer female pups were born in litters exposed to 80 ppm, while significantly more male pups were born in litters exposed to 20 ppm. Total arsenic levels differed between tissues with the highest levels in the brain and kidney in PND1 offspring. Levels were higher on PND1 than PND21, and there were few sex differences. The dose-response relationships in PND1 tissues were curvilinear, but in PND21 liver and kidney tissues, arsenic levels in control animals were significantly higher than levels in exposed animals. The tissue and age-specific disposition suggests that common biomarkers such as blood and urinary arsenic are not accurate predictors of levels in sensitive organs such as the brain.

Identification of Nearctic black flies using DNA barcodes (Diptera: Simuliidae).

DNA barcoding has gained increased recognition as a molecular tool for species identification in various groups of organisms. In this preliminary study, we tested the efficacy of a 615-bp fragment of the cytochrome c oxidase I (COI) as a DNA barcode in the medically important family Simuliidae, or black flies. A total of 65 (25%) morphologically distinct species and sibling species in species complexes of the 255 recognized Nearctic black fly species were used to create a preliminary barcode profile for the family. Genetic divergence among congeners averaged 14.93% (range 2.83-15.33%), whereas intraspecific genetic divergence between morphologically distinct species averaged 0.72% (range 0-3.84%). DNA barcodes correctly identified nearly 100% of the morphologically distinct species (87% of the total sampled taxa), whereas in species complexes (13% of the sampled taxa) maximum values of divergence were comparatively higher (max. 4.58-6.5%), indicating cryptic diversity. The existence of sibling species in Prosimulium travisi and P. neomacropyga was also demonstrated, thus confirming previous cytological evidence about the existence of such cryptic diversity in these two taxa. We conclude that DNA barcoding is an effective method for species identification and discovery of cryptic diversity in black flies.

Elevated glutamate and NMDA disrupt production of the second messenger cyclic GMP in the early postnatal mouse cortex.

The second messenger cyclic guanosine monophosphate (cGMP) plays many roles during nervous system development. Consequently, cGMP production shows complex patterns of regulation throughout early development. Elevated glutamate levels are known to increase cGMP levels in the mature nervous system. A number of clinical conditions including ischemia and perinatal asphyxia can result in elevated glutamate levels in the developing brain. To investigate the effects of elevated glutamate levels on cGMP in the developing cortex we exposed mouse brain slices to glutamate or N-methyl D-aspartate (NMDA). We find that at early postnatal stages when the endogenous production of cGMP is high, glutamate or NMDA exposure results in a significant lowering of the overall production of cGMP in the cortex, unlike the situation in the mature brain. However, this response pattern is complex with regional and cell-type specific exceptions to the overall lowered cGMP production. These data emphasize that the response of the developing brain to physiological disturbances can be different from that of the mature brain, and must be considered in the context of the developmental events occurring at the time of disturbance.

Isolation and characterization of 11 microsatellite loci from the black fly, Simulium negativum (Diptera: Simuliidae).

Eleven polymorphic microsatellite loci were isolated and developed from the black fly, Simulium negativum, a member of the Simulium arcticum sibling species complex. The observed heterozygosity of the 11 loci ranged from 0.03 to 0.83. The number of alleles per locus ranged from 8 to 19. Significant linkage disequilibria were encountered only for the primer pairs BF7-1 with BF7-5 and BF6-32 with BF7-16. Presumably, these microsatellite loci can be used to study genetic structure within the entire S. arcticum complex.

Arsenic exposure disrupts neurite growth and complexity in vitro.

Arsenic has neurotoxic effects on both central and peripheral components of the mature nervous system. There is increasing evidence that exposure to arsenic is also toxic to the developing nervous system and can result in decreased cell division and increased apoptosis in cultured developing neurons. However, the effects of arsenic on subsequent neuron growth and morphology remain unclear. In the present study we used differentiating PC12 cells to investigate the effects of sodium arsenite on the early stages of neurite production and growth. We find that arsenic has concentration- and time-dependent effects on initial neurite outgrowth in vitro. Exposure to low micromolar levels of sodium arsenite for five days results in reduced neurite production, outgrowth and complexity in newly differentiating PC12 cells. Furthermore, we find that exposure of more mature PC12 cells to arsenite can inhibit further neurite development. These results suggest that exposure to arsenic can disrupt early stages of neuron differentiation by altering the normal progression of morphological development and could potentially contribute to compromised long term functioning of neurons.

Developmental appearance of cyclic guanosine monophosphate (cGMP) production and nitric oxide responsiveness in embryonic mouse cortex and striatum.

The second messenger cyclic guanosine monophosphate (cGMP) regulates multiple aspects of both structural development and physiological function in the developing nervous system. Recent in vitro experiments have shown that cGMP also modulates the response of developing vertebrate neurons to guidance molecules. This has led to the proposal that in vivo cGMP plays a critical role in directing the outgrowth of the apical dendrites of developing neurons in the cerebral cortex. Despite this proposed role, the onset, localization, and dynamics of cGMP production in the embryonic cortex are unknown. To investigate the potential contribution of cGMP in the embryo, we have used a pharmacological and immunohistochemical approach to test whether the endogenous production of cGMP, and the capacity to produce cGMP in response to nitric oxide (NO), in the cerebral cortex is compatible with the proposed developmental roles for cGMP. We find that cortical cGMP production and NO sensitivity are regionally and developmentally regulated. Cortical cGMP production begins at E15, later than in the ganglionic eminences, becomes high in the cortical plate but not the ventricular zone, and is dependent on nitric oxide synthase activity. Furthermore, although radially migrating neurons were not NO responsive until they reached the cortical plate, NO exposure revealed an additional population of tangentially migrating presumptive interneurons from the ganglionic eminences with the capacity to produce cGMP. These results provide a new level of understanding of the stage and cell type specific regulation of the NO/cGMP pathway during embryonic development.